Volume 142

Number 3

August 6, 2010

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Noncoding RNAs
Orchestrate p53
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Leading Edge
Cell Volume 142 Number 3, August 6, 2010

IN THIS ISSUE
STEM CELL SELECT

ANALYSIS
347 Stitching Together Cross-border Research C. Macilwain

CORRESPONDENCE
350 Successes of Genome-wide Association Studies R.J. Klein, X. Xu, S. Mukherjee,
J. Willis, and J. Hayes
351 Strategies for Genetic Studies of Complex Diseases K. Wang, M. Bucan, S.F.A. Grant,
G. Schellenberg, and H. Hakonarson

353 Response: Why It Is Time to Sequence J. McClellan and M.C. King

PREVIEWS
356 Fyn-Tau-Amyloid: A Toxic Triad C. Haass and E. Mandelkow

358 Noncoding RNAs: The Missing ‘‘Linc’’ A.M. Barsotti and C. Prives
in p53-Mediated Repression
360 Stem Cells and DNA Damage: Persist or Perish? A.A. Lane and D.T. Scadden

362 Mitochondrial Matrix Reloaded with RNA T. Endo, K. Yamano, and T. Yoshihisa

MINIREVIEW

364 A MAP for Bundling Microtubules C.E. Walczak and S.L. Shaw

PERSPECTIVE
368 The Pioneer Round of Translation: L.E. Maquat, W.-Y. Tarn, and O. Isken
Features and Functions

SNAPSHOT

496 Nonhomologous DNA End Joining (NHEJ) M.R. Lieber and T.E. Wilson
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Articles
Cell Volume 142 Number 3, August 6, 2010
375 Direct Reprogramming of Fibroblasts
into Functional Cardiomyocytes
by Defined Factors
387 Dendritic Function of Tau Mediates
Amyloid-b Toxicity in
Alzheimer’s Disease Mouse Models
398 Single-Stranded DNA Transposition
Is Coupled to Host Replication
409 A Large Intergenic Noncoding RNA
Induced by p53 Mediates Global Gene
Repression in the p53 Response
420 A Minimal Midzone Protein Module
Controls Formation and Length of
Antiparallel Microtubule Overlaps
433 Insights into Antiparallel Microtubule
Crosslinking by PRC1, a Conserved
Nonmotor Microtubule Binding Protein
444 Aurora Kinases and Protein Phosphatase 1
Mediate Chromosome Congression
through Regulation of CENP-E
456 PNPASE Regulates RNA Import
into Mitochondria
468 Plzf Regulates Germline Progenitor
Self-Renewal by Opposing mTORC1
M. Ieda, J.-D. Fu, P. Delgado-Olguin,
V. Vedantham, Y. Hayashi, B.G. Bruneau,
and D. Srivastava
L.M. Ittner, Y.D. Ke, F. Delerue, M. Bi, A. Gladbach,
€
J. van Eersel, H. Wolfing, B.C. Chieng, M.D.J. Christie,
I.A. Napier, A. Eckert, M. Staufenbiel, E. Hardeman,
€
and J. Gotz
B. Ton-Hoang, C. Pasternak, P. Siguier,
C. Guynet, A.B. Hickman, F. Dyda, S. Sommer,
and M. Chandler
M. Huarte, M. Guttman, D. Feldser, M. Garber,
M.J. Koziol, D. Kenzelmann-Broz, A.M. Khalil,
O. Zuk, I. Amit, M. Rabani, L.D. Attardi, A. Regev,
E.S. Lander, T. Jacks, and J.L. Rinn
P. Bieling, I.A. Telley, and T. Surrey
R. Subramanian, E.M. Wilson-Kubalek, C.P. Arthur,
M.J. Bick, E.A. Campbell, S.A. Darst, R.A. Milligan,
and T.M. Kapoor
Y. Kim, A.J. Holland, W. Lan, and D.W. Cleveland
G. Wang, H.-W. Chen, Y. Oktay, J. Zhang, E.L. Allen,
G.M. Smith, K.C. Fan, J.S. Hong, S.W. French,
J.M. McCaffery, R.N. Lightowlers, H.C. Morse III,
C.M. Koehler, and M.A. Teitell
R.M. Hobbs, M. Seandel, I. Falciatori, S. Rafii,
and P.P. Pandolfi
(continued)
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480 Myc-Nick: A Cytoplasmic Cleavage M. Conacci-Sorrell, C. Ngouenet, and R.N. Eisenman
Product of Myc that Promotes a-Tubulin
Acetylation and Cell Differentiation

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494 SIRT1 Suppresses b-Amyloid Production G. Donmez, D. Wang, D.E. Cohen, and L. Guarente
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On the cover: When p53 is active in cells, it, like an orchestra conductor, coordinates the
activation and repression of many cellular players. Among these are long intergenic noncod-
ing RNAs (lincRNAs) that contribute to the balanced modulation of transcription in the p53
response. Huarte et al. (pp. 409–419) show that lincRNA-p21, represented as a baton, is a di-
rect transcriptional target of p53 and is necessary for the repression of many target genes,
particularly those involved in apoptosis downstream of p53 activation. Artwork by Lauren
Solomon, John Rinn, Maite Huarte, and Sigrid Hart, Broad Institute; adapted from artwork
by iStockphoto.
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Leading Edge

In This Issue

Reprogramming’s Got the Beat

PAGE 375
Reprogramming of fibroblasts to induced pluripotent stem cells suggested that a somatic cell could be reprogrammed into
alternative fates. Ieda et al. now report that a combination of three developmental transcription factors, Gata4, Mef2c, and
Tbx5, rapidly and efficiently reprograms postnatal cardiac or dermal fibroblasts directly into cardiomyocyte-like cells without
passing through a cardiac progenitor state. Thus, reprogramming of endogenous or explanted fibroblasts might provide
a source of cardiomyocytes for regenerative approaches.

Fyn-ally, the Missing Link between Tau and Ab

PAGE 387
The peptide amyloid-b (Ab) and the tau protein are both found in toxic aggre-
gates in the brains of Alzheimer’s disease (AD) patients. In this issue, Ittner
et al. reveal a mechanism by which tau may collaborate with Ab in mediating
AD pathogenesis. The authors show that tau, generally thought of as an axonal
protein, targets Fyn kinase to dendritic spines, where it phosphorylates NMDA
receptors. This leads to increased excitotoxicity, which boosts the toxic effects
of Ab on neurons. Disruption of tau-mediated targeting of Fyn in a mouse model
of AD decreases excitotoxicity via NMDA receptors and ameliorates other path-
ological features associated with Ab.

p53 Gets Linc-ed In

PAGE 409
Mammalian genomes encode numerous noncoding RNAs, including a class of large intergenic noncoding RNAs (lincRNAs)
associated with p53. In this issue, Huarte et al. show that lincRNA-p21 is a direct p53 transcriptional target that also mediates
p53-dependent transcriptional repression through its physical association with hnRNP-K. These results reveal a new
aspect to the p53 transcriptional response and suggest that lincRNAs may serve as key regulatory hubs in transcriptional
pathways.

Lagging Strand Takes the Lead in Transposition

PAGE 398
For most transposons, excision and insertion require double-stranded
templates. In one recently identified family of bacterial insertion sequences
(IS200/IS605), however, the cutting and pasting is done with only a single-
stranded DNA segment. Ton-Hoang et al. now show that these single strands
are preferentially excised and inserted from the lagging strand of replicating
DNA, coupling transposition to replication fork passage. In addition to identifi-
cation of a novel transposition pathway, the unique excision and insertion prop-
erties of the IS200/IS605 family may make them useful tools for probing the
in vivo structures of ssDNA segments.

A-PRC-iating Midzone Dynamics

PAGE 420 and PAGE 433
During cell division, microtubules are arranged in the mitotic spindle to segre-
gate the duplicated chromosomes. In anaphase, tightly bundled antiparallel microtubules in the spindle center form the mid-
zone structure. Using an in vitro reconstitution approach, Bieling et al. show how PRC1, an antiparallel microtubule bundler,
and the processive motor kinesin-4 form the minimal module needed to dynamically organize the core structure of the verte-
brate midzone. Related work from Subramanian et al. provides detailed insight into how PRC1 functions. Using structural and
biophysical approaches, they find that PRC1 dimers contain both structured and unstructured domains that crosslink anti-
parallel microtubules, which allow the protein to track along the overlap at the midzone without substantially resisting relative
filament sliding.

Cell 142, August 6, 2010 ª2010 Elsevier Inc. 335

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Chromosomes Turn on a Phosphate
PAGE 444
Proper orientation of chromosomes in the mitotic spindle ensures accurate
segregation and guards against aneuploidy, a common feature of human
cancers. In this issue, Kim at al. demonstrate that CENP-E, a kinetochore motor
protein, is phosphorylated at a single site by Aurora kinases at the spindle poles
to promote chromosome congression. CENP-E is subsequently dephosphory-
lated by PP1 at the outer kinetochore, enabling bistable orientation. These find-
ings explain how spatially regulated phosphorylation can control chromosome
movements during mitosis.

Protecting the Family Jewels

PAGE 468
The mTORC1-signaling pathway is a critical regulator of cell growth. Aberrant
mTORC1 activation is associated with stem cell depletion through poorly char-
acterized targets. Now, Hobbs et al. demonstrate that Plzf, a transcription factor implicated in germline maintenance,
opposes mTORC1 pathway activity in spermatogonial progenitor cells (SPCs). Further, they show that elevated mTORC1
activity inhibits response of SPCs to GDNF, a niche-derived growth factor required for self-renewal. This study provides
important insight into mechanisms of germline maintenance and defines a model by which mTORC1 activity is detrimental
to stem cell function.

RNA Takes the Autobahn to the Mitochondria

PAGE 456
RNA import into mammalian mitochondria is poorly understood compared
with protein import. Here, Wang and colleagues report the identification of
a mammalian mitochondrial RNA import factor, the enzyme PNPASE. They
find a 20 nucleotide stem-loop RNA structure that can mediate PNPASE-
dependent mitochondrial RNA import. PNPASE-dependent imported RNAs
regulate processing of long mitochondrial RNA transcripts and the translation
of electron transport chain proteins for respiration. The study identifies a
component of the mammalian mitochondrial RNA import pathway and a poten-
tial new approach for selectively targeting RNAs to mitochondria.

Mini-Myc Goes Nonnuclear

PAGE 480
Myc family proteins are nuclear transcriptional regulators that control cell
growth, proliferation, and apoptosis. Here, Conacci-Sorrell et al. report the characterization of Myc-nick, a truncated form
of Myc generated by calpain cleavage of full-length Myc. Myc-nick lacks nuclear localization and DNA binding regions and
is predominantly cytoplasmic. Myc-nick interacts with microtubules and the GCN5 acetyltransferase to promote a-tubulin
acetylation, changes in cell morphology, and terminal differentiation. Proteolytic cleavage may provide a functional switch
from the nuclear, proproliferation form of Myc to a cytoplasmic, prodifferentiation form.

Cell 142, August 6, 2010 ª2010 Elsevier Inc. 337

#ALLåFORå0APERS
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Leading Edge

Stem Cell Select

Lineage commitment requires stem cells to loosen their grip on pluripotency, sequentially picking and
choosing among competing genetic programs to realize specific cell fates. New findings described in this
issue’s Stem Cell Select address the impact of history and cellular memory on differentiation events and
reveal critical molecular mediators of stem cell reprogramming and pluripotency.

A Remembrance of Tissues Past

According to two recent reports (Kim et al., 2010; Polo et al., 2010),
how you achieve stem cell pluripotency has unexpected conse-
quences. Kim et al. show that reprogrammed pluripotent cells from
mice created by somatic cell nuclear transfer differ from those
created through the expression of transcription factor cocktails.
Their findings suggest that cells reprogrammed by somatic cell
nuclear transfer bear greater similarity to embryonic stem cells
than induced pluripotent stem (iPS) cells reprogrammed with defined
factors, that is, at least prior to extensive passaging. Delving into the
molecular basis for this difference reveals that iPS cells retain
residual DNA methylation signatures reflecting their cell type of
Osteogenic colonies from fibroblast-derived
induced pluripotent stem cells stained with alizarin
origin. Similar observations are made by Polo et al. in their examina-
red to detect calcium deposits. Image courtesy of tion of the gene expression patterns and epigenetic marks of
K. Kim. a collection of iPS cells derived from different mouse tissues. The
two reports show that iPS cells retain a form of epigenetic memory
that makes it possible to identify which tissue iPS cells come from and, more importantly, biases their return
to that tissue type upon differentiation. Kim et al. show that further interventions, such as treatment of iPS cells
with chromatin-modifying agents or repeating the cycle of differentiation and reprogramming, alter the impact of
this epigenetic memory, whereas Polo et al. report that the epigenetic memory is dissipated by continuous
passaging of the cells in culture. These findings highlight the notion that pluripotency is not a singular condition
but a diverse spectrum of states. They also suggest that a cell’s history is not easily erased, bringing to mind the
well-known quote from William Faulkner: ‘‘The past is never dead. It’s not even past.’’
K. Kim et al. (2010). Nature. Published online July 19, 2010. 10.1038/nature09342.
J. M. Polo et al. (2010). Nat. Biotechnol. Published online July 19, 2010. 10.1038/nbt1667.

DNA Repair Gives Germ Cells a Fresh Start

DNA methylation is one of the cell’s most stable epigenetic marks.
Hajkova et al. (2010) now provide evidence that base excision repair
(BER) is actively engaged in the removal of these marks in mouse
primordial germ cells (PGCs). On their journey to totipotency,
PGCs go through a dramatic transformation in their chromatin land-
scape, with a major reorganization in their nuclear architecture and
changes in histone and DNA modifications. In cells undergoing these
reprogramming events, the authors observe an activation of BER
pathways, coincident with the removal of methylated cytosines. Chromatin-bound XRCC1 (green) in the male
The activation of BER is also temporally linked to the occurrence pronucleus of the zygote (shown) and in
of single-stranded DNA (ssDNA) breaks, a step in BER. Consistent primordial germ cells suggests the presence of
with a direct role of BER in DNA demethylation, inhibitors of BER single-stranded DNA breaks at the time of DNA
demethylation. Image courtesy of P. Hajkova.
interfere with the removal of methyl-cytosine from the paternal
pronucleus of the zygote, another setting in which active DNA demethylation is reported to occur. In relation
to the recent papers by Kim et al. (2010) and Polo et al. (2010) discussed above, future work may assess whether
promotion of this BER pathway could be used to augment existing methods for the generation of induced plurip-
otent stem cells.
P. Hajkova et al. (2010). Science 329, 78–81.

Cell 142, August 6, 2010 ª2010 Elsevier Inc. 339

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A Chronicle of Differentiation Foretold
How do events in embryonic stem cells set the stage for tissue-specific expression later in development? Liber
et al. (2010) follow the molecular events at an enhancer specific for pre-B cell differentiation to show how it is
primed in embryonic stem cells (ESCs) for activation at a later stage. The core of the pathway uncovered involves
a handover between the transcription factor Sox2, which binds the l5-VpreB1 enhancer in ESCs, and Sox4,
which binds the same region in pro-B cells. In ESCs, Sox2 promotes histone 3 lysine 4 di- and trimethylation,
which are activating histone marks, and modulates the recruitment of the Foxd3, a factor that maintains the
enhancer and the surrounding regions in a repressed state. At the pro-B cell stage, the Sox and Fox binding sites
cooperate (the former with Sox4 bound) to fully activate transcription, thereby enhancing expression of l5,
a protein that acts as a critical surrogate for the immunoglobulin light chain during B cell differentiation. The
model proposed by the authors is that factors in ESCs establish active epigenetic marks that then cooperate
with tissue-specific factors to drive transcription during differentiation. Future work is likely to address whether
ESC factors have a more general role in gene priming in this and other lineage commitment events.
D. Liber et al. (2010). Cell Stem Cell 7, 114–126.

Holding Back a Natural Killer Instinct

Becoming a T cell from a hematopoietic progenitor means avoiding
the temptations of other possibilities along the way, including B cell,
macrophage, dendritic cell, and natural killer (NK) cell fates. Three
recent papers reveal a transcription factor needed for T cell precur-
sors to take the final step of commitment (Ikawa et al., 2010; Li et al.,
2010a; Li et al., 2010b). They show that in the absence of this factor,
Bcl11b, would-be T cells can instead be redirected to a natural killer
cell fate. NK cells are lymphocytes that directly kill cells recognized
T cell lineage commitment depends on the tran- as non-self. Li et al. (2010a) demonstrate that Bcl11b is needed
scription factor Bcl11b. Image courtesy of L. Li both for T lineage commitment and to limit self-renewal, as it
and E.V. Rothenberg. promotes the downregulation of both NK cell genes and regulatory
genes that are characteristic of stem and progenitor cells. Future
work may explore which of the regulated genes are direct targets of Bcl11b. Potential functional consequences
of reprogramming T cell progenitors are explored by Li et al. (2010b), who show that even mature T cells can be
converted to NK cells by the loss of Bcl11b, and these reprogrammed cells share with native NK cells the
capacity to hinder tumor establishment in a mouse model of lung metastasis. The findings of Ikawa et al. may
serve as a starting point for efforts to identify the in vivo signals that direct this lineage decision or that maintain
progenitors in a multipotent state. They show in culture that an arrest in T cell development can be promoted by
interleukin-7 (IL-7), thereby maintaining their myeloid and natural killer cell potential, due to failure to induce
Bcl11b. A topic for future examination is to determine how signaling by IL-7 or other cytokines intersects with
Bcl11b at the decision point for T cell commitment.
T. Ikawa et al. (2010). Science 329, 93–96.
L. Li et al. (2010a). Science 329, 89–93.
P. Li et al. (2010b). Science 329, 85–89.

Robert P. Kruger

Cell 142, August 6, 2010 ª2010 Elsevier Inc. 341

“As financial resources become more scarce,
it is more critical to identify research and
researchers who are the most productive and
on the right track.”
Peter Brimblecombe
Professor, Atmospheric Chemistry
School of Environmental Sciences
University of East Anglia, UK

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 015.A1.0101.F.US © 2009 Eppendorf AG.
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 Expand your stem cell library and save
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Leading Edge

Analysis

Stitching Together Cross-border Research

European research has been hampered by fragmented national research programs. Is joint
programming the answer? Colin Macilwain investigates.

The nations of the European Union (EU) In the last 2 years, however, a new project would focus on neurodegenerative
spent €80 billion ($100 billion) last year on fix has been proposed for the problem: disease research, which is particularly
public nonmilitary research and develop- “joint programming” between national weak and fragmented in Europe (Figure
ment, yet European science still seems research agencies. The idea is to get 1). The European Commission’s research
to have a quality gap compared with the interested nations to band together directorate estimates that US spending in
US. For example, the EU produces 33% of and agree on a detailed strategy for a this area ($856 million, or €527 million, in
research papers published annually world- given research field and then pick-and- 2007) is almost ten times that of Europe
wide but garners only 34% of citations, choose which elements of that strategy ($93 million, or €57 million).
compared with the US, which publishes to collaborate on. “Joint programming Alzheimer’s disease researcher Bart
29% of papers but earns 41% of citations is critical to the future, but it is still in De Strooper of KU Leuven in Belgium
(http://www.nsf.gov/statistics/seind08/). gestation,” says Frank Gannon, former agrees that neurodegenerative dis-
Policymakers believe that one reason for director of the European Molecular Biol- ease research is lagging in Europe. “My
this quality shortfall is the fragmentation ogy Organization and current member impression is that in the United States,
of research spending in Europe. Accord- of the European Research Area Board, much more of a vision has been devel-
ing to the European Commission based which advises the European Commis- oped with regard to problems of aging,
in Brussels, 85% of public research funds sion. “From my point of view, it is cur- and Alzheimer’s in particular,” he says,
in Europe are distributed through sepa- rently the most crucial, single thing that pointing to collaborations such as the
rate national programs run by the EU’s 27 we have to put right.” Alzheimer’s Disease Neuroimaging Ini-
member states. Many think that the way to Last December, the Council of Ministers tiative (http://www.loni.ucla.edu/ADNI/),
get more bang for their euro is to tie these representing the EU member states con- which is supported by several institutes
national activities more closely together. firmed that the first joint programming pilot of the National Institutes of Health (NIH)
Multiple attempts to unite different
European research programs have
failed, however. The €7 billion that the
European Commission allocates for
research annually through its Frame-
work Programme is supposed to nur-
ture cross-border collaborations but
does so one project at a time. And
other efforts—including the long-
established European Cooperation
in Science and Technology (COST)
scheme and plans in the Framework
6 Programme (which ran from 2002
to 2006) for “integrated projects” and
“networks of excellence”—tried and
failed to link the national research pro-
grams together. All of these efforts have
foundered on a mixture of bureaucracy,
nationalism, inertia, and the reluctance
of top researchers, who are able to get
funding in their own countries, to get
involved. “It is very difficult for member
states to come together on a common
basis,” concedes Enda Connolly, chief
executive of Ireland’s Health Research
Board, which distributes €40 million Figure 1. Europe’s Research Landscape
annually for biomedical research in Ire- Brain disease research in Europe, including the study of neurodegenerative diseases such as Alzheimer’s
and Parkinson’s, is weakly coordinated across Europe and receives less investment compared with the
land. “They are all locked into their own US. (x axis, degree of coordination; y axis, spending in Europe relative to the US). Source: European
programs.” Commission, 2008 (ec.europa.eu/research/press/2008/pdf/com_2008_468_en.pdf).

Cell 142, August 6, 2010 ©2010 Elsevier Inc.  347

and the private sector. “We in Europe
look to the US and are happy if some of
us are incorporated in their initiatives,” he
says. “We should be much more active
on the international scene.”
The idea of a combined approach
to boost neurodegenerative disease
research was first proposed in 2008,
when France held the rotating presi-
dency of the EU and President Nicolas
Sarkozy sought to push aging issues
higher up Europe’s research agenda.
In July of that year, the European Com-
mission issued a paper advocating joint
programming as a generic approach to
improving coordination among national
research bodies. Meanwhile, medical
research agencies in France, Germany,
and the UK were already seeking ways
to strengthen their respective involve-
ment in an area of biology where the US
has a pronounced lead (http://www.mrc.
ac.uk/Utilities/Documentrecord/index.
htm?d=MRC004898).
Philippe Amouyel, an epidemiologist
at the University of Lille in France, led
a working group established in 2008 to
look into the idea of joint programming
in the discipline and is now chair of the
board of the EU Joint Programme for
Neurodegenerative Disease. (Connolly
also sits on the initiative’s five-person
management board.) Initially supported
by INSERM, the French biomedical
research agency, the working group now
has a small €2 million European Com-
mission grant to cover its administration
costs.
The Joint Programme has appointed
a fifteen-member scientific advisory
board, which met in Stockholm in April
and will now draw up a 5 year strate-
gic plan for neurodegenerative disease
research in Europe. The board is made
up of five social scientists, five clini-
cians, and five biologists: Jesus Avila
of the University of Madrid, Bart De
Strooper, John Hardy of University Col-
lege, London, Leszek Kaczmarek of the
Polish Academy of Sciences and the
chairman, Thomas Gasser of the Uni-
versity of Tubingen. There is no budget
yet to implement the plan, but officials
involved in the discussions say that it is
likely to involve an investment of about
€200 million over 5 years, mainly from
national funding bodies. “For the very
first time,” says Amouyel, “we’ll have
348  Cell 142, August 6, 2010 ©2010 Elsevier Inc.
a common view in Europe of what we
need to do in neurodegenerative dis-
ease research.”
Most of the research supported will
be in basic neurobiology, including
sequencing the complete genomes of
patients to find risk genes, the devel-
opment and standardization of disease
biomarkers, and developing better ani-
mal models of these diseases. “The big
problem is that we don’t have a pipeline
for new therapies, so we need to better
understand the fundamental pathophys-
iology of these diseases,” says Rob
Buckle, program manager for neurosci-
ences and mental health at the UK Medi-
cal Research Council (MRC). “This is an
opportunity to do things in a different
way in Europe.” Buckle says he hopes
that the Joint Programme will do work
that could be relevant to the treatment of
several neurological diseases, including
Parkinson’s and motor neuron disease,
as well as Alzheimer’s. “Mechanistically,
there’s a lot of overlap between these
disorders,” he says.
Gasser, a neurologist who studies
Parkinson’s disease, says the scien-
tific advisory board will confer broadly
before publishing its research plan in
summer 2011. Three meetings early next
year, involving interested biologists, cli-
nicians, and social scientists, respec-
tively, will help the process along. Gas-
ser adds that there is a “huge political
will” to make the Joint Programme work
but notes that its directed approach “will
never displace bottom-up, undirected
basic research that can give us com-
pletely new insights.”
There are already some examples
of the kinds of collaboration that might
proceed under the Joint Programme. On
June 29th, for example, the UK MRC,
the German Centre for Neurodegenera-
tive Diseases (DZNE), and the Canadian
Institutes of Health Research (CIHR)
announced a £3 million ($4.6 million) col-
laboration on methods, technologies,
and data sharing in neurodegenerative
disease research. (Canada’s involvement
reflects the MRC’s desire to cooperate
with partners outside Europe who have
relevant expertise.) And Amouyel says
that some test projects may go ahead
under the Joint Programme before the
strategic plan’s completion, in areas such
as genomics, the standardization of bio-
markers, developing new therapeutics,
and infrastructure for large clinical trials.
“They could get going by the end of the
year,” he says, adding that the initiative
is moving quickly. “To get something
from concept to practical action in three
years is really new in European research
policy.”
De Strooper adds that as a researcher
in Belgium, he sees advantages in col-
laborating with larger countries—France,
Germany, and the UK—that are starting
initiatives in neurodegenerative disease
research. He says that he hopes the
Joint Programme will help researchers to
do animal modeling and drug screening,
obtain microRNA profiles of patients,
and do deep sequencing and annota-
tion of expression profiles for Alzheim-
er’s and other diseases. Alzheimer’s
researcher and geneticist John Hardy
says he hopes that the program can
help researchers to find better biomark-
ers for neurodegenerative diseases and
to use rapid, full-genome sequencing of
patients to find the risk genes for them.
But he admits that it is too early to know
how it will unfold. “You get involved in the
process, but you never know if there’s
going to be a good outcome,” he says.
“There is a genuine need for it. But you
do worry that the optimism behind the
programme will end up clashing with the
hard reality of budget cuts.”
Although the joint programming pilot
has been broadly welcomed, many
experienced researchers and research
administrators have questioned whether
the joint programming approach has
enough backing or momentum to have
much impact on fragmented European
research. Critics charge that the overall
approach is ill-defined, inadequately pro-
moted, and—most of all—underfinanced.
Physicist and European Research Advi-
sory Board member Jerzy Langer of the
Polish Academy of Sciences says he
fears that it won’t get much further than
a long line of prior Commission efforts to
get national research agencies to work
more closely together. “I’m not saying
it is wrong,” he says. “But any initiative
that starts from the bureaucracy tends to
go nowhere.” Langer contends that joint
programming still has little constituency
among researchers. “The key question,”
he says, “is when, and in what capacity,
real researchers get involved.”
 “There is serious added value to be
achieved if we can use joint program-
ming to get national funding used more
effectively,” says Ian Halliday, president
of the European Science Foundation in
Strasbourg. “But politically and organi-
zationally, this is not going to be easy.”
And observers of the joint programming
pilot say that the MRC and INSERM are
already frustrated by the need to accom-
modate participation by the 24 EU nations
who have signed up for the pilot—many
of whom cannot contribute much in the
way of cutting-edge neurobiology. Some
suggest that participants will have to
learn to do what physicists have done at
CERN, the successful European particle
physics center in Switzerland, and find
ways of accommodating partners who
are less scientifically advanced.
Buckle denies that this is a problem
for the MRC, arguing that the Joint Pro-
gramme for Neurodegenerative Disease
will be an “umbrella” for the sharing of
information and will allow “different con-
stellations” of nations to work together
on different areas of interest. “Everyone
involved will be signed up to the same
top-level objectives,” he says, “but
everyone will have different interpreta-
tions of how to best achieve them.”
Backers of joint programming say it
can work because it is the EU member
states, through the Council of Ministers,
who have endorsed the approach and
have pledged to see it through. “For the
first time, member states are coming
together at the ministerial level to iden-
tify jointly areas where public research
can contribute to tackling Europe’s
major societal problems,” said Maire
Geoghegan-Quinn, the newly appointed
European Commissioner for research,
in a statement. “It is precisely because
it is underpinned by a high-level, strate-
gic and structured process—and most
importantly of all, by real political will—
that joint programming is a very big step
forward.”
The Commission’s approach to the
idea is finely nuanced, however, because
if joint programming is seen as a Com-
mission project, member states will
view it as a means of getting Commis-
sion money. So Commission officials are
orchestrating it from behind the scenes,
hoping that national governments will
take the actions needed to provide
money and drive it forward. But there are
hints that large-scale Commission fund-
ing to support joint programming could
become available under the next phase
of the Framework Programme, FP8,
which starts in 2014.
Governments are certainly watching
the pilot with interest. In April, the Com-
mission recommended three further
pilots—in food security, healthy diets,
and cultural heritage conservation. In
tough economic times, at least in theory,
joint programming could help to reduce
duplication enabling research funds to
be spent more efficiently. “In a way, a
shrinking budget is an opportunity,” says
Cell 142, August 6, 2010 ©2010 Elsevier Inc.  349
Amouyel, “because you have to allocate
resources more efficiently.” And in fields
such as neurodegenerative disease,
where even the largest national research
agencies cannot cover every aspect, the
joint programming approach may help.
Yet the odds remain stacked against
it making much difference: EU member
states control five-sixths of Europe’s
research budget, and they want to spend
it on their own scientists. A 2009 study
by Eurohorcs, the federation of national
research councils, found that 14 out of
32 of the national agencies surveyed had
legal prohibitions on supporting work
outside their borders (http://www.era.
gv.at/attach/EUROHORCs.pdf). At the
larger agencies, around 5% of the budget
is usually devoted to collaborations with
European partners (some small agen-
cies, such as the Foundation for Polish
Science and the Greek National Hellenic
Research Foundation, spend proportion-
ally more). The perceived political draw-
backs of spending dwindling research
funds “abroad” remain daunting, however.
And there is a strong imperative within
national research agencies to hold on to
control of what they have. “There’s always
going to be people at national agencies
who fear that they could lose out” from
joint programming, agrees Connolly. “But
there’s very strong commitment at the
political level, and in the European Com-
mission, to making this happen. Member
states are coming to see that they can’t
do everything on their own.”
Colin Macilwain
Edinburgh, UK
DOI 10.1016/j.cell.2010.07.031

Successes of Genome-wide
Association Studies
In a recent Essay in Cell, McClellan and
King argue that genomic resequencing
rather than genome-wide association
studies (GWAS) will be necessary to
understand the genetic basis of common
disease (McClellan and King, 2010). Like
the authors, we too are excited about the
potential for emerging sequencing tech-
nologies to facilitate discoveries that
explain the missing heritability of com-
mon diseases. However, we disagree
with the implication that GWAS have not
been successful to date. Instead, we
propose that insofar as the goal of these
studies is to understand the etiology of
heritable diseases, GWAS have provided
numerous tantalizing clues for us biolo-
gists to decipher. Rather than disprove
the common disease/common variant
hypothesis, we find that results from
GWAS support the contention that com-
mon polymorphisms do directly contrib-
ute to disease risk, validating the linkage
disequilibrium-based GWAS approach
for helping to identify variants underlying
disease. Although we do not dismiss the
likelihood that rare variants also contrib-
ute to common diseases, we expect that
whole-genome sequencing approaches
will show that the full spectrum of alleles,
from rare to common, play important
roles in disease etiology. Here, we argue
that the existence of common disease-
causing polymorphisms is not inconsis-
tent with population genetic theory and
that actual results from GWAS suggest
that the reported associations represent
real biology rather than false positives.
The contention that deleterious alleles
that cause human diseases are common
in the population may seem paradoxical,
but several mechanisms can explain how
such pathogenic alleles can overcome
negative selective pressure. First, accu-
mulating evidence demonstrates that
there is balancing selection in which a
certain allele confers susceptibility to one
disease while simultaneously conferring
protection from another. The best known
example is heterozygosity for sickle cell
anemia, which affords protection against
malaria. GWAS have identified other
350  Cell 142, August 6, 2010 ©2010 Elsevier Inc.
Correspondence
such instances, for example, the TCF2
(or HNF1B) gene where alternate alleles
are risk factors for type 2 diabetes and
prostate cancer (Gudmundsson et al.,
2007). More generally, several loci where
alleles have opposite effects on the risk of
developing type 1 diabetes and Crohn’s
disease have been reported (Wang et al.,
2010), and it is likely that more examples
of balancing selection are yet to be dis-
covered.
The argument that common pathogenic
variants must have withstood selective
pressure throughout human history is
predicated on the assumption that mod-
ern humans developed in the same envi-
ronment that we exist in today. However,
due to the rapid acceleration of human
development in the recent evolution-
ary timeframe, numerous environmental
changes have occurred that may impact
the risk of complex common diseases.
For instance, variants that were ben-
eficial in the past may well have turned
against their carriers as human lifestyles
changed. The “thrifty gene hypothesis”
suggests that variants that predispose
to type 2 diabetes and obesity may have
conferred a selective advantage in times
of famine (Neel, 1962). However, in devel-
oped countries, where food tends to be
in overabundance, type 2 diabetes and
obesity have become common diseases.
Furthermore, existing neutral variation
may manifest positive or negative effects
as new environmental modifiers come into
play. A set of single-nucleotide polymor-
phisms (SNPs) associated with lung can-
cer and located at a locus encoding the
nicotinic acetylcholine receptor appears
to have a stronger effect on lung cancer
risk in smokers born long ago relative to
those born more recently (Landi et al.,
2009); this effect has been attributed to
changes in the composition of cigarettes
over time. This demonstrates that recent
environmental changes can alter the
disease-influencing effect of a common
variant. These examples are likely to be
only the tip of the iceberg of phenotypic
effects modulated by gene-environment
interactions.
Leading Edge
The fact that most SNPs identified by
GWAS do not lie in coding regions or other
known regulatory elements is expected
from the study design and is not evidence
of false positives. The assumption under-
lying the design of GWAS and choice of
genotyped SNPs is that the true functional
allele will be nearby and correlated with
the initial SNP through linkage disequi-
librium. When one considers linkage dis-
equilibrium, there is an observed excess of
GWAS hits that influence promoter regions
or change the protein-coding sequence
of a gene and a relative paucity of hits in
intergenic regions (Hindorff et al., 2009).
Moreover, many disease-associated SNPs
identified by GWAS are located in genes or
pathways previously known or suspected
to play a role in disease etiology. Recent
GWAS for Alzheimer’s disease, Crohn’s
disease, type 1 diabetes, and type 2 dia-
betes have rediscovered SNP associations
previously reported from candidate gene
studies. The demonstration that GWAS
can identify common disease suscepti-
bility variants provides a positive control
(Hindorff et al., 2009). More generally, the
functional pathways of GWAS-identified
genes often make sense; for instance,
numerous inflammatory genes have been
implicated by GWAS in inflammatory bowel
disease (Hindorff et al., 2009). We have
found a similar concordance between the
literature and GWAS hits in our own work.
In a GWAS for age-related macular degen-
eration, an SNP in complement factor H
strongly associates with disease risk; this
is consistent with previous suggestions
that the complement pathway plays a role
in disease etiology (Klein et al., 2005). Sim-
ilarly, using GWAS, we identified a germ-
line variant in the intron of the JAK2 gene
that is associated with myeloproliferative
neoplasms; JAK2 is known to harbor acti-
vating oncogenic somatic mutations in this
disease (Kilpivaara et al., 2009). One would
not expect such correlations between
GWAS findings, genic regions, and known
disease biology if these findings were ran-
domly distributed false positives due to
population stratification or other causes.
As these associations are likely to be real,
the most logical and parsimonious expla-
nation is that, in general, GWAS success-
fully identify disease-associated variants
and that variants found through GWAS tag
regions important for the biology of these
diseases.
 In light of this, it is likely that GWAS hits
found in intergenic regions far from known
genes are true associations whose biol-
ogy is not yet understood, rather than
false positives. The human genome is
incompletely annotated. Regions where
GWAS associations have been found, but
no known genes are located, could easily
harbor unidentified new genes or regula-
tory elements. For instance, the authors
point to the colon and prostate cancer risk
SNP rs693267 located 335 kb upstream
from the MYC gene on chromosome 8q24
(McClellan and King, 2010). This locus has
been shown to physically interact with
MYC and is associated with enhanced
Wnt signaling. Therefore, although the biol-
ogy of this locus is not fully understood,
it suggests a paradigm where intergenic
disease-associated SNPs alter enhancer
elements, either directly or through linkage
disequilibrium, and therefore cause differ-
ential regulation of disease-related genes.
This observation leads to a broader
point: A lack of biological understanding
of how these disease-associated vari-
ants are pathogenic does not mean that
there is no biology to discover. Although
our understanding of the mechanisms
by which disease risk loci contribute to
pathogenesis currently lags behind the
pace at which new loci are discovered,
promising stories continue to emerge. To
continue the previous example, although
no definitive correlation between the
rs6983267 genotype and MYC expression
has been demonstrated, MYC is known to
be tightly regulated and the right develop-
mental time point may need to be exam-
Strategies for Genetic
Studies of Complex Diseases
In a recent Essay published in Cell,
McClellan and King discussed genetic
heterogeneity and the potential role of
rare genetic variants in complex human
diseases (McClellan and King, 2010).
These important issues, in particu-
lar the application of high-throughput
ined to see such a correlation. Although
further work is necessary to uncover the
elusive mechanism by which the SNP
confers risk, we propose that the existing
evidence supports rather than refutes this
SNP as a true cancer risk allele. Another
example is a non-protein-coding region
of chromosome 9q21 in which SNPs
have been robustly associated with arte-
rial disease. A recent paper reported that
targeted deletion of an orthologous region
in mouse interferes with cis-regulation of
nearby genes (Cdkn2a/Cdkn2b) and may
influence vascular cell proliferation (Visel
et al., 2010). As a third example, an intronic
type 2 diabetes risk SNP (rs7903146) was
recently found to overlap with a region of
islet cell-selective chromatin, and the two
alleles of rs7903146 correlate with the
open/closed chromatin state of the region
(Gaulton et al., 2010). Thus, understanding
the mechanisms by which GWAS loci con-
tribute to disease will require considerable
effort and time. We take this not as a sign
that the common disease-common vari-
ant model has failed but rather that a chal-
lenge exists for the scientific community—
a challenge that must be addressed with
both traditional experimental genetics and
innovative new approaches.
Robert J. Klein,1,* Xing Xu,1 Semanti
Mukherjee,1 Jason Willis,1 and James
Hayes1
1
Program in Cancer Biology and Genetics,
Memorial Sloan-Kettering Cancer Center,
New York, NY 10065, USA
*Correspondence: kleinr@mskcc.org
DOI 10.1016/j.cell.2010.07.026
sequencing techniques to discover
disease genes, are highly relevant to
genetics researchers. However, the
authors allocated a substantial propor-
tion of their efforts to being critical of
the utility of genome-wide association
studies (GWAS). These particular sec-
Cell 142, August 6, 2010 ©2010 Elsevier Inc.  351
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tions of the Essay may lead to misin-
terpretation of published studies by us
and others. For the broad readership of
Cell and for the scientific community in
general, we highlight our concerns in
this Correspondence.
The authors refer to the fact that most
single-nucleotide polymorphisms (SNPs)
detected in GWAS reside in intergenic
regions and consequently challenge the
utility and reliability of GWAS with the
question: “How did genome-wide asso-
ciation studies come to be populated by
risk variants with no known function?”
When addressed in the proper context,
however, it is well established that GWAS
do not attempt to identify functional
SNPs but rather “tag” the approximate
location of disease variants, typically
down to 100 kb or less. This is made
possible due to the linkage disequilib-
rium (LD) patterns characterized by the
International HapMap project, that is, the
correlation of genotypes between the
yet-to-be-determined underlying disease
variant and neighboring SNPs. Indeed,
the vast majority of SNPs used in GWAS
are of unknown biological function, due
to the fact that most SNPs reside outside
of coding regions and that the manufac-
turers of the SNP arrays selected SNPs
from the HapMap to facilitate efficient
tagging across the genome, that is, the
priority was information capture rather
than putative function. Furthermore,
noncoding SNPs identified by GWAS
may reveal intergenic regulatory ele-
ments that are critical to understand,
and it is now up to the genetics com-
munity to develop approaches to interro-
gate the function of regulatory variants.
The authors did refer to LD as a potential
explanation for noncoding variants yield-
ing association in GWAS, but they failed
to recognize that the design of GWAS is
not to directly interrogate causal variants
in the first instance. For example, the two
GWAS that we have conducted for sickle
cell anemia and hearing loss (Dickson et
al., 2010) yielded top hits in intergenic
regions, but these are in close proximity
to the causal genes (HBB and GJB2) that
were already well-established before the
GWAS era. These studies demonstrate
the reliability of GWAS for identifying the
approximate locations of disease genes
by noncoding SNPs. In addition, they
represent two vivid examples of how
GWAS can work by leveraging LD.
McClellan and King have also attrib-
uted many published GWAS hits to
population stratification. In the absence
of scientific support or statistical deriva-
tion, they claim that “an odds ratio of 3.0,
or even of 2.0 depending on population
allele frequencies” would be robustly
interrogated by GWAS. The vast major-
ity of published GWAS loci therefore
fall below the threshold for “popula-
tion stratification.” However, compared
to candidate gene association studies,
352  Cell 142, August 6, 2010 ©2010 Elsevier Inc.
the beauty of whole-genome SNP data
is that inflation of test statistics due to
population substructure can be read-
ily identified and adjusted. Populations
do not differ by just one or two SNPs;
instead, they differ at many loci such that
whole-genome data aid in identifying
stratification, including extremely fine­
scale subpopulations among Europeans
(Novembre et al., 2008). The GWAS com-
munity has established methods to deal
with population stratification, and these
methods effectively adjust for common
variants without controversy. There are
certainly challenges with the analyses of
rare variants, hypervariable variants, or
interrogation of recently admixed popu-
lations, which are all active topics of cur-
rent research. There are now standard
practices to handle population stratifica-
tion in GWAS, such as genomic control,
EigenStrat, and multidimensional scal-
ing. Furthermore, family-based study
designs have the advantage of pro-
tecting against stratification. Thus, the
GWAS community has developed ways
to address population stratification, and
odds ratios of 1.5, 1.2, or even 1.1 have
proven to be bona fide signals rather than
artifacts of population stratification.
The Essay’s authors go on to claim
that the autism locus reported by our
group (Wang et al., 2009) is a “false posi-
tive” due to population stratification, but
the study was driven by family-based
cohorts both at the discovery and repli-
cation stages, with case-control cohorts
further supporting the finding. Here,
McClellan and King ignore the fact that
family-based analysis is robust against
stratification and have mistakenly con-
sidered GWAS hits as “false positives”
if allele frequencies varied across Euro-
pean or HapMap populations. For exam-
ple, they claim that our reported autism-
associated SNP, rs4307059 (Wang et
al., 2009), was a “particularly dramatic
example of the perils of cryptic popula-
tion stratification.” Their reasoning is that
the frequency of the proposed risk variant
varies from 0.21 to 0.77 across European
populations and that it is monomorphic in
African populations. However, they used
data from the Human Genome Diver-
sity Project that examined 938 samples
from 51 worldwide populations (Coop
et al., 2009), including 7 samples from
Tuscany (allele frequency = 0.77) and 15
samples from Orkney (allele frequency =
0.21). When dealing with whole-genome
data, small sample sizes often lead to
biased estimates of allele frequencies,
which we have now proven to be the
case in this instance. We queried the
101 Tuscan samples included in the
HapMap 3 project (http://www.sanger.
ac.uk/humgen/hapmap3) and found
that the allele frequency is 0.41, which
is very similar to what we reported for
our European American cohort (0.39
in Wang et al., 2009) and substantially
different from 0.77 quoted by McClel-
lan and King. Furthermore, even if the
allele frequency measures are accurate,
McClellan and King should not claim this
SNP as “hypervariable” without appro-
priate control experiments to compare
it with other SNPs. They may not appre-
ciate that the vast majority of SNPs do
have variable allele frequencies between
populations; in fact, 44% of SNPs on the
Illumina array (http://hgdp.uchicago.edu/
Browser_tracks/FST) have more extreme
population divergence (measured by
Fst values) than rs4307059. Similarly,
another SNP, which has been replicated
for its association with type 1 diabetes
(Hakonarson et al., 2007) in over 20 inde-
pendent studies, is ranked in the 29th
percentile for Fst value. As such, it is our
interpretation that the authors took a ran-
dom SNP from the middle of a ranked list
and inappropriately claimed it as a “par-
ticularly dramatic” example of “popula-
tion stratification.” Extending this further,
we also wish to point out that replication
of GWAS signals depends on many fac-
tors, including the power of the study,
the sample ascertainment scheme or
diagnostic criteria, and the heterogene-
ity of the study population (for example,
sporadic versus familial cases). For neu-
ropsychiatric diseases, it is well known
that replication can be difficult with small
sample sizes, as is the case for the study
specifically referred to by the authors
(Weiss et al., 2009), which interrogated
258 families with autism for replica-
tion that were not part of AGRE (Autism
Genetic Resource Exchange). Never-
theless, the autism locus on 5p14.1 has
been replicated in a family-based GWAS
without case-control comparisons (Ma et
al., 2009) and has been associated with
communication-spectrum phenotypes
through quantitative trait associations
in 7313 children from a UK birth cohort,
eliminating concerns of population strati-
fication (St Pourcain et al., 2010).
Like McClellan and King, we expect
that with the development of high-
throughput sequencing technologies,
whole-genome sequencing will be an
invaluable tool for the study of rare
genetic variants contributing to com-
plex diseases. However, the apparent
importance of rare variants does not
discount the contribution of common
variants; the concerns about population
stratification should not be overstated
and certainly should not be presented
as an argument to discredit many pub-
lished GWAS signals with an odds ratio
of less than 2. These sections within
an otherwise excellent Essay must be
countered so that Cell readers have a
more balanced view of the current state
of the field.
Response
Why It Is Time to Sequence
In response to our Essay “Genetic Het-
erogeneity of Human Disease” (McClellan
and King, 2010), Wang et al. and Klein et al.
challenge our critique of GWAS findings.
Both Correspondence suggest that our
conclusions lack an understanding of the
principles of the common variant-common
disease model and its application in GWAS
methodology. We respectfully disagree,
and in fact our Essay directly addressed
many of their points. We are happy to clar-
ify further. The issue is not ignorance of
GWAS methodology; the issue is reconcil-
ing GWAS findings with population genet-
ics, evolution, and biology.
There is no dispute that common
genetic variants influence human traits.
Alleles with the strongest influences on
human traits are associated with benign
phenotypes, such as hair color and
eye color, that vary across individuals.
Alleles for these traits lie in coding and
known regulatory regions (Hindorff et
al., 2009). Such variants have been influ-
enced by selection in human evolution.
Kai Wang,1 Maja Bucan,2 Struan F.A.
Grant,1,3 Gerard Schellenberg,4 and
Hakon Hakonarson1,3,*
1
Center for Applied Genomics, Children’s
Hospital of Philadelphia
2
Department of Genetics
3
Department of Pediatrics
4
Department of Pathology and Laboratory
Medicine
University of Pennsylvania, Philadelphia, PA
19104, USA
*Correspondence: hakonarson@chop.edu
DOI 10.1016/j.cell.2010.07.025
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alleles that truly influence disease does
not imply that all GWAS findings are
valid. To date, published GWAS have
reported more than 500 single-nucle-
otide polymorphisms (SNPs) associated
with various diseases and traits, 88% of
which lie in introns or intergenic regions,
and for which the median odds ratio for
effect size is 1.33 (Hindorff et al., 2009).
Few of these associations have biologi-
cal support.
In order to identify alleles that influence
disease using GWAS, two fundamen-
tal criteria must be met: (1) a common
Cell 142, August 6, 2010 ©2010 Elsevier Inc.  353
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variant influencing the trait in question
must truly exist in the population being
studied; and (2) the genotyped markers
used in association analyses must either
include the causal variant or be in link-
age disequilibrium (LD) with the causal
variant in the population being stud-
ied. Common “risk SNPs” detected by
GWAS could in theory be in LD with rare
disease-causing alleles, if by chance
rare causal alleles are disproportionately
linked with a common variant (Dickson et
al., 2010).
In addressing our critique, both
Wang et al. and Klein et al. acknowl-
edge that GWAS methodologies are
designed to detect SNPs primarily
found in intergenic or intronic regions,
given the construction of standard SNP
arrays. They argue that the risk SNPs
implicated by GWAS are not expected
to be causal but instead are in LD with
true causal variants. Wang et al. main-
tain that standard GWAS methodolo-
gies adequately control for population
stratification. Further, they note that
a large proportion of HapMap SNPs
vary widely across populations, and
therefore it is not surprising that highly
variable SNPs emerge as risk vari-
ants. These issues are at the crux of
the debate and will be the focus of our
response.
Genetic heterogeneity across human
populations limits GWAS methodology
and may result in the identification of “risk
alleles” that are neither likely to be func-
tional nor likely to be in LD with alleles that
are. The genetic architecture of human
disease is shaped by the same evolu-
tionary forces that impact the human
genome. Genetic architecture is dic-
tated by four factors: mutation, selection,
migration, and drift. Negative selection
globally reduces variation at missense
mutations, particularly in genes associ-
ated with disease (Barreiro et al., 2008).
Frequencies of missense alleles (i.e.,
nonsynonymous SNPs) are less diverse
across populations than are frequencies
of synonymous SNPs, SNPs in untrans-
lated regions (UTRs), and intronic SNPs
because selection reduces interpopula-
tion variation for alleles influencing phe-
notypic expression (Garte, 2010). Popula-
tion differences in genic regions, primarily
at nonsynonymous SNPs and 5`UTR vari-
ants, may persist as the result of selec-
tion for adaptive responses or by chance
(Barreiro et al., 2008). For alleles influ-
encing phenotypes, selection constrains
overall variation in allele frequency while
also influencing specific patterns of varia-
tion defined by geographic ancestry.
Many SNPs, inversions/deletions
(indels), and short tandem repeats
vary widely in allele frequency among
populations. This is especially true for
variants that are not in coding or regu-
latory regions. Such alleles vary with
population clusters in patterns more
consistent with neutral drift and migra-
tion rather than with selection (Coop et
al., 2009). The colonization of the world
by modern humans was carried out
by a series of founder populations with
subsequent rapid expansion of popula-
tion size. Neutral alleles emerging at the
forefront of these expansions “surfed”
waves of population growth. Variations
in allele frequencies across populations
stem from differences in the timing of the
variant’s emergence in the expansion.
Intergenic or intronic SNPs that vary
widely in frequency among populations
are most likely neutral alleles reflect-
ing the history of human migration.
Wide variations in allele frequency also
354  Cell 142, August 6, 2010 ©2010 Elsevier Inc.
characterize many “risk alleles” identi-
fied by GWAS, including, for example,
rs4307059 reported to be associated
with autism (Wang et al., 2009), which is
a major focus of the Correspondence by
Wang et al. We suggest that associations
based on such highly variable SNPs are
often artifacts of cryptic population strat-
ification. Wang et al. argue that standard
GWAS strategies have been adopted to
control for population stratification. How-
ever, these methods control by person,
not by SNP. Because populations from
large geographic areas (e.g., Europe) are
genetically heterogeneous, outlier SNPs
that vary widely among subgroups of
such populations are not excluded by
these methods and often drive positive
associations.
Wang et al. also assert that the asso-
ciation of rs4307059 and autism was
driven by family-based cohorts, which
are robust to stratification. However,
no SNPs reached genome-wide signifi-
cance in their study using a family-based
design (Wang et al., 2009). Significance
was obtained by pooling subjects from
both family-based and case-control
samples, including thousands of unre-
lated cases and controls. Analyses
only comparing affected to unaffected
persons within families will be robust
to stratification; but comparing a mixed
series of related and unrelated cases to
controls can exacerbate stratification
due to the clustering of shared ances-
tries in the affected group.
In arguing for rs4037059 as a risk vari-
ant for autism, Wang et al. dismiss the
negative results of a replication study
(Weiss et al., 2009) due to small sample
size. Yet, they cite another study (Ma et al.,
2009) as supporting an association with
this genomic region (5p14.1), even though
no SNPs in this study met genome-wide
significance and rs4037059 was not even
nominally significant in the discovery
sample. Variable weak associations of
different SNPs across the same genomic
region in different cohorts do not consti-
tute replication.
Both Klein et al. and Wang et al. suggest
that the vast majority of GWAS risk alleles
are in LD with causal mutations, and that
intergenic and intronic risk variants rep-
resent regulatory elements. In principle,
either or both of these hypotheses could
be true. However, thus far, virtually no
such mutations or elements have been
found by following up on GWAS findings.
Wide variations in allele frequency across
populations argue against shared hap-
lotype structure, which is necessary to
detect causal variants in LD.
Common variants influencing traits or
disease must withstand selection in every
generation in order to be maintained at
polymorphic frequencies worldwide.
Regulatory elements are impacted by
the same evolutionary forces as coding
regions. Klein et al. discuss alternative
methods whereby otherwise deleterious
alleles are maintained in the population,
citing both well-known and speculative
examples of balancing selection and
gene-environment interactions. How-
ever, as is the case for much of this dis-
cussion, a confirmed biological mecha-
nism for one illness does not mean that
all GWAS findings can be attributed to
similar mechanisms.
Klein et al. suggest that a common
risk allele may have been under neu-
tral or positive selection in early human
history, thus explaining its worldwide
prevalence and maintenance in the
population despite the association with
disease. Yet evolution is an ongoing
process. If a common variant decreases
reproductive fitness during modern his-
tory, coinciding with the vast expansion
of the human population, the frequency
of the variant will decline. Klein et al.
also suggest that many common risk
variants, either the identified SNP or
a causal variant in LD, operate by vir-
tue of some yet-to-be-determined bal-
ancing selection or gene-environment
interaction. This is obviously possible
but needs to be demonstrated for any
given variant. In the presence of a true
balancing selection or gene-environ-
ment interaction, variation in allele fre-
quencies will correlate with geographic
variation in disease prevalence and is
likely to offer clues about specific envi-
ronmental exposures or risk factors.
The best characterized examples of
balancing selection are found in spe-
cific geographic populations sharing
an environmental exposure, such as an
infectious disease. In these examples,
e.g., sickle cell disease, risk alleles are
maintained at a much higher rate in the
exposed population than in populations
from other parts of the world.
 It is a leap of theoretical faith to infer that
SNPs with no known function and variable
weak associations across different disease
cohorts represent prima facie evidence of
regulatory function, gene-environment
interaction, or balanced selection. We are
not suggesting that such phenomena do
not occur. Deciphering such mechanisms
will be a major scientific focus over the next
decade. However, for the vast majority of
SNPs identified by GWAS, these mecha-
nisms are speculative in the absence of
biological evidence.
Many of the examples cited by Klein et
al. and Wang et al. in support of GWAS
suffer from the same limitations that we
highlighted in our Essay (McClellan and
King, 2010). For example, as Klein et al.
describe, there is robust epidemiologi-
cal data in support of individuals with
diabetes having a lower risk for prostate
cancer. However, associations between
putative risk/protective SNPs in HNF1B
and JAZF1 and the two illnesses vary
across cohorts, and these variants do
not mediate the relationship between the
two diseases (Stevens et al., 2010).
We understand that many believe that
most GWAS findings are valid. Ultimately,
the debate hinges on how definitive one
views variable results with statistically
highly significant p values and very small
effects that diminish with further study.
Currently, GWAS results fail to explain
the vast majority of genetic influence
on any human illness. Further, most risk
variants implicated by GWAS have no
demonstrated biological, functional, or
clinical relevance for disease.
We suggest that biological relevance
needs to be established before assert-
ing that positive correlations from GWAS
are equivalent to disease risk. Such evi-
dence must address the functional sig-
nificance of the SNP, or a variant in LD
with the SNP, rather than arguing for the
appeal of a nearby gene. In their argu-
ments, Wang et al. and Klein et al. simply
restate GWAS principles, which serves
to reify, not prove, the model. We make a
plea for more rigorous analysis. Science
ultimately advances by evidence.
Jon McClellan1 and
Mary Claire King2,*
1
Department of Psychiatry
2
Department of Medicine and Department
of Genome Sciences
University of Washington, Seattle, WA
98195, USA
*Correspondence:
mcking@u.washington.edu
DOI 10.1016/j.cell.2010.07.027
Cell 142, August 6, 2010 ©2010 Elsevier Inc.  355
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 Leading Edge

Previews

Fyn-Tau-Amyloid: A Toxic Triad

Christian Haass1,2,* and Eckhard Mandelkow3,4,*
1
German Center for Neurodegenerative Diseases (DZNE), 80336 Munich, Germany
2
Adolf Butenandt Institute, Ludwig Maximilian University, 80336 Munich, Germany
3
Max Planck Unit for Structural Molecular Biology, 22607 Hamburg, Germany
4
German Center for Neurodegenerative Diseases (DZNE), 23175 Bonn, Germany
*Correspondence: chaass@med.uni-muenchen.de (C.H.), mand@mpasmb.desy.de (E.M.)
DOI 10.1016/j.cell.2010.07.032

The axonal protein tau and amyloid β-peptide (Aβ) are key players in the pathogenesis of Alzheim-
er's disease, and tau mediates Aβ toxicity, but it is not clear how. Ittner et al. (2010) now report an
unexpected physiological function for tau in neuronal dendrites that may explain how tau mediates
Aβ toxicity.

Alzheimer’s disease (AD) is a devastat-
ing public health problem for our aging
societies. Although it is well established
that amyloid β-peptide (Aβ) forms toxic
oligomers in the brain (Haass and Sel-
koe, 2007), it is not clear how Aβ initiates
the amyloid cascade and causes the
death of neurons. Tau, an axonal protein,
seems to be an executor of Aβ toxicity
even though it is localized to axons, and
Aβ toxicity is primarily triggered through
interactions of Aβ oligomers with den-
dritic spines (Haass and Selkoe, 2007).
Tau binds to microtubules, a process that
is prevented by its abnormal phosphory-
lation during AD pathogenesis. Loss of
microtubule binding by tau is thought to
cause the disassembly of microtubules
followed by the aggregation and depo-
sition of tau in pathogenic neurofibril-
lary tangles. Although amyloid plaques
and tau tangles are prominent markers
of AD, one of the first and most obvious
pathological abnormalities observed
in brain tissue from AD patients is the
relocalization of tau from axons to the
somatodendritic compartment of neu-
rons (Figure 1) (Ballatore et al., 2007).
During brain development, tau and other
microtubule-associated proteins are ini-
tially distributed ubiquitously throughout
neurons, but then, as differentiation pro-
gresses, tau becomes sorted into axons
(Figure 1). In AD and other neurodegen-
erative diseases involving tau (termed
“tauopathies”), this neat sorting pattern
breaks down (Ballatore et al., 2007),
perhaps because abnormal phospho-
rylation of tau enables it to detach from
microtubules and diffuse rapidly into
other neuronal compartments (Konzack
et al., 2007). In this issue of Cell, Ittner
et al. (2010) now shed light on an unex-
pected dendritic function for normal tau
that suggests how tau may mediate Aβ
toxicity.
Most research on tau function has
focused on whether it is important for
microtubule stabilization, neurite out-
growth, or the formation of tracks for
cargo transport along axons. The cata-
strophic redistribution of tau to the soma
and dendrites of neurons in AD sug-
gests that an efficient sorting mechanism
normally keeps tau localized to axons.
However, such “polarized” sorting like
other cellular sorting pathways is never
completely efficient, thus enabling small
amounts of tau to become localized to
the somatodendritic compartment even
under physiological conditions. This
led Ittner and colleagues to propose a
microtubule-independent physiological
function for tau that regulates signaling in
dendritic spines. Their research was ini-
tially triggered by accumulating evidence
that subacute seizures occur not only in
transgenic mouse models of AD, but also
in AD patients (Palop and Mucke, 2009).
Interestingly, tau deficiency decreases
seizures induced by the Aβ-mediated
overstimulation of excitatory N-methyl-D-
aspartate (NMDA) receptors and improves
survival in a transgenic AD mouse model
(Roberson et al., 2007).
It is well established that tau binds not
only to microtubules but also to several
nonreceptor tyrosine kinases including
Fyn through its N-terminal domain (Lee
et al., 1998). This enables tau to seques-
ter Fyn and to alter its localization in the
neuron. Fyn phosphorylates subunit 2 of
the NMDA receptor, resulting in stabili-
zation of this receptor’s interaction with
PSD95, a scaffolding protein in the den-
dritic spines of neurons. This stabiliza-
tion, in turn, strengthens signaling by the
excitotoxic neurotransmitter glutamate,
which enhances Aβ toxicity (Figure 1). A
tau-dependent increase in Fyn in den-
dritic spines could boost excitotoxic sig-
naling, and, conversely, sequestration
of Fyn by tau or downregulation of tau
expression could mitigate excitotoxicity.
To address whether this is the case, Itt-
ner and coworkers first generated trans-
genic mice that overexpressed a variant of
tau (∆tau) that lacked the C terminus and
thus could bind to Fyn but not to micro-
tubules. Interestingly, ∆tau was completely
excluded from dendrites and accumulated
within the soma (Figure 1). Moreover, ∆tau
efficiently competed with endogenous tau
for binding to Fyn, resulting in sequestra-
tion of Fyn in the soma. Similarly, loss of
tau also prevented postsynaptic targeting
of Fyn, and loss of Fyn in the dendrites
was enhanced when ∆tau was expressed
in mice deficient in normal tau. This sug-
gests that the targeting of Fyn to dendrites
depends on normal tau, a surprising find-
ing for a protein believed to act only in
axons. But how tau-based sorting of Fyn
occurs and whether other proteins are
required remain unclear.
Fyn phosphorylates NMDA receptor
subunit 2 at tyrosine 1472 and stabilizes
the interaction of this subunit with the
postsynaptic protein PSD95 (Nakazawa et
al., 2001). However, when Fyn is trapped
within the soma by expression of ∆tau or
reduced expression of endogenous tau,
there is decreased phosphorylation of

356  Cell 142, August 6, 2010 ©2010 Elsevier Inc.

the NMDA receptor, the interaction of the
receptor with PSD95 is destabilized, and
excitotoxic signaling decreases. Excito-
toxicity has been implicated in Aß-medi-
ated toxicity, and a reduction in tau ame-
liorates the pathological action of Aβ in
the AD transgenic mouse model (Rober-
son et al., 2007). Similarly, overexpression
of ∆tau in the AD mice prevented seizures
and improved survival; indeed, these ben-
efits were enhanced when expression of
endogenous tau was reduced. Moreover,
the AD mice overexpressing ∆tau showed
improvements in memory, suggesting
that Aβ toxicity was reduced when Fyn
levels decreased in the dendritic spines.
This was independent of Aβ production or
the overall amyloid plaque load.
Notably, there is an interesting twist
to the Fyn-tau-microtubule story. In oli-
godendrocytes, tau is necessary for
the outgrowth of cell processes and for
transport of Fyn, which is important for
myelinating neurons. Expression of the
N-terminal domain of tau alone causes
abnormal sorting of Fyn, resulting in poor
myelination of neurons and seizures in
rodents (Klein et al., 2002). Thus, there are
two completely distinct settings for this
potentially toxic triad, which implicates
abnormal tau in seizure disorders as well
as in neurodegenerative diseases.
But can the Fyn-tau connection in den-
dritic spines be exploited to develop new
therapeutic strategies for treating AD? A
peptide that blocks phosphorylation of
NMDA receptors by Fyn protects neu-
rons from excitotoxic damage (Aarts et
al., 2002). Strikingly, when Ittner and col-
leagues treated their transgenic AD mice
with this peptide, memory deficits were
ameliorated and there was improved
survival, similar to the results when Fyn
was sequestered by ∆tau.
The Ittner et al. study may provide
a missing link between extracellular
deposits of Aβ and intracellular tau and
pinpoints tau and Fyn as possible medi-
ators of Aβ toxicity. Although the idea is
tantalizing, the actual colocalization of
tau and Fyn within dendritic spines and
their modes of action remain to be shown
under physiological conditions. A major
surprise of this study is that a normal
physiological function of tau, regarded
as an axonal protein, mediates Aβ tox-
icity at dendritic spines. Is this physio-
logical function of tau affected during the
Figure 1. A Toxic Triad in Alzheimer’s Disease.
Shown is the neuronal localization of tau protein (red) and Fyn kinase (blue) under physiological and
pathological conditions.
(Left) Normal tau is primarily located in axons but also interacts with Fyn kinase and targets it to den-
drites. Fyn kinase phosphorylates subunit 2 of the NMDA receptor in dendritic spines, which results
in stabilization of the receptor’s interaction with the postsynaptic density protein PSD95, leading to
enhanced excitotoxicity. Excitotoxicity is known to increase the toxic effects of oligomers of Aβ (dark
purple) on neurons.
(Middle) Overexpression of a ∆tau variant lacking the C terminus, which binds to Fyn kinase but not to
microtubules, results in the sequestration of Fyn in the soma, preventing Fyn from reaching the dendrites.
Consequently, Fyn-mediated phosphorylation of NMDA receptors is decreased and Aβ-mediated toxicity
is reduced (pale purple).
(Right) Enhanced redistribution of abnormally hyperphosphorylated tau from axons to the somatoden-
dritic compartment during AD pathogenesis may increase tau-dependent sorting of Fyn to the dendrites,
boosting excitotoxic signaling and increasing the toxic effects of Aβ (black) on neurons.
earliest stages of AD? This may be the ates the transport of Fyn to dendrites. If
case, as redistribution of full-length tau that transport mechanism is disturbed,
(and possibly tau fragments) to the som- what other neuronal functions would be
atodendritic compartment occurs well affected? Clearly, not only Fyn, but also
before the formation of neurofibrillary other signaling molecules containing
tangles. The abnormal sorting of tau to SH3 domains bind to the PXXP motifs
the somatodendritic compartment may in the N-terminal domain of tau and of
be due to changes in signaling cascades related microtubule-associated proteins;
that alter kinase and phosphatase activi- but we do not know whether inhibition
ties in those dendritic spines affected by of this binding would have deleterious
Aβ, resulting in local changes in tau sort- effects. Also, is ∆tau just a scavenger
ing and cytoskeletal rearrangements. that when overexpressed sequesters
But do tau and Fyn still interact after tau Fyn? Could proteins other than tau per-
redistribution, given that the Fyn bind- form this activity in vivo? The enhanced
ing site on tau can be phosphorylated phenotypes upon reduction of endoge-
leading to disruption of this binding? If nous tau seem to speak against this pos-
they do, then relocalized tau may boost sibility. The provocative work of Ittner et
Fyn action in dendrites and hence phos- al. raises new challenging questions, but
phorylation of NMDA receptors, thus also brings us a significant step closer
enhancing excitotoxic signaling and to understanding AD pathophysiology.
increasing the sensitivity of neurons to Hopefully, these findings will help in the
Aβ (Figure 1). However, before targeting design of new therapeutic strategies for
the tau-Fyn interaction for therapeutic reducing the synaptic dysfunction and
purposes, we need to know more about neuronal loss of AD and other neurode-
the mechanism by which tau medi- generative diseases.
Cell 142, August 6, 2010 ©2010 Elsevier Inc.  357
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Anthony M. Barsotti1 and Carol Prives1,*
1
Department of Biological Sciences, Columbia University, New York, NY 10027, USA
*Correspondence: clp3@columbia.edu
DOI 10.1016/j.cell.2010.07.029
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scriptional activation of microRNAs but
also their processing into mature, active
forms (Figure 1, middle) (Shi et al., 2010).
Now, Huarte, Rinn, and their colleagues
(Huarte et al., 2010) add an exciting new
route through which p53 executes wide-
spread gene repression, specifically by
activating a long intergenic RNA (Figure
1, bottom).
LincRNAs are large RNA molecules
(primary transcript ≥5 kb) that are evo-
lutionarily conserved across mammalian
genomes. Although these RNAs are tran-
scribed by RNA polymerase II, 5′capped,
and polyadenylated like normal mRNAs,
they do not code for proteins (Guttman
et al., 2009). Previous work by the Rinn
group suggested that lincRNAs may
repress transcription by targeting chro-
matin-modifying complexes to specific
genomic loci (Khalil et al., 2009).
In their new work, Huarte et al. (2010)
sought to identify specific lincRNAs that
operate within the p53 pathway. They con-
structed a tiling microarray designed to
detect the expression of ?400 lincRNAs.
They then incubated this array with RNA
isolated from two mouse cell of this interaction. Indeed, a
lines engineered with control- large percentage of genes
lable expression of p53. These commonly inhibited by p53
experiments uncovered sev- and lincRNA-p21 also require
eral lincRNAs that are poten- hnRNP-K for their repression.
tial targets of p53, and the Further, hnRNP-K is recruited
authors focused on lincRNA- to promoters for genes that
p21, named for its proximity are downregulated by the
to the p53-target gene p21. p53/lincRNA-p21/ hnRNP-
Importantly, they confirmed K axis, and this recruitment
that the human ortholog of the depends on lincRNA-p21.
mouse lincRNA-p21 is con- Taken together, these results
served in both sequence and imply that hnRNP-K plays
regulation. a significant and direct role
To ascertain the role of Figure 1. Activation for Repression’s Sake in executing gene repres-
lincRNA-p21 within the p53 Activation of p53 in response to stress triggers expression of three distinct sion mediated by p53 and
pathway, the authors induced classes of targets that silence gene expression in different ways, resulting in lincRNA-p21.
different cellular outcomes.
the p53 response pathway (Top) The p21 protein inhibits cyclin-dependent kinases, thereby activating Supporting this model,
and then silenced either p53 the Rb family of proteins and inhibiting the E2F transcription factors. This previous studies found that
or lincRNA-p21. By compar- ultimately halts progression of the cell cycle. hnRNP-K is present in a
(Middle) The microRNA miR-34a degrades or inhibits translation of the mRNAs
ing microarrary data under of genes important for both cell-cycle progression and survival. repressive complex with the
these two conditions, the (Bottom) A new class of p53 targets, exemplified by the long intergenic non- linker histone H1.2. Moreover,
authors identified a large set coding RNA lincRNA-p21, is activated by p53 and then inhibits gene expres- this complex blocks chromatin
sion (Huarte et al., 2010). Although the exact mechanism of gene repression
of genes that are derepressed is unclear, lincRNA-p21 interacts with heterogeneous ribonucleoprotein K acetylation and subsequent
in response to disruption of (hnRNP-K) to downregulate a large group of genes that are repressed by p53, transcriptional activation by
both p53 and lincRNA-p21. including several that are important for cell survival. p53 and the transcription fac-
Moreover, these specific tor complex p300 (Kim et al.,
genes are highly represented in the by p53 is crucial for p53-facilitated cell 2008). In contrast, however, Moumen et
group of genes normally repressed by death (Ho and Benchimol, 2003). Further, al. (2005) found that hnRNP-K cooperates
p53. This is a significant finding because Huarte et al. show that the activation of with p53 as a coactivator of transcription
it implicates lincRNA-p21 as a potent certain proapoptotic genes by p53, such in response to DNA damage. In the system
downstream mediator of p53-dependent as Noxa and Perp, depends on lincRNA- used by Huarte and colleagues, hnRNP-K
transcriptional repression that acts on a p21. Therefore, lincRNA-p21 may also and lincRNA-p21 have independent roles
very large scale. play an important role in determining the as well, with hnRNP-K sharing an over-
As mentioned, p21 itself is also thought promoter selectivity of p53 for gene acti- lapping set of target genes with p53 that
to inhibit numerous target genes of p53. vation in addition to directing the repres- are not regulated by lincRNA-p21. Thus, it
Nevertheless, disruption of lincRNA-p21 sion of target genes. is not clear that lincRNA-p21 and hnRNP-
does not alter levels of p21 mRNA or Next, Huarte and colleagues delved K are more closely related than p53 and
protein. Furthermore, genes regulated into the mechanism by which lincRNA- hnRNP-K. Nevertheless, the large amount
by lincRNA-p21 do not appear to overlap p21 represses transcription. With great of data generated by the new microarray
with those identified in previous studies satisfaction, they identify hnRNP-K as analyses supports a more widespread
as targets of p21. These results suggest a key protein partner for lincRNA-p21. role for hnRNP-K in p53-dependent gene
that lincRNA-p21 acts independently of Best known for its role in chromatin repression than in p53-dependent gene
p21. remodeling and transcriptional regula- activation.
In response to stress, p53 initiates a tion (initiation, elongation, and termina- The Huarte et al. findings raise several
cellular program that often results in cell- tion), hnRNP-K binds to a wide range of key questions. Does the binding part-
cycle arrest or cell death. Despite the molecules, including DNA, RNA, protein ner of hnRNP-K determine whether it
large common set of genes regulated by kinases, and factors that remodel chro- acts as a positive or negative regulator
p53 and lincRNA-p21, Huarte, Rinn, and matin. Thus, hnRNP-K also participates of transcription? For example, does the
coworkers found that silencing of lin- in diverse processes, such as RNA interaction between p53 and hnRNP-K
cRNA-p21 blocks programmed cell death splicing, mRNA stability, and translation serve as a platform for other coactiva-
(i.e., apoptosis) but not cell-cycle arrest. (Bomsztyk et al., 2004). tors, whereas the association between
LincRNA-p21 represses the expression After validating the specificity of the lincRNA-p21 and hnRNP-K favors for-
of several important prosurvival factors, association between lincRNA-p21 and mation of a repressive complex? This
which may help to explain this interesting hnRNP-K and identifying the region of model would indeed explain the seem-
phenotype. This hypothesis is supported the RNA necessary for their binding, ingly contradictory roles of hnRNP-K
by previous studies demonstrating that the authors again turned to microar- with regard to p53 activity. Further, how
the repression of antiapoptotic genes ray analyses to assess the importance does lincRNA-p21 influence the binding

Cell 142, August 6, 2010 ©2010 Elsevier Inc.  359

of hnRNP-K to specific genomic loci?
Is there functional significance to the
remarkable proximity (?15 kb) of lin-
cRNA-p21 to the p21 gene? If these loci
are regulated interdependently, they may
act as a key molecular switch between
life and death. Finally, given the impor-
tance of lincRNA-p21 to p53-dependent
cell death, is lincRNA-p21 mutated in
cancer? Answers to each of these ques-
tions will certainly enrich our under-
standing of the functional relationship
between p53 and this powerful class of
regulatory molecules, lincRNAs.
References
Barsotti, A.M., and Prives, C. (2009). Oncogene
28, 4295–4305.
Bomsztyk, K., Denisenko, O., and Ostrowski, J.
(2004). Bioessays 26, 629–638.
Guttman, M., Amit, I., Garber, M., French, C., Lin,
M.F., Feldser, D., Huarte, M., Zuk, O., Carey, B.W.,
Cassady, J.P., et al. (2009). Nature 458, 223–227.
Ho, J., and Benchimol, S. (2003). Cell Death Differ.
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Huarte, M., Guttman, M., Feldser, D., Garber, M.,
Koziol, M., Kenzelmann-Broz, D., Khalil, A., Zuk, O.,
Amit, I., Rabani, M., et al. (2010). Cell, this issue.
Khalil, A.M., Guttman, M., Huarte, M., Gar-
ber, M., Raj, A., Rivea Morales, D., Thomas, K.,
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chim. Biophys. Acta 1805, 218–225.

Stem Cells and DNA Damage:

Persist or Perish?
Andrew A. Lane1,2,* and David T. Scadden1,2,*
1
Center for Regenerative Medicine, Cancer Center, Massachusetts General Hospital, Boston, MA 02114, USA
2
Harvard Stem Cell Institute, Department of Stem Cell and Regenerative Biology, Harvard University, Boston, MA 02114, USA
*Correspondence: aalane@partners.org (A.A.L.), dscadden@mgh.harvard.edu (D.T.S.)
DOI 10.1016/j.cell.2010.07.030

Stem cells repopulate tissues after injury while also renewing themselves, but this makes them
vulnerable to genotoxic damage. Mohrin et al. (2010) and Milyavsky et al. (2010) now show that
mouse and human hematopoietic stem cells make opposing decisions about whether to die or to
persist in response to DNA damage.

Stem cells have the immense responsi-
bility of maintaining tissue and organism
homeostasis over the lifetime of an indi-
vidual. As such, stem cells are speculated
to have evolutionary characteristics that
offer protection against acute insults,
allowing them to survive and to repopu-
late their tissues in the short term. How-
ever, they must also act as self-renew-
ing guardians of the genome to ensure
maximal integrity of the genomic code
for future stem cells and their mature tis-
sue progeny. The hematopoietic (blood)
system is perhaps the best studied tis-
sue in terms of its hierarchical develop-
ment from a small number of long-term
stem cells that are relatively quiescent,
to progenitors that proliferate and dif-
ferentiate, and then to mature blood cell
lineages that are produced by the billion
each day. Hematopoietic stem cells are
thought to be resistant to injury including
DNA damage, which may be related to
their specific gene expression programs,
epigenetic factors, or exogenous protec-
tion by the stem cell “niche.” Two new
reports in Cell Stem Cell from the labora-
tories of Emmanuelle Passegué (Mohrin
et al., 2010) and John Dick (Milyavsky et
al., 2010) further our understanding of
how hematopoietic stem cells respond
to radiation-induced DNA damage.
So how do quiescent stem cells han-
dle genotoxic insults? Mohrin et al. (2010)
found that murine hematopoietic stem
and progenitor cells (HSPCs)—defined as
bone marrow cells expressing the mark-
ers: lineage − /c-Kit+/Sca-1+/Flk2−—were
more resistant to apoptosis induced
by a specific dose of ionizing radiation
than were more differentiated progenitor
cells (Figure 1). The unique DNA damage
response of mouse HSPCs involves the
tumor suppressor protein p53 and is lost
when stem cells are forced out of qui-
escence and into the cell cycle by treat-
ment with chemotherapy or cytokines.
Not only are quiescent HSPCs poised
to resist apoptosis as evidenced by their
antiapoptotic gene expression program,
but they are also able to repair their DNA
by nonhomologous end joining (NHEJ).
Repair of DNA damage through homolo-
gous recombination (which has a lower
error rate than NHEJ) requires that cells
enter the cell cycle; thus, quiescent stem
cells must rely on NHEJ as an alterna-
tive. The reliance of quiescent adult tis-
sue stem cells on NHEJ for the repair of
DNA damage may in fact be a general

360  Cell 142, August 6, 2010 ©2010 Elsevier Inc.

phenomenon in mice, given umbilical cord blood HSPCs
the similar conclusions of a have a different biology than
recent study using hair follicle the bone marrow HSPCs of
stem cells as a model system mouse. However, it is tempt-
(Sotiropoulou et al., 2010). ing to see the different find-
Unfortunately, Mohrin et al. ings in the light of evolution,
(2010) also uncover a down- that is, as a reflection of the
side to short-term radioresis- different challenges faced by
tance and rapid DNA repair mammals with different life
through the error-prone NHEJ spans and ages of reproduc-
pathway. Spectral karyotyping tive maturity.
revealed gross chromosomal Recent elegant studies from
aberrations in irradiated mouse Bondar and Medzhitov (2010)
HSPCs, and some of the same and Marusyk et al. (2010)
abnormal cytogenetic find- demonstrate that competitive
ings persisted in the progeny selection takes place within
of irradiated HSPCs trans- tissue stem cell populations.
planted into mouse recipients. These authors found that irra-
Furthermore, despite their diated p53-deficient HSPCs
resistance to apoptosis imme- in the mouse have an initial
diately after injury, irradiated survival advantage but that
HSPCs were unable to con- Figure 1. Hematopoietic Cell Responses to DNA Damage long-term fitness is balanced
tribute to long-term sustained (Top) Quiescent murine hematopoietic stem and progenitor cells (HSPCs) by complex interactions with
are poised to survive DNA damage induced by low-level ionizing radiation
hematopoiesis when seri- through a DNA repair process called nonhomologous end joining (NHEJ), neighboring HSPCs and the
ally transplanted into mouse which tends to be error prone (Mohrin et al., 2010). In contrast, mouse HSPCs relative levels of p53 and DNA
recipients. Such events would and committed progenitors (CP) progressing through the cell cycle are more damage in stem cells and their
be of obvious risk to a long- likely to undergo apoptosis or repair their DNA using higher-fidelity homolo-
gous recombination. Although the short-term consequence of HSPC survival
neighbors. The studies from the
lived organism as serial expo- is maintenance of tissue integrity in the face of injury, long-term consequenc- Passegué and Dick labs indi-
sure of stem cells to genotoxic es include genomic rearrangements that persist and HSPCs with a diminished cate that hematopoietic cells
agents could readily result in functional capacity. within a tissue have adopted
(Bottom) In contrast, compared to more committed progenitors, the default
leukemia or aplasia. program for damaged human HSPCs is to undergo apoptosis. However, a de- different means of handling
Does the human hemato­ crease in p53 rescues human HSPCs from apoptosis immediately after low- DNA damage depending on
poietic system accept the level irradiation (Milyavsky et al., 2010). Despite interspecies differences in their differentiation stage. That
the short-term response to radiation, the long-term functional consequences
same tradeoff between stem of avoiding apoptosis for both mouse and human HSPCs include persistent mouse and human stem cells
cell survival in the short term DNA damage and decreased self-renewal capacity. The delicate balance be- may have acquired or under-
versus accumulation of del- tween tissue survival and the DNA damage response therefore could predis- gone selection for distinct
pose surviving HSPCs to future malignant transformation.
eterious mutations in the long responses to ionizing radiation
term? In a companion study, is a reasonable notion. How-
Milyavsky et al. (2010) addressed this ent mice. Therefore, a short-term gain ever, it remains to be seen which specific
question in human umbilical cord blood in survival could be achieved by human molecular mechanisms that differ between
cells. They observed an enhanced sen- hematopoietic stem cells as found in the stem cells and progenitors, or between
sitivity to apoptosis induced by low-dose mouse, but the default setting for irradi- stem cells of different species, lead to
ionizing radiation in these cells compared ated human HSPCs is an increase in p53 these distinctive traits. We now have a set
to more differentiated cells. In contrast expression resulting in apoptosis. of reagents with which to discover and
to the findings of Mohrin et al. in the The differences between these two understand how such important yet differ-
mouse, these authors noted that human studies may have a technical basis: mark- ent tissue stem cell traits have evolved.
hematopoietic stem and early multipotent ers for stem and progenitor populations There are other practical implications
progenitor cells were poised for apopto- are more refined in the mouse than in the of the Passegué and Dick reports. Sec-
sis in response to DNA damage. Survival human so somewhat different stem and ondary myelodysplasia and leukemia are
and the clonogenic and reconstitutive progenitor cell populations may have believed to arise from DNA damage to
capacity of the irradiated human HSPCs been analyzed. In addition, slightly differ- HSPCs from the radiation or chemother-
were rescued by blocking p53 expression ent doses of radiation were used. In the apy given to treat a primary malignancy.
or by overexpression of the antiapoptotic in vivo experiments of Milyavsky et al., Mohrin et al. show intriguing evidence that
factor Bcl-2. However, irradiated human human HSPCs from umbilical cord blood NHEJ activity and chromosomal aberra-
HSPCs lacking p53 were unable to sus- were transplanted into the mouse bone tions decrease when HSPCs are induced
tain hematopoiesis and showed evidence marrow niche, which may have provided to enter the cell cycle prior to irradiation.
of persistent DNA double-strand breaks less efficient survival signals for human Interestingly, a parallel evolving hypoth-
when serially transplanted into recipi- cells than for mouse cells. Also, human esis in the study of cancer stem cells sug-

Cell 142, August 6, 2010 ©2010 Elsevier Inc.  361

gests that activating leukemia stem cells
from quiescence prior to chemotherapy
may result in more efficient elimination of
these cancer-repopulating cells (Saito et
al., 2010). An unexpected benefit of such
a prestimulation strategy may be that nor-
mal hematopoietic stem cells activated
from quiescence would simultaneously
be protected from accumulating long-
term DNA damage. However, as shown
by Milyavsky and colleagues, stem cell
escape from acute damage, particularly if
it involves a decrease in p53 activity, may
lead to long-term deleterious effects on
stem cell fitness and repopulating ability.
The interplay between the response to
acute injury and long-term fitness needs
to be more fully understood and will
require both laboratory models and the
thoughtful correlative study of stem cells
from patients receiving genotoxic chemo-
therapy. Understanding these events may
point the way to methods for preserving
short-term tissue reconstitution while
maintaining long-term cell and genomic
integrity.
References
Bondar, T., and Medzhitov, R. (2010). Cell Stem
Cell 6, 309–322.
Marusyk, A., Porter, C.C., Zaberezhnyy, V., and
DeGregori, J. (2010). PLoS Biol. 8, e1000324.
Milyavsky, M., Gan, O.I., Trottier, M., Komosa, M.,
Tabach, O., Notta, F., Lechman, E., Hermans, K.G.,
Eppert, K., Konovalova, Z., et al. (2010). Cell Stem
Cell 7. Published online July 7, 2010. 10.1016/j.
stem.2010.05.016.
Mohrin, M., Bourke, E., Alexander, D., Warr,
M.R., Barry-Holson, K., Le Beau, M.M., Mor-
rison, C.G., and Passegué, E. (2010). Cell Stem
Cell 7. Published online July 7, 2010. 10.1016/j.
stem.2010.06.014.
Saito, Y., Uchida, N., Tanaka, S., Suzuki, N., Tomi-
zawa-Murasawa, M., Sone, A., Najima, Y., Takagi,
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Sotiropoulou, P.A., Candi, A., Mascré, G., De Cler-
cq, S., Youssef, K.K., Lapouge, G., Dahl, E., Se-
meraro, C., Denecker, G., Marine, J.C., and Blan-
pain, C. (2010). Nat. Cell Biol. 12, 572–582.

Mitochondrial Matrix Reloaded with RNA

Toshiya Endo,1,* Koji Yamano,1 and Tohru Yoshihisa2,3
1
Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
2
Research Center for Materials Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
3
Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
*Correspondence: endo@biochem.chem.nagoya-u.ac.jp
DOI 10.1016/j.cell.2010.07.024

Although mitochondrial biogenesis requires the import of specific RNAs, the pathways and cellular
machineries involved are only poorly understood. Wang et al. (2010) now find that polynucleotide
phosphorylase in the intermembrane space of mammalian mitochondria facilitates import of
several RNAs into the mitochondrial matrix.

Mitochondria, the power plants of the
eukaryotic cell, are bound by two mem-
branes and contain 1000–1500 different
proteins and tens of RNAs. Most of the
genes that encode mitochondrial pro-
teins are found in the nuclear genome
and thus are translated in the cytosol
and then imported into mitochondria.
The pathways and machineries required
for protein import into mitochondria
have been extensively studied and are
highly conserved among fungi, plants,
and mammals (Endo and Yamano 2009;
Chacinska et al., 2009). The mitochon-
drial matrix also contains several kinds of
noncoding RNAs that are also imported
from the cytosol. However, in contrast to
protein translocation, the mechanisms
that mediate import of RNAs into mito-
chondria remain enigmatic (Salinas et al.,
2008; Lithgow and Schneider, 2010). In
this issue of Cell, Wang et al. (2010) shed
light on this question, revealing that poly-
nucleotide phosphorylase (PNPase) is a
much sought after component of the RNA
import apparatus in mammalian cells.
PNPases comprise an evolutionally
conserved enzyme family (found in bac-
teria, plants, flies, and mammals but not
in yeast) that has 3′→5′ exoribonuclease
and RNA-polymerase activities (Chen et
al., 2007). Although bacterial PNPases
are cytosolic, eukaryotic PNPases are
mainly localized in mitochondria or chlo-
roplasts. Prior work has established
how PNPases get to the intermembrane
space (IMS). After crossing the mito-
chondrial outer membrane via the trans-
locase of outer mitochondrial membrane
40 (TOM40) complex, the PNPase pre-
cursor engages with the translocase of
the inner membrane 23 (TIM23) complex
(Figure 1) (Chen et al., 2006; Rainey et al.,
2006). After the PNPase presequence is
removed by matrix processing pepti-
dase (MPP), an AAA protease Yme1 in
the inner membrane pulls PNPase into
the IMS, where PNPase assembles into
a trimeric complex (Figure 1).
Wang et al. now assess the function of
mammalian PNPase by tissue-specific
disruption of the PNPase gene in mouse
hepatocytes. They find that mitochondria
from hepatocytes deficient in PNPase
display defects in oxidative phosphory-
lation (OXPHOS), the major ATP-gener-
ating metabolic pathway of respiration.
This defect is shown to arise from the
failure in the processing of polycistronic
mitochondrial mRNAs encoding the

362  Cell 142, August 6, 2010 ©2010 Elsevier Inc.

subunits of the OXPHOS complexes. But
how does PNPase in the IMS affect the
processing of RNAs in the matrix?
A hint to the answer came from the
observation that the RNA component
of mammalian mitochondrial RNase
P is markedly decreased by PNPase
depletion. Given that RNase P medi-
ates processing of mitochondrial trans-
fer RNAs (tRNAs), which are encoded
between open reading frames for
OXPHOS components, failure in tRNA
excision also impairs maturation of the
OXPHOS subunit transcripts. In addi-
tion, expression of human PNPase in
yeast mitochondria, which do not pos-
sess PNPase, enhances the import effi-
ciency of heterologous human RNase P
RNA, 5S ribosomal RNA, and the RNA
component of MRP, a ribonucleopro-
tein that mediates mitochondrial DNA
replication. Similar PNPase-dependent
import in vitro is confirmed with mito-
chondria isolated from PNPase-defi-
cient mouse liver or embryonic fibro-
blasts. These findings suggest that
PNPase in the IMS is a component of
the RNA import system in human mito-
chondria.
What is the mechanism of RNA import
that is facilitated by PNPase? In yeast and
plant mitochondria, substrate tRNAs are
recognized by the mitochondrial surface
components including the import recep-
tor Tom20, and then move through the
Tom40 channel or voltage-dependent
anion channel (VDAC). This movement
probably involves several mechanisms,
including a piggyback mechanism in
which the RNA substrate is associated
with an appropriate escort protein (Sali-
nas et al., 2008; Lithgow and Schneider,
2010). Although no receptor or translo-
cation channel for RNA import is known
for mammalian mitochondria, the pres-
ent study reveals that PNPase binds to
substrate RNAs with a specific stem-
loop structure. When grafted onto other
nonsubstrate RNAs, this stem-loop can
direct the RNA into yeast mitochondria
containing human PNPase. Therefore,
after crossing the outer membrane by an
unknown mechanism, substrate RNAs
with a specific stem-loop structure are
recognized by PNPase, which subse-
quently allows the transfer of a subset of
RNAs to downstream components of the
import pathway. The binding of PNPase
Figure 1. PNPase Facilitates the Import of RNAs into the Mitochondrial Matrix
The polynucleotide phosphorylase (PNPase) precursor is imported into the intermembrane space with
the aid of the translocase of outer mitochondrial membrane 40 (TOM40) complex, the translocase of
the inner mitochondrial membrane (TIM23) complex, matrix processing peptidase (MPP), and AAA
protease Yme1. The trimeric PNPase promotes the import of RNAs from the cytosol into the matrix.
Mitochondrial mRNAs are processed in the matrix and their translation products are assembled into
the oxidative phosphorylation (OXPHOS) complexes I–IV. The OXA complex integrates proteins from
the matrix into the inner membrane. OM, outer membrane; IM, inner membrane. Heat shock protein
70 (Hsp70) is shown as an import motor for presequence-containing proteins. AAC, ADP-ATP carrier;
DΨ, membrane potential.
to substrate RNAs may also contribute to place center stage. Will the unfolding
the unidirectional translocation of RNAs drama of RNA translocation in mitochon-
across the outer membrane via a trap- dria reveal common principles or expose
ping mechanism or by preventing their a diversity of organism specific mecha-
backward movement to the cytosol. nisms?
The identity of the import chan-
nel in the inner membrane is unknown, References
although Wang et al. find that translo-
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RNAs across the inner membrane (Fig-
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the inner membrane import channel may
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723–730.
mitochondrial heat shock protein 70, as
an import motor for presequence-con- Lithgow, T., and Schneider, A. (2010). Philos. Trans.
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many of the components of the yeast and Rainey, R.N., Glavin, J.D., Chen, H.-W., French,
human RNA import pathway remain to S.W., Teitell, M.A., and Koehler, C.M. (2006). Mol.
be identified, the efficient in vitro system Cell. Biol. 26, 8488–8497.
developed by Wang et al. should acceler- Salinas, T., Duchêne, A.-M., and Maréchal-Drouard,
ate the pace of discovery. L. (2008). Trends Biochem. Sci. 33, 320–329.
The study by Wang et al. casts a spot-
Wang, G., Chen, H.-W., Oktay, Y., Zhang, J., Allen,
light on mitochondrial RNA import, with E.L., Smith, G.M., Fan, K.C., Hong, J.S., French,
mammalian PNPase taking its rightful S.W., McCaffery, J.M., et al. (2010). Cell, this issue.
Cell 142, August 6, 2010 ©2010 Elsevier Inc.  363

A MAP for Bundling Microtubules
Claire E. Walczak1,* and Sidney L. Shaw2,*
1
Medical Sciences
2
Department of Biology
Indiana University, Bloomington, IN 47405, USA
*Correspondence: cwalczak@indiana.edu (C.E.W.), sishaw@indiana.edu (S.L.S.)
DOI 10.1016/j.cell.2010.07.023
Microtubules assemble into arrays of bundled filaments that are critical for multiple steps in cell
division, including anaphase and cytokinesis. Recent structural and functional studies, including
two papers in this issue of Cell (Bieling et al., 2010; Subramanian et al., 2010), demonstrate how
the MAP65 protein PRC1 crosslinks microtubules and cooperates with kinesin motors to control
the dynamics and size of bundled regions.
The microtubule cytoskeleton is a remarkable structure that
can adopt diverse architectures uniquely suited to the indi-
vidual needs of a particular cell type or process. For example,
in vertebrate cells, the mitotic spindle, which separates chro-
mosomes during anaphase, contains two antiparallel arrays
of microtubules with their minus ends anchored at opposing
centrosomes and their plus ends overlapping to form a bundle
of crosslinked filaments in the middle of the spindle (Figure 1,
bottom inset). In contrast, plant (angiosperm) cells do not pos-
sess discrete microtubule organizing centers (i.e., centrioles)
but instead rely primarily on specific interactions between
microtubules to organize the filaments into crosslinked arrays
(Ehrhardt, 2008).
The variety of microtubule structures observed across differ-
ent cell types requires a diverse group of proteins to assemble,
stabilize, and dynamically control these microtubule arrays.
Microtubule-associated proteins (MAPs), which include both
molecular motors and nonmotor proteins, regulate the global
properties of microtubule structures by moving and crosslinking
filaments. Although much is known about these individual pro-
teins, key questions remain about how they interact to control
the size, shape, and dynamics of microtubule arrays. Now, two
studies in this issue of Cell (Bieling et al., 2010; Subramanian et
al., 2010) demonstrate how the MAP65 protein PRC1 (protein
regulator of cytokinesis 1) independently bundles microtubules
into antiparallel arrays and works with two motors, kinesin-4
and kinesin-5, to control the global properties of these overlap-
ping regions. Together, these papers suggest a model for how
microtubule bundles can persist despite the action of numer-
ous motor proteins acting along them.
Microtubule Structure and Dynamics
Microtubules are linear polymers inside the cell composed of α/β-
tubulin heterodimers arranged head to tail into protofilaments. The
protofilaments, typically 13, associate laterally to form a hollow
tube with substantial flexural rigidity and inherent structural polar-
ity, described as having plus and minus ends (Figure 1). Microtu-
bules exhibit dynamic instability (Mitchison and Kirschner, 1984)
wherein individual microtubules within a population interconvert
between states of growth and shortening. In general, microtubule
plus ends are more dynamic than minus ends.
364  Cell 142, August 6, 2010 ©2010 Elsevier Inc.
Leading Edge
Minireview
The polarity of microtubules within a bundle of overlapping
filaments is critical to the action of motor proteins that slide
filaments past each other and drive the movement of cargoes,
such as chromosomes, on these microtubules. For example,
during late anaphase, the overlapping regions of microtubules
at the center of the mitotic spindle elongate as the microtubules
push the spindle poles to opposite sides of the cell. However,
to separate daughter cells during cytokinesis, this overlapping
region shortens and forms a dense, compact array of anti-
parallel microtubules, called the midzone. How does the cell
specify the size of the overlapping region in a bundle and man-
age the timing and position of its remodeling? To answer these
questions requires a better understanding of the key molecular
players that govern the formation of microtubules.
MAP65 Family of Microtubule Crosslinking Proteins
One major class of proteins that crosslink microtubules into
arrays is the MAP65/Ase1/PRC1 family. Biochemical studies
with plant extracts identified the first members of this family
as 65 kD proteins capable of bundling microtubules (Chang-
Jie and Sonobe, 1993). Subsequent studies demonstrated that
MAP65 proteins crosslink microtubules in vitro with a spacing
between microtubule filaments of 25 nm (Chan et al., 1999),
consistent with in situ observations of microtubule bundles in
plants. A genetic screen identified the yeast ortholog of MAP65
as Ase1, which is required for properly elongating the spindle
during anaphase (Pellman et al., 1995). Ase1 was later shown
to be a homodimer that also bundles microtubules in vitro
(Schuyler et al., 2003), an activity that is critical for its role in
sliding microtubule filaments past each other during anaphase.
The vertebrate ortholog of MAP65/Ase1 is PRC1, and in mam-
malian cells, PRC1 regulates the organization of the central
spindle during cytokinesis (Jiang et al., 1998; Mollinari et al.,
2002). Phosphorylation of PRC1 by cyclin dependent kinase 1
(Cdk1) negatively regulates the crosslinking activity of PRC1,
which limits the bundling of microtubules by PRC1 until late
stages of mitosis when they are needed for cytokinesis (Zhu
et al., 2006).
Like other MAP65 proteins, PRC1 and Ase1 are not molec-
ular motors themselves but instead work in concert with
motor proteins to organize arrays of microtubules. In fis-

sion yeast, Ase1 dynamically controls the overlap of bundles
by coordinating with the kinesin-14 motor klp2 (Janson et
al., 2007). In mammalian cells, PRC1 is transported to the
midzone of the spindle by Kif4 (kinesin family member 4), a
kinesin-4 motor protein that is critical for positioning chro-
mosomes and for cytokinesis in multiple organisms (Glotzer,
2009; Hornick et al., 2010). Disruption of either PRC1 or Kif4
perturbs the localization of the other protein, making it diffi-
cult to elucidate whether the Kif4 motor recruits PRC1 to the
microtubules or whether loss of PRC1 disrupts the localiza-
tion of the central spindle and thus Kif4. Nevertheless, PRC1
is required to set up the central spindle before a number of
other kinesins locate to the spindle. These kinesins include
motors involved in finishing the assembly of the central spin-
dle and in cytokinesis, including mitotic kinesin-like protein
1 (MKLP-1) and 2 and Kif14 (Glotzer, 2009; Hornick et al.,
2010). Nevertheless, it is still unknown how PRC1 interacts
with these motor proteins to direct the size, shape, and sta-
bility of the central spindle.
Kinesin-4 Limits Microtubule Overlap Length
To understand how PRC1 organizes microtubule arrays, Biel-
ing and colleagues (2010) developed a total internal reflection
fluorescence (TIRF) microscopy assay in which microtubule
seeds are attached to a microscope slide and mixed with
Figure 1. PRC1 Controls Microtubule
­Assembly
(A) Protein regulator of cytokinesis 1 (PRC1) can
initiate crosslinking of dynamic microtubules (MT)
that interact in an antiparallel fashion (top). Kine-
sin-4 is a molecular motor directed at the plus
ends of microtubules; it accumulates in the re-
gion where microtubules crosslink as their plus
ends grow (middle). The interaction of kinesin-4
with PRC1 increases the dwell time of kinesin-4
on microtubules, which in turn limits the length
of the overlap region by blocking microtubule
growth at the plus ends (bottom) (Bieling et al.,
2010).
(B) Kinesin-5 is also a molecular motor directed at
the plus ends of microtubules, but kinesin-5 can
slide microtubules past each other (top). When
kinesin-5 is added to crosslinked microtubules
(middle), PRC1 maintains the crosslinks despite
the sliding action (bottom) (Subramanian et al.,
2010).
(C) PRC1 forms a homodimer that interacts
through its central spectrin domains with two mi-
crotubules to crosslink the antiparallel filaments.
Although PRC1 is shown to associate predomi-
nantly with α-tubulin, the current resolution of
the structures presented by Subramanian et al.
(2010) is not sufficient to distinguish between
binding to α- or β-tubulin.
tubulin dimers to polymerize dynamic
microtubules. They then added PRC1
proteins labeled with fluorescent tags
to crosslink the microtubules (Figure
1A). The PRC1 proteins bound prefer-
entially to microtubules that overlap at
antiparallel regions, showing decisively
that PRC1 alone is sufficient to cross-
link antiparallel microtubules.
Knowing that PRC1 interacts directly with Kif4 (Xklp1 in Xeno-
pus), Bieling et al. next determined how Xklp1 alters the forma-
tion of microtubule bundles by PRC1. Previous studies showed
that a truncated version of Xklp1 could inhibit both growth and
shrinkage of microtubules at particular ends (Bringmann et al.,
2004), suggesting that Xklp1 regulates the dynamics of micro-
tubules within bundles. Indeed, bundles of antiparallel microtu-
bules still formed when Xklp1 and PRC1 were added together
to the polymerizing microtubules, but, remarkably, the bundles
grew to a fixed size when Xklp1 was present. The authors show
that this limit in bundle length is due to cessation of growth
at the plus end of the microtubules and that the steady-state
length of the overlapping region depends on the concentration
of Xklp1 (Figure 1A).
These findings uncover an elegant and simple system capable
of self-organizing into a bundle of microtubules with a defined
length. Moreover, they show that only two additional proteins,
PRC1 and Xklp1, are required for making stable microtubule
bundles from highly dynamic polymers, with PRC1 generating
the bundles and Xklp1 controlling their length.
To understand how Xklp1 “monitors” and regulates the
size of the overlap region between microtubules, Bieling et al.
next measured how Xklp1 changes the dynamics of individual
microtubules that are not crosslinked. They found that Xklp1
Cell 142, August 6, 2010 ©2010 Elsevier Inc.  365
alone blocks the growth of microtubules but only at much
higher concentrations of Xklp1 than were required to limit the
length of microtubule bundles in the presence of PRC1. Biel-
ing et al. then demonstrate that the ability of Xklp1 to limit the
bundle length of crosslinked microtubules requires the proces-
sive motility of Xklp1. This suggests that Xklp1 motility plays an
active role in bundle architecture rather than simply acting to
regulate microtubule growth.
The key connection, however, came when the authors found
that the presence of PRC1 actually increases the time that Xklp1
stays within the overlapping region of two microtubules (i.e.,
the dwell time). Together with the previous results, this sug-
gests a model in which PRC1 forms antiparallel crosslinks of
microtubules and recruits Xklp1 within the crosslinked regions,
where it walks toward the plus ends of microtubules (Figure
1B). The effective concentration of Xklp1, which depends on the
concentration of PRC1, determines the extent to which Xklp1
blocks microtubule growth and thus the steady-state length of
the microtubule bundle. Thus, together Xklp1 and PRC1 deter-
mine the size and stability of the overlapping zone between
microtubules. Control of bundle length is vital because the
central spindle must elongate during late anaphase but then
shrink to a shorter size to form the midzone during cytokinesis.
Changes in PRC1 binding or the activity of Xklp1 during these
morphological transitions may provide a mechanism to control
the morphology and function of the central spindle.
PRC1 Forms Compliant Crosslinks between Microtubules
The above study illustrates how a stable bundle of microtu-
bules of a fixed size can form in the presence of two proteins
known to function at the midzone in vertebrate cells. However,
the work does not explain how PRC1 behaves under conditions
where other forces may be acting on the bundles. For example,
molecular motors actively slide crosslinked microtubules past
each other during the late stages in mitosis.
To characterize how PRC1 and its crosslinking activity
modulates or affects the sliding of bundled microtubules, Sub-
ramanian et al. (2010) take advantage of elegant microscopy
assays they developed in an earlier study to visualize microtu-
bule filaments sliding past each other in vitro by the kinesin-5
motors (Kapitein et al., 2005) (Figure 1B). Kinesin-5 proteins are
important for establishing the bipolarity of spindles during the
early stages of mitosis by actively sliding apart microtubules
of opposite polarity. Furthermore there is evidence that these
molecular motors slide antiparallel microtubules apart during
late anaphase (i.e., anaphase B).
Using this assay, Subramanian et al. now find that PRC1
displays two distinct behaviors in the presence of kinesin-5.
In certain cases, the concentration of PRC1 in the overlap
region and, thus the bundling length, stay constant while one
microtubule slides relative to another filament in the cross-
linked region. In the second case, the length of the crosslinked
region reduces at a rate similar to that at which the filaments
slide past each other. Although in this case PRC1 still tracked
the microtubule overlap zone, the crosslinking protein did not
reduce the velocity at which filaments slide even when excess
PRC1 was present. Together, these results demonstrate that
the crosslinks by PRC1 do not significantly resist the sliding
366  Cell 142, August 6, 2010 ©2010 Elsevier Inc.
motion of the microtubules by the kinesin-5 motors. This is
important because during cytokinesis these crosslinks must
remain in place to preserve the organization of the microtu-
bules in the midzone. At the same time, the crosslinks must
allow the microtubules to slide past each other to achieve and
maintain complete segregation of the chromosomes.
PRC1 Has a Spectrin-like Microtubule-Binding Domain
How does PRC1 maintain the structure of midzone bundles
while simultaneously allowing the overlap region to adjust to
the changing architecture of the central spindle? To answer
this question requires a better understanding of the structural
and biophysical aspects of how PRC1 bundles microtubules.
PRC1 has three prominent domains: the N-terminal domain,
which mediates homodimerization; the central domain, which
contains the major site for binding to microtubules; and the
C-terminal domain, which regulates the interaction with micro-
tubules (Figure 1C). Using time-lapse TIRF microscopy, Subra-
manian et al. found that PRC1 diffuses one-dimensionally along
the microtubule lattice for an average of 7 s, and the C-terminal
regulatory domain enhances this association. These findings
support the idea that binding of PRC1 to microtubules is medi-
ated by both the central microtubule-binding domain and the
unstructured C-terminal region. This latter domain contains a
large number of positively charged residues (i.e., lysines and
arginines), which is a common feature of regions that interact
with microtubules.
To gain further insight into how PRC1 interacts with micro-
tubules, Subramanian and colleagues determined the X-ray
crystal structure of the central microtubule-binding domain
of PRC1 and cryo-electron microscopy (cryo-EM) reconstruc-
tions of PRC1 fragments bound to microtubules. They found
that the central domain of PRC1 consists of a three-helix bun-
dle (?70 Å long) with connecting loops between the helices.
A cluster of highly conserved and positively charged residues
exists at the interface between α helix 1 and α helix 2 within
this helical bundle. Mutation of these residues diminished but
did not abolish microtubule binding by PRC1, further support-
ing the hypothesis that PRC1 possesses two major surfaces
that contact microtubules.
Interestingly, this domain shares structural homology with
the spectrin domains found in actin-binding proteins (Djinovic-
Carugo et al., 2002). Spectrin domains are not required for
interaction with actin filaments; instead, they typically link
together different functional domains of actin-binding proteins.
The present findings by Subramanian and colleagues identify
a new role for spectrin domains in regulating the microtubule
cytoskeleton.
Cryo-EM reconstructions of microtubules interacting with a
truncated fragment of PRC1, which includes the homodimeriza-
tion domain and the spectrin domain but not the C-terminal
domain, revealed that PRC1 crosslinks nearly all microtubules
in an antiparallel manner with a spacing of ?35 nm between
filaments (Figure 1C). Notably, the PRC1 molecules bound to
crosslinked microtubules were more structured than those
bound to a single microtubule. This suggests that crosslink-
ing itself converts PRC1 from an inherently flexible molecule to
a rigid one, which may enhance PRC1’s crosslinking activity.
In both cases, the cryo-EM data indicate that PRC1 interacts
with one α/β-tubulin heterodimer and extends as a single rod
shape almost perpendicular to the microtubule lattice (Figure
1C). Remarkably, the crystal structure of the spectrin domain
of PRC1 fits nicely into the cryo-EM density with the conserved
basic residues between α helix 1 and α helix 2 residing at the
microtubule surface. The binding site of PRC1 on the microtu-
bule surface is slightly displaced relative to where many motor
proteins interact, providing a possible clue for how motors and
crosslinking proteins can bind simultaneously to the same sur-
face of the microtubule.
The above studies clearly define the key microtubule-binding
element of PRC1, but the single-molecule studies using trun-
cated derivatives of PRC1 suggest that PRC1 has a second
microtubule-binding domain at its C terminus or this C-termi-
nal domain somehow regulates the interaction of the spectrin
domain with the microtubule surface. To address this ques-
tion, Subramanian and colleagues obtained a second cryo-EM
reconstruction of a PRC1 construct containing the spectrin
microtubule-binding domain and the C-terminal region. This
fragment bound in a similar position on the microtubule lat-
tice as the PRC1 construct containing only the dimerization
and spectrin domains (Subramanian et al., 2010). Therefore,
the most attractive hypothesis is that the C-terminal domain
enhances the affinity of PRC1 with microtubules rather than
forming a distinct second site for microtubule binding.
Future Perspectives
The two current papers by Bieling et al. and Subramanian et
al. provide critical insight into how inherently dynamic micro-
tubules are organized into functional sub-assemblies, which
are fundamental to multiple biological systems. It is remark-
able that just two proteins are sufficient to reconstitute the
morphological subassembly of the spindle midzone. However,
it is essential to remember that these proteins do not work in
isolation in vivo but rather function in the complex milieu of
the central spindle. The finding that PRC1 induces bundles of
microtubules that remain compliant to the action of kinesin-5
is key for understanding how the microtubules in the midzone
slide apart while still maintaining an organized structure. It
will be interesting to add kinesin-5 to the mixture of Xklp1 and
PRC1 to see how the system responds to regulators of both
microtubule growth and microtubule sliding, a situation that
more closely reconstitutes the physiological one.
The work presented here also opens the doors to crucial
structure-function and signaling studies on the PRC1 family
of proteins. The identification of residues that clearly form the
attachment site to microtubules will allow for the engineering of
mutations in PRC1 that modulate the strength of its interaction
with microtubules. Previous work has shown that phosphoryla-
tion regulates PRC1’s interaction with the central spindle (Fu et
al., 2007). It is interesting that those phosphorylation sites map
to the unstructured C-terminal domain of PRC1 that is positively
charged and shown to enhance microtubule binding. Interest-
ingly, another study recently found that phosphorylation of
an unstructured region of the kinetochore attachment protein
Hec1 also controls its affinity for microtubules (Guimaraes et
al., 2008). Thus, phosphorylation of unstructured domains may
play a general role in regulating the affinities of microtubule-
binding proteins. Finally, understanding how distinct structural
modifications of the PRC1 protein affect the morphology of
microtubule cytoskeletal arrays in vivo will be an important
avenue of future research endeavors.
Acknowledgments
The authors thank Clive Lloyd, Yixian Zheng, and Stephanie Ems-McClung
for helpful discussions. The authors are supported by National Institutes of
Health grant GM059618 to C.E.W. and National Science Foundation grant
0920555 to S.L.S.
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 Leading Edge

Perspective

The Pioneer Round of Translation:

Features and Functions
Lynne E. Maquat,1,* Woan-Yuh Tarn,1,3 and Olaf Isken2,*
1
Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester,
Rochester, NY 14642, USA
2
Institute for Virology and Cell Biology, University of Lübeck, 23562 Lübeck, Germany
3
Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan
*Correspondence: lynne_maquat@urmc.rochester.edu (L.E.M.), isken@molbio.uni-luebeck.de (O.I.)
DOI 10.1016/j.cell.2010.07.022

In mammalian cells, newly synthesized mRNAs undergo a pioneer round of translation that is
important for mRNA quality control. Following maturation of messenger ribonucleoprotein particles
during and after the pioneer round, steady-state cycles of mRNA translation generate most of the
cell’s proteins. Translation factors, RNA-binding proteins, and targets of signaling pathways that
are particular to newly synthesized mRNAs regulate critical functions of the pioneer round.

Introduction
In mammalian cells, newly synthesized transcripts are subject
to a series of nuclear processing steps to become mature tem-
plates for protein synthesis (Moore and Proudfoot, 2009). The
5′ ends of these transcripts acquire a 5′-m7GpppN cap struc-
ture (where N is the first transcribed nucleotide) while being
elongated by RNA polymerase II. The cap first binds to the
cap-binding protein (CBP) heterodimer CBP80-CBP20 (CBC),
which supports the pioneer round of mRNA translation (Isken
and Maquat, 2008). This round involves the loading of one or
more ribosomes, depending on the efficiency of translation ini-
tiation and the length of the open translational reading frame
(Isken and Maquat, 2008; Isken et al., 2008). The cap subse-
quently binds to the eukaryotic translation initiation factor 4E
(eIF4E), which directs steady-state rounds of mRNA translation
(Isken and Maquat, 2008).
Although CBC-bound mRNAs are precursors to eIF4E-
bound mRNAs, the two messenger ribonucleoprotein par-
ticles (mRNPs) differ in significant ways (Figure 1). For exam-
ple, spliced CBC-bound mRNAs differ from the eIF4E-bound
mRNAs that derive from them because they are associated
with one or more exon-junction complexes (EJCs) of proteins.
By the time eIF4E replaces CBC at the mRNA cap, EJCs are no
longer detectable, largely because most reside within the cod-
ing region of mRNAs and therefore are displaced by translat-
ing ribosomes during the pioneer round (Gehring et al., 2009;
Sato and Maquat, 2009). As another example, the poly(A) tails
of CBC-bound mRNAs are associated with the mostly nuclear
but shuttling poly(A)-binding protein N1 (PABPN1) and the pri-
marily cytoplasmic but likewise shuttling PABPC1; in contrast,
eIF4E-bound mRNAs do not detectably bind to PABPN1, the
replacement of which by PABPC1 is promoted by the pioneer
round of translation (Sato and Maquat, 2009).
Despite these and other differences (see below), both CBC-
bound and eIF4E-bound mRNAs most likely engage in similar
mechanisms of translation initiation, elongation, and termina-
tion. Therefore, it is not surprising that both CBC-bound and
eIF4E-bound mRNAs use many of the same translation initiation
factors. These factors include not only PABPC1, which data indi-
cate is important for activating the translation of both mRNPs,
but also eIF4G, eIF3, eIF4B, eIF4A, eIF2 (Figures 1 and 2; Isken
and Maquat, 2008), and undoubtedly many other factors that
work in conjunction with ribosomes to synthesize proteins.
Although both mRNPs support protein synthesis and can be tar-
geted for translational activation or repression, the purpose for
so doing is distinct: the translation of CBC-bound mRNAs pro-
vides a means to control the quality of gene expression; in con-
trast, the translation of eIF4E-bound mRNAs generates the bulk
of cellular proteins (Isken and Maquat, 2008). Here, we discuss
our growing understanding of how cells maintain the specialized
functions of each mRNP via associations with particular transla-
tion factors, RNA-binding proteins, and signaling targets.
The Pioneer Round of Translation Supports Nonsense-
Mediated mRNA Decay
In higher eukaryotes, the vast majority of genes contain multi-
ple introns that are removed from the primary transcript by the
process of splicing. Splicing may be accompanied by routinely
made mistakes so as to result in mRNAs that contain a prema-
ture termination codon (McGlincy and Smith, 2008). Premature
termination codons can also arise during splicing as a con-
sequence of a conditionally regulated process, as exemplified
by those pre-mRNAs whose splice site usage is influenced by
the encoded RNA-binding protein (McGlincy and Smith, 2008).
Nonsense-mediated mRNA decay is a translation-dependent
mRNA surveillance pathway that detects and eliminates tran-
scripts containing premature termination codons and thus
have the potential to be deleterious by virtue of the truncated
proteins they encode (see, e.g., Rebbapragada and Lykke-
Andersen, 2009).
During the pioneer round of translation, nonsense-mediated
mRNA decay is thought to be triggered by the first ribosome
that translates newly processed CBC-bound mRNAs and
arrives at a premature termination codon (or a normal termi-
nation codon) that is situated more than ?50–55 nucleotides
upstream of an EJC-bearing exon-exon junction, although

368  Cell 142, August 6, 2010 ©2010 Elsevier Inc.

Figure 1. Pioneer and Steady-State Translation Initiation Complexes
Shown is a CBP80-CBP20 (CBC)-bound mRNP from the pioneer round of translation and an eIF4E-bound mRNP from steady-state translation. CBC-bound
mRNAs direct pioneer rounds of translation, whereas eIF4E-bound mRNAs, which derive from the remodeling of CBC-bound mRNAs, support the bulk of cel-
lular protein synthesis. CBC-bound mRNAs are associated with at least one exon-junction complex (EJC) (provided they are the product of pre-mRNA splicing)
and the poly(A)-binding proteins PABPN1 and PABPC1. PYM, which interacts with EJC components and the small 40S ribosomal subunit (not shown), and
SKAR (S6 kinase 1 ALY-REF-like target), a component of EJCs, may help to activate the pioneer round of translation. eIF3e, a non-core subunit of the eukaryotic
translation initiation factor eIF3, may regulate the translation of a specific set of CBC-bound mRNAs. CTIF (CBP80-CBP20-dependent translation initiation fac-
tor) interacts directly with CBP80, as does eIF4G. It is currently unclear whether CTIF is the sole eIF4G-like molecule or if eIF4G also functions during pioneer
rounds. eIF4G has been proposed to form a complex with poly(A)-bound PABPC1 to circularize and promote the translation of CBC-bound mRNAs, similar to
how poly(A)-bound PABPC1 circularizes and promotes the translation of eIF4E-bound mRNAs. Importin (IMP)-β binds to IMP-α (which is a stable constituent of
cap-bound CBC) and augments the translation-independent replacement of CBC by eIF4E. In contrast, the pioneer round of translation promotes the removal
of EJCs and of associated RNA-binding proteins such as SF2/ASF (which also activates the pioneer round; not shown) and the replacement of PABPN1 by
PABPC1. The insets depict how the SURF complex (comprising the PIK-related protein kinase SMG1, UPF1, eRF1, and eRF3) assembles together with an
80S stalled ribosome at a premature termination codon of CBC-bound mRNA. SMG1 subsequently phosphorylates UPF1 upon UPF1 and SMG1 binding to a
downstream EJC during the process of nonsense-mediated mRNA decay. AUG, translation initiation codon; STOP, normal termination codon.

there are exceptions to this rule. The CBC plays a critical role
in nonsense-mediated mRNA decay not only because it com-
prises the mRNP that harbors EJCs but also because CBP80
interacts directly with the nonsense-mediated mRNA decay
factor, up-frameshift 1 (UPF1), enhancing the efficiency of this
process (Isken and Maquat, 2008). In short, nonsense-medi-
ated mRNA decay is thought to involve recognition of prema-
ture termination codons by the SURF complex, which consists
of the UPF1 phosphoinositide 3-kinase (PIK)-related protein
kinase called SMG1, UPF1, and the two translation termina-
tion factors referred to as eukaryotic release factor (eRF)1 and
eRF3 (Figure 1; Kashima et al., 2006; Isken and Maquat, 2008).
After recognition of the premature termination codon, SMG1
and UPF1 join the EJC that resides downstream of the pre-
mature termination codon. Notably, the interaction between
CBP80 and UPF1 promotes nonsense-mediated mRNA decay
at two sequential steps (Hwang et al., 2010). The first is the
association of SMG1-UPF1 with eRF1-eRF3 at a premature
termination codon to form the SURF complex. The second is
the association of SMG1-UPF1 with a downstream EJC, which
results in SMG1-mediated UPF1 phosphorylation. Subse-
quently, phosphorylated UPF1 elicits translational repression
by binding to the eIF3 constituent of the 43S preinitiation com-
plex that is poised at the translation initiation codon (Isken et
al., 2008). Phosphorylated UPF1 also promotes the recruitment
of mRNA decay activities (Isken et al., 2008). The importance

Cell 142, August 6, 2010 ©2010 Elsevier Inc.  369

Figure 2. Signaling Pathways and Translation
Shown are the signaling pathways that regulate the pioneer round of translation (top) and subsequent steady-state translation (bottom). Under condi-
tions that activate the mTOR signaling pathway, mTORC1 becomes activated and may bind to the translation initiation factor eIF3 associated with
CBP80-CBP20 (CBC)-bound mRNA resulting in the phosphorylation and dissociation of activated S6 kinase 1 (S6K1). The EJC component SKAR (S6
kinase 1 ALY-REF-like target) then recruits phosphorylated (i.e., activated) S6K1. Activated S6K1 next mediates the phosphorylation of SKAR and other
downstream effectors, including possibly eIF4B and PDCD4 (programmed cell death factor 4), that also associate with CBC-bound mRNA and thereby
promote the pioneer round of translation for spliced mRNAs. The Cdc42-dependent phosphorylation of S6K1 also may promote the binding of CBC to
cap structures, which would further stimulate the pioneer round. Another CBC-bound mRNA constituent, SF2/ASF, binds to the RNA export receptor TAP
and also triggers S6K1 signaling via mTORC1 in ways that promote CBC-bound mRNA translation. Moreover, PYM interacts with the EJC core proteins
Y14-MAGOH and with the 40S ribosomal subunit to somehow promote the translation of spliced mRNAs presumably during and certainly beyond the
pioneer round.
(Left) The boxes summarize the translational regulation of CBC-bound or eIF4E-bound mRNAs under various cell-growth conditions. Under conditions of heat
shock, hypoxia, or serum starvation, the pioneer round of translation is favored over steady-state translation; steady-state translation is largely inhibited by
phosphorylation of eIF2α (not shown) or by binding of 4E-BP1 to eIF4E.

of the CBC to nonsense-mediated mRNA decay is corrobo-
rated by the finding that decay is still restricted to CBC-bound
mRNAs in the case of mRNAs containing premature termina-
tion codons that initiate translation in a cap-independent mode
from an internal ribosome entry site (IRES), provided that IRES
function depends on eIF3 (Isken and Maquat, 2008).
CBC-bound mRNAs form polysomes that are smaller than
the polysomes associated with their eIF4E-bound counterparts,
consistent with the replacement of CBC by eIF4E taking place on
polysomes during the pioneer round of translation. This finding,
and data demonstrating that nonsense-mediated mRNA decay
involves a step of translational repression that targets eIF3, indi-
cate that CBC-bound mRNAs, like eIF4E-bound mRNAs, are
generally loaded with more than one ribosome, eIF3, and other
well-characterized translation initiation factors.
Specialized Factors of the Pioneer Round of ­Translation
The process of translation can be divided into four phases—
initiation, elongation, termination, and ribosome recycling.
The majority of regulation occurs during the initiation phase
(Sonenberg and Hinnebusch, 2009). There is considerably
more known about the translation of eIF4E-bound mRNAs than
the translation of CBC-bound mRNAs, which is a relatively
recent discovery. Nevertheless, there are likely to be many
more similarities than differences between the translation of
the two mRNPs, as available data have borne out. Factors
that specifically regulate the pioneer round of translation will
likely target, directly or via another protein, the CBC, the EJC,
PABPN1, or other constituents that do not typify eIF4E-bound
mRNA, just as factors that specifically regulate steady-state
translation often target eIF4E.

370  Cell 142, August 6, 2010 ©2010 Elsevier Inc.

PYM
Compared to nonspliced mRNA, it is well established that the
process of pre-mRNA splicing endows spliced mRNA with a
higher translational capacity during both pioneer and steady-
state rounds of translation (Moore and Proudfoot, 2009). The abil-
ity of splicing to promote translation appears to be mediated at
least in part by EJCs, which data indicate are dynamic rather than
either static or homogenous complexes. Different EJC constitu-
ents may augment different steps of translation initiation (Lee et
al., 2009). For example, PYM may move between mRNA-bound
EJCs and translationally active ribosomes: PYM can interact with
the EJC core proteins Y14-MAGOH and, via a separate domain,
with the 40S ribosomal subunit and the 48S preinitiation complex
to somehow promote the translation of spliced mRNAs during
and beyond the pioneer round (Diem et al., 2007). The finding that
PYM detectably coimmunoprecipitates with CBP80 but not eIF4E
not only is consistent with the idea that PYM is present during,
and gone after, the pioneer round of translation but also raises
the possibility that PYM constitutes a fraction of ribosomes that
may specifically function during pioneer rounds. For example, by
coupling ribosome recruitment to EJC disassembly, PYM may
ensure the preferential recruitment of newly synthesized CBC-
bound mRNAs over eIF4E-bound mRNAs to the translational
machinery. Alternatively, translation factors that are unique to
CBC-bound mRNAs and/or EJCs could recruit ribosomes bound
by PYM for the pioneer round. Along these lines, PYM is recruited
to those newly exported mRNAs of Kaposi’s sarcoma-associated
herpesvirus that are intronless and thus devoid of EJCs, by the
viral open reading frame 57 protein (ORF57). ORF57 interacts with
PYM and enhances viral mRNA translation via the PYM-mediated
recruitment of preinitiation complexes (Boyne et al., 2010). Nota-
bly, the recent report that PYM is an EJC disassembly factor that
inhibits nonsense-mediated mRNA decay when overexpressed,
i.e., when a fraction of PYM exists free of an association with 40S
ribosomal subunits so as to promiscuously dissociate EJCs (Geh-
ring et al., 2009), is compatible with the idea that EJC constituents
can activate translation.
SKAR
The EJC constituent called SKAR, for S6 kinase 1 (S6K1) ALY-
REF-like target, may also mark spliced CBC-bound mRNAs
with a translational status that is distinct from the translational
status of eIF4E-bound mRNAs and provides another example
of how EJCs positively regulate the pioneer round of transla-
tion. The mammalian target of the rapamycin (mTOR) signaling
pathway promotes cellular translation depending on the pres-
ence of growth factors, the absence of stress, and the availabil-
ity of nutrients or other energy sources (Ma and Blenis, 2009).
mTOR is a PIK-related protein kinase that, together with raptor
and LST8, forms the mTOR complex 1 (mTORC1). Interestingly,
signaling through mTORC1 modulates the translation of both
CBC-bound mRNAs and eIF4E-bound mRNAs by distinct but
undoubtedly overlapping mechanisms (Figure 2).
In the case of eIF4E-bound mRNAs, the recruitment of acti-
vated mTORC1 by eIF3 into the proximity of the eIF4E-binding
protein 1 (4E-BP1) activates translation by mediating the phos-
phorylation of 4E-BP1 and S6K1. 4E-BP1 phosphorylation results
in 4E-BP1 dissociation from mRNA-bound eIF4E, which activates
translation by allowing eIF4G to interact with and thereby stabilize
the association of eIF4E, eIF4A, and PABPC1 with mRNA. S6K1
phosphorylation results in the dissociation of activated S6K1 from
mRNA-bound eIF3. Released S6K1 promotes scanning of the 43S
preinitiation complex to the AUG translation initiation codon and,
therefore, translation by phosphorylating both the eIF4A activator
eIF4B and the eIF4A inhibitor and tumor suppressor programmed
cell death 4 (PDCD4).
In the case of CBC-bound mRNAs, activated mTORC1 may be
recruited by eIF3 and, additionally, SF2/ASF (see below), promot-
ing the phosphorylation-induced activation of S6K1 and then the
dissociation of activated phosphorylated S6K1. EJC-bound SKAR
then recruits activated S6K1 to CBC-bound spliced mRNAs, and
SKAR-bound S6K1 promotes the pioneer round of translation by
allowing for the phosphorylation of SKAR and other downstream
effectors that could include eIF4B and PDCD4, which also asso-
ciate with CBC-bound mRNAs (Ma et al., 2008). The finding that
SKAR regulates cell growth (Richardson et al., 2004) raises the
interesting question of how the mTORC1-induced translation of
all or a subset of CBC-bound spliced mRNAs is transduced to a
functionally significant increase in total-cell protein synthesis.
SF2/ASF
The serine-arginine-rich protein SF2/ASF plays a central role in
recruiting the splicing machinery to pre-mRNA splice sites and
can either enhance or inhibit splicing. Like PYM, SF2/ASF coim-
munoprecipitates with CBP80 but not detectably with eIF4E
(Sato et al., 2008). Data indicate that SF2/ASF, when bound to
exonic sequences, can travel as a constituent of CBC-bound
mRNPs from nuclei into the cytoplasm, where it has the ability
to promote the pioneer round of translation (Sato et al., 2008).
Evidence indicates that SF2/ASF recruits a number of transla-
tional activators to mRNAs, including the RNA export receptor
TAP (Sato et al., 2008) and mTORC1 (Michlewski et al., 2008).
Also, like PYM, SF2/ASF promotes mRNA translation beyond
the pioneer round (Sato et al., 2008; Michlewski et al., 2008).
CBP80
Another effector of the pioneer round could be CBP80 as the
binding of CBC to cap structures is stimulated by growth fac-
tors during the G1/S phase of the cell cycle, activated forms of
the Ras proto-oncogene protein, and the Rho-GTPase Cdc42.
Cdc42 has been shown to transduce extracellular signals from
G-coupled protein receptors, integrins, and growth factor
receptors at least in part by binding to S6K1 in a GTP-depen-
dent manner. Binding facilitates recognition of the Cdc42-
S6K1 complex by upstream S6K-activating kinases (Chou and
Blenis, 1996; Wilson and Cerione, 2000). Moreover, signaling
from Cdc42 to CBP80 is transmitted in part by the S6K-cata-
lyzed phosphorylation of CBP80, although the biological con-
sequence remains unknown (Ma and Blenis, 2009).
As exemplified above, it appears that an efficient pioneer
round of translation is followed by efficient steady-state
cycles of translation. Growth factors may augment cellu-
lar protein synthesis not only by promoting CBC binding to
caps but also by expediting the replacement of CBC by eIF4E
(see below). Despite not yet knowing mechanistic details, it is
clear that activating or inhibiting pioneer rounds of translation
depending on cellular growth conditions is likely to provide
an important checkpoint mechanism for the bulk of cellular
protein synthesis.
Cell 142, August 6, 2010 ©2010 Elsevier Inc.  371
Importin-α
Regulated binding of CBC to mRNA caps is additionally influ-
enced by importin-α, which stably associates with cap-bound
CBC (Sato and Maquat, 2009). Cytoplasmic importin-β inter-
acts directly with importin-α and CBP20, thereby promoting the
replacement of CBC by eIF4E at mRNA caps (Figure 1; Dias et
al., 2009; Sato and Maquat, 2009). In view of the recent structure
of an importin-α-CBC-cap complex and a structural model of
the importin-β-importin-α-CBC complex (Dias et al., 2009), it will
be possible to study how signaling pathways, and the resulting
posttranslational modifications of importin-α, importin-β, and/
or CBC, might regulate importin-α or importin-β binding to cap-
associated CBC to either stabilize or disrupt the interaction of
CBC with mRNA caps. When considering the critical role of CBC
in assuring the quality of gene expression through nonsense-
mediated mRNA decay, and that CBC can be replaced by eIF4E
even when the pioneer round of translation is inhibited (Sato and
Maquat, 2009), it is important that timing the pioneer round rela-
tive to the replacement of CBC by eIF4E be coordinated so that
the pioneer round of translation largely takes place first.
eIF3e
Mammalian eIF3, which is a large protein complex consisting of
?13 subunits, has multiple functions in stimulating translation
initiation (Sonenberg and Hinnebusch, 2009). The eIF3 subunit
eIF3e (also called p48/INT6) coimmunoprecipitates with CBP80
as well as the EJC constituent and nonsense-mediated mRNA
decay factor UPF2, but it does not detectably coimmunoprecipi-
tate with eIF4E and, like all eIF3 subunits that have been tested,
is essential for nonsense-mediated mRNA decay (Morris et al.,
2007; Isken et al., 2008). eIF3e, which is a core subunit of mam-
malian eIF3 that is not present in all species, appears to contribute
to the recruitment of ribosome-bound eIF3 to mRNA by directly
associating with eIF4G (Sonenberg and Hinnebusch, 2009). How-
ever, at least in fission yeast, eIF3e is not critical for global pro-
tein synthesis, and there exist distinct eIF3 complexes that differ
in their non-core subunits. Interestingly, those complexes that
contain eIF3e associate with a restricted set of mRNAs, some of
which are implicated in the response to cell stress (Zhou et al.,
2005). Therefore, it is possible that a specialized eIF3e-containing
eIF3 complex, if present in mammals, could specifically function
during pioneer rounds of translation by recruiting a defined set of
newly synthesized mRNAs to the translational machinery. By so
doing, this specialized complex could route those mRNAs that
are targets of nonsense-mediated mRNA decay to this pathway
and activate the steady-state translation of those that are not non-
sense-mediated mRNA decay targets.
CTIF
Another initiation factor that is central to steady-state transla-
tion is eIF4G, which binds directly to cap-associated eIF4E and
serves critical roles as a scaffold for eIF3, PABPC1, and eIF4A.
eIF4G, which also binds directly to CBC, is thought to function in
a similar fashion during pioneer rounds of translation. Recently,
an eIF4G-like protein called CBP80-CBP20-dependent transla-
tion initiation factor (CTIF) has been implicated specifically during
pioneer rounds. CTIF interacts directly with CBP80 as well as pre-
mRNAs, coimmunoprecipitates with eIF3 in an RNase-resistant
manner, and functions in the translation of CBC-bound mRNAs;
however, it plays no detectable role in the translation of eIF4E-
372  Cell 142, August 6, 2010 ©2010 Elsevier Inc.
bound mRNAs (Kim et al., 2009). CTIF could promote the recruit-
ment of ribosomes to newly synthesized CBC-bound mRNAs. As
one possibility, CTIF, which contains a middle domain of eIF4G
(MIF4G) but lacks the eIF4E-binding domain of eIF4G, may func-
tionally replace eIF4G during pioneer rounds if, for example, it
(possibly together with CBP80, which contains three MIF4Gs)
serves as an eIF4G-like scaffold for CBC-bound mRNPs. Alter-
natively, CTIF may function in addition to eIF4G considering that
CTIF lacks the PABPC1- and eIF4A-binding domains that typify
eIF4G and that another MIF4G-containing protein, called SLIP1,
interacts directly with eIF4G to promote the translation of histone
mRNA (Cakmakci et al., 2008). Moreover, data indicate that eIF4G
functions during pioneer rounds of translation. Regardless, as is
possible for eIF3, the use of translation initiation factors that are
unique to and activate pioneer rounds could be advantageous by
shortening the time span between transcriptional activation and
the start of protein production.
The Pioneer Round during Cell Stress
The bulk of cellular cap-dependent translation is rapidly downreg-
ulated by signaling pathways in response to most physiological
and environmental stressors as part of a repertoire of events that
mitigate cellular damage and promote cell survival (Figure 2). This
global downregulation during, for example, viral infection, amino
acid starvation, hypoxia, or heat shock is often accompanied by
eIF2α phosphorylation. eIF2α phosphorylation decreases the
translation of the majority of CBC-bound as well as eIF4E-bound
mRNAs by limiting ternary complex abundance while mediating
the selective translation of a subset of cellular mRNAs that ini-
tiate translation in a mechanism that depends on an upstream
open reading frame. These mRNAs encode specific transcription
factors that help the cell adapt to stress and ultimately restore
translation (Holcik and Sonenberg, 2005). In addition to eIF2α
phosphorylation, stress-induced proteolysis of translation initia-
tion factors can also compromise cellular translation.
There are variations to this theme that result in the differen-
tial regulation of pioneer rounds of translation relative to sub-
sequent rounds of translation. For example, the preferential
translation of CBC-bound mRNAs is maintained during serum
starvation and heat shock, when the translation of eIF4E-bound
mRNAs is compromised (Oh et al., 2007b; Marín-Vinader et al.,
2006). Serum starvation does not result in eIF2α phosphoryla-
tion but instead activates 4E-BP1.
As another example, during early phases of hypoxia, activa-
tion of the endoplasmic reticulum kinase PERK leads to eIF2α
phosphorylation and the translational inhibition of both CBC-
bound and eIF4E-bound mRNAs (Oh et al., 2007a). However,
during late stages of hypoxia, eIF2α dephosphorylation allows
for the resumption of CBC-bound mRNA translation (Oh et al.,
2007a). Concomitantly, in an eIF2α-independent pathway, dis-
ruption of eIF4E-eIF4G-eIF4A and sequestration of eIF4E by
dephosphorylated 4E-BP1 and the eIF4E-transporter (which
moves eIF4E into the nucleus and processing bodies) maintain
the repression of eIF4E-bound mRNA translation (Koritzinsky
et al., 2006; Lee et al., 2008). Conceivably, the restoration of
CBC-bound mRNA translation as a first step toward transla-
tional normalcy promotes cell survival after stress by allowing
for the surveillance of newly synthesized mRNAs. Furthermore,
the renewed engagement of newly made mRNAs with the
translational machinery could speed up the cellular response
after stress.
Spatial Regulation of the Pioneer Round of Translation
Proper embryonic patterning and development as well as neu-
ronal function often involve proteins that bind to the 3′ untrans-
lated regions (3′ UTRs) of particular mRNAs in a sequence-spe-
cific manner to couple mRNA translational activation and mRNA
localization (Sonenberg and Hinnebusch, 2009). By so doing, an
expression gradient of the encoded morphogens, in the case of
oocytes, or of proteins that maintain synaptic function or plastic-
ity, in the case of neurons, is spatially established.
There have been many reports of translational repression
occurring prior to mRNA localization so as to inhibit ectopic
protein production from unlocalized mRNAs. If the sole mecha-
nism of translational repression targets eIF4E, then an mRNA
should have undergone the pioneer round of translation prior
to repression although, as noted above, it is possible for eIF4E
to replace CBC without a pioneer round. In theory, other mech-
anisms of repression might target both CBC-bound mRNA and
eIF4E-bound mRNA.
Examples of translational repression include sequestrating
mRNAs into translationally silenced particles, as Bruno does
for oskar mRNA in Drosophila; recruiting the CCR4 deadeny-
lase to shorten poly(A) tail length, as Bicaudal-C does for its
own mRNA and certain other germline mRNAs in Drosophila;
and blocked joining of the 60S ribosomal subunit to the 48S
preinitiation complex, as Zipcode-binding protein 1 (ZBP1,
also called IMP1) does for β-actin mRNA at the leading lamel-
lipodia of fibroblasts or neurite growth cones (Besse and
Ephrussi, 2008; Sonenberg and Hinnebusch, 2009). Notably,
the findings that ZBP1 associates cotranscriptionally with
β-actin mRNA (Besse and Ephrussi, 2008), and RNP granules
undergoing ZBP1-mediated transport contain CBP80 and EJC
constituents but lack detectable eIF4E (Jønson et al., 2007),
suggest that ZBP1-mediated translational repression targets
CBC-bound transcripts.
To date, studies of arc mRNA and other specific mRNAs in
mammalian neurons provide the best examples of translational
repression that targets CBC-bound mRNAs (Giorgi et al., 2007).
arc mRNA, which harbors two 3′ UTR introns, is a natural tar-
get of nonsense-mediated mRNA decay: When the pioneer
round of translation terminates normally, arc mRNA undergoes
nonsense-mediated mRNA decay. The arc gene is transcrip-
tionally induced upon intense synaptic activation, and the
resulting mRNA is translated and thus targeted for nonsense-
mediated mRNA decay, once it localizes to activated synapses
in a mechanism that depends on glutamatergic receptors. By
essentially limiting ARC protein synthesis to the pioneer round
of translation at activated synapses, improper protein synthe-
sis at inopportune times and cellular locations is prevented.
These findings are consistent with data indicating that CBP80-
bound mRNAs migrate to spines when glutamatergic recep-
tors of rat dendrites are stimulated (di Penta et al., 2009) and
thus have yet to undergo the pioneer round of translation. At
present, which signaling factors control these pioneer rounds
of translation in neurons remain unknown.
Features of the pioneer translation initiation complex also
appear to be important for proper localization of oskar mRNA
to the posterior pole in Drosophila oocytes. For example, splic-
ing of the first intron of oskar pre-mRNA (or another intron at
the site of the first intron) is required for oskar mRNA localiza-
tion, presumably by virtue of the resulting EJC in conjunction with
nearby mRNA sequences (Hatchet and Ephrussi, 2004). In fact,
demonstrated roles for the EJC constituents eIF4AIII, Tsunami-
Magonashi, and Barentz (the latter two of which are orthologous
to mammalian Y14-MAGOH and MLN51, respectively) in oskar
mRNA localization and their colocalization with oskar mRNA at
the posterior pole (Besse and Ephrussi, 2008) indicate that the
pioneer round of translation and removal of the first EJC occur
after the regulatory steps of translational repression and local-
ization. As noted above, these regulatory steps may involve the
RNA-binding protein Bruno. As oskar mRNA is also translation-
ally repressed by Bruno-mediated recruitment of the Cup protein,
which targets eIF4E (Besse and Ephrussi, 2008), there appear
to be multiple pathways to translationally silence oskar mRNA.
In fact, a number of mRNAs are repressed at different steps of
translation when bound by CBC or eIF4E.
MicroRNAs
It was recently reported that the microRNA (miRNA) miR-2 medi-
ates translational repression, which interferes with the interac-
tion between eIF4E and eIF4G in a way that is predicted to have
no consequence to the pioneer round of translation (Zdanow-
icz et al., 2009). Therefore, mRNA caps bound by eIF4E could
represent another signature target used by cells to differentially
regulate the translation of CBC-bound mRNA relative to eIF4E-
bound mRNA. Nevertheless, other mechanisms of miRNA-
mediated translational repression can function simultaneously
to repress the translation of CBC-bound mRNA. This became
evident through the recent demonstration that (1) Ago2, which
is known to inhibit the translation of its target mRNAs, is loaded
onto both CBC-bound mRNAs and eIF4E-bound mRNAs, (2)
the loading of endogenous microRNA-induced silencing com-
plexes (miRISCs) onto the 3′ UTRs of CBC-bound mRNAs that
contain a premature termination codon abrogates nonsense-
mediated mRNA decay, and (3) several natural substrates of
nonsense-mediated mRNA decay are stabilized by miRISC-
mediated translational repression (Choe et al., 2010). It will be
interesting to determine if factors that are specific for CBC-
bound mRNA and the pioneer round of translation are recog-
nized during miRNA-mediated translational repression.
Future Directions
Mammalian cells have evolved two mRNP templates for protein
synthesis that manifest a precursor-product relationship and
serve distinct functions. Although these two templates share
many constituent proteins that homogenize their regulation, at
least some unique features impart a means for their differential
control. Examples also exist of distinct regulatory mechanisms
that coordinate CBC-bound mRNA translation and eIF4E-bound
mRNA translation by targeting factors that are particular to each
mRNP. As eIF4E-bound mRNA supports the bulk of cellular protein
synthesis, it is the logical target for a rapid response to changes
in physiological conditions. However, CBC-bound mRNA holds
Cell 142, August 6, 2010 ©2010 Elsevier Inc.  373
the unique capability of enhancing subsequent rounds of transla-
tion, downregulating further translation, or completely stopping
not only CBC-supported mRNA surveillance but also eIF4E-
supported protein production. Future studies are expected to
illuminate molecular aspects of known and unforeseen regulatory
mechanisms.
Acknowledgments
We thank Chenguang Gong and Hanae Sato for helpful comments and ac-
knowledge NIH R01 grants GM074593 and GM59614 (L.E.M.), the National
Science Council of Taiwan (W.-Y.T.), and start-up funds from the University of
Lübeck (O.I.) for support.
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Direct Reprogramming of Fibroblasts
into Functional Cardiomyocytes
by Defined Factors
Masaki Ieda,1,2,3,6,* Ji-Dong Fu,1,2,3 Paul Delgado-Olguin,1,2,4 Vasanth Vedantham,1,5 Yohei Hayashi,1
Benoit G. Bruneau,1,2,4 and Deepak Srivastava1,2,3,*
1Gladstone Institute of Cardiovascular Disease
2Department of Pediatrics
3Department of Biochemistry and Biophysics
4Cardiovascular Research Institute
5Department of Medicine
University of California, San Francisco, San Francisco, CA 94158, USA
6Present address: Departments of Cardiology and of Clinical and Molecular Cardiovascular Research, Keio University School of Medicine,
Shinanomachi 35, Shinjuku-ku, Tokyo 160-8582, Japan
*Correspondence: ieda@cpnet.med.keio.ac.jp (M.I.), dsrivastava@gladstone.ucsf.edu (D.S.)
DOI 10.1016/j.cell.2010.07.002
SUMMARY
The reprogramming of fibroblasts to induced plurip-
otent stem cells (iPSCs) raises the possibility that
a somatic cell could be reprogrammed to an alterna-
tive differentiated fate without first becoming a stem/
progenitor cell. A large pool of fibroblasts exists in
the postnatal heart, yet no single ‘‘master regulator’’
of direct cardiac reprogramming has been identified.
Here, we report that a combination of three develop-
mental transcription factors (i.e., Gata4, Mef2c, and
Tbx5) rapidly and efficiently reprogrammed post-
natal cardiac or dermal fibroblasts directly into differ-
entiated cardiomyocyte-like cells. Induced cardio-
myocytes expressed cardiac-specific markers, had
a global gene expression profile similar to cardio-
myocytes, and contracted spontaneously. Fibro-
blasts transplanted into mouse hearts one day after
transduction of the three factors also differentiated
into cardiomyocyte-like cells. We believe these find-
ings demonstrate that functional cardiomyocytes
can be directly reprogrammed from differentiated
somatic cells by defined factors. Reprogramming
of endogenous or explanted fibroblasts might pro-
vide a source of cardiomyocytes for regenerative
approaches.
INTRODUCTION
Heart disease is a leading cause of adult and childhood mortality.
The underlying pathology is typically loss of cardiomyocytes that
leads to heart failure or improper development of cardiomyo-
cytes during embryogenesis that leads to congenital heart
malformations. Because postnatal cardiomyocytes have little
or no regenerative capacity, current therapeutic approaches
are limited. Embryonic stem cells possess clear cardiogenic
potential, but efficiency of cardiac differentiation, risk of tumor
formation, and issues of cellular rejection must be overcome
(Ivey and Srivastava, 2006; Laflamme et al., 2007; Nussbaum
et al., 2007; van Laake et al., 2008). The ability to reprogram
fibroblasts into induced pluripotent stem cells (iPSCs) with four
defined factors might address some of these issues by providing
an alternative source of embryonic-like stem cells (Takahashi
and Yamanaka, 2006). However, generating sufficient iPSC-
derived cardiomyocytes that are pure and mature and that can
be delivered safely remains challenging (Zhang et al., 2009).
The human heart is composed of cardiomyocytes, vascular
cells, and cardiac fibroblasts. In fact, cardiac fibroblasts com-
prise over 50% of all the cells in the heart (Baudino et al.,
2006; Camelliti et al., 2005; Snider et al., 2009). Cardiac fibro-
blasts are fully differentiated somatic cells that provide support
structure, secrete signals, and contribute to scar formation
upon cardiac damage (Ieda et al., 2009). Fibroblasts arise from
an extracardiac source of cells known as the proepicardium,
and do not normally have cardiogenic potential (Snider et al.,
2009). The large population of endogenous cardiac fibroblasts
is a potential source of cardiomyocytes for regenerative therapy
if it were possible to directly reprogram the resident fibroblasts
into beating cardiomyocytes. Unfortunately, although embryonic
mesoderm can be induced to differentiate into cardiomyocytes
(Takeuchi and Bruneau, 2009), efforts to accomplish this in
somatic cells have thus far been unsuccessful, and to our knowl-
edge, no ‘‘master regulator’’ of cardiac differentiation, like MyoD
for skeletal muscle (Davis et al., 1987), has been identified
to date.
The generation of iPSCs suggests that a specific combination
of defined factors, rather than a single factor, could epigeneti-
cally alter the global gene expression of a cell and allow greater
plasticity of cell type than previously appreciated. Consistent
Cell 142, 375–386, August 6, 2010 ª2010 Elsevier Inc. 375
A C Control αMHC-GFP explant Figure 1. Screening for Cardiomyocyte-
Off On
FACS 10 5 0% 0%10 5
68% 0% Inducing Factors
αMHC promoter GFP αMHC promoter GFP
10 4 10
4
(A) Schematic representation of the strategy to test
TFs 10 3 10
3 candidate cardiomyocyte-inducing factors.

Thy1
10 2 10 2
(B) Morphology and characterization of fibroblast-
Cardiac fibroblasts Cardiomyocytes 0
100% 0%
0
31.4% 0.6% like cells migrating from aMHC-GFP heart
2 3 4 5
0 102 103 10 4 10 5
0 10 10 10 10
explants. Phase contrast (left), GFP (middle), and
αMHC-GFP
B D Thy-1 immunostaining (right). Insets are high-
6 magnification views. See also Figure S1.

Explant
*

αMHC-GFP+
5 (C) Thy-1+/GFP cells were FACS sorted from

cells (%)
4
3 ** explant cultures for reprogramming.
2 (D) Summary of FACS analyses for a-MHC-GFP+
1 * cells. Effect on GFP+ cell induction with 14 factors
0

Control

Baf60c
Gata4
Hand2
Hopx
Hrt2
Isl1
Mef2c
Mesp1
Myocd
Nkx2.5
Pitx2c
Smyd1

Tbx5
14 factors

Srf
Phase αMHC-GFP Thy1 or the removal of individual factors from the pool of
14 factors (n = 3). Removal of Baf60c, Hand2,
14 factors - 1 Hopx, Hrt2, or Pitx2c did not decrease the percent
E of GFP+ cells and were excluded for further anal-
10 4 10
4
10 4 10 4
Control 14 factors 14 factors - Gata4 14 factors - Pitx2c yses. See also Figure S2.
10
3
10
3
10 3 10
3
(E) FACS plots for analyses of GFP+ cells. GFP+
100% 97.3% 98.9% 93.4% cells were analyzed 1 week after 14 factor trans-
10 2 10 2 10 2 10 2
duction. The number of GFP+ cells were reduced
10
1 0% 10 1
1.7% 10 1
0.5% 10
1 5.1% by removal of Gata4, but increased by removal of
PI

Pitx2c from 14 factors.

10
0
10
0
10
0
10
0
(F–H) Effect on GFP+ cell induction of the removal
100 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10
0
10 1 10 2 10 3 10 4 100 10 1 10 2 10 3 10 4
of individual factors from the pool of 9 (F), 6 (G), or 5
αMHC-GFP
(H) factors (n = 3). Factors that did not decrease
F G H efficiency upon removal were excluded from
20
* 20 20 **
** further study.
αMHC-GFP+

αMHC-GFP+

αMHC-GFP+

15 ** 15 15
cells (%)

cells (%)

cells (%)

(I) GFP+ (20%) cells were induced from fibroblasts

10 **** * 10 * * * 10
*
* * by the combination of four factors, Gata4, Mef2c,
5
* 5 * * 5 Mesp1, and Tbx5. Representative data are shown
0 0 0 in each panel. PI, propidium iodine. All data are pre-
Control
9 factors
6 factors
Gata4
Mef2c
Mesp1
Myocd
Nkx2.5
Tbx5
Control

9 factors
Gata4
Isl1
Mef2c
Mesp1
Myocd
Nkx2.5
Smyd1

Tbx5
14 factors

Srf

Control
6 factors
5 factors
Gata4
Mef2c
Mesp1
Myocd
Tbx5

sented as means ± SD. *p < 0.01; **p < 0.05 versus

relevant control. Scale bars represent 100 mm.
9 factors - 1 6 factors - 1 5 factors - 1
See also Figures S1 and S2.
I 10 4 10
4

Control Gata4/Mef2c/Mesp1
10
3
10
3
/Tbx5

10 2
100% 76.5%
10 2
RESULTS
10
1 0% 10
1 20.6%
PI

10
0
10
0
10 0 10 1 10 2 10 3 10 4
αMHC-GFP
with this, the bHLH transcription factor, Neurogenin 3, in combi-
nation with Pdx1 and Mafa, can efficiently reprogram pancreatic
exocrine cells into functional b cells in vivo, although the exocrine
cells were known to have some potential to become islet cells
in vitro and share a common parent cell with islet cells (Baeyens
et al., 2005; Zhou et al., 2008). A combination of three factors,
Ascl1, Brn2, and Myt1l, converts dermal fibroblasts to functional
neurons (Vierbuchen et al., 2010), although the degree of global
reprogramming of the neurons is unknown.
In this study, we examined whether key developmental
cardiac regulators could reprogram cardiac fibroblasts into car-
diomyocytes. We found that out of a total of 14 factors, a specific
combination of three transcription factors, Gata4, Mef2c, and
Tbx5, was sufficient to generate functional beating cardiomyo-
cytes directly from mouse postnatal cardiac or dermal fibro-
blasts and that the induced cardiomyocytes (iCMs) were globally
reprogrammed to adopt a cardiomyocyte-like gene expression
profile.
Screening for Cardiomyocyte-
100 101 10 2 10 3 10 4
Inducing Factors
We developed an assay system in
which the induction of mature cardiomyo-
cytes from fibroblasts could be analyzed quantitatively by
reporter-based fluorescence-activated cell sorting (FACS) (Fig-
ure 1A). To accomplish this, we generated aMHC promoter-
driven EGFP-IRES-puromycin transgenic mice (aMHC-GFP), in
which only mature cardiomyocytes expressed the green fluores-
cent protein (GFP) (Gulick et al., 1991). We confirmed that only
cardiomyocytes, but not other cell types such as cardiac fibro-
blasts, expressed GFP in the transgenic mouse hearts and in
primary cultured neonatal mouse cardiac cells (Figure S1 avail-
able online).
To have enough cardiac fibroblasts for FACS screening, we
obtained GFP cardiac fibroblasts from neonatal aMHC-GFP
hearts by explant culture. Fibroblast-like cells migrated from
the explants after 2 days and were confluent after 1 week. The
migrating cells did not express GFP, but expressed Thy1 and
vimentin, markers of cardiac fibroblasts (Figure 1B and data
not shown) (Hudon-David et al., 2007; Ieda et al., 2009). To avoid
contamination of cardiomyocytes, we filtered the cells by cell

376 Cell 142, 375–386, August 6, 2010 ª2010 Elsevier Inc.

strainers to remove heart tissue fragments and isolated Thy1+/
GFP cells by FACS (Figure 1C). Using FACS, we confirmed that
Thy1+/GFP cells did not express cardiac troponin T (cTnT),
a specific sarcomeric marker of differentiated mature cardio-
myocytes (Figure S1) (Kattman et al., 2006). With these proce-
dures, we had no cardiomyocyte contamination in the fibroblast
culture and could generate greater than twice the number of
cardiac fibroblasts than by conventional fibroblast isolation tech-
niques (Ieda et al., 2009).
To select potential cardiac reprogramming factors, we used
microarray analyses to identify transcription factors and epige-
netic remodeling factors with greater expression in mouse
cardiomyocytes than in cardiac fibroblasts at embryonic day
12.5 (Ieda et al., 2009). Among them, we selected 13 factors
that exhibited severe developmental cardiac defects and
embryonic lethality when mutated (Figure S2). We also included
the cardiovascular mesoderm-specific transcription factor
Mesp1 because of its cardiac transdifferentiation effect in
Xenopus (David et al., 2008). We generated individual retrovi-
ruses to efficiently express each gene in cardiac fibroblasts
(Figure S2).
We transduced Thy1+/GFP neonatal mouse cardiac fibro-
blasts with a mixture of retroviruses expressing all 14 factors
or with DsRed retrovirus (negative control) (Hong et al., 2009).
We did not observe any GFP+ cells in cardiac fibroblasts 1 week
after Ds-Red retrovirus infection or 1 week of culture without any
viral infection. In contrast, transduction of all 14 factors into fibro-
blasts resulted in the generation of a small number of GFP+ cells
(1.7%), indicating the successful activation of the cardiac-
enriched aMHC gene in some cells (Figures 1D and 1E).
To determine which of the 14 factors were critical for activating
cardiac gene expression, we serially removed individual factors
from the pool of 14. Pools lacking five factors (Baf60c, Hand2,
Hopx, Hrt2, and Pitx2c) produced an increased number of
GFP+ cells, suggesting they are dispensable in this setting
(Figures 1D and 1E). Of note, removing Gata4 decreased the
percentage of GFP+ cells to 0.5%, and removing Pitx2c
increased it to 5%. Removal of the five factors listed above
resulted in an increase in the percentage of GFP+ cells to 13%
(Figure 1F). We conducted three further rounds of withdrawing
single factors from nine-, six-, and five-factor pools, removing
those that did not decrease efficiency upon withdrawal, and
found that four factors (Gata4, Mef2c, Mesp1, and Tbx5) were
sufficient for efficient GFP+ cell induction from cardiac fibro-
blasts (Figures 1F–1H). The combination of these four factors
dramatically increased the number of fibroblasts activating the
aMHC-GFP reporter to over 20% (Figure 1I).
Gata4, Mef2c, and Tbx5 Are Sufficient for
Cardiomyocyte Induction
Next, we examined the expression of cTnT by FACS. We found
that 20% of GFP+ cells expressed cTnT at high enough levels
to detect by FACS 1 week after the four-factor transduction.
Again removing individual factors from the four-factor pool in
transduction, we found that Mesp1 was dispensable for cTnT
expression (Figures 2A and 2B). In contrast, we did not observe
cTnT+ or GFP+ cells, when either Mef2c or Tbx5 was removed.
Removal of Gata4 did not significantly affect the number of
GFP+ cells, but cTnT expression was abolished, suggesting
Gata4 was also required. Whereas the combination of two
factors, Mef2c and Tbx5, induced GFP expression but not
cTnT, no combination of two factors or single factor induced
both GFP and cTnT expression in cardiac fibroblasts (Figure 2C).
These data suggested that the combination of three factors,
Gata4, Mef2c, and Tbx5, is sufficient to induce cardiac gene
expression in fibroblasts.
We found that 30% of GFP+ cells expressed cTnT 1 week after
the three-factor transduction. Next, to confirm our screening
results, we transduced cardiac fibroblasts with three factors
(Gata4, Mef2c, and Tbx5, hereafter referred to as GMT) plus
Nkx2-5, a critical factor for cardiogenesis but excluded by our
initial screening. Surprisingly, adding Nkx2-5 to GMT dramati-
cally inhibited the expression of GFP and cTnT in cardiac
fibroblasts. We also transduced cardiac fibroblasts with the
combination of Baf60c, Gata4, and Tbx5, which can transdiffer-
entiate noncardiogenic mesoderm to cardiomyocytes in mouse
embryos (Takeuchi and Bruneau, 2009). We found that this
combination did not efficiently induce cTnT or GFP expression
above that of Tbx5 alone, confirming our screening results
(Figure 2D).
To determine if other cardiac genes were enriched in GFP+
cells, we sorted GFP+ cells and GFP cells 7 days after transduc-
tion with GMT and compared gene expression of cardiomyo-
cyte-specific genes, Myh6 (a-myosin heavy chain), Actc1
(cardiac a-actin), Actn2 (actinin a2), and Nppa (natriuretic
peptide precursor type A) by quantitative RT-PCR (qPCR). We
found that these cardiac genes were upregulated significantly
more in GFP+ than in GFP cells (Figure 2E). Next, we used
immunocytochemistry to determine if cardiac proteins were
expressed in GFP+ cells. Despite the detection of cTnT in only
30% of GFP+ cells, most GFP+ cells induced with the three fac-
tors expressed sarcomeric a-actinin (a-actinin) and had well-
defined sarcomeric structures, similar to neonatal cardiomyo-
cytes (Figure 2F; Figure S1). In addition to a-actinin, some
GFP+ cells also expressed cTnT and ANF (atrial natriuretic
factor), indicating GFP+ cells expressed several cardiomyo-
cyte-specific markers (Figures 2G and 2H). We also confirmed
that neither GFP+ nor GFP cells expressed smooth muscle or
endothelial cell markers (Figure S2), suggesting specificity of
GMT effects.
Induced Cardiomyocytes Originate from Differentiated
Fibroblasts and Are Directly Reprogrammed
We next isolated neonatal cardiac fibroblasts by the conven-
tional fibroblast isolation method in which hearts were digested
with trypsin and plated on plastic dishes (Ieda et al., 2009).
More than 85% of the cells expressed Thy1, and we isolated
Thy1+/GFP cells by FACS to exclude cardiomyocyte contami-
nation (Figure 3A). Fibroblasts transduced with GMT expressed
GFP, cTnT, and actinin after 1 week at the same level as fibro-
blasts isolated from explant cultures (Figures 3B and 3C). Similar
results were obtained on introduction of GMT into adult cardiac
fibroblasts, with full formation of sarcomeric structures (Fig-
ure 3D; Figure S2).
To determine if the induced cardiomyocyte-like cells (iCMs)
were arising from a subpopulation of stem-like cells, we
Cell 142, 375–386, August 6, 2010 ª2010 Elsevier Inc. 377
 A Control 4 factors 4 f - Gata4 4 f - Mef2c 4 f - Mesp1 4 f - Tbx5
Figure 2. Combination of Three Transcrip-
10
4

0% 0%
10
4

0.7% 4.3%
10 4
0.1% 0.5%
10 4
0.2% 0.1%
10
4

1.0% 6.5%
10
4

0.7% 0% tion Factors Induces Cardiac Gene Expres-

10
3
10
3
10
3
10
3
10
3
10 3
sion in Fibroblasts
(A) FACS analyses for a-MHC-GFP and cardiac
cTnT

10 2 10 2 10 2 10 2 10 2 10 2

10
1
10
1
10 1 10
1
10 1 10
1 Troponin T (cTnT) expression. Effects of the
10
0 100% 0% 10
0 70.3% 24.7% 10
0 70.9% 28.3% 10
0 98.6% 1.2 % 10
0 72.7% 19.7% 10
0 98.9% 0.3% removal of individual factors from the pool of four
100 10 1 10 2 10 3 10 4 10 0 101 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 100 101 10 2 103 10 4 100 101 10 2 103 10 4 100 10 1 10 2 10 3 10 4
factors on GFP+ and cTnT+ cell induction.
αMHC-GFP (B) Quantitative data of GFP+ cells and cTnT+ cells
B C
30 (%) 30
(%) in (A) (n = 3).
αMHC-GFP+ cells
25 25 (C) Effect of the transduction of pools of three, two,
* cTnT+ cells
20 20 and one factors on GFP+ and cTnT+ cell induction
15 15 (n = 3).
10 10 (D) FACS analyses for a-MHC-GFP and cTnT
expression. Effects of GMT plus Nkx2.5 and
* ** ** ** ** **
5 5
0
* ** ** 0 Baf60c/Gata4/Tbx5 transduction are shown.
Control

4 factors

Gata4

Mef2c

Mesp1

Tbx5

3 factors
Control

Gata4

Mef2c

Gata4

Mef2c
Tbx5

Tbx5
(E) The mRNA expression in GFP+ and GFP cells
7 days after GMT transduction was determined by
4 factors - 1 factor 3 factors - 1 1 factor qPCR (n = 3).
(F) Immunofluorescent staining for GFP, a-actinin,
D 3 factors 3 f + Nkx2.5 Baf60c/Gata4/Tbx5 F αMHC-GFP α-actinin Merged
10
4
10
4
10
4
and DAPI. The combination of the three factors,
0.8% 4.0% 0.3% 0.9% 0.3% 0.5%
10
3
10
3
10 3 GMT, induced abundant GFP, and a-actinin
expression in cardiac fibroblasts 2 weeks after
cTnT

10 2 10 2 10 2

10
1
10
1
10 1
transduction. High-magnification views in insets
0 82.2% 13.0% 0 96.4% 2.4% 0 96.5% 2.7% control show sarcomeric organization. See also Figures
10 10 10
100 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4
S1 and S2.
αMHC-GFP (G) Induced cardiomyocytes expressed cTnT by
immunocytochemistry with clear sarcomeric orga-
E
Relative mRNA expression

250 * (αMHC-GFP+ vs. αMHC-GFP- cells) nization 4 weeks after transduction. Insets are
200 Myh6 3 factors high-magnification views.
Actc1 (H) Induced cardiomyocytes expressed ANF at
150
* Actn2 perinuclear sites 2 weeks after transduction.
100 Nppa All data are presented as means ± SD. *p < 0.01
50
* * versus relevant control. Scale bars represent
0 3 factors 100 mm.
See also Figures S1 and S2.
αMHC-GFP cTnT Merged αMHC-GFP ANF Merged
G H

(Figures 3E–3G). Like cardiac fibroblasts,

tail-tip fibroblast-derived GFP+ cells ex-
pressed a-actinin and had well-defined
sarcomeric structures (Figure 3H; Fig-
ure S3), suggesting noncardiac fibro-
blasts can also be reprogrammed into
cardiomyocytes by GMT induction.
analyzed c-kit expression (Beltrami et al., 2003) in the Thy1+/ These results excluded the possibility that the iCMs arose from
GFP cells. Most c-kit+ cells coexpressed Thy1, whereas 15% contamination of cardiomyocytes or cardiac progenitors before
of Thy1+ cells expressed c-kit, which is consistent with a previous cardiac induction in the fibroblast population.
report of cardiac explant-derived cells (Davis et al., 2009). We We also determined whether the reprogramming of fibroblasts
isolated GFP /Thy1+/c-kit+ cells and GFP /Thy1+/c-kit cells to differentiated cardiomyocytes was a direct event or if the
by FACS and transduced each population of cells with GMT. fibroblasts first passed through a cardiac progenitor cell fate
We found 2–3-fold more cardiomyocyte induction in GFP / before further differentiation. To distinguish between these two
Thy1+/c-kit cells than in GFP /Thy1+/c-kit+ cells (Figure S3). possibilities, we used mice expressing Isl1–yellow fluorescent
These results suggest that most of the iCMs originated from protein (YFP) obtained by crossing Isl1-Cre mice and R26R-
a c-kit-negative population. EYFP mice (Srinivas et al., 2001) (Figure S3). Isl1 is an early
We then sought to more definitively exclude the possibility of cardiac progenitor marker that is transiently expressed before
rare cardiac progenitors giving rise to iCMs. We tested the cardiac differentiation. If iCMs generated from fibroblasts
potential of mouse tail-tip dermal fibroblasts to generate iCMs. passed through a cardiac progenitor state, they and their
We found that sorted Thy1+/GFP tail-tip dermal fibroblasts descendants would permanently express YFP (Laugwitz et al.,
transduced with GMT expressed GFP at the same level as 2005). We isolated Isl1-YFP /Thy1+cells from Isl1-YFP heart
GMT-transduced cardiac fibroblasts, although the percentage explants by FACS and transduced the cells with GMT. The
of cTnT+ cells was less than cardiac fibroblast-derived iCMs resulting cTnT+ cells did not express YFP in significant numbers,

378 Cell 142, 375–386, August 6, 2010 ª2010 Elsevier Inc.

A Control αMHC-GFP CF B Control Gata4/Mef2c/Tbx5 Figure 3. Induced Cardiomyocytes Origi-
10 4 10 4
10 5 0% 0% 10 5 86.6% 0% 0% 0% 1.9% 6.5% nate from Differentiated Fibroblasts and
10
3
10
3 Are Directly Reprogrammed
10 4 10 4
(A) Cardiac fibroblasts (CF) isolated by the conven-
10 2 10 2
10
3
10 3 tional isolation method. Most cells were positive
for Thy1, and Thy-1+/GFP cells were sorted by

cTnT
Thy1

1
2 2 10 1 10
10 10
0 0
FACS for transduction.
100% 0% 13.2% 0.1% 0 100% 0% 0
73.3% 18.2%
2 3 4 5 2 3 4 5
10
100 10 1 10 2 10 3 10 4
10
10 0 10 1 10 2 10 3 10 4
(B) FACS analyses for aMHC-GFP and cTnT
0 10 10 10 10 0 10 10 10 10
expression in cardiac fibroblasts isolated in (A)
αMHC-GFP αMHC-GFP
1 week after transduction by GMT.
C D Adult αMHC-GFP Explant (C) Immunofluorescent staining for GFP, a-actinin,
αMHC-GFP CF-derived iCMs Gata4/Mef2c/Tbx5
10 4

3.5%
and DAPI in the GMT induced cardiomyocytes
5.2%
10
3
originated from (A).
(D) Cardiac fibroblasts isolated from adult aMHC-
10 2
cTnT GFP hearts were transduced with three factors.
10 1
See also Figure S2.
αMHC-GFP α-Actinin Merged (E) Thy-1+/GFP tail-tip dermal fibroblasts (TTFs)
0
73.9% 17.4%
10
100 10 1 10 2 10 3 10 4
were sorted by FACS for transduction.
αMHC-GFP (F) FACS analyses for GFP and cTnT expres-
E F G sion in TTFs isolated in (E) 1 week after GMT
αMHC-GFP TTF Gata4/Mef2c/Tbx5
transduction.
88.4% 0%
10 4 30 (%) αMHC-GFP+ cells
10 5 1.5% 2.5%
25 cTnT+ cells (G) Quantitative data of GFP+ cells and cTnT+ cells
10 3
10
4
20 indicated in (F) (n = 3 in each group).
10 3
10 2 15 (H) Immunofluorescent staining for GFP, a-actinin,
cTnT
Thy1

10 and DAPI in TTF-derived iCMs. See also Figure S3.

2 10 1
10
0
5 * (I) Isl1-YFP /Thy1+cells were sorted from Isl1-Cre/
11.6% 0% 74.5% 21.5%
2 3 4 5
10
0
0 1 2 3 4
0 Rosa-YFP heart explants and transduced with
0 10 10 10 10 10 10 10 10 10 CF TTF
GMT. See also Figure S3.
αMHC-GFP αMHC-GFP One week
(J) The vast majority of cTnT+ cells induced from
H I Isl1-YFP /Thy1+cells was negative for YFP.
αMHC-GFP TTF-derived iCMs Control Isl1-YFP Explant
10 5 0% 0% 35.5% 27.5%
(K) Mesp1-YFP /Thy1+cells were sorted from
10 5
Mesp1-Cre/Rosa-YFP TTFs and transduced with
4 4
10 10
GMT. See also Figure S3.
10
3
10
3
(L) All cTnT+ cells induced from Mesp1-YFP /
Thy1

Thy1+cells were negative for YFP.

10 2
10 2

αMHC-GFP α-Actinin Merged 0 0 All data are presented as means ± SD. *p < 0.01
100% 0% 19.4% 17.5%
0 10
2
10
3
10
4
10
5 0 102 10 3 10 4 10 5
versus relevant control. Scale bars represent
Isl1-YFP 100 mm. See also Figure S3 for analyses of c-kit+
cells.
J Gata4/Mef2c/Tbx5 K Mesp1-YFP TTF L 10 4
Gata4/Mef2c/Tbx5
10 4
0.4% 87.7% 0.2% 5.7% 0% See also Figures S2 and S3.
8.6% 10 5

3
10 3 10
4
10

10 2 10 2
10 3
cTnT

Thy1

cTnT

10 1 2 10 1
10

89.7% 1.3%
0
11.9% 0.2% 94.3% 0%
3L). The resulting cTnT+ cells did not
0 0
10 10
100 10 1 10 2 10 3 10 4 0 102 10 3 10 4 10 5 100 101 10 2 10 3 10 4 express YFP, suggesting that the iCMs
Isl1-YFP Mesp1-YFP Mesp1-YFP were not converted into the cardiac
mesoderm cell state for reprogramming,
but rather they were directly reprog-
suggesting that the iCMs were not first reprogrammed into Isl1+ rammed into differentiated cardiomyocytes by the three factors
cardiac progenitor cells (Figures 3I and 3J). Moreover, these (Figure 3L).
results provided supportive evidence that the iCMs did not orig-
inate from a rare population of cardiac progenitor cells that might Induced Cardiomyocytes Resemble Postnatal
exist in neonatal hearts. Cardiomyocytes in Global Gene Expression
Whereas Isl1 marks most early cardiac progenitors, a subpop- We next analyzed the time course of cardiomyocyte induction
ulation of cardiac progenitors remains Isl1 negative. Mesp1 is from cardiac fibroblasts. GFP+ cells were detected 3 days after
the earliest pan-cardiovascular progenitor cell marker that is induction and gradually increased in number up to 20% at day
transiently expressed in nascent mesoderm before further 10 and were still present after 4 weeks (Figure 4A). GFP+ cells
cardiovascular differentiation (Figure S3) (Saga et al., 1999). were less proliferative than GFP cells and, over time, decreased
We therefore generated Mesp1-YFP mice by crossing Mesp1- in percentage relative to the total number of cells. Importantly, the
Cre and R26R-EYFP mice to determine if iCMs were reprog- percentage of cTnT+ cells among the a-MHC-GFP+ iCMs and the
rammed into early cardiac mesoderm before further differentia- intensity of cTnT expression increased significantly over time
tion. We isolated Mesp1-YFP /Thy1+ tail-tip dermal fibroblasts (Figures 4B and 4C). We sorted GFP+ cells at 1, 2, and 4 weeks
by FACS and transduced the cells with GMT (Figures 3K and after transduction with GMT and compared cardiac gene

Cell 142, 375–386, August 6, 2010 ª2010 Elsevier Inc. 379

A B Percent of cTnT+ cells in αMHC-GFP+ iCMs Figure 4. Gene Expression of Induced Car-
diomyocytes Is Globally Reprogrammed
Percent (%) of total cells

4
Control 4
1 week 4
2 weeks 4
4 weeks
(A) The percent of aMHC-GFP+ cells after GMT
10 10 10 10
30 αMHC-GFP+ cells
25
* 10
3
100% 10
3
59.2% 10
3
55.9% 10
3
51% transduction (n = 3). The number of GFP+ cells
20
* * 10 2 10 2 10 2 10 2
was counted by FACS at each time point and
15
** divided by the number of plated cells.
10 10
1 0% 10
1 31.8% 10 1
35.5% 10
1 45.1%
5 (B) FACS analyses of percent of cells with cTnT
(vs. 3d)
0 10
0

10 0 10 1 10 2 10 3
10
10 4
0

100 101 10 2 10 3
10
10 4
0

10 0 101 10 2 10 3
10
10 4
0

10 0 10 1 10 2 103 10 4
expression among aMHC-GFP+ iCMs. Note that
0 1 3 7 10 14 28
Time (days)
cTnT
cTnT + cell number and cTnT intensity were both
increased over time (n = 4).
C Relative
D Relative mRNA expression (C) Quantitative data of cTnT intensity in (B) (n = 4).
2000
CF
cTnT intensity Actc1 Myh6 Ryr2 (D) Actc1, Myh6, Ryr2, Gja1, and Col1a2 mRNA
1600
* 1.0 1.0 1.0 iCMs (1W)
0.8 0.8 0.8 expression in cardiac fibroblasts (CF), induced
iCMs (2W)
1200 * cardiomyocytes (iCMs) (1 week (W), 2 W, 4 W after
0.6 0.6 0.6 iCMs (4W)
800 * * * transduction), and neonatal cardiomyocytes (CM),
0.4 0.4 0.4 CM
400 0.2 ** 0.2 * 0.2 determined by qPCR (n = 3).
*
(vs. 1W) ND ND ND (E) Heatmap image of microarray data illustrating
0 0 0 0
0 1 2 4 differentially expressed genes among CF, a-
Time (weeks) 1.0 Gja1 16 Col1a2
(vs. 1W iCMs) MHC-GFP , iCMs (FACS sorted 2 and 4 weeks
0.8 12 after transduction), and CM (n = 3 in each group).
0.6
* 8 The scale extends from 0.25- to 4-fold over
0.4
4
mean ( 2 to +2 in log2 scale). Red indicates
0.2
increased expression, whereas green indicates
0 0 *
decreased expression. Group 1 includes the
E genes upregulated only in CM, and group 2
includes the genes upregulated in CM and 4W-
iCMs compared to CF. Lists of genes are shown
CF 2W GFP- 4W GFP- 2W iCMs 4W iCMs CM
in Table S1 and Table S2. All data are presented
as means ± SD. *p < 0.01; **p < 0.05 versus rele-
vant control. See also Figure S4 for endogenous
and exogenous expression of reprogramming
factors and Table S1 and Table S2 for differentially
expressed genes.
See also Tables S1 and S2 and Figure S4.

1
We next compared the progressive
global gene expression pattern of iCMs,
neonatal cardiomyocytes, and cardiac
2
fibroblasts by mRNA microarray anal-
yses. We sorted GFP+ cells and GFP
FDR-adj p<0.0001 in at least one comparison cells 2 and 4 weeks after GMT transduc-
tion. The iCMs at both stages were similar
to neonatal cardiomyocytes, but were
expression with cardiac fibroblasts and neonatal cardiomyo- distinct from GFP cells and cardiac fibroblasts in global gene
cytes. The cardiomyocyte-specific genes, Actc1, Myh6, Ryr2 expression pattern (Figure 4E). We found that functionally impor-
(ryanodine receptor 2), and Gja1 (connexin43), were significantly tant cardiac genes were upregulated significantly more in 4 week
upregulated in a time-dependent manner in GFP+ cells, but iCMs than in 2 week iCMs, including Pln (phospholamban),
were not detected in cardiac fibroblasts by qPCR (Figure 4D). Slc8a1 (sodium/calcium exchanger), Myh6, Sema3a (sema-
Col1a2 (collagen 1a2), a marker of fibroblasts, was dramatically phorin 3a), Id2 (inhibitor of DNA binding 2), and Myl2 (myosin, light
downregulated in GFP+ cells from 7-day culture to the same level polypeptide 2, regulatory, cardiac, slow, also known as MLC2v)
as in cardiomyocytes. These data indicated that the three factors (Table S1). Conversely, some genes were downregulated more
induced direct conversion of cardiac fibroblasts to cardiomyo- in 4 week iCMs than in 2 week iCMs (Table S1). The array analyses
cytes rapidly and efficiently, but full maturation was a slow also identified genes that were upregulated more in neonatal car-
process that occurred over several weeks. Total gene expression diomyocytes than in 4 week iCMs or cardiac fibroblasts (group 1
of the three reprogramming factors was upregulated 6- to 8-fold in in Figure 4E), including Bmp10 (bone morphogenetic protein 10),
iCMs over neonatal cardiomyocytes. However, only endogenous Erbb4 (v-erb-a erythroblastic leukemia viral oncogene homolog
expression of Gata4 was upregulated in iCMs to the same level as 4), Irx4 (Iroquois related homeobox 4), and Atp1a2 (ATPase,
in neonatal cardiomyocytes, whereas endogenous Mef2c and Na+/K+ transporting, a 2 polypeptide) (Table S2). We also identi-
Tbx5 expression was lower in iCMs than in cardiomyocytes, fied genes that were expressed to a greater extent in both cardi-
potentially reflecting negative autoregulatory loops (Figure S4). omyocytes and 4 week iCMs than in fibroblasts (group 2 in

380 Cell 142, 375–386, August 6, 2010 ª2010 Elsevier Inc.

A B Figure 5. Fibroblasts Are Stably Reprog-
CF iCMs (CF) TTF iCMs (TTF) Cardiac cells
Nppa Myh6 rammed into iCMs by Gata4, Mef2c, and
H3K27me3 H3K4me3
20 5 Tbx5
4
* ** (A) The promoters of Actn2, Ryr2, and Tnnt2 were
15
Actn2

3 CF analyzed by ChIP for trimethylation status of

10
2 histone H3 of lysine 27 or 4 in cardiac fibroblasts
5 * 1
** (CF), CF-derived iCMs, tail-tip fibroblasts (TTF),
Fold enrichment/IgG

0 0
20 5 αMHC- TTF-derived iCMs, and neonatal cardiac cells.
GFP- Data were quantified by qPCR.
15 4
(B) The promoters of Nppa and Myh6 were
Ryr2

3
10 ** analyzed with bisulfite genomic sequencing for
2
5
** * 1
iCMs
DNA methylation status in CF, a-MHC-GFP cells,
0 0 a-MHC-GFP+ iCMs (FACS sorted 4 weeks after
15 5 transduction), and neonatal CM. Open circles indi-
4 cate unmethylated CpG dinucleotides; closed
10
Tnnt2

3 circles indicate methylated CpGs.

2 CM
5 * 1
* * (C) Schematic representation of the strategy to
0
* 0
test expression kinetics of the doxycycline (Dox)-
inducible lentiviral system.
C WT TTF
+Dox -Dox (D) Wild-type TTFs were infected with pLVX-tetO-
1 day 6 days GFP and pLVX-rtTA and imaged before (off Dox),
1 day after Dox addition, and at time points after
infection
Dox withdrawal (–Dox). All images were taken
5´-LTR tetO GFP 3´-LTR
using constant exposure times and identical
5´-LTR CMV rtTA 3´-LTR
camera settings.
(E) Schematic representation of the strategy to
D Off Dox 1d, +Dox 1d, -Dox 3d, -Dox 6d, -Dox determine temporal requirement of Gata4/Mef2c/
Tbx5 in reprogramming. Thy1+/GFP TTF were
GFP

infected with the pLVX-tetO-GMT and pLVX-rtTA

lentiviruses, and Dox was added for 2 weeks and
thereafter withdrawn for 1 week.
(F) Immunofluorescent staining for GFP, a-actinin,
E Off On and DAPI in iCMs 2 weeks after lentiviral infection
αMHC promoter GFP αMHC promoter GFP and Dox induction.
αMHC-GFP TTF +Dox iCMs -Dox iCMs (G) Immunofluorescent staining for GFP, a-actinin,
2 weeks 1 week and DAPI 1 week after Dox withdrawal. iCMs
infection maintained a-MHC GFP expression and had
5´-LTR tetO Gata4/Mef2c/Tbx5 3´-LTR
a-actinin positive sarcomeric structures. High-
5´-LTR CMV rtTA 3´-LTR
magnification views in insets show sarcomeric
organization. Representative data are shown in
F +Dox G +Dox -Dox each panel. All data are presented as means ±
SD. *p < 0.01; **p < 0.05 versus relevant control.
Scale bars represent 100 mm.

αMHC-GFP α-actinin Merged αMHC-GFP α-actinin Merged

Figure 4E), including Actc1, Myl7 (myosin, light polypeptide 7, iCMs, and neonatal cardiac cells by chromatin immunoprecipita-
regulatory, also known as MLC2a), Tnnt2 (troponin T2, cardiac), tion, followed by qPCR (Figure 5A). After reprogramming,
Tbx3 (T-box 3), and Srf (serum response factor) (Table S2). H3K27me3 was significantly depleted at the promoters of all
Thus, iCMs were similar, but not identical, to neonatal cardiomyo- the genes analyzed in iCMs, reaching levels comparable to those
cytes, and the reprogramming event was broadly reflected in in cardiac cells, whereas H3K4me3 increased on the promoter
global gene expression changes. regions of Actn2 and Tnnt2 in iCMs, as compared with cardiac
fibroblasts. Ryr2 had similar levels of H3K4me3 in iCMs as in
Fibroblasts Are Epigenetically Reprogrammed fibroblasts, suggesting that its activation reflects the resolution
to a Cardiomyocyte-like State by Gata4/Mef2c/Tbx5 of a ‘‘bivalent’’ chromatin mark (Bernstein et al., 2006). These
To determine if iCMs have gained a cardiomyocyte-like chro- results suggested that cardiac fibroblast-derived iCMs gained
matin state, we analyzed the enrichment of histone modifications a chromatin status similar to cardiomyocytes at least in some
in the promoter regions of the cardiac-specific genes Actn2, cardiac specific genes. Intriguingly, H3K27me3 levels were
Ryr2I, and Tnnt2. We analyzed the enrichment of trimethylated higher in tail-tip fibroblasts than cardiac fibroblasts on all three
histone H3 of lysine 27 (H3K27me3) and lysine 4 (H3K4me3), genes analyzed and, despite a significant reduction upon
which mark transcriptionally inactive or active chromatin, reprogramming to iCMs, remained somewhat higher than in
respectively (Li et al., 2007), in cardiac fibroblasts, 4 week cardiac cells and cardiac fibroblast-derived iCMs.

Cell 142, 375–386, August 6, 2010 ª2010 Elsevier Inc. 381

 The DNA methylation status of specific loci also reflects the
stability of the reprogramming event and we therefore investi-
gated such changes during reprogramming from cardiac fibro-
blasts to iCMs. We performed bisulfite genomic sequencing in
the promoter regions of Nppa and Myh6 in cardiac fibroblasts,
4 week GFP cells, iCMs, and neonatal cardiomyocytes. Both
promoter regions were hypermethylated in cardiac fibroblasts
and GFP cells, as expected from the cardiomyocyte-specific
expression of these genes, but were comparatively demethy-
lated in iCMs, similar to neonatal cardiomyocytes (Figure 5B).
These results indicated that reprogramming by Gata4, Mef2c,
and Tbx5 induced epigenetic resetting of the fibroblast genome
to a cardiomyocyte-like state.
To further assess the stability of the reprogramming event, we
generated a doxycycline-inducible lentiviral system in which
transgene expression of the reprogramming factors was con-
trolled by doxycycline administration. We first transduced wild-
type tail-tip fibroblasts with a mixture of lentiviruses containing
pLVX-tetO-GFP and pLVX-rtTA to determine the expression
kinetics of this system (Figure 5C). We confirmed that the
majority of fibroblasts infected with both viruses expressed
GFP within 1 day after doxycycline induction, and the GFP
expression was instantly diminished by withdrawal of doxycy-
cline and disappeared within 6 days (Figure 5D). Thy1+/GFP
tail-tip fibroblasts were harvested from aMHC-GFP neonatal
mice, transduced with a pool of lentiviruses containing inducible
Gata4, Mef2c, and Tbx5, along with pLVX-rtTA, and subse-
quently treated with doxycycline (Figure 5E). We found that
aMHC-GFP expression was induced from tail-tip fibroblasts
after doxycycline administration and that the iCMs had well-
defined sarcomeric structures marked with an anti-a-actinin
antibody after 2 weeks of culture (Figure 5F). Doxycycline was
withdrawn after 2 weeks of culture, and cells were subsequently
cultured without doxycycline for 1 week to fully remove exoge-
nous expression of the reprogramming factors (Figure 5E). The
iCMs maintained aMHC-GFP expression and had sarcomeric
structures after doxycycline withdrawal, suggesting that the
fibroblasts were stably reprogrammed into iCMs after 2 weeks
exposure to Gata4, Mef2c, and Tbx5 (Figure 5G).
Induced Cardiomyocytes Exhibit
Spontaneous Contraction
To determine if iCMs possessed the functional properties char-
acteristic of cardiomyocytes, we analyzed intracellular Ca2+
flux in iCMs after 2–4 weeks of culture. Around 30% of cardiac
fibroblast-derived iCMs showed spontaneous Ca2+ oscillations
and their frequency was variable, resembling what was observed
in neonatal cardiomyocytes (Figures 6A, 6B, and 6D; Movie S1).
We observed that tail-tip dermal fibroblast-derived iCMs also
exhibited spontaneous Ca2+ oscillations, but the oscillation
frequency was lower than that of cardiomyocytes and cardiac
fibroblast-derived iCMs (Figures 6C and 6E; Movie S2).
In addition to the characteristic Ca2+ flux, cardiac fibroblast-
derived iCMs showed spontaneous contractile activity after
4–5 weeks in culture (Movies S3 and S4; Figure S5). Single-cell
extracellular recording of electrical activity in beating cells
revealed tracings similar to the potential observed in neonatal
cardiomyocytes (Figure 6F). Intracellular electrical recording of
382 Cell 142, 375–386, August 6, 2010 ª2010 Elsevier Inc.
iCMs displayed action potentials that resembled those of adult
mouse ventricular cardiomyocytes (Figure 6G). Thus, the reprog-
ramming of fibroblasts to iCMs was associated with global
changes in gene expression, epigenetic reprogramming, and
the functional properties characteristic of cardiomyocytes.
Transplanted Cardiac Fibroblasts Transduced
with Gata4/Mef2c/Tbx5 Reprogram In Vivo
To investigate whether GMT-transduced cardiac fibroblasts can
be reprogrammed to express cardiomyocyte-specific genes in
their native environment in vivo, we harvested GFP /Thy1+
cardiac fibroblasts 1 day after viral transduction and injected
them into immunosuppressed NOD-SCID mouse hearts. GMT-
infected cells did not express GFP at the time of transplantation
(Figure 4A). Cardiac fibroblasts were infected with either the
mixture of GMT and DsRed retroviruses or DsRed retrovirus
(negative control) to be readily identified by fluorescence.
Cardiac fibroblasts infected with DsRed did not express a-acti-
nin or GFP, confirming cardiomyocyte conversion did not
happen in the negative control (Figures 7A and 7B). Despite
being injected into the heart only 1 day after viral infection,
a subset of cardiac fibroblasts transduced with GMT and DsRed
expressed GFP in the mouse heart within 2 weeks (Figure 7B).
Importantly, the GFP+ cells expressed a-actinin and had sarco-
meric structures (Figure 7C). These results suggested that
cardiac fibroblasts transduced with Gata4, Mef2c, and Tbx5
can reprogram to cardiomyocytes within 2 weeks upon trans-
plantation in vivo.
DISCUSSION
Here we demonstrated that the combination of three transcrip-
tion factors, Gata4, Mef2c, and Tbx5, can rapidly and efficiently
induce cardiomyocyte-like cells from postnatal cardiac and
dermal fibroblasts. iCMs were similar to neonatal cardiomyo-
cytes in global gene expression profile, electrophysiologically,
and could contract spontaneously, demonstrating that func-
tional cardiomyocytes can be generated from differentiated
somatic cells by defined factors. Although much refinement
and characterization of the reprogramming process will be
necessary, the findings reported here raise the possibility of
reprogramming the vast pool of endogenous fibroblasts that
normally exists in the heart into functional cardiomyocytes for
regenerative purposes.
The three reprogramming factors, Gata4, Mef2c, and Tbx5,
are core transcription factors during early heart development
(Olson, 2006; Srivastava, 2006; Zhao et al., 2008). They interact
with one another, coactivate cardiac gene expression (e.g.,
Nppa, Gja5 [Cx40], and Myh6), and promote cardiomyocyte
differentiation (Bruneau et al., 2001; Garg et al., 2003; Ghosh
et al., 2009; Lin et al., 1997). Gata4 is considered a ‘‘pioneer’’
factor and might open chromatin structure in cardiac loci (Cirillo
et al., 2002), thus allowing binding of Mef2c and Tbx5 to their
specific target sites and leading to full activation of the cardiac
program. Although the reprogramming event appears stable at
the epigenetic level, as marked by histone methylation and
DNA methylation, the global gene expression of iCMs is similar
but not identical to neonatal cardiomyocytes. Whether they are
A C Rhod-3 intensity Figure 6. Induced Cardiomyocytes Exhibit
Rhod-3 intensity (Cardiac fibroblast-derived iCMs) (Tail tip fibroblast-derived iCMs) Spontaneous Ca2+ Flux, Electrical Activity,
and Beating
(A and B) Cardiac fibroblast (CF)-derived iCMs
showed spontaneous Ca2+ oscillation with varying
1s 1s 1s
frequency (A), similar to neonatal cardiomyocytes
(B). Rhod-3 intensity traces are shown.
B Rhod-3 intensity (Neonatal cardiomyocytes) (C) Tail-tip dermal fibroblast (TTF)-derived iCMs
showed spontaneous Ca2+ oscillation with lower
frequency. The Rhod-3 intensity trace is shown.
(D) Spontaneous Ca2+ waves observed in CF-
1s 1s derived a-MHC-GFP+ iCMs (white dots) or
neonatal cardiomyocytes (arrows) with Rhod-3 at
Ca2+ max and min is shown. Fluorescent images
D Cardiac fibroblast-derived iCMs Neonatal cardiomyocytes correspond to the Movie S1.
(E) Spontaneous Ca2+ oscillation observed in the
TTF-derived a-MHC-GFP+ iCMs with Rhod-3 at
Ca2+ max and min is shown. Fluorescent images
correspond to the Movie S2.
αMHC-GFP Ca2+ max Ca2+ min Ca2+ max Ca2+ min (F) Spontaneously contracting iCMs had electrical
activity measured by single cell extracellular elec-
trodes. Neonatal cardiomyocytes showed similar
E Tail tip fibroblast-derived iCMs
electrical activity.
(G) Intracellular electrical recording of CF-derived
iCMs cultured for 10 weeks displayed action
potentials that resembled those of adult mouse
ventricular cardiomyocytes. Representative data
αMHC-GFP Ca2+ max Ca2+ min are shown in each panel (n = 10 in A–F, n = 4 in
G). See also Figure S5 and Movies S1, S2, S3
F Cardiac fibroblast-derived iCMs G and S4.
Cardiac fibroblast-derived iCMs See also Movies S1, S2, S3, and S4 and Figure S5.
extracellular electrical recording
0 mV
2 mV

20 mV

1s
50 ms
ming factors. Remarkably, reprogram-
Neonatal cardiomyocytes ming of cardiac fibroblasts to myocytes
Adult mouse ventricular cardiomyocytes
extracellular electrical recording occurred in a relatively short period,
with the first GFP+ cells appearing at
day 3, in contrast to iPSC reprogram-
2 mV

0 mV
ming, which typically takes 10–20 days
1s
and occurs with much lower efficiency
20 mV

(<0.1%) (Takahashi and Yamanaka,

50 ms 2006). Despite the early initiation of
reprogramming, the process appears to
continue for several weeks, with progres-
more similar to adult ventricular cardiomyocytes or other sive changes in gene expression, contractile ability, and electro-
subpopulations remains to be determined. Additional epigenetic physiologic maturation.
regulators, microRNAs, or signaling proteins may be leveraged Although many questions remain regarding the mechanisms
to increase the efficiency and robustness of the reprogramming of reprogramming, we were able to genetically test the ‘‘route’’
event. Furthermore, other combinations of factors likely also of cell fate alteration. Our findings suggest that cardiomyocytes
induce cardiac reprogramming, much like the experience in the were directly induced from cardiac fibroblasts without reverting
iPSC field. to a cardiac progenitor cell state, which may explain the rapid
Several lines of evidence suggest that the iCMs we describe early reprogramming process. This conclusion was supported
here originated from differentiated fibroblasts. We found that by the absence of Isl1-Cre-YFP or Mesp1-Cre-YFP activation
any potential rare cardiac ‘‘progenitor-like’’ cells, marked by during the process of reprogramming, which would have marked
c-kit or Isl1, were dispensable for cardiomyocyte induction any cells that transiently expressed Isl1 or Mesp1 (Laugwitz
(Beltrami et al., 2003). Furthermore, the high efficiency of et al., 2005; Saga et al., 1999).
cardiac induction (up to 20%) does not favor the interpretation The ability to reprogram endogenous cardiac fibroblasts into
that rare stem or progenitor cells were the origin of induced cardiomyocytes has many therapeutic implications. First, the
cardiomyocytes. Most importantly, the ability to reprogram avoidance of reprogramming to pluripotent cells before cardiac
dermal fibroblasts into iCMs supports the conclusion that differentiation would greatly lower the risk of tumor formation
cardiac progenitors are not the target cells for the reprogram- in the setting of future cell-based therapies. Second, large

Cell 142, 375–386, August 6, 2010 ª2010 Elsevier Inc. 383

A DsRed-cell injected DsRed-cell injected DsRed-cell injected
DsRed α-actinin Merged
B
DsRed-cell injected DsRed-cell injected DsRed-cell injected
DsRed αMHC-GFP Merged
GMT/DsRed-cell injected GMT/DsRed-cell injected GMT/DsRed-cell injected
DsRed αMHC-GFP Merged
C GMT-cell injected GMT-cell injected GMT-cell injected
α-actinin αMHC-GFP Merged
amounts of an individual’s own fibroblasts can be grown from
a cardiac biopsy or skin biopsy in vitro for transduction with
the defined factors, followed by delivery of cells to damaged
hearts. Third, and most promising, is the potential to introduce
the defined factors, or factors that mimic their effects, directly
into the heart to reprogram the endogenous fibroblast popula-
tion, which represents more than 50% of the cells, into new car-
diomyocytes that can contribute to the overall contractility of the
heart. Our observation that injection of fibroblasts into the heart
only 1 day after induction of Gata4/Mef2c/Tbx5 resulted in
reprogramming of the transplanted cells suggests that this
may be possible. Future studies in human cells and advances
in safe delivery of defined factors will be necessary to advance
this technology for potential regenerative therapies.
EXPERIMENTAL PROCEDURES
Generation of aMHC-GFP, Isl1-YFP, and Mesp1-YFP Mice
To generate aMHC-GFP mice, EGFP-IRES-Puromycin cDNA was subcloned
into the expression vector containing a-myosin heavy chain promoter (Gulick
et al., 1991). Pronuclear microinjection and other procedures were performed
according to the standard protocols (Ieda et al., 2007). PCR primers are listed
in the Extended Experimental Procedures. Isl1-YFP mice were obtained by
crossing Isl1-Cre mice and R26R-EYFP mice (Srinivas et al., 2001). Mesp1-
YFP mice were obtained by crossing Mesp1-Cre mice and R26R-EYFP mice
(Saga et al., 1999).
384 Cell 142, 375–386, August 6, 2010 ª2010 Elsevier Inc.
Figure 7. Transplanted Cardiac Fibroblasts Trans-
duced with Gata4/Mef2c/Tbx5 Can Be Reprog-
rammed to Cardiomyocytes In Vivo
(A) DsRed infected cardiac fibroblasts (DsRed-cell) were
transplanted into NOD-SCID mouse hearts 1 day after
infection and cardiac sections were analyzed by immuno-
cytochemistry after 2 weeks. Transplanted fibroblasts
marked with DsRed did not express a-actinin (green).
(B) Cardiac fibroblasts infected with DsRed or Gata4/
Mef2c/Tbx5 with DsRed (GMT/DsRed-cell) were trans-
planted into NOD-SCID mouse hearts 1 day after infection
and visualized by histologic section. Note that a subset of
GMT/DsRed cells expressed a-MHC-GFP. Data were
analyzed 2 weeks after transplantation.
(C) Gata4/Mef2c/Tbx5-transduced cardiac fibroblasts
(GMT-cell) were transplanted into mouse hearts and histo-
logic sections analyzed. A subset of induced GFP+ cells
expressed a-actinin (red) and had sarcomeric structures.
Insets are high-magnification views of cells indicated by
arrows. Data were analyzed 2 weeks after transplantation.
Representative data are shown in each panel (n = 4 in each
group). Scale bars represent 100 mm. Note that GMT/
DsRed or GMT-infected cells did not express GFP at the
time of transplantation (Figure 4A).
Cell Culture
For explant culture, isolated neonatal or adult mouse
hearts were minced into small pieces less than 1 mm3 in
size. The explants were plated on gelatin-coated dishes
and cultured for 7 days in explant medium (IMDM/20%
FBS) (Andersen et al., 2009). Migrated cells were har-
vested and filtered with 40 mm cell strainers (BD) to avoid
contamination of heart tissue fragments. aMHC-GFP /
Thy1+, Isl1-YFP /Thy1+, aMHC-GFP /Thy1+/c-kit , or
aMHC-GFP /Thy1+/c-kit+ live cells (as defined by the
lack of propidium iodine staining) were isolated using FACS Aria 2 (BD Biosci-
ences). For conventional isolation of neonatal cardiac fibroblasts, hearts were
digested with 0.1% trypsin and plated on plastic dishes (Ieda et al., 2009).
For isolation of tail-tip fibroblasts, tails were digested with 0.1% trypsin and
plated on plastic dishes. Attached fibroblasts were cultured for 7 days and
aMHC-GFP /Thy1+ or Mesp1-YFP /Thy1+ cells were sorted and cultured in
DMEM/M199 medium containing 10% FBS at a density of 104/cm2. Cells
were transduced by retroviruses or lentiviruses after 24 hr.
Isolation of Cardiomyocytes
To isolate cardiomyocytes, neonatal aMHC-GFP+ ventricles were cut into
small pieces and digested with collagenase type II solution (Ieda et al.,
2009). A single-cell suspension was obtained by gentle triturating and passing
through a 40 mm cell strainer. aMHC-GFP+ live cells were isolated by FACS
Aria 2. To obtain cardiac cells, cells were plated on gelatin-coated plastic
dishes and treated with Ara C (Sigma) to inhibit nonmyocyte proliferation.
Molecular Cloning and Retroviral/Lentiviral Infection
Retroviruses or inducible lentiviruses containing the cardiac developmental
factors were generated as described and as detailed in the Extended Experi-
mental Procedures (Kitamura et al., 2003; Takahashi and Yamanaka, 2006).
The pMXs-DsRed Express retrovirus infection in cardiac fibroblasts resulted
in >95% transduction efficiency (Hong et al., 2009).
FACS Analyses and Sorting
For GFP expression analyses, cells were harvested from cultured dishes and
analyzed on a FACS Calibur (BD Biosciences) with FlowJo software. For
aMHC-GFP/cTnT expression, cells were fixed with 4% PFA for 15 min, per-
meabilized with Saponin, and stained with anti-cTnT and anti-GFP antibodies,
followed by secondary antibodies conjugated with Alexa 488 and 647 (Katt-
man et al., 2006).
For aMHC-GFP /Thy1+, Isl1-YFP /Thy1+, and Mesp1-YFP /Thy1+ cell
sorting, cells were incubated with PECy7 or APC-conjugated anti-Thy1
antibody (eBioscience) and sorted by FACS Aria 2 (Ieda et al., 2009).
For aMHC-GFP /Thy1+/c-kit and aMHC-GFP /Thy1+/c-kit+ cell sorting,
PECy7-conjugated anti-Thy1 and APC-conjugated anti-c-kit antibodies
(BD) were used. We used bone marrow cells as a positive control for c-kit
staining.
Cell Transplantation
Fibroblasts were harvested 1 day after retroviral infection. A left thoracotomy
was carried out in NOD-SCID mice, and 106 cultured cells were injected
into the left ventricle. After 1–2 weeks, the hearts were excised for
immunohistochemistry.
Histology and Immunocytochemistry
Cells or tissues were fixed, processed and stained with antibodies against
numerous proteins in standard fashion as detailed in the Extended Experi-
mental Procedures.
Quantitative RT-PCR
Total RNA was isolated from cells, and qRT-PCR was performed on an ABI
7900HT (Applied Biosystems) with TaqMan probes (Applied Biosystems),
which are listed in the Extended Experimental Procedures. To quantify endog-
enous-specific transcripts and both endogenous and transgene common tran-
scripts, primers were designed using Vector NTI, and SYBR green technology
was used. Primer information is available on request. mRNA levels were
normalized by comparison to Gapdh mRNA.
Microarray Analyses
Mouse genome-wide gene expression analyses were performed using
Affymetrix Mouse Gene 1.0 ST Array. aMHC-GFP+ cardiomyocytes were
collected by FACS. Three-factor transduced GFP+ cells and GFP cells
were collected by FACS after 2 and 4 weeks of culture. Cardiac fibroblasts
were also collected after 4 weeks of culture. RNA was extracted using Pico-
Pure RNA Isolation (Arcturus). Microarray analyses were performed in triplicate
from independent biologic samples, according to the standard Affymetrix
Genechip protocol. Data were analyzed using the Affymetrix Power Tool
(APT, version 1.8.5). See the Extended Experimental Procedures for additional
statistical methods.
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitations were performed on cardiac fibroblasts, tail-
tip dermal fibroblasts, iCMs, and neonatal cardiac cells. Immunoprecipitations
were done using the Imprint Chromatin Immunoprecipitation Kit (Sigma)
following the manufacturer instructions. Antibodies against H3K27me3 and
H3K4me3 were from Active motif, and normal rabbit IgG was from Cell
Signaling Technology. Primer sequences for qPCR custom TaqMan gene
expression assays (Applied Biosystems) are listed in the Extended Experi-
mental Procedures.
Bisulfite Genomic Sequencing
Bisulfite treatment was performed using the Epitect Bisulfite Kit (QIAGEN)
according to the manufacturer’s recommendations. PCR primers are listed
in the Extended Experimental Procedures. Amplified products were cloned
into pCR2.1-TOPO (Invitrogen). Ten randomly selected clones were
sequenced with the M13 forward and M13 reverse primers for each gene.
2+
Ca Imaging
Ca2+ imaging was performed according to the standard protocol. Briefly, cells
were labeled with Rhod-3 (Invitrogen) for 1 hr at room temperature, washed,
and incubated for an additional 1 hr to allow de-esterification of the dye.
Rhod-3-labeled cells were analyzed by Axio Observer (Zeiss) with MiCAM02
(SciMedia).
Electrophysiology
After 4 week transduction with GMT, the electrophysiological activities of iCMs
were analyzed using extracellular electrode recording with an Axopatch 700B
amplifier and the pClamp9.2 software (Axon Instruments). iCMs were visually
identified by GFP expression and spontaneous contraction. Glass patch
pipettes, with typical resistances of 2–4 MU, were directly attached on single
GFP+ cells for extracellular recording in Tyrode’s bath solution. For recording
intracellular action potentials, single GFP+ cells were held at 70 mV
membrane potential with a stimulation of 0.1–0.5 nA for 5 ms to elicit
a response after 10-week transduction with GMT.
Statistical Analyses
Differences between groups were examined for statistical significance using
Student’s t test or ANOVA. p values of <0.05 were regarded as significant.
ACCESSION NUMBERS
Microarray data have been submitted and can be accessed by the Gene
Expression Omnibus (GEO) accession number GSE22292.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, five
figures, two tables, and four movies and can be found with this article online
at doi:10.1016/j.cell.2010.07.002.
ACKNOWLEDGMENTS
We are grateful to members of the Srivastava lab, to K. Tomoda for critical
discussions and comments on the manuscript, to J. Olgin and C. Ding for elec-
trophysiology assistance, to Z. Yang and K. Worringer for help with lentivirus
experiments, to Y. Huang for cell transplantation experiments, to B. Taylor,
G. Howard, and S. Ordway for editorial assistance and manuscript prepara-
tion, to C. Barker and L. Ta in the Gladstone Genomics core, to C. Miller and
J. Fish in the Gladstone Histology core, to A. Holloway in the Gladstone Bioin-
formatics core, and to S. Elmes in the Laboratory for Cell Analysis in UCSF. We
also thank S. Yamanaka for helpful discussions and providing pMXs-DsRed
Express plasmid, T. Kitamura for Plat-E cells, J. Robbins for a-MHC promoter
plasmid, T.M. Jessell for Isl1-Cre mice, and F. Costantini for R26R-EYFP mice.
M.I. and Y.H. are supported by a grant from the Uehara Memorial Foundation.
V.V. is supported by grants from the GlaxoSmithKline Cardiovascular
Research and Education Foundation and the NIH/NHLBI. P.D.-O. was a post-
doctoral scholar of the California Institute for Regenerative Medicine. D.S. and
B.G.B are supported by grants from NHLBI/NIH and the California Institute for
Regenerative Medicine. The J. David Gladstone Institutes received support
from a National Center for Research Resources Grant RR18928-01. D.S. is
a member of the Scientific Advisory Board of iPierian, Inc., and RegeneRx.
B.G.B. is a member of the Scientific Advisory Board of iPierian Inc.
Received: February 18, 2010
Revised: May 18, 2010
Accepted: June 25, 2010
Published: August 5, 2010
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Dendritic Function of Tau Mediates
Amyloid-b Toxicity in
Alzheimer’s Disease Mouse Models
Lars M. Ittner,1,6,* Yazi D. Ke,1,6 Fabien Delerue,1 Mian Bi,1 Amadeus Gladbach,1 Janet van Eersel,1 Heidrun Wölfing,1
Billy C. Chieng,2 MacDonald J. Christie,2 Ian A. Napier,2 Anne Eckert,3 Matthias Staufenbiel,4 Edna Hardeman,5
and Jürgen Götz1,*
1Alzheimer’s and Parkinson’s Disease Laboratory
2Neuropharmacology Laboratory
Brain and Mind Research Institute, University of Sydney, Sydney NSW 2050, Australia
3Neurobiology Research Laboratory, Psychiatric University Clinic, University of Basel, Basel CH-4025, Switzerland
4Novartis Institutes for BioMedical Research, Basel CH-4002, Switzerland
5Neuromuscular and Regenerative Medicine Unit, University of New South Wales, Sydney NSW 2052, Australia
6These authors contributed equally to this work

*Correspondence: littner@med.usyd.edu.au (L.M.I.), jgoetz@med.usyd.edu.au (J.G.)

DOI 10.1016/j.cell.2010.06.036

SUMMARY where familial mutations have been identified in the tau-encod-

Alzheimer’s disease (AD) is characterized by
amyloid-b (Ab) and tau deposition in brain. It has
emerged that Ab toxicity is tau dependent, although
mechanistically this link remains unclear. Here, we
show that tau, known as axonal protein, has
a dendritic function in postsynaptic targeting of the
Src kinase Fyn, a substrate of which is the NMDA
receptor (NR). Missorting of tau in transgenic mice
expressing truncated tau (Dtau) and absence of tau
in tau
/
mice both disrupt postsynaptic targeting
of Fyn. This uncouples NR-mediated excitotoxicity
and hence mitigates Ab toxicity. Dtau expression
and tau deficiency prevent memory deficits and
improve survival in Ab-forming APP23 mice, a model
of AD. These deficits are also fully rescued with
a peptide that uncouples the Fyn-mediated interac-
tion of NR and PSD-95 in vivo. Our findings suggest
that this dendritic role of tau confers Ab toxicity at
the postsynapse with direct implications for patho-
genesis and treatment of AD.
INTRODUCTION
Alzheimer’s disease (AD) is characterized by two hallmark
lesions, amyloid-b (Ab) plaques and neurofibrillary tangles
(NFTs) (Ballatore et al., 2007). Ab is derived from the amyloid-b
precursor protein (APP) by proteolytic cleavage (Haass et al.,
1992; Selkoe, 1997). The major constituent of NFTs is tau,
a microtubule (MT)-associated protein (Goedert et al., 1988). In
the course of AD, tau becomes phosphorylated, forming aggre-
gates that deposit as NFTs and neuropil threads (Geschwind,
2003). Tau can also form aggregates in the absence of an overt
Ab pathology, for example in frontotemporal dementia (FTD),
ing MAPT gene (Ballatore et al., 2007). Evidence that tau
pathology in AD is induced by Ab comes from our previous
observation that intracerebral Ab injections exacerbate hyper-
phosphorylation of tau and NFT formation in transgenic mice
that express FTD mutant P301L tau (Götz et al., 2001b). A similar
finding was obtained by crossing transgenic mice with NFT and
plaque pathologies (Lewis et al., 2001).
Ab-plaque formation along with memory impairment and tau
pathology with increased phosphorylation, in the absence of
deposition and NFT formation, has been reproduced in several
transgenic mouse lines that express human APP together with
pathogenic mutations identified in familial AD (Götz and Ittner,
2008; Hsiao et al., 1996; Mucke et al., 2000; Sturchler-Pierrat
et al., 1997). In one of these, PDAPP, tau deficiency (tau
/
)
was shown to rescue lethality and memory deficits by an uniden-
tified mechanism (Roberson et al., 2007).
Tau is known as axonal protein that regulates MT stability
and MT-dependent processes (Dixit et al., 2008; Drechsel
et al., 1992; Lee et al., 1988), while Ab likely exerts toxicity
at the postsynapse (Selkoe, 2002; Shankar et al., 2008;
Zhao et al., 2006). Although in AD, hyperphosphorylated tau
accumulates in the somatodendritic compartment of neurons
(Ballatore et al., 2007), given the spatial separation it remains
unknown how tau is involved in mediating Ab toxicity when AD
is initiated.
Seizures characterize several APP transgenic strains (Minkevi-
ciene et al., 2009; Palop et al., 2007; Palop and Mucke, 2009) and
have been associated with AD; the extent of their contribution to
pathology, however, remains to be established (Minkeviciene
et al., 2009; Palop et al., 2007; Palop and Mucke, 2009). Excito-
toxicity results from overactivation of N-methyl-D-aspartate
(NMDA) receptors (NRs). Interestingly, tau reduction decreases
susceptibility to excitotoxic seizures in vivo, which may explain
the concomitant improvement of the PDAPP phenotype (Rober-
son et al., 2007). How tau prevents excitotoxic damage at
a molecular level is not understood.

Cell 142, 387–397, August 6, 2010 ª2010 Elsevier Inc. 387

A B wt ∆tau74 Figure 1. Truncated Tau Is Excluded from
Fyn binding site Dendrites in Dtau74 Mice
htau40 (A) The longest human tau isoform (htau40; 441 aa)
1 441
is composed of an amino-terminal projection
mThy1.2 ∆tau cx hp
domain (PD), the microtubule-binding (MTB)
1 255
domain with four repeats (gray boxes), and the car-
PD MTB C’ boxy-terminal tail (C0 ). Dtau transgenic mice
express only the PD of tau under control of the
wt ∆tau74 am neuronal mThy1.2 promoter. Dtau lacks the MTB
C kD domain and therefore the MT-binding and aggre-
50
Tau-5 37 gation properties of full-length tau, but contains
50
a Fyn binding site.
HT7 37 D ∆tau74 pR5 (B) Expression pattern of Dtau in Dtau74 brains.
Immunohistochemistry (IHC) with a human tau-
Gapdh specific antibody (HT7; brown) reveals Dtau
S expression within several brain regions, including
HT7 DAPI

4
(fold of wt)

wt
tau levels

3 ∆tau74 hippocampus (hp), cortex (cx), and amygdala (am).

2 (C) Western blotting of wild-type and Dtau74
1
D hippocampal extracts reveals endogenous murine
tau (50 kD) in all and Dtau (37 kD) only in transgenic
0
total endogenous samples. Quantification shows comparable levels
tau tau of endogenous tau, while endogenous tau and
Dtau levels add up to 2.4-fold increased total levels
in Dtau74 compared to WT mice.
(D) IHC of the hippocampal CA1 region reveals that in Dtau74 mice, Dtau localizes to the soma (S) but is excluded from dendrites (D), whereas expression of P301L
mutant full-length tau in pR5 mice results in a somato-dendritic localization of transgenic tau (HT7; reactive with Dtau and pR5 tau, but not endogenous tau,
in red). The scale bar represents 50 mm.
Error bars represent the standard error. See also Figure S1.

Tau interacts via its amino-terminal projection domain (PD)
with the kinase Fyn (Figure 1A) (Lee et al., 1998). Fyn phosphor-
ylates the NR subunit 2 (NR2) to facilitate interaction of the NR
complex with the postsynaptic density protein 95 (PSD-95)
(Nakazawa et al., 2001; Rong et al., 2001; Tezuka et al., 1999),
linking NRs to synaptic excitotoxic downstream signaling (Salter
and Kalia, 2004). Disruption of the NR/PSD-95 interaction
prevents excitotoxic damage in cultured neurons and a rat
model of stroke, without affecting synaptic NMDA currents
(Aarts et al., 2002). Reduction of Fyn in APP transgenic mice
prevents Ab toxicity, while overexpression enhances it (Chin
et al., 2005; Chin et al., 2004).
To address how tau confers Ab toxicity, we generated trans-
genic mice (Dtau74) that express only the amino-terminal projec-
tion domain (PD) of tau and crossed them with Ab-forming
APP23 and tau
/
mice. We found that tau has an important
dendritic function, as in Dtau74 and tau
/
mice, postsynaptic
Fyn localization is reduced, resulting in reduced NR phosphory-
lation, destabilized NR/PSD-95 interaction, and protection from
excitotoxicity.
RESULTS
Truncated Tau Is Excluded from Dendrites
Tau comprises an amino-terminal projection domain, an MT
binding (MTB) domain that mediates interaction with MTs
(Butner and Kirschner, 1991; Lee et al., 1988) and is essential
for tau aggregation (Crowther et al., 1989; Ksiezak-Reding and
Yen, 1991) and a carboxy-terminal tail region (Figure 1A). We
generated truncated (Dtau) transgenic mice that express the
projection domain of tau in neurons, intended to compete with
functions of endogenous tau. Four phenotypically normal lines
expressed Dtau throughout the brain (Figure 1B) at comparable
levels, with line Dtau74 expressing the transgene at 1.4-fold
higher levels than endogenous tau (Figure 1C). Expression of
Dtau neither affected levels nor distribution of endogenous tau
(Figure 1C and Figures S1A–S1C available online). Consistent
with previous in vitro findings (Maas et al., 2000), Dtau localized
to the cell membrane, as indicated by coimmunostaining with
cadherin and subcellular fractionation of membranes (Figures
S1D and S1E). In AD and also full-length P301L mutant tau trans-
genic pR5 mice, tau is hyperphosphorylated and redistributed
into the somatodendritic compartment (Figure 1D) (Götz et al.,
2001a). In contrast to full-length tau, Dtau, while in the soma,
was virtually excluded from dendrites (Figure 1D). In pR5
mice, tau becomes progressively hyperphosphorylated and
insoluble, and eventually the mice develop NFTs. Surprisingly,
Dtau in Dtau74 mice is hardly phosphorylated at all (Figure S1F).
Postsynaptic Targeting of Fyn Is Tau Dependent
Different from full-length human tau in pR5 mice, in the absence
of an MTB domain, Dtau fails to interact with MTs, as determined
by MT precipitation from hippocampi (Figure 2A). However, Dtau
contains motifs that mediate interaction with the Src kinase Fyn,
as shown in vitro (Lee et al., 1998). Accordingly, Fyn can be coim-
munoprecipitated with Dtau from Dtau74 hippocampi in vivo,
using a human tau-specific antibody (HT7) (Figure 2B). Immuno-
precipitation (IP) with tau-specific antibodies to epitopes not
present on the Dtau construct reveals a significantly reduced
interaction of Fyn with endogenous tau (Figure 2B). Likewise,
IP with Fyn antibodies shows a reduced interaction with endog-
enous tau in Dtau74 mice (Figure 2B). Together, this suggests
a dominant negative effect of Dtau on the normal interaction of
Fyn and endogenous tau. A similar effect on the Fyn/Tau

388 Cell 142, 387–397, August 6, 2010 ª2010 Elsevier Inc.

 A C Figure 2. Dtau Impairs Tau-Dependent
p R 74

p R 74

74
Dendritic Targeting of the Src Kinase Fyn
au

au

au
5

5
pR

wt ∆tau74
∆t

∆t

∆t
human tau (A) Dtau from Dtau74 mice does not interact with
Tau-5 murine tau
S
microtubules. Endogenous murine tau, but not
∆tau Dtau, precipitates with microtubules in extracts
tubulin from Dtau74 mice. In contrast, both full-length
actin
D human and endogenous murine tau precipitate
extract no MT MT prec with microtubules in extracts from pR5 mice.

Fyn Drebrin
(B) Expression of Dtau results in a 74% ± 6%
B
74
u -/-
au

(n = 8; *p < 0.01) reduced interaction of Fyn with

5
pR
∆t
t

-/- -/-
ta
w

human tau
tau ∆tau74.tau endogenous murine tau (mtau) compared to the
mTau S wild-type (wt), as revealed by coimmunoprecipita-
murine tau
input

HT7 ∆tau tion (coIP) with antibodies to endogenous murine

Fyn tau (mTau). In Dtau74 mice, Fyn instead coimmu-
Gapdh noprecipitates with Dtau, as revealed by antibody
D HT7. Similarly, in pR5 mice, Fyn precipitates with
IP: mTau

mTau
full-length human tau (htau). CoIP with Fyn anti-
Fyn bodies predominantly pulled down endogenous
tau in WT, Dtau in Dtau74, and full-length human
mTau D
IP: HT7

40 tau in pR5 mice. No precipitation was observed

HT7 * * from tau
/
tissue.
intensity (AFU)

wt
Fluorescence

30
Fyn * ∆tau74 (C) Fyn accumulates in cell bodies in Dtau74,
20 tau-/-
Fyn
* * tau
/
, and Dtau74.tau
/
mice. While Fyn staining
IP: Fyn

∆tau74.tau-/-
mTau 10
* (red) colocalizes with dendritic drebrin (green) in
HT7 0 WT CA1 neurons, Fyn staining is evident in the
soma dendrites
soma (S) and is reduced in the dendrites (D) of
Dtau74 and tau
/
neurons. The insets show
E wt ∆tau74 tau-/- F wt ∆tau74 tau-/- G wt ∆tau74 tau-/- higher magnification of dendritic staining. The
IP:Fyn Fyn
scale bar represents 50 mm.
Fyn
420
(D) Quantification of fluorescence intensity of Fyn
Gapdh p -Fyn PSD-95
staining in cell bodies and dendrites shows accu-
p531-Fyn
1.0 1.0
mulation of Fyn in cell bodies of Dtau74, tau
/
,
(fold of wt)
(fold of wt)

and Dtau74.tau
/
mice (n = 15, *p < 0.0001).

Fyn
Fyn

0.5 0.5
* * (E) Total Fyn levels are not reduced in Dtau74 and
0 0 tau
/
mice. Western blots of hippocampal
u -/-

u -/-
∆t w t

∆t wt
74

74

extracts from WT, Dtau74, and tau
/
brains

au

au
ta

ta

show comparable levels of Fyn, normalized to

Gapdh (n = 6).
(F) Phosphorylation of activating (Y420) and inactivating (Y531) sites of immunopurified Fyn from WT, Dtau74, and tau
/
brains is similar.
(G) Hippocampal synaptosomal preparations reveal reduced levels of Fyn in Dtau74 and tau
/
postsynapses compared to the WT (n = 6, *p < 0.005).
Error bars represent the standard error. See also Figure S2.

interaction was obtained by overexpression of full-length tau in
pR5 mice (Figure 2B). Given the dendritic exclusion of Dtau in
Dtau74 in contrast to full-length tau in pR5 mice (Figure 1D),
we speculated that the aberrant Dtau/Fyn interaction might
affect the normal intracellular distribution of Fyn. Immunohisto-
chemistry showed that Fyn colocalized with drebrin in wild-
type (WT) brain, consistent with postsynaptic targeting, while in
Dtau74 brains it accumulated in the soma, an effect enhanced
by crossing of Dtau and tau
/
(Figures 2C and 2D). Together
with reduced dendritic Fyn staining, this suggests impaired
postsynaptic targeting of Fyn. To determine the role of tau in
dendritic localization of Fyn, we also analyzed tau
/
mice. Fyn
also accumulated in the soma (Figures 2C and 2D), suggesting
that postsynaptic targeting of Fyn is, at least in part, tau depen-
dent. This is consistent with reduced localization of Fyn-DsRED
in primary hippocampal neurons either from tau
/
mice or mice
coexpressing Dtau (Figure S2). Interestingly, further truncation of
Dtau shows that the Fyn-interactive motif, PXXP (Lee et al.,
1998), is critical for Fyn localization.
Despite changes in the localization of Fyn, its total levels
and activity were comparable in Dtau74, tau
/
, and WT mice,
as determined by total and phosphorylation site-specific anti-
Fyn antibodies (Figures 2E and 2F). To quantify changes in the
subcellular localization of Fyn, we prepared synaptosomes
from WT, Dtau74, and tau
/
hippocampi. Consistent with the
immunohistochemical findings of reduced postsynaptic target-
ing, levels of synaptic Fyn were reduced by 73% and 62% in
Dtau74 and tau
/
mice, respectively, compared to WT controls
(Figure 2G). Taken together, both the presence of Dtau and
absence of endogenous tau impair synaptic localization of Fyn.
Uncoupled NMDA Receptors and PSD-95 in Dtau
and tau
/
Synapses
The postsynaptic NR subunit NR2b is a known substrate of
Fyn (Nakazawa et al., 2001). NR2b phosphorylation at Y1472
strengthens the NR/PSD-95 interaction (Rong et al., 2001). In
both Dtau74 and tau
/
mice, Y1472 phosphorylation is signifi-
cantly reduced compared to the WT, while total levels of NR1,
NR2a, and NR2b are unaffected (Figure 3A). To determine
whether this affects the stability of NR/PSD-95 complexes,
we performed coimmunoprecipitations (coIPs). Markedly less
NR1, NR2a, and NR2b coimmunoprecipitated with PSD-95

Cell 142, 387–397, August 6, 2010 ª2010 Elsevier Inc. 389

 tau-/- total pH6 pH8 SDS Figure 3. Destabilized NMDA Receptors in
A wt ∆tau74 C
the Postsynaptic Density of Dtau74 and
NR1 Gap43
tau
/
Mice

Y1472 (fold of wt)

NR2a 1.0 SNAP25
(A) Levels of NR subunits NR1, NR2a and NR2b,
NR2b * PSD-95
0.5 * and PSD-95 are comparable in extracts from WT,
Y1472 mTau Dtau74, and tau
/
brains, whereas phosphoryla-
0
PSD-95 wt Fyn tion of NR2b at the Fyn site, Y1472, that is known
∆tau74
tau-/- NR1 to stabilize NR/PSD-95 complexes (Roche et al.,
Gapdh
NR2a 2001), is significantly reduced in Dtau74 and
tau
/
than in the WT (n = 6, *p < 0.005).
NR2b
(B) PSD-95 antibodies coimmunoprecipitate much
B wt ∆tau74 tau-/- D wt ∆tau74 tau-/- less NR subunits NR1, NR2a, and NR2b from
IP:PSD-95 1.0 NR1 Dtau74 and tau
/
than from WT hippocampi.
NR1

0.5 * * NR2a
Similarly, coimmunoprecipitation (coIP) of Fyn
NR1 0
1.0 with PSD-95 is reduced in Dtau74 and tau
/

pH8
NR2a

NR2a NR2b
0.5 * * compared to WT hippocampi, while that of nNOS
NR2b 0 Fyn
1.0 and Homer was unaffected. Endogenous murine
NR2b

mTau 0.5
* * SNAP25 Tau (mTau) coprecipitates with PSD-95 from WT
WB 0
HT7 1.0
NR1 hippocampi, while much less mTau, but no Dtau
mTau

0.5 * (HT7), is recovered from Dtau74 hippocampi.

Fyn NR2a
mTau is absent in tau
/
coIPs, consistent with
0
1.0
nNOS * * SDS
Fyn

0.5 NR2b tau deficiency. (n = 3, *p < 0.005.)

0
Homer Fyn (C) Sequential extraction of synaptosomes. Puri-
PSD-95 fied WT synaptosomes were further fractionated
with buffers of increasing stringency (pH6 <
E wt ∆tau74 tau-/- ∆tau74.tau-/- G pH8 < SDS), to purify proteins that are stably asso-
6 80
ciated with the PSD (Phillips et al., 2001). Brain
EPSC (NMDA/AMPA)

extracts (total) are loaded for comparison. NR

Mini EPSC (pA)

5
60
4 subunits NR1, NR2a and NR2b, PSD-95, tau,
3 40
and Fyn are purified in the SDS fraction, consistent
2
1
20 with strong anchoring in the PSD. Soluble proteins,
50 pA
0 0 such as GAP43 and proteins that are not (such as
u -/-

u -/-

u -/-

u -/-

50 ms
t

74

74
w

SNAP25) or less stably associated with the PSD,

ta

.ta

ta

.ta
au

au

H
74

74
∆t

∆t

are extracted with less stringent pH 6 and pH 8

au

au

100 5
∆t

∆t

F wt ∆tau74 tau-/- ∆tau74.tau-/- buffers, respectively.

EPSC (% NR2b)

Frequency (s-1)

75 4

50 pA
3 (D) SDS fractions from synaptosomes show that
50
2 stable anchoring of NRs in the PSD is reduced in
50 ms
25 1 Dtau74 and tau
/
mice. While NRs are recovered
0 0 in the SDS fraction of WT synaptosomes, they are
u -/-

u -/-

u -/-

u -/-
t

74

74
w

primarily found in the pH 8 and hardly at all in SDS

ta

.ta

ta

.ta
au

au
74

74
∆t

∆t

fractions from Dtau74 and tau
/
mice.

au

au
∆t

∆t

(E) Representative traces of AMPAR- (gray) and

NR- (black) mediated components of electrically
evoked (e) EPSCs in CA1 hippocampal neurons from WT, Dtau74, tau
/
, and Dtau74.tau
/
mice (average of 12 sweeps per neuron) normalized with AMPAR-
mediated component. Neurons were voltage clamped and held at +40 mV. AMPAR-mediated eEPSCs are inverted for clarity. There is no significant difference of
NMDA/AMPA ratios between genotypes (n = 19–20).
(F) Representative traces of eEPSCs (average of 12 sweeps per neuron) separating total NMDAR-mediated and NR2b subunit-mediated components
(black traces, total NR eEPSC minus component in CP-101,606 [5 mM]) normalized to the amplitude of the total NR eEPSC. Neurons were voltage clamped
at +20 mV. NR2b EPSCs were obtained by subtraction of EPSCs generated in CP101 606 (5 mM) from total NR-mediated EPSCs, i.e., before CP applications.
There is no significant difference in the percentage of NR2b component between genotypes (n = 18–20).
(G and H) Mean amplitude (G) and rate (s
1) (H) of AMPAR-mediated mEPSCs (recorded in 1 mM TTX) were unaffected in Dtau74, tau
/
, and Dtau74.tau
/
mice
(n = 10–12).
Error bars represent the standard error. See also Figure S3.

from Dtau74 and tau
/
compared to WT extracts, consistent buffers of increasing stringency (Phillips et al., 2001). In line with
with a decreased interaction of NR and PSD-95 in both strains a strong PSD association in WT synaptosomes, NR subunits
(Figure 3B). The PSD-95-interacting proteins Homer and were mostly found in the SDS fraction (Figure 3C and 3D). In
nNOS, however, were coimmunoprecipitated to a similar extent contrast, they were markedly reduced in Dtau74 and tau
/
,
from WT, Dtau74, and tau
/
brains, suggesting intact interac- appearing instead in earlier fractions, suggestive of a weakened
tions. The NR/PSD-95 interaction facilitates stable anchoring anchoring in the PSD (Figure 3D). Interestingly, endogenous
of NRs in the postsynaptic density (PSD) (Roche et al., 2001). tau that was enriched by synaptosome preparation, recovered
Therefore, we next extracted purified synaptosomes from WT, in WT SDS fractions and coimmunoprecipitated with PSD-95
Dtau74, and tau
/
mice that show similar levels of NR subunits, from WT, and to a lesser degree from Dtau74, brains (Figures
but reduced NR2b phosphorylation at Y1472 (Figure S3A), using 3B and 3C and Figure S1C).

390 Cell 142, 387–397, August 6, 2010 ª2010 Elsevier Inc.

A B Figure 4. Dtau Expression Improves
4
100 APP23.∆tau74.tau-/- Memory and Ameliorates Premature
APP23.∆tau74.tau+/-
Survival (% alive mice)

APP23.tau-/- Mortality of APP23 Mice

Errors (T-maze)
3
APP23.tau+/-
APP23.∆tau74 (A) APPswe transgenic APP23 mice (n = 76) present
Acquisition
75 2 with a pronounced premature mortality that is * 2h
24h
**
** ** ameliorated by reducing tau levels in APP23.tau+/

**
1
**** ** **
** ** ** (n = 41, p < 0.001) and even more in APP23.tau
/

APP23
mice (n = 108, p < 0.0001). Expression of Dtau
50 0
improves the survival of APP23.Dtau74 mice

-/-

-/-

u -/-
t

74

74
0 2 4 6 8 10

P2
u

u
au

au
ta

ta

.ta
Age (months) (orange, n = 43, p < 0.01) similar to APP23.tau+/
.

AP

3.
∆t

∆t

74
P2
3.

au
P2

AP

∆t
AP
-/-

Interestingly, combination of Dtau expression with

3.
P2
C D 1.5
u
.ta

AP
(fold of APP23)
74

74

hAPP mRNA

tau reduction completely rescues APP23.Dtau74.-

-
u -/
au

au

1.0
∆t

∆t
ta

tau+/
(purple, n = 38, p < 0.0001) and

3.

3.

3.
3
P2

P2

P2

P2

0.5
G APP23
AP

AP

AP

AP

APP23.Dtau74.tau
/
(red, n = 52, p < 0.0001)

t
w

0.0
APP23.∆tau74
Fyn
mice from lethality.
NR2b E 300 90
(B) Improved memory acquisition of APP23.D
Aβ1-40 (ng/mg)

Aβ 1-42 (ng/mg)

Y1472 200 60
tau74, APP23.tau
/
, and APP23.Dtau74.tau
/

PSD-95 100 30
APP23.tau APP23.∆tau74.tau compared to APP23 mice in the T maze, 2 and 24 -/- +/-
0 0
2.0 3.0 hr after a five-trial acquisition, at 8 months of age.
Y1472 (fold of wt)
Fyn (fold of wt)

*
1.5
2.0 While WT, Dtau74, and tau
/
mice only make few
1.0
*
* 1.0
* * F 100
errors during the trials, memory deficits of APP23
per section

0.5 **
Plaques

** 75
0
0
25
mice are obvious from the continuously high
-/-
APP23 APP23.tau
APP23.∆tau74 APP23.∆tau74.tau 0 +/- numbers of errors made during the entire test. In
contrast, both APP23.Dtau74, APP23.tau
/
, and
APP23.Dtau74.tau
/
mice presented with WT-
like numbers of errors (n = 8, *p < 0.05, **p < 0.01).
(C) In synaptosomal preparations obtained from 4-month-old APP23, both Fyn levels and NR2b phosphorylation at Y1472 are increased as compared to wild-
type (wt) mice (n = 6, *p < 0.05). However, in synaptosomes from APP23.Dtau74 and APP23.tau
/
, and even more in APP23.Dtau74.tau
/
, both levels of Fyn
and NR2b phosphorylation are significantly lower than in APP23 mice (n = 6, *p < 0.05, **p < 0.01). Representative western blots from three independent exper-
iments are shown.
(D–G) Dtau expression and tau deficiency do not affect APP mRNA expression, Ab levels, or plaque burden.
(D) Levels of APP mRNA are not altered in APP23 mice in the presence of Dtau or when tau is absent (tau
/
).
(E) Ab1–40 and Ab1–42 levels are comparable in APP23, APP23.Dtau74, and APP23.tau
/
mice.
(F and G) Thioflavine S staining (green) reveals Ab plaques (arrows; insets) at similar numbers (F) and with similar morphology (G) in APP23 mice, independent of
coexpression of Dtau or tau reduction.
Error bars represent the standard error. See also Figure S4.

The organization of NRs within the PSD is important for coor- contribute to toxicity in mice; however, primary disease-related
dinated signal transduction (Kim and Sheng, 2004). Hence, alter- effects are attributed to Ab, as suggested by reverted deficits
ations of NRs in Dtau74 and tau
/
mice may affect synaptic in Ab-immunized APP models (Röskam et al., 2010) and absence
currents. Therefore, we determined excitatory postsynaptic of seizure-induced hippocampal remodeling in APP transgenic
currents (ESPCs) in acute hippocampal slices from WT, mice with low Ab levels (Palop et al., 2007). Excitotoxicity has
Dtau74, tau
/
, and Dtau74.tau
/
mice. In Dtau74, tau
/
, been linked to premature lethality in APP transgenic mice (Chis-
and Dtau74.tau
/
mice, we found np significant changes in hti et al., 2001; El Khoury et al., 2007; Leissring et al., 2003;
synaptic currents (Figure 3E). Similarly, no significant reduction Roberson et al., 2007). Hence, we speculated that in Dtau74
emerged in the contribution of NR2b-containing NRs to ESPCs alterations in NR/PSD-95 interaction might similarly rescue the
in Dtau74, tau
/
, and Dtau74.tau
/
mice (Figure 3F). Baseline early lethality that characterizes APPswe mutant APP23 mice
miniature amplitudes and frequency were also comparable (Figures S4A and S4B) (Sturchler-Pierrat et al., 1997). APP23
(Figures 3G and 3H and Figures S3B–S3D). Taken together, mice have high Ab levels already at a very young age (Kuo
these data indicate that both expression of Dtau or tau deficiency et al., 2001; Van Dam et al., 2003), eventually forming plaques
reduces the interaction of NRs with PSD-95 without affecting and presenting with neuronal loss and memory deficits (Calhoun
synaptic NR levels and currents. et al., 1998; Kelly et al., 2003; Sturchler-Pierrat et al., 1997).
When we crossed APP23 either with Dtau74 or tau
/
mice
Dtau Expression Prevents Premature Lethality (Figure S4C), this caused both a significantly delayed onset of
and Memory Deficits in APP23 Mice mortality and an improved overall survival (Figure 4A). Whereas
It has been shown previously that perturbing the interaction of any rescue (either on a tau
/
background or by expressing
NRs with PSD-95 had no effect on NR-mediated currents but Dtau) was partial, expression of Dtau on a heterozygous or
reduced the resilience of neurons to NMDA-mediated excitotox- homozygous tau-deficient background rescued lethality
icity (Aarts et al., 2002). Interestingly, excitotoxicity has been completely, suggesting complementary beneficial effects of
proposed to contribute to Ab toxicity in PDAPP mice, which tau deficiency and Dtau expression on survival (Figure 4A).
was reduced when the mice were crossed onto a tau
/
back- In contrast, crossing of APP23 mice with pR5 mice with an
ground (Roberson et al., 2007). APP expression per se may increased dendritic accumulation of tau (Figure 1D) (Götz et al.,

Cell 142, 387–397, August 6, 2010 ª2010 Elsevier Inc. 391

2001a), resulted in increased premature lethality, with no survival
beyond 4 months of age (Figure S4D). Interestingly, both Fyn
A 7 B 8 *
levels and Y1472 phosphorylation of NR2b are increased in 6
*

Seizure Severity
pR5 synaptosomes (Figure S4E). Because of possible confound- 6

Latency (min)
5
ing effects of APP overexpression and Ab formation in APP **
4
mutant mouse strains, we used also primary neurons treated ** *** 4
with Ab, in the absence of APP overexpression, as a model. In 3
tau
/
and Dtau74-expressing neurons, acute Ab toxicity was 2
wt
2 ∆tau74
markedly reduced (Figure S2E). Interestingly, deletion of the tau-/-
Fyn-interacting motif, PXXP (Lee et al., 1998), from Dtau abro- 1 ∆tau74.tau-/-
gated the protective effect. 0 0

-/-

u- /-
∆t wt
74
We next determined whether Dtau expression or tau reduction 1 2 3 4 5 6 7

au tau
.ta
au
Seizure Severity

74
also improves memory functions in APP23 mice. Memory defi-

∆t
cits were both improved to WT levels in APP23.Dtau74
and APP23.tau
/
mice using the water T maze (Figure 4B).
Consistent with the findings in Dtau and tau
/
mice, both
C 7 D 8 APP23 * *
synaptic Fyn levels and NR2b phosphorylation were reduced 6 *
APP23.∆tau74 *

Seizure Severity
in APP23.Dtau74 and APP23.tau
/
and even more so in ** 6 *

Latency (min)
5
APP23.Dtau74.tau
/
synaptosomes, while they were increased
in APP23 compared to WT brains (Figure 4C). Interestingly, in 4 ***
4
APP23 mice, neither Dtau expression nor tau reduction affected 3
human APP messenger RNA (mRNA) levels (Figure 4D), Ab levels
2 2
(Figure 4E), or plaque burden (Figures 4F and 4G). Similarly,
1 APP23.tau-/-
phosphorylation of endogenous tau was comparable in APP23 APP23.∆tau74.tau-/-
and APP23.Dtau74 mice (data not shown). Taken together, 0 0
expression of Dtau in APP23 or crossing of APP23 with tau
/

74 u -/-
u- /-
AP .∆t 23
3. 2 74

1 2 3 4 5 6 7
au ta
.ta
3 P
P2 P au
P2 AP

Seizure Severity
∆t 3.

mice reduces Fyn-mediated NR2b phosphorylation, attenuates

premature mortality, and improves memory deficits without
AP

changing Ab levels or plaque load.

AP

Dtau Reduces Susceptibility to Excitotoxic Seizures E 7 F 8 wt + vehicle

wt + MK801
Ab-induced aberrant excitatory neuronal activity may contribute 6 *
Seizure Severity

to the deficits that characterize AD mouse models (Busche et al., 6

Latency (min)

5
2008; Palop and Mucke, 2009). APP23 mice show spontaneous *
seizures (Lalonde et al., 2005), similar to other APP transgenic 4
4
strains (Minkeviciene et al., 2009; Palop et al., 2007; Palop and 3
Mucke, 2009). Hence, reduced mortality of APP23.Dtau74 and
2
APP.tau
/
mice may be related to a reduced susceptibility to 2
APP23 + vehicle
excitotoxic seizures. We therefore first induced convulsions in 1
APP23 + MK801
Dtau74, tau
/
, Dtau74.tau
/
, and WT mice using the g-amino- 0 0
butyrate (GABA) antagonist pentylenetetrazole (PTZ). Seizure wt APP23 1 2 3 4 5 6 7
severity was significantly reduced in Dtau74, tau
/
, and vehicle MK801
Seizure Severity
Dtau74.tau
/
compared to the WT (Figure 5A), while the latency
to develop severe convulsion increased (Figure 5B). Next, we Figure 5. Dtau Expression Reduces Susceptibility to Excitotoxic
induced seizures in APP23, APP23.Dtau74, APP23.tau
/
, and Seizures
APP23.Dtau74.tau
/
mice. APP23 mice presented with a (A) When excitotoxic seizures were induced by i.p. injection of PTZ (50 mg/kg),
reduced convulsion latency and showed the most severe seizure mean seizure severity was significantly reduced in both Dtau74, tau
/
, and
Dtau74.tau
/
compared to WT mice (n = 10, **p < 0.01, ***p < 0.001).
response, with the lowest survival rate (1/11) and all mice reach-
(B) Similarly, the latency to more severe seizure stages is increased in Dtau74,
ing status epilepticus (n = 11) (Figure 5C). However, when APP tau
/
, and Dtau74.tau
/
mice.
expression was combined with Dtau expression or tau defi- (C) In APP23 mice, PTZ-induced seizures are mostly lethal (10 of 11), whereas
ciency, this significantly decreased seizure severity, reduced in APP23.Dtau74, APP23.tau
/
, and APP23.Dtau74.tau
/
seizure severity is
fatality, and increased convulsion latency (Figures 5C and 5D). markedly reduced (n = 10, *p < 0.05, **p < 0.01, ***p < 0.001).
The double mutant Dtau74.tau
/
prevented severe seizures (D) APP23.Dtau74, APP23.tau
/
, and APP23.Dtau74.tau
/
mice show an
better than Dtau74 or tau
/
alone, on both WT and APP23 back- increased latency to more severe seizures compared to APP23 mice.
(E and F) Pretreatment of WT or APP23 mice with MK801 (0.1 mg/kg) reduced
grounds, in agreement with the survival data (Figure 4A). Inter-
seizure severity (n = 8, p < 0.05) (E) and increased latency to more severe
estingly, we found a similar degree of protection from PTZ- seizures (F).
induced seizures as in Dtau74, tau
/
, APP23.Dtau74, or Error bars represent the standard error.
APP23.tau
/
mice when we pretreated WT and APP23 mice,

392 Cell 142, 387–397, August 6, 2010 ª2010 Elsevier Inc.

 Figure 6. Peptide-Driven Uncoupling of the

AA

9c
t

2B
2B
en

A D E NR/PSD-95 Interaction Reduces Ab Toxicity

74
m

R
R
t

u -/-
en

-N
-N
at

au
is
tre

ys
and Improves Survival and Memory of
m

t
t

∆t
Ta
Ta

ta
h

w
at

al
e-

as
7
tre

an
PI
pr

PSD-95 APP23 Mice

1h 1h 5’ 6
24h NR1 (A) Twenty-day-old primary cortical neurons were

Seizure Severity
5

input
control NMDA Aβ
B PI/Hoechst Tau-54 *
pretreated with 100 nM Tat-NR2B9c peptide,
which disrupts the NR/PSD-95 interaction (Aarts
vehicle

3
Gapdh
2 et al., 2002), prior to treatment with the toxins
PSD-95 NMDA, Ab, H2O2, and staurosporine. Twenty-
1

IP
Tat-NR2B9c Tat-NR2BAA

NR1 four hours after treatment, cell death was deter-

0

t-N AA

9c
mined by propidium iodide (PI) uptake. Control

2B
Ta 2B
R
R
cells were pretreated with vehicle or 100 nM Tat-

-N
F

t
p

Ta
m
NR2BAA (inactive peptide).

ed
d
pu

ge

ov
ch mp

re mp
an
B

.v.

m
O
(B and C) Tat-NR2B9c (bottom row) significantly

pu

pu
i.c
D

6wks 28d 28d H reduces toxicity of NMDA and Ab to cortical

C 4mo 8mo neurons, as indicated by lower numbers of PI-
100 ## ## ## control G 4
positive cells (red; arrows) compared to pretreated
## ## ## H2 O 2 *
##
## ##
## Staurosporine 100 vehicle (top row) or Tat-NR2BAA (middle row)

Errors (T-maze)
## 3
Percentage survival

80 ## NMDA
Aβ (1μM) Tat-NR2B9c controls. Nuclei were stained with Hoechst
% of dying cells

A β (0.1μM) 75
#
Aβ (1μM) + NMDA (n=17)
60 Aβ (0.1μM) + NMDA 2 (blue). Treatment with H2O2, staurosporine,
# Tat-NR2BAA
50 (n=9) NMDA, Ab, or NMDA/Ab causes significant cell
40 * **
pump
1st pump

* 25 aCSF (n=11) 1 death (#p < 0.005, ##p < 0.0001), which for
* * NMDA- and Ab-treated neurons is reduced by
nd

20 *
2

* 0 0 pretreatment with Tat-NR2B9c, but not for H2O2-

0 30 60 240 ed

9c

SF
0 days after implantation and staurosporine-treated neurons (*p < 0.05,
2B
at

aC
vehicle Tat-NR2BAA Tat-NR2B9c
tre

R
-N
un

**p < 0.01). One hundred cells each were counted

t
Ta

in three independent experiments. The scale bar

represents 25 mm.
(D) Immunoprecipitation (IP) with a PSD-95 antibody from hippocampus of WT mice that were i.c.v. infused with Tat-NR2B9c and Tat-NR2BAA for 1 week, and
from untreated WT, Dtau74, and tau
/
mice. Less NRs were coimmunoprecipitated upon Tat-NR2B9c, but not Tat-NR2BAA treatment. The reduction was
comparable to Dtau74 and tau
/
mice.
(E) One week of i.c.v. infusion of Tat-NR2B9c reduced PTZ-induced seizure severity significantly, compared to inactive Tat-NR2BAA (n = 10, *p < 0.01).
(F) APP23 mice treated with vehicle (artificial cerebrospinal fluid [aCSF]) alone or together with Tat-NR2B9c or Tat-NR2BAA, using osmotic mini pumps. DOB,
date of birth.
(G) Survival of APP23 mice upon i.c.v. delivery of Tat-NR2B9c (n = 17) is markedly improved compared to vehicle (aCSF)-treated (n = 11, p < 0.01) and Tat-
NR2BAA-treated (n = 9, p < 0.05) controls. Gray boxes indicate time of drug delivery from two consecutively implanted pumps.
(H) Tat-NR2B9c-treated APP23 mice show markedly improved memory functions at 4 and 8 months after initiating treatment, compared to aCSF-treated or age-
matched untreated APP23 mice (n = 8, n = 4 for aCSF, *p < 0.05, **p < 0.01).
Error bars represent the standard error.

respectively, with the NR-antagonist MK801 (Figures 5E and 5F). death, consistent with shared signaling pathways mediating their
Hence, reduced susceptibility to excitotoxicity is consistent with toxicity (Figures 6B and 6C). Cell death induced by NMDA and
a reduced NR contribution and may contribute to reduced Ab, both separate and in combination, was significantly reduced
mortality in APP23 mice in the presence of Dtau or absence of by preincubation with Tat-NR2B9c, but not when induced by
endogenous tau. hydrogen peroxide or staurosporine (Figures 6B and 6C). Tat-
NR2BAA had no protective effects. Hence, perturbing the NR/
Targeted Uncoupling of NR and PSD-95 Prevents PSD-95 interaction with Tat-NR2B9c ameliorates Ab-mediated
Premature Death and Memory Seficits in APP23 Mice toxicity in vitro.
Provided that disturbed NR/PSD-95 complexes with a reduced Next, we tested in vivo whether APP23 mice would also benefit
dendritic Fyn localization in Dtau74 and tau
/
mice contribute from treatment with Tat-NR2B9c. A single dose of this peptide
to improved memory functions and survival of APP23.Dtau74 has previously been shown to confer virtually complete protec-
and APP23.tau
/
mice, targeted perturbation of the NR/PSD- tion from excitotoxic damage in a rat model of stroke (Aarts
95 interaction, independent of tau or Fyn, should also decrease et al., 2002). First, we determined whether sufficient NR/
Ab toxicity. Therefore, we treated primary cortical cultures with PSD-95 uncoupling was achieved by intracerebroventricular
the Tat-NR2B9c peptide composed of carboxy-terminal amino (i.c.v.) Tat-NR2B9c treatment, using osmotic minipumps. We
acids of NR2b (including Y1472) fused to a HIV1-Tat peptide to delivered either Tat-NR2B9c or Tat-NR2BAA for 1 week and
achieve cell membrane permeability (Figure 6A). Tat-NR2B9c then performed coIP with a PSD-95 antibody (Figure 6D). This
has been shown previously to protect from NMDA-induced exci- revealed a reduced NR/PSD-95 interaction upon Tat-NR2B9c,
totoxicity (Aarts et al., 2002; Kornau et al., 1995). As a negative but not Tat-NR2BAA, treatment. The level of reduction was
control, we included Tat-NR2BAA in which critical amino acids similar to that found in Dtau74 and tau
/
brains (Figure 6D).
were replaced by alanine (Aarts et al., 2002; Kornau et al., Sufficient uptake of peptides by the brain was further confirmed
1995). NMDA and Ab both induced pronounced cell death, while by protection from PTZ-induced seizures by Tat-NR2b9c, but
a combined NMDA/Ab treatment did not further increase cell not Tat-NR2BAA (Figure 6E). Next, we implanted minipumps

Cell 142, 387–397, August 6, 2010 ª2010 Elsevier Inc. 393

A wt B ∆tau74
Ca2+ Ca2+
NMDAR NMDAR
NR2b NR2b
PSD PSD
P
Fyn
P
Fyn
PSD-95 Tau PSD-95 Tau
dendritic spine
∆tau
(B) In Dtau74 mice, Dtau is excluded from entering dendrites. Since Fyn interacts with Dtau (red bar) in the cell body of neurons, it is therefore trapped and less
localized to dendrites. Also, phosphorylation of NR2b and the interaction of NRs and PSD-95 are markedly reduced. Hence, excitotoxic downstream signaling is
uncoupled from NRs and their stability within the PSD is reduced. As NR-mediated currents are not dependent on this interaction, they are not affected.
(C) As for Dtau74 mice, in tau
/
mice, tau-dependent localization of Fyn to the postsynapse is also markedly reduced. NR2b phosphorylation and the interaction
of NRs and PSD-95 are decreased. Thus, excitotoxic downstream signaling is uncoupled from NRs, and their stability within the PSD is reduced. Again, NR-medi-
ated currents are not affected.
into 6-week-old APP23 mice for i.c.v. delivery of artificial cere-
brospinal fluid (aCSF), with and without Tat-NR2B9c or Tat-
NR2BAA (Figure 6F). Mice in the aCSF and Tat-NR2BAA control
groups died frequently (7 of 11 and 4 of 9, respectively), whereas
only 1 of 17 mice died in the Tat-NR2B9c group (Figure 6E).
Finally, we tested whether Tat-NR2B9c-treatment has long-
term effects on memory in APP23 mice. The T maze revealed
comparable memory deficits in age-matched aCSF-treated
and untreated APP23 mice (Figure 6F). However, treatment
with Tat-NR2B9c resulted in a significantly improved perfor-
mance. Thus, perturbing NR/PSD-95 interaction is sufficient
to prevent premature lethality and memory deficits in APP23
mice.
DISCUSSION
Dendritic Localization of Fyn Is Tau-Dependent
Our data reveal a dendritic function of the ‘‘axonal’’ protein tau, in
targeting the kinase Fyn to the dendrite (Figure 7). We also found
an association of tau with the PSD complex by using coIP, PSD
purification, and immunohistochemistry with enhanced antigen
retrieval. It is important to note that levels of tau in the dendritic
compartment are much lower than in axons, suggesting that
under physiological conditions a major function of tau is in axonal
MT stabilization and regulation of MT-dependent processes
(Dixit et al., 2008; Weingarten et al., 1975). Here, we show that
the additional role of tau in dendrites becomes pivotal in disease,
in particular in mediating early Ab toxicity.
In both Dtau74 and tau
/
mice, dendritic targeting of Fyn is
significantly reduced, as revealed by immunohistochemistry
and synaptosomal purification and confirmed in primary
neurons. In Dtau74 mice, this is due to a competition of Dtau
with endogenous tau in the interaction with Fyn. Both the abun-
dance of Dtau in the cell body and its exclusion from dendrites
394 Cell 142, 387–397, August 6, 2010 ª2010 Elsevier Inc.
C tau-/- Figure 7. Simplified Scheme of the
Proposed Mechanism Underlying Reduced
Ca2+ Excitotoxicity in Dtau74 and tau
/
Mice
NMDAR Compared to the Wild-Type
NR2b
(A) Postsynaptic NMDA receptors (NRs) are het-
PSD eromeric complexes predominantly formed by
P
Fyn
subunits NR1, NR2A, and NR2B. The Src kinase
PSD-95
Fyn localizes to the postsynapse in a tau-depen-
dent manner and associates with the postsynaptic
density (PSD; gray box), where it phosphorylates
(P) the NR subunit NR2b at Y1472 in the extreme
carboxy terminus. This phosphorylation facilitates
the interaction of NRs with the scaffolding protein
PSD-95. This interaction increases the stability of
NRs within the PSD and couples NRs to excito-
toxic downstream signaling (skull). NR-mediated
currents (ESPC trace), however, do not depend
on this NR/PSD-95 interaction. Whether tau is
associated with the PSD via Fyn or another inter-
action partner remains to be elucidated.
result in ‘‘trapping’’ of Fyn in the soma. Tau
/
mice, in compar-
ison, show a similar accumulation of Fyn, suggesting that post-
synaptic Fyn targeting requires tau. This difference in mediating
aberrant sorting of Fyn between Dtau74 and tau
/
mice
(Figure 7) may explain the additive effects on seizure suscepti-
bility and survival in Dtau.tau
/
crosses. Reduced levels of
postsynaptic Fyn in Dtau74 and tau
/
mice are associated
with reduced phosphorylation of the Fyn-substrate NR2b at
Y1472. Consistent with a critical role of Y1472 phosphorylation
in facilitating the interaction of NRs with PSD-95 (Rong et al.,
2001), this complex is reduced and destabilized in Dtau74 and
tau
/
brains. Whether the Fyn-mediated stabilization of NR/
PSD-95 complexes in the PSD under physiological conditions
involves a direct interaction with tau and what the exact mecha-
nism(s) of tau-mediated dendritic Fyn localization are remains to
be established.
In a rat model of stroke, targeted disruption of the NR/PSD-95
interaction prevented excitotoxic damage and reduced the
lesion size (Aarts et al., 2002). Consistent with this, reduced
NR/PSD-95 complexes in Dtau74 and tau
/
mice were associ-
ated with a reduced susceptibility to excitotoxicity. Interestingly,
NR-mediated currents were not affected in Dtau74 and tau
/

mice, which is in line with normal synaptic activity upon treat-
ment with Tat-NR2B9c (Aarts et al., 2002). Normal NR-mediated
currents in Dtau74 and tau
/
mice may be explained by
reduced, but not totally depleted, synaptic Fyn in Dtau74 and
tau
/
mice, comparable to the situation in heterozygous fyn-
deficient mice that have no overt deficits (Yagi et al., 1993), while
in homozygous fyn-deficient mice these are pronounced (Grant
et al., 1992).
Dtau and tau
/
Prevent Deficits of APP23 Mice
Excitotoxicity is increasingly recognized as a mechanism of how
Ab exerts toxicity in AD. Accordingly, we found that crossing
of Dtau74 and tau
/
mice, both characterized by reduced
susceptibility to excitotoxicity, with Ab-forming APP23 mice
ameliorated premature mortality and memory deficits of APP23
mice. In contrast, early lethality was more pronounced in
APP23 mice crossed with pR5. Similarly, tau deficiency or Dtau
expression conferred protection from Ab-induced toxicity in
primary neuronal cultures. However, Ab levels and plaque forma-
tion, as well as endogenous tau phosphorylation (in APP23.D
tau74), were comparable in APP23 mice, suggesting an alterna-
tive mechanism for protection. Interestingly, in APP23 mice we
found both increased postsynaptic Fyn and Y1472 phosphoryla-
tion of NR2b that was completely reverted in APP23.Dtau74 and
APP23.tau
/
mice. Further reduction of post-synaptic Fyn in
APP23.Dtau74.tau
/
mice suggests additional tau-indepen-
dent mechanisms in dendritic Fyn localization, which are partially
competed with by Dtau. Consistent with a role for Fyn in Ab
pathology, Fyn transgenic mice present with seizures and
premature mortality (Kojima et al., 1998). This is exacerbated in
Fyn/APPmut double-transgenic mice (Chin et al., 2004). More-
over, APP-associated mortality is reduced on a fyn
/
back-
ground (Chin et al., 2004). Hence, our findings in APP23.Dtau74
and APP23.tau
/
mice are consistent with previous data (Chin
et al., 2005; Chin et al., 2004). Furthermore, they are in line
with the recent observation that crossing of PDAPP mice onto
a tau
/
background reverses Ab-associated defects (Roberson
et al., 2007).
Mechanistically, our data suggest that stable NR/PSD-95
complex formation is required for Ab toxicity in APP23 mice.
This is likely to contribute to disease together with other tau-
dependent and -independent mechanisms of Ab toxicity. In
support of our findings, we used a tau/Fyn-independent
approach to disrupt this interaction, by delivering the Tat-
NR2B9c peptide to young APP23 mice. This peptide has been
shown to protect from excitotoxicity in vitro and in vivo.
We show specifically that perturbing the NR/PSD-95 interac-
tion with the Tat-NR2B9c peptide improves survival and
memory functions of APP23 mice. The data suggest that disrup-
tion of the NR/PSD-95 interaction is sufficient to prevent
Ab toxicity involving NR signaling. Remarkably, Tat-NR2B9c-
treated APP23 mice survived long term, suggesting that treat-
ment within a short therapeutic window is sufficient to prevent
lethality.
In summary, we reveal a dendritic role for the ‘‘axonal’’
protein tau in postsynaptic targeting of Fyn. This involves inter-
action of Fyn with the tau projection domain (Lee et al., 1998).
Accordingly, dominant negative effects of Dtau expression or
tau deficiency result in reduced postsynaptic Fyn, decreased
phosphorylation of its substrate NR2b and instability of NR/
PSD-95 complexes (in Dtau74 and tau
/
mice). Importantly,
this additional function of tau appears to be pivotal for medi-
ating Ab toxicity, in that premature lethality, memory deficits,
and seizure susceptibility of APP23 mice were mitigated in
APP23.Dtau74 and APP23.tau
/
mice. Hence, reduction of
tau levels or targeting of tau-dependent mechanisms, such as
the Fyn-mediated interaction of NRs and PSD-95, are suitable
strategies in the treatment of AD and related disorders, high-
lighting tau as an attractive drug target, in addition to Ab
(Ashe, 2007).
EXPERIMENTAL PROCEDURES
Animals
APP23 and pR5 transgenic and tau
/
mice have been generated previously
(Götz et al., 2001a; Sturchler-Pierrat et al., 1997; Tucker et al., 2001). The
generation of Dtau74 mice is described in the Extended Experimental Proce-
dures. Two- to three-month-old mice were analyzed in age- and sex-matched
groups, unless stated otherwise. All animal experiments were approved by the
Animal Ethics Committee of the University of Sydney.
Histology, Western Blotting, IP, and Synaptosome Preparation
Detailed protocols are provided in the Extended Experimental Procedures.
Electrophysiology
Electrophysiological recording were done in acute hippocampal slices
obtained from 4- to 8-week-old wild-type, Dtau74, tau
/
, and Dtau74.tau
/

mice as described in detail in the Extended Experimental Procedures.
Experimental Seizures
Seizures were induced by intraperitoneal (i.p.) injection of (50 mg/kg body
weight) pentylenetetrazole (PTZ; Sigma) as described (Roberson et al.,
2007). Where indicated, mice were injected i.p. with (0.1 mg/kg body weight)
MK801 (Sigma) 30 min prior to PTZ administration. Mice were video moni-
tored, and seizure severity was rated by an independent, blinded person, as
follows: 0, no seizures; 1, immobility; 2, tail extension; 3, forelimb clonus; 4,
generalized clonus; 5, bouncing seizures; 6, full extension; and 7, death.
i.c.v. Treatment with Osmotic Pumps
Six-week-old APP23 and WT mice were anesthetised with ketamine/xylazine,
and i.c.v. delivery cannulas (Alzet; brain infusion kit #3 with one spacer) were
implanted with a stereotaxic frame (KOPF Instruments) at the following
coordinates according to the bregma: AP,
0.25 mm; ML, 1 mm; and DV,

2.5 mm. Osmotic mini pumps (Alzet; model #1004) were filled with aCSF
(Alzet) with and without Tat-NR2B9c or Tat-NR2BAA peptide (750 mM) and
equilibrated in 0.9% NaCl at 37 C for 48 hr. They were attached to the i.c.v.
cannula tubing and subcutaneously implanted at the back. After 28 days,
the pumps were replaced with a second batch of pumps via a small skin inci-
sion for another 28 days. Then, they were removed and the tubing was ligated.
Statistics
Statistics was done with the Prizm 4 software (GraphPad) with Student’s t or
two-way ANOVA test. Values are given as mean ± standard error.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures and
four figures and can be found with this article online at doi:10.1016/j.cell.
2010.06.036.
ACKNOWLEDGMENTS
The authors thank Yves-Alain Barde for providing tau
/
mice and him as well
as Hanns Möhler and Nikolas Haass for helpful comments. This research was
supported by grants from the University of Sydney, National Health and
Medical Research Council, Australian Research Council, and Deutsche
Forschungsgemeinschaft. J.G. is a Medical Foundation Fellow.
Received: December 14, 2009
Revised: April 6, 2010
Accepted: May 28, 2010
Published online: July 22, 2010
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Single-Stranded DNA Transposition
Is Coupled to Host Replication
Bao Ton-Hoang,1,* Cécile Pasternak,2 Patricia Siguier,1 Catherine Guynet,1 Alison Burgess Hickman,3 Fred Dyda,3
Suzanne Sommer,2 and Michael Chandler1,*
1Laboratoire de Microbiologie et Génétique Moléculaires, Centre National de Recherche Scientifique, Unité Mixte de Recherche 5100,

118 Route de Narbonne, F31062 Toulouse Cedex, France

2Université Paris-Sud, Centre National de Recherche Scientifique, Unité Mixte de Recherche 8621, Laboratoire de Recherche Correspondant

du Commissariat à l’Energie Atomique 42V, Institut de Génétique et Microbiologie, Bâtiment 409, Orsay, France
3Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,

MD 20892, USA
*Correspondence: bao.tonhoang@ibcg.biotoul.fr (B.T-H.), michael.chandler@ibcg.biotoul.fr (M.C.)
DOI 10.1016/j.cell.2010.06.034

SUMMARY genetic tools in identifying specific gene regulatory regions by

DNA transposition has contributed significantly to
evolution of eukaryotes and prokaryotes. Insertion
sequences (ISs) are the simplest prokaryotic trans-
posons and are divided into families on the basis
of their organization and transposition mechanism.
Here, we describe a link between transposition of
IS608 and ISDra2, both members of the IS200/
IS605 family, which uses obligatory single-stranded
DNA intermediates, and the host replication fork.
Replication direction through the IS plays a crucial
role in excision: activity is maximal when the ‘‘top’’
IS strand is located on the lagging-strand template.
Excision is stimulated upon transient inactivation of
replicative helicase function or inhibition of Okazaki
fragment synthesis. IS608 insertions also exhibit an
orientation preference for the lagging-strand tem-
plate and insertion can be specifically directed to
stalled replication forks. An in silico genomic
approach provides evidence that dissemination of
other IS200/IS605 family members is also linked to
host replication.
INTRODUCTION
DNA transposition involves movement of discrete DNA seg-
ments (transposons) from one genomic location to another.
It occurs in all kingdoms of life and has contributed significantly
to evolution of eukaryotes and prokaryotes. Transposable
elements can represent a significant proportion of their host
genomes (Biémont and Vieira, 2006). They have been particularly
well studied in bacteria where they are major motors of broad
genome remodeling, play an important role in horizontal gene
transfer, and can sequester and transmit a variety of genes
involved in accessory cell functions, such as resistance to anti-
microbial agents, catabolism of unusual compounds, and path-
ogenicity, virulence, or symbiosis. They are also important as
insertion and are being developed as delivery systems for gene
therapy applications.
A variety of structurally and mechanistically distinct enzymes
(transposases) have evolved to carry out transposition by several
different pathways (Turlan and Chandler, 2000; Curcio and
Derbyshire, 2003). They all possess an endonuclease activity
allowing them to cleave, excise, and insert transposon DNA
into a new location. Depending on the system (Curcio and Derby-
shire, 2003), different types of nucleophile can be used by trans-
posases to attack a phosphorus atom of a backbone phospho-
diester bond and cleave DNA. These include water (generally
activated by enzyme-bound metal ions), a hydroxyl group at
the 50 or 30 end of a DNA strand, or a hydroxyl group of an amino
acid of the transposase itself, such as serine or tyrosine.
Many mobile DNA elements move using a ‘‘cut-and-paste’’
mechanism by excision of a double-stranded copy from one
genomic location and insertion at another. Recently, a family of
bacterial insertion sequences (ISs), the IS200/IS605 family, has
been found that uses a completely different pathway and an
unusual transposase with a catalytic tyrosine (a Y1 transposase).
Studies of one member, IS608 (Figure 1A), provided a detailed
picture of their transposition (Ton-Hoang et al., 2005; Ronning
et al., 2005; Guynet et al., 2008; Barabas et al., 2008). In vitro,
this requires single-stranded DNA (ssDNA) substrates and is
strand specific: only the ‘‘top’’ strand is recognized by the
element-encoded transposase, TnpA, and is cleaved and trans-
ferred, whereas the ‘‘bottom’’ strand does not transpose. Exci-
sion of the top strand as a transposon circle with joined left
and right ends is accompanied by rejoining of the DNA flanks.
The circle junction then undergoes TnpA-catalyzed integration
into an ssDNA target in a sequence-specific reaction. Insertion
involves transfer of both the 50 and 30 ends of the single-strand
circle junction into the ssDNA target. The left (50 ) IS608
end always inserts specifically just 30 of the tetranucleotide,
50 -TTAC-30 (Kersulyte et al., 2002), which is also essential for
subsequent transposition (Ton-Hoang et al., 2005).
The obligatorily single-stranded nature of IS200/IS605 trans-
position in vitro raises the possibility that it is limited in vivo by
the availability of its ssDNA substrates. A number of cellular

398 Cell 142, 398–408, August 6, 2010 ª2010 Elsevier Inc.


Figure 1. IS608 Excision Depends on the Direction of Replication
(A) IS608 organization and a simplified transposition model. Gray arrows, tnpA
and tnpB orfs; red and blue boxes, left (LE) and right (RE) ends (color code
retained throughout).
(Ai) Schematized single-stranded IS608 showing secondary structures of LE
and RE, the flanking TTAC, and cleavage positions at the ends (vertical black
arrows).
(Aii) Excision and ssDNA circle formation with an RE-LE junction and a sealed
donor joint (black line) retaining TTAC.
(Aiii) TnpA brings together the transposon junction with a new TTAC-carrying
target (dotted black line). Vertical black arrows, points of cleavage and strand
transfer.
(Aiv) IS608 insertion into the target.
(B) Orientation of the IS608 derivative with respect to replication direction. The
disposition of the IS608 active (top) strand with respect to replication direction
is shown when the fork approaches from one direction (left) when it is part of
the lagging-strand template or the other (right) when it is part of the leading
strand. This is described in more detail in Figure S1 and its legend.
(C) Excision measured directly in vivo by the appearance of ‘‘donor joint’’ plas-
mids, deleted for the IS608 derivative. Left: pBS102 with the active IS608
processes generate or occur using ssDNA, including DNA repair,
natural transformation, conjugative plasmid transfer, single-
stranded phage infection, and replication (where the DNA
serving as the template for Okazaki fragment synthesis on the
lagging strand of the replication fork is single stranded).
Here, we investigate the link between IS608 transposition and
the availability of ssDNA during replication in vivo. Our results
demonstrate that transposition of the IS200/IS605 family is
closely integrated into the host cell cycle and takes advantage
of the presence of ssDNA on the lagging-strand template at
the replication fork for dissemination. We also show that IS608
transposition is affected by perturbing the fork: transitory inacti-
vation of crucial replication proteins increased excision from the
lagging-strand template, and stalling of the fork resulted in inser-
tions directed to the lagging strand of the blocked fork.
Our results also suggest that insertion and excision of the
related element, ISDra2, also depends on the lagging-strand
template in its host, the radiation-resistant bacterium Deinococ-
cus radiodurans, and that this dependency can be abolished
with irradiation. We have extended our analysis to a number of
related IS200/IS605 elements in a variety of sequenced bacterial
genomes. The results of this in silico analysis are also consistent
with a strong bias of insertion into the lagging-strand template in
these organisms. Together, the results demonstrate the impor-
tance of the lagging-strand template for IS608 and ISDra2
activity and suggest that all IS200/IS605 family members have
evolved a mode of transposition that exploits ssDNA at the repli-
cation fork.
RESULTS
IS608 Excision Depends on the Direction of Replication
To investigate whether replication direction affects IS608 trans-
position, we used a plasmid assay in E. coli to monitor the exci-
sion step of transposition (Ton-Hoang et al., 2005). In this assay,
the IS-carrying plasmids included an IS608 derivative in which
the tnpA and tnpB genes (Figure 1A) were replaced by a chloram-
phenicol resistance (CmR) cassette (Figure 1C). In one case, the
active (top) IS608 strand was located in the lagging-strand
template (pBS102), and in the second, the replication origin
was inverted (Figures 1B and 1C), placing the transposionally
active top strand on the leading-strand template (pBS144).
A second compatible plasmid supplied TnpA in trans under
strand as part of the lagging-strand template. Right: pBS144 with the active
IS608 strand as part of the leading-strand template. Ori, pBR322 origin of repli-
cation; Cm, bla, SpSm, chloramphenicol, b-lactamase and, streptomycin/
spectinomycin resistance genes; Plac, lac promoter; tnpA-his, C-terminal
his6-tagged tnpA gene. Directions of DNA replication and transcription are
indicated. Agarose gel (0.8%) showing separation of plasmid DNA from over-
night cultures of strains carrying pBS102 + pBS21 (lane 2) and pBS144 +
pBS121 (lane 3). Lane 1, 1 kb standard.
(D) Mating-out assays. Left-hand column, transposon donor plasmid; middle,
presence or absence of TnpA (relevant plasmids in parentheses); right,
measured transposition frequencies (standard error in parentheses; n > 3).
Plasmids pBS102ter1 and pBS144ter1 carry a single set of origin proximal
terminators; pBS102ter2 and pBS144ter2 carry two sets of terminators flank-
ing both transposon ends (Extended Experimental Procedures).
See also Figure S1A.
Cell 142, 398–408, August 6, 2010 ª2010 Elsevier Inc. 399
control of plac (Figure 1C). After overnight growth, IS excision
was monitored by detection of reclosed donor backbone mole-
cules from which the IS had been deleted.
As shown in Figure 1C, when the IS608 active strand was
located on the lagging-strand template, the donor backbone
species could be clearly identified along with the parental
plasmid and the plasmid used to supply transposase (lane 2).
However, when the replication origin was inverted and the active
IS strand was located on the leading strand, formation of the
excised donor backbone species was only barely detectable
(lane 3).
This effect of replication direction on IS608 transposition was
also observed in mating-out assays (Galas and Chandler, 1982)
that measure overall transposition frequency. We monitored
transposition was monitored by following movement of IS608
from a nonmobilizable donor plasmid into a conjugative plas-
mid. When the IS608 active strand was on the lagging-strand
template (Figure 1D, line 2), the transposition frequency was
5.6 3 105, but when it was on the leading-strand template,
the frequency dropped 27-fold to 2.1 3 106 (line 3). To ensure
that this was not due to possible changes in transcription
resulting from the inversion introduced during cloning to
switch the orientation of the replication origin (where bla was
inverted together with ori), we inserted transcriptional termina-
tors (Simons et al., 1987) on either one (lines 4–5) or both (lines
6–7) sides of the IS608 derivatives to insulate them from
impinging transcription. In these cases, the observed effect of
replication direction on transposition frequency remained
unchanged.
Effect of Mutant Primase and Replicative Helicase
on IS Excision
Since IS608 excision is sensitive to replication direction, we
asked whether it was affected by perturbation of the replication
fork. We used two temperature-sensitive replication mutants:
dnaG308ts, encoding a mutant DNA primase, DnaG (Wechsler
and Gross, 1971), and dnaB8ts, encoding a mutant of the essen-
tial replication fork DNA helicase, DnaB (Carl, 1970). Replication
in these mutants occurs at 30 C but is interrupted after a shift to
the restrictive temperature of 42 C. Inhibition of either DnaG or
DnaB activity is expected to increase the amount of ssDNA at
the fork, principally on the lagging-strand template (Louarn,
1974; Fouser and Bird, 1983; Belle et al., 2007) (see also the
Discussion).
To test the effect of inhibition of DnaG and DnaB activities, we
used a genetic screen in which a b-lactamase gene is interrupted
by an IS608 derivative (pAM1, Figure S2; Experimental Proce-
dures). TnpA-catalyzed precise excision (using TnpA provided
in trans) results in reconstitution of the b-lactamase gene
(Figure 2A) and the appearance of ampicillin resistant (ApR)
colonies.
As shown in Figures 2Bi, 2Bii, and 2Biii for the wild-type and
dnaB and dnaG mutants, respectively, after overnight growth
at 30 C (a permissive temperature) without TnpA induction, the
frequency of ApR colonies was low for the wild-type and mutant
hosts (column a). Induction of TnpA expression resulted in
a nearly 3-fold increase in excision with little difference between
wild-type, dnaBts, and dnaGts strains (column b). However,
400 Cell 142, 398–408, August 6, 2010 ª2010 Elsevier Inc.
when the growth protocol was modified to include a 30 min
temperature shift to 42 C (and further incubation of 3 hr at
30 C to allow replication to recover), excision increased about
10- and 7-fold in the dnaB and dnaG mutants, respectively,
compared to the wild-type (column d). Omission of the 42 C
step resulted in indistinguishable basal levels for wild-type and
mutant hosts (column c). Thus, inactivation of DnaB helicase
function or inhibition of initiation of Okazaki fragment synthesis
with a dnaGts mutation stimulated the excision step of transpo-
sition, consistent with the notion that ssDNA at the replication
fork is a substrate for IS excision.
Effect of Transposon Size on Excision
If excision occurs at the replication fork and requires ssDNA, it
seemed possible that IS length might influence excision
frequency since the probability that both IS ends are within the
single-stranded region of the lagging-strand template should
decrease with increasing IS length. We therefore examined the
effect of IS length in the excision assay by using a set of IS608
derivatives with varying spacing between the left end (LE) and
right end (RE) and found that excision decreased strongly as
a function of increasing IS length and that these frequencies
were strongly modified in a strain carrying a dnaGts mutation.
Increasing the IS length from 0.3 to 2 kb, resulted in a 7- to
10-fold decrease in excision frequency with a log-linear relation-
ship, consistent with the notion that excision occurs more effi-
ciently when both ends are located within the short single
lagging-strand region of about 1.5–2 kb upstream of the first
complete Okazaki fragment at the replication fork (see Johnson
and O’Donnell, 2005, for review). As the IS length was further
increased, excision decreased only slightly, at least up to a trans-
poson length of 4 kb (Figure 2C) with a possible inflection
between 1.5 and 2 kb.
The length of ssDNA on the lagging-strand template depends
on the initiation frequency of Okazaki fragment synthesis, in turn
determined by DnaG. Progressive inactivation of DnaG activity
by growth of the dnaGts mutant at increasing but sublethal
temperatures should reduce this frequency and increase the
mean length of ssDNA at the replication fork upstream of the first
complete Okazaki fragment. We therefore analyzed excision of
the IS608-derived transposons in wild-type and dnaGts mutants
at different temperatures (Figure 2D). While profiles were indis-
tinguishable for the wild-type strain at 30 C and 33 C, the
dnaGts mutant exhibited a generally higher excision frequency
at 30 C and showed a lower length dependent slope, revealing
that the replication fork is affected by the mutation even at the
normal permissive temperature. However, an inflection still
appeared to occur. At 33 C, excision increased significantly,
particularly for the longer transposons.
To further examine this, we cloned the wild-type dnaG allele
downstream of the tnpAIS608 gene in the TnpA-providing plasmid
so that both were under control of the same promoter (Experi-
mental Procedures). When this plasmid, pBS179, was intro-
duced into the dnaGts strain, it clearly suppressed the dnaGts
defect at 33 C (Figure 2E). Moreover, when introduced into the
wild-type dnaG strain, the excision frequencies were even
further reduced and the length-dependant slope was increased
(Figure 2F).
Figure 2. Excision Frequency: Effects of dnaBts and dnaGts Mutants and IS Length
(A) Schematic of the excision assay. The relevant features of the generic IS-carrying donor plasmid, pAM1, and the product after IS excision, DpAM1, are shown
together with the plasmid used to supply transposase, pBS135. These are described in detail in Figure S1 and its legend.
(B) Transitory inactivation of DnaB or DnaG. Stimulation factors for excision from wild-type, dnaBts, and dnaGts strains: i, ii, and iii, respectively. Values are
normalized to those of an overnight culture without IPTG at 30 C for each strain (0.8 3 102, 0.6 3 102, and 2.2 3 102 corresponding to column a), overnight
cultures diluted at 30 C with IPTG for 4 hr (column b), TnpA induction at 30 C (45 min) and incubation at 30 C (3 hr 30 min) without IPTG (column c), and TnpA
induction at 30 C (45 min) followed by a shift to 42 C without IPTG (30 min) and then at 30 C (3 hr) (column d).
(C) Effect of IS length on excision frequency. IS608 derivatives are 0.3, 0.5, 0.8, 1.1, 1.4, 1.9, 3, and 4 kb long. The curve shows results expressed as ApRTpRKmR /
TcRKmR with DH5a at 37 C. Error bars are the standard deviation (SD) of more than four independent experiments.
(D) Effect of dnaG on IS length-dependant excision. MC4100, 30 C (dark blue) and 33 C (yellow); MC4100dnaGts, 30 C (light blue); and MC4100dnaGts+dnaGwt,
33 C (purple).
(E) Effect of DnaGwt overexpression on excision frequency in dnaGts strains. MC4100dnaGts, 33 C (purple), and MC4100dnaGts+dnaGwt, 33 C (light blue).
(F) Effect of DnaGwt overexpression on excision frequency in wild-type strains. MC4100, 33 C (yellow), and MC4100+dnaGwt, 33 C (purple).
See also Figure S1B.

IS608 Insertion into the E. coli Chromosome lagging-strand template, they should occur in one orientation
The circular E. coli chromosome replicates bidirectionally from on one side of ori and in the opposite orientation on the other.
the replication origin, oriC. If IS608 insertions target the To test this, we isolated IS608 insertions in the E. coli

Cell 142, 398–408, August 6, 2010 ª2010 Elsevier Inc. 401


Figure 3. Insertions into the E. coli Chromosome and into a Resident
Plasmid
(A) Experimental system. The temperature-sensitive transposon donor
plasmid, pBS156 (middle), and the plasmid used for supplying TnpA,
pBS135 (left), are shown together with a schematic of the host chromosome
(right). Growth at the nonpermissive temperature results in loss of pBS156
and IS insertion into the chromosome or pBS135. This is described in detail
in Figure S1 and its legend.
(B) Chromosomal insertions. The chromosome is shown linearized at its
terminus. Red spot, origin of replication, ori; rrn, ribosomal RNA genes; black
arrows, positions of insertions (Table S1). Multiple insertions are indicated by
a number above the larger arrows. Replication occurs in both directions into
the left and right replicores, blue and orange, respectively: the lagging-strand
template of the left (bottom) and right (top) replicores. Fisher’s exact test gives
a p value of 7.122e6.
(C) Plasmid insertions. Replication of the p15A-based plasmid occurs from left
to right. Orange triangle, origin of replication; KmR, tnpA, and lacIQ, kanamycin
resistance, transposase and lac repressor genes, respectively. Arrows indi-
cate their direction of transcription. Vertical arrowheads show the position of
the insertions obtained from the same experiments as in (A) above.
See also Figure S1C and Table S1.
chromosome using a temperature-sensitive plasmid as the IS
donor and supplying TnpA in trans (Figure 3A). Insertions were
localized by an arbitrary PCR procedure (Experimental Proce-
dures) followed by DNA sequencing. We observed a dramatic
skew in strand specificity of IS608 insertion relative to the origin
of replication, oriC.
Insertions either in the left or right replicores were in the
orientation expected for transposition into the lagging-strand
template (Figure 3B). It is interesting to note that while insertions
were distributed around the chromosome, many appeared in the
vicinity of the highly transcribed rRNA genes (Table S1).
We also obtained insertions into the TnpA donor plasmid in the
same experiment. In contrast to the chromosome, this plasmid
replicates unidirectionally and the IS608 insertion pattern was
completely (Figure 3C) different. All occurred into only one
402 Cell 142, 398–408, August 6, 2010 ª2010 Elsevier Inc.
strand, the lagging-strand template, a result that has strong
statistical support (Figure 3 legend).
In all cases, both plasmid and chromosome insertions
occurred 30 to a TTAC target, as expected. As these are distrib-
uted equally on both strands of the chromosome and the TnpA
donor plasmid (data not shown), the observed strand biases
for insertion cannot be explained by a bias in target sequence
distribution.
Targeting Stalled Plasmid Replication Forks:
Tus-Ter System
Since our accumulating data suggested that IS608 targets
ssDNA at the replication fork, we asked whether insertion could
also be observed into forks stalled at a predefined location. For
this, we used the E. coli Tus/Ter system in which a Ter site, when
bound by the protein Tus, strongly reduces replication fork
progression through Ter in the nonpermissive orientation (Ternp)
(see Bierne et al., 1994) but not in the opposite, permissive,
orientation (Terp) (Neylon et al., 2005).
The assay (Figure 4A and Figure S1) used a suicide conjuga-
tion system (Demarre et al., 2005) in which the unidirectionally
replicating target plasmid was carried by a recipient strain with
an inactivated chromosomal tus gene. The target plasmid
carried a functional tus gene and a Ter site in either the permis-
sive or nonpermissive orientation (Figures 4B and Extended
Experimental Procedures). The plasmid-based tus gene was
transitorily induced only for the duration of the experiment to
avoid plasmid loss. We also inserted a stretch of DNA with
several spaced copies of the TTAC target tetranucleotide on
both strands upstream of the Ter sites.
Mapping of the IS insertion sites revealed that all occurred at
TTAC sequences and were distributed over the entire length of
both target plasmids, largely in an orientation expected for inser-
tion into the lagging-strand template (Figure 4B, black arrow-
heads). The overall distribution was the same regardless of Ter
site orientation. As for the other target plasmids used here, we
confirmed that TTAC sequences were distributed roughly
equally on both DNA strands (data not shown).
An additional set of insertions was observed in the target
plasmid carrying Ternp when compared to that with Terp (Figures
4B and 4C). To precisely map these, we used PCR analysis with
primers complementary to a sequence upstream of the Ter sites
and either LE (for lagging-strand insertions) or RE (for leading-
strand insertions). When the target plasmid carried Ternp, ampli-
fication products from four independent experiments (Figure 4C,
lanes 1–4) revealed insertions adjacent to the Ter site on the
lagging-strand template (Figure 4B, filled red arrowheads; Fig-
ure 4C), although occasionally an insertion was observed on
the opposite strand (lane 5). Although there was a degree of vari-
ability in the insertion distribution between experiments (com-
pare lanes 1–4), a consistently strong signal was obtained from
the TTAC site located at 63 bp from Ternp, suggesting that this
is the preferred insertion site. However, insertions were also
observed at the closest site, located only 26 bp from Ter
(Figure 4C, lanes 1, 3, and 4).
With Terp, no strong Tus-dependent insertions were observed
(Figure 4D). In four independent experiments we only once iso-
lated an insertion in this region (in the plasmid population from

Figure 4. IS608 Insertions into Tus/Ter Stalled Replication Forks
(A) Experimental design. The suicide conjugative IS-carrying donor plasmid,
pBS167b, is shown (top). It cannot propagate in the recipient, and IS insertions
were monitored into the Ter-carrying target plasmid, pBS172-3, as described
in detail in Figure S1 and its legend. Plasmids are described in the Extended
Experimental Procedures.
(B) Insertions with Ter in permissive and nonpermissive orientations. Replica-
tion from ori is from left to right; gray boxes, araC repressor, tus, and bla
genes; *, Ter-proximal TTAC sequences; horizontal red arrowhead, Ternp
(nonpermissive, pBS172); horizontal black arrowhead, Terp (permissive,
pBS173); black vertical arrowheads, IS608 insertions; red vertical arrowheads,
multiple insertions upstream of Ternp or an insertion inside of Terp. The position
of the oligonucleotides used to localize insertions is also shown.
(C) Detailed analysis of insertions in the Ternp region. PCR amplification prod-
ucts were separated on a 5% acrylamide gel. The distribution and orientation
of insertions close to Ter were determined in four independent experiments
(LE, lanes 1–4; RE, lanes 5–8). Distribution of TTAC on the lagging-strand
and leading-strand template are shown in the cartoon (left and right, respec-
tively).
(D) Detailed analysis of insertions in the Terp region. Identical experiment as
shown in (C) with Terp.
See also Figure S1D.
experiment 1, lane 1), and this had occurred into a TTAC site
within the Ter sequence itself rather than immediately upstream.
Thus, the lagging strand of replication forks blocked by Tus is
a preferential target for IS608 insertions and these can occur in
close proximity to the Tus binding site, Ter.
Another IS200/IS605 Family Member: ISDra2
ISDra2 is an IS200/IS605 family member from the highly radiation
resistant D. radiodurans. It has a similar organization to IS608
and inserts specifically 30 to a TTGAT pentanucleotide (Islam
et al., 2003). Like IS608, it transposes using ssDNA intermedi-
ates (Pasternak et al., 2010). We had observed specific stimula-
tion of ISDra2 transposition upon g- or UV-irradiation (Mennecier
et al., 2006; Pasternak et al., 2010) related to the availability of
ssDNA generated during radiation-induced fragmentation and
reassembly of the host genome, called extended synthesis
dependent strand annealing (ESDSA) (Zahradka et al., 2006).
To determine whether spontaneous ISDra2 transposition is
influenced by host chromosome replication, we measured
ISDra2 excision in D. radiodurans as a function of replication
direction through the IS. D. radiodurans replication is thought
to be bidirectional (Hendrickson and Lawrence, 2006). We con-
structed D. radiodurans strains with an ISDra2 derivative,
ISDra2-113 (Pasternak et al., 2010), placed in one or the other
orientation at the same chromosomal location (Extended Exper-
imental Procedures). Excision of these ISs reconstitutes a func-
tional TcR gene. Consistent with our results with IS608, no exci-
sion (<4.8 3 109; Figure 5A) was detected without transposase,
whereas in the presence of TnpAISDra2, relatively high excision
levels (2.1 3 103) were observed when the IS derivative was
in one orientation but was reduced about 30-fold to 6.7 3 105
in the other (Figure 5A).
However, the orientation bias of excision was lost in g-irradi-
ated D. radiodurans strains: after g-irradiation, excision of
ISDra2-113 reached the same and maximal level of about 2 3
102 for both orientations relative to the direction of replication
(Figure 5A). Since there should be no strand bias in generating
ssDNA after irradiation during ESDSA, the lack of excision and
insertion bias in irradiated D. radiodurans suggests that insertion
targets are no longer limited to those rendered accessible during
the replication.
Finally, we isolated 23 independent spontaneous insertions of
ISDra2-113 in D. radiodurans chromosome I by scoring for TcR
CmR colonies. All occurred into a TTGAT target and exhibited
an orientation bias (18/23) on the lagging-strand template (Fig-
ure 5B and Table S2) without irradiation. In contrast, 21 inser-
tions isolated from g-irradiated D. radiodurans occurred essen-
tially equally on both strands (Figure 5C and Table S3).
Genomic Analysis Reveals Orientation Bias
in Other IS200/IS605 Family Members
In view of the strong orientation bias observed for IS608 and
ISDra2 insertions, we wondered whether similar patterns might
be found in other bacterial genomes as vestiges of members
of the IS200/IS605 family occur widely (ISfinder: http://www-is.
biotoul.fr). We identified several genomes with a significant
number of copies of various members of this family and deter-
mined the orientation of insertion relative to the origin of
Cell 142, 398–408, August 6, 2010 ª2010 Elsevier Inc. 403

Figure 5. Excision and Insertion of ISDra2-113 in Deinococcus radio-
durans
(A) ISDra2-113 excision frequencies depend on whether the active (top) IS-
Dra2-113 strand is located on the lagging- or leading-strand template. The
middle column indicates whether TnpA was provided and whether the cells
were subjected to g-irradiation. Strain constructions are presented in the
Extended Experimental Procedures.
(B) Spontaneous ISDra2-113 insertions into the D. radiodurans chromosome I.
The origin (ori, red ellipse) and direction of replication of the D. radiodurans
chromosome was assumed to be that proposed by Hendrickson and Law-
rence (2006). All insertions occurred 30 to TTGAT pentanucleotide sequences.
Detailed mapping is presented in Table S2. Fisher’s exact test gave a p value of
0.01108. Multiple insertions are indicated by a number above the larger
arrows.
(C) ISDra2-113 insertions into the D. radiodurans chromosome I after g-irradi-
ation. Detailed mapping is presented in Table S3. Fisher’s exact test gave
a p value of 0.1344, suggesting that the pattern is random.
See also Tables S2 and S3.
replication (as defined by GC skew). The genomic ISs exhibited
a strong orientation bias, supporting our notion that the lagging-
strand template provides the most attractive ssDNA for IS
targeting.
The results for representative bacterial genomes are shown in
Figure 6. For example, S. enterica CT18 carries 27 full copies of
IS200 (Deng et al., 2003) (Figure 6A). Thirteen of these are on
one replicore and 14 on the other, while all but five occur in
the orientation expected for insertion into the lagging-strand
template. Another example is Yersinia pseudotuberculosis
(IP31758) (Chain et al., 2004) (Figure 6C) with 16 full IS1541
copies (nine in the left and seven in the right replicore), 13 of
these in an orientation consistent with insertion into the
lagging-strand template. Photobacterium profundum SS9 (Vezzi
et al., 2005; Figure 6D) carries 28 ISPpr13 copies distributed
between its two chromosomes, with 26 in the expected orienta-
tion, and in Shewanella woodyi with 13 copies of ISShwo2, all
except one are in the expected orientation (Karpinets et al.,
2010; Figure 6E). Fisher’s exact test provided strong statistical
support (Figure 6 legend).
Interestingly, the distribution of the 42 IS1541 copies in
Y. pestis (Microtus) (Figure 6F) and the 50 copies of it in Y. pestis
404 Cell 142, 398–408, August 6, 2010 ª2010 Elsevier Inc.
(Pestoides F) (Figure 6G) are clearly different and show a complex
pattern with no apparent relationship to the replication direction.
However, these Y. pestis strains revealed aberrant GC skews
resulting from a series of inversions and rearrangements
throughout the chromosome (Song et al., 2004; Garcia et al.,
2007). When these are taken into account, an astoundingly
close correlation emerges between GC skew and IS1541
orientation.
Similar reasoning may explain those insertions that initially
appear in contradiction with a lagging-strand template targeting
preference. For example, of the five IS200 insertions in S. enter-
ica strain CT18 oriented in the opposite direction (Figure 6A), one
is located in a short chromosome region with an inverted GC-
skew compared to the neighboring sequences. This implies
that this region has undergone inversion and that the associated
IS insertion likely had occurred prior to this event. The insertion
pattern observed in S. enterica (Ty2) (Deng et al., 2003;
Figure 6B) is quite similar to that of S. enterica CT18; both carry
an identical number of IS200 copies. The differences in IS200
distribution between these two strains can be entirely explained
by a large interreplicore inversion between S. enterica (Ty2) and
S. enterica CT18. These genomic results strongly support the
idea that insertion into the lagging-strand template of the chro-
mosomes of their host is a general characteristic of IS200/
IS608 family members.
DISCUSSION
The transposition pathway of IS200/IS605 IS family members
involves ssDNA substrates and intermediates. In vitro IS excision
requires both transposon ends to be single stranded, and inser-
tion of the excised single-stranded circular transposon interme-
diate requires access to an ssDNA target (Guynet et al., 2009).
We now demonstrate that excision and insertion of two family
members, IS608 and ISDra2, occur preferentially in the lagging-
strand DNA template in vivo.
Excision is dependent on replication direction through the IS: it
is high when the active IS strand is on the lagging-strand
template but substantially lower when part of the leading-strand
template. We also examined insertions of a plasmid-localized
IS608 into normally replicating E. coli chromosomes and sponta-
neous insertions of ISDra2 into D. radiodurans chromosome I.
They were largely directed to the lagging-strand template, result-
ing in a skew of strand-specific insertion on either side of the
replication origin in these bidirectionally replicating chromo-
somes. For a unidirectionally replicating E. coli plasmid, inser-
tions occurred into only one strand. Importantly, the orientation
effect for ISDra2 insertion was abolished when transposition
was triggered by g-irradiation, accompanied by an increase in
transposition frequency, consistent with the observation that
g-irradiation induces a repair pathway resulting in massive
amounts of ssDNA with no obvious strand bias.
IS608 insertions into the E. coli chromosome were fairly evenly
distributed but a significant number occurred in the highly tran-
scribed rrn genes (which are oriented in the sense of replication),
suggesting that high transcription levels might also provide
accessible ssDNA for IS608 insertion, e.g., by generating R loops
or by affecting replication fork passage. Replication in E. coli
Figure 6. Orientation of IS200/IS605 Family Members in Bacterial Genomes
Overall GC skew (G – C / G + C) is indicated in blue and orange. Black line, calculated GC skew with Artemis (http://www.sanger.ac.uk/Software/Artemis/) with
a 10 kb window; red ellipse, origin of replication; vertical arrow heads (above or below the genome depending on their orientation), individual ISs.
(A) S. enterica (typhi) CT18 (NC_003198); IS200, 709 bp (Fisher’s exact test p value, 0.002054).
(B) S. enterica (typhi) Ty2 (NC_004631); IS200, 709 bp (p value, 0.004799).
(C) Y. pseudotuberculosis IP31758 (NC_009708); IS1541, 708–709 bp (p value, 0.02144).
(D) P. profundum SS9 (NC_006371 and NC_006370); ISPr13, 596 bp (chromosome 1 p value, 0.0005139; chromosome 2 p value, 0.002806).
(E) S. woodyi (NC_010506); ISShwo2, 613 bp (p value, 0.02297).
(F) Y. pestis Microtus 91001 (NC_005810); IS1541, 711 bp (p value, 1.139e06).
(G) Y. pestis Pestoides F (NC_009381); IS1541D, 711 bp (p value, 2.479e06).
In (F) and (G), p values were calculated using the potential number of target sequences taking into account the regions of GC skew inversion.

has been estimated to be approximately 20-fold faster than is
transcription (800 nt/s versus 20 to 50 nt/s) (Kornberg and Baker,
1992). Replication forks may stall at transfer RNA and other
highly expressed genes, possibly because transcription
complexes collide ‘‘head on’’ with the forks. Replication forks
also stall upon codirectional encounters with RNA polymerase
(Elı́as-Arnanz and Salas, 1997; Mirkin et al., 2006), possibly as
a result of a trapped RNA polymerase not readily displaced
from DNA by fork progression.
That ssDNA at the replication fork facilitates IS608 transposi-
tion is reinforced by studies which perturb the fork using temper-
ature sensitive DnaG (primase) or DnaB (helicase) mutants.
DnaG inactivation prevents initiation of Okazaki fragment syn-
thesis, increasing the average length of ssDNA upstream of the
first complete Okazaki fragment on the lagging-strand template.
(Louarn, 1974; Fouser and Bird, 1983). DnaB inactivation results
in accumulation of large amounts of ssDNA mostly likely arising
from degradation of both the nascent DNA and leading-strand
template or from uncoupled leading-strand synthesis (Belle
et al., 2007). We observed a significant stimulation of IS608 exci-
sion after transitory inactivation of both dnaGts and dnaBts
mutants.
If excision occurs at the replication fork and requires ssDNA,
the probability that both ends are within the single-stranded
region of the lagging-strand template should decrease with
increasing IS length, and thus the size of the IS should influence
excision frequency exactly as we observe. Moreover, the effi-
ciency of excision was generally higher in the dnaGts mutant
even at the permissive growth temperature of 30 C and the slope
of the curve was less steep. At the sublethal temperature of
33 C, excision was even higher and the length dependence
even less marked. This is consistent with an increase in ssDNA
length of on the lagging-strand template resulting from a lower
Okazaki fragment initiation frequency in the dnaGts mutant
even at temperatures permissive for growth and suggests that
the slope is a function of the ssDNA length available on the
lagging-strand template.
We also investigated the effect of DnaG overproduction.
Expression of a cloned wild-type dnaG gene not only sup-
pressed the dnaGts phenotype but also resulted in an even

Cell 142, 398–408, August 6, 2010 ª2010 Elsevier Inc. 405

more pronounced length dependence in the wild-type back-
ground, suggesting that, as observed in vitro (Zechner et al.,
1992; Sanders et al., 2010), DnaG concentration controls the
frequency of initiation and Okazaki fragment size. More impor-
tantly, this result would suggest that DnaG is not saturating at
the normal replication fork in vivo.
While the slopes of the curves presumably reflect the length of
ssDNA on the lagging-strand template, the explanation for the
apparent inflection of the curves for the longer transposons is
less clear. It is possible that we are observing the combined
effects of two phenomena: for example the initial probability
that both ends are in a single-stranded form and the probability
that both ends find each other. Further analysis is required to
determine the exact reason behind this behavior.
Although, for simplicity, we describe the lagging-strand
template as single stranded at the fork, it is important to note
that it is not naked but protected by proteins such as single-
strand binding protein (Ssb) (Shereda et al., 2008). This implies
that the transposition machinery can access the DNA through
the protecting protein and raises the question of whether TnpA
can recognize Ssb or other components of the replisome. Exper-
iments to investigate this are in progress.
We also used the natural Tus/Ter replication fork termination
system (Neylon et al., 2005; Kaplan and Bastia, 2009) to examine
whether blocked forks might also attract IS608 insertions. The
Tus-Ter complex forms a transient barrier to the replicative heli-
case, DnaB, when a fork arrives in the nonpermissive direction
(Neylon et al., 2005). When provided with appropriate target tet-
ranucleotides, Tus-dependent IS608 insertions readily occurred
close (26–77 nt) to the Ter site on the lagging-strand template,
consistent with nucleotide resolution mapping of the terminated
nascent DNA in vitro and in vivo showing that the final lagging-
strand primer sites are arrested 50–70 nucleotides upstream of
Ter (Hill and Marians, 1990; Mohanty et al., 1998). While our
data are consistent with the idea that stalled forks favor IS608
insertion, we do not yet know whether the ssDNA substrates
are directly available at blocked forks or are generated during
their processing, e.g., during replication restart or repair of ds
breaks caused by replication arrest (Michel et al., 1997; Bierne
and Michel, 1994).
To determine whether other IS200/IS605 family members
might locate suitable ssDNA substrates for transposition, we
annotated several complete bacterial genomes for ISs and iden-
tified several that harbor multiple copies of these family
members. In the majority, these ISs showed a similar orientation
bias to IS608 and ISDra2 relative to replication direction. Thus,
targeting of the ssDNA available on the lagging-strand template
appears to be a general theme among IS200/IS605 family
members.
Replication direction and therefore identification of the lagging
strand is generally implied from GC skew, the preference for G
over C on the leading strand thought to be the result of differen-
tial repair (Lobry, 1996; Grigoriev, 1998). More strictly speaking,
we observed that IS orientation was correlated with the GC skew
of the region into which they were inserted rather than with repli-
cation direction per se, suggesting that the insertions predated
the genome rearrangements whose scars are revealed by
changes in GC skew. This has two important implications: either
406 Cell 142, 398–408, August 6, 2010 ª2010 Elsevier Inc.
that transposition is infrequent or that transposition events
become genetically fixed in the population.
IS200/IS605 family members are not alone in showing asym-
metric strand preference in insertion. Other transposable
elements such as Tn7 and IS903 also appear to do so (Peters
and Craig, 2001; Hu and Derbyshire, 1998). Tn7 is targeted to
replication forks during conjugative plasmid transfer and inserts
in a specific orientation. The transposon-encoded TnsE protein
is instrumental in targeting the transpososome to the junction
between single- and double-stranded DNA by interaction with
the b clamp component of the replication apparatus (Parks
et al., 2009; Chandler, 2009). Insertion of Tn7 into a replication
fork likely occurs within the dsDNA covered by an Okazaki frag-
ment. IS903 insertion bias in the conjugative F plasmid might
also reflect targeting to the conjugative replication fork. It is
worthwhile noting that IS10 and IS50 have also exploited host
replication: both are activated by transient formation of hemime-
thylated DNA after fork passage (Roberts et al., 1985; Yin et al.,
1988; Dodson and Berg, 1989).
However, there are major mechanistic differences between
Tn7, IS903, and members of the IS200/IS605 family, suggesting
that different pathways are at play. Perhaps most importantly,
Tn7 transposes using a dsDNA intermediate in contrast to the
ssDNA species used by IS608 and ISDra2. For Tn7 and many
other transposons with dsDNA intermediates, strand transfer
into a dsDNA target occurs using the 30 OH groups on each com-
plementary strand at each end. Insertion of the first strand into
a single-strand target would leave a fatal break. Thus, whereas
an overarching theme in DNA transposition may be the exploita-
tion of replication forks, different elements do so in different ways.
The unique excision and insertion properties of the IS200/
IS605 family may make them useful tools for probing ssDNA
structures in vivo. The relationship between excision and IS
length might be used to determine the effect of various factors
on the state of the replication fork in vivo. For example, treat-
ments leading to reduced fork velocity, Okazaki fragment
synthesis, uncoupling of lagging from leading-strand synthesis,
or simply forks blocked by different factors could all be probed
using an excision assay as an experimental readout. We are
aware that the topology of the replication fork of small, multicopy
plasmids may differ in some respects from that of the chromo-
some, and this system may also be used to explore these
possible differences. We are at present testing these possibilities
experimentally.
EXPERIMENTAL PROCEDURES
Bacterial Strains and Media
Bacterial strains are described in the Extended Experimental Procedures; see
also Figure S2 and Table S4. E. coli cultures were grown in Luria broth (LB) sup-
plemented where necessary with chloramphenicol (Cm, 10 or 30 mg/ml), kana-
mycin (Km, 20 mg/ml), ampicillin (Ap, 100 mg/ml), gentamycin (Gm, 5 mg/ml),
spectinomycin (Sp, 30 mg/ml) and streptomycin (Sm, 20 mg/ml), tetracycline
(Tc, 15 mg/ml), or 2,6-Diaminoheplanediole acid (DAP, 0.006%). D. radiodurans
media and growth conditions were as described (Pasternak et al., 2010; Bona-
cossa de Almeida et al., 2002).
Plasmids
Details of plasmid constructions are available upon request; see Figure S3,
Table S4, and the Extended Experimental Procedures for schematics of
plasmids used and additional information. The dnaG-carrying plasmid pBS179
was constructed by insertion of a DNA fragment carrying the wild-type dnaG
allele with its natural ribosome binding site isolated by PCR with flanking
oligonucleotides, dnaGN, and dnaGC into the SphI site of transposase
supplying plasmid downstream of the tnpA gene, placing both genes under
control of plac. The clone was verified by sequencing and complements the
dnaGts allele.
Excision Assay with pAM1 Derivatives
Effect of dnaG308 and dnaB8 on Excision
E. coli MG4100 wild-type and dnaG308 and dnaB8 mutant strains were grown
overnight at 30 C, the permissive temperature, in LB+KmTc, and cultures were
diluted at 30 C and grown with 0.5 mM IPTG for 4 hr or 45 min. IPTG was
removed by centrifugation and cells were resuspended in prewarmed medium
at 42 C for 30 min and incubated at 30 C for 3 hr. As a control, the 42 C step
was omitted. Cells were harvested and dilutions plated on LA+KmTc and
LA+KmTcAp.
Effect of Transposon Length
E. coli DH5a harboring pBS135 and pAM1-derivative plasmids was grown
overnight at 37 C in LB+KmTc. Cultures were diluted in fresh medium at
37 C with KmTc and 0.5 mM IPTG to induce TnpA expression. Cells were
harvested after 5 hr and dilutions plated on LA+KmTc and LA+KmTcAp.
Effect of Transposon Length in the dnaG308 Mutant Strain
Overnight cultures of MC4100 and MC4100 dnaGts were grown at 30 C and
diluted in fresh medium at 30 C or 33 C containing KmTc and 0.5 mM IPTG.
Cells were harvested after 5 hr and dilutions plated on LA+KmTc and
LA+KmTcAp at 30 C.
Tus/Ter Experiments
Overnight donor and recipient strains were grown in LB+DAP+SpSmCm
and LB+GmKmAp, respectively. Glucose (0.5%) was added to fully repress
the para used to drive Tus expression. Cultures were diluted in fresh LB medium
without antibiotics (with DAP for donor cells). At an OD600 of 0.5, donor
cells were incubated without agitation, the recipient was diluted to an OD600
of 0.15, and TnpA was induced with 0.5 mM IPTG. After 60 min, Tus
was induced for 30 min by addition of 0.08% arabinose. Donor and recipient
strains were mixed, incubated for 2 hr and plated on Cm-containing LA plates
supplemented with 0.5% glucose. Bulk plasmid DNA was isolated and the
distribution of insertions in the population was analyzed by PCR with
six sets of primers: B248, B249, tus as forward primers, and LE and RE
as reverse primers (Extended Experimental Procedures) to determine the
insertion orientation. PCR amplification used Phusion DNA Polymerase
(Finnzyme) in GC buffer as follows: 30 s 98 C, 35 cycles of 10 s 98 C, 30 s
64 C, and 1 min 72 C. Fifty nanograms of bulk plasmid DNA were used for
each reaction.
Sequencing IS608 Insertions in the E.coli Chromosome
IS608 insertions into the E. coli chromosome and coresident plasmid pBS135
were sequenced by arbitrary PCR as described (Guynet et al., 2009).
Measurement of In Vivo Spontaneous and g-Induced Excision
Frequencies of ISDra2-113
Excision frequencies were determined with individual CmR TcS colonies puri-
fied from strains GY14302 or GY14303 (Extended Experimental Procedures)
grown with or without TnpA expression in trans as described (Pasternak
et al., 2010). The mean and standard deviations were calculated from six inde-
pendent experiments. ISDra2 insertion sites were mapped by arbitrary-primed
(AP)-PCR with DyNazyme EXT DNA polymerase (Finnzymes) as described
(Extended Experimental Procedures).
Statistical Analysis of Insertion Orientation
This was performed with Fisher’s exact test with the ‘‘R’’ statistical package
(http://www.r-project.org/) taking into account the number of potential target
sites on the leading and lagging strands. For the Yersinia species, the number
of potential target sites and of insertion sites was calculated taking into
account GC skew inversion.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, three
figures, and four tables and can be found with this article online at doi:10.1016/
j.cell.2010.06.034.
ACKNOWLEDGMENTS
We would like to thank members of the ‘‘Mobile Genetic Elements’’ group,
A. Bailone, P. Polard, and D. Lane for discussions, G. Coste and B. Marty
for expert technical assistance, L. Lavatine for guiding us through the
mysteries of statistics, A. Varani for identifying a representative oligonucleotide
sequence used in the PCR mapping for the D. radiodurans genome, and the
Institut Curie for the use of the 137Cs irradiation system. This work was sup-
ported by: intramural funding from the Centre National de Recherche Scienti-
fique (France), in its later stages by ANR grant Mobigen (M.C. and S.S.), Euro-
pean contract LSHM-CT-2005-019023 (M.C.), and the Commissariat à
l’Energie Atomique and Electricité de France (France; S.S.). At the National
Institutes of Health, this work was supported by the Intramural Program of
the National Institute of Diabetes and Digestive and Kidney Diseases.
Received: December 23, 2009
Revised: April 3, 2010
Accepted: May 17, 2010
Published: August 5, 2010
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A Large Intergenic Noncoding RNA
Induced by p53 Mediates Global Gene
Repression in the p53 Response
Maite Huarte,1,2,* Mitchell Guttman,1,3 David Feldser,3,4 Manuel Garber,1 Magdalena J. Koziol,1,2
Daniela Kenzelmann-Broz,5,6 Ahmad M. Khalil,1,2 Or Zuk,1 Ido Amit,1 Michal Rabani,1 Laura D. Attardi,5,6 Aviv Regev,1,3
Eric S. Lander,1,3,7 Tyler Jacks,3,4 and John L. Rinn1,2,*
1The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
2Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
3Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
4The Koch Institute for Integrative Cancer Research, Cambridge, MA 02139, USA
5Department of Radiation Oncology
6Department of and Genetics

Stanford University School of Medicine, Stanford, CA 94305, USA

7Department of Systems Biology, Harvard Medical School, Boston, MA 02114, USA

*Correspondence: mhuarte@broadinstitute.org (M.H.), jrinn@broadinstitute.org (J.L.R.)

DOI 10.1016/j.cell.2010.06.040

SUMMARY 4 trimethylated (H3K4me3) promoter region and histone 3-lysine

Recently, more than 1000 large intergenic noncoding
RNAs (lincRNAs) have been reported. These RNAs
are evolutionarily conserved in mammalian genomes
and thus presumably function in diverse biologi-
cal processes. Here, we report the identification of
lincRNAs that are regulated by p53. One of these
lincRNAs (lincRNA-p21) serves as a repressor in
p53-dependent transcriptional responses. Inhibition
of lincRNA-p21 affects the expression of hundreds
of gene targets enriched for genes normally re-
pressed by p53. The observed transcriptional repres-
sion by lincRNA-p21 is mediated through the phys-
ical association with hnRNP-K. This interaction is
required for proper genomic localization of hnRNP-
K at repressed genes and regulation of p53 mediates
apoptosis. We propose a model whereby transcrip-
tion factors activate lincRNAs that serve as key
repressors by physically associating with repressive
complexes and modulate their localization to sets of
previously active genes.
INTRODUCTION
It has become increasingly clear that mammalian genomes
encode numerous large noncoding RNAs (Mercer et al., 2009;
Ponting et al., 2009; Mattick, 2009; Ponjavic et al., 2007). It
has been recently reported the identification of more than 1000
large intergenic noncoding RNAs (lincRNAs) in the mouse
genome (Carninci, 2008; Guttman et al., 2009). The approach
to identify lincRNAs was by searching for a chromatin signature
of actively transcribed genes, consisting of a histone 3-lysine
36 trimethylation (H3K36me3) corresponding to the elongated
transcript (Guttman et al., 2009). These lincRNAs show clear
evolutionary conservation, implying that they are functional
(Guttman et al., 2009; Ponjavic et al., 2007).
In an attempt to understand the potential biological roles of
lincRNAs, a method to infer putative function based on correla-
tion in expression between lincRNAs and protein-coding genes
was developed. These studies led to preliminary hypotheses
about the involvement of lincRNAs in diverse biological pro-
cesses, from stem cell pluripotency to cell-cycle regulation
(Guttman et al., 2009). In particular, we observed a group of
lincRNAs that are strongly associated with the p53 transcrip-
tional pathway. p53 is an important tumor suppressor gene
involved in maintaining genomic integrity (Vazquez et al., 2008).
In response to DNA damage, p53 becomes stabilized and trig-
gers a transcriptional response that causes either cell arrest or
apoptosis (Riley et al., 2008).
The p53 transcriptional response involves both activation and
repression of numerous genes. While p53 is known to transcrip-
tionally activate numerous genes, the mechanisms by which p53
leads to gene repression have remained elusive. We recently
reported evidence that many lincRNAs are physically associated
with repressive chromatin modifying complexes and suggested
that they may serve as repressors in transcriptional regulatory
networks (Khalil et al., 2009). We therefore hypothesized that
p53 may repress genes in part by directly activating lincRNAs,
which in turn regulate downstream transcriptional repression.
Here, we show that lincRNAs play a key regulatory role in the
p53 transcriptional response. By exploiting multiple independent
cell-based systems, we identify lincRNAs that are transcriptional
targets of p53. Moreover, we find that one of these p53-activated
lincRNAs—termed lincRNA-p21—serves as a transcriptional
repressor in the p53 pathway and plays a role in triggering apo-
ptosis. We further demonstrate that lincRNA-p21 binds to

Cell 142, 409–419, August 6, 2010 ª2010 Elsevier Inc. 409

A +/- Cre Doxorubicin
Figure 1. Several LincRNAs Are p53 Tran-
scriptional Targets
(A) Experiment layout to monitor p53-dependent
LSL/LSL
p53 mRNAs transcription. p53-restored (+Cre) or not restored
MEFs
0 3 6 9 (
Cre) p53LSL/LSL MEFs were treated with
DNA damage time (hours)
500 nM dox for 0, 3, 6, and 9 hr (upper left).
+ Cre KRAS (p53LSL/LSL) tumor cells were treated with
LSL/LSL
hydroxytamoxifen for p53 restoration for 0, 8, 16,
KRAS p53 LincRNAs
24, 40, or 48 hr (lower left). RNA was subjected
Lung Tumor 0 8 16 24 40 48
time (hours)
microarray
to microarray analysis of mRNAs and lincRNAs.
p53 restoration profiling (B and C) Venn diagrams showing the number of
shared and distinct mRNAs (B) or lincRNAs (C)
B C induced in a p53-dependent manner in the MEF
MEF MEF or KRAS systems.
MEF & KRAS MEF & KRAS
645 KRAS 995 38 KRAS 32 (D and E) mRNAs (D) and lincRNAs (E) activated by
289 11 p53 induction (FDR < 0.05) in MEF or KRAS
system. Colors represent transcripts above (red)
D E or below (blue) the global median scaled to
0h 3h 6h 9h 0h 8h 16h 24h 40h48h 0h 3h 6h 9h 0h 8h 16h 24h 40h48h
8-fold activation or repression, respectively.
(F) Promoter region, conserved p53 binding
motif, promoter orientation, and p53-dependent
645 mRNAs

995 mRNAs

32 lincRNAs
38 lincRNAs

fold induction in reporter assays of lincRNA

promoters induced in a p53-dependent manner
(values are average of at least three biological
replicates [±STD]; p values are determined by
t test).
(G) p53-dependent induction of lincRNA pro-
-8 +8 -8 +8 moters requires the consensus p53 binding ele-
ments. Relative firefly luciferase expression
driven by promoters with p53 consensus motif
F induction (lincRNA-p21, lincRNA-Mkln1) or with deleted
Name H3K4me3 sequence strand fold induction P value
motif (DlincRNA-p21 and DlincRNA-Mkln1) in
lincRNA-Mkln1 chr6:31061148-31061169 GAACATGCCTGGCACTTGCAAA - 13.5 +/-1.6 5.9E-06
p53-restored p53LSL/LSL (p53+/+) or p53LSL/LSL
lincRNA-p21 chr17:29214831-29220756 GGCCTTGCCCGGGCTTGCCT - 24.6 +/-2.3 1.3E-05
cdkn1a chr17:28818329-28822000 CTGGGCATGTCTGGGCAGCGATC + 16.8 +/-1.2 3.7E-05 (p53
/
) cells. Values are relative to p53
/
and
normalized by renilla levels (average of three repli-
cates ±STD).
G H (H) p53 specifically binds to p53 motifs in lincRNA
147 promoters. p53 ChIP enrichment in p53+/+ and
(Normalized Firefly Luciferase Activity)

30
117 p53
/
MEFs on regions with p53 motifs (lincRNA-
Relative Reporter Induction

25 35 p21, lincRNA-Mkln1, Cdkn1a) or two irrelevant

Relative Fold Enrichment

+/+ +/+
p53 30 p53
20 -/- -/- regions (controls). Enrichment values are relative
(p53 ChIP/IgG)

p53 p53
25
15 20
to IgG and average of 3 replicates (±STD).
15 See also Figure S1 and Table S1.
10
10
5
5
0 0
l
21

n1

a
21

n1

n1

l-1

l-2
ro
n1

n1
p2
nt

ro

ro
kl
p

kl

kl
dk

dk
A-

A-

co

A-

nt

nt
M

M
C

co

co
A-

A-
N

A-
N
cR

cR

cR
N

N
cR

cR

cR
lin

in

lin
Δl

lin

in

lin
Δl

hnRNP-K. This interaction is required for proper localization of experimental systems that allow us to monitor gene expression
hnRNP-K and transcriptional repression of p53-regulated genes. changes at different times after p53 induction (Ventura et al., 2007).
Together, these results reveal insights into the p53 transcrip- The first system uses mouse embryonic fibroblasts (MEFs)
tional response and lead us to propose that lincRNAs may serve derived from mice where the endogenous p53 locus is inacti-
as key regulatory hubs in transcriptional pathways. vated by insertion of a transcriptional termination site flanked
by loxP sites (LSL) in the first intron. This endogenous p53 locus
RESULTS (p53 LSL/LSL) is restorable by removal of the stop element by Cre
recombination (Ventura et al., 2007). The p53 LSL/LSL MEFs were
Numerous LincRNAs Are Activated treated with AdenoCre virus expressing the Cre recombinase to
in a p53-Dependent Manner reconstitute the normal p53 allele or AdenoGFP control virus to
As a first attempt to dissect the functional mechanisms of maintain the inactive p53 LSL/LSL allele. Then we compared the
lincRNAs, we focused on a strong association in the expression transcriptional response between the p53-reconstituted and
patterns of certain lincRNAs and genes in the p53 pathway p53 LSL/LSL MEFs after 0, 3, 6, and 9 hr of DNA damage treatment
(Guttman et al., 2009). In order to determine whether these with doxorubicin (we will refer to this system as ‘‘MEFs’’)
lincRNAs are regulated by p53, we employed two independent (Figure 1A). The second system uses a lung tumor cell line

410 Cell 142, 409–419, August 6, 2010 ª2010 Elsevier Inc.

derived from mice expressing an oncogenic K-Ras mutation
(K-RasG12D) and a restorable p53 knockout allele (p53 LSL/LSL),
similar to that described above (D.F. and T.J., unpublished data).
We compared the transcriptional response at different times
(0, 8, 16, 24, 40, and 48 hr) after restoration of p53 expression
by Cre recombination (Experimental Procedures) (we will refer
to this system as ‘‘KRAS’’) (Figure 1A).
To assess the transcriptional responses in each of these
systems, we isolated total RNA at each time point before
and after p53 restoration and performed DNA microarray anal-
ysis to monitor protein-coding gene expression levels. In the
MEF system and KRAS systems, we identified a total of 1067
(645 activated, 422 repressed) and 1955 (995 activated, 960
repressed) genes, respectively, that were regulated in a p53-
dependent manner (false discovery rate [FDR] < 0.05) (Figures
1B and 1D and Table S1, parts A and B, available online). The
sets of p53-induced genes identified in the two systems showed
significant (p < 10
8) overlap, including such canonical p53
targets as Cdnk1a, Mdm2, Perp, and Fas (Table S1, parts A
and B). There are also a number of p53-induced genes unique
to each system, likely reflecting specific properties of each
cell-type (Levine et al., 2006) (Figure S1C and Table S1, parts
A and B).
Functional analysis of the classes of genes that are enriched
among the genes regulated by p53 in both the MEF and KRAS
systems showed strong enrichment for known p53-regulated
processes, such as the cell cycle and apoptosis (Figures S1A
and S1B). Moreover, gene set enrichment analysis (GSEA) (Sub-
ramanian et al., 2005) of previously published microarray anal-
yses revealed a significant overlap with the p53 regulated genes
identified here (Table S1, parts H and I). Together these results
demonstrate that these two systems are largely reflective of
canonical p53 transcriptional responses.
We next examined lincRNAs regulated by p53 in these two
systems across the same time course, by analogously using
a custom tiling microarray representing 400 lincRNAs and
analyzing the data with previously described statistical methods
(Guttman et al., 2009) (Experimental Procedures). We found 38
and 32 lincRNAs induced by p53 in the MEF and KRAS systems,
respectively (Figures 1C and 1E and Table S1, parts C–G). Inter-
estingly, 11 lincRNAs were induced by p53 in both model
systems (Figure 1C and Table S1, part C), many more than
expected by chance (p < 10
6). These results confirm that, in
a manner similar to canonical p53 protein coding gene targets,
numerous lincRNAs are temporally regulated by p53.
LincRNAs Are Direct Transcriptional Targets of p53
We sought to identify lincRNAs that might be canonical p53
target genes. As a first approach, we examined the promoters
of p53-induced lincRNA for enrichment of evolutionarily con-
served p53-binding motifs (Garber et al., 2009) (Extended
Experimental Procedures). The promoters of the p53-induced
lincRNAs were highly enriched for conserved p53 motifs relative
to the promoters of all lincRNAs (p < 0.01). We selected two
lincRNAs whose promoter regions contain highly conserved
canonical p53-binding motifs (el-Deiry et al., 1992; Funk et al.,
1992); we termed these lincRNA-p21 and lincRNA-Mkln1 (with
the names referring to the neighboring gene). We next performed
transcriptional reporter assays for these lincRNAs. Specifically,
we cloned their promoters (as defined by the H3K4me3 peaks
[Guttman et al., 2009]) into a luciferase reporter vector (Experi-
mental Procedures) and transfected the constructs along with
a vector to normalize transfection efficiency. Both promoters
showed significant induction of firefly luciferase in p53-wild-
type but not in p53 null cells (p < 0.01) (Figures 1F and 1G).
To determine whether the canonical p53-binding motif is
required for the observed transactivation, we repeated these
experiments in the absence of the p53-binding motif. Mutant
promoters resulted in the abolition of the observed transactiva-
tion for both lincRNA-p21 and lincRNA-Mkln1 in p53+/+ cells
(Figure 1F). Finally, we performed chromatin immunoprecipita-
tion (ChIP) experiments to determine whether p53 directly binds
to the sites containing the consensus motifs in vivo. Indeed, p53
is bound to the site containing the consensus motif in the
promoters of both lincRNA-p21 and lincRNA-Mkln1 in p53+/+
but not p53
/
MEFs treated with doxorubicin, and it is not
enriched at negative control sites of irrelevant regions (Figure 1H
and Extended Experimental Procedures). Together, these
results demonstrate that lincRNA-p21 and lincRNA-Mkln1 are
bona fide p53 transcriptional targets.
LincRNA-p21 Is Induced by p53 in Different Cell Systems
We were intrigued by the p53 transcriptional target lincRNA-p21,
which resides 15 Kb upstream of the gene encoding the critical
cell-cycle regulator Cdkn1a (also known as p21), a canonical
transcriptional target of p53 (Riley et al., 2008) (Figures 2A–2C,
Figure S2A, and Table S1, parts C–E). Given the proximity of
lincRNA-p21 to the Cdkn1a gene, we sought to ensure that the
lincRNA transcript is distinct from that of the Cdkn1a gene. To
this end, we cloned the full-length transcriptional unit of lincRNA-
p21 using the 50 and 30 RACE method (Schaefer, 1995); the
transcript contains two exons comprising 3.1 Kb (Figure 2B).
In support of lincRNA-p21 being an independent transcript,
lincRNA-p21 is transcribed in the opposite orientation from the
Cdkn1a gene. Furthermore, the analysis of chromatin structure
in mouse embryonic stem cells (mESCs) (Mikkelsen et al., 2007)
indicates that these are distinct genes with distinct promoters
(Figure S2A).
We next examined the transcriptional regulation of lincRNA-
p21 in two additional cancer-derived cell lines. Specifically, we
irradiated p53LSL/LSL mice to induce lymphomas and sarcomas
and then restored p53 expression in tumor-derived cells (Exper-
imental Procedures) (Ventura et al., 2007). In cells derived in both
tumor types, lincRNA-p21 was strongly induced after p53 resto-
ration. Moreover, the induction of lincRNA-p21 followed similar
kinetics as those of p53 and Cdnk1a, consistent with lincRNA-
p21 being a primary transcriptional target of p53 (Figure 2D
and Figure S2B).
We further investigated the orthologous lincRNA-p21 locus in
the human genome. We first mapped the promoter (H3K4me3
domain) of lincRNA-p21 to human genome. Interestingly, this
region corresponds to one of four intergenic p53-binding sites
identified from a study by Wei et al. (2006) (Figure 2B) performing
p53 ChIP followed by sequencing. Next, we mapped the
lincRNA-p21 exonic structures to the human genome to deter-
mine whether this region is expressed and induced by DNA
Cell 142, 409–419, August 6, 2010 ª2010 Elsevier Inc. 411
A 75 kb Figure 2. LincRNA-p21: A p53 Target Gene
Induced in Different Tumor Models
(A) Schematic representation of the chromosomal
Chr17 Sfrs3 lincRNA-p21 Cdkn1a location of the lincRNA-p21 gene locus. Arrow-
heads indicate the orientation of transcription.
(B) Promoter and transcript structure of lincRNA-
B C p21 gene locus. Chromatin structure at the
lincRNA-p21 locus is shown as mESC ChIP-Seq
p53 motif

GA G A G C TG
A C T C C GGGC A TC 6
human lincRNA-p21 data (Mikkelsen et al., 2007); for each histone
modification (green, H3K4me3; blue, H3K36me3),

Relative RNA level

G G C

A C
G
A C G
T C
T T T G A A
T T
T C
A A G G T
G T A C G G
CT C
T
5
T C
T T
G
T A A A C
T
C T C A A A

PET overlap ChIP-seq results are plotted as number of DNA

4 Untreated
density
Dox
fragments obtained at each position relative to
* 153 bp
500 bp 3
the genomic average. Red stars indicate the posi-
PETS 2
tion of the p53-binding motif. The promoter region
1
where p53 ChIP-PET fragments (black segments)
0
RT-PCR -RT map is enlarged (Wei et al., 2006). PET overlap

H3K4Me3
[ *
density (gray) and p53 motif sequence are shown.
The structure of the full-length lincRNA-p21 is rep-
resented with red boxes as exons and arrowed

H3K36Me3
[ lines as the intronic sequence.
(C) Human lincRNA-p21 is induced by DNA
damage. Relative RNA levels of human lincRNA-

RNA
[ lincRNA-p21
3073 bp
p21 determined by qRT-PCR (RT-PCR) or qPCR
(
RT) from untreated human fibroblasts or 500 nM
DOX-treated for 14 hr. PCR primers map on the
human region orthologus to the first exon of the
mouse gene.
D LUNG TUMOR SARCOMA LYMPHOMA (D) LincRNA-p21 is induced by p53 in different
Relative RNA level

tumor cell lines. LincRNA-p21, p53, and Cdkn1a

Relative RNA level

Relative RNA level

2 1.2 1.2
1.7 1 1 relative RNA levels at different times after p53
1.3 0.8 0.8 lincRNA-p21
1 0.6 0.6 Cdkn1a restoration.
p53
0.7 0.4 0.4 Values in (C) and (D) are the median of four tech-
0.3 0.2 0.2
nical replicates (±STD).
0 0 0
0 8 16 24 40 48 0 12 24 36 48 0 12 24 36 48 72 See also Figure S2.
time after p53 restoration time after p53 restoration time after p53 restoration
(hours) (hours) (hours)

damage in human fibroblasts. Indeed, qRT-PCR showed that the pool was effective at knocking down its intended target genes
orthologous 50 exon region (adjacent to observed p53 ChIP in p53LSL/LSL restored MEFs (Figures 3A and 3B). We then
binding site by Wei et al.) of human lincRNA-p21 is expressed used microarray analysis to examine the broader transcriptional
and strongly induced in human fibroblasts upon DNA damage consequences of knockdown of p53 and lincRNA-p21. We iden-
(Figure 2C and Extended Experimental Procedures). tified 1520 and 1370 genes that change upon knockdown of p53
Collectively, these results provide evidence that both the and lincRNA-p21, respectively (relative to nontargeting control
human and mouse lincRNA-p21 promoters are bound by p53 siRNA, FDR < 0.05). We observed a remarkable overlap of
resulting in transcriptional activation in response to DNA 930 genes in both the lincRNA-p21 and p53 knockdowns, vastly
damage. Moreover, lincRNA-p21 is induced by p53 in diverse more than would be expected by chance (p < 10
200) (Figure 3C,
biological contexts, including multiple different tumor types Figure S3A, and Table S2). Strikingly, 80% (745/930) of the
(Figure 2D and Figure S2B), suggesting that lincRNA-p21 plays common genes are derepressed in response to both p53 and
a role in the p53 pathway. lincRNA-p21 knockdown, much higher proportion than ex-
pected by chance (p < 10
10) (Figure 3C and Table S2) when
LincRNA-p21 as a Repressor in the p53 Pathway compared to all genes affected by the p53 knockdown (Fig-
We next investigated the consequence of the loss of lincRNA- ure S3A). This observation suggests that lincRNA-p21 partici-
p21 function in the context of the p53 response. We reasoned pates in downstream p53 dependent transcriptional repression.
that, if lincRNA-p21 plays a role in carrying out the p53 transcrip- To demonstrate that the observed derepression upon
tional response, then inhibition of lincRNA-p21 would show lincRNA-p21 knockdown is indeed p53 dependent and is not
effects that overlap with inhibition of p53 itself. To test this due to off target effects of the RNAi-mediated knockdown, we
hypothesis, we performed RNA interference (RNAi)-mediated performed several additional experiments and analyses. First,
knockdown of lincRNA-p21 and p53 separately and monitored we repeated the knockdown experiments with four individual
the resulting changes in mRNA levels by DNA microarray siRNAs targeting lincRNA-p21, transfected separately rather
analysis. than in a pool and confirmed the derepression effect on select
Toward this end, we first designed a pool of small interfering target genes (Figure S3F and Table S2). Second, we confirmed
RNA (siRNA) duplexes targeting lincRNA-p21, a pool targeting that the same genes that were derepressed in the lincRNA-p21
p53 or nontargeting control sequences. We validated that each and p53 knockdown experiments correspond to genes that are

412 Cell 142, 409–419, August 6, 2010 ª2010 Elsevier Inc.

A B C Figure 3. LincRNA-p21 Is a Global
Repressor of Genes in the p53 Pathway
1.2 siRNA (A) RNAi-mediated knockdown of lincRNA-p21
Relative RNA level

1
p53 l NA and p53. Relative RNA levels determined by
0.8 lincRNA-p21 tro cR 1 p53
on lin -p2 53 p53 lincRNA
0.6 c p 1520
&
lincRNA-p21 -p21 qRT-PCR in p53-reconstitued p53LSL/LSL MEFs
0.4 p53 930 1370
transfected with the indicated siRNAs and treated
0.2
βActin with DOX (median of four technical replicates
0
siRNA siRNA siRNA
control lincRNA-p21 p53
±STD).
(B) p53 protein levels after lincRNA-p21 and p53
knockdown from cells treated as in (A). bActin
D levels are shown as loading control.
MEF KRAS
(C) Many genes are corepressed by lincRNA-p21
0.4
* 0.4 and p53. Top: Venn diagram of differentially ex-
*
Enrichment Score

Enrichment Score

0.2 0.2 80% pressed genes (FDR < 0.05) upon p53 knockdown
0.0 0 (left) or lincRNA-p21 knockdown (right); cells were
-0.2
treated as in (A) and subjected to microarray anal-
-0.2
Repressed Induced Repressed Induced ysis. Bottom: expression level of genes in lincRNA-
-0.4 by p53 by p53 -0.4
by p53 by p53
20%
p21 and p53 siRNA-treated cells relative to control
* FDR<0.001 * FDR<0.002 siRNA experiments. Expression values are dis-
siRNA siRNA siRNA P< 10 -10
control p53 lincRNA-p21
played in shades of red or blue relative to the
global median expression value across all experi-
-8 +8
ments (linear scale).
E p53 (D) Genes derepressed by lincRNA-p21 and p53
knockdown overlap with the genes repressed by
p53 restoration in the MEF and KRAS systems.
The black line represents the observed enrichment
LinRNA-p21
score profile of genes in the lincRNA-p21/p53
derepressed gene set to the MEF or KRAS gene
sets, respectively.
Cabc1 Fas Bax Phka2 Noxa Perp Stat3 Atf2 Bcl2l3 G2e3 Vcan Mtap4 Mapk3 CyclinD2 Cyclin G Cdk4 Reprimo Mdm2 Cdkn1a Cdkn1b Cdkn2a Wt1 Brca1
(E) Genes corregulated by lincRNA-p21 and p53
APOPTOSIS CELL CYCLE ARREST
are part of the p53 biological response. Examples
of genes affected by lincRNA-p21 and/or p53
siRNA-knockdown (FDR < 0.05). Downregulated
and upregulated genes are indicated with blue
arrows and red lines respectively.
See also Figure S3 and Table S2.

normally repressed upon p53 induction in both the KRAS and increase in viability after DNA damage of cells treated with
MEF systems, in the absence of RNAi treatment (GSEA FDR < siRNAs targeting either lincRNA-p21 or p53 compared to those
0.002) (Figure 3D). Third, we demonstrated that enforced treated with the control siRNA pool (Figures 4A and 4B). The
expression of lincRNA-p21 (Experimental Procedures) also per- increase in viability was greater for knockdown of p53, but was
turbed the expression of genes that are normally regulated by still highly significant for knockdown of lincRNA-p21 (p < 0.01).
p53 in both the KRAS and MEF systems (GSEA FDR < 0.01) We observed similar results when using three individual siRNA
(Figure S3H). Finally, we repeated the siRNA experiments in duplexes targeting lincRNA-p21, as well as two different control
the absence of p53 (dox/-AdCre) and demonstrated that dere- siRNA pools (Figure 4B and Figures S4A–S4C). These results
pression of these genes did not occur upon siRNA-mediated suggest that lincRNA-p21 plays a physiological role in regulating
knockdown in the absence of p53 (Figure S3I). Collectively, cell viability upon DNA damage in this system, although they do
these results indicate that lincRNA-p21 acts to repress many not discriminate whether the effect is due to misregulation of the
genes in p53-dependent transcriptional response. cell cycle or apoptosis.
To distinguish between these two possibilities, we first exam-
lincRNA-p21 Regulates Apoptosis ined whether cell-cycle regulation in response to DNA damage is
The activation of the p53 pathway has two major phenotypic affected by knockdown of p53 and lincRNAp-21. Specifically,
outcomes: growth arrest and apoptosis (Levine et al., 2006). we assayed 5-bromo-2-deoxyuridine (BrdU) incorporation and
Consistent with this, our microarray analysis demonstrates that propidium iodide staining of the cells by fluorescence-activated
p53 and lincRNA-p21 both regulate a number of apoptosis cell sorting (FACS) analysis. Consistent with the ability of p53
and cell-cycle regulator genes (Figure 3E, Figure S3G, and to inhibit cell-cycle progression, knockdown of p53 caused a
Table S2, parts A and B). Thus, we aimed to determine the phys- significant increase in BrdU incorporation in response to DNA
iological role of lincRNA-p21 in these processes. damage (p < 0.01). In contrast, knockdown of lincRNA-p21
Toward this end, we used RNAi-mediated knockdown of showed no significant changes in either BrdU levels or in the
lincRNA-p21 in dox-treated or untreated primary MEFs. We simi- percentages of cells in any of the cell-cycle phases (S, G1, or
larly performed RNAi-mediated knockdown of p53 (as a positive G2) with or without dox treatment (Figure 4C). These results
control) or used the nontargeting siRNA pool (as a negative suggest that lincRNA-p21 does not substantially contribute to
control) under the same conditions. We observed a significant cell-cycle arrest upon DNA damage.

Cell 142, 409–419, August 6, 2010 ª2010 Elsevier Inc. 413

A B C Figure 4. LincRNA-p21 Is Required for
untreated
6
untreated
4 Proper Apoptotic Induction
Relative cell number

Fold change relative

(A) Increased cell viability of lincRNA-p21-

to siRNA control
5 G1
3
4 G2
siRNA p53 S depleted cells. Relative number of siRNA-trans-
3 2
siRNA control fected MEFs treated with 400 nM DOX from
2 siRNA lincRNA-p21
siRNA control-1 siRNA control-2 1
1 24 hr after transfection (right) or untreated (left)
0 0 62 20 18 59 2417 55 22 23
0 24 48 72 siRNA siRNA siRNA determined by MTT assay.
time post transfection (hours) control lincRNA-p21 p53
(B) Knockdown of lincRNA-p21 with individual
DOX siRNAs increases cell viability. Images of MEFs
siRNA p53-1 siRNA p53-2 4 treated with different individual siRNAs after

Fold change relative

DOX

to siRNA control
Relative cell number

2.0
3 G1 * 48 hr of DOX treatment (72 hr after transfection).
1.6 G2
siRNA p53 2 S (C) LincRNA-p21 knockdown doesn’t affect cell-
1.2
siRNA control
0.8 siRNA lincRNA-p21 1 cycle regulation. Relative cell numbers in each
0.4
siRNA siRNA 0 57 34 9 56 33 11 29 44 27 cell-cycle phase determined by FACS of BrdU
0 lincRNA-p21-1 lincRNA-p21-2 siRNA siRNA siRNA
0 24 48 72 control lincRNA-p21 p53
incorporation and PI staining of MEFs treated as
time post transfection (hours)
in (A). Numbers inside bars represent percentages
D E of cells in each phase.
30 (D) LincRNA-p21 knockdown causes a decrease
in cellular apoptosis. p53-reconstituted p53LSL/LSL

% of apoptotic cells
25
siRNA control-1 siRNA control-2 siRNA p53 20
1.81 14.6 1.9 14.3 1.9 5.96
MEFs transfected with three individual siRNAs
15
10
* * targeting lincRNA-p21 (bottom), two independent
5 control siRNAs (upper left and middle) or a siRNA
0 pool targeting p53 (upper right). Twenty-four hours
7-AAD
7-AAD
7-AAD

si p53
lin
si

si NA
after transfection, cells were treated with 400 nM

R
R tro

R -p
cR
81.9 1.64 83.1 1.26 90.9 1.26
co

N
N l

N 2

A
n
A

A 1
Annexin-V Annexin-V Annexin-V
F doxorubicin and 14 hr later were harvested and
siRNA lincRNA-p21-1 siRNA lincRNA-p21-2 siRNA lincRNA-p21-3 subjected to FACS analysis. The x axis represents
siRNA
1.87 9.39 1.8 6.56 1.93 5.5
Annexin-V and the y axis 7-AAD staining. The
21 A
-p RN
l
ro

cp
3
nt

percentage of cells in each quadrant is indicated.

p5
co

lin

Cleaved (E) Decreased apoptosis caused by lincRNA-p21

caspase 3
7-AAD
7-AAD
7-AAD

knockdown. Percentage of Annexin-V-positive

87.1 1.61 91.3 0.92 92.49 1.35 βActin
Annexin-V Annexin-V Annexin-V cells (FACS) at 38 hr after transfection (14 hr of
G 400 nM DOX treatment) in MEFs treated as in (A).
DOX
(F) LincRNA-p21 knockdown in p53-reconstituted
untreated
2.5 p53LSL/LSL MEFs causes decrease in Caspase 3
Relative cell number

25
Relative cell number

20 control vector
2
control vector
cleavage. Levels of cleaved Caspase 3 or control
15 lincRNA-p21
1.5
lincRNA-p21 bActin in p53 reconstituted-p53LSL/LSL MEFs
10 1
treated with the indicated siRNA pools and
5 0.5
500 nM DOX for 14 hr.
0 0
0 24 48 72 0 24 48 72 (G) Decreased cell viability caused by lincRNA-p21
time after selection (hours) time after selection (hours)
overexpression. Relative numbers of LKR cells
overexpressing lincRNA-p21 or control plasmid
determined by MTT assay.
H DOX I DOX
8
(H) Overexpression of lincRNA-p21 causes cellular
% of apoptotic cells

7 * 70
60
control vector
apoptosis under DNA damage induction. Per-
6 50 lincRNA-p21
% of cells

5 centage of Annexin-V-positive LKR cells overex-

4 40
3 30
2 20 pressing lincRNA-p21 or control vector treated
1 10 with 500 nM DOX.
0 0
control lincRNA-p21 G1 G2 S (I) LincRNA overexpression doesn’t affect cell-
cycle regulation. Cell-cycle analysis of DOX-
treated LKR cells overexpressing lincRNA-p21 or
control plasmid.
All values are the average of 3 biological replicates
(±STD). * p < 0.01 relative to controls.
Also see Figure S4.

We then examined the impact of lincRNA-p21 and p53 knock- p21 would result in an increased apoptosis. Indeed, lincRNA-
downs on apoptosis. To this end, we assayed the proportion of the p21 overexpression in a lung cancer cell line harboring a KRAS
cell population undergoing apoptosis by measuring Annexin-V by mutation (referred to as LKR) and in NIH/3T3 MEFs caused a sig-
FACS analysis. We observed a significant decrease in the number nificant decrease in cell viability (Experimental Procedures, Fig-
of apoptotic cells after DNA damage in both the lincRNA-p21 and ure 4G, and Figure S4E). This decrease in viability was due to
p53 depleted cells relative to the siRNA control (p < 0.01) (Figures increased apoptosis in response to DNA damage (p < 0.01) and
4D and 4E). We also observed a decrease in Caspase 3 cleavage not to an effect in cell-cycle regulation (Figures 4H and 4I and
after knockdown of both p53 or lincRNA-p21, relative to controls Figure S4G). Together, these results demonstrate a reproducible
(Figure 4F). We next sought to determine whether, conversely to and similar reduction of apoptotic cells in response to DNA
lincRNA-p21 knockdown, the enforced expression of lincRNA- damage in both lincRNA-p21 and p53 knockdown experiments.

414 Cell 142, 409–419, August 6, 2010 ª2010 Elsevier Inc.

A Biotinylated
B se p2
1
D Figure 5. LincRNA-p21 Physically Interacts
RNA en A-
its A
an RN cRN with hnRNP-K
lin Native RIP
7 (A) Schematic representation of RNA pulldown

Fold Enrichment (RIP/IgG)

Nuclear 6
experiments to identify associated proteins. Bioti-
lincRNA-p21
extract 5 βActin
4
nylated lincRNA-p21 or antisense RNA were incu-
Gapdh
3 bated with nuclear extracts, targeted with strepta-
2 vidin beads, washed, and associated proteins
1
60 kDa hnRNP-K resolved in a gel. Specific bands were cutout and
0
E identified by mass spectrometry.
(B) LincRNA-p21 and hnRNP-K specifically
X-linked RIP interact in vitro. SDS-PAGE gel of proteins bound
Streptavidin 40 to lincRNA-p21 (right lane) or antisense RNA (left

Fold Enrichment (RIP/IgG)

lincRNA-p21
beads p53
30 βActin
lane). The highlighted region was submitted for
mass spectrometry identifying hnRNP-K as the
C 20
hnRNP-K band unique to lincRNA-p21.
10 (C) Western blot analysis of the specific associa-
Mass tion of hnRNP-K with lincRNA-p21. A nonspecific
Nono 0
Spectrometry
hnRNP-K hnRNP-K protein (NONO) is shown as a control.
antibody 1 antibody 2
(D) Association between endogenous lincRNA-
p21 and hnRNP-K in the nucleus of DNA damaged
MEFs in native conditions. RNA Immunoprecipita-
F 1 2 3 4 5 6 7 8 9 10 G tion (RIP) enrichment is determined as RNA asso-
16 * *
* * ciated to hnRNP-K IP relative to IgG control.
% apoptoptotic cells

hnRNP-K
12
(E) Physical association between lincRNA-p21 and
8 hnRNP-K after chemical crosslinking of life cells.
RNA
4 hnRNP-K was immunoprecipitated from nuclear
extracts of formaldehyde-crosslinked DNA-dam-
0
5’ 3’ length
aged MEFs, and associated RNAs were detected
ve

5’

Fu

3’

3’

1 778 nt
ct

77

26

18
ll
or

le
8

33

89

2 938 nt by RT-qPCR. The relative enrichment is calcu-

ng
nt

nt

nt
th

3 1459 nt
4 2125 nt
hnRNP-K Apoptosis
lated as in (D) and is the median of three techni-
5 3073 nt
6 3073 nt (antisense) Interaction Induction cal replicates of a representative experiment
7 2844 nt 5’ 778 nt + - (±STD).
8 2633 nt Full length + +
9
10
1889 nt
735 nt 3’ 2633 nt - - (F) LincRNA-p21 binds hnRNP-K through its 50
3’ 1889 nt - - terminal region. RNAs corresponding to dif-
ferent fragments of lincRNA-p21 or its antisense
sequence (middle and bottom) were treated as in (A) and associated hnRNP-K was detected by western blot (top).
(G) Percentage of Annexin-V-positive LKR cells overexpressing the indicated lincRNA-p21 fragments or empty vector as control (average of three replicates
[±STD]). * p < 0.001.
See also Figure S5.

Although MEFs typically respond to DNA damage by under-
going cell-cycle arrest rather than apoptosis (Kuerbitz et al.,
1992), several additional lines of evidence are consistent with
the observed apoptosis phenotype in response to knockdown
on p53 and lincRNA-p21. First, certain critical cell-cycle regula-
tors, such as Cdkn1a/p21, Cdkn2a, and Reprimo, are regulated
by p53 but not lincRNA-p21. For example, knockdown of
lincRNA-p21 perturbs neither the transcript levels of Cdkn1a/
p21 nor the protein stability (Figure S3E); this may explain why
lincRNA-p21 knockdown is insufficient to cause a cell-cycle
phenotype, yet the p53 knockdown is. Second, we observed
that both lincRNA-p21 and p53 knockdowns resulted in the
repression of apoptosis genes (Noxa and Perp) and derepres-
sion of cell survival genes (Bcl2l3, Stat3, and Atf2, among others)
(Figure 3E and Table S2). Moreover, the decrease of apoptotic
cells in response to knockdown of lincRNA-p21 was comparable
to that caused by knockdown of p53 (Figures 4D and 4E and
Figures S4D and S4E). Third, the apoptosis phenotype is depen-
dent on the dosage of dox-induced DNA damage (Figure S4D).
Thus, the apoptosis response is both p53 dependent and
lincRNA-p21 dependent, with this dependence confirmed in
multiple cell types and conditions (Figures 4B, 4D, 4F, and 4H
and Figures S4A–S4C). Collectively, these observations demon-
strate that lincRNA-p21 plays an important role in the p53-
dependent induction of cell death.
LincRNA-p21 Functions through Interaction
with hnRNP-K
We next wanted to investigate the mechanism by which
lincRNA-p21 mediates transcriptional repression. We have
recently reported that many lincRNAs regulate gene expression
through their interaction with several chromatin regulatory com-
plexes (Khalil et al., 2009). Thus, we hypothesized that lincRNA-
p21 could affect gene expression in a similar manner.
To test this, we first performed nuclear fractionation experi-
ments and confirmed that lincRNA-p21 is enriched in the nucleus
(Figure S5A). We next sought to identify proteins that are associ-
ated with lincRNA-p21 by an RNA-pulldown experiment. Specif-
ically, we incubated in vitro-synthesized biotinylated lincRNA-
p21 and antisense lincRNA-p21 transcripts (negative control)
with nuclear cell extracts and isolated coprecipitated proteins
with streptavidin beads (Experimental Procedures). We resolved
the RNA-associated proteins on a SDS-PAGE gel, cut out the
bands specific to lincRNA-p21, and subjected them to mass
spectrometry (Figures 5A and 5B). In all six biological replicates,
mass-spectrometry analysis identified heterogeneous nuclear

Cell 142, 409–419, August 6, 2010 ª2010 Elsevier Inc. 415

ribonucleoprotein K (hnRNP-K) as specifically associated with
the sense (but not antisense) strand of lincRNA-p21. We inde-
pendently verified this interaction by western blot analysis
(Figure 5C). hnRNP-K has been shown to play various roles in
the p53 pathway (Kim et al., 2008; Moumen et al., 2005). Interest-
ingly, among these roles, Kim et al. (2008) demonstrated that
hnRNP-K is a component of a repressor complex that acts in
the p53 pathway, consistent with our evidence that lincRNA-
p21 plays a role in global repression in this pathway.
To further validate the interaction between lincRNA-p21 and
hnRNP-K in our cell-based systems, we performed RNA immu-
noprecipitation (RIP) with an antibody against hnRNP-K from
nuclear extracts of MEFs subjected to DNA damage. We
observed an enrichment of lincRNA-p21 (but not other unrelated
RNAs) with hnRNP-K antibody as compared to the nonspecific
antibody (IgG control) (Figure 5D). We further performed analo-
gous RIP experiments with formaldehyde crosslinked cells
followed by stringent washing conditions (Ule et al., 2005) to
rule out potential nonspecific interactions. Consistent with
a bona fide interaction, we observed a greater and very signifi-
cant enrichment of lincRNA-p21 in the hnRNP-K RIP relative to
the IgG control RIP with two hnRNP-K different antibodies
(Figure 5E).
We further performed deletion-mapping experiments to deter-
mine whether hnRNP-K interacts within a specific region of
lincRNA-p21. To this end, we carried out RNA pulldown experi-
ments with truncated versions of lincRNA-p21 followed by
western blot detection of bound hnRNP-K. These analyses
identified a 780 nt region at the 50 end of lincRNA-p21 required
for the interaction with hnRNP-K (Figure 5F). Interestingly, RNA
folding analyses of this region based on sequence conservation
and compensatory changes across 14 mammalian species
(Hofacker, 2003) predict a highly stable 280 nt structure of
lincRNA-p21 with deep evolutionary conservation (Figures S5B
and S5C). Together, the RNA pulldown, native RIP, crosslinked
RIP, and deletion mapping results demonstrate a specific asso-
ciation between hnRNP-K and lincRNA-p21.
We next sought to determine the functional relevance of the
interaction between lincRNA-p21 and hnRNP-K. To do so, we
monitored the ability of different truncated versions of lincRNA-
p21 to induce cellular apoptosis when overexpressed in LKR
cells (Experimental Procedures). This revealed that the deletion
of the 50 end of lincRNA-p21, which mediates the hnRNP-K inter-
action, abolishes the ability of lincRNA-p21 to induce apoptosis
(Figure 5G). Interestingly, the 780 nt fragment at the 50 end of
lincRNA-p21 alone does not induce apoptosis, indicating that
this fragment is required but not sufficient for lincRNA-p21-medi-
ated cellular apoptosis.
We hypothesized that hnRNP-K is required for proper tran-
scriptional repression of target genes shared between p53 and
lincRNA-p21. If so, knockdown of hnRNP-K should result in
derepression of these shared targets. We tested this hypothesis
by performing siRNA-mediated knockdown of hnRNP-K,
lincRNA-p21, and p53 in p53-restored p53LSL/LSL MEFs, treating
the cells with dox and profiling the changes in gene expression
by microarray analysis.
Consistent with our previous data, we observed a strong over-
lap of 582 genes affected in the hnRNP-K, lincRNA-p21, and p53
416 Cell 142, 409–419, August 6, 2010 ª2010 Elsevier Inc.
knockdowns (FDR < 0.05) (Figure 6 and Figure S6). Remarkably,
83% of these common genes were derepressed in all three
knockdown experiments (Figure 6A and Figure S6D). The genes
previously identified as coregulated by lincRNA-p21 and
p53 also were strongly enriched (GSEA FDR < 10
4) among
those regulated by hnRNP-K (Figure 6B and Table S3). Thus,
lincRNA-p21 and hnRNP-K play roles in repressing a significant
common set of genes in the p53-dependent response to DNA
damage.
We further reasoned that if hnRNP-K is involved in the repres-
sion of genes corepressed by p53 and lincRNA-p21, then
hnRNP-K might also be physically bound to the promoters of
these genes. To test this, we performed ChIP experiments with
antibodies against hnRNP-K, followed by hybridization to DNA
tiling microarrays covering 30,000 gene promoters. We identified
1621 promoter regions with significant occupancy by hnRNP-K
(FDR < 0.05) (Figure 6 and Table S3). Notably, these promoter
regions exhibit a significant overlap with genes that were differ-
entially expressed upon hnRNP-K knockdown (GSEA FDR <
0.001) (Figure S6E). Moreover, hnRNP-K localizes to a significant
fraction (FDR < 0.001) of the genes corepressed by lincRNA-p21
and p53 (Figure 6C), suggesting that these are primary sites of
hnRNP-K regulation.
We next wanted to determine whether lincRNA-p21 plays
a role in hnRNP-K localization at promoters of p53-repressed
genes. To this end, we determined whether siRNA-mediated
knockdown of lincRNA-p21 affected the localization of hnRNP-K
after induction of p53. Specifically, we performed hnRNP-K
ChIP in dox-treated p53-restored p53LSL/LSL MEFs transfected
with either siRNAs targeting lincRNA-p21 or nontargeting con-
trol siRNAs. These experiments revealed that the depletion of
lincRNA-p21 causes a significant reduction in the association
of hnRNP-K at the promoter regions of genes that are normally
repressed in a lincRNA-p21- and p53-dependent fashion, as
determined by ChIP-qPCR (Figure 6D). Specifically, 12 of the
15 tested promoter regions exhibited loss of hnRNP-K enrich-
ment, in two biological replicate experiments, upon depletion
of lincRNA-p21. Thus, hnRNP-K is bound to the promoters of
genes that are normally repressed in a p53- and lincRNA-p21-
dependent manner, and this localization requires lincRNA-p21.
Collectively, our results indicate that lincRNA-p21 is a direct
p53 transcriptional target in response to DNA damage, acts to
repress genes that are downregulated as part of the canonical
p53 transcriptional response, is necessary for p53 dependent
apoptotic responses to DNA damage in our cell-based systems,
and functions at least in part through interaction with hnRNP-K
by modulating hnRNP-K localization.
DISCUSSION
It is clear that mammalian genomes encode numerous large
noncoding RNAs (Carninci, 2008; Guttman et al., 2009; Mattick,
2009; Ponjavic et al., 2007). Here, we demonstrate that
numerous lincRNAs are key constituents in the p53-dependent
transcriptional pathway. Moreover, we observed that some of
these lincRNAs are bound by p53 in their promoter regions and
sufficient to drive p53-dependent reporter activity that requires
A Figure 6. LincRNA-p21 and hnRNP-K Corepress Genes in the p53
lincRNA- Transcriptional Response
hnRNP-K
p21
582 (A) Many genes are coregulated by p53, lincRNA-p21, and hnRNP-K. Genes
affected by knockdown of lincRNA-p21, p53, or hnRNP-K in p53-restored-
p53 DNA-damaged p53LSL/LSL MEFs determined by microarray analysis (FDR <
0.05). Shades of red or blue represent expression values relative to global
median across experiments. Percentages of up- and downregulated genes
are indicated.
17% (B) Genes repressed by lincRNA-p21 are significantly enriched in genes
repressed by p53 and hnRNPK. GSEA comparing the genes upregulated on
knockdown of LincRNA-p21 and those upregulated upon knockdown of p53
582 genes

(left) or hnRNP-K (right). The black line represents the observed enrichment
score profile of genes in the lincRNA-p21 gene set to the p53 or hnRNP-K
83%
gene sets, respectively.
(C) hnRNP-K associates to promoters of genes corepressed by lincRNA-p21
and p53. Examples of promoters of genes repressed by p53 and lincRNA-
p21 (G2e3, Mtap4, Suv39h1, and Vcan) or repressed by lincRNA-p21
siRNA siRNA siRNA siRNA -80 but not p53 (Rb1) bound by hnRNP-K (blue) determined by ChIP-chip of
P< 10
B p53 lincRNA-p21 hnRNP-K control hnRNP-K in dox-trated p53-reconstituted p53LSL/LSL MEFs (FDR < 0.05).
-8 +8 Cdkn2a and Wt1 are negative controls (gray).
(D) hnRNP-K binding to lincRNA-p21 and p53 corepressed genes is depen-
Enrichment Score

Enrichment Score

p53 hnRNP-K
* dent on lincRNA-p21. Relative enrichment of hnRNP-K (ChIP-qPCR) in the
0.5 * 0.5 indicated promoter regions in p53-reconstituted p53LSL/LSL MEFs transfected
with siRNA lincRNA-p21 or siRNA control and dox-treated determined by
0.0 0.0
ChIP-qPCR (representative of two biological replicates shown ±STD).
* FDR<.001 *FDR<.001 (E) Proposed models for the function of licRNA-p21 in the p53 transcriptional
C response. Induction of p53 activates the transcription of lincRNA-p21 by
+3
binding to its promoter (upper left). LincRNA-p21 binds to hnRNP-K, and this
lincRNA-p21
hnRNP-K interaction imparts specificity to genes repressed by p53 induction (upper right).
/p53
bound 0
repressed
-2 See also Figure S6 and Table S3.
G2e3
P<0.001
Relative Binding Enrichment (hnRNP-K ChIP/IgG)

+3 +3

-2
0 0 the consensus p53-binding motif, suggesting that these
-2
Mtap4 Suv39h1 lincRNAs are bona fide p53 transcriptional targets.
+3 +3 Having discovered multiple lincRNAs in the p53 pathway, we
0 0 decided to focus on one such lincRNA in particular: lincRNA-
-2 -2
Rb1 Vcan p21. Intrigued by its properties (genomic location upstream of
+3 +3 p21, p53-dependent activation requiring the consensus p53
0 0 motif, which is bound by p53 and conserved p53-dependent
-2
-2
Cdkn2a Wt1
activation of this gene in both human and mouse cell-based
systems), we explored the functional roles of lincRNA-p21. Our
D studies revealed a role for lincRNA-p21 in a p53-dependent
115
45 39
apoptotic response after DNA damage.
14 35 20 31 We further observed that siRNA-mediated inhibition of lincRNA-
26
12 siRNA control
p21 affects the expression of hundreds of gene targets that are
Relative Fold Enrichment

siRNA lincRNA-p21 enriched for genes normally repressed by p53 in both the MEF
(hnRNPK ChIP/IgG)

10

8
and RAS cell-based systems. Strikingly, the vast majority of these
common target genes are derepressed upon inhibition of either
6
p53 or lincRNA-p21—suggesting that lincRNA-p21 functions as
4
a downstream repressor in the p53 transcriptional response.
2
We gained mechanistic clues into how lincRNA-p21 functions
0
to repress such a large subset of the p53 transcriptional
Vc

Jm

Zb

At

Lp

Pd

Zf

G a
xc

us

b1

sp

dk

ap
tp

p3
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p
an

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lim
jd

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25

n2

dh
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86
3

response by biochemical experiments that identified a specific

2

E interaction between lincRNA-p21 and hnRNP-K. This interaction

p53
POL II
is supported by RNA-pulldown, native RIP, crosslinked RIP, and
deletion-mapping experiments. Moreover, we identified a 780 nt
50 region of lincRNA-p21 that is required for hnRNP-K binding
hnRNP-K hnRNP-K and subsequent induction of apoptosis. Interestingly, this region
is much more highly conserved than the remainder of the tran-
? script. This suggests that patches of conservations, previously
determined to be unique to lincRNAs, (Guttman et al., 2009),
lincRNA-p21
may point to functional elements for binding interactions within

Cell 142, 409–419, August 6, 2010 ª2010 Elsevier Inc. 417

lincRNAs (as was also recently determined for Xist binding to
PRC2) (Zhao et al., 2008).
hnRNP-K is known to interact with other repressive complexes
such as the Histone H1.2 or members of the Polycomb-group
(PcG) (Kim et al., 2008; Denisenko and Bomsztyk, 1997). The
physical interaction between lincRNA-p21 and hnRNP-K is likely
required for lincRNA-p21-mediated gene repression, as loss of
hnRNP-K function results in the derepression of the same genes
that are repressed by both p53 and lincRNA-p21. Importantly,
genome-wide ChIP-chip analysis revealed hnRNP-K binding at
the promoters of these corepressed gene loci, suggestive of direct
regulation by hnRNP-K and lincRNA-p21. Moreover, we observed
a lincRNA-p21 dependent binding of hnRNP-K at several of these
corepressed promoter regions. While hnRNP-K has been previ-
ously shown to activate one gene in the p53 pathway (Moumen
et al., 2005), our analyses suggest that it plays a much more
widespread role in repression. Together, these results implicate
lincRNA-p21 as an important repressor in the p53 pathway, by
interacting with and modulating the localization of hnRNP-K.
Our results raise the possibility that many transcriptional
programs (beyond the p53-pathway) may involve inducing
protein factors that activate specific sets of downstream genes
and lincRNAs that repress previously active sets of genes. The
notion of a noncoding RNA being involved in silencing-specific
gene loci is consistent with our recent observation that many
lincRNAs (including lincRNA-p21) bind to chromatin complexes
(such as PRC2) and are required to mediate repression at key
gene loci (Khalil et al., 2009). Moreover, there are several exam-
ples of lincRNAs involved in repression of known target genes—
including HOTAIR-dependent repression of HOXD genes (Rinn
et al., 2007) and XIST, AIR, and KCNQ1OT1, involved in genomic
imprinting and silencing of several genes in cis (Nagano et al.,
2008; Pandey et al., 2008; Zhao et al., 2008).
The precise mechanism by which lincRNA-p21 contributes to
repression at specific loci remains to be defined. Various possi-
bilities include that (1) lincRNA-p21 might direct a protein
complex to specific loci by Crick-Watson base pairing; (2)
lincRNAs might act by forming DNA-DNA-RNA triple-helical
structures, which do not require Crick-Watson base-pairing,
such as reported for a large noncoding RNA that forms a
triple-helix upstream of the Dihydrofolate Reductase (DHFR)
promoter resulting in repression of DHFR (Martianov et al.,
2007); or (3) lincRNAs might alter the binding specificity of
DNA-binding proteins (such as hnRNP-K) to influence their target
preference (Figure 6D). Further experiments are needed to
distinguish between these and other possibilities.
Aside from the general interest in gene regulation, we note that
lincRNA-p21 and several other lincRNAs function in an important
pathway for cancer. It is tempting to speculate that other
lincRNAs may also play key roles in numerous other tumor-
suppressor and oncogenic pathways, representing a hitherto
unknown paradigm in cellular transformation and metastasis.
It will be important for future studies to determine whether
lincRNAs genes can serve as tumor suppressor genes or
oncogenes.
In summary, lincRNAs may point to new mechanisms of gene
regulation, components in disease pathways and potential
targets for the development of therapies.
418 Cell 142, 409–419, August 6, 2010 ª2010 Elsevier Inc.
EXPERIMENTAL PROCEDURES
Cell Lines and In Vivo Models
KRAS lung tumor-derived cell lines were isolated from individual tumors (D.F.
and T.J., unpublished data). Isolation of matched p53+/+ and p53
/
MEFs,
p53LSL/LSL MEFs, lymphomas, and sarcomas, and p53 restoration were
done as described (Ventura et al., 2007). Primary WT MEFs and NIH/3T3
MEF cells were purchased from ATCC. Transfection, infection, and treatment
conditions are described in the Extended Experimental Procedures.
Promoter Reporter Assays
LincRNA promoters were cloned into pGL3-basic vector (Promega), and motif
deletions were performed by mutagenesis. p53-reconstituted or control
p53LSL/LSL MEFs were transfected with 800 ng pGL3 and 30 ng TK-Renilla
plasmid per 24-well. Twenty-four hours later, cells were treated with 500 nM
dox for 13 hr, and cell extracts were assayed for firefly and renilla luciferase
activities (Promega E1910).
LincRNA and Gene-Expression Profiling and Informatic Analyses
RNA isolation, lincRNA expression profiling, and ChIP-chip analyses (Nimble-
gen arrays), as well as Affymetrix gene-expression profiling and analyses, were
performed as described (Guttman et al., 2009) (Extended Experimental Proce-
dures). Structure predictions were performed using the Vienna RNA package
(Hofacker, 2003).
Antibodies
The following antibodies were used: anti-p53, Novocastra (NCL-p53-CM5p)
(western blot) and Vector Labs (CM-5) (ChIP); anti-hnRNP-K, Santa Cruz
Biotechnology (sc-25373) (western blot) and Abcam (Ab70492 and Ab39975)
(ChIP and RIP); and control rabbit IgG Abcam (Ab37415-5) (RIP and ChIP-
chip).
Viability and Apoptosis Assays and Cell-Cycle Analysis
MTT assays were performed with Cell Proliferation Kit I from Roche
(11465007001). For apoptosis quantification, the Apoptosis Detection Kit I
from BD Biosciences (cat#559763) and FACS (van Engeland et al., 1996)
were used. Cell-cycle analysis was performed as described (Brugarolas
et al., 1995).
Cloning, RNA Pulldown, Deletion Mapping, RIP, and ChIP
50 and 30 RACE cloning of lincRNA-p21 were performed from total RNA of dox-
treated MEFs with RLM-RACE Kit (Ambion). RNA pulldown and deletion
mapping were performed as described (Rinn et al., 2007) with 1 mg mESC
nuclear extract and 50 pmol of biotinylated RNA. Mass spectrometry was per-
formed as described (Shevchenko et al., 1996). Native RIP was carried out as
described (Rinn et al., 2007). For crosslinked RIP, cells were crosslinked with
1% formaldehyde, antibody incubated overnight, recovered with protein G
Dynabeads, and washed with RIPA buffer. After reverse crosslink, RNA was
analyzed by qRT-PCR. p53 ChIP and hnRNP-K ChIP experiments were per-
formed as previously described (Rinn et al., 2007) (Extended Experimental
Procedures).
RNA Interference and LincRNA-p21 Overexpression
siRNA transfections were done with 75 nM siRNA and Lipofectamine 2000
(Invitrogen). For overexpression, lincRNA-p21 or truncated forms were cloned
into the pBABE vector. After transfection, cells were selected with 2 mg/ml
puromycin.
ACCESSION NUMBERS
The accession number for the full-length mouse lincRNA-p21 sequence
reported in this paper is HM210889 (bankit1350506). All primary data are avail-
able at the Gene Expression Omnibus (GSE21761).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, six
figures, and four tables and can be found with this article online at doi:10.
1016/j.cell.2010.06.040.
ACKNOWLEDGMENTS
We would like to thank Loyal A. Goff (Massachusetts Institute of Technology
[MIT]) for bioinformatic support, Nadya Dimitrova (MIT) for input on the manu-
script, David Garcia (MIT) for experimental assistance, and Sigrid Hart (Broad
Institute) for illustration support. J.L.R. is a Damon Runyon-Rachleff, Searle,
and Smith Family Foundation Scholar. J.L.R. and A.R. are Richard Merkin
Foundation Scholars. This work was supported by the National Institutes of
Health (NIH) Director’s New Innovator Award, Smith Family Foundation,
Damon Runyon Cancer Foundation, Searle Scholar Program, and NIH
1R01CA119176-01.
Received: October 6, 2009
Revised: April 6, 2010
Accepted: June 3, 2010
Published online: July 29, 2010
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A Minimal Midzone Protein Module
Controls Formation and Length of
Antiparallel Microtubule Overlaps
Peter Bieling,1,2 Ivo A. Telley,1 and Thomas Surrey1,*
1CellBiology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
2Present address: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco,
CA 94158, USA
*Correspondence: surrey@embl.de
DOI 10.1016/j.cell.2010.06.033
SUMMARY
During cell division, microtubules are arranged in a
large bipolar structure, the mitotic spindle, to segre-
gate the duplicated chromosomes. Antiparallel mic-
rotubule overlaps in the spindle center are essential
for establishing bipolarity and maintaining spindle
stability throughout mitosis. In anaphase, this anti-
parallel microtubule array is tightly bundled forming
the midzone, which serves as a hub for the recruit-
ment of proteins essential for late mitotic events.
The molecular mechanism of midzone formation
and the control of its size are not understood. Using
an in vitro reconstitution approach, we show here
that PRC1 autonomously bundles antiparallel micro-
tubules and recruits Xklp1, a kinesin-4, selectively to
overlapping antiparallel microtubules. The proces-
sive motor Xklp1 controls overlap size by overlap
length-dependent microtubule growth inhibition.
Our results mechanistically explain how the two
conserved, essential midzone proteins PRC1 and
Xklp1 cooperate to constitute a minimal protein
module capable of dynamically organizing the core
structure of the central anaphase spindle.
INTRODUCTION
The various architectures adopted by the microtubule cytoskel-
eton are determined by distinct combinations of mechanochem-
ical protein activities. How global properties of a complex
cytoskeletal system, for instance characteristic shape and size,
emerge from the local biochemical properties of the constituting
molecules is an open question. During cell division, the microtu-
bule cytoskeleton forms the mitotic spindle to segregate the
duplicated set of chromosomes (Walczak and Heald, 2008).
Spindles consist of two antiparallel arrays of microtubules that
overlap in the spindle center, an organization that ensures
spindle function. At anaphase onset, the central spindle reorga-
nizes to form the midzone, a dense specialized array of antipar-
allel microtubules (Glotzer, 2009; Khmelinskii and Schiebel,
420 Cell 142, 420–432, August 6, 2010 ª2010 Elsevier Inc.
2008). The midzone size in metazoans is typically 2–3 mm in
length (Mastronarde et al., 1993). Several proteins essential for
spindle elongation, faithful chromosome segregation, and pro-
per cytokinesis relocalize selectively to the central spindle as a
consequence of changes in their phosphorylation patterns in
anaphase: PRC1 (protein required for cytokinesis 1), kinesin-4,
the centralspindlin complex (containing a kinesin-6), the chro-
mosome passenger complex, and several other molecular
motors, nonmotor microtubule-bundling proteins, and regula-
tors (Glotzer, 2009). How the organization of the midzone is de-
termined by the midzone proteins is, however, not understood.
The conserved vertebrate-bundling protein PRC1 (Jiang et al.,
1998; Kurasawa et al., 2004; Mollinari et al., 2002; Zhu and Jiang,
2005) and its orthologs (Ase1 in yeasts [Loiodice et al., 2005;
Schuyler et al., 2003], SPD-1 in C. elegans [Verbrugghe and
White, 2004], Feo in D. melanogaster [Verni et al., 2004], and
MAP65 in plants [Muller et al., 2004]) have emerged as key
players for midzone formation. Ase1 in budding yeast has been
proposed to be the core component of the spindle midzone.
Genetic experiments have demonstrated that localization of all
midzone proteins depends on Ase1, whose localization is inde-
pendent of most other midzone proteins (Khmelinskii et al.,
2007). Interestingly, fission yeast Ase1 has been shown to bind
with some preference to antiparallel versus parallel microtubule
bundles in vitro (Janson et al., 2007; Kapitein et al., 2008), indi-
cating that recognition of antiparallel microtubules is an autono-
mous property of Ase1 in yeast.
In contrast to yeast, PRC1 in higher eukaryotes interacts with
motor proteins of the kinesin-4 type. These chromokinesin
motors relocalize to the central spindle in anaphase (Kurasawa
et al., 2004; Kwon et al., 2004; Lee and Kim, 2004; Powers
et al., 2004; Vernos et al., 1995; Zhu and Jiang, 2005). Correct
localization of both PRC1 and kinesin-4 to the midzone depend
on each other (Kurasawa et al., 2004; Zhu and Jiang, 2005; Zhu
et al., 2005). However, whether kinesin-4 transports PRC1 to the
midzone (Zhu and Jiang, 2005) or PRC1 selectively binds the
central region of the anaphase spindle thereby recruiting kine-
sin-4 (Kurasawa et al., 2004) is presently unclear. Importantly,
it is unknown how the size of the midzone is determined.
Whereas absence of PRC1 results in complete loss of midzone
microtubule bundles in mammalian cells (Kurasawa et al.,
2004; Mollinari et al., 2002; Zhu and Jiang, 2005), absence of
kinesin-4 leads to increased anaphase spindle length (Kurasawa
et al., 2004; Zhu and Jiang, 2005). Interestingly, a fragment of
Xenopus kinesin-4 has been shown to decrease microtubule
growth and shrinkage rates in vitro (Bringmann et al., 2004), an
activity different from other regulators of microtubule dynamics
(Howard and Hyman, 2007). Hence, kinesin-4 might contribute
to midzone formation by regulating midzone microtubule
dynamics. The minimal set of protein activities required for
specific organizational features of the spindle midzone and the
logic behind the combination of the required activities are,
however, unknown.
We have developed an in vitro reconstitution approach to
investigate the combinatorial action of purified full-length
Xenopus kinesin-4 (Xklp1) and Xenopus PRC1 on microtubule
organization and dynamics. We show that Xklp1 is recruited to
antiparallel microtubule overlaps by PRC1, which autonomously
discriminates between parallel and antiparallel microtubules with
high selectivity. Xklp1 ensures the maintenance of microtubule
overlap size by selectively inhibiting the growth of overlapping
microtubules in a manner that is dependent on overlap length.
Our results demonstrate that only two proteins are necessary
and sufficient for the selective formation and for adaptable length
control of antiparallel microtubule overlaps in vitro, as required
for the formation of the midzone in vivo.
RESULTS
PRC1 Selectively Binds to Antiparallel Microtubule
Overlaps with Nanomolar Affinity
We developed a fluorescence microscopy assay to visualize
dynamic microtubule encounters in vitro. We attached short,
Alexa 568- and biotin-labeled, stabilized microtubule seeds
under flow to glass slides, which were functionalized with a
NeutrAvidin-biotin-polyethylene glycol layer. The majority of
the seeds aligned, and microtubules growing from these seeds
in the presence of Alexa 568–tubulin occasionally encountered
each other in a plus end-to-plus end configuration (Figure 1A).
Multicolor time-lapse TIRF microscopy revealed that antiparallel
microtubules connected in the presence of 5 nM purified PRC1–
Alexa 647 as soon as their plus ends encountered, forming an
antiparallel microtubule overlap (Figure 1B, Movie S1 available
online). Interestingly, PRC1–Alexa 647 strongly accumulated in
the growing antiparallel overlap region under conditions at which
it did not localize significantly to individual microtubules or to
parallel microtubule pairs (Figure 1C, Movie S1). This demon-
strates that vertebrate PRC1 does not require an additional
factor to selectively target antiparallel microtubule configura-
tions. Autonomous binding to antiparallel microtubules, there-
fore, appears to be conserved between PRC1 orthologs from
vertebrates and yeast (Janson et al., 2007).
The length of antiparallel microtubule overlaps increased
linearly with time after the microtubule encounter (Figure 1D,
top) indicating constant average overlap growth velocity. Over-
lap length as measured from plus end to plus end of the two
antiparallel microtubules increased approximately twice as fast
(4.9 ± 0.9 mm/min, mean ± standard deviation [SD]) as the length
of single microtubules that were not part of an antiparallel micro-
tubule pair (2.3 ± 0.3 mm/min) (Figure 1D, bottom), demonstrating
that the growth velocity of two microtubules of an antiparallel
overlap is similar to that of individual microtubules and therefore
not significantly affected by the strong accumulation of PRC1.
To investigate the mechanism by which vertebrate PRC1
specifically recognizes antiparallel overlaps, we quantified its
affinity and selectivity of binding to distinct microtubule configu-
rations. We measured the average fluorescence intensity of
PRC1–Alexa 647 in antiparallel overlaps as a function of the
PRC1–Alexa 647 concentration (Figure 1E, Figure S1A). PRC1
binding to antiparallel overlaps was strong and positively coop-
erative with a microscopic dissociation constant of 7.6 ± 0.4 nM
(mean ± standard error of the mean [SEM]) and a Hill coefficient
of 1.9 ± 0.1 (Figure 1E). Under standard imaging conditions, we
did not detect any accumulation of PRC1 on individual microtu-
bules or between parallel microtubules even at the highest PRC1
concentration used (31.5 nM) (Figure S1B). We estimate that
PRC1 binds at least 28 times more strongly to antiparallel
microtubule pairs than to individual or parallel microtubules
(Extended Experimental Procedures), indicating considerably
higher selectivity for antiparallel microtubules than previously
reported for Ase1 from fission yeast (Janson et al., 2007).
To investigate the mechanism of this high binding selectivity,
we imaged single PRC1 molecules at higher temporal resolution.
Many binding events of individual PRC1–Alexa 647 molecules
were detected in antiparallel overlaps (Figure 1F, right), whereas
hardly any events were observed in parallel microtubule pairs
(Figure 1F, left). Individual PRC1 molecules were not statically
bound but diffused in antiparallel overlaps (Figures S1C and
S1D). Despite the high affinity for antiparallel microtubules,
turnover of PRC1 in antiparallel overlaps was fast with a single-
molecule half-life of around 1 s (Figure 1G, Figure S1E).
Therefore, selective binding to antiparallel microtubules is an
intrinsic property of the individual PRC1 dimer and it is very
dynamic. This appears to distinguish PRC1 from Ase1 whose
moderate selectivity for antiparallel microtubule binding was
suggested to be a consequence of the formation of slowly
dissociating oligomers (Kapitein et al., 2008).
Reconstitution of a Minimal Midzone with Stable
Antiparallel Microtubule Overlaps In Vitro
We next tested the additional effect of Xenopus kinesin-4 Xklp1
on microtubule overlap formation. In the presence of both 5 nM
purified PRC1–Alexa 647 and 15 nM purified Xklp1–GFP (Fig-
ure S2), antiparallel microtubules connected to each other as
soon as their plus ends encountered, forming an antiparallel
microtubule overlap that remarkably reached a constant size
after awhile (Figure 2A, Movie S2, and Movie S3). PRC1–Alexa
647 and also Xklp1–GFP were observed to strongly accumulate
selectively in antiparallel but not parallel overlap regions (Fig-
ure 2B, Movie S2). Xklp1 had only a minor positive effect on
the amount of overlap-associated PRC1 (Figure 2C). In contrast,
no Xklp1 was observed in the absence of PRC1 in antiparallel
microtubule overlaps (Figures 2D–2F). Microtubules often failed
to form a tight bundle in the presence of Xklp1 only (Movie S4),
a behavior never observed when PRC1 was present.
These TIRF microscopy data indicate that Xklp1 interacts
directly with PRC1, which we confirmed by pull-down experi-
ments (Figure 2G, lane 2). Consequently, an N-terminal motor
Cell 142, 420–432, August 6, 2010 ª2010 Elsevier Inc. 421
 PRC1 Figure 1. PRC1 Preferentially Binds to and
A evanescent field bundling protein/complex B
microtubules Diffuses in Antiparallel Microtubule Over-
Alexa568-Tubulin
microtubule seed 0:00 laps
(A) Scheme of the experimental setup. Dynamic
0:23
microtubules were grown in the presence of free
0:47 Alexa 568-labeled tubulin (light blue) and fluores-
cently labeled midzone proteins (red) from stabi-
1:10 lized microtubule seeds (dark blue) attached to a
anti-parallel overlap PEG-passivated glass surface by means of
1:33
biotin–NeutrAvidin (yellow and green) links. Micro-
1:57 tubules occasionally encountering each other in a
plus end-to-plus end configuration forming an
antiparallel microtubule overlap and fluorescently
C PRC1 D
scheme microtubules PRC1 microtubules labeled midzone proteins were observed by multi-
antiparallel overlap color time-lapse TIRF microscopy.
15 free microtubules
(B) Time sequence of overlaid TIRF microscopy

length [µm]
10
images of PRC1–Alexa 647 (red, 5 nM) and a
dynamic Alexa 568–microtubule pair (blue) form-
5 ing an antiparallel overlap taken at the indicated
0
times in min:s. The time-lapse was recorded at 1
0 1 2 3 frame per 4.67 s. Scale bar, 10 mm.
time [min]

growth velocity [µm/min]

E F parallel overlap
fluor. signal in AP overlap
(C) TIRF images showing several microtubules
6 growing from immobilized microtubule seeds, re-
corded 4–9 min after start of the experiment
4
(top), and a kymograph (time-space plot) of a
2 selected microtubule pair (bottom) in the presence
0 of PRC1–Alexa 647. The kymograph begins
0 1 2
time [min]
3 1 min after start of the experiment. Left: sche-
matic illustration of the microtubule configurations
antiparallel overlap visible in the image and kymograph: microtubule

scheme
segments (gray, plus ends labeled with ‘‘+’’)
2
growing from seeds (white) form antiparallel (red)
microtubule image
[x104 au]

before time-lapse or parallel (green) overlaps. Parallel overlaps

form from parallel seed bundles. Right: Overlaid
1
and single-channel TIRF microscopy images and
kymographs for PRC1–Alexa 647 and Alexa 568
0 microtubules as indicated. Concentrations and
0 10 20 30 frame rate as in (B). Horizontal scale bars, 10 mm.
PRC1 concentration [nM]
G 100 kymograph of
PRC1 time-lapse
Vertical scale bar, 1 min.
(D) Average length (top) and average instantaneous
growth velocity (bottom) of antiparallel microtubule
overlaps (red, n = 7) and of individual microtubules
1 − CDF

10-1 not part of a pair from the same experiment (blue,

n = 14) forming in the presence of PRC1–Alexa
647 as a function of time. For antiparallel microtu-
microtubule image bule overlaps t = 0 is the moment of the plus end-
10-2
0 2 4 6 8 10 12 after time-lapse to-plus end encounter. Error bars are standard
dwell time [s] PRC1 (single molecules)
deviation (SD). Concentrations are as in (B).
microtubules
(E) Average fluorescence signal of PRC1–Alexa
647 bound to antiparallel microtubule overlaps
plotted against the total PRC1–Alexa 647 concentration. Data (black points) were fitted either using the Hill equation (black, straight line) or a hyperbolic function
(red, dotted line). At least a total of 100 mm of antiparallel overlap were evaluated per condition. Error bars are standard error of the mean (SEM).
(F) Single-molecule imaging of PRC1–Alexa 647 in a parallel (left) and an antiparallel (right) microtubule pair. Top: schematic representation of the pairs. Below:
kymographs, positioned between TIRF microscopy images of microtubule overlaps before (top) and after (bottom) fast recording of individual PRC1–Alexa 647
molecules, showing the behavior of individual PRC1–Alexa 647 molecules (green). The PRC1–Alexa 647 concentration was 50 pM. Frame rate was 1 frame per
100 ms. Horizontal scale bar, 10 mm. Vertical scale bar, 5 s.
(G) Dwell time distribution plotted as 1  cumulative distribution function (CDF) of individual PRC1–Alexa 647 molecules in antiparallel overlaps (black, n = 687).
The red dashed curve is a biexponential fit with characteristic times t1 = 0.91 ± 0.02 s and t2 = 4.47 ± 0.24 s (mean ± 95% confidence interval [CI]), and relative
amplitudes 87% t1 and 13% t2. The black dashed curve is a monoexponential fit. The mean bleaching time for Alexa 647 was 32.0 ± 4.5 s. Consequently t1 is
practically unaffected by bleaching and the corrected t2 is 5.2 s.
See also Figure S1 and Movie S1.

fragment, Xklp506–GFP, that does not interact with PRC1 (Fig- interaction between Xklp1 and PRC1 is required for PRC1-depen-
ure 2G, lane 3) failed to accumulate in antiparallel microtubule dent recruitment of Xklp1 to antiparallel microtubule overlaps.
overlaps despite the presence of PRC1–Alexa 647, as observed The most striking observation in the presence of both midzone
by TIRF microscopy (Figure 2H, right). This shows that a direct proteins was, however, that the growth of microtubule plus ends

422 Cell 142, 420–432, August 6, 2010 ª2010 Elsevier Inc.

being part of an antiparallel overlap slowed down until it ceased
completely in all observed cases (n > 100) (Figure 2A, kymograph
in 2B, 2H, left, Movie S2, Movie S3). The growth of overlap
microtubules gradually stopped within about 1 min after the
end-to-end encounter (Figure 2I, green). In contrast, individual
microtubules not engaged in overlaps in the same experiment
continued growing with constant velocity of 2.1 ± 0.3 (mean ±
SD) mm/min (Figure 2I, blue). Inhibition of antiparallel microtubule
overlap growth was a consequence of the simultaneous pres-
ence of PRC1 and full-length Xklp1. The presence of neither
PRC1 alone (Figures 1B–1D) nor Xklp1 alone (Figure 2F) had a
significant effect on microtubule growth in antiparallel overlaps.
Hence, stable antiparallel overlaps with a constant size of several
micrometers formed only in the presence of both midzone pro-
teins (Figures 2A, 2B, and 2I; Movie S3). Such antiparallel micro-
tubule overlaps maintained a constant size over long periods of
time despite the complete inhibition of microtubule growth. This
is remarkable, given that slowdown of microtubule growth
usually results in frequent transitions to microtubule depolymer-
ization (catastrophes) (Arnal et al., 2004; Janson et al., 2003;
Walker et al., 1988).
Our results show that a ‘‘minimal midzone’’ can be reconsti-
tuted in vitro with dynamic microtubules and two proteins only:
PRC1 is necessary and sufficient to recognize and bundle
antiparallel microtubules. Xklp1 alone does not bind to antipar-
allel overlaps but is instead recruited by overlap-associated
PRC1. This recruitment results in local inhibition of microtubule
growth without causing microtubule depolymerization, leading
to antiparallel microtubule overlaps of constant size.
Xklp1 Inhibits Turnover of Tubulin at the Growing
Microtubule End
To better understand how Xklp1 affects microtubule dynamics,
we measured the effect of varying concentrations of Xklp1
alone on the dynamics of individual microtubules. Similar to an
N-terminal fragment of Xklp1 (Bringmann et al., 2004), full-length
Xklp1 alone reduced the growth velocity of microtubule plus
ends in a concentration-dependent manner (Figures 3A and 3B).
This effect could only be observed at Xklp1 concentrations of
several hundred nanomolar, well above the Xklp1 concentra-
tions in the lower nanomolar range that lead to inhibition of
microtubule growth in antiparallel overlaps in the presence of
PRC1. Interestingly, the catastrophe frequency remained very
low at increasing Xklp1 concentrations (Figure 3C), despite the
strong reduction in microtubule growth velocity, which is ex-
pected to lead to more frequent catastrophes (Arnal et al.,
2004; Janson et al., 2003; Walker et al., 1988). The effect of
Xklp1 on the catastrophe frequency is more evident when
comparing microtubules growing with similar velocities (Fig-
ure 3D). The catastrophe frequencies of microtubules in the
presence of Xklp1, plotted as a function of their growth velocities
(Figure 3D, red), are significantly lower than those of microtu-
bules growing at similar velocities in the absence of Xklp1 at
reduced tubulin concentrations (Figure 3D, blue). This shows
that Xklp1 inhibits catastrophes, explaining why local inhibition
of microtubule growth by PRC1-recruited Xklp1 in antiparallel
microtubule pairs (Figures 2A, 2B, and 2I) does not result in
destabilization of the overlap.
To gain a better understanding of the effect of Xklp1 on the
microtubule catastrophe frequency, we measured the microtu-
bule growth velocity at different tubulin concentrations with
and without 300 nM Xklp1 (Figure 3E). This allowed us to deter-
mine the association and dissociation rate constants of guano-
sine-50 -triphosphate (GTP)-tubulin at the growing plus end
(Drechsel et al., 1992; Walker et al., 1988). We found that
300 nM Xklp1 reduced the on-rate of GTP-tubulin by a factor
of about two and the off-rate by a factor of around three (Fig-
ure 3F). This shows that Xklp1 has the unique property of making
microtubules less dynamic by reducing the overall turnover of
GTP-tubulin at growing microtubule ends, distinguishing it
from other known mitotic regulators of microtubule dynamics
(Brouhard et al., 2008; Helenius et al., 2006; Varga et al., 2006).
Dynamic Control of Antiparallel Microtubule Overlap
Length
To investigate how the inhibitory effect of Xklp1 on tubulin turn-
over affects the length of antiparallel microtubule overlaps in the
presence of PRC1, we varied the Xklp1:PRC1 ratio in antiparallel
microtubule encounter experiments, leaving the PRC1 concen-
tration constant (Figure 4A, Movie S5). At Xklp1:PRC1 ratios lower
than 1, microtubule plus-end growth persisted over long times
(>10 min) after formation of an antiparallel overlap, albeit at greatly
reduced speeds as compared to microtubules that were not part
of an overlap (Movie S5). At Xklp1:PRC1 ratios R1, microtubule
plus ends within the antiparallel overlap stopped growing soon
after their initial encounter leading to stable antiparallel overlaps
with a distinct characteristic steady-state length for each
Xklp1:PRC1 ratio (Figure 4B). The average steady-state overlap
length decreased with increasing Xklp1 concentration, ranging
from 4.2 mm to 1.2 mm for ratios from 1 to 20, respectively (Fig-
ure 4B). Thus the concentration of Xklp1 determines the steady-
state length of the antiparallel microtubule overlap.
Next, we investigated whether the steady-state overlap
lengths could reversibly respond to changes in the Xklp1 con-
centration (Figure 4C). We preformed short overlaps at high
Xklp1:PRC1 ratios, followed by reduction of the Xklp1 concen-
tration, keeping the PRC1 and tubulin concentrations constant.
Strikingly, we observed that microtubules of short preformed
overlaps started to grow again until they had reached the length
characteristic for the new Xklp1:PRC1 ratio (Figures 4C–4E,
Movie S6). These results demonstrate that the Xklp1:PRC1 ratio
can dynamically control the steady-state overlap length.
Why do higher Xklp1:PRC1 ratios result in shorter antiparallel
overlaps? The major contribution of Xklp1 in determining overlap
length could be either its inhibitory effect on microtubule growth
or, alternatively, a consequence of Xklp1 sliding apart antiparallel
microtubules, similar to kinesin-5 (Kapitein et al., 2005). Indeed,
we observed that microtubules engaged in antiparallel overlaps
frequently buckled over time (Figure S3A), indicative of plus-
end-directed microtubule sliding. The average sliding length
(Extended Experimental Procedures) was, however, in all cases
short as compared to the overlap length at steady state (Figure 4B).
A plot of the individual sliding lengths of all microtubule pairs
against their individual overlap length also did not reveal any corre-
lation (Figure S3B). Therefore, antiparallel microtubule sliding
does not significantly contribute here to overlap length control.
Cell 142, 420–432, August 6, 2010 ª2010 Elsevier Inc. 423
 Xklp1 + PRC1
A microtubules C I
20

PRC1 signal in AP overlap

0:00

growth velocity [µm/min]

antiparallel overlap
7.5 free microtubules
0:23 15 6

[x103au]

length [µm]
0:47 10 5 4
1:10
5 2.5 2
1:33
0 0 0
1:57 - Xklp1 + Xklp1 0 1 2 3 0 1 2 3
time [min] time [min]

Xklp1 + PRC1
B scheme microtubules PRC1 Xklp1 microtubules

D Xklp1 E G Beads Input

microtubules M 1 2 3 M 1 2 3 M
250 kDa
8
Xklp1 signal in AP overlap

0:00 Xklp1-GFP
150 kDa

0:23 6
100 kDa
[x103au]

PRC1-SNAP
0:47 Xklp1506-GFP
4 75 kDa

1:10
2
tubulin 50 kDa
1:33
0 37 kDa
1:57 +PRC1 - PRC1

F Xklp1 H Xklp1 + PRC1 Xklp1506+ PRC1

scheme microtubules Xklp1 microtubules microtubules microtubules

Figure 2. A Minimal Protein System Forms Stable Antiparallel Overlaps In Vitro

(A) Time sequence of overlaid TIRF microscopy images of PRC1–Alexa 647 (red, 5 nM) and Xklp1–GFP (green, 15 nM) in a dynamic Alexa 568-labeled microtubule
pair (blue) forming an antiparallel overlap at the indicated times in min:s.
(B) TIRF images, recorded 4–9 min after the start of the experiment, showing several microtubules growing from immobilized microtubule seeds (top) and a kymo-
graph of a selected microtubule pair (bottom). Left: schematic illustration of the microtubule configurations. Color coding as in Figure 1C. Right: overlaid and
single-channel TIRF microscopy images and kymographs for PRC1–Alexa 647, Xklp1–GFP, and Alexa 568 microtubules as indicated. Concentrations as in (A).
(C) Average fluorescence intensity signal of PRC1–Alexa 647 bound to antiparallel microtubule overlaps in the presence (n = 45 overlaps) or absence (n = 18
overlaps) of Xklp1–GFP, obtained from intensity line scans. Error bars are SEM.
(D) Time sequence of overlaid TIRF images of 15 nM Xklp1–GFP (green) and a dynamic Alexa 568-microtubule pair (blue) forming an antiparallel overlap taken at
the indicated times in min:s.

424 Cell 142, 420–432, August 6, 2010 ª2010 Elsevier Inc.

A Figure 3. Xklp1 Inhibits Turnover of Tubulin at the
Growing Microtubule End
(A) Representative kymographs of Alexa 568-labeled
microtubules growing in the absence or presence of
Xklp1–GFP (not shown) at the indicated concentrations.
Concentration of soluble tubulin is 17.5 mM. The frame
rate was 1 frame per 3 s. Horizontal scale bar, 10 mm.
Vertical scale bar, 1 min.
(B) Average microtubule growth velocity and (C) catas-
trophe frequency plotted against the total Xklp1–GFP
concentration. Sixty microtubules from two independent
experiments were analyzed per condition.
0 nM Xklp1 300 nM Xklp1 600 nM Xklp1
(D) Catastrophe frequency as a function of microtubule
growth velocity. Red data points are from experiments
B C D with 17.5 mM tubulin and varying Xklp1–GFP concentra-
3 3 tions, as shown in (B) and (C). Blue data points are from
growth velocity [µm/min]

-Xklp1
catastrophe frequency
catastrophe frequency

10 +Xklp1 experiments with varying tubulin concentrations (7, 12,

[10-2 events/min]
[10-2 events/min]

2 2 and 17.5 mM) in the absence of Xklp1–GFP.

(E) Average growth rate in the absence (blue circles) or in
5 the presence of 300 nM Xklp1–GFP (red circles) as a func-
1 1
tion of total tubulin concentration. A multiple linear regres-
sion model with a categorical factor g (+Xklp1: g = 0,
0 0 0 Xklp1: g = 1) revealed that both the abscissa and the
0 200 400 600 0 200 400 600 0 1 2 3
Xklp1 concentration [nM] Xklp1 concentration [nM] growth velocity [µm/min] slope were significantly different (p < 0.001, see Experi-
mental Procedures). The resulting regression lines repre-
sent vg = kon c – koff with the net assembly rate vg in tubulin
E F
dimers per second and the time constants for association
150

20

-Xklp1 kon koff

+Xklp1 [µM-1 s-1] [ s-1] kon in s1mM1 and dissociation koff in s1 of GTP-tubulin
10
growth rate [dimers/s]

4.03 ± 0.04 9.60 ± 0.85 at the growing microtubule end. Inset: regression lines
0

- Xklp1
100

[3.951 ; 4.101] [8.626 ; 10.571] and their 95% confidence intervals (dashed) at the origin,
-10

0 4 8
1.86 ± 0.02 3.08 ± 0.49 showing that not only kon (slope) but also koff (intersection
+ 300 nM Xklp1
[1.820 ; 1.906] [2.125 ; 4.027] with abscissa) is different for the two conditions. 185 to
50

210 microtubules from two independent experiments were

analyzed for each tubulin concentration per condition.
(F) Values for kon and koff (mean ± SEM, with 95% confi-
0

dence in brackets) as obtained from fits in (E).

0 5 10 15 20 25 30 35
tubulin concentration [µM]
Error bars in (B)–(E) are SD.

Xklp1 Is a Processive Motor estingly, accumulation of Xklp1 at these free microtubule ends
To elucidate the mechanism by which Xklp1 sets the size of also resulted in inhibition of microtubule growth (Movie S7).
antiparallel microtubule overlaps, we inspected the behavior This suggests that (1) it is the plus-end-associated fraction
of the motor more closely. Occasionally, antiparallel overlaps of Xklp1 that inhibits microtubule growth and (2) Xklp1 can
formed by lateral encounters between one microtubule end target the plus end by means of processive motility over con-
and one microtubule segment distant from the growing end siderable distances. This is surprising, given that an N-terminal
(Figure 5A). In such cases, Xklp1–GFP loaded onto the overlap fragment of Xklp1 has been proposed to be at most weakly
region by PRC1 also accumulated at the plus end of the processive based on enzymatic activity assays (Bringmann
microtubule extending beyond the overlap (Figure 5A). Inter- et al., 2004).

(E) Average fluorescence intensity signal of Xklp1–GFP bound to the antiparallel microtubule overlap in the presence (n = 42 overlaps) or absence of PRC1–Alexa
647, as obtained from intensity line scans. Error bars are SEM.
(F) Kymograph of the microtubule pair shown in (D). Color code as in (B).
(G) Pull-down of Xklp1 by PRC1–SNAP immobilized on beads. Coomassie-stained SDS-gel shows fractions bound to the beads after incubation with different
input solutions. Lanes 1: negative control with beads lacking PRC1–SNAP incubated with a mixture of 2 mM full-length Xklp1–GFP, 2 mM truncated Xklp1506–GFP,
and 5 mM soluble tubulin. Only weak nonspecific binding of the two Xklp1 proteins is seen. Lanes 2 and 3: beads with immobilized PRC1–SNAP incubated with
either 2 mM full-length Xklp1–GFP (lanes 2) or truncated 2 mM Xklp1506–GFP (lanes 3) in the presence of 5 mM soluble tubulin.
(H) Representative kymographs of antiparallel microtubule overlaps (blue) in the presence of PRC1–Alexa 647 (red, 5 nM) and either full-length Xklp1–GFP
(green, 15 nM) (left) or truncated Xklp1506–GFP (green, 15 nM) (right).
(I) Average length (left) and average instantaneous growth velocity (right) of antiparallel overlaps (green, n = 9) and individual microtubules that are not part of a pair
from the same experiment (blue, n = 18) formed in the presence of PRC1 and full-length Xklp1 as a function of time (conditions as in A and B). For comparison, the
length of microtubule overlaps formed in the presence of only PRC1 (from Figure 1D) is shown (red dashed line). t = 0 is the moment of plus end-to-plus end
encounter. Error bars are SD. Concentrations are as in (A). Horizontal scale bars are 10 mm. Vertical scale bars are 1 min. The frame rate for all time-lapse movies
was 1 frame per 4.67 s. Kymographs start 1 min after the start of the experiment.
See also Figure S2 and Movie S2, Movie S3, and Movie S4.

Cell 142, 420–432, August 6, 2010 ª2010 Elsevier Inc. 425

A B Figure 4. Dynamic Control of Antiparallel Overlap
Xklp1 PRC1 microtubles
Length
overlap length
20 : 1 sliding length (A) Representative triple-channel TIRF microscopy images
Xklp1 : PRC1 of PRC1–Alexa 647 (red, 5 nM) and Xklp1–GFP (green,
4

length [µm]
100, 25, and 5 nM) at PRC1:Xklp1 ratios as indicated in
a dynamic Alexa 568–microtubule pair (blue) forming an
5:1
antiparallel overlap. Images were recorded 7–12 min after
Xklp1 : PRC1
2 start of the experiment. Scale bar, 10 mm.
(B) Average overlap length (red) or average sliding length
1:1 (blue) as a function of the Xklp1:PRC1 ratio. Error bars
Xklp1 : PRC1
0
are SD. Overlap lengths and sliding lengths were
1 2 3 5 10 20 measured for at least 15 overlaps from at least three
Xklp1:PRC1 ratio experiments per condition after steady state had been
reached. Yellow shading indicates the regime where no
steady-state length was established.
imaging
dilution

C SS 1 overlap growth SS 2 D (C) Reversibility experiment: Antiparallel microtubule over-

Xklp1 PRC1 microtubles laps were allowed to form at 100 nM Xklp1–GFP and 5 nM
PRC1–Alexa 647 (Xklp1:PRC1 ratio 20:1) and to reach
00:00 their steady-state length (black dot with SD bars in red
overlap length [µm]

area). Subsequently, Xklp1–GFP was diluted to 5 nM

4
01:22 keeping the PRC1–Alexa 647 and tubulin concentrations
constant (new Xklp1:PRC1 ratio 1:1, details in Experi-
02:44 mental Procedures). Quantification of overlap lengths
2
versus time for three examples (dark green, blue, and
red dots in green area) shows that after 6 min the over-
03:06
laps reach a new steady-state length characteristic for
0 the new, lower Xklp1 concentration (purple area).
0 3 6 9
time [min] 04:28 (D) Example time series of overlaid triple-channel TIRF
E microscopy images at the indicated times (min:s) after
06:00 Xklp1–GFP dilution and (E) corresponding kymographs
showing a representative growing overlap. The frame rate
was 1 frame per 4.67 s. Colors and horizontal scale bar as
in (A). Vertical scale bar, 1 min. See also Figure S3 and
Movie S5 and Movie S6.
merge PRC1 Xklp1

We therefore imaged single molecules of full-length Xklp1–GFP to the other microtubule of the pair (Figure 6A, left, Figure S5A,
on stabilized, individual microtubules in the absence of PRC1. We Movie S8). This highly mobile behavior of the motor was very
observed that at low ionic strength Xklp1–GFP dimers indeed different from that of single PRC1–Alexa 647 molecules (5 pM)
moved processively (Figure 5B) with an average run length of 1.2 in the presence of excess PRC1–Alexa 488 (5 nM) in the overlap,
mm (Figure 5C) and an average velocity of 0.8 mm/s (Figure 5F, which only diffused for very short distances (Figure 6A, right). The
red). Reduction in ionic strength was necessary because binding dwell time distribution of Xklp1–GFP in PRC1-containing antipar-
of Xklp1 to individual microtubules was very weak. Processivity allel overlaps was roughly exponential (Figure 6B, black dots)
was an intrinsic property of the dimerized motor domain, as with a calculated average dwell time of 5.9 s (dashed red line).
demonstrated by imaging a GFP-labeled motor fragment (Figures This demonstrates that Xklp1 dwells longer in a PRC1-decorated
5D–5G). Run length (0.9 mm, Figure 5E) and velocity (0.9 mm/s, antiparallel overlap as compared to on individual microtubules in
Figure 5F, black) of Xklp1506–GFP were in a similar range as the the absence of PRC1 at low ionic strength (Figure 6B, gray
values for the full-length motor. The longer dwell times of the circles), and also longer than the individual PRC1 molecules in
full-length motor (Figure S4A) are probably a consequence of the antiparallel overlap (Figure 6B, blue dots). This shows that
a secondary microtubule-binding site (Figure S4B). These results PRC1 increases the residence time of Xklp1 in the overlap prob-
demonstrate that Xklp1 is a processive motor, suggesting that it ably by offering additional binding sites. Mean squared displace-
might measure the overlap length by processive movement. ment (MSD) analysis confirmed that Xklp1 was very mobile in
PRC1-containing overlaps (Figure 6C, black dots), in contrast
Xklp1 Determines Overlap Length by a Processive to slowly diffusing PRC1 molecules (Figure 6C, blue dots;
Overlap Exploration Mechanism Figure S1C). However, Xklp1 explored shorter distances in
To test the hypothetical mechanism of processive overlap PRC1-containing overlaps than on individual microtubules in
length measurement, we imaged single full-length Xklp1–GFP the absence of PRC1 under low ionic strength conditions
molecules (500 pM) in the presence of excess PRC1–Alexa 647 (Figure 6C, gray dots). The MSD curve of Xklp1 in antiparallel over-
(5 nM) in dynamic antiparallel microtubule pairs at high time reso- laps could be well described using a ‘‘persistent random walk’’
lution. Strikingly, Xklp1–GFP was very mobile in PRC1-containing model (Figure 6C, dashed red line, Extended Experimental Proce-
antiparallel overlaps. Xklp1–GFP showed processive movement dures) (Othmer et al., 1988; Tranquillo and Lauffenburger, 1987). A
often abruptly changing direction, indicating a transition from one fit to the data yielded a characteristic persistence time between

426 Cell 142, 420–432, August 6, 2010 ª2010 Elsevier Inc.

A Xklp1 PRC1 microtubles scheme
Figure 5. Xklp1 Is a Processive Motor
(A) Time sequence (min:s) of multichannel TIRF micros-
copy images of PRC1–Alexa 647 (red, 5 nM) and Xklp1–
GFP (green, 15 nM) in a dynamic Alexa 568-labeled
+ microtubule pair (blue) forming an antiparallel overlap by
a lateral encounter between one microtubule plus end
and one microtubule segment distant from its growing
end, as illustrated in the scheme (right). Scale bar, 5 mm.
+
(B) Representative kymograph of individual full-length
Xklp1–GFP dimers (green, 2.5 nM) on an individual stabi-
00:00 00:47 01:33 02:20 03:07 lized microtubule (red) at low ionic strength (see Extended
Experimental Procedures) imaged at high time resolution
(10 frames/s). Horizontal scale bars, 10 mm. Vertical
B Xklp1 microtubles
D Xklp1506 microtubles
F scale bar, 5 s.
35
Xklp1 (C) Histogram of the run length distribution of full-length
30 fit, v = 0.82 µm/s Xklp1–GFP with a monoexponential fit (red dashed);
Xklp1506
MSD [µm2] 25 fit, v = 0.93 µm/s mean run length was 1.20 mm (95% CI in brackets).
20 (D) Representative kymograph of individual Xklp1506–GFP
15 dimers (green, 20 nM) on an individual taxol-stabilized
microtubule (red) at very low ionic strength conditions
10
(see Extended Experimental Procedures). Imaging and
5
bars as in (B).
0
0 1 2 3 4 5 6 (E) Histogram of the run length distribution of Xklp1506–
time [s] GFP with a monoexponential fit (red dashed); mean run
C E G length was 0.91 mm (95% CI in brackets).
400 300 300
Xklp1 Xklp1506 (F) Mean squared displacement (MSD) curve of full-length
L = 1.20 µm L = 0.91 µm
300 [1.122 ; 1.273] [0.860 ; 0.967] Xklp1–GFP (red) and truncated Xklp1506–GFP (black) and
Xklp1
200 200 Xklp1506 parabolic fits (dashed lines, see Experimental Proce-
counts

counts
counts

dKin401
200 dures). Error bars are SEM.
(G) Histograms of the initial brightness of Xklp1–GFP,
100 100
100 Xklp1506–GFP, and dimeric Kin401–GFP as a control (Telley
et al., 2009), indicating that both fragment and full-length
0 0 0
Xklp1 are dimeric.
0 5 10 15 20 0 5 10 15 20 2 4 6 8 10 12 14 16 18 20 See also Figure S4 and Movie S7.
run length [µm] run length [µm] intensity [102 au]

directional switches of 2.3 s and a velocity of 0.5 mm/s for average exploratory range limits the maximum size of the
Xklp1 in the overlap. For time intervals larger than the persistence steady-state antiparallel microtubule overlap length to 5–6 mm
time, the movement of Xklp1 was effectively diffusive. (Figure 4B, Figure 6D). This defines a processive exploration
To estimate which distances Xklp1 can explore in PRC1- mechanism for antiparallel microtubule overlap length control.
decorated antiparallel microtubule overlaps, we calculated the
distribution of average exploratory distances (Figure 6D, black DISCUSSION
dots, Extended Experimental Procedures). This analysis reveals
that Xklp1 can explore average lengths of up to 6 mm. This is The Mechanism of Antiparallel Overlap Generation
in strong contrast to the low exploratory distances of PRC1 and Length Control by PRC1/Kinesin-4 Explains
(Figure 6D, blue dots), which can only move for distances of up Anaphase Spindle Characteristics In Vivo
to 0.2 mm. Strikingly, the average exploratory distances of We have elucidated a molecular mechanism capable of selective
individual Xklp1 molecules coincide well with the range of formation and adaptable length control of antiparallel microtubule
steady-state lengths of antiparallel microtubule overlaps (Fig- overlaps by two conserved midzone proteins. Central to this
ure 4B). This suggests that, by processive movement, Xklp1 rea- mechanism is the combination of four distinct molecular activities
ches the plus ends of antiparallel overlapping microtubules, (Figure 7): (1) bundling of microtubules with high selectivity for
where it can modulate microtubule dynamics. To test whether antiparallel orientation by PRC1, (2) recruitment of kinesin-4 by
the processive nature of Xklp1’s motility is indeed central to the PRC1 to antiparallel overlaps, and (3) processive microtubule
mechanism of steady-state length establishment, we replaced plus-end-directed motility together with (4) an inhibitory effect
adenosine-50 -triphosphate (ATP) by adenosine-50 -diphosphate on microtubule growth by the motor protein kinesin-4. The combi-
(ADP) in the TIRF microscopy experiment, thereby preventing nation of these activities constitutes a minimal two-component
processive motility of Xklp1 but not its recruitment by PRC1. We system capable of formation of a minimal midzone in vitro.
observed that under these conditions the two midzone proteins Our in vitro reconstitution explains why loss of either PRC1 or
failed to produce stable steady-state overlap lengths (Figure S6). kinesin-4 leads to nonequivalent defects in midzone formation
Instead, overlap microtubules continued growing despite the in vivo (Kurasawa et al., 2004; Mollinari et al., 2002; Zhu and
presence of PRC1 and Xklp1 in the overlap. We conclude that Jiang, 2005). We show that selective antiparallel microtubule
processive overlap exploration allows Xklp1 to reach and modu- crosslinking is an intrinsic property of vertebrate PRC1, an
late the behavior of microtubule plus ends and that its finite activity which appears to be evolutionarily conserved (Janson

Cell 142, 420–432, August 6, 2010 ª2010 Elsevier Inc. 427

A Xklp1 in overlap PRC1 in overlap Figure 6. Xklp1 Determines Antiparallel Microtu-
bule Overlap Length by Processive Overlap
scheme Exploration
(A) Single-molecule imaging of either individual Xklp1–
microtubule/PRC1 image
before time-lapse GFP molecules (500 pM, green) in a PRC1–Alexa 647
(5 nM, blue) containing antiparallel microtubule overlap
(red) (right panel) or individual PRC1–Alexa 647 molecules
(5 pM, green) in a PRC1–Alexa 488 (5 nM, blue) containing
antiparallel microtubule overlap (red) (left panel). Scheme
(top) and triple-channel TIRF microscopy images of the
microtubule pair before (upper image) and after (lower
image) fast time-lapse imaging of individual Xklp1–GFP
or PRC1–Alexa 647 molecules, as illustrated in a kymo-
graph (middle). The frame rate was 10 s1. Horizontal
kymograph of single-
molecule time-lapse scale bar, 10 mm. Vertical scale bar, 5 s.
(B) Dwell time distribution (shown as 1 – CDF) of single
Xklp1–GFP molecules moving in antiparallel microtubule
overlap regions (conditions as in A, right; black dots, n =
226) and a monoexponential fit (dashed red); mean dwell
time is 5.85 ± 0.12 s (mean ± 95% CI). For comparison,
the distributions for Xklp1–GFP on single taxol-microtu-
bules (gray circles, under conditions as in Figure 5B) and
for PRC1–Alexa 647 in a PRC1–Alexa 488-containing anti-
parallel overlap (blue dots, under conditions as in A, left)
microtubule/PRC1 image are shown.
after time-lapse
(C) Mean squared displacement (MSD) curves for the same
Xklp1 (single molecules) PRC1 (single molecules) conditions as shown in (B), and a fit to the data of Xklp1–
microtubules microtubules
GFP in antiparallel overlaps using the ‘‘persistent random
PRC1 (bulk protein in overlap) PRC1 (bulk protein in overlap)
walk’’ model, yielding persistence time Tp = 2.31 ± 0.68 s
and mean velocity v = 0.52 ± 0.06 mm/s (mean ± 95% CI,
30 Xklp1 in overlap
B 100 C Xklp1 on single MT
see Experimental Procedures). Error bars are SEM.
Xklp1 in overlap 25 PRC1 in overlap (D) Calculated probability distribution of the average
Xklp1 on single MT exploratory distance (black dots, see Extended Experi-
PRC1 in overlap 20
MSD [µm2]

mental Procedures) of Xklp1–GFP molecules in antiparallel

1−CDF

10-1
15 overlaps and the predicted probability curve (dashed red)
calculated from the persistent random walk model using
10
the parameter values as obtained from the fits in (B) and
5 (C). For comparison, calculated average exploratory
0 distance distributions are plotted for Xklp1–GFP on single
10-2
0 5 10 15 20 25 30 0 2 4 6 8 10 12 14 16 stabilized microtubules and PRC1–Alexa 647 in overlaps
dwell time [s] time [s] (as in B and C). Inset: semi-logarithmic plot of average
D 1.0
100 exploratory distances.
See also Figure S5, Figure S6, and Movie S8.
calculated probability

0.8 10-1

0.6
10-2
0.4
0 2 4 6 8 10 Xenopus kinesin-4 Xklp1 binds only weakly to
Xklp1 in overlap microtubules. However, by interacting with
Xklp1 on single MT
0.2 PRC1 in overlap PRC1, Xklp1 is recruited to antiparallel microtu-
0
bule overlaps, reminiscent of selective recruit-
0 2 4 6 8 10 ment of sliding motors of the kinesin-5 and kine-
avg. exploratory distance [µm]
sin-6 families by Ase1 in budding yeast and
fission yeast, respectively (Fu et al., 2009; Khme-
et al., 2007). Autonomous bundling of antiparallel microtubules linskii et al., 2009). This recruitment results in the local inhibition of
places PRC1 at the core of the activities essential for midzone microtubule plus-end growth in antiparallel overlaps by Xklp1,
formation and explains why PRC1 is absolutely required for the leading to a defined steady-state overlap length. This explains
bundling and stabilization of antiparallel microtubule overlaps, why the midzone unnaturally extends but half-spindles remain
as indicated by the complete loss of central anaphasic microtu- connected upon loss of kinesin-4 in vivo, resulting in long, distorted
bule bundles in the absence of PRC1 in vivo (Kurasawa et al., anaphase spindles (Kurasawa et al., 2004; Zhu and Jiang, 2005).
2004; Mollinari et al., 2002; Zhu and Jiang, 2005).
Vertebrate PRC1 has previously been shown to interact with Processive Overlap Exploration by Kinesin-4 Ensures
kinesin-4 during anaphase, an interaction necessary for proper Adaptive Overlap Length Control
midzone organization (Kurasawa et al., 2004; Zhu and Jiang, Xklp1 reduces the dynamicity of microtubules by lowering the
2005). Here, we establish a hierarchy of interactions: full-length turnover of tubulin at the growing microtubule end. On individual

428 Cell 142, 420–432, August 6, 2010 ª2010 Elsevier Inc.


MT seed growth
Recognition
- + MT pair
+ -
PRC1 single MT
+ -
Recruitment - +
+
- from an extended region near the end of stabilized microtubules
Xklp1
+ -
Processive - +
- ‘‘antenna mechanism’’ to be effective under conditions where
Exploration +
+ -
STOP
Growth -
Inhibition STOP - why Xklp1 is a fast motor in comparison to other mitotic kinesins.
+ -
Figure 7. A Two-Component System Controls Antiparallel Microtu-
bule Overlaps
Model illustrating schematically the activities that lead in their combination to
antiparallel microtubule overlap formation and length control (see Discussion).
microtubules this activity can be observed in vitro only at highly
elevated Xklp1 concentrations (several hundred nanomolar) well
above physiological levels (Bieling et al., 2010a). In contrast,
recruitment of Xklp1 to antiparallel microtubule overlaps by
PRC1 results in complete inhibition of microtubule growth
already at lower, close-to physiological concentrations at which
individual microtubules remain unaffected in their growth. As
a consequence, specific recruitment of Xklp1 ensures the local
inhibition of growth of only those microtubules engaged in anti-
parallel, PRC1-decorated microtubule overlaps. Activation of
the PRC1/kinesin-4 system, through dephosphorylation at
anaphase onset, therefore results in tight bundling and growth
inhibition of antiparallel microtubule overlaps in the central
spindle (Kurasawa et al., 2004; Mollinari et al., 2002; Zhu and
Jiang, 2005), whereas microtubules residing outside of the mid-
zone are likely not affected by the PRC1/kinesin-4 system.
Xklp1 motors move processively with velocities of around
0.5 mm/s within the PRC1-decorated antiparallel microtubule
overlap (Figure 6C, Figure S5B). The drag force exerted by
PRC1 on Xklp1 can be estimated to be low (Extended Experi-
mental Procedures) compared to typical stall forces of kinesins
(Carter and Cross, 2005; Svoboda and Block, 1994), explaining
why Xklp1 is slowed down only mildly by interacting with
PRC1. This explains why Xklp1 efficiently explores the antipar-
allel overlap instead of generating substantial microtubule sliding
forces. This contrasts Xklp1 to antiparallel microtubule sliding
motors like kinesin-5, which reside relatively static between
the sliding microtubules (Kapitein et al., 2005; Kapoor and
Mitchison, 2001; Miyamoto et al., 2004; Uteng et al., 2008).
How does processive movement of Xklp1 within the antipar-
allel overlap region result in length control? Accumulation of
Xklp1 at growing microtubule ends (Figure 5A) is likely to be
responsible for the dose-dependent inhibition of microtubule
growth (Figure 4). The targeting of Xklp1 to growing microtubule
ends is reminiscent of the manner by which the microtubule de-
polymerizing kinesins Kip3 (kinesin-8) (Varga et al., 2006, 2009)
and MCAK (kinesin-13) (Helenius et al., 2006) reach the ends of
artificially stabilized microtubules. Both proteins are ‘‘collected’’
in vitro either by directional (Kip3) or diffusive (MCAK) motility.
Xklp1 (kinesin-4) is also collected from an extended region,
however, in this case from the microtubule overlap by processive
bidirectional motility (Figure 6A, Figure S5A). For such an
microtubules grow, it is important that the velocity of Xklp1 in
the overlap (0.5 mm/s [Figure 6C, Figure S5B]) is considerably
faster than the velocity with which the microtubules elongate
(35 nm/s in our in vitro experiments and 200 nm/s in vivo
[Rusan et al., 2001; Tournebize et al., 1997]). This might explain
The lowest Xklp1 concentration (5 nM) required to form antipar-
allel microtubule overlaps with stable steady-state lengths
resulted in overlaps of around 5 mm (Figure 4B). This length is in
remarkably good agreement with the maximal exploratory
distance of individual Xklp1 molecules in PRC1-enriched antipar-
allel overlaps (Figure 6D). This suggests that the threshold amount
of Xklp1 required to induce a complete stop of microtubule growth
is reached at this concentration by exploiting the entire explor-
atory range of Xklp1 molecules in the overlap. At higher concen-
trations, the threshold amount is already reached by ‘‘collecting’’
from shorter overlaps, leading to characteristic overlap lengths as
a function of the Xklp1 concentration. In Xenopus egg extract, the
concentration of Xklp1 is approximately 100 nM (Bieling et al.,
2010a). Our experiments predict that such concentrations lead
to overlap lengths of 2 mm (Figure 4B), which is in agreement
with a midzone length of 2–3 mm as measured in vivo (Kurasawa
et al., 2004; Mastronarde et al., 1993; Zhu and Jiang, 2005).
The PRC1/kinesin-4 system offers adaptive, self-regulatory
control of overlap size by introducing a negative feedback
between overlap length and microtubule growth. Shortening
the microtubule overlaps as a consequence of motor-driven
microtubule sliding would result in lowered amounts of kinesin-
4 at the microtubule ends because the motor could be collected
now only from shorter distances. Similar to our reversibility
experiment where we reduced the global concentration of
Xklp1 (Figures 4C–4E), the system would be predicted to dynam-
ically respond to antiparallel sliding resulting in shortening
overlaps by initiating microtubule elongation. Local, selective
inhibition of microtubule growth by kinesin-4 in antiparallel
overlaps at conditions that globally favor microtubule elongation
therefore allows for a dynamic control of overlap length.
Antiparallel Microtubule Overlaps in Yeasts
and Metazoans
Maintenance of stable antiparallel microtubule overlaps in the
spindle center requires coordination between microtubule
(de)polymerization and microtubule sliding. In particular, spindle
elongation during anaphase B must not compromise spindle
stability by potentially reducing the length of the antiparallel
Cell 142, 420–432, August 6, 2010 ª2010 Elsevier Inc. 429
overlap region. Several proposals have been made for mecha-
nisms to locally regulate microtubule dynamics in the anaphase
spindle (Cheerambathur et al., 2007; Gardner et al., 2008; Pear-
son et al., 2006). Interestingly, recent work in fission yeast has
linked the conserved microtubule-binding protein CLASP to
the stabilization of antiparallel microtubule bundles (Bratman
and Chang, 2007). Ase1-dependent recruitment of CLASP to
antiparallel microtubule overlaps has been suggested to prevent
microtubule depolymerization past the overlap by inducing
microtubule rescue (Bratman and Chang, 2007). A similar
PRC1-dependent stabilizing function of CLASP for antiparallel
microtubule overlaps during anaphase appears also to exist in
cultured human cells (Liu et al., 2009).
Although local induction of microtubule rescue might explain
how overlap stability is ensured, it does not readily explain how
microtubule overgrowth is prevented and thus how a defined
overlap size is established. This is especially the case for meta-
zoan systems in which microtubule growth velocities are high
(Kinoshita et al., 2001). Our in vitro experiments have demon-
strated how the PRC1/kinesin-4 system can control antiparallel
overlap length at conditions that globally favor microtubule elon-
gation. It might be the combination of inhibition of microtubule
growth by kinesin-4 and the promotion of rescues by CLASP
that ensures the stabilization and length control of antiparallel
microtubule overlaps in metazoans. The adaptive nature of this
mechanism would allow for independent regulation of antiparallel
microtubule sliding activities at different stages of anaphase.
In contrast to metazoan anaphase spindles, the length of
antiparallel microtubule overlaps does not appear to be tightly
controlled in yeast. Microtubule plus ends are dynamic in fission
yeast anaphase spindles as well as in bundled interphase arrays
(Sagolla et al., 2003). The slower microtubule growth velocities
in yeast might be one reason for less stringent control of the
overlap size in this organism. In addition, yeast uses the proces-
sive motor kinesin-8 (Klp5/6 in fission yeast and Kip3 in budding
yeast) as the major microtubule plus-end-destabilizing factor
(Cottingham et al., 1999; Gupta et al., 2006; West et al., 2001).
Length-dependent destabilization of microtubules by kinesin-8
(Tischer et al., 2009; Varga et al., 2006) might be sufficient for
a less stringent control of overlap stability. This might explain
the absence of kinesin-4 from both fission and budding yeast.
During interphase, fission yeast also uses antiparallel microtu-
bule overlaps as means to organize the microtubule cytoskeleton.
Recently, a model for the formation of such overlaps was
proposed based on the combined action of the sliding motor
Klp2 (a kinesin-14) and Ase1 (Janson et al., 2007). In this model,
antiparallel microtubules slide with respect to each other driven
by minus-end-directed motors targeted selectively to microtu-
bule plus ends. Sliding stops either as a consequence of plus
ends growing beyond the overlap region or possibly by friction ex-
erted by overlap-enriched Ase1 (Janson et al., 2007). This leads to
the formation of antiparallel microtubule overlaps with inverted
polarity and dynamically varying sizes, a mechanism that differs
from that of tight overlap size control during metazoan anaphase.
Conclusion
We have reconstituted a two-component system necessary and
sufficient to form antiparallel microtubule overlaps with precisely
430 Cell 142, 420–432, August 6, 2010 ª2010 Elsevier Inc.
defined length in vitro, an essential process for proper midzone
formation in metazoan anaphase spindles. The in vitro reconsti-
tution allowed us to dissect the mechanism of autonomous
antiparallel microtubule recognition by PRC1 and of local micro-
tubule growth inhibition and overlap length control by kinesin-4.
In the future, the extension of our reconstitution approach by
including other midzone proteins promises to yield further mech-
anistic understanding of aspects of the organization and function
of the late mitotic spindle.
EXPERIMENTAL PROCEDURES
Dynamic Microtubule Overlap Assay
A flow chamber assembled from a biotin-PEG functionalized glass coverslip
and a PLL-PEG passivated glass slide separated by two stripes of double-
sided tape (Bieling et al., 2010b) was filled with a series of solutions: (1)
Pluronic F-127 and k-casein, (2) NeutrAvidin and k-casein, (3) buffer, (4) short
GMP–CPP stabilized, biotinylated brightly labeled microtubule seeds (contain-
ing 26% Alexa Fluor 568-labeled tubulin), (5) buffer. Microtubule growth was
initiated by flowing in dimly labeled tubulin (containing 10% Alexa Fluor
568-labeled tubulin) together with midzone proteins at the indicated concen-
trations. The final assay buffer was 80 mM K-PIPES, pH 6.8, 85 mM KCl,
85 mM K-acetate, 4 mM MgCl2, 1 mM GTP, 2 mM MgATP, 1 mM EGTA,
10 mM b-mercaptoethanol, 0.25% [wt/wt] Brij-35, 0.1% methyl cellulose,
100 mg/ml b-casein, and oxygen scavengers. If not stated otherwise, the
concentrations of proteins were 5 nM PRC1–Alexa 647, 15 nM Xklp1–GFP
or Xklp1506–GFP, and 17.5 mM dimly labeled tubulin.
To test the reversibility of antiparallel microtubule overlap formation, over-
laps were allowed to form in the presence of 5 nM PRC1–Alexa 647, 100 nM
Xklp1–GFP (PRC1:Xklp1 ratio of 1:20), and 17.5 mM dimly labeled tubulin for
7–10 min at 28 C ± 1 C. Then the PRC1:Xklp1 ratio was changed to 1:1 by
flowing in a solution with only 5 nM Xklp1–GFP, leaving all other concentrations
constant.
Multicolor time-lapse imaging using TIRF microscopy was always
performed at 28 C ± 1 C.
Single-Molecule Imaging
Single full-length Xklp1–GFP molecules were imaged at a concentration of
2.5 nM on individual paclitaxel-stabilized, biotinylated, and fluorescently
labeled microtubules (5% Alexa 568-labeled tubulin) in low ionic strength
buffer (80 mM K-PIPES, pH 6.8, 85 mM K-acetate, 4 mM MgCl2, 2 mM MgATP,
1 mM EGTA, 10 mM b-mercaptoethanol, 10 mM paclitaxel, 0.25% [wt/wt]
Brij-35, 100 mg/ml b-casein, and oxygen scavengers). Xklp1506–GFP at 20
nM, or Kin401–GFP at 0.1 nM as a control, was imaged in very low ionic strength
buffer (12 mM K-PIPES, pH 6.8, 2 mM MgCl2, 2 mM MgATP, 1 mM EGTA,
10 mM b-mercaptoethanol, 10 mM paclitaxel, 100 mg/ml b-casein, and oxygen
scavengers).
For imaging individual PRC1–Alexa 647 molecules in dynamic antiparallel
microtubule overlaps, the PRC1–Alexa 647 concentration was lowered to 50
pM or to 5 pM in the presence of 5 nM PRC1–Alexa 488. For imaging individual
Xklp1–GFP molecules in dynamic, PRC1-containing antiparallel microtubule
overlaps, the Xklp1–GFP concentration was lowered to 500 pM.
Data Analysis
The movements of single fluorescent molecules were analyzed using auto-
mated particle tracking (Kalaimoscope, TransInsight, Dresden, Germany)
(Telley et al., 2009). For dwell time distributions, the cumulative distribution
function (CDF) of data was calculated using the ‘‘ecdf’’ function in Matlab
(MathWorks), and either a monoexponential or a biexponential function was
fitted to (1–CDF) data using a ‘‘least-squares’’ approach. Histograms of run
lengths were fitted with a monoexponential function as described (Telley et al.,
2009). MSD curves were calculated and fitted using the function v2 Dt2 + 2D
Dt + offset, with the mean velocity v and the diffusion coefficient D. For the
case of Xklp1 in the overlap the MSD curve was fitted to a ‘‘persistent
random walk’’ model (Othmer et al., 1988; Tranquillo and Lauffenburger,
1987): MSD = 2v2Tp [Dt – Tp (1 – exp(–Dt/Tp)] with the persistence time Tp and
mean velocity v, and hence v2Tp the effective diffusion coefficient. The
‘‘average exploratory distance’’ distribution in microtubule overlaps was
calculated by combining information from the dwell time distribution and the
MSD curve in antiparallel microtubule overlaps, thereby eliminating time.
Microtubule dynamics was determined by kymograph analysis using ImageJ.
The growth versus tubulin concentration relationship in the presence or
absence of Xklp1 was analyzed using a multiple linear regression model with
a categorical factor g (+Xklp1: g = 0, –Xklp1: g = 1) and y = b1 + m1 x + g
(b2 + m2 x). Generally, this analysis provides a significance test for differences
in the slope m and abscissa b of two linear relationships. Here, the slope and
abscissa represent the kinetic parameters kon and koff, respectively, of the
microtubule dynamics.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, six
figures, and eight movies and can be found with this article online at doi:10.
1016/j.cell.2010.06.033.
ACKNOWLEDGMENTS
We thank Vladimir Rybin for analytical ultracentrifugation, Jan Ellenberg,
Elmar Schiebel, Johanna Roostalu, and Scott Hansen for critically reading
the manuscript, and the DFG, HFSPO, the European Commission (STREP
‘‘Active Biomics’’), and the Swiss National Science Foundation for financial
support.
Received: January 4, 2010
Revised: April 19, 2010
Accepted: June 7, 2010
Published: August 5, 2010
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Note Added in Proof
In a manuscript by Subramanian et al., which appears in this issue of Cell, the
authors arrive at similar conclusions regarding the nature of microtubule cross-
links formed by PRC1.
Insights into Antiparallel Microtubule
Crosslinking by PRC1, a Conserved
Nonmotor Microtubule Binding Protein
Radhika Subramanian,1 Elizabeth M. Wilson-Kubalek,2 Christopher P. Arthur,2 Matthew J. Bick,3 Elizabeth A. Campbell,3
Seth A. Darst,3 Ronald A. Milligan,2 and Tarun M. Kapoor1,*
1Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA
2Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
3Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065, USA
*Correspondence: kapoor@mail.rockefeller.edu
DOI 10.1016/j.cell.2010.07.012
SUMMARY
Formation of microtubule architectures, required for
cell shape maintenance in yeast, directional cell
expansion in plants and cytokinesis in eukaryotes,
depends on antiparallel microtubule crosslinking
by the conserved MAP65 protein family. Here, we
combine structural and single molecule fluorescence
methods to examine how PRC1, the human MAP65,
crosslinks antiparallel microtubules. We find that
PRC1’s microtubule binding is mediated by a struc-
tured domain with a spectrin-fold and an unstruc-
tured Lys/Arg-rich domain. These two domains, at
each end of a homodimer, are connected by a linkage
that is flexible on single microtubules, but forms well-
defined crossbridges between antiparallel filaments.
Further, we show that PRC1 crosslinks are compliant
and do not substantially resist filament sliding by
motor proteins in vitro. Together, our data show
how MAP65s, by combining structural flexibility and
rigidity, tune microtubule associations to establish
crosslinks that selectively ‘‘mark’’ antiparallel over-
lap in dynamic cytoskeletal networks.
INTRODUCTION
The dynamic reorganization of microtubule networks plays a crit-
ical role in diverse biological processes, including cell migration,
neuronal transport and cell division. It is now clear that different
cytoskeletal architectures arise from the interplay between
motor proteins, which can crosslink and move microtubules rela-
tive to one another, and nonmotor microtubule associated
proteins (MAPs), which can crosslink microtubules to stabilize
specific orientations (Glotzer, 2009; Manning and Compton,
2008). While we have good biophysical and structural models
for motor proteins that crosslink microtubules, much less is
known about nonmotor microtubule crosslinking proteins.
Several nonmotor MAPs that crosslink microtubules (e.g.,
MAP65, NuMA, NuSAP and Mia1p) are now known to play
important roles in dividing and nondividing cells (Ribbeck et al.,
2006; Sasabe and Machida, 2006; Schuyler et al., 2003; Thadani
et al., 2009; Zeng, 2000). Current models for the functions of
these proteins are based on cellular localizations and loss-of-
function studies. However, we lack any structural data to explain
how microtubule crosslinking is achieved by these MAPs.
Recently, there have been several advances in our under-
standing of the structure of nonmotor MAPs. Among the best
characterized class of MAPs are the +TIP proteins (e.g.,
XMAP215, EB1 and CLIP170), which can dynamically track the
growing end of a microtubule. Microtubule binding in these
proteins is mediated by calponin-homology, CAP/Gly or TOG
domains (Slep and Vale, 2007). Similarly, structural work on
Ndc80, a conserved mitotic MAP, has revealed how a calpolin-
homology domain may be used to establish kinetochore-micro-
tubule associations during cell division (Ciferri et al., 2008; Wei
et al., 2007; Wilson-Kubalek et al., 2008). However, due to lack
of similarity in primary sequence, it is not very likely that these
structural models will shed light on nonmotor MAPs that can
crosslink two microtubules.
As a step toward developing structural models for how non-
motor MAPs may crosslink microtubules, we focused on the
conserved MAP65 family, which plays key roles in microtubule
organization in eukaryotes. Since their initial discovery in bud-
ding yeast, microtubule crosslinking functions of the MAP65
proteins have been shown to be required for cell shape mainte-
nance in yeast cells, directional cell expansion in plants and
formation of the central spindle in eukaryotes (Chan et al.,
1999; Jiang et al., 1998; Loiodice et al., 2005; Yamashita et al.,
2005). Currently, at least three activities have been ascribed to
these proteins. First, MAP65s can selectively crosslink microtu-
bules in an antiparallel orientation (Gaillard et al., 2008; Loiodice
et al., 2005). Second, these nonmotor crosslinkers can oppose
filament movements driven by motor proteins. For example,
Ase1, the fungal MAP65, is proposed to antagonize kinesin-14
driven filament sliding required for organizing microtubules
during interphase (Janson et al., 2007). Third, these crosslinking
proteins can recruit signaling proteins or kinesins to the microtu-
bule structures they stabilize. For example, the recruitment
of Polo-like kinase to the central spindle during cytokinesis is
mediated via interactions with PRC1, the human MAP65 (Neef
Cell 142, 433–443, August 6, 2010 ª2010 Elsevier Inc. 433
 Figure 1. Single-Molecule Analysis of Microtubule Binding by PRC1
(A) Schematic of PRC10 s domain organization and a guide for constructs used
in the fluorescence microscopy assays (purple: coiled-coil domain; green:
microtubule binding domain; black: C-terminal domain).
(B) Fluorescence intensity analysis of two PRC1 constructs, GFP-PRC1-FL
(aa: 1–620; intensity = 2.5 3 104 ± 0.9 3 104, N = 469) and GFP-PRC1-NS
(aa: 1–466; intensity = 2.0 3 104 ± 0.8 3 104, N = 156). Dimeric-Eg5-GFP
(Intensity = 2.5x104 ± 1.0x104, N = 377) and tetrameric-Eg5-GFP (Intensity =
4.2x104 ± 2.2 3 104, N = 290) were used as references. Intensities are reported
as mean ± SD.
(C–H) Single molecule TIRF assay was used to examine the association of
PRC1 constructs (green) with microtubules (orange) immobilized on a glass
surface. (C) Schematic for assay showing the two constructs, GFP-PRC1-FL
and GFP-PRC1-NSDC (aa 1–486). Single frames showing two-color overlays
(top) and associated kymographs (below) of GFP-PRC1-FL (D and E) or
GFP-PRC1-NSDC (F and G). (H) Distribution of microtubule association life-
times for GFP-PRC1-FL (blue) and GFP-PRC1-NSDC (red).
(I–K) Microtubule association of GFP-PRC1-NSDC under different ionic
strength conditions. Representative kymographs from assays at 0.75x motility
buffer (I), motility buffer (J), motility buffer+20 mM KCl. (K) The scale bar repre-
sents 1.5 mm, 10 s. See also Figure S1 and Figure S3.

et al., 2007) and kinesin-5 driven microtubule sliding during

anaphase depends on Ase1 (Khmelinskii et al., 2009). Currently,
we do not have a structural framework to explain how these
MAPs specifically crosslink antiparallel microtubules. Moreover,
the activities of MAP65s have not been reconstituted in the pres-
ence of motor proteins to test if MAP65s resist filament sliding by
motor proteins or if their main function is to act as ‘‘marks’’ that
recruit other proteins to regions of antiparallel microtubule
overlap in dynamic networks.
Here we show, using single molecule fluorescence microscopy
assays, X-ray crystallography and electron microscopy that
PRC1 uses structured and unstructured domains to bind micro-
tubules. These domains at each end of a PRC1 homodimer are
connected by a linker that adopts a rigid conformation only when
crosslinking microtubules. We also show, in assays combining
TIRF and fluorescent speckle microscopy (FSM), that PRC1
does not substantially resist filament sliding by kinesin-5. Based
on these results, we propose a model for how a crosslinking MAP
can achieve specific and compliant crosslinking of microtubules
by balancing structural rigidity and flexibility.

RESULTS

Structured and Unstructured Domains Mediate

Microtubule Binding in PRC1
PRC1, like other Map65 family proteins, has a modular architec-
ture with an N-terminal coiled-coil domain, a central region that
can mediate microtubule binding, and a C-terminal regulatory
domain (Figure 1A). While the central domain is thought to
be required for microtubule binding, the contributions of the
C-terminal domain remain poorly characterized. To address
this, we analyzed the microtubule binding activity of PRC1 using
two approaches, a TIRF microscopy assay to examine the prop-
erties of single molecules and a microtubule cosedimentation
assay to analyze equilibrium binding.
For visualizing PRC1 molecules by fluorescence microscopy
we expressed and purified recombinant GFP-tagged full-length
PRC1 in bacteria. We found that C-terminal tags on PRC1

434 Cell 142, 433–443, August 6, 2010 ª2010 Elsevier Inc.

resulted in constructs that were highly unstable and therefore
used a construct with GFP fused to the N-terminus of PRC1
(GFP-PRC1-FL). We first examined the oligomerization state of
single GFP-PRC1-FL molecules. Analysis of the intensities of
single fluorescent spots of GFP-PRC1-FL immobilized on a glass
coverslip, when compared to intensities of known dimeric and
tetrameric reference constructs, indicated that GFP-PRC1-FL
is a dimer (Figure 1B), similar to the yeast homolog, Ase1 (Kapi-
tein et al., 2008a; Schuyler et al., 2003). We also confirmed that
untagged PRC1 is a homo-dimer and has an extended confor-
mation (see below, and data not shown). Previous studies have
suggested that PRC1 exists as a homo-tetramer, but gel filtration
chromatography alone can sometimes be unreliable for such
analyses of proteins with elongated structures (Zhu et al.,
2006). To examine the interaction of single PRC1 molecules
with a microtubule, we used streptavidin to attach X-rhoda-
mine-labeled, biotinylated microtubules to a glass surface. After
blocking the glass surface, we added low concentrations of
GFP-PRC1-FL (20 pM) (Figure 1C). Similar to what is observed
for Ase1, we found that single GFP-PRC1-FL molecules diffuse
in 1-D along the microtubule surface (Figure S1D and 1E; (Kapi-
tein et al., 2008a)). Analysis of time-lapse sequences using
kymographs showed that individual GFP-PRC1-FL molecules
can maintain associations with the microtubule lattice that can
last several seconds (t1/2 = 7 ± 0.4 s; Figure 1H).
To examine the contribution of the C terminus of PRC1 to
microtubule binding, we first generated a construct comprised
of just the N-terminal dimerization and the microtubule-binding
domain (aa 1–466, hereafter GFP-PRC1-NS). Intensity distribu-
tion of GFP-PRC1-NS indicated that this construct, like the
full-length protein, is a dimer (Figure 1B). We were unable to
detect microtubule associations of single molecules of GFP-
PRC1-NS at the concentrations needed to resolve single
molecules using TIRF microscopy (<10 nM; data not shown), Figure 2. Microtubule Cosedimentation Assays to Determine Equi-
suggesting that residues in the C terminus domain contribute librium Dissociation Constants for PRC10 s Microtubule-Binding
Domains
to microtubule binding. Secondary structure algorithms predict
(A) Schematic for constructs used in this assay: PRC1-S (aa 341–466) and
that the C-terminal domain in PRC1 is likely to be unstructured. PRC1-SC (aa 341–620). SDS-PAGE analysis of cosedimentation assays for
Consistent with this prediction, we find that PRC1’s C terminus PRC1-SC (B) and PRC1-S (C). Arrows in (B) indicate C terminus proteolysis
is prone to proteolysis. A highly susceptible cleavage site resides products in PRC1-SC. Bands marked with red boxes in (B) indicate the relative
between two Lys/Arg-rich basic clusters in this domain (aa. Asn tubulin concentration at which 50% of the different PRC1-SC truncation prod-
500; Figure S1 available online). As basic amino acids are often ucts cosediment with microtubules. (D) Band intensities from the gels in (C)
were used to determine fraction protein bound, and plotted against microtu-
implicated in mediating interactions with the negatively charged
bule concentration (n = 3, mean ± SD). The data were fit to a modified Hill equa-
microtubules surface, we generated a GFP-fused PRC1 con- tion to determine Kd’s (PRC1-S: 3.3 ± 1.8 mM; PRC1-SC: 0.6 ± 0.3 mM).
struct with one of the two basic-residue clusters (aa 1–486,
hereafter GFP-PRC1-NSDC). Under TIRF microscopy condi-
tions we could readily observe microtubule binding by single domain to PRC1’s microtubule interaction. For these experi-
GFP-PRC1-NSDC molecules. Diffusion on the microtubule was ments we generated PRC1 constructs lacking the dimerization
apparent for the subset of binding events that lasted several domain so that we could exclude the potentially complex effects
seconds (Figures 1F and 1G). Analysis of the life-times of micro- of filament crosslinking/bundling on this analysis (Figure 2A).
tubule association indicated that this construct has a higher These PRC1 constructs were confirmed to be monomeric by
microtubule unbinding rate than the full-length protein (Fig- size exclusion chromatography (data not shown). The microtu-
ure 1H, lifetime is less than the lower limit of reliable event bule binding affinity (Kd) of a construct comprising of the central
detection, < 3 s). Increasing salt concentrations reduced the domain and the C terminus (aa 341–620, named PRC1-SC) was
GFP-PRC1-NSDC binding lifetimes, consistent with a charge- 0.6 ± 0.3 mM (Figures 2B and 2D). A construct comprising of the
dependent microtubule-binding interaction mediated by central domain alone (aa 341–466, hereafter named PRC1-S)
PRC1’s C terminus (Figures 1I–1K). had a 5-fold weaker binding (Kd: 3.3 ± 1.8 mM; Figures 2C
We next carried out microtubule cosedimentation assays for and 2D). Interestingly, we observe cooperative microtubule
an independent analysis of the contribution of the C-terminal binding by PRC1-SC (Hill coefficient of 3 ± 1). This suggests

Cell 142, 433–443, August 6, 2010 ª2010 Elsevier Inc. 435

that the C terminus may be responsible for interactions between
PRC1 molecules on the microtubule lattice analogous to what
has been previously reported for Ase1 (Kapitein et al., 2008a).
Together, these data indicate that PRC1’s microtubule binding
is mediated by two regions, one that is predicted to be structured
and another, unstructured region, rich in Lys/Arg residues.
PRC1’s Microtubule-Binding Domain Has a Spectrin Fold
To determine the structural basis of microtubule binding by
PRC1, we focused on its central microtubule-binding domain.
As this domain has no obvious homology to any known microtu-
bule binding motif, we used X-ray crystallography to determine
its structure. Nonisomorphous crystals of the PRC1-S construct
were obtained from both the native protein, which diffracted to
1.75 Å resolution, and the selenomethionyl-substituted protein,
which diffracted to 2.0 Å resolution. The structure of the seleno-
methionyl-substituted protein was solved by single-wavelength
anomalous dispersion, refined, and, subsequently used as
a model to solve the structure of the native protein using molec-
ular replacement (Table S1). We found that the microtubule-
binding domain of PRC1 is an 70 Å long 3-helix bundle (labeled
helix-1, -2, and -3), with connecting loops (labeled loop1-2 and
loop2-3) and N- and C- termini at opposite ends (Figure 3A
and Figures S2A and S2B). Residues from all three helices
contribute to a hydrophobic core which dominates the interface
between the three helices (Figure 3B). This core is flanked on
both ends of the helix bundle by two salt bridges mediated by
conserved residues (Figure 3C).
Using DALI, we found that PRC1-S has high structural similarity
to spectrin domains (Figure 3D), motifs commonly found in
proteins associated with the actin cytoskeleton (e.g., alpha-
actinin, spectrin and dystrophin; (Djinovic-Carugo et al., 2002)).
However, a spectrin fold has not thus far been found in any other
known MAP. Interestingly, in actin-binding proteins, the elon-
gated and rigid triple-helix of a spectrin domain does not directly
mediate actin binding, but acts as a spacer between canonical
actin-binding motifs, such as calponin homology domains.
Therefore, it appears that the spectrin fold has been evolutionarily
‘recycled’ and re-engineered to act as a microtubule interacting
domain in the Map65 protein family. These findings establish
a new role for the spectrin motif as a microtubule-binding domain.
A Conserved Basic Region Forms the Microtubule-
Binding Surface in the Spectrin Domain of PRC1
As it was unclear how the spectrin fold would bind microtubules,
we analyzed the electrostatic surface potential and the conser-
vation of surface residues for this domain. A map of the electro-
static surface potential shows a positively charged region
comprising of loop1-2 and residues from all three helices that
are proximal to this loop in the three dimensional structure
(Figure 3E). Interestingly, a cluster of highly conserved residues
is also located within this region (Figure 3F), suggesting that
the junction of helix-1 and helix-2 could form the microtubule
binding region in the spectrin fold. To test this hypothesis we
generated constructs of PRC1’s spectrin domain in which basic
residues in this region were mutated to alanine (Figure S2C).
Each of these constructs was characterized to be monomeric
by size-exclusion chromatography (Figure S2D). We used micro-
436 Cell 142, 433–443, August 6, 2010 ª2010 Elsevier Inc.
tubule cosedimentation assays to compare microtubule binding
of mutant and wild-type PRC1-S constructs (Figure 3G). We
found that mutation of each of the basic residues in the most
conserved region reduced microtubule binding 2- to 4-fold,
though none of the individual mutations abolished microtubule
binding (Figure 3H). To test that the observed microtubule
binding is not simply a nonspecific electrostatic interaction, we
tested two other constructs. First, we made a C terminus trunca-
tion in PRC1-SC (labeled del453–466; aa 341–452) to remove
three surface exposed lysines on helix-3. Second, we generated
a construct in which a lysine residue on helix-2 (K407), distal to
the potential microtubule binding site, was mutated to alanine.
Both of these constructs show less than 50% reduction in
microtubule binding. From this analysis, we propose that the
spectrin domain in PRC1 uses a basic surface comprising of
conserved residues at one end of the triple helix bundle to
mediate microtubule binding (Figure 3F, circle).
Spectrin Domain Fits with an Optimal Orientation
into the Cryo-EM Density Map of the PRC1-Microtubule
Complex
To investigate the interaction of PRC1 with the microtubule
lattice we examined the structure of dimeric PRC1 bound to
single microtubules by cryo-electron microscopy (Cryo-EM)
and helical image analysis. As PRC1-FL extensively bundles
microtubules at concentrations needed for this analysis, we
used our findings from single molecule experiments to engineer
a construct PRC1-NSDC (aa 1–486; Figure 4A), which was
more suitable for this analysis (Figure 1A). This construct was
confirmed to be dimeric by HPLC Size Exclusion Chromatog-
raphy/Laser Light Scattering Analysis (Figure S3), and found to
retain sufficient binding to fully decorate single microtubules
without extensive bundling.
Diffraction of individual cryo-EM images of PRC1-NSDC
bound to microtubules showed an 80 Å layer line, indicating
that one PRC1-NSDC molecule binds a a/b-tubulin heterodimer.
The 3D reconstruction of microtubule bound PRC1-NSDC
showed a single rod-shaped density corresponding to PRC1-
NSDC, protruding approximately perpendicular to the microtu-
bule lattice (microtubule side view, Figure 4B; top view,
Figure 4C). Interestingly, the PRC1 density in the reconstruction
is of the same size as PRC1’s spectrin domain. The optimal fit of
the spectrin domain crystal structure to the reconstructions
oriented the conserved, basic residues at the junction of helix-1
and helix-2 toward the microtubule lattice (Figure 4D and 4E).
This binding model is also consistent with our site-directed
mutagenesis analysis (Figure 3H). Comparison of the 3D recon-
struction of this complex with cryo-EM structures of motor
proteins (e.g kinesin and dynein’s microtubule-binding domain)
bound to microtubules suggests that the binding site for PRC1
partially overlaps with, but is not identical to, the microtubule-
binding surface used by motor proteins (Carter et al., 2008;
Figure S4B). Surprisingly, we see no extra density distal to the
microtubule surface that would correspond to the N-terminus
coiled-coil dimerization domain in PRC1-NSDC. This indicates
that the coiled-coil domain is likely to be flexible and can
have more than one conformation when bound to a single
microtubule.
Figure 3. PRC10 s Conserved Microtubule-Binding Domain Adopts a Spectrin Fold
(A) Ribbon diagram shows the overall structure. Five disordered residues in the loop between helix-1 and 2 are indicated by dots.
(B) Hydrophobic residues from helix-1 (cyan), helix-2 (yellow) and helix-3 (green), which form the core of the triple helix bundle, are highlighted (sticks).
(C) Salt bridges between charged residues from helix-1 (cyan), helix-2 (yellow) and helix-3 (green) are indicated (spheres).
(D) Overlay of PRC10 s spectrin domain (orange) with its closest structural homolog, which is a spectrin repeat in Plectin (blue, PDB = 2odu-A, Z-score = 7.7,
rmsd = 3.4 Å calculated using DALI).
(E) Surface representation showing electrostatic potential of the spectrin domain in PRC1 (Red to blue is 10 kbT to +10 kbT, as calculated using APBS).
(F) Residues with low (cyan), intermediate (white), and high (magenta) conservation on the surface of the PRC10 s spectrin domain. The labeled residues were
selected for mutagenesis studies.
(G) SDS-PAGE analysis of microtubule cosedimentation assays of PRC1-S mutants at 27 mM tubulin.
(H) Band intensities from (G) were quantified to determine the fraction of PRC1 bound to microtubules (n = 3, error bars indicate SE). See also Figure S2.

To further understand the PRC1-microtubule interaction, we
obtained a second EM reconstruction with a monomeric
construct that lacked most of the oligomerization domain but
contained the spectrin domain and the entire C terminus
(aa 303–620). The observed density for this construct overlaps
with the PRC1-NSDC density close to the microtubule lattice
(Figures S4C–S4E). We also observe some additional density
distal to the microtubule surface which is not observed in
PRC1-NSDC. It is possible that this density corresponds to
PRC1’s C terminus folding back to interact with the spectrin
domain or to the amino acids at PRC1’s N-terminus that may
be ordered when this construct binds microtubules. As we do
not observe any additional density past the C terminus of the
spectrin domain that is close to the microtubule lattice, we favor
the possibility that the Lys/Arg-rich domain is disordered even
when PRC1 is bound to a microtubule. Together, these data

Cell 142, 433–443, August 6, 2010 ª2010 Elsevier Inc. 437


Figure 4. The Spectrin Domain Fits into the Cryo-EM Density Map of
the PRC1-Microtubule Complex with an Optimal Orientation
(A) Schematic comparing full-length PRC1 to the construct PRC1-NSDC
(aa 1–486) used for Cryo-EM analysis.
(B) Surface rendered side-view of 3D EM density map of the microtubule-
PRC1-NSDC complex.
(C) Top view of cryo-EM density map of undecorated tubulin (gold) superim-
posed with the microtubule-PRC1-NSDC complex (purple).
(D and E) (D) Side- and (E) top-view of the crystal structure (cyan ribbon
diagram) docked into the PRC1 density (purple mesh) protruding from an
a/b tubulin dimer (gold). Residues R377 and K387, which are involved in micro-
tubule binding, are indicated in red and black respectively. Microtubule
polarity indicated in (B) was determined by comparison with a 3D EM structure
of a dynein-microtubule complex, which has a well defined polarity (Carter
et al., 2008). See also Figure S4.
show that PRC1’s spectrin domain is highly ordered and less
flexible than the other domains when associated with a single
microtubule.
Cryo-Electron Tomography Reveals Ordered PRC1
Crosslinks between Two Antiparallel Microtubules
Helical reconstructions suggest that PRC1 may not be an inher-
ently rigid molecule when bound to one microtubule. It is difficult
to explain how a flexible protein with two microtubule interaction
surfaces at opposite ends of dimer can achieve the crosslinking
specificity reported for MAP65 proteins (Gaillard et al., 2008;
Janson et al., 2007). The only other structural analysis of cross-
linking by the MAP65 family is a cryo-EM study of the plant
homolog of PRC1, which show that the protein can form dense
crossbridges between two microtubules (Gaillard et al., 2008).
438 Cell 142, 433–443, August 6, 2010 ª2010 Elsevier Inc.
Figure 5. The Dimerization Domain in PRC1 Is Ordered When Cross-
linking Two Microtubules
(A) Schematic highlights the section viewed in the cryotomographic recon-
structions shown in (B) and (C), which encompasses the two microtubules
and the PRC1 crosslinks.
(B and C) Slices through cryotomographic 3D reconstructions of microtubules
crosslinked by PRC1-NSDC. Slices represent the central area of the microtu-
bules and the bound PRC1-NSDC. The top and bottom of the microtubules are
excluded in these views. Examples of microtubule pairs with dense (B) and
sparse (C) PRC1 occupancy. Insets show 4-fold enlargements of the cross-
links between microtubules. Small red arrows in the inset highlight individual
cross-bridges. Blue arrow in (B) indicates a PRC1 molecule that does not
form crosslinks with another microtubule. Long red arrows in (B) indicate the
polarity of microtubules, portions of which are highlighted in blue for clarity.
The top two microtubules in (B) share PRC1 crossbridges as do the bottom
two microtubules, however, the middle two microtubules are separated by
another microtubule (dotted blue line) that is positioned below the plane and
only the top of its tubulin lattice can be observed.
However, the conformational differences between crosslinking
and noncrosslinking molecules could not be visualized in this
study. It also remains unknown whether establishing ordered
crossbridges is a property of single MAP65 molecules or results
from protein-protein interactions that may be involved in PRC1’s
co-operative microtubule binding (Figure 2D). To address these
questions, we employed cryo-electron tomography to obtain
a higher resolution structure of two microtubules crosslinked
by PRC1 (Figure 5A). For this analysis we used the PRC1
construct we had used for helical reconstruction (PRC1-NSDC;
Figure 4A). 3D reconstructions from cryo-electron tomography
of microtubules densely crosslinked by PRC1-NSDC shows
rod-like striations connecting two microtubules (Figure 5B
and inset). From analyzing the overall direction of crosslinking
protein density, we estimated that 90% (n = 20) of the cross-
linked microtubules were antiparallel. This shows that the C
terminus truncation construct has antiparallel crosslinking spec-
ificity similar to full-length MAP65 and Ase1 (Gaillard et al., 2008;
Schuyler et al., 2003). We find that the average distance between
two crosslinked filaments was 35 nm (±2 nm; n = 20) and the
crossbridge angle of PRC1 linkages was 70 (±5; n = 15) rela-
tive to the microtubule surface. As helical reconstruction shows
that the spectrin domain in PRC1 protrudes almost perpendic-
ular to the microtubule surface, the crossbridge orientation likely
results from 20 hinges between the oligomerization and the
spectrin domains.
Consistent with what is seen from the helical reconstruction
analysis, the tomograms show that the PRC1 densities observed
on the microtubule surface distal to crosslinked microtubule (i.e.,
the noncrosslinking PRC1 molecules, Figure 5B, blue arrow)
were less ordered than those between two filaments. This indi-
cates that noncrosslinking and crosslinking PRC1 molecules
have distinct conformations.
We next examined if the regular crossbridge geometry and the
intermicrotubule distances observed are retained in regions of
sparse PRC1 decoration between crosslinked microtubules.
We find that single PRC1 molecules still form crossbridges
with orientations similar to those observed in regions of dense
crosslinks (Figure 5C and inset). Interestingly, we found that
PRC1 crosslinks were not observed in regions where filament
spacing was significantly greater than 35 nm, indicating that
PRC1 molecules exhibit specificity for a narrow range of inter-
microtubule distances. These findings agree with our observa-
tions in fluorescence microscopy assays in which GFP-PRC1-
FL accumulation is seen only at a narrow range of microtubule
crosslinking angles (unpublished data). Together, our data indi-
cate that although the dimerization domain is not entirely rigid
when PRC1 is bound to a single microtubule, this domain in indi-
vidual PRC1 molecules can adopt a specific conformation upon
crosslinking two microtubules. The rigidity in the linker, together
with oriented binding by the spectrin domain, can allow PRC1 to
be a selective crosslinker of antiparallel microtubules aligned
with a narrow range of interfilament spacing.
PRC1 Is a Compliant Crosslinker of Antiparallel
Microtubules
Our analysis of microtubule binding by the structured and
unstructured domains in PRC1 shows that these interactions
have moderate affinity. Together with the observed 1-D diffusion
of single PRC1 molecules on microtubules, these data predict
that PRC1 crosslinks may not substantially resist filament sliding
by motor proteins. To test this hypothesis, we devised an in vitro
assay to visualize near-simultaneously PRC1 localization and
motor-protein driven sliding of pairs of crosslinked microtubules.
We attached biotinylated microtubules to a glass surface via
streptavidin. These microtubules also incorporated low levels
of fluorescent tubulin for analysis of relative microtubule motion
using fluorescent speckle microscopy (FSM). GFP-PRC1-FL
was added to the immobilized microtubules, followed by nonbio-
tinylated microtubules to generate a ‘sandwich’ comprised of
two microtubules crosslinked by PRC1 (Figure 6A). As would
be expected, based on studies of Ase1, we find that GFP-
PRC1-FL shows a 10 ± 4 -fold preference for regions where
two microtubule overlap relative to regions of single microtu-
bules (N = 15; Figures S5A–S5C; (Kapitein et al., 2008a)). To drive
the relative sliding of microtubules in this ‘sandwich’, we added
kinesin-5, a well characterized motor protein needed for cell
division.
Under these conditions we observed two types of events.
First, kinesin-5 could move a shorter microtubule relative to
a longer filament in the ‘sandwich’ such that the amount of
overlap tracked by GFP-PRC1, remained unchanged (Figures
6B–6D). Second, we observed events in which the microtubule
overlap in the ‘sandwich’ reduced at the rate of filament motion
(Figure 6E and Figures S5D and S5E) and GFP-PRC1 dynami-
cally tracked the microtubule overlap zone (Figure 6F). Remark-
ably, in this set of events, the velocity of relative filament sliding
by kinesin-5 did not increase as the extent of microtubule over-
lap reduced (Figures 6G and 6H). Fluorescence intensity analysis
indicated that the number of PRC1-crosslinks in the overlap
region decreased proportionately (data not shown). Analysis of
all data show that kinesin-5’s sliding velocity reduced only
2.4 fold over an 18-fold change in PRC1 concentration
(Figure 6I). At higher PRC1 concentrations in this assay extensive
microtubule bundling was observed and it was very difficult to
reliably detect pairs of crosslinked microtubules. The few events
that could be analyzed under these conditions indicated that
a further reduction in kinesin-5 driven filament sliding was not
observed as PRC1 concentration was increased (Figure S5F-I).
We estimated the ratio of PRC1 and kinesin-5 at microtubule
overlap zones in our assays. Using GFP-kinesin-5 at concentra-
tions similar to the un-tagged kinesin-5 used in the above
assays, we found that microtubule sliding was driven by a few
motor protein molecules, and unlike PRC1, kinesin-5 did not
show a preference for microtubule overlap zones (data not
shown and (Hentrich and Surrey, 2010)). The average fluores-
cence intensity of single GFP-PRC1-FL molecules was then
used to determine the number of GFP-PRC1-FL molecules at
microtubule overlap regions. These data indicate that the ratio
of GFP-PRC1-FL to kinesin-5 molecules at microtubule overlap
zones was 25 (total solution concentration: GFP-PRC1-FL =
0.54 nM, kinesin-5 = 1.8 nM). This shows that crosslinks formed
by multiple PRC1 molecules do not substantially resist the rela-
tive microtubule movement driven by fewer kinesin-5 molecules.
Discussion
Members of the MAP65 family organize microtubules by prefer-
entially crosslinking filaments with an antiparallel orientation. In
this study, we provide a structural framework for how three
structurally distinct domains in human MAP65 (PRC1) are
combined to achieve selective and efficient crosslinking of anti-
parallel microtubules, as would be needed during the self-orga-
nization of dynamic cytoskeletal networks.
The conserved microtubule-binding domain in PRC1 has
a spectrin fold. First identified in spectrin, a constituent of the
membrane skeleton, this fold has since been seen in several
actin crosslinking proteins such as alpha-actinin and dystrophin
(Djinovic-Carugo et al., 2002). In these proteins, spectrin-
domains appear as repeated units that connect other actin-
binding domains and regulate properties of the cytoskeletal
Cell 142, 433–443, August 6, 2010 ª2010 Elsevier Inc. 439

Figure 6. PRC1 Crosslinks Do Not Substantially Resist the Relative
Sliding of Two Microtubules by Kinesin-5
(A) Schematic illustrating the assay used to examine the effect of PRC1 (green)
on kinesin-5 (cyan)-mediated relative sliding of two microtubules (orange),
when extent of overlap between microtubules is unchanged. Near-simulta-
neous dual-mode microscopy was used to image microtubules (via wide-field
fluorescent speckle microscopy) and GFP-PRC1-FL (via Total Internal Reflec-
tion Fluorescence (TIRF) microscopy).
(B) Frames from a time-lapse sequence (1 min interval) show GFP-PRC1-FL
(green) enriched at regions where two crosslinked microtubules (red) overlap.
(C) Corresponding kymograph shows the surface-attached static microtubule
(vertical streaks) and a moving microtubule (diagonal streaks, 16.5 nm/s).
(D) Kymograph shows that the GFP-PRC1-FL decorated region moves at the
velocity of the moving microtubule.
440 Cell 142, 433–443, August 6, 2010 ª2010 Elsevier Inc.
structures. In PRC1, this domain appears to have evolved to
mediate microtubule binding, an entirely different role for this
protein fold. Further, the spectrin fold is unrelated to the other
protein folds (e.g Cap/Gly motifs and calponin-homology
domains) that are known to mediate interactions with the micro-
tubule lattice. It has been proposed that some protein surfaces
are evolutionarily selected to act as a versatile protein interaction
platform. This is best exemplified in the Fc domain of IgG, which
is structurally adapted to interact with several different protein
scaffolds (DeLano et al., 2000). Similarly, it appears that the
microtubule surface has evolved to bind a large variety of unre-
lated structural motifs and thereby accommodate diverse
MAPs that can carry out a wide range of functions (Amos and
Schlieper, 2005).
Microtubule binding by the spectrin domain is augmented by
a Lys/Arg rich unstructured domain in PRC1. Synergistic microtu-
bule binding by structured and unstructured domains have also
been seen in other MAPs such as Ndc80 (Guimaraes et al.,
2008; Miller et al., 2008), suggesting that this feature may be
a frequent adaptation in MAPs. There are also at least two other
functions ascribed to these unstructured microtubule-binding
domains. First, these unstructured domains are often sites of
phospho-regulation (Holt et al., 2009). Interestingly, Cdk1 phos-
phorylation sites in PRC1 map to the unstructured Lys/Arg
domain (Zhu et al., 2006). Our data suggest that these phosphor-
ylations would directly attenuate microtubule affinity by reducing
the net positive charge of the domain. Dephosphorylation of these
residues would activate PRC1’s microtubule binding at
anaphase, as would be needed for PRC1’s functions during the
final stages of cell division. Second, as has been suggested for
Ndc80, these domains are proposed to allow a mode of attach-
ment which does not significantly resist microtubule movement.
We find that PRC1 does not strongly oppose microtubule sliding
by kinesin-5, indicating that moderate binding affinities and diffu-
sive microtubule interactions of PRC1 can permit microtubule
movement while maintaining attachment. This result is also
consistent with the reported diffusion constant of Ase1 which indi-
cates that > 100 crosslinker molecules would be needed to
generate a resistive force greater than 1.5 pN, which is in the range
of the force required to inhibit kinesin-5 movement (Bormuth et al.,
2009; Kapitein et al., 2008a; Korneev et al., 2007; Valentine and
Block, 2009). This would be relevant in vivo, when microtubule
bundling by Ase1 and sliding by kinesin-5 are both required for
spindle elongation in anaphase B (Khmelinskii et al., 2009).
Crossbridges between two PRC1-crosslinked microtubules
are formed by PRC1’s dimerization domain. These are seen to
project at an angle of 70 relative to the microtubule lattice.
Binding at a fixed angle relative to the microtubule lattice has
(E) Schematic illustrating the assay when overlap between microtubules
decreases during relative microtubule sliding.
(F–H) Frames from a time-lapse sequence (2 min interval) (F) and corresponding
kymographs showing microtubule movement (7 nm/s) (G) and GFP-PRC1-FL
localization to overlap region that reduces due to relative sliding of filaments (H).
(I) Velocity distributions for kinesin-5 driven microtubule sliding at 1.8 nM
kinesin-5 and 0.034 (V = 23.1 ± 6.3 nm/s, N = 43), 0.14 nM (V = 17.7 ±
3.5 nm/s, N = 39), and 0.54 nM GFP-PRC1-FL (V = 9.7 ± 3.1 nm/s, N = 41).
Velocities are reported as mean ± SD. The scale bars represent 1.5 mm,
100 s. See also Figure S5.

Figure 7. PRC1 Is a Compliant, Microtubule-Overlap Tracking
Protein that Tunes Structural Rigidity to Specifically Crosslink Two
Antiparallel Microtubules
(A) A model for how PRC1 can align microtubules into antiparallel arrays. The
spectrin domain in PRC1 can make oriented contacts with the microtubule to
decode filament polarity. The unstructured domain acts to enhance the
binding affinity, while allowing diffusion along microtubules. The dimerization
domain is not entirely rigid on a single microtubule but adopts a specific
conformation when crosslinking two microtubules.
(B) The rigidity and flexibility of the different domains in PRC1 can facilitate
sorting of randomly oriented microtubules into antiparallel arrays and allow
PRC1 to function as a selective ‘mark’ for microtubule overlap regions.
also been reported for Ndc80 (Wilson-Kubalek et al., 2008) but
the implications of defined projection angles for any microtu-
bule-binding protein is thus far unknown. The crossbridge also
determines the intermicrotubule spacing between two microtu-
bules. This intermicrotubule distance could also affect the ability
of motor proteins to bind and slide PRC1 crosslinked microtu-
bules, providing an additional mechanism for activating or
deactivating specific motors at these structures. For example,
the reported length of kinesin-5 motor is approximately 95 nm,
which is 2-fold greater than the 37 nm intermicrotubule spacing
of PRC1 crosslinked microtubules (Kashina et al., 1996). Hence,
the reduction in the velocity of microtubule sliding by kinesin-5
seen in our experiments at high PRC1 concentrations could
result from the inability of kinesin-5 to bind properly and
efficiently walk along both microtubules with dense PRC1 cross-
links. Such modulation of motor activity through control of
interfilament spacing has been proposed to affect the magnitude
of active forces generated in striated muscles during muscle
contraction (Millman, 1998).
Based on our results, we propose a structural model for how
polarity-specific crosslinking is mediated by PRC1 (Figures 7A
and 7B). The ability of a microtubule associated protein to distin-
guish parallel and antiparallel filaments relies on two factors.
First, PRC1 molecules must decode filament polarity when
bound to one microtubule. Our results show that the spectrin
domain is the most ordered region in a PRC1-microtubule com-
plex and uses a well-defined surface for microtubule binding.
This suggests that this domain makes contacts with the microtu-
bule lattice that decode filament orientation. Second, the micro-
tubule polarity needs to be transmitted across the linker to
the second spectrin domain in the PRC1 homodimer. Our data
show that the dimerization domain in PRC1 has a single confor-
mation when crosslinking two microtubules. The structural
rigidity of this domain is likely to be responsible for PRC1’s selec-
tivity for antiparallel microtubules. Though specificity can be
achieved by oriented binding of the spectrin domain and rigidity
in the linker domain, the weak microtubule binding affinity of the
spectrin domain alone does not explain the extensive filament
bundling induced by PRC1. To increase binding and conse-
quently crosslinking efficiency, PRC1 uses an unstructured posi-
tively charged domain for maintaining long-lived associations
with microtubules. Additionally, the flexibility in the linker domain
of PRC1, which appears to have more than one conformation
when bound to one microtubule, may allow for an initial contact
with a second microtubule that could have a wide-range of
orientations. Relative to a highly rigid structure, such flexibility
could increase the crosslinking efficiency of PRC1 molecules.
These features would enable PRC1 to stay associated with the
first microtubule encountered, explore its length by 1-D diffu-
sion, and thereby increase the probability of capturing a second
filament for establishing antiparallel linkages between two
microtubules.
Many cellular processes require recognition of a ‘‘mark’’ at
precise locations. Post-translational modifications such as ubiq-
uitination and methylation are some of the common marking
mechanisms for proteins and DNA in cells. In the microtubule
cytoskeleton, the +TIP-proteins track and identify microtubule
growing microtubule plus-ends (Akhmanova and Steinmetz,
2008). Similarly, by recognizing specific microtubule geometries
and forming compliant crosslinks, the MAP65 proteins can mark
antiparallel microtubule overlap during the self-organization of
dynamic cytoskeletal networks.
EXPERIMENTAL PROCEDURES
Crystallization of PRC1-S
PRC1-S (341-466) was concentrated to 50 mg/ml in motility buffer (80 mM
PIPES (pH 6.8), 1 mM MgCl2, 1 mM EGTA) and 150 mM KCl and crystallized
by hanging drop vapor diffusion at 4 C using an equal volume of protein
sample and crystallization solution consisting of 0.1 M CHES (pH 9.5) and
30%–35% PEG 3350. Crystals (needles of dimension 25 X 25 X 300 mm)
appeared between 1 - 3 weeks. Microseeding was used to obtain crystals of
the selenomethionyl derivative of the construct. Crystals were frozen in liquid
nitrogen after a 5 min soak in a solution comprising of the crystallization solu-
tion with 5%–10% v/v glycerol. Both native and SAD datasets were collected
along single needles at NE-CAT beamline 24ID-E at APS at the peak selenium
wavelength 0.97918 Å and processed as described in the supplement.
Cryo-EM and Image Analysis
Microtubules were polymerized as previously described (Wilson-Kubalek
et al., 2008). Microtubules (0.166 mg/ml in motility buffer) were applied to
plasma cleaned C-flat grids and incubated with PRC1-NSDC (0.53 mg/ml) or
PRC1-SC’ (0.9 mg/ml). The sample was then vitrified in liquid ethane, using
a manual plunger. The data sets were collected using a GATAN cryoholder
Cell 142, 433–443, August 6, 2010 ª2010 Elsevier Inc. 441
and a FEI Tecnai F20 transmission electron microscope equipped with a Gatan
Ultrascan 4000 3 4000 pixel CCD camera. Images of the decorated microtu-
bules were recorded in low-dose conditions (<10 e/Å) at a magnification of
29,000 and a nominal defocus range from 1.5 to 2.0 mm. For image analysis
of the PRC1-microtubule complexes, PRC1-decorated 15-protofilament
microtubules were selected from CCD images. 3D reconstructions were calcu-
lated using Phoelix, essentially as described elsewhere (Whittaker et al., 1995).
3D EM Reconstructions
Surface representations of side- and top- views of the cryo-EM reconstruc-
tions (Figure 4 and S4) were produced using the Chimera software package
(Pettersen et al., 2004). To interpret the PRC1 densities protruding from the
microtubule density, we manually docked the crystal structure of the spectrin
domain into the EM density reconstructions using Chimera.
Cryotomography
Tilt-series images were acquired using the Serial EM software package (Mas-
tronarde, 2005) on a Tecnai F20 microscope through a range of +/ 50 at 1.5
increments. The specimen was subjected to a total dose of 100 electrons.
The tomographic image reconstruction was performed using the IMOD soft-
ware package (Kremer et al., 1996).
Fluorescence Microscopy
All experiments were performed on an instrument described previously (Kapi-
tein et al., 2008b). GFP was imaged with TIRF illumination from a 491-nm laser
source (Cobalt Calypso 50; Solamere Technology) and X-rhodamine was
imaged with wide-field illumination. For all experiments, oxygen scavenging
mix (OS) comprising of 25 mM glucose, 40 mg/ml glucose oxidase, 35 mg/ml
catalase and 0.5% beta-mercaptoethanol was included in the final buffer.
Kinesin-5/PRC1 sliding experiments were performed as described previ-
ously with some modifications (Kapitein et al., 2008b). Briefly, X-rhodamine-
labeled biotinylated taxol-stabilized microtubules were immobilized on the
coverslip by first coating the surface with biotinylated BSA followed by strepta-
vidin. After a brief incubation with casein to block nonspecific binding to
the surface, GFP-PRC1-FL was added and allowed to bind the immobilized
microtubules. This was followed by addition of X-rhodamine-labeled microtu-
bules which generated a ‘sandwich’ with two microtubules crosslinked by
PRC1. After washing the solution microtubules, the final assay mix comprising
of GFP-PRC1-FL and/or kinesin-5 in motility buffer supplemented with
80 mM KCl, 2 mM MgCl2, 1 mg/mL k-casein, 1 mM MgATP, 20 mM taxol and
OS mix was added to the flow chamber. TIRF and wide-field images were
acquired near simultaneously with 0.2 s exposure, 0.1 s-1 frame rate and EM
gain set to 100 and 200 for TIRF and wide-field respectively. Each pair of moving
microtubule was analyzed by kymographs using MetaMorph (MDS Analytical
Technologies). Sliding velocities were calculated from the slopes of the diag-
onal streaks generated by the movement of one speckled microtubule over
an immobilized microtubule. In a few rare events (<5%), we observed an abrupt
2-fold change in microtubule movement velocity in the kymographs. Based
on our analyses of kinesin-5 sliding alone (without PRC1), we believe this is
due to kinesin-5 switching between walking on both microtubules it crosslinks
to walking on only one of the two microtubules. This change in motility mode
leads to a 2-fold reduction in relative filament sliding. In these cases, both
velocities were measured and are included in the histograms shown.
ACCESSION NUMBERS
Coordinates have been deposited with PDB IDs 3NRX (native crystal structure)
and 3NRY (SelenoMet crystal structure) and EMDB accession codes
EMD-5205 (EM map of PRC1 [aa 1-486]) and EMD-5212 (EM map of PRC1
[aa 303-620]).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, five
figures, and one table and can be found with this article online at doi:
10.1016/j.cell.2010.07.012.
442 Cell 142, 433–443, August 6, 2010 ª2010 Elsevier Inc.
ACKNOWLEDGMENTS
We thank K. Rajashankar (Argonne Advanced Photon Source) and Deena Oren
(Rockefeller University Structural Biology Resource Center (SBRC)) for tech-
nical assistance. T.M.K. is grateful to the NIH (GM65933 and GM65933-S1)
for support. R.S. is a recipient of the Rockefeller Women and Science postdoc-
toral fellowship. R.A.M. acknowledges support from the NIH (GM52468). We
also acknowledge the National Resource for Automated Molecular Micros-
copy (NIH RR17573), the SBRC (NIH 1S10RR022321-01), and the Keck
Facility at Yale University (NIH 1S10RR023748-01) for instrument use.
Received: May 10, 2010
Revised: June 21, 2010
Accepted: July 6, 2010
Published: August 5, 2010
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Note Added in Proof
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formed by PRC1.
Cell 142, 433–443, August 6, 2010 ª2010 Elsevier Inc. 443
Aurora Kinases and Protein Phosphatase 1
Mediate Chromosome Congression
through Regulation of CENP-E
Yumi Kim,1,2,4 Andrew J. Holland,1,4 Weijie Lan,1 and Don W. Cleveland1,3,*
1Ludwig Institute for Cancer Research
2Department of Molecular and Cellular Biology, University of California, Berkeley, CA 94720, USA
3Department of Cellular and Molecular Medicine
University of California, San Diego, La Jolla, CA 92093, USA
4These authors contributed equally to this work
*Correspondence: dcleveland@ucsd.edu
DOI 10.1016/j.cell.2010.06.039
SUMMARY
Opposing roles of Aurora kinases and protein phos-
phatase 1 (PP1) during mitosis have long been sug-
gested. Here, we demonstrate that Aurora kinases
A and B phosphorylate a conserved residue on the
kinetochore motor CENP-E. PP1 binds CENP-E via
a motif overlapping this phosphorylation site and
binding is disrupted by Aurora phosphorylation.
Phosphorylation of CENP-E by the Auroras is en-
riched at spindle poles, disrupting binding of PP1
and reducing CENP-E’s affinity for individual micro-
tubules. This phosphorylation is required for CENP-
E-mediated towing of initially polar chromosomes
toward the cell center. Kinetochores on such chro-
mosomes cannot make subsequent stable attach-
ment to spindle microtubules when dephosphoryla-
tion of CENP-E or rebinding of PP1 to CENP-E is
blocked. Thus, an Aurora/PP1 phosphorylation
switch modulates CENP-E motor activity as an
essential feature of chromosome congression from
poles and localized PP1 delivery by CENP-E to the
outer kinetochore is necessary for stable microtu-
bule capture by those chromosomes.
INTRODUCTION
Accurate chromosome segregation during mitosis requires the
bipolar attachment of duplicated chromosomes to spindle
microtubules emanating from opposite poles. Each time a cell
divides, a specialized proteinaceous structure called the kineto-
chore assembles on the surface of each centromere, and it is
the kinetochore that binds to spindle microtubules and directs
chromosome motion during mitosis (Cleveland et al., 2003).
Microtubule capture by the kinetochore is a stochastic process.
Initial kinetochore attachment is often mediated via an interac-
tion with the lateral surface of a microtubule, and kinetochores
attached in this manner undergo rapid, dynein-mediated pole-
444 Cell 142, 444–455, August 6, 2010 ª2010 Elsevier Inc.
ward motion (Rieder and Alexander, 1990). Although some
chromosomes achieve biorientation without being transported
to the spindle pole, dynein-mediated transport is an important
mechanism to collect chromosomes to a common microtu-
bule-dense region, where kinetochores have a greater chance
of promoting efficient chromosome alignment.
Congression of polar-localized chromosomes to a metaphase
position is powered by a processive, plus end-directed kineto-
chore motor CENP-E (Kinesin-7) (Kapoor et al., 2006; Kim
et al., 2008). In various cell types and organisms, removal or
inhibition of CENP-E leads to a failure in complete metaphase
chromosome alignment, with a few unattached chromosomes
found close to the spindle poles (Putkey et al., 2002; Wood
et al., 1997; Yao et al., 2000; Yucel et al., 2000). Even the kinet-
ochores that do become bioriented and fully aligned in the
absence of CENP-E stably bind only half as many microtubules
(Putkey et al., 2002). Our finding that CENP-E possesses a highly
flexible and very long coiled-coil (Kim et al., 2008) raises the
possibility that, while it can work advantageously for initial
capture, CENP-E may also contribute, in part, to the inappro-
priate attachments of kinetochores. Indeed, the process of
capturing spindle microtubules by kinetochores is prone to
errors. Undesirable attachment frequently occurs in early prom-
etaphase, with a single kinetochore capturing microtubules from
both spindle poles (merotelic attachment), or both sister kineto-
chores attached to the same pole (syntelic attachment) (Cimini
and Degrassi, 2005). These improper kinetochore attachments,
if not resolved, can lead to chromosome missegregation and
aneuploidy (Holland and Cleveland, 2009).
Correction of aberrant kinetochore attachment requires
a conserved Ser/Thr kinase Aurora/Ipl1 (Lampson et al., 2004;
Tanaka et al., 2002). While budding yeast has a single Aurora
kinase Ipl1, metazoans express at least two Aurora kinases,
Aurora A and B. Like Ipl1, Aurora B is a component of the chromo-
some passenger complex (together with INCENP, Survivin, and
Borealin/Dasra) and is targeted to the inner centromere from
prophase to metaphase (Ruchaud et al., 2007). Aurora B is
thought to aid chromosome biorientation by destabilizing the
kinetochore-microtubule interaction of improperly attached
chromosomes (Cimini et al., 2006). Several proteins directly
involved in microtubule capture at the kinetochore, including
Dam1 in budding yeast and the core kinetochore microtubule
binding components in metazoans (Ndc80 and KNL1), are known
Aurora B substrates (Cheeseman et al., 2002, 2006; Gestaut
et al., 2008; Liu et al., 2010), and phosphorylation by Aurora B
has been shown to decrease the affinity of these proteins for
microtubules (Cheeseman et al., 2006; Gestaut et al., 2008).
Despite the high sequence similarity with Aurora B, Aurora A
plays distinct roles during mitosis. Localized to the centrosomes
during interphase and at the spindle poles during mitosis, Aurora
A has been implicated in promoting mitotic entry and is required
for centrosome maturation and separation (Marumoto et al.,
2005). Inhibition of Aurora A has also been reported to cause
chromosome congression defects (Hoar et al., 2007; Kunitoku
et al., 2003; Marumoto et al., 2003); however, how Aurora A
acts to promote chromosome alignment is unknown.
Genetic evidence in yeast (Francisco et al., 1994) and in verte-
brates (Emanuele et al., 2008; Liu et al., 2010) suggest that the
Aurora kinase activity is opposed by the ubiquitous Ser/Thr phos-
phatase, protein phosphatase 1 (PP1/Glc7). In vertebrates, PP1
isoforms a and g can be detected at outer kinetochores (Trinkle-
Mulcahy et al., 2006; Trinkle-Mulcahy et al., 2003), and PP1 has
been shown to stabilize kinetochore-microtubule attachment by
counteracting Aurora B kinase activity (Liu et al., 2010; Sassoon
et al., 1999). Recently, the non-essential yeast protein Fin1 and
conserved kinetochore protein KNL1 have been identified to target
some PP1 to yeast and vertebrate kinetochores, respectively
(Akiyoshi et al., 2009; Liu et al., 2010). However, whether the kinet-
ochore possesses multiple docking modules for PP1 is not known.
Phosphorylation of the C-terminal tail of CENP-E by Cdk1,
MAPK, or Mps1 has been previously proposed either to regulate
CENP-E motor activity prior to its binding to kinetochores (Espeut
et al., 2008) or inhibit a microtubule binding site in the tail that may
link anti-parallel, midzone microtubules in anaphase (Liao et al.,
1994; Zecevic et al., 1998). Additionally, among a proteomic
screen of 260 mitotic phosphoproteins, CENP-E was identified
to be multiply phosphorylated during mitosis (Nousiainen et al.,
2006). However, the significance of the phosphorylations of
CENP-E has not been established. Using purified components,
selective inhibitors and a phosphospecific antibody, here we
demonstrate that Aurora kinases, both A and B, phosphorylate
a single conserved residue close to the CENP-E motor domain.
We also identify a docking motif for PP1 that overlaps the site
of phosphorylation and demonstrate that PP1 binding to CENP-E
is disrupted by Aurora-mediated phosphorylation. Our findings
establish an Aurora/PP1 phosphorylation switch that is required
not only for congression of polar chromosomes through modula-
tion of the intrinsic motor properties of CENP-E, but also for
subsequent stable biorientation of those chromosomes by
CENP-E’s delivery of PP1 to the outer kinetochore.
RESULTS
A Conserved Phosphorylation Site Near the Neck
Domain of CENP-E Is Phosphorylated by Aurora
A and B In Vitro
In searching for the origin of the one-dimensional diffusion found
in CENP-E motility (Kim et al., 2008), we identified a highly
conserved stretch of basic residues downstream of the CENP-E
coiled-coil neck (Figure 1A). Consisting of four or more consec-
utive arginines or lysines, this basic stretch and the following
threonine (422 in human and 424 in Xenopus laevis) are
conserved in almost all the eukaryotes that possess a clear
CENP-E homolog. Interestingly, the conserved threonine resides
in a consensus motif for phosphorylation by Aurora kinase
(Cheeseman et al., 2002) and has been previously mapped as
a phosphorylation site in a mass spectrometry-based proteomic
screen of mitotic spindles (Nousiainen et al., 2006).
To test whether CENP-E T422 (424 in Xenopus laevis) is phos-
phorylated by Aurora kinases, we performed in vitro kinase
assays using purified Aurora kinases and portions of Xenopus
CENP-E as a substrate. Xenopus Aurora B, together with its
activator INCENP, phosphorylated both full-length and a motor
fragment of CENP-E (CENP-E1-473). However, Aurora B failed
to phosphorylate CENP-E1-473 in which threonine 424 had
been converted to alanine (Figure 1B). Xenopus CENP-E
T424 was also readily phosphorylated by Aurora A (Figures
S1A and S1B available online), confirming that the conserved
threonine located close to the CENP-E motor domain is phos-
phorylated by both Aurora A and B in vitro. The stoichiometry
of CENP-E1-473 phosphorylation by Aurora A saturated at two
moles of PO4 per mole of CENP-E1-473, most likely with an
additional phosphorylation site located C-terminal to T424, as
a shorter CENP-E1-415 fragment was not phosphorylated by
either Aurora kinase (Figure S1B).
Aurora A and B Contribute to Phosphorylation
of CENP-E T422 in Cells
To examine the phosphorylation of CENP-E T422 in vivo, a rabbit
polyclonal antibody was generated against a phosphopeptide of
human CENP-E surrounding T422 (Figure S1C). The affinity
purified anti-pT422 antibody recognized recombinant human
CENP-E1-429 only in the presence of active kinase (Figure 1C)
and recognition of phosphorylated Xenopus CENP-E1-428 by
the anti-pT422 antibody was abolished by the mutation T424A
(Figure S1D). The anti-pT422 antibody also recognized wild-
type (WT) CENP-E immunoprecipitated from nocodazole-
arrested human (DLD-1) cells, but not CENP-E containing a
T422A mutation or WT CENP-E that had been incubated with
l-phosphatase (Figure 1D). Together, these results demonstrate
that the anti-pT422 antibody specifically recognizes CENP-E
phosphorylated at T422.
To establish whether Aurora A or B phosphorylates CENP-E
T422 in cells, we took advantage of the anti-pT422 antibody
and a series of small molecule inhibitors that specifically inhibit
either one or both of the Aurora kinases. As expected, treatment
with the dual Aurora kinase inhibitor VX-680 (Harrington et al.,
2004) abolished phosphorylation of the Aurora A substrate
Transforming acidic coiled-coil 3 (p-TACC3) and the Aurora B
substrate histone H3 (p-Histone H3) (Figure 1E). VX-680 treat-
ment abolished phosphorylation of CENP-E at T422, whereas
treatments with an Aurora A specific inhibitor (MLN8054) or an
Aurora B specific inhibitor (ZM447439) (Ditchfield et al., 2003;
Manfredi et al., 2007) resulted in only a partial reduction in
T422 phosphorylation, indicating that inhibition of either Aurora
kinase alone is not sufficient to eliminate the phosphorylation
Cell 142, 444–455, August 6, 2010 ª2010 Elsevier Inc. 445
A 1 324 Figure 1. A Conserved Threonine Close to
CENP-E motor core neck the Motor Domain of CENP-E Is Phosphory-
335 345 355 365 375 385 395 405 415 425
lated by Aurora A and B on the Kinetochores
Human VNE VS TDE AL L KR YRKE I MD L KKQL EE VS - - L E TR AQAMEKDQL AQL L EEKD L L QK VQNEK I EN L TRML VTSSS L T L QQE L K AKRKRR VTWC L G
of Unaligned Chromosomes
Mouse VNE VSNDE AL L KR YRRE I AD L RKQL EE VN - - TK TR AQEMEKDQL AQL L DEKD L L QK VQDEK I NN L KRML VTSSS I AL QQE L E I KRKRR VTWC YG (A) Alignment of CENP-E protein sequences using
Chicken VNE VL DDD AL L KR YRKE I L D L KKQL EE VS - - MK TQ I H AMEKHQL AH L L EEKNS L QKMQEDR I RN L TEML VTS AS FSSKQN AK ARRRRR VTWAPG
Xenopus VNE VL DDE AL L KR YRKE I L D L KKQL EN L ESSSE TK AQAMAKEEH TQL L AE I KQL HKEREDR I WH L TN I VVASSQES - QQDQR VKRKRR VTWAPG ClustalW algorithm. a-helical coiled-coil in the
Drosophila VNEMVSD ATMMKR L ERE I K VL KDK L AEEE - - - - - - - - - - - - - - - - - RKNENQQK VEH L ERQ I KHDMHK I I CGHS L SDKGQQ - - - - KRRR TWCP T
a d a d a d a d a d a d Paircoil score CENP-E neck were predicted for human CENP-E
0
Neck linker α-helical coiled-coil using Paircoil (Berger et al., 1995). Heptad repeat
0.5 Phosphothreonine
positions (a and d) in the coiled-coil are indicated.
B Aurora B kinase assay C Aurora B kinase assay E (B) In vitro kinase assays using Aurora B/INCENP
to phosphorylate Xenopus full-length CENP-E
4A

4A

WT
KD
Aurora B: -
2

2
T4

T4

or CENP-E1-473 with or without T424A mutation.

E

E
73

73

3
-47

-47
- NP-

- NP-

HCENP-E1-429
E 1-4

E 1-4

Coomassie
Coomassie staining of purified proteins and auto-
E1

E1

(49 kDa)
E

E
XC

XC
XC

XC

XC
XC

kDa kDa
radiogram showing incorporation of g-32P ATP.
pT422 Input
175 175 (C) Coomassie staining and immunoblot for pT422
HCENP-E
83 83
using human CENP-E1-429 incubated with wild-
A

type (WT) or kinase dead (KD) Aurora B.

22

62
WT

WT

62
D
T4

MycLAP-CENP-E:
MycLAP-CENP-E

λ-PPase: - - + IP
(D) CENP-E immunoprecipitates from nocoda-
(340 kDa)

47.5 47.5
Myc zole-treated DLD-1 cells expressing either WT or
IP
pT422
T422A MycGFP-CENP-E were blotted with the
Inhibit
32
P Coomassie pT422 antibody. Half the WT immunoprecipitate
was treated with l-phosphatase.
DNA Tubulin ACA pT422 Merge
F (E) Nocodazole-arrested DLD-1 cells expressing
MycGFP-CENP-E were treated with Aurora kinase
prometa
Early

inhibitors and MG132 and blotted for P-TACC3

(Aurora A substrate), P-Histone H3 (Aurora B
substrate), and tubulin (loading control). CENP-E
immunoprecipitates using a Myc antibody were
prometa
Late

blotted with the pT422 antibody.

(F) PtK2 cells were stained for DNA (Blue), Tubulin
(Green), ACA, and pT422 (Red).
(G) Nocodazole-arrested HeLa cells were treated
prometa

with VX-680 and MG132 and stained for CENP-E

Late

(Green), pT422 (Red) and DNA (Blue).

(H) pT422 fluorescence intensity was normalized
to the total CENP-E fluorescence. Plots show the
G CENP-E pT422 Merge H mean of > 15 cells per condition from two indepen-
Normalized to pT422 to

dent experiments. ***p < 0.0001 by t test. Error

total CENP-E ratio

1.2 n>15
***
DMSO
Noc +

1.0 bars represent SEM. See also Figure S1 and

0.8 Figure S2.
0.6

0.4
VX-680
Noc +

0.2

0
DMSO VX680

of CENP-E T422. However, when cells were treated with the outer kinetochore protein Bub1 (Figure S2A). Kinetochore-
MLN8054 and ZM447439 together to inhibit both Aurora A localized pT422 disappeared following depletion of CENP-E by
and B, phosphorylation of T422 was completely inhibited (Fig- siRNA (Figure S2B), confirming the specificity of the pT422 stain-
ure 1E). Thus, we conclude that both Aurora A and B contribute ing at kinetochores. Inhibition of Aurora kinases with VX-680
to the phosphorylation of CENP-E at T422 in vivo. sharply reduced kinetochore-localized pT422 signal (Figure 1G).
When normalized to the total level of CENP-E at the kinetochore
Phosphorylation of CENP-E T422 Is Enriched (which is also reduced in VX-680 treated cells (Ditchfield et al.,
on Kinetochores Close to the Spindle Poles 2003)), a > 90% reduction in T422 phosphorylation was seen
In unperturbed PtK2 cells, pT422 staining was uniformly detect- following VX-680 treatment (Figure 1H), demonstrating that
able at individual kinetochores in early prometaphase, which kinetochore-localized CENP-E is a substrate for Aurora kinases
colocalized with the centromere components recognized by in vivo.
autoantisera containing centromere antibodies (ACA) (Figure 1F).
The kinetochore-localized pT422 signal was reduced on chro- Aurora-Mediated Phosphorylation of CENP-E T422
mosomes congressed to the equator of the cells, but remained Reduces Its Affinity for Microtubules
enriched at the kinetochores of unaligned chromosomes that To determine if phosphorylation of T422 affects the motor
are close to the spindle poles (Figure 1F). In nocodazole-treated properties of CENP-E, we phosphorylated T424 of Xenopus
HeLa cells, the pT422 antibody recognized a large crescent CENP-E motor (CENP-E1-473) and measured CENP-E’s microtu-
around kinetochore pairs, which colocalized with CENP-E and bule-stimulated ATPase activity in the presence of an increasing

446 Cell 142, 444–455, August 6, 2010 ª2010 Elsevier Inc.

A ATPase assays B Microtubule pelleting assays (2 mM MgADP) Figure 2. Phosphorylation of T424 of Xeno-
pus CENP-E by Aurora Kinase Reduces
15 n=3 Microtubule (μM): 0 0.125 0.25 0.5 1 2 5 CENP-E’s Affinity for Microtubules and
ATPase rate (s-1)

S P S P S P S P S P S P S P Reduces Its Run Length In Vitro

10 XCE1-473 MT (A) ATPase rates of Xenopus CENP-E1-473 and
XCE1-473 phosphorylated CENP-E1-473 measured with
MT
5 Aurora A increasing concentrations of microtubules. Plots
T424A XCE1-473
show the mean of three independent experiments
0
MT and error bars represent SEM. kcat and KmMT
0 1 2 3 4 5 T424A XCE1-473 MT values are represented ± SE.
Microtubules (μM) Aurora A
(B) Equilibrium binding of WT or T424A CENP-E1-473

CENP-E bound (%)

C 3 mM MgADP, 33 ˚C
XCE1-473 XCE1-473 + Aurora A
70
(incubated with or without Aurora A) to microtu-
60
50 WT bules in the ADP state. S, supernatant; P, pellet.
40 WT + Aurora A Percent of CENP-E1-473 bound is shown below
30
T424A (Xenopus) (n = 3; Error bars represent SD).
20
10
T424A + Aurora A (C) Kymographs showing diffusive motion of
0 CENP-E1-473-RFP preincubated with or without
Aurora A in 3 mM MgADP at 33 C.
0 1 2 3 4 5
Microtubule (μM)
(D) Duration of binding (t) was distributed exponen-
tially, and the mean binding time (tmean) was
D XCE1-473 F XCE1-473 determined by fitting the data into a cumulative
XCE1-473 + Aurora A XCE1-473 + Aurora A
distribution function (exp [-t/tmean]). Inset shows
1-cumulative probability

17 sec 1-cumulative probability 1.6 μm 1-cumulative probability of CENP-E1-473-RFP

time

1 12 sec 1 1.2 μm
Frequency of events (%)

Frequency of events (%)

binding time plotted on a log scale. The tmean is

0.1
17 ± 0.13 s for CENP-E1-473-RFP, n = 231 and
0.1
60
70
50
60
12 ± 0.07 s for CENP-E1-473-RFP plus Aurora A,
0.01 0.01

50 40
40
0.001
0 25 50 75 100
0.001
0 2 4 6 8 10 n = 240. Values represented ± SE of the curve fit.
30
30 Duration of binding (sec) Run length (μm)
20
Probability < 0.0001 that the duration of binding
20
10 10 for CENP-E1-473-RFP plus Aurora A was distrib-
112 sec 0 0 uted the same as the duration of binding for
0 20 40 60 80 100 0 1 2 3 4 5 6 7 8
2 μm
Duration of binding (sec) Run length (μm) CENP-E1-473-RFP (by Kolmogorov-Smirnov Test).
(E) Kymographs showing processive motion of
E 3 mM MgATP, 33 ˚C G CENP-E1-473-RFP in the presence of 3 mM MgATP
XCE1-473 XCE1-473 + Aurora A XCE1-473 at 33 C. See Movie S1.
time

(F) Run length of CENP-E1-473-RFP was determined

5 μm

by fitting the data into a cumulative distribution func-

Microtubules

tion. Inset shows 1-cumulative probability of CENP-

E1-473-RFP run length plotted on a log scale. Mean
run length is 1.6 ± 0.02 mm for CENP-E1-473-RFP,
n = 337 and 1.2 ± 0.02 mm for CENP-E1-473-RFP
plus Aurora A, n = 294. See Movie S1. Probability <
112 sec
0.05 that the run lengths for CENP-E1-473-RFP
2 μm
plus Aurora A were distributed the same as the
CENP-E1-473-RFP run lengths (by Kolmogorov-
Smirnov Test). Values represent ± SE of the curve fit.
(G) Accumulated CENP-E (green) at the end of the
microtubules (red). See also Figure S3.

concentration of microtubules (Figure 2A). The maximal ATP and the gliding speed (data not shown), it is likely that the phos-
turnover rate (kcat) was not affected by Aurora A phosphorylation phorylation of T424 reduces CENP-E’s microtubule affinity
(13 ± 0.6 s-1 for CENP-E1-473, n = 3; 14 ± 1.3 s-1 for CENP-E1-473 primarily in its ADP bound state without affecting the rate-limiting
plus Aurora A, n = 3) (Figure 2A). However, the concentration of step in CENP-E enzymatic cycle (Woehlke et al., 1997). To test
microtubules required to reach the half maximal ATPase rate this hypothesis, the extent of Xenopus CENP-E1-473 binding to
(KmMT) was increased by > 3 fold following phosphorylation microtubules was determined with or without prior phosphoryla-
(0.17 ± 0.03 mM for CENP-E1-473, n = 3; 0.64 ± 0.15 mM tion by Aurora kinase (Figure 2B). Phosphorylation of WT
CENP-E1-473 plus Aurora A, n = 3) (Figure 2A). CENP-E1-473 by Aurora A reduced the amount of CENP-E that
KmMT reflects CENP-E’s affinity for microtubules. In the cosedimented with microtubules by
50% with a corresponding
absence of microtubules, kinesins are tightly bound to ADP in 50% increase in apparent Kd(MT) (2 mM for CENP-E1-473; 3 mM for
solution and the rate of ADP release is extremely low (Hackney, CENP-E1-473 plus Aurora A). By contrast, Aurora A did not affect
1988). However, binding of ADP-bound kinesin to microtubules microtubule binding of T424A CENP-E1-473 (apparent Kd(MT) of
greatly accelerates the rate of ADP release, and the kinesin 3.5 mM T424A CENP-E1-473; 3.4 mM for T424A CENP-E1-473
proceeds to complete its enzymatic cycle. Since phosphorylation plus Aurora A), confirming that phosphorylation at T424 reduces
of CENP-E increased KmMT without significantly affecting kcat the affinity of CENP-E for microtubules in the ADP state.

Cell 142, 444–455, August 6, 2010 ª2010 Elsevier Inc. 447

A Figure 3. Phosphorylation of CENP-E at
T422 Is Required for Chromosome Align-
MycGFP Motor Discontinuous coild-coil KT MT ment
tag domain targeting binding
(A) Diagram of MycLAP-CENP-E transgene with
Myc GFP
previously identified phosphorylation sites. Splice
T422 S454 S611 S1211 T1268 S2509 S2555 variations exist within the CENP-E coiled-coil
S2512 S2558
S2543 domain and phosphorylation sites are numbered
with respect to their position in the CENP-E clone
B CENP-E siRNA targeted siRNA resistant used in this study.
against the 3'UTR of CENP-E transgene (B) Schematic of replacing endogenous CENP-E
endogenous CENP-E induced with Tet with a siRNA resistant transgene.
(C) Immunoblot showing depletion of CENP-E by
Time (h): 0 12 24 36 48 60 72 80 siRNA.
(D) Immunoblot showing knockdown with control
Plate cells Transfect Replate Add Tet (GAPDH) or CENP-E siRNA in cells expressing
siRNA cells Begin filming/ various MycLAP-CENP-E transgenes.
harvest protein (E) Box and whisker plots showing the time spent in
for immunoblot mitosis for cells expressing MycLAP-CENP-E
following transfection of control (GAPDH, green) or
CENP-E (red) siRNA. >90 cells per condition are
C D W tal MycGFP-CENP-E
plotted from at least three independent experiments.
(F) Immunofluorescence images of cells in which

A
n

or

22
re

Control siRNA C-E

A
T

ig
endogenous CENP-E has been replaced with WT
Pa

9A

T4
10
R
siRNA
Control siRNA: + - + - + - + - + - + - or T422A MycGFP-CENP-E. GFP (green); Tubulin
Dilution (%): 100 50 30 20 10 100 kDa CENP-E siRNA: - + - + - + - + - + - + kDa (red). See also Figure S4, Figure S5, Figure S6, Movie
CENP-E Myc S2, and Movie S3.
275 275
Tubulin 55 CENP-E
275
Tubulin 55
require ATP hydrolysis (Kim et al., 2008).
E F WT T422A Following phosphorylation, the duration
of CENP-E1-473-RFP binding to microtu-
n>90 bules was shortened by
30% in the
Time in mitosis (min)

DNA

presence of ADP (t = 17 ± 0.13 s for

CENP-E1-473-RFP, n = 231; t = 12 ± 0.07 s
for CENP-E1-473-RFP plus Aurora A,
n = 240) (Figure 2D), consistent with the
GFP/Tubulin

observation that phosphorylation of

Parental WT Rigor 10A 9A
MycGFP-CENP-E
Phosphorylation Reduces CENP-E Processivity
Total Internal Reflection Fluorescence (TIRF) microscopy was
used to determine how Aurora phosphorylation affects properties
of individual CENP-E molecules. Xenopus CENP-E1-473 was
tagged with the monomeric, photostable red fluorescent protein
TagRFP-T (CENP-E1-473-RFP) (Shaner et al., 2008). Oregon
Green 488-labeled GMPCPP microtubules were tethered to
a coverslip in a flow chamber (Figure S3A) and CENP-E1-473-
RFP was added in the presence of apyrase to induce rigor
binding. As expected, CENP-E1-473-RFP was stably bound in
the absence of nucleotides, and fluorescence signals were
photobleached in one or two steps 89% of the time (75 double
steps and 68 single step; n = 160), consistent with a dimeric state
for the CENP-E1-473 motor (Figure S3B). When CENP-E1-473-RFP
was introduced into the flow chamber in a buffer containing ADP,
both phosphorylated and unphosphorylated CENP-E1-473-RFP
remained loosely bound to microtubules without displaying
directional motility (Figure 2C), supporting our previous observa-
tion that CENP-E motility contains a diffusive mode that does not
T424 reduces CENP-E’s affinity to micro-
T422A tubules in the ADP bound state.
Processivity of CENP-E in the presence
of ATP was reduced after phosphoryla-
tion on T424, with run lengths of phosphorylated CENP-E1-473-
RFP on individual microtubules 25% shorter than those of the
unphosphorylated motor (1.6 ± 0.02 mm for CENP-E1-473-RFP,
n = 337; 1.2 ± 0.02 mm for CENP-E1-473-RFP plus Aurora A,
n = 294) (Figures 2E and 2F). Importantly, once reaching a micro-
tubule end using its plus end-directed motility, individual
CENP-E dimers did not immediately dissociate (Figure 2G), but
remained bound there for 5.8 s (±0.8 SEM, n = 26), a feature
previously observed for several other processive kinesins
(Case et al., 1997; Okada and Hirokawa, 1999; Varga et al.,
2006).
Phosphorylation of CENP-E T422 Is Required
for Chromosome Congression
CENP-E is phosphorylated during mitosis on at least ten sites
(Figure 3A) (Nousiainen et al., 2006; Zecevic et al., 1998), albeit
the significance of these phosphorylations has not been tested.
To determine the consequence of preventing CENP-E phos-
phorylation in human cells, we developed a strategy to replace

448 Cell 142, 444–455, August 6, 2010 ª2010 Elsevier Inc.

endogenous CENP-E with phosphorylation defective transgenes
(Figure 3B). Full-length CENP-E fused at the N-terminus to
a MycGFP epitope tag (Figure 3A) was integrated at a predefined
genomic locus in DLD-1 cells using FRT/Flp-mediated recombi-
nation and expression was induced by addition of tetracycline
(Figure S4A). Time-lapse microcopy revealed that the subcellular
distribution of WT MycGFP-CENP-E closely mirrored that of
endogenous CENP-E, localizing to kinetochores after nuclear
envelope breakdown and relocating to the spindle midzone in
anaphase and to the midbody during cytokinesis (Figure S5A).
Transfection of siRNA targeting the 30 untranslated region of
CENP-E mRNA depleted endogenous CENP-E by
90% across
the population, yielding it undetectable at the kinetochores of
most mitotic cells (Figure 3C and data not shown). As expected,
depletion of CENP-E extended the average duration of mitosis
(to 221 min) compared to control transfected cells (71 min)
(Figures 3D and 3E). Importantly, this delay was largely rescued
by the expression of MycGFP-CENP-E (producing an average of
100 min in mitosis). Replacing endogenous CENP-E with a rigor
mutant (T93N) (Figure 3D) strongly exacerbated the mitotic delay
(to 506 min on average) with a few chromosomes chronically
misaligned near the spindle poles (Figure 3E), confirming our
previous finding that the motor activity of CENP-E is essential
for metaphase chromosome alignment (Kim et al., 2008).
Replacement of endogenous CENP-E with a variant (10A) with
all 10 phosphorylation sites abolished (by substitution to
alanines) produced a robust mitotic delay (505 min on average).
On the other hand, abolishing phosphorylation of the nine sites
other than T422 (9A) (Figure 3D) had little effect on mitotic
progression (114 min on average in mitosis) (Figure 3E). Surpris-
ingly, preventing phosphorylation of T422 alone was sufficient to
produce a substantial mitotic delay (425 min on average),
demonstrating that of these 10 CENP-E phosphorylation sites,
phosphorylation at T422 makes the largest contribution to timely
mitotic progression.
Replacing endogenous CENP-E with the T422A mutant
prevented complete metaphase chromosome alignment, with
a few chromosomes remaining close to the spindle poles in
> 85% of cells (Figure 3F, Figures S4B–S4D, and Figure S5B),
a phenotype highly reminiscent of that observed with diminished
levels of CENP-E (Weaver et al., 2003). Phosphorylation of T422
was not required for the kinetochore recruitment of CENP-E
(Figures S6A and S6B). To eliminate the possibility that mutation
of T422 caused defects other than simply preventing phosphor-
ylation, we created an additional CENP-E phosphodeficient
mutant, in which two arginines in the Aurora consensus motif
were converted to lysines (RR:KK) (Figure S6C). Mutation of
RR:KK did not abolish the epitope of the pT422 antibody (data
not shown). However, recombinant Xenopus CENP-E1-428
carrying the RR:KK mutation could not be efficiently phosphory-
lated by Aurora A and B in vitro (Figure S6D and data not shown)
and the RR:KK mutant was not phosphorylated on T422 in
human cells (Figure S6E). Indeed, replacing endogenous
CENP-E with the RR:KK mutant caused a mitotic delay (Fig-
ure S6F) similar to that observed with the T422A mutant with
a few chromosomes remaining close to the spindle poles, con-
firming that phosphorylation of CENP-E at T422 is required for
chromosome congression.
CENP-E Phosphorylation by Aurora Is Required
for Congression of Polar Chromosomes
CENP-E has been implicated in powering chromosome congres-
sion by transporting mono-oriented chromosomes to the spindle
equator along mature kinetochore fibers of already bioriented
chromosomes (Kapoor et al., 2006). To test whether phosphory-
lation of T422 is required for this process, we adopted a method
to enrich mono-oriented, polar chromosomes (Kapoor et al.,
2006; Lampson et al., 2004) in cells in which endogenous
CENP-E was replaced with the WT or T422A MycLAP-CENP-E
(Figures 4A and 4B). Cells were first treated with monastrol
to generate monopolar spindles with a high frequency of synteli-
cally-attached chromosomes and released from monastrol in
the presence of an Aurora kinase inhibitor (ZM) to allow bipolar
spindles to form while preserving improper kinetochore attach-
ments. Following the removal of ZM, congression of mal-
oriented chromosomes was assessed (Figures 4A and 4B). As
a control, cells were treated in parallel with DMSO to determine
the extent of chromosome misalignment in an unperturbed
mitosis. The enrichment of improper kinetochore attachments
significantly increased the number of polar chromosomes in cells
defective in T422 phosphorylation, but not in cells expressing
WT CENP-E (Figures 4C–4F). Live cell imaging demonstrated
that, following reactivation of the Aurora kinases, improperly
attached chromosomes were frequently moved to either spindle
pole in cells expressing WT or T422A CENP-E (Figure S7A–S7C).
However, these chromosomes remained closely associated with
those poles in cells expressing T422A CENP-E (Figure S7C),
establishing that phosphorylation of CENP-E on T422 by Aurora
kinases is required for the congression of polar chromosomes.
Aurora-Mediated Phosphorylation of CENP-E T422
Opposes Direct Binding of CENP-E to the Catalytic
Subunit of PP1
Following CENP-E T422 is a highly conserved tryptophan, thereby
producing a RRVTW sequence that conforms to the docking motif
for protein phosphatase 1 (PP1) (Figure 5A) (Hendrickx et al., 2009).
Indeed, our mass spectrometry analysis of tandem affinity purified
CENP-E from mitotic human cells identified the catalytic subunit of
PP1 to be associated with CENP-E (data not shown) and PP1 was
also present in CENP-E immunoprecipitates from nocodazole-
arrested DLD-1 cells (Figure 5D). The interaction between CENP-E
and PP1 is direct, as recombinant CENP-E motor (CENP-E1-473)
was recovered together with PP1g in a pulldown experiment using
Microcystin-beads (Figure 5B). Recovery of a stoichiometric (1:1)
complex of CENP-E and PP1 required addition of > 5 molar excess
of CENP-E over PP1, indicating a weak affinity between CENP-E
and PP1. Further, CENP-E with a W425A substitution had mark-
edly reduced binding to PP1 (Figure 5B), demonstrating that the
interaction between CENP-E and PP1 is mediated through the
PP1 docking motif. To test whether phosphorylated T422 is
a substrate for PP1, phosphorylated CENP-E1-473 was incubated
with either PP1g or PP1g preinactivated with the inhibitor Micro-
cystin (Figure 5C). Monitoring of CENP-E’s phosphorylation status
with the pT422 antibody revealed that PP1g rapidly dephosphory-
lated CENP-E T422 (Figure 5C).
Previous reports have shown that phosphorylation of serine or
threonine overlapping the PP1 docking motif impairs the binding
Cell 142, 444–455, August 6, 2010 ª2010 Elsevier Inc. 449
A Figure 4. CENP-E Phosphorylation by
DMSO MG
DMSO MG1 3 2 Aurora Is Required for Congression of Polar
3 hr 1 .5 hr Chromosomes
(A) Drug treatment scheme to enrich mono-
Mon ZM+MG MG oriented chromosomes near spindle poles.
Monast rol ZM + MG1 3 2 MG1 3 2
(B) Diagram illustrating attachment status of
2 hr 1 hr 1 .5 hr
Fix for IF chromosomes following treatment with drug
regime in (A).
(C and D) Immunofluorescence images of drug-
Monast rol ZM + MG1 3 2 MG1 3 2
B treated cells in which endogenous CENP-E was
replaced with either WT or T422A MycLAP-
CENP-E. KNL-1 (green); DNA (red).
? (E) Percentage of cells from (C) and (D) with polar
chromosomes.
? (F) Number of polar chromosomes in cells from (C)
and (D) displaying chromosome misalignment.
Bars represent the mean of four independent
experiments. ***p < 0.0001 by t test. Error bars
DMSO MG Mon ZM+MG MG represent SEM. See also Figure S7 and Movie S4.
C D
WT T422A WT T422A

in vivo significance of the dephosphoryla-

tion of CENP-E T422 by PP1, we microin-
DNA DNA DNA DNA
KNL1 KNL1 KNL1 KNL1 jected rhodamine-labeled pT422 anti-
bodies into HeLa cells stably expressing
histone H2B-YFP. Consistent with our
E DMS O MG F ***
Cells with polar chromosomes (%)

100 Mon ZM+MG MG

immunofluorescence analysis (Figure 1F),
Number of polar chromosomes

DMS O MG the microinjected rhodamine-labeled

CENP-E siRNA Mon ZM+MG MG
80 pT422 antibody was virtually absent
CENP-E siRNA
from aligned kinetochores, but accumu-
60 lated to high levels at the kinetochores of
chromosomes positioned close to the
40 spindle poles (Figure 6B). Microinjection
of the pT422 antibody substantially de-
20
layed the duration of mitosis compared
0 to control injected cells (average 125 min
WT T422A WT T422A for rhodamine-labeled rabbit IgG injected
MycGFP-CENP-E MycGFP-CENP-E cells; 619 min for rhodamine-labeled
pT422 injected cells) (Figures 6C–6E).
Interestingly, antibody-mediated preser-
to PP1 (Hubbard and Cohen, 1989). Given that CENP-E T422 is vation of phosphorylation on CENP-E T422 promoted dynamic
overlapped by a consensus motif for Aurora kinases and chromosome movements distinct from the chromosome
a conserved motif for PP1 binding, we tested whether Aurora behaviors observed when T422 phosphorylation is abolished
phosphorylation at T422 disrupts PP1’s binding to CENP-E. (Figure S5B). Polar chromosomes congressed to the equator of
Following in vivo inhibition of T422 phosphorylation with the the cell, but most failed to make stable microtubule attachments
pan Aurora inhibitor VX-680, the amount of PP1 associated and fell back out of the spindle equator or continued to move
with CENP-E was dramatically increased (Figure 5D). Moreover, forward to the other pole (Figure 6C). Consistently, the microin-
phosphorylation of CENP-E1-473 by Aurora A resulted in a jected pT422 antibody remained enriched on the kinetochores

10-fold reduction in the binding of CENP-E to the catalytically of chromosomes juxtaposed to the metaphase plate that did
inactive (D64N) PP1g in vitro (Figure 5E), demonstrating that not form stable microtubule attachments (Figure 6B). Thus,
Aurora-mediated phosphorylation of CENP-E T422 opposes despite CENP-E-mediated congression of chromosomes to the
direct binding of CENP-E to PP1. proximity of the spindle equator, stable kinetochore attachment
(and subsequent biorientation) does not occur when dephos-
Dephosphorylation of CENP-E T422 by PP1 Is Required phorylation of CENP-E by PP1 is blocked.
for Stable Biorientation of the Chromosomes
Congressed by CENP-E DISCUSSION
The pT422 antibody inhibited PP1-mediated dephosphorylation
of Xenopus CENP-E1-473 at T424 (the position homologous to Here, we demonstrate that phosphorylation by Aurora kinases
T422 in human CENP-E) in vitro (Figure 6A). Thus, to test the of a conserved residue (T422) close to the CENP-E motor

450 Cell 142, 444–455, August 6, 2010 ª2010 Elsevier Inc.

A 420 425 B Input (1/10) PP1-pulldown Figure 5. Aurora Phosphorylation of CENP-E
Xenopus K RKRR VTWAP T422 Opposes Direct Binding of CENP-E to
WT W425A WT W425A
Human K RKRR VTWC L PP1
Mouse KRKRR VTWC Y CENP-E1-473 1x 2x 5x 1x 2x 5x - 1x 2x 5x 5x 1x 2x 5x 5x
(A) CENP-E T422 overlaps the conserved PP1
Chicken RRRRR VTWAP PP1γ - - - - - - + + + + - + + + - kDa
Drosophila - - KRRR TWCP
docking motif (R/K-R/K-V-X-F/W).
CENP-E1-473 57 (B) Coomassie staining of the PP1-bound
PP1 docking motif
complexes purified with Microcystin-agarose. 1,
PP1γ 40
Aurora 2, or 5 molar excess of WT or W425A Xenopus
phosphorylation CENP-E1-473 was incubated with Xenopus PP1g.
(C) In vitro phosphatase assay using PP1g (with or
without Microcystin) and phosphorylated Xenopus
C Aurora A D MycGFP-CENP-E
1-473 1-473 CENP-E1-473 as substrate. Immunoblot shows
CENP-E p-CENP-E IP: IgG Myc phosphorylation of T422 and coomassie stain
t = 0 min
30 min t = 30 min

0
shows purified proteins.

SO
-68
PP1 PP1 + Microcystin (D) Nocodazole-arrested DLD-1 cells expressing

DM
VX
kDa
MycGFP-CENP-E were treated with VX-680 and
Time (min): 0 30 35 40 45 60 35 40 45 60 Myc MG132 before harvesting. CENP-E immunoprecip-
275
57 kDa
itates were blotted for pT422 and PP1.
pT422
pT422 (E) Coomassie staining of the PP1-bound
1-473 275
CENP-E complexes purified with Microcystin-agarose. 1, 2
Coomassie
PP1 36 or 5 molar excess of WT CENP-E1-473 was preincu-
Aurora A
bated with either WT or kinase-dead Aurora A
before incubating with catalytically inactive
E (D64N) PP1g.
Input (1/10) PP1-pulldown
+ KD AurA + AurA + KD AurA + AurA
1x 2x 5x 1x 2x 5x - 1x 2x 5x 5x 1x 2x 5x 5x CENP-E1-473
- - - - - - + + + + - + + + - D64N PP1γ

CENP-E
1-473
Aurora A
KD Aurora A
D64N PP1γ
domain is essential to promote the congression of polar chro-
mosomes and dephosphorylation of this site is required for
the stable biorientation of these kinetochores. Aurora-medi-
ated phosphorylation of this site regulates the intrinsic motor
properties of CENP-E and disrupts the binding of the
opposing phosphatase PP1 to CENP-E, thereby establishing
a bistable phosphoswitch for regulation of CENP-E (see sche-
matic in Figure 7).
The Aurora phosphorylation site on CENP-E is adjacent to its
coiled-coil neck, next to several conserved positively charged
amino acids. Phosphorylation at T422 diminishes the basic
charge of what we propose to be an electrostatic tether directly
involved in microtubule binding (analogous to similar domains
identified in other kinesin family members [Okada and Hirokawa,
1999; Ovechkina et al., 2002; Thorn et al., 2000]). Consistently,
phosphorylation at T422 reduces CENP-E’s affinity for microtu-
bules and allows the motor to dissociate more readily during
processive runs. Phosphorylation of CENP-E 422 is highest on
the kinetochores close to the spindle poles. Since Aurora A is
concentrated at the poles, it is likely to be responsible for phos-
phorylation of T422 on such polar-oriented chromosomes.
Aurora phosphorylation reduces the proportion of time that
each motor molecule is bound unproductively to the many
dynamic astral microtubules nucleated near the pole (Figure 7B).
Phosphorylation-dependent reduction in CENP-E residence
time on an individual microtubule of a kinetochore fiber, on the
other hand, will be of little consequence, as rapid rebinding to
an adjacent microtubule is likely, given the high local concentra-
tion of parallel microtubules that comprise the fiber (Figure 7C).
Thus, Aurora-mediated destabilization of
CENP-E tethering to individual spindle
microtubules yields a variant of kinetic
proofreading (Hopfield, 1974), with local,
destabilized attachment as a means to
eliminate incorrect initial attachments, while allowing productive
CENP-E-powered movement along a kinetochore microtubule
bundle.
A requirement for Aurora A in modulating CENP-E offers
a mechanistic explanation for prior reports that Aurora A inhibi-
tion causes chromosome misalignment with a few chromo-
somes found close to the spindle poles (Hoar et al., 2007;
Kunitoku et al., 2003; Marumoto et al., 2003). Although Aurora
A-mediated phosphorylation of the centromere-specific histone
H3 variant CENP-A has previously been proposed to promote
chromosome congression (Kunitoku et al., 2003), we conclude
that CENP-E is the kinetochore substrate whose Aurora
A-dependent phosphorylation is directly required for chromo-
some congression. For Aurora B, the absence of tension exerted
on mono-oriented polar kinetochores and the juxtaposed posi-
tion of sister kinetochores on syntelically attached chromo-
somes would bring it in close proximity to the highly elongated
and flexible CENP-E, allowing Aurora B phosphorylation to
modulate processivity of CENP-E attached to kinetochores
with reduced tension (Figures 7A and 7B). Further, Aurora
B-dependent phosphorylation in and around the inner centro-
meres of sister kinetochores would also be expected to prefer-
entially destabilize any incorrect attachments made by the
230 nm long CENP-E to microtubules that reach across the
inter-kinetochore space.
Recent evidence has demonstrated that KNL1, one of the core
microtubule binding components thought to be responsible for
end-on attachment at metazoan kinetochores (Cheeseman
et al., 2006), binds PP1 on chromosomes aligned at metaphase.

Cell 142, 444–455, August 6, 2010 ª2010 Elsevier Inc. 451

A B Figure 6. Dephosphorylation of CENP-E
pT422-Rhod Ab injected cells
Aurora A T422 by PP1 Is Required for the Stable Bio-
1-473 1-473
Histone H2B-YFP pT422-Rhod Ab rientation of Chromosomes Congressed
CENP-E p-CENP-E
from a Spindle Pole by CENP-E

Projection
t = 0 min t = 30 min

+pT422 +Rabbit IgG (A) Phosphorylated Xenopus CENP-E1-473 (1 mM)

antibody was preincubated with rabbit IgG or the pT422
1-473 1-473 antibody (3 mM) and subsequently mixed with
t = 60 min p-CENP-E p-CENP-E
PP1g (0.2 mM). Phosphorylation of T422 was
PP1 PP1 determined using rhodamine-labeled pT422 anti-

45° rotation
Boil and probe with pT422-Rhod body and visualized using the rhodamine fluoro-
Time (min): 0 30 60 65 70 75 90 60 65 70 75 90 phore. Anti-Myc immunoblot shows the CENP-
E1-473 loading.
pT422-Rhod 49 kDa
(B) Reconstructed Z-sections of rhodamine-
Myc labeled pT422 antibody-injected live HeLa cells
expressing Histone H2B-YFP. Histone H2B-YFP
C Histone H2B-YFP pT422-Rhod Ab (purple); pT422-Rhod antibody (green).
Rabbit IgG-Rhod

0:00 0:05 0:10 1:00 1:15 1:20 2:25 (C) Time-lapse images of antibody-microinjected
(green) HeLa cells stably expressing Histone
Inject:

H2B-YFP (purple). Numbered arrows track the

movement of four individual chromosomes which
congress to the equator, but fail to stably biorient.
Stills are taken from Movie S5 and Movie S6.
(D) Box and whisker plots showing the time spent
0:00 3:20 3:50 4:00 4:50 5:05
3 3
in mitosis for microinjected cells. Uninjected cells
1 1 1 in the same field of view were also examined. For
pT422-Rhod Ab

each antibody, >120 microinjected cells were

2 2 2
Inject:

observed from two independent experiments. (E)

5:20 7:25 7:35 8:15 8:45 8:50 Bar graphs showing the percentage of abnormal
mitosis for the cells observed in (D).
3 4 4
4

D evidence implicates dephosphorylation

E
of the core microtubule-binding proteins 100
Time in mitosis (min)

Abnormal mitosis (%)

1000
80 (e.g., KNL1 and the four member Ndc80
750
60
complex) by CENP-E-bound PP1 as an
500 essential step in reversing their prior inac-
40
250
tivation by Aurora-dependent phosphor-
20
ylation.
0
Non-Inj Inj Non-inj
o Inj
0
Non-Inj Inj Non-Inj Inj Finally, the spatial regulation of CENP-
Ab injected: Rabbit IgG-Rhod pT422-Rhod Ab injected: Rabbit IgG-Rhod pT422-Rhod E by Aurora kinases and PP1 may provide
an insight into the classic observation that
Binding is through a motif for PP1 docking with an overlapping phosphorylation controls the directionality of two opposing
Aurora phosphorylation site (Liu et al., 2010), a situation similar kinetochore motors on isolated chromosomes (Hyman and
to what we now report for CENP-E. Thus, the vertebrate kineto- Mitchison, 1991). To coordinate prometaphase chromosome
chore has evolved multiple modules for recruiting PP1, with movement, this phosphorylation-dependent switch must turn
recruitment by KNL1 and CENP-E each providing different func- off the minus end-directed motor and turn on the plus end-
tions. Blocking KNL1 recruitment of PP1 increased the number directed motor at the spindle poles. Here, we have shown that
of kinetochores without cold stable microtubules and decreased the plus-end directed motor properties of CENP-E (and binding
the level of PP1 recruited to kinetochores. Nevertheless, it did of PP1) are altered by a gradient of Aurora kinase activity
not affect congression or chromosome alignment, but did lead emanating from the spindle poles. This provides spatial informa-
to an unexplained inhibition of cell growth (Liu et al., 2010). In tion within the mitotic spindle to regulate CENP-E activity
contrast, we have now shown that once CENP-E tows initially according to the position of chromosome.
misoriented chromosomes to the cell center, its subsequent
dephosphorylation and rebinding of PP1 is essential for stable EXPERIMENTAL PROCEDURES
microtubule attachment to the kinetochores on these chromo-
somes (Figure 7D). Thus, we propose a model in which CENP-E Constructs
powers chromosome movement away from the high Aurora The full-length human CENP-E open reading frame was cloned into a pcDNA5/
FRT/TO based vector (Invitrogen) modified to contain an amino-terminus
activity at poles and then exploits its flexible coiled-coil and
Myc-LAP epitope tag. The LAP tag consists of GFP-TEV-S-peptide as previ-
plus end-directed motility to deliver PP1 phosphatase activity ously described (Cheeseman et al., 2004). TagRFP-T (a kind gift from Roger
within its
230 nm reach at the outer kinetochore (Figures 7E Tsien, UCSD) was cloned into pET23d vector (Novagen) containing Xenopus
and 7F). For the kinetochores on these chromosomes, our CENP-E (aa1-473). This cloning strategy generates a 16 amino acid-long linker

452 Cell 142, 444–455, August 6, 2010 ª2010 Elsevier Inc.

 Phosphorylation by Aurora B
Centromere protein E
+
A P P P D P
P P
P P
Cytoplasmic dynein P P
P P PP1
Aurora B PP1 +
activity
PP1
P
+
P
Protein phosphatase 1
+
- + - - + - K-fiber
PP1

Dynein-dependent poleward movement

-
KMN network PP1-mediated dephosphorylation of
Aurora A along a laterally attached microtubule;
+ + CENP-E away from polar Aurora A
Ndc80 complex activity microtubule binding by KMN network
Mis12 is inhibited by phosphorylation
complex
KNL1
ATP, Aurora A
Phosphate B P P E
P PP1 PP1
P

Kinetochore
P P

Centrioles
P P
+ -
K-fiber + P

P - - P
P PP1
Microtubules + PP1 - + - + P
P
Aurora A phosphorylation of CENP-E PP1 bound to CENP-E dephosphorylates
+ at pole releases PP1 + Ndc80 and KNL1, enabling their end-on
microtubule attachment

PP1
C P
P F
P P
PP1 + -

Kinetochore
P PP1
P
P
P
P
+ PP1

K-fiber + PP1
PP1

+ -
- - - +
Processive tracking of p-CENP-E only Stable K-fiber attachment by PP1-bound
+ along a K-fiber
+ KNL1/ Mis12 and Ndc80 complex

Figure 7. A Model of CENP-E Regulation by Aurora Kinases and PP1

(A) Unattached chromosomes are translocated poleward along a single astral microtubule in a dynein-dependent manner (Rieder and Alexander, 1990).
(B) Aurora A phosphorylates CENP-E at T422 near the spindle poles, releasing PP1 from CENP-E. Phosphorylation of CENP-E provides selectivity toward
bundles of kinetochore microtubules (K-fiber) for CENP-E to glide along.
(C) Our evidence here demonstrates that CENP-E powers the congression of polar chromosomes to the spindle equator along the K-fiber of an already bioriented
chromosome, as earlier proposed (Kapoor et al., 2006).
(D) As chromosomes congress, kinetochores move away from the Aurora A gradient concentrated at the spindle poles and CENP-E is dephosphorylated.
(E) Dephosphorylated CENP-E recruits a high local concentration of PP1 to the outer kinetochores of chromosomes it has translocated away from a pole. CENP-E
delivered PP1 is essential for stable, microtubule capture by the kinetochores of these towed chromosomes through its dephosphorylation of key components at
the kinetochore-microtubule interface (e.g., Ndc80 and KNL1), thereby increasing their affinity for microtubules.
(F) Upon biorientation, kinetochore substrates become separated from the inner centromeric Aurora B. Kinetochore-recruited PP1 through its direct binding to
KNL1 and CENP-E stabilizes kinetochore-microtubule interactions.

(MASMTGGGGMGRLR) between CENP-E and TagRFP-T. All human and
Xenopus CENP-E mutants were generated by site-directed mutagenesis
(QuickChange, Stratagene).
Cell Culture and siRNA Treatment
Cells were maintained at 37 C in a 5% CO2 atmosphere in Dulbecco’s
modified eagle medium (DMEM) containing 10% tetracycline free fetal bovine
serum (Clontech), 100 U/ml penicillin, 100 U/ml streptomycin and 2 mM L-
glutamine. For siRNA treatment, 1.5 3 105 cells were plated in a 6-well plate
and duplexed siRNAs were introduced using Oligofectamine (Invitrogen).
siRNAs directed against CENP-E (50 -CCACUAGAGUUGAAAGAUA-30 ) and
GAPDH (50 -UGGUUUACAUGAUCCAAUA-30 ) were purchased from Dharma-
con. Cells were processed for immunofluorescence microscopy or live cell
imaging 48 hr after transfection.
Generation of Stable Cell Lines
Stable DLD-1, H2B-RFP cell lines expressing CENP-E were generated using
the FRT/Flp-mediated recombination as described previously (Holland et al.,
2010). Small molecules were used at the following final concentrations: noco-
dazole, 0.2 mg/ml; taxol, 10 mM; monastrol, 20 mM; S-Trityl-L-cysteine, 5 mM;
MG132, 20 mM; ZM447439 (Tocris Bioscience), 3 mM; VX-680 (Selleck)
0.5 mM and MLN8054 (a kind gift of Patrick Eyers), 0.25 mM. All small molecules
were from Sigma-Aldrich unless otherwise specified.
Immunofluorescence Microscopy
Cells were pre-extracted for 90 s in MTSB (100 mM PIPES [pH 6.8], 0.1%
Triton X-100, 0.1 mM CaCl2, 1 mM MgCl2) and fixed in 2% formaldehyde in
MTSB. Cells were blocked in 2.5% FBS, 0.2 M glycine, 0.1% Triton X-100 in
PBS for 1 hr. For the pT422 staining, cells were extracted and fixed in the pres-
ence of 500 nM Microcystin-LR (EMD). Antibody incubations were conducted
in blocking solution for 1 hr. DNA was detected using DAPI and cells were
mounted in ProLong (Invitrogen). Images were collected using a DeltaVision
Core system (Applied Precision) controlling an interline charge-coupled device
camera (Cooldsnap, Raper). Kinetochore signal intensity was determined
using MetaMorph (Molecular Devices), by measuring integrated fluorescence
intensity with a 10 3 10 pixel square. Background signal was subtracted
from an area adjacent to the kinetochore. The mean integrated fluorescence
intensity of at least 10 kinetochore pairs per cell was calculated. Antibodies
used are specified in the Extended Experimental Procedures.
Single Molecule Assays
CENP-E single molecule assays were performed as previously described (Kim
et al., 2008) with the following modifications. Slides and 22 3 22-mm square

Cell 142, 444–455, August 6, 2010 ª2010 Elsevier Inc. 453

coverslips were silanized as described (Helenius et al., 2006). A flow chamber
was incubated with 50 mg/ml of a rat monoclonal anti-tubulin antibody (YL1/2,
Serotec) for 5 min, followed by 1% Pluronic F-127 (Invitrogen) in BRB80 for
15 min and Oregon Green 488-labeled GMPCPP microtubules for 10 min.

0.2 mg/ml of Xenopus CENP-E1-473-RFP was incubated with 50 mg/ml of
Aurora A in 20 mM Tris (pH 7.5), 25 mM KCl, 1 mM MgCl2, 1mM DTT,
0.1 mM MgATP for 15 min at room temperature and diluted to
0.5 nM before
imaging in motility buffer (25 mM K-PIPES, [pH 6.9], 5 mM MgCl2, 1 mM EGTA,
1 mM DTT, 0.25% Brij35, 0.5 mg/ml casein, 4.5 mg/ml glucose, 0.2 mg/ml
glucose oxidase, 0.35 mg/ml catalase, 0.5% bME) containing either 3 mM
MgATP or 3 mM MgADP. Frames were captured every 500 ms with 200 ms
exposure, and the typical duration of imaging was 2–3 min. Note, that since
imaging was performed at an elevated temperature (33 C) and in higher
MgCl2, the speed of CENP-E movement was faster than that measured at
room temperature in our previous study (Kim et al., 2008).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures,
Supplemental References, seven figures, and six movies and can be found
with this article online at doi:10.1016/j.cell.2010.06.039.
ACKNOWLEDGMENTS
The authors would like to thank Stephen Taylor, Patrick Eyers, Todd Stuken-
berg, and Arshad Desai for providing reagents. This work was supported by
a grant (GM29513) from the National Institutes of Health to D.W.C., who
receives salary support from the Ludwig Institute for Cancer Research.
A.J.H. is supported by a European Molecular Biology Organization (EMBO)
Long-Term Fellowship.
Received: October 27, 2009
Revised: April 26, 2010
Accepted: June 14, 2010
Published: August 5, 2010
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Cell 142, 444–455, August 6, 2010 ª2010 Elsevier Inc. 455
PNPASE Regulates RNA Import
into Mitochondria
Geng Wang,1 Hsiao-Wen Chen,4 Yavuz Oktay,1 Jin Zhang,5 Eric L. Allen,5 Geoffrey M. Smith,5 Kelly C. Fan,5
Jason S. Hong,5 Samuel W. French,5 J. Michael McCaffery,6 Robert N. Lightowlers,7 Herbert C. Morse III,8
Carla M. Koehler,1,2,* and Michael A. Teitell2,3,5,*
1Department of Chemistry and Biochemistry
2Molecular Biology Institute
3Jonsson Comprehensive Cancer Center, Broad Stem Cell Research Center, California NanoSystems Institute, and Center for Cell Control

University of California at Los Angeles, Los Angeles, CA 90095, USA

4Center for Molecular and Mitochondrial Medicine and Genetics, University of California at Irvine, Irvine, CA 92697, USA
5Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
6Integrated Imaging Center, Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA
7Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon the Tyne, UK
8Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville,

MD 20852, USA
*Correspondence: koehler@chem.ucla.edu (C.M.K.), mteitell@mednet.ucla.edu (M.A.T.)
DOI 10.1016/j.cell.2010.06.035

SUMMARY somes, with mammalian mitochondria importing several

RNA import into mammalian mitochondria is consid-
ered essential for replication, transcription, and
translation of the mitochondrial genome but the
pathway(s) and factors that control this import are
poorly understood. Previously, we localized polynu-
cleotide phosphorylase (PNPASE), a 30 / 50 exoribo-
nuclease and poly-A polymerase, in the mitochon-
drial intermembrane space, a location lacking
resident RNAs. Here, we show a new role for PNPASE
in regulating the import of nuclear-encoded RNAs
into the mitochondrial matrix. PNPASE reduction
impaired mitochondrial RNA processing and polycis-
tronic transcripts accumulated. Augmented import of
RNase P, 5S rRNA, and MRP RNAs depended on
PNPASE expression and PNPASE–imported RNA
interactions were identified. PNPASE RNA process-
ing and import activities were separable and a mito-
chondrial RNA targeting signal was isolated that
enabled RNA import in a PNPASE-dependent
manner. Combined, these data strongly support an
unanticipated role for PNPASE in mediating the trans-
location of RNAs into mitochondria.
INTRODUCTION
Much is understood about the mechanisms that regulate
nuclear-encoded protein import into mitochondria (Chacinska
et al., 2009). By contrast, much less is known about the factors
that regulate mitochondrial RNA import. Almost every organism
with mitochondria imports tRNAs and aminoacyl-tRNA synthe-
tases (Alfonzo and Soll, 2009; Duchene et al., 2009). The number
of imported tRNAs ranges from one in yeast to all in trypano-
different tRNAs both in vitro and in vivo (Kolesnikova et al.,
2004; Rubio et al., 2008). Recently, microRNAs were isolated
from mitochondria (Kren et al., 2009) and the nuclear-encoded
5S rRNA was identified as one of the most abundant RNAs in
human mitochondria (Entelis et al., 2001; Smirnov et al., 2008).
RNase MRP and RNase P enzyme complexes localize and
function in mammalian mitochondria and may contain RNAs
that are encoded within the nucleus. RNase MRP functions
as a site-specific endoribonuclease involved in primer RNA
cleavage during the replication of mitochondrial DNA (Chang
and Clayton, 1987). Mammalian RNase P functions in the
processing of tRNAs during the maturation of mitochondrial
transcripts that encode oxidative phosphorylation (OXPHOS)
components. Large polycistronic RNA transcripts are generated
from the heavy and light strand promoters in mammalian mito-
chondria (Bonawitz et al., 2006). tRNAs often separate the
coding regions for OXPHOS subunits and are processed and
removed by RNase P (Doersen et al., 1985). Earlier studies
showed that the RNA component of RNase P was imported
into mammalian mitochondria (Doersen et al., 1985) and the
RNase P RNA and processing activity has been copurified
from mitochondria (Puranam and Attardi, 2001). By contrast,
RNase P RNA is encoded in the mitochondrial genome in
Saccharomyces cerevisiae (Hollingsworth and Martin, 1986).
Recently, an additional RNase P enzyme, consisting of three
protein subunits, has been purified from human mitochondria.
This alternative RNase P enzyme processes single tRNA 50
precursor sequences in vitro without an RNA component
(Holzmann et al., 2008; Walker and Engelke, 2008). The identifi-
cation of two enzymes with RNase P activity in mammalian
mitochondria warrants further investigation.
Critical open questions about RNA import into mammalian
mitochondria include the selectivity of RNAs, the factor(s) target-
ing RNA from the cytosol, and the translocation pathway(s)
across the mitochondrial membranes (Duchene et al., 2009).

456 Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc.

Different import pathways have been proposed for a subset of
precursors in different species and the details of the components
involved remain ill-defined. For example, the TOM and TIM
protein import complexes have been implicated in the import
of tRNALys in yeast mitochondria (Tarassov et al., 1995). By
contrast, a protein import pathway-independent mechanism
may exist and involve a 600 kDa multi-subunit RNA import
complex (RIC) in Leishmania (Mukherjee et al., 2007). Thus,
inconsistencies among RNA import systems suggest a critical
need to learn more about the nature of the RNAs that are
imported and the factor(s) mediating this import.
Previously we showed an unexpected location in the mito-
chondrial intermembrane space (IMS) for mammalian polynucle-
otide phosphorylase (PNPASE) (Chen et al., 2007; Chen et al.,
2006; Rainey et al., 2006), a 30 / 50 exoribonuclease and
poly-A polymerase that uses phosphorolysis to degrade RNA
(Yehudai-Resheff et al., 2001). This was a surprise because
We expected that PNPASE would instead localize in the RNA-
abundant mitochondrial matrix. Therefore, initial studies on
mammalian PNPASE focused on a general role in maintaining
mitochondrial homeostasis, potentially by regulating adenine
nucleotide levels (Chen et al., 2006; Portnoy et al., 2008). Here,
we show that PNPASE has a central role in augmenting the
import of small RNA components required for DNA replication
and RNA processing into the mitochondrial matrix. We suggest
that PNPASE regulates adenine nucleotide levels and mitochon-
drial homeostasis at least partly by regulating RNA import to
control the abundance of the electron transport chain (ETC)
components.
RESULTS
PNPASE Forms a Trimer in Yeast and Mammalian
Mitochondria
To examine PNPASE in the IMS, a coimmunoprecipitation (IP)
assay was performed to identify potential binding partners. A
6XHis-Protein-C (HisPC) tag was added to the C terminus of
PNPASE and stable PNPASE-HisPC expressing HEK293 cells
were generated. The PNPASE-HisPC protein was detected in
isolated mitochondria by immunoblot (Figure S1A available
online). PNPASE-HisPC was isolated from mitochondria using
sequential purification over Ni2+ and Protein-C affinity columns,
followed by elution, and copurifying proteins were identified by
Sypro Ruby staining (Figure S1B) and liquid chromatography-
tandem mass spectrometry (LC-MS/MS) (data not shown). All
of the identified bands originated from PNPASE, suggesting
that PNPASE lacks partner proteins in vivo. Bands of lower
molecular weight than the 85 kDa of PNPASE monomers
were likely degradation products. The assembly state of
PNPASE was also investigated. Mitochondria from yeast ex-
pressing human PNPASE (Rainey et al., 2006) were detergent
solubilized and separated on blue-native (BN) gels. Immunoblot
showed PNPASE in a complex of 240 kDa (Figure S1C), similar
to the trimeric complex of endogenous mouse hepatocyte
PNPASE (Chen et al., 2006) and bacterially-expressed human
PNPASE (French et al., 2007). PNPASE-HisPC isolated from
HEK293 mitochondria also migrated in a similarly-sized complex
(Figure S1D). Overall, PNPASE assembled identically in yeast
and mammalian mitochondria into a homo-oligomeric
complex, consisting of a trimer or a ‘dimer of trimers’ (Symmons
et al., 2002). These results strongly suggest that PNPASE
assembles and may function similarly in yeast and mammalian
mitochondria.
Pnpt1 Knockout Cells Show Altered Mitochondrial
Morphology and Impaired Respiration
We used several approaches to determine the function of
PNPASE in mitochondria. First, the gene encoding PNPASE
(Pnpt1) was knocked out (KO) in C57BL/6 mice (Figure S2 and
Figure S3). Homozygous Pnpt1neo-flox mice, in which exon 2
was flanked by loxP recombination sites, were viable and fertile.
A complete KO of Pnpt1 exon 2 was generated by crossing
CMVCRE expressing mice with Pnpt1WT/neo-flox heterozygotes
followed by inter-crossing the Pnpt1WT/KO progeny. Pnpt1KO/KO
mice were embryonic lethal (Figure 1A).
Our prior cell line studies showed that RNAi targeting PNPASE
reduced ATP production by OXPHOS and slowed cell growth
(Chen et al., 2006). Recently, a viable liver KO of the COX10
gene, which is required for cytochrome c oxidase and ETC func-
tion, was produced, suggesting that targeted disruption of Pnpt1
in hepatocytes might be tolerated (Diaz et al., 2008). Therefore,
a liver-specific KO (HepKO) of Pnpt1 was generated by the
cross AlbCRE/WT/Pnpt1neo-flox/neo-floxx AlbWT/WT/Pnpt1neo-flox/neo-flox,
which produced fertile progeny at the expected frequency
(Figure S2C). Quantitative real-time PCR (QPCR) from HepKO
liver showed reduced Pnpt1 transcripts containing targeted
exon 2 compared with those containing untargeted exon 28
(Figure 1B). PNPASE protein expression was also markedly
reduced in HepKO liver compared with sex-matched littermate
WT liver (Figure 1B). The expression of Pnpt1 transcripts and
PNPASE protein in HepKO liver likely arises from heterogeneous
liver elements. Also, the AlbCRE deleting strain is hepatocyte-
specific but incomplete (Postic and Magnuson, 2000). The
physiology of these mice will be reported elsewhere.
The ultrastructure of HepKO liver mitochondria was investi-
gated by transmission electron microscopy (TEM). Rather than
displaying ordered, linear cristae with convolutions as in WT
mitochondria, the HepKO mitochondria showed disordered
circular and smooth IM cristae (Figure 1C), similar to mitochon-
dria that are impaired for OXPHOS (Mandel et al., 2001) and to
Pnpt1 RNAi mammalian cell lines (Chen et al., 2006). These
and prior results suggesting that reduced PNPASE caused a
decrease in ATP production prompted the evaluation of O2
consumption from HepKO liver mitochondria. Oxygen electrode
studies showed a 1.5- to 2-fold decrease in the activity of
Complex IV and Complexes II+III+IV when normalized to citrate
synthase activity in HepKO compared to WT mitochondria
(Figure 1D). Combined, these data establish an in vivo role for
PNPASE in mitochondrial morphogenesis and respiration
through an unknown mechanism.
PNPASE Is Required for the Processing
of Mitochondrial RNA Transcripts
The data showing decreased respiration in HepKO mitochon-
dria suggested a reduction in functional ETC complexes. There-
fore, RNA processing and translation were examined in cells
Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc. 457

with decreased PNPASE. HEK293 cells with > 75% reduced
PNPASE expression were generated by RNAi, followed by
mitochondrial RNA (mtRNA) transcript quantification using
QPCR normalized to cytosolic GAPDH RNA. All mtRNAs tested
were reduced in Pnpt1 RNAi cells compared to WT cells
(Figure 1E). Proteins translated from mtRNAs were decreased
in HEK293 Pnpt1 RNAi cells (data not shown) and HepKO liver
cells (Figure 2A), strongly suggesting that a decrease in
functional ETC complexes was responsible for decreased
respiration.
The processing of polycistronic mtRNAs was investigated
because reduced PNPASE could cause an accumulation of large
precursor transcripts, resulting in reduced ETC proteins. Tran-
script processing requires RNase P excision of the tRNAs
between ETC gene coding regions. Primers were designed to
test processing between adjacent Cox1 and Cox2 transcripts
that are separated by tRNAser and tRNAasp (Figure 2B). The
primer set Cox1f and Cox1r generates a 450-bp fragment
whereas the primer pair Cox1f and Cox2r generates a 900-bp
fragment when tRNAser and tRNAasp are not excised from large
458 Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc.
Figure 1. Deletion of Pnpt1 in Hepatocytes
Impairs Mitochondrial Function
(A) Breeding strategy (top) and results (bottom) for
attempting to generate a PNPASE KO mouse.
(B) Hepatocyte-specific Pnpt1 KO (HepKO) in
4-week old mice. Top: QPCR for liver Pnpt1
expression using an exon 2 – exon 3 primer pair
versus a primer pair within exon 28. Bottom:
PNPASE immunoblot from 4-week old WT and
HepKO mouse livers.
(C) HepKO mitochondria have altered cristae. Left:
TEM of 6-week old littermate livers shows circular,
smooth HepKO IM cristae in contrast to linear,
stacked cristae of WT mitochondria. Right: Anal-
ysis of cristae morphology in which a single normal
cristae within a mitochondrion was scored as
normal. Indet = indeterminate.
(D) Decreased respiration in isolated HepKO mito-
chondria. Oxygen consumption (nmol/min/mg
protein) for ETC complexes IV and II+III+IV was
measured using an O2 electrode. Mitochondrial
mass was determined by citrate synthase (CS)
activity using a spectrophotometer. Respiratory
activities are shown normalized to CS activity.
(E) Decreased mature mtRNAs in HEK293 cells
with RNAi to PNPT1. Transcripts were quantified
relative to cytosolic GAPDH expression by QPCR
from HEK293 cells 7d post-infection (nadir) with
scramble (Scr) or PNPT1 RNAi retroviral
constructs. See also Figure S1, Figure S2, and
Figure S3.
precursor transcripts. Isolated mitochon-
drial RNAs were examined by RT-PCR. A
900-bp fragment was detected from
HepKO but not from WT liver mitochon-
dria (Figure 2B). Similar results were
obtained using the same primers in
PNPASE KO mouse embryonic fibro-
blasts (MEFs) (Figure S3 and Figure S4A).
To query RNA processing at a second site, primers were gener-
ated for adjacent Cox2 and Atp8/6 loci, separated by tRNAlys.
Again, polycistronic transcripts accumulated in the PNPASE
KO MEFs (Figure S4B). The sizes of Cox1 and Cox3 transcripts
were investigated using specific probes and Northern blot
(Figure 2C). In addition to the mature Cox1 and Cox3 transcripts,
a range of larger precursor transcripts was seen in HepKO liver
cells. Also, the mature 0.9-kb Cox3 transcript was more
abundant in WT than HepKO liver. The abundance of mitochon-
dria-encoded COX3 and ND6 proteins was determined
(Figure 2D). In HepKO liver mitochondria, the steady-state
abundance of PNPASE was decreased by 2-fold compared
to the WT, similar to a 2-fold decrease for COX3 and ND6
proteins. Controls TOM40, MORTALIN, TIM23, and BAP37
showed that the amount of nuclear-encoded mitochondrial
proteins, and therefore the mitochondrial mass, was similar
between HepKO and sex-matched WT littermate liver cells.
Thus, the processing of polycistronic mtRNAs was impaired in
mitochondria with reduced PNPASE, resulting in fewer mature
mtRNAs and reduced ETC complexes.

RNase P RNA Binds to PNPASE and May Function
in PNPASE-Dependent mtRNA Processing
Although an in vitro form of the RNase P enzyme can function
without nucleic acid (Holzmann et al., 2008; Walker and Engelke,
2008), the RNA component of RNase P localizes to mitochondria
(Puranam and Attardi, 2001) and has not been excluded from
mtRNA processing in vivo (Holzmann et al., 2008). Therefore,
the abundance of RNase P RNA in HepKO liver mitochondria
was determined by RT-PCR and QPCR (Figure 3A). Reproduc-
ibly, RNase P RNA was decreased by 75% in HepKO versus
WT liver mitochondria, suggesting that PNPASE may help import
and/or stabilize RNase P RNA. Therefore, we determined
whether RNase P RNA directly bound to PNPASE in HEK293
cells stably expressing dual-tagged PNPASE-HisPC. Isolated
mitochondria were treated with nuclease and tagged PNPASE
was purified. RNase P RNA was amplified by RT-PCR and
copurified with PNPASE (Figure 3B, lane 4). Importantly, control
IMS-localized TIM23-HisPC in stably-expressing HEK293 cells
did not bind RNase P RNA (Figure 3B, lane 2). PNPASE also
Figure 2. HepKO Liver Mitochondria Do Not
Efficiently Process mtRNA Precursors
(A) In organello protein synthesis. WT and HepKO
mitochondria (100 mg) were treated with micro-
coccal nuclease S7, and in organello translation
was performed using [35S]-MET. TOM40 immuno-
blot shows equivalent mitochondria in each assay.
(B) RNA was isolated from WT and HepKO liver
mitochondria followed by DNase I treatment to
remove contaminating DNA. RT-PCR was per-
formed for Cox1 and Cox2 with primers shown in
the schematic (upper) and separated on a 1.5%
agarose gel.
(C) Northern blot of mtRNA from WT and HepKO
mouse liver mitochondria using a Cox1 or Cox3
DNA probe. Asterisks mark larger precursor
mtRNAs and the arrow shows the mature mtRNA.
(D) Steady-state expression of nuclear- and mito-
chondrial-encoded proteins in WT and HepKO
liver mitochondria. Equivalent nuclear-encoded
protein expression shows that HepKO reduced
mitochondria-encoded protein expression was
not due to differing mitochondrial content between
WT and HepKO liver cells. See also Figure S4.
did not adventitiously bind RNA because
the RNA transcripts for Cox1, GAPDH,
mitochondrial 12S rRNA and mitochon-
drial tRNAtrp were not bound to PNPASE
(Figure 3B). Furthermore, PNPASE-
HisPC, but not TIM23-HisPC, bound
in vitro transcribed and imported RNase
P and MRP RNAs, but not control mito-
chondrial RNA, in cross linking IP assays
(Figure S5A) Thus, RNase P and MRP
RNA bound specifically to PNPASE.
A protein-only RNase P complex was
shown to process the 50 -terminus of a
single mitochondrial tRNAlys (Holzmann
et al., 2008; Walker and Engelke, 2008).
However, most mammalian mitochondrial tRNA genes are
grouped together in the mitochondrial genome and lack an
intervening pre-sequence, causing them to be expressed with
the ETC genes in polycistronic transcripts. Therefore, we
examined whether protein-only RNase P can efficiently process
paired mitochondrial tRNAs, as must occur in vivo. As done
previously (Holzmann et al., 2008), mitoplast lysates were
untreated or treated with micrococcal nuclease, followed by
inactivation with EGTA and EDTA. tRNAlys or abutting tRNAs,
tRNAhistRNAser, were then added to the lysates. Nuclease-
treated mitoplast lysates cleaved the 50 -precursor sequence of
the single and abutting tRNA substrates as efficiently as the
lysates without treatment, as shown before (Figure 3C). By
contrast, nuclease-treated lysates were impaired in cleaving
the two abutting tRNAs into individual tRNAs, strongly sug-
gesting an additional nucleic acid component is required for
efficient processing. Interestingly, mitoplast lysates from
HepKO liver showed the same defect on abutting tRNA matura-
tion as the nuclease-treated WT mitoplast lysates (Figure 3D).
Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc. 459

Furthermore, the in vivo processing and separation of an
endogenous paired tRNAhistRNAser substrate was inhibited in
HepKO compared to WT liver mitochondria, whereas a linked
12 s rRNA-tRNAval substrate was processed equivalently (Fig-
ure S5B). These results, combined with prior data (Puranam and
Attardi, 2001), suggest protein-only and RNase P RNA-containing
RNase P complexes coexist in mitochondria, with efficient tRNA
processing requiring PNPASE-dependent RNase P RNA.
PNPASE Augments the Import of RNase P, 5S rRNA,
and MRP RNAs into Yeast Mitochondria
Similar PNPASE complexes in yeast and mammalian mitochon-
dria (Chen et al., 2006) (Figure S1) suggest yeast as a model for
studying the import of nuclear-encoded RNAs. Importantly, added
human PNPASE did not alter yeast mitochondrial morphology,
rate of proliferation, or extent of cell death, supporting this choice
460 Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc.
Figure 3. RNase P RNA Binds to PNPASE
and May Function in PNPASE-Dependent
tRNA Processing
(A) Left: RNA was isolated from WT and HepKO
liver mitochondria following nuclease treatment.
RT-PCR was performed with primers that
amplify nuclear-encoded RNase P RNA (212 bp).
Right: QPCR analysis of RNase P RNA expression
relative to TOM40 protein in isolated mitochondria.
(B) PNPASE-HisPC (PNP) or TIM23-HisPC (TIM23)
was purified from stably transfected HEK293 cells.
Candidate interacting RNAs that copurified in the
final eluate with PNPASE-HisPC and TIM23-
HisPC were identified by primer-specific RT-
PCR. T is the total lysate (0.3% of the reaction)
before mitochondrial purification and B is the
bound fraction. Note that only RNase P RNA
bound to PNPASE-HisPC (lane 4).
(C) Mitoplast extract (10 mg) was prepared from
nuclease-treated, intact WT liver mitochondria.
The extract was then treated with nuclease (+),
as indicated, and then inactivated with EDTA
and EGTA. The nuclease-treated or untreated
extract was incubated with abutted tRNAs
(tRNAHistRNASer) or a single tRNA (tRNALys) at
25 C for 10 or 30 min. RNA was separated on an
urea-acrylamide gel and detected by autoradiog-
raphy. A MORTALIN immunoblot shows equiva-
lent mitoplast extract in each assay.
(D) Mitoplast extract was prepared from nuclease-
treated, intact WT or HepKO liver mitochondria.
The enzymatic assay was performed as described
for panel (C). See also Figure S5.
(Figure S6). Mitochondria isolated from
WT yeast or yeast expressing human
PNPASE were incubated with in vitro tran-
scribed human RNase P RNA in import
buffer. The reaction was treated with
nuclease to remove nonimported RNA
followed by RNA isolation and RT-PCR.
RNase P RNA abundance was increased
in mitochondria containing PNPASE com-
pared to WT mitochondria (Figure 4A). This
RNA increase was specific for certain RNAs because cytosolic
GAPDH RNA was not increased in the same mitochondria
(Figure 4B). Osmotic shock was used to identify the location of
the imported RNase P RNA (Koehler et al., 1998). Mitochondria
were incubated in hypotonic buffer to rupture the outer membrane
and the mitoplasts (P, pellet fraction that contains the matrix and
IM) and the supernatant (S, contains the soluble IMS contents)
were separated by centrifugation (Figure 4C). RNase P RNA was
detected by RT-PCR and was localized in the mitochondrial
matrix. Detergent exposed the matrix to verify that the nuclease
degraded the RNase P RNA. To confirm that osmotic shock did
not disrupt the IM, antibodies against cytochrome b2 (cyt b2;
IMS) and a-ketoglutarate dehydrogenase (KDH; matrix) showed
that cyt b2 was sensitive to protease in the IMS, but KDH was
resistant to protease until the IM was lysed with Triton X-100.
Thus, RNase P RNA import was augmented, or RNase P RNA

was stabilized, in the yeast mitochondrial matrix when exogenous
PNPASE was present in the IMS.
To confirm the RT-PCR results and assay other imported
RNAs, we performed the in vitro RNA import assay with yeast
mitochondria and radiolabeled human RNAs (Figure 4D). Two
different RNA volumes were used and the imported RNA was
isolated and separated on a urea-acrylamide gel followed by
autoradiography. RNase P, 5S rRNA, and MRP RNAs showed
augmented import or stability in mitochondria expressing
PNPASE relative to control mitochondria (Figure 4D). Again,
this increase was RNA-type specific as PNPASE did not
augment GAPDH RNA levels. When the mitochondrial
membrane potential was dissipated, the RNase P RNA level
was not increased in this assay system (Figure 4E).
PNPASE Mutations that Inactivate RNA Processing
Do Not Affect RNA Import or Stability
Specific point mutations in conserved regions of PNPASE impair
its RNA exonuclease or 30 poly-A polymerase activity in
Figure 4. PNPASE Augments RNase P, 5S
rRNA, and MRP RNA Import into Yeast Mito-
chondria
(A) Upper: In vitro transcribed human RNase P
RNA was incubated with yeast mitochondria
expressing human PNPT1 (PNP) or an empty
vector (Vec) control. Nonimported RNA was
digested with nuclease and the imported RNA
was detected by RT-PCR. PNPT1-expressing
mitochondria without added RNase P RNA was
included as a specificity control for import and
RT-PCR (lane 2: Std, 1% of the reaction). Lower:
Control showing equivalent total mitochondrial
nucleic acid in each reaction.
(B) Upper: As in panel (A), with cytosolic human
GAPDH RNA used as a substrate. Middle- Control
showing equivalent total mitochondrial nucleic
acid in each reaction. Lower: Western blot
showing PNPASE expression and PORIN immu-
noblot showing equivalent mitochondria in each
import assay.
(C) After import as in panel (A), mitochondria
were subjected to osmotic shock, fractionated
by centrifugation into soluble (S) and pellet (P)
fractions, followed by proteinase K and nuclease
additions where indicated. The pellet fraction
was solubilized with Triton X-100 to expose the
matrix. Localization was determined by RT-PCR
for RNase P RNA and immunoblot for KDH (matrix)
and cyt b2 (IMS) proteins.
(D) Upper: Radiolabeled RNase P, MRP, 5S rRNA,
and GAPDH human RNAs were in vitro transcribed
and then incubated with yeast mitochondria
expressing PNPASE or an empty vector control.
Nonimported RNA was digested with nuclease,
followed by RNA isolation, separation on a urea-
acrylamide gel, and autoradiography. Import reac-
tions were repeated with 13 and 23 amounts of
RNA. Lower: Control showing equivalent total
mitochondrial nucleic acid in each reaction.
(E) Upper: As in panel A except that the mitochon-
drial membrane potential (Dc) was dissipated prior
to import. Lower: Control showing equivalent total
mitochondrial nucleic acid in each reaction.
biochemical assays (Portnoy et al., 2008), although the effects
of these mutations on PNPASE in vivo are unknown. To deter-
mine whether the RNA import or stabilization activity of PNPASE
was separable from its RNA processing activities, RNase P RNA
import was studied when different PNPASE mutants were
expressed in yeast mitochondria (Figure 5A). The point mutants
generated and tested were based on results from Schuster and
colleagues (Portnoy et al., 2008). Mutants D135G and S484A
lacked poly-A polymerase and RNA degradation activities
in vitro. Mutant D544G and double mutant R445E/R446E
showed enhanced in vitro poly-A polymerase activity but com-
promised degradation activity. Of the four mutants, PNPASE
S484A and R445E/R446E supported the import or stabilization
of RNase P RNA, whereas mutants D135G and D544G were
defective in this function (Figure 5A). The abundance of WT
and the four mutant PNPASE proteins were similar between
yeast strains and all of the PNPASE proteins assembled into
240 kDa complexes without impairment (Figure 5A, lower
panel). This contrasts with the expectation that mutant D135G
Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc. 461

would fail to form a trimeric complex from prior studies (Portnoy
et al., 2008). Therefore, the mitochondrial RNA import or stabili-
zation function of PNPASE is separable from its poly-A poly-
merase or exoribonuclease activities.
To determine whether PNPASE augmented either RNA import
or stabilization in mitochondria, the enzymatic properties of
the WT and S484A mutant protein were examined with respect
to RNA turnover in vitro and in isolated yeast mitochondria.
For in vitro studies, WT and S484A PNPASE were immunoprecip-
itated from yeast mitochondria and tested in an in vitro degrada-
tion assay with radiolabeled RNase P RNA (Figure 5B). WT
PNPASE degraded the RNase P RNA, but the S484A mutant
was impaired, consistent with prior results (Portnoy et al., 2008).
In addition, radiolabeled RNase P RNA was imported into mito-
chondria, followed by incubation at 25 C for up to 90 min
(Figure 5C). The internalized RNase P RNA was separated on
a urea-acyrlamide gel and quantified during this time course using
a phosphorimager. The rate of degradation of RNase P RNA was
462 Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc.
Figure 5. PNPASE Mutations that Inactivate
RNA Processing Do Not Affect RNA Import
or Stability
(A) Upper: Schematic for the positions of point
mutations made in the PNPASE protein. Listed
are the in vitro effects of mutations on 30 poly-
merase and RNA degrading activities, from (Port-
noy et al., 2008). Middle: Import reactions were
performed as in Figure 4A. Radiolabeled RNase
P RNA was incubated with isolated yeast mito-
chondria expressing an empty vector or the listed
PNPASE constructs. Lower panels: Immunoblot of
WT and point mutant PNPASE yeast transfectants
used in panel A import assay. A PORIN immuno-
blot confirms the colocalization of PNPASE WT
and mutants in yeast mitochondria. The assembly
state of WT and point mutant PNPASE was deter-
mined by solubilization with 1% digitonin and
separation on a 6%–16% BN gel, followed by
PNPASE immunoblot.
(B) Upper: WT and S484A PNPASE IPs from yeast
mitochondria were used to analyze RNA degrada-
tion properties. Lower: WT or S484A mutant
PNPASE was incubated with radiolabeled RNase
P RNA for 10 min at 25 C to assess degradation
activity. The asterisk marks degradation products.
(C) Left: Following in vitro import of radiolabeled
RNase P RNA and nuclease treatment to remove
nonimported RNA, mitochondria were incubated
for up to 90 min at 25 C and aliquots removed at
the indicated time points. The RNA was then
resolved by urea-acrylamide gel electrophoresis.
Right: RNase P RNA that was not degraded was
quantified using FX imager; n = 3.
similar for degradation competent WT
and incompetent mutant PNPASE pro-
teins, supporting a role for PNPASE in
augmenting the import of specific RNAs
into the mitochondrial matrix. This result
further supports PNPASE localizing to
the IMS because a greater amount of
WT PNPASE imported into the matrix could cause a relative
increase in the rate of turnover of matrix localized RNase P RNA.
A Predicted Stem-Loop RNA Structure Mediates
PNPASE-Dependent RNA Import
Results showing PNPASE-dependent RNA import into mito-
chondria (Figure 3, Figure 4, and Figure 5) do not exclude an
indirect role. To establish a direct PNPASE role, a systematic
search was used to identify PNPASE-dependent RNA import
sequences. Primers were designed to generate distinct
segments of the 340 nucleotide (nt) RNase P RNA full length
sequence. RPf1 lacked the 50 70 nt, RPf2 lacked the 50 140 nt,
and RPr1 lacked the 30 148 nt of WT PNPT1 (Figure 6A). Import
assays were performed using full length or truncated in vitro
transcribed RNase P RNAs (Figures 6B and 6C). Augmented
RPf1 and RPr1 import into yeast mitochondria depended upon
PNPASE, as did the full length RNase P RNA. In striking contrast,
RPf2 was not efficiently imported into yeast mitochondria,

implicating the sequence between nt 71 and 140 in PNPASE-
augmented RNA import. To further refine this import signal,
RNA sequences lacking the 50 86 (RPf3) or 102 (RPf4) nts
were generated (Figure 6A). Augmented RPf3 and RPf4 import
into yeast mitochondria was PNPASE-dependent (Figure 6D),
further implicating an import signal between nt 103 and 140.
The most obvious, predicted secondary structure of RNase P
RNA in this region was a 20 nt stem-loop (Figure 6F). Interest-
ingly, a similarly-predicted stem-loop structure was also identi-
fied in MRP RNA. To determine whether one or both stem-loop
structures could mediate mitochondrial targeting of non-
imported GAPDH RNA, each 20 nt stem-loop sequence was
fused to the 50 -terminus of the GAPDH RNA, which is not
imported (Figure 4D and Figure 7D). Strikingly, the RNase P
and MRP stem-loop structures licensed the PNPASE-depen-
dent import of GAPDH RNA into yeast mitochondria (Figure 6E).
By contrast, a control random 20nt sequence could not mediate
this import. Human mitochondrial tRNAtrp with the RNase P RNA
step-loop structure, but not tRNAtrp itself, was imported into
Figure 6. A Stem-Loop Structure Mediates
PNPASE-Dependent RNA Import
(A) Schematic depiction of human RNase P RNA
and deletion fragments.
(B) Import of full length RNase P RNA into yeast
mitochondria expressing PNPASE (PNP) or
control (Vec) vectors, as in Figure 4A.
(C) Import of the indicated RNase P RNA frag-
ments.
(D) Import of RNase P RNA fragments RPf3 and
RPf4.
(E) Import of human GAPDH mRNA or GAPDH
mRNA with control (CR), MRP RNA, or RNase P
RNA 20 nt sequences fused to the 50 end, as
shown in panel (F).
(F) The secondary structures and sequences of
mitochondrial RNA targeting signals in RNase P
(RP) and MRP (MRP) RNAs. A random sequence
(CR) was used as a control.
(G) Isolated mitochondria from HEK293 cells
stably expressing IMS-localized PNPASE-HisPC
or TIM23-HisPC (control) dual-tagged proteins
were incubated with [32P]-CTP labeled CR-tRNAtrp
or RP-tRNAtrp, followed by UV-cross linking,
tag-IP, separation by SDS-PAGE, and autoradiog-
raphy. See also Figure S6.
isolated mouse liver mitochondria, with
the tRNAtrp-PNPASE interaction cap-
tured using UV-cross linking (Figure 6G).
These results strongly implicate the
structural specificity of mitochondrial
RNA import (Figure 6F) and the direct
involvement of PNPASE in this process.
PNPASE Augments RNA Import
into Yeast and Mammalian
Mitochondria In Vivo
To explore in vivo RNA import into mito-
chondria, a construct was generated in
which the human RNase P RNA was expressed from the yeast
NME1 promoter (Figure 7A). When expressed in control yeast,
RNase P RNA localized to mitochondria, consistent with
nuclear-encoded RNA import by a PNPASE-independent mech-
anism, since yeast normally lack PNPASE. By contrast, RNase P
RNA import increased by 2-fold in mitochondria from yeast
expressing PNPASE compared with control cells (Figures 7A
and 7B). Importantly, an RNA similar in size to RNase P RNA
(340 nt), HOT13, that is translated in the cytosol and imported
as a protein into mitochondria, was not localized to mitochon-
dria. Also, mitochondrial-encoded RPM1, which codes for the
yeast homolog of RNase P RNA, was sequestered in the mito-
chondrion at a level equivalent to control yeast mitochondria,
as expected. These data indicate that PNPASE augments the
import of RNase P RNA into yeast mitochondria in vivo. Finally,
replacement of the human RNase P RNA stem-loop sequence
with the 20nt random sequence blocked augmented RNase P
RNA import into yeast mitochondria in vivo (Figure S7), confirm-
ing the role of the stem-loop in PNPASE-regulated import.
Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc. 463

PNPASE augmented import of RNase P RNA into yeast
mitochondria is nonphysiologic. Therefore, we developed WT,
PNPASE KO, WT expressing human PNPASE, and PNPASE
KO expressing human PNPASE MEFs for import assays.
PNPASE abundance in each MEF line was confirmed by
immunoblot (Figure 7C). Radiolabeled RNase P RNA was not
imported into mitochondria from the PNPASE KO MEFs, but
was imported into mitochondria that contained mouse and/or
human PNPASE. The in vitro import of RNase P, MRP, 5S
rRNA, and GAPDH RNAs was also tested in liver mitochondria
isolated from the HepKO mouse and WT littermates. Again,
RNase P, 5S rRNA, and MRP RNAs were imported into mito-
chondria expressing PNPASE, whereas cytosolic GAPDH RNA
was not imported (Figure 7D). As expected, more than half of
the imported MRP RNA was processed into the mature
130 nt form (Figure 7D, S7) (Chang and Clayton, 1987). By
contrast, mitochondrial RNA import was severely compromised
in HepKO liver mitochondria. Combined, these results strongly
support PNPASE as the first RNA import factor that mediates
the translocation of specific RNAs into the mammalian
mitochondrial matrix.
464 Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc.
Figure 7. PNPASE Augments RNA Import
into Yeast and Mammalian Mitochondria In
Vivo
(A) Upper: Human RNase P RNA yeast expression
construct is driven by the RPM1 RNA promoter,
NME1. Lower: Mitochondria from yeast express-
ing human RNase P RNA and either PNPASE
(PNP) or an empty vector (Vec) were isolated and
treated with nuclease. RNA was then isolated
from the total cell lysate or from nuclease-treated
mitochondria (Mito) and analyzed by primer-
specific RT-PCR.
(B) QPCR for Cox1 and RNase P RNAs isolated
from mitochondria in panel (A), normalized to the
total mitochondrial RNA obtained.
(C) Radiolabeled, in vitro transcribed RNase P
RNA was imported into mitochondria from MEF
cell lines WT (expressing mouse PNPASE,
mPNP), Pnpt1 knockout (KO), PNPT1 overexpres-
sion (expressing mPNP and hPNP), or Pnpt1
knockout plus PNPT1 overexpression (expressing
hPNP). Upper panel is an immunoblot for mouse
and human PNPASE expression. Middle panel is
an immunoblot of b-ACTIN, a loading control.
Lower panel is an autoradiogram of RNase P
RNA import into isolated MEF mitochondria.
(D) Radiolabeled, in vitro transcribed RNAs were
incubated with WT or HepKO liver mitochondria
for 10 min at 25 C. Nonimported RNA was
removed with nuclease, followed by RNA isolation
and separation on a urea-acrylamide gel. Import
reactions were repeated with 13 and 23 amounts
of synthesized RNAs. TOM40 immunoblot pro-
vides a mitochondrial loading control. See also
Figure S7.
DISCUSSION
In contrast to the protein translocation, very little is understood
about the import of specific RNAs into the mitochondrion. A
confounder is that the spectrum of RNAs imported and the
import factors and mechanisms seem to vary greatly among
different organisms. At an extreme is mammalian mitochondria,
in which despite strong evidence for RNA import (Alfonzo and
Soll, 2009; Tarassov et al., 1995), no factors have thus far been
identified. Here we implicate PNPASE as the first RNA import
factor for mammalian mitochondria. Our results show that
PNPASE KO disrupts mitochondrial morphology and respiration
in mouse liver cells, at least partially by inhibiting the import of
RNAs that control the transcription and translation of the ETC
proteins. Our data also suggest that a nucleic acid component
of the RNase P RNA processing complex, possibly RNase P
RNA (Puranam and Attardi, 2001), is imported in vivo to process
linked tRNAs in long mitochondrial transcripts. PNPASE medi-
ated RNA delivery into the mitochondrial matrix and this import
was augmented over background. Strikingly, PNPASE RNA
import and RNA processing functions were separable and
predicted stem-loop structures were identified in two imported
RNAs that could transfer PNPASE-dependent import potential
to nonimported RNAs. Combined, these results open a new
chapter for studies into the pathway(s) and mechanism(s) of
RNA import into mammalian mitochondria.
A key question that this study presages is how PNPASE
regulates the import of specific cytosolic RNAs. For this, the
location of PNPASE first needs to be considered. Previously,
we localized PNPASE to the IMS. Carbonate extraction studies
also indicated that PNPASE was bound to the IM facing the
IMS (Chen et al., 2006; Rainey et al., 2006). However, more
recently, others have shown a weak interaction with the
matrix-localized RNA helicase hSUV3, suggesting a matrix
localization for PNPASE (Szczesny et al., 2009; Wang et al.,
2009). Our sub-fractionation studies were highly reproducible
and, in contrast, we favor the idea that hSUV3 bound to PNPASE
after all of the mitochondrial sub-compartments were exposed
during purification. The methods used to identify this interaction
do not exclude this possibility and we have already shown an
unanticipated interaction between PNPASE and the cytosol-
localized oncoprotein TCL1 using similar limited resolution
methods (French et al., 2007). Our failure to again identify a
hSUV3 and PNPASE interaction using an ultra-sensitive dual-
tag expression and purification system (Claypool et al., 2008)
further supports this interpretation (Figure S1). An alternative
explanation that we cannot exclude or confirm with current
methodologies is that a small amount of PNPASE could get
into the matrix and interact with hSUV3.
A second interesting area opened by the current results is to
determine how PNPASE controls RNA import into mitochondria.
In concept, PNPASE could import RNAs from the cytosol into
the IMS and then pass this RNA to another protein or complex
that would assist it through the IM into the matrix (Figure 4C).
Interestingly, PNPASE augments import in yeast, which does
not have a PNPASE homolog, indicating a distinct RNA import
mechanism that PNPASE can augment directly or indepen-
dently. In mouse liver and MEF mitochondria, PNPASE KO is
incomplete because cells without PNPASE are nonviable, so it
is unclear whether it is absolutely essential for all RNA import
or whether the amount detected in import assays is still mediated
by the minimal residual amount of PNPASE present required
for cell survival. Furthermore, it is not clear if this imported
RNA in PNPASE KO mitochondria is stuck in the IMS and not
in the matrix, which could provide further insight for the detailed
function of PNPASE in RNA import.
PNPASE has two external domains (KH and S1) that bind RNA
near the opening of a central processing pore in a trimeric
complex (Carpousis, 2002; Symmons et al., 2000). It is not clear
whether the same domains are used indiscriminately or in some
distinct manner to trigger PNPASE RNA processing versus
import functions. It is possible that the stem-loop structures
identified in RNase P and MRP RNAs interact with PNPASE
in a manner that triggers only import rather than processing.
Interestingly, GAPDH RNA can be a target of PNPASE degrada-
tion activity in vitro (French et al., 2007), although when either
of the identified stem-loop structures is appended to the
50 -terminus, GAPDH RNA is efficiently imported into mitochon-
dria (Figure 6E). Indeed, RNA structural elements regulate
PNPASE function in chloroplasts and prokaryotes (Lisitsky
et al., 1996; Yehudai-Resheff and Schuster, 2000) and a stem-
loop structure protects the RNA from degradation by PNPASE
in chloroplasts (Yehudai-Resheff et al., 2001). A detailed dissec-
tion of what constitutes a trigger sequence for processing versus
import activities is clearly warranted. Finally, since the overall
sequence homology between the identified stem-loop struc-
tures on RNase P and MRP RNAs is not high, binding interactions
between specific RNAs and PNPASE may be stronger or
weaker, allowing or inhibiting detection by standard in vitro
techniques such as IP, which could make the verification of
additional candidate imported RNAs challenging without the
functional import assay system for validation.
Studies and important applications that use RNA import in
mammalian cells have been hampered by the inconsistent
requirements between systems and their study in vitro versus
in vivo. Although the exact mechanism for how PNPASE
augments and licenses RNA import is not yet known, the identifi-
cation of PNPASE represents the first receptor-like component
that binds RNA in mammalian cells to mediate RNA import into
the mitochondrial matrix. This finding, along with the identification
of import signal sequences, should open up additional studies to
determine what other pathway components are involved and
what the RNA sequence or structure rules tell us about how
PNPASE may decipher between processing and import.
EXPERIMENTAL PROCEDURES
Protein and RNA Purification
Purification of the PNPASE-HisPC protein complex was performed as before
(Claypool et al., 2008). For protein-RNA interactions, mitochondria (1 mg/ml)
were solubilized in lysis buffer (300 mM NaCl, 10 mM imidazole, 10% glycerol,
0.25% Triton X-100, 2 mM DTT, 20 mM HEPES [pH 6.6]) containing protease
inhibitor (Roche) and RNase inhibitor (Invitrogen). Insoluble material was
removed by spinning and extracts transferred to Eppendorf tubes. 50 ml of
Ni2+NTA resin (QIAGEN) was incubated in 1 ml lysis buffer with 100 mg/ml
ssDNA for 1h at 4 C. The resin was then mixed with the mitochondrial lysates
in the presence of 100 mg/ml ssDNA for 1 hr at 4 C. After incubation, the resin
was washed 103 with lysis buffer containing RNase inhibitor. The protein-RNA
complex was eluted with elution buffer (300 mM NaCl, 10 mM imidazole, 10%
glycerol, 0.25% Triton X-100, 20 mM citrate [pH 5.5]) containing RNase
inhibitor. RNA was isolated from the eluate using TRIzol reagent (Invitrogen).
Isolation of Mitochondrial RNA and DNA
Mitochondria (1 mg/ml) were treated with 25 mg/ml of micrococcal nuclease S7
in nuclease buffer (0.6 M Sorbitol, 20 mM MgCl2, 5 mM CaCl2, 20 mM Tris
[pH 8.0]) for 30 min at 27 C. The reaction was stopped by addition of 20 mM
EGTA. Mitochondria were collected and solubilized in SDS buffer (100 mM
NaCl, 1% SDS, 20 mM Tris pH 7.4) at 65 C for 5 min. RNA was purified using
TRIzol reagent, and treated with RNase-free DNase I (Roche) for 1h at 37 C.
DNase I was inactivated by heating at 65 C for 10 min. Phenol-chloroform
(EM Science) extractions were used for DNA purification from the mitochon-
drial lysates.
In Vitro Transcription
RNAs were synthesized as before (Portnoy et al., 2008). For radiolabeled RNA
synthesis, [32P]-CTP (MP Biochemicals) was incorporated. The RNAs were
purified using TRIzol reagent.
RNA Import Assay
Yeast mitochondria were isolated from cells grown in selection medium until
stationary phase and mammalian mitochondria were isolated as previously
described (Chen et al., 2006; Rainey et al., 2006). In vitro RNA import assays
Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc. 465
were performed in a 200-ml volume containing 0.5 mg of RNA, 100 mg of mito-
chondria, 0.6 M sorbitol, 2 mM KH2PO4, 50 mM KCl, 10 mM MgCl2, 2.5 mM
EDTA, 5 mM L-methionine, 1 mg/ml BSA, 5 mM ATP, 2 mM DTT, 5 mM
NADH, 50 mM HEPES, [pH 7.1], at RT for 10 min. Mitochondria were spun
at 11,000 x g for 5 min and washed once with wash buffer (0.6 M sorbitol,
20 mM Tris, [pH 8.0]). Mitochondria were spun again and resuspended in
200 ml nuclease buffer containing 25 mg/ml of micrococcal nuclease S7 and
incubated for 30 min at 27 C. Mitochondria were collected and solubilized in
SDS buffer at 65 C for 5 min. RNA was purified using TRIzol reagent. For
import into mammalian mitochondria, 0.25 M sucrose instead of 0.6 M sorbitol,
and 20 mM succinate instead of 5 mM NADH, were used. For import with
radiolabeled RNA, the purified RNAs were analyzed by urea-acrylamide gel
and autoradiography.
RNA Degradation Assay
The RNA processing activity of WT and mutant PNPASE was done as before
(Portnoy et al., 2008). [32P]-RNA was incubated with the corresponding
proteins in buffer E (20 mM HEPES, [pH 7.9], 60 mM KCl, 12.5 mM MgCl2,
0.1 mM EDTA, 2 mM DTT, and 17% glycerol, 0.1 mM Pi) at 25 C for 5 min.
Following incubation, the RNA was isolated and analyzed by urea-acrylamide
gel and autoradiography.
Additional Procedures
Osmotic shock was performed by incubating mitochondria for 30 min on ice in
0.03 M sorbitol and 20 mM HEPES-KOH, (pH 7.4) (Claypool et al., 2008). Blue
native gel electrophoresis was performed on a 6%–16% linear polyacrylamide
gradient using 50 mg of digitonin solubilized material (Chen et al., 2006).
Northern blotting was performed as previously described (Tollervey, 1987).
Total mtRNA was separated on a 12% agarose-formaldehyde gel and
transferred to a nylon membrane. Hybridization was carried out with
[32P]-dCTP (MP Biochemicals) labeled DNA probes. In organello protein
synthesis assays were performed as before with minor changes (Stuart and
Koehler, 2007). 100 mg mouse liver mitochondria were incubated in 100 ml
translation buffer (250 mM sucrose, 100 mM KCl, 1 mM MgCl2, 10 mM Tris
(pH 7.4), 10 mM K2HPO4 (pH 7.4), 10 mM glutamate, 10 mM malate, 5mM
NADH, 1 mM ADP, 1 mg/ml BSA, 100 mg/ml emetine, 100 mg/ml cyclohexi-
mide, and 30 mM of amino acid mix without methionine) with 5 ml of L-[35S]
methionine (MP Biochemicals) at 37 C for 30 min. The mitochondria were
then precipitated and proteins resolved by 12% SDS PAGE.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures and
seven figures and can be found with this article online at doi:10.1016/j.cell.
2010.06.035.
ACKNOWLEDGMENTS
We thank Michelle Husain for expertise in TEM. Supported by NIH grants
R01GM061721 and R01GM073981 (C.M.K.), R01CA90571 (M.A.T.) and
PN2EY018228 (Roadmap for Medical Research Nanomedicine Initiative)
(M.A.T.), and K22CA120147 (S.W.F.), the Intramural Research Program of
the NIH, National Institute of Allergy and Infectious Diseases (H.C.M. III), the
Muscular Dystrophy Association 022398 (C.M.K.), the American Heart Associ-
ation 0640076N (C.M.K.), and the California Institute of Regenerative Medicine
(RS1-00313 and RB1-01397, M.A.T.). C.M.K. is an Established Investigator of
the American Heart Association and M.A.T. is a Scholar of the Leukemia and
Lymphoma Society.
Received: December 22, 2009
Revised: March 20, 2010
Accepted: May 13, 2010
Published: August 5, 2010
466 Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc.
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Cell 142, 456–467, August 6, 2010 ª2010 Elsevier Inc. 467
Plzf Regulates Germline Progenitor
Self-Renewal by Opposing mTORC1
Robin M. Hobbs,1 Marco Seandel,2 Ilaria Falciatori,3 Shahin Rafii,3 and Pier Paolo Pandolfi1,*
1Cancer Genetics Program, Beth Israel Deaconess Cancer Center, Departments of Medicine and Pathology, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, MA 02115, USA
2Department of Surgery
3Howard Hughes Medical Institute, Department of Genetic Medicine
Weill Cornell Medical College, New York, NY 10065, USA
*Correspondence: ppandolf@bidmc.harvard.edu
DOI 10.1016/j.cell.2010.06.041
SUMMARY
Hyperactivity of mTORC1, a key mediator of cell
growth, leads to stem cell depletion, although the
underlying mechanisms are poorly defined. Using
spermatogonial progenitor cells (SPCs) as a model
system, we show that mTORC1 impairs stem cell
maintenance by a negative feedback from mTORC1
to receptors required to transduce niche-derived
signals. We find that SPCs lacking Plzf, a transcrip-
tion factor essential for SPC maintenance, have
enhanced mTORC1 activity. Aberrant mTORC1 acti-
vation in Plzf / SPCs inhibits their response to
GDNF, a growth factor critical for SPC self-renewal,
via negative feedback at the level of the GDNF
receptor. Plzf opposes mTORC1 activity by inducing
expression of the mTORC1 inhibitor Redd1. Thus, we
identify the mTORC1-Plzf functional interaction as
a critical rheostat for maintenance of the spermato-
gonial pool and propose a model whereby negative
feedback from mTORC1 to the GDNF receptor
balances SPC growth with self-renewal.
INTRODUCTION
Maintenance of a wide array of adult tissues is dependent on the
presence of a resident stem cell pool with self-renewal potential
that generates differentiating progeny. Factors regulating the
balance between stem cell self-renewal and differentiation
ensure tissue homeostasis whereas disruption of these regula-
tory mechanisms can lead to tissue degeneration or cancer (Ito
et al., 2009). One factor central to stem cell homeostasis is
mammalian TOR complex 1 (mTORC1), a signaling complex
that promotes protein translation and cell growth by phosphory-
lating components of the translation machinery (Ma and Blenis,
2009). mTORC1 is regulated in response to diverse stimuli
including nutrient availability, energy status, growth factors,
and cellular stress. Persistent mTORC1 activation in certain
tissues leads to increased proliferation but subsequent exhaus-
tion of the stem cell compartment, demonstrating that aberrantly
468 Cell 142, 468–479, August 6, 2010 ª2010 Elsevier Inc.
activated mTORC1 is detrimental to stem cell maintenance
(Castilho et al., 2009; Gan and DePinho, 2009; Yilmaz et al.,
2006). It is proposed that inappropriate mTORC1 activation
drives stem cell depletion through aberrant translation of down-
stream targets and subsequent activation of tumor suppressive/
fail-safe mechanisms resulting in cellular senescence or apo-
ptosis (Ito et al., 2009). However, the molecular mechanisms
and targets of mTORC1 in this context are currently unknown.
Interestingly, inhibition of mTORC1 also extends organism life-
span (Harrison et al., 2009; Schieke and Finkel, 2006), consistent
with the notion that declining stem cell potential underlies aging
(Rossi et al., 2008).
Undifferentiated germline cells of the testis (spermatogonial
progenitor cells; SPCs) are formed from gonocytes during post-
natal development of the mouse testis and possess self-renewal
potential (de Rooij and Russell, 2000). A major advance in the
study of male germline biology was the development of culture
systems allowing long-term SPC expansion in vitro while main-
taining stem cell potential. Key to this was the observation that
mice heterozygous for the glial cell-derived neurotrophic factor
(GDNF) cytokine gene had a depletion of SPC activity (Meng
et al., 2000). GDNF is produced by Sertoli cells within the testis,
and signals via the GFRa1/c-Ret receptor to promote SPC self-
renewal and growth through activation of Src family kinases and
Akt (Lee et al., 2007b; Oatley et al., 2007). Culture of SPCs with
GDNF plus a variety of additional factors (including basic fibro-
blast growth factor; bFGF) preserves self-renewal capabilities
and allows essentially unlimited cell expansion while maintaining
in vivo differentiation potential; assessed by the ability to repopu-
late depleted recipient testis (Kanatsu-Shinohara et al., 2003;
Kubota et al., 2004; Seandel et al., 2007). Although some cellular
signaling pathways involved in SPC self-renewal have been
described, it remains unclear how SPCs integrate signals from
general mitogenic stimuli with those required for self-renewal
to balance stem cell maintenance and differentiation.
A limited number of cell intrinsic factors have also been impli-
cated in SPC function, foremost among which is promyelocytic
leukemia zinc finger (PLZF). PLZF was identified from the trans-
location breakpoint in t(11;17) acute promyelocytic leukemia
(Chen et al., 1993) and encodes a transcription factor belonging
to the POZ-Krüppel (POK) family. PLZF binds DNA through
carboxy-terminal Krüppel-type zinc fingers and recruits histone
deacetylases (HDACs) via an amino-terminal POZ domain (David
et al., 1998). Recruitment of HDACs to target promoters can
result in gene repression although PLZF is also able to activate
gene expression (Doulatov et al., 2009; Labbaye et al., 2002).
Male mice lacking Plzf expression undergo progressive germ
cell loss and testis atrophy with age causing infertility (Buaas
et al., 2004; Costoya et al., 2004). Plzf is expressed by SPCs
and is needed in a cell autonomous fashion for maintenance
of the germ lineage. A male patient with biallelic PLZF loss-of-
function and gonad hypoplasia has been recently reported
(Fischer et al., 2008), emphasizing the role played by PLZF in
germ cell biology.
SPC maintenance is dependent on Plzf plus key growth
factors such as GDNF. As mTORC1 is activated in response to
growth factor signaling we hypothesized that both Plzf and
mTORC1 are involved in the GDNF response of SPCs and that
Plzf and mTORC1 may crosstalk. To assess the roles of Plzf
and mTORC1 in GDNF-dependent SPC maintenance, we devel-
oped systems for isolation and culture of SPCs from wild-type
(WT) and Plzf / prepubertal mice (subsequent to formation
of the SPC pool and prior to overt germ cell depletion in the
Plzf / mouse). We find that Plzf opposes mTORC1 activity
and define a cellular signaling network controlling SPC homeo-
stasis whereby mTORC1 integrates mitogenic signals received
by SPCs and determines their sensitivity to self-renewal stimuli.
RESULTS
Isolation, Culture, and Comparative Analysis
of Plzf +/+ and Plzf / SPCs
The testis is composed of a heterogeneous mix of mitotic and
meiotic germ cells plus multiple types of somatic cells. SPCs
are rare within the testis and systems for their purification are
poorly developed. We therefore sought to take advantage of
the expression of Plzf in SPCs to enrich for this cell type. Plzf-
expressing cells were identified from juvenile testis by intracel-
lular staining and flow cytometry using a newly developed
monoclonal antibody (Figure 1A). Our aim was to correlate the
Plzf-expressing population to sets of cell surface markers that
would allow subsequent purification of those live cells. By adapt-
ing markers able to enrich for stem cell activity from cryptorchid
testis (Kubota et al., 2003; Shinohara et al., 2000), we found
that the Plzf-positive population was av-integrin negative and
expressed low levels of Thy-1 (Figure 1B). Negative selection
for av-integrin combined with Thy-1 positive selection allowed
isolation of Plzf-positive cells and minimized somatic cell
contamination (Figures S1A and S1B available online) (Virtanen
et al., 1986). c-Kit expression is associated with SPC differentia-
tion (Schrans-Stassen et al., 1999) and the avneg Thy-1low popu-
lation was composed primarily of c-Kit negative cells with
a smaller c-Kit positive fraction (Figure 1C, left). The c-Kitneg cells
were largely in G1/G0 phase of cell cycle whereas the c-Kitpos
population contained more cells in S plus G2 phases (Figure 1C,
right). As SPCs are more quiescent than differentiating sper-
matogonia (Takubo et al., 2008), the avneg Thy-1low population
contains both SPCs (c-Kitneg, quiescent) and cells undergoing
differentiation (c-Kitpos, proliferating). To confirm that the avneg
Thy-1low c-Kitneg fraction was enriched for stem cells, we
compared the ability of avneg Thy-1low c-Kitneg and unfractio-
nated cells to repopulate recipient depleted testis (Ogawa
et al., 1997). Donor cells were isolated from transgenic mice
expressing GFP from the b-actin promoter, allowing visualization
of donor-derived colonies (Figure 1D, left). Numbers of GFP-
positive colonies were scored 2 months posttransplant (Fig-
ure 1D, right), representing numbers of engrafted stem cells.
Stem cell activity was enriched 25-fold in the avneg Thy-1low
c-Kitneg population compared to unfractionated cells. As the
avneg Thy-1low c-Kitneg fraction represents 3%–5% of total
cells (1/25th of testis cellularity) from 10 to 14 days postnatal
testis (Figures 1B, 1C, and 1G), a 25-fold enrichment of stem
cell activity indicates that almost all stem cells are contained
within this fraction. Given the number of colonies obtained
from avneg Thy-1low c-Kitneg cells (Figure 1D) and an estimated
10% engraftment efficiency (Nagano et al., 1999), there are
1265 stem cells per 105 cells in this fraction (1 in 80 cells
have stem cell potential).
We next analyzed prepubertal Plzf +/+ and Plzf / testis by
flow-cytometry to determine if Plzf / mice display aberrant
SPC activity before overt germ cell depletion. Although the char-
acteristic av/Thy-1 flow profile was present in juvenile Plzf /
testis (2 and 3 weeks postnatal), the avneg Thy-1low population
was depleted relative to the WT (Figure 1E and Figure S1C)
and the percentage of c-Kitpos cells within the Plzf / avneg
Thy-1low fraction was increased (Figure 1F and Figure S1D).
The decreased frequency and numbers of avneg Thy-1low c-Kitneg
cells in Plzf / testis (Figure 1G) confirms that Plzf loss is detri-
mental to SPCs whereas the increase in c-Kitpos cells within the
avneg Thy-1low fraction is suggestive of increased differentiation
commitment. As Plzf directly represses c-Kit expression
(Filipponi et al., 2007), this can be reflective of a de-repression
of the c-Kit locus. However, the Plzf / avneg Thy-1low fraction
still contains c-Kitneg cells (Figure 1F and Figure S1D), indicating
that aberrant c-Kit expression is unlikely to fully explain the
Plzf / phenotype. Additionally, both Plzf +/+ and Plzf / avneg
Thy-1low c-Kitneg cells show substantial enrichment in expres-
sion of the SPC-associated factors Pou5f1/Oct4, Ngn3, Bcl6b,
and Pou3f1 (Figure 1H and Figure S1E) (Oatley et al., 2006;
Ohbo et al., 2003; Wu et al., 2010; Yoshida et al., 2006), confirm-
ing the identity of this fraction. We also noted that Plzf / avneg
Thy-1low cells had decreased levels of b1 and a6 integrins but
equivalent levels of CD9 compared to controls (Figures S1F
and S1G) (Kanatsu-Shinohara et al., 2004b; Shinohara et al.,
1999). Our data indicate that SPCs from juvenile Plzf / mice
have an impaired ability to maintain an undifferentiated state,
which precedes overt germ cell loss.
We next attempted in vitro culture of isolated Plzf +/+ and
Plzf / SPCs, adapting previously developed culture systems
(Kubota et al., 2004; Seandel et al., 2007). As anticipated, germ
cell colony-forming activity under SPC culture conditions was
essentially entirely contained within the avneg Thy-1low fraction
(Figure S2A). We successfully derived SPC lines from both
Plzf +/+ and Plzf / avneg Thy-1low cells, which grew as discrete
colonies on mouse embryonic fibroblast (MEF) feeder cells
(Figure 2A) and maintained their cell surface marker identity
during exponential growth over 1 year of culture (Figure S2B).
WT SPC lines expressed Plzf (Figure 2B) whereas Plzf / SPC
Cell 142, 468–479, August 6, 2010 ª2010 Elsevier Inc. 469

tionated testis cells), SPCs (av-integrin negative, Thy-1 low, c-Kit negative), early differentiating spermatogonia (av-integrin negative, Thy-1 low, c-Kit positive),
late differentiating spermatogonia (av-integrin negative, Thy-1 negative, c-Kit positive), and somatic cells (av-integrin positive). mRNA levels are normalized to
those of b-actin and standard deviations from duplicate reactions are shown.
See also Figure S1.
lines were more difficult to establish (not shown) consistent with
a role for Plzf in SPC function. Early passages of Plzf / SPCs
had a slower growth rate than WT cells (Figures S2C and S2D)
but could be maintained long-term and growth became more
comparable to WT cells at later passages (Figure 5C). The ability
to culture Plzf / SPCs seemed counterintuitive, but the gener-
ation of these cultures became critical for further dissection of
Plzf function and to the solution of this apparent contradiction.
An additional indicator of SPC potential is the ability to derive
embryonic stem-like cells from the cultured lines (multipotent
adult spermatogonial-derived stem cells; MASCs) (Kanatsu-
470 Cell 142, 468–479, August 6, 2010 ª2010 Elsevier Inc.
+/+
Figure 1. Isolation and Analysis of Plzf
and Plzf / SPCs
(A) Detection of Plzf-expressing spermatogonia
from juvenile testis (10–14 days postnatal) by intra-
cellular staining and flow cytometry using anti-Plzf
antibody.
(B) Plzf expressing cells correlate to a discrete av-
integrin negative, Thy-1 low population by flow cy-
tometry. CD45 marker excludes contaminating
leukocytes.
(C) Left panel: flow cytometric analysis of c-Kit
within the Plzf-expressing av-integrin negative,
Thy-1 low population. Right panels: cell cycle
status of c-Kit negative and positive populations
plus percentage of cells in G1/S/G2 cell cycle
phases from a representative sample.
(D) Left panels: seminiferous tubules repopulated
with donor GFP-positive av integrin negative,
Thy-1 low, c-Kit negative cells in testis transplant
assay. Fluorescent image (top) and brightfield
image of same recipient testis (bottom) are shown.
Right panel: numbers of GFP positive colonies ob-
tained from av integrin negative, Thy-1 low, c-Kit
negative (sorted) and unsorted testis cells in recip-
ient testes 2 months posttransplant. Data is pre-
sented as mean number of colonies per 1 3 105
donor cells together with standard error of the
mean (SEM). Fold enrichment of stem cell activity
in sorted populations is indicated (***p < 0.0001).
Values are averaged from two independent exper-
iments. Thirteen recipient testes were analyzed for
sorted populations and 12 for unsorted.
(E) Representative av-integrin/Thy-1 flow profiles
of Plzf +/+ and Plzf / littermate mouse testes at
2 weeks postnatal age. The av-integrin negative,
Thy-1 low gate, containing Plzf-positive cells in
WT testis is indicated.
(F) Representative flow cytometry analysis of c-Kit
expression within the av-integrin negative, Thy-1
low testis cell fractions from (E).
(G) Quantification of frequency and absolute
numbers of av integrin negative, Thy-1 low, c-Kit
negative testis cells from mice of the indicated
postnatal ages and Plzf genotypes (n = 3 per geno-
type and age group, **p < 0.002, *p < 0.02).Graphs
represent mean values ± SEM.
(H) Quantitative RT-PCR analysis of Pou5f1 mRNA
expression in testis cell populations from juvenile
Plzf +/+ and Plzf / littermate mice: total (unfrac-
Shinohara et al., 2004a; Seandel et al., 2007). We observed
spontaneous formation of MASC colonies from two of seven
WT lines (Figure S2E). MASC formation was not observed in
four Plzf / SPC lines, possibly indicating a defective capability
to generate MASCs. The multipotent capabilities of MASC lines
were verified by teratoma formation assay (Figure S2F). On SPC
to MASC conversion, expression of the pluripotency-associated
factors Pou5f1/Oct4, Sox2 and Nanog were substantially
increased whereas Plzf expression was lost (Figure S2G and
data not shown) (Seandel et al., 2007). Formation of MASCs indi-
cates that SPC potential is maintained in our culture system,

whereas the apparent inability of Plzf / SPCs to form MASCs
can be consistent with their defective function.
Plzf / SPCs Show Enhanced mTORC1 Activity
From analysis of cultured SPC lines, we noticed that Plzf / cells
were physically larger than WT SPCs, measured by the FSC
parameter of flow cytometry (Figures 2C and 2D). Alterations in
cell size are associated with changes in activity of mTORC1
acting through its downstream targets S6Kinase1 (S6K1), which
phosphorylates ribosomal S6 protein (RPS6), and 4EBP1 (Fingar
et al., 2002; Ruvinsky et al., 2005). Given the role of mTORC1 in
hematopoietic stem cells (Gan and DePinho, 2009), we consid-
ered that aberrant mTORC1 activity in Plzf / SPCs could
contribute to their defective maintenance. Importantly, Plzf /
SPCs had elevated levels of phosphorylated RPS6 compared
to WT cells, confirming increased mTORC1 activity (Figure 2E
and Figure 3C). Inhibition of mTORC1 with rapamycin decreased
the size of Plzf / SPCs to that of WT cells suggesting that
Figure 2. Increased mTORC1 Pathway
Activity in Plzf / SPCs
(A) SPC lines established from culture of av-integ-
rin negative, Thy-1 low cells from juvenile Plzf +/+
and Plzf / littermate mice immunostained for
the germ cell marker Mvh and counterstained
with DAPI to detect DNA. Scale bar represents
100 mm.
(B) Cultured SPCs of the indicated genotype
analyzed for Plzf expression by intracellular stain-
ing and flow cytometry (left panel) and by western
blot (right panel). The germ cell marker Mvh plus
actin are used as loading controls.
(C) Analysis of SPC size by flow cytometry.
Forward-scatter (FSC) profiles of two indepen-
dently derived sets of littermate Plzf +/+ and
Plzf / SPC lines (1 and 2) are shown.
(D) Quantification of flow cytometry profiles from
(C) showing averaged mean FSC with standard
deviation and p value.
(E) Control (DMSO) and rapamycin-treated Plzf +/+
and Plzf / SPCs were fixed and permeabilized
48 hr post-treatment and levels of Phospho-
RPS6 analyzed by flow cytometry.
(F) Normalization of increased Plzf / SPC size by
mTORC1 inhibition. FSC profiles of Plzf / SPCs
treated with DMSO (control) or rapamycin for
48 hr compared to untreated Plzf +/+ SPCs.
(G) Overlay of representative av-integrin negative,
Thy-1 low, c-Kit negative testis cell fraction FSC
profiles from Plzf +/+ and Plzf / littermate mice
treated from 10 days postnatal age for a period
of 1 week with vehicle or rapamycin.
(H) Quantification of relative mean FSC values of
av-integrin negative, Thy-1 low, c-Kit negative
testis cells of mice treated as in (G) (n = 4 per geno-
type and treatment group, *p < 0.05, **p < 0.01).
Horizontal bars represent mean values.
See also Figure S2.
elevated mTORC1 activity was responsible for the size increase
(Figure 2F). Rapamycin was confirmed to inhibit RPS6 phos-
phorylation in Plzf / SPCs (Figure 2E). Increased mTORC1
activity in Plzf / SPCs was associated with elevated levels of
cellular protein (Figure S2H), consistent with the role of mTORC1
in regulating protein translation. Importantly, freshly isolated
Plzf / SPCs were also physically larger (Figures 2G and 2H)
and rapamycin treatment of Plzf / mice normalized SPC size
(Figures 2G and 2H). Together, our data indicate that Plzf inhibits
mTORC1 activation in SPCs.
Growth Factor-Mediated Regulation of mTORC1 in SPCs
Given the possibility that mTORC1 activity can regulate SPC
function, we next defined key regulatory inputs of mTORC1 in
WT SPCs. Growth factor signaling represents a major activating
input of mTORC1 (Ma and Blenis, 2009; Shaw and Cantley,
2006) and mTORC1 inhibition interferes with mitogenic stimuli
causing cell cycle arrest (Brown et al., 1994). Indeed, rapamycin
Cell 142, 468–479, August 6, 2010 ª2010 Elsevier Inc. 471

suppressed SPC colony growth and caused an accumulation of
cells in G1 phase of the cell cycle (Figures S3A and S3B).
Growth factor receptors activate mTORC1 by signaling
through phosphoinositide 3-kinase (PI3K)/Akt and Ras/Erk
MAPK, which inhibit the TSC1/TSC2 complex (Huang and
Manning, 2008); TSC1/TSC2 negatively regulates mTORC1 via
its GTPase-activating protein activity toward the small G protein
Rheb. Treatment of WT SPCs with PI3K/Akt or Erk MAPK
pathway inhibitors significantly reduced levels of phosphory-
lated RPS6 (Figure 3A), indicating that efficient mTORC1 activa-
tion in SPCs requires both PI3K/Akt and Erk MAPK. Inhibition of
Src family kinases, implicated in the GDNF response of SPCs
(Oatley et al., 2007), had modest effects on phospho-RPS6
(Figure 3A). Therefore, despite the ability of Src family kinases
to activate PI3K/Akt downstream GDNF (Encinas et al., 2001;
Oatley et al., 2007), this SPC self-renewal pathway couples inef-
ficiently to mTORC1.
SPCs are maintained with a cocktail of growth factors
(Kanatsu-Shinohara et al., 2003; Seandel et al., 2007) and are
responsive to others (Hamra et al., 2007); thus we next assessed
contributions of these factors to mTORC1 activation in WT
SPCs. We found that GDNF triggered a modest phosphorylation
of RPS6 whereas other mitogens (including bFGF) induced much
472 Cell 142, 468–479, August 6, 2010 ª2010 Elsevier Inc.
Figure 3. Upstream Pathways and Growth Factors
Regulating mTORC1 in SPCs
(A) WT SPCs fed with complete SPC medium containing
inhibitors to the indicated signaling pathways were har-
vested 5 hr after treatment and analyzed by western blot
for the indicated proteins and phospho (P)-proteins.
(B) WT SPCs were starved overnight in basal SPC medium
lacking cytokine supplements. The indicated cytokines
were then added and SPCs harvested 20 min later then
analyzed by western blot. Cytokines were used at the
concentrations present in complete SPC medium. Neure-
gulin1 (NRG1) is not a standard supplement for SPC
medium although SPCs are responsive to it (see text).
(C) Western blot analysis of two independently derived
sets of littermate Plzf +/+ and Plzf / SPC lines (1 and 2)
under steady-state conditions is shown to the left. Quanti-
fication of the relative levels of phospho-Akt, Erk, and
RPS6 (corrected to the total levels of respective proteins)
are shown in panels on the right.
See also Figure S3.
higher levels of phospho-RPS6 (Figure 3B). The
relative abilities of GDNF and bFGF to activate
mTORC1 correlated to their efficiency of Akt
and Erk activation (Figure 3B). However, this
correlation did not hold true for epidermal
growth factor (EGF) stimulation, possibly re-
flecting additional pathways by which EGF acti-
vates mTORC1 (Fan et al., 2009). In summary,
the self-renewal signal GDNF poorly activates
mTORC1 when compared to more general
mitogens for SPCs (bFGF, EGF), in agreement
with the Src kinase inhibition data (see above).
However, we note that although GDNF is
considered the key SPC self-renewal signal,
SPC expansion and self-renewal in vitro requires a combination
of GDNF plus other factors (e.g., bFGF) that stimulate mTORC1
more efficiently (Lee et al., 2007b).
Plzf Uncouples mTORC1 Activity from Growth Factor
Signaling in SPCs through Redd1 Modulation
Given that mTORC1 activity in SPCs is regulated by growth
factors present in the culture medium and resultant PI3K/Akt
and Erk-MAPK activation, we next assessed whether the
elevated mTORC1 activity of Plzf / SPCs correlated with an
enhanced growth factor response. However, in comparison to
WT cells, the Akt and Erk activities of steady-state Plzf /
SPCs were decreased whereas phospho-RPS6 was increased
(Figure 3C), indicating that the elevated mTORC1 activity is not
due to increased activity of growth factor pathways.
We next considered alternative mechanisms by which
mTORC1 activity would be increased in Plzf / SPCs. Cellular
stress-response pathways provide additional regulatory inputs
resulting in mTORC1 inhibition when conditions such as low
energy availability or hypoxia require temporary arrest of cell
growth (Huang and Manning, 2008). We hypothesized that
mTORC1 hyperactivity in Plzf / SPCs could be due to reduced
activity of stress response pathways. Accordingly, we first

appropriate luciferase reporter together with PLZF constructs or vector control as indicated. Reporter activities were normalized to that of TK-Renilla and empty
vector luciferase reporter controls (*p < 0.001). Mean values from triplicate wells and associated standard deviations are indicated.
assessed levels of upstream regulatory proteins as stress
pathways converge on mTORC1 at the level of TSC1/TSC2.
However, we did not find significant differences in levels of
Tsc1, Tsc2, Rheb, or the mTOR kinase itself in Plzf / SPCs
compared to WT cells (Figure 4A).
By contrast, we noticed that levels of Redd1 (also known as
Ddit4, Rtp801, and Dig2) were substantially lower in Plzf /
SPCs compared to controls, at both protein and mRNA levels
(Figures 4A and 4B). Redd1 is induced by multiple types of cell
stress (e.g., hypoxia, DNA damage) and by developmental
signals and inhibits mTORC1 through regulation of TSC1/TSC2
(Brugarolas et al., 2004; Corradetti et al., 2005; Ellisen et al.,
2002). As a role for Redd1 in SPCs is not described, we analyzed
the distribution of Redd1 expression in isolated spermatogonial
Figure 4. Plzf Regulates Expression of the
mTORC1 Pathway Inhibitor Redd1
(A) Western blot analysis for components of the
mTORC1 pathway in two independently derived
sets of littermate Plzf +/+ and Plzf / SPC lines
(1 and 2) under steady-state conditions.
(B) Quantitative RT-PCR analysis of Redd1 mRNA
expression in independently derived Plzf +/+ and
Plzf / SPC lines. mRNA levels are normalized
to those of b-actin and standard deviations from
duplicate reactions are shown.
(C) Quantitative RT-PCR analysis of Redd1 mRNA
expression in spermatogonial fractions from
juvenile Plzf +/+ and Plzf / littermate mice:
SPCs (av-integrin negative, Thy-1 low, c-Kit nega-
tive), early differentiating spermatogonia (av-
integrin negative, Thy-1 low, c-Kit positive), late
differentiating spermatogonia (av-integrin nega-
tive, Thy-1 negative, c-Kit positive). mRNA levels
are normalized to those of b-actin and standard
deviations from duplicate reactions are shown.
Dashed line indicates Redd1 expression levels in
unfractionated WT testis cells.
(D) WT SPCs infected with control shRNA (against
GFP) or two independent shRNA constructs
against Redd1 were analyzed by western blot for
the indicated proteins. Quantification of relative
levels of phospho-RPS6 (corrected to total RPS6
levels) is shown under respective lanes. Cells
were under steady-state culturing conditions
(see also Figure S4).
(E) Top panel: Redd1 promoter (from translation
start site at +1 to 2 kb upstream) with location of
previously described transcription factor binding
sites. The promoter is divided into proximal and
distal regions (PP and DP, respectively). Positions
of ChIP amplicons are indicated. Bottom panel:
ChIP assay. Quantitative PCR for Redd1 promoter
regions from WT SPC chromatin pulled down
with Plzf antibody. Fold enrichment is shown rela-
tive to background of chromatin pulled down from
Plzf / SPCs with the same antibody. Standard
deviations from duplicate PCR reactions are
indicated.
(F) Luciferase reporter assay with constructs con-
taining the PP+DP Redd1 promoter regions or PP
alone. 293HEK cells were transfected with the
fractions and found a relative enrichment of Redd1 mRNA
in SPCs (Figure 4C). We also confirmed that freshly isolated
Plzf / SPCs had lower Redd1 expression compared to controls
(Figure 4C). Furthermore, shRNA knockdown of Redd1 in WT
SPCs increased mTORC1 activity (Figure 4D and Figure S4).
Together, our data suggest that Plzf opposes mTORC1 by main-
taining Redd1 expression and that Redd1 is a key mTORC1
regulator in SPCs.
Plzf Is a Transcriptional Activator of the mTORC1
Inhibitor Redd1 in SPCs
Redd1 expression was decreased at the mRNA level in
Plzf / SPCs. We therefore tested whether Plzf could directly
induce Redd1 expression. We first performed a chromatin
Cell 142, 468–479, August 6, 2010 ª2010 Elsevier Inc. 473

immunoprecipitation (ChIP) assay to assess whether Plzf was
bound to the Redd1 promoter in SPCs. We scanned a 2-kb
region upstream Redd1 that contains binding sites for multiple
transcription factors known to regulate Redd1 expression
(Figure 4E) (Ellisen et al., 2002; Lin et al., 2005; Shoshani et al.,
2002). Importantly, we could detect Plzf association with distal
promoter (DP) regions but not proximal promoter (PP) regions
(Figure 4E), indicating that Plzf regulates Redd1 expression
through recruitment to the DP. To confirm this we performed
luciferase assays with REDD1 promoter constructs containing
both DP+PP elements and the PP element alone (Figure 4F).
In agreement with our ChIP results, PLZF activated the DP+PP
construct but not the PP construct. Further, deletion of the
PLZF POZ domain prevented PP+DP reporter activation, indi-
cating a requirement for this protein-protein interaction domain.
We conclude that Plzf directly activates Redd1 through the DP to
inhibit mTORC1 in SPCs.
Active mTORC1 Inhibits SPC Self-Renewal Pathways
via a Negative Feedback Loop
As Plzf / SPCs show increased mTORC1 but decreased Akt
activities (Figure 3C), we considered that negative feedback
474 Cell 142, 468–479, August 6, 2010 ª2010 Elsevier Inc.
Figure 5. A Negative Feedback Loop from
mTORC1 to the GDNF Receptor Is Activated
in Plzf / SPCs
(A) Plzf +/+ and Plzf / SPCs starved overnight in
basal SPC medium supplemented with DMSO
(vehicle control) or rapamycin were stimulated
with 10ng/ml GDNF for 20 min before harvesting
for western blot analysis.
(B) Quantification of relative levels of phospho-Akt
and phospho-RPS6 (corrected to total levels of
respective proteins) from (A).
(C) Plzf +/+ and Plzf / SPCs were plated in
medium containing decreasing concentrations of
GDNF, harvested one week later and counted to
determine fold cell recovery as an indicator of
growth. The concentration of GDNF was varied
from 4 ng/ml (left bars) to 1 ng/ml (right bars). Stan-
dard deviations from duplicate wells are indicated
(**p < 0.01, *p < 0.02).
(D) Quantitative RT-PCR analysis of GDNF
receptor components in Plzf +/+ and Plzf /
SPCs treated with DMSO (vehicle control) or rapa-
mycin for 48 hr. mRNA levels are normalized to
those of b-actin and standard deviations from
duplicate reactions are shown.
(E) Flow cytometry analysis of Gfra1 levels from
SPC lines treated as in (D).
(F) Quantitative RT-PCR analysis of GDNF
receptor components in av-integrin negative,
Thy-1 low, c-Kit negative testis cell fractions
pooled from 2-week-old Plzf +/+ and Plzf /
mice. mRNA levels are normalized to those of
b-actin and standard deviations from duplicate
reactions are shown.
See also Figure S5.
from mTORC1 could prevent Plzf / cells from activating Akt
in response to growth factors. In cells lacking TSC1/TSC2,
increased mTORC1-signaling induces a negative feedback
loop from the mTORC1 downstream target S6K to upstream
signaling components causing inhibition of PI3K/Akt (Harrington
et al., 2004; Shah et al., 2004). Therefore, we tested whether
aberrant mTORC1 activity inhibited the response of Plzf /
SPCs to GDNF, a growth factor required for SPC self-renewal,
which could explain the maintenance defect of Plzf / SPCs.
We compared the response of starved Plzf +/+ and Plzf /
SPCs to GDNF in the absence or presence of rapamycin to
vary mTORC1 activity (Figures 5A and 5B). Consistent with
previous data, Plzf / SPCs showed lower levels of basal
and GDNF-stimulated Akt activity when compared to WT
SPCs, indicating a substantially reduced ability of Plzf / cells
to activate PI3K/Akt. However, on rapamycin treatment, the
ability of Plzf / SPCs to activate Akt in response to GDNF
was significantly increased and became comparable to that of
WT cells. This rescue of GDNF responsiveness by rapamycin
indicates that a negative feedback response from aberrantly
activated mTORC1 suppresses the response of Plzf / SPCs
to GDNF. Indeed, if GDNF levels in the media were reduced,
Plzf / SPC growth was inhibited more substantially than that of
the WT cells (Figure 5C). A decreased sensitivity to GDNF due
to negative feedback from mTORC1 can explain the defect in
Plzf / SPC maintenance in vivo when levels of GDNF are
limiting (Meng et al., 2000) and how Plzf / SPCs can be
cultured in vitro when GDNF is supplied to excess. Importantly,
GDNF expression in Plzf / testis was equivalent to that of
the WT (Figure S5), ruling out noncell autonomous defects in
production of niche factors.
Activated mTORC1 Suppresses Expression of GDNF
Receptor Components
We next sought to determine the mechanism by which activated
mTORC1 inhibits response of Plzf / SPCs to GDNF. Negative
feedback from mTORC1 to PI3K/Akt was originally character-
ized in the context of insulin responsiveness and signaling
through IRS1/2 proteins (Harrington et al., 2005). Although IRS
proteins have been implicated in activation of PI3K by the
GDNF receptor component c-Ret (Hennige et al., 2000), we
noticed that expression of the GDNF receptor components
GFRa1 and c-Ret in SPCs were responsive to rapamycin
(Figures 5D and 5E). Cultured Plzf / SPCs expressed lower
levels of GFRa1 and c-Ret compared to WT cells (correlating
with reduced GDNF-responsiveness) but on mTORC1 inhibition,
expression of these GDNF receptor components was increased
back to WT levels. Expression of GFRa1 and c-Ret were also
lower in freshly isolated Plzf / SPCs compared to WT SPCs
(Figure 5F), confirming that Plzf loss disrupts GDNF receptor
expression in vivo. Our data suggest that aberrantly activated
mTORC1 inhibits the response of Plzf / SPCs to GDNF by
opposing expression of the receptor. Interestingly, mTORC1
inhibits upstream signaling events in MEFs through transcrip-
tional inhibition of the PDGF receptor (Zhang et al., 2007). In
SPCs, negative feedback between mTORC1 and the GDNF
receptor will lead to loss of self-renewal when mTORC1 is hyper-
activated, such as occurs on loss of Plzf expression (Figure S6A).
This feedback loop would inversely couple cell growth to self-
renewal, so that on expansion of the stem cell pool by mitogenic
stimulation and resultant mTORC1 activation, response of the
cells to self-renewal signals is inhibited, restricting stem cell
numbers. Interestingly, GDNF activates mTORC1 weakly in
SPCs when compared to other mitogens (e.g., bFGF) (Figure 3B),
suggesting that GDNF would trigger a limited activation of the
negative feedback response whereas bFGF could cause more
extensive inhibition of GDNF signaling via mTORC1.
Rapamycin Treatment Attenuates the SPC Maintenance
Defect of Plzf / Mice and Increases SPCs in WT
Control Mice
As inhibition of aberrant mTORC1 activity in Plzf / SPCs in vitro
rescues their response to GDNF, we next asked whether
mTORC1 inhibition in vivo could rescue the Plzf / SPC mainte-
nance defect. We initiated rapamycin treatment of Plzf / and
WT control mice during early stages of postnatal development
(10 days postnatal) and continued daily treatment for 1 week,
a regimen able to normalize the increased size of Plzf / SPCs
(Figures 2G and 2H). Subsequent analysis of SPC compartment
status demonstrated that rapamycin increased both frequency
and numbers of avneg Thy-1low c-Kitneg cells in Plzf / testis,
approaching those values found in vehicle-treated WT controls
(Figures 6A and 6B). Rapamycin treatment of Plzf / mice
increased both the frequency of avneg Thy-1low cells and normal-
ized the increased percentage of c-Kitpos cells within this fraction
compared to controls (Figure 6A). We conclude that aberrant
mTORC1 activation can explain, at least in part, the SPC mainte-
nance defect of Plzf / mice.
Intriguingly, rapamycin also increased the frequency of SPCs
in WT mice (Figure 6B), suggesting that mTORC1 inhibition
stimulates SPC function in a WT setting. Indeed, in WT SPCs,
rapamycin enhanced Akt activation in response to GDNF
(Figures 5A and 5B) and increased GDNF receptor expression
(Figures 5D and 5E). Importantly, prolonged rapamycin treat-
ment of juvenile WT mice increased the number of cells with
high levels of Plzf expression in the testis (Figures 6C and 6D
and Figure S6B) and enhanced GDNF receptor expression
(Figure S6C), suggesting that physiological mTORC1 activity
controls size and function of the SPC pool in vivo.
DISCUSSION
Disruption of genes encoding for negative regulators of the PI3K/
Akt/mTORC1 pathway (e.g., Pten, Tsc1, and Pml) leads to loss of
hematopoietic stem cell (HSC) quiescence and subsequent
exhaustion. Rapamycin prevents HSC depletion, demonstrating
that aberrant mTORC1 activation is responsible for stem cell loss
(Chen et al., 2008; Gan et al., 2008; Ito et al., 2008; Yilmaz et al.,
2006). The negative effects of mTORC1 on HSC maintenance
have been attributed to its downstream targets in cell growth
pathways (Chen et al., 2008) and aberrant mTORC1 activity
elicits activation of tumor suppressive mechanisms that can
contribute to stem cell failure (Figure S6D) (Alimonti et al.,
2010; Lee et al., 2007a). By studying Plzf function in SPCs, we
now implicate negative feedback responses from the mTORC1
pathway to receptors required to transduce niche-derived self-
renewal signals in the loss of stem cell potential (Figure S6E).
HSC maintenance is also linked to niche signals acting
through growth factor receptors (Arai et al., 2004; Yoshihara
et al., 2007) thus it will be of interest to translate our findings
into the hematopoietic system. In addition, the decreased sensi-
tivity of Plzf / SPCs to GDNF mimics a reduced production of
niche-signals; the decline in stem cell numbers in aging mouse
testis is due partly to declining niche function and GDNF produc-
tion (Ryu et al., 2006; Zhang et al., 2006), thus loss of Plzf expres-
sion can represent a premature aging phenotype. Our studies
therefore provide further insight into the association between
stem cell maintenance, mTORC1, and aging (Harrison et al.,
2009; Rossi et al., 2008).
A correlation between aging and cancer is also apparent, likely
due to the accumulation of multiple genetic hits over time for
tumor development (Rossi et al., 2008). As stem cells are resi-
dent long-term in tissues and possess self-renewal capability
(a feature of cancer stem cells), they are suggested to be the
source of many cancers. However, loss of tumor suppressors
(e.g., Pten, Pml) trigger stem cell depletion (Ito et al., 2009) and
subsequent cancer development is associated with additional
genetic hits (Guo et al., 2008). It is proposed that oncogenic
Cell 142, 468–479, August 6, 2010 ª2010 Elsevier Inc. 475

hits in stem cells elicit activation of fail-safe mechanisms that
have to be evaded to allow cancer development (Figure S6D).
These fail-safe mechanisms prevent propagation of clones of
cells derived from stem cells with oncogenic mutations and the
consequent risk of acquiring additional mutations that lead to
cancer. The mTORC1 hyperactivation that occurs in response
to loss of tumor suppressors such as Pten and Pml is likely
a trigger of this fail-safe mechanism as rapamycin prevents
stem cell exhaustion. Based on our new model, mTORC1
hyperactivation in response to oncogenic stimulation may also
desensitize the cancer stem cell to niche-signals that are
required for self-renewal; thus additional mutations supporting
niche-independent self-renewal could allow cancer to develop
(Figure S6E).
476 Cell 142, 468–479, August 6, 2010 ª2010 Elsevier Inc.
Figure 6. mTORC1 Inhibition Attenuates
SPC Maintenance Defect of Plzf / Mice
(A) Top panels: representative av-integrin/Thy-1
flow profiles of testis cells from Plzf +/+ and
Plzf / mice treated with vehicle or rapamycin
from 10 days postnatal age for a period of
1 week and analyzed the day after completing
treatment. Bottom panels: Flow cytometry anal-
ysis of c-Kit expression within the corresponding
av-integrin negative, Thy-1 low fractions.
(B) Quantification of frequency and absolute
numbers of av integrin negative, Thy-1 low, c-Kit
negative testis cells from mice of the indicated
genotypes treated with Vehicle or rapamycin as
in (A) (n R 5 per genotype and treatment group,
**p < 0.02, *p < 0.05). Graphs indicate mean
values ± SEM.
(C) WT juvenile mice (2–3 weeks postnatal) were
treated daily for 2 weeks with rapamycin or vehicle.
Testes were harvested and subject to immunohis-
tochemistry for Plzf. Representative images are
203.
(D) Quantification of cells showing strong positivity
for Plzf in seminiferous tubules of testis from (C),
representing SPCs. Results from duplicate exper-
iments (1 and 2) are shown together with SEM
(**p < 0.01).
(E) Illustration of the effects of aberrant mTORC1
activity or inhibition of physiological mTORC1
activation with rapamycin on SPC numbers and
function.
See also Figure S6.
PLZF was first identified from involve-
ment in acute promyelocytic leukemia
(APL) (Chen et al., 1993). Thus, our model
of Plzf-dependent SPC maintenance
can have implications for leukemia patho-
genesis. The ability of Plzf to inhibit
mTORC1 through Redd1 induction may
be relevant in this context given that
perturbed PLZF function and elevated
mTORC1 activity are associated with
leukemia development (He et al., 2000;
Martelli et al., 2007; McConnell and Licht,
2007). In addition, REDD1 is involved in
leukemic and myeloid cell differentiation (Gery et al., 2007;
Nishioka et al., 2009), suggesting that this gene can be a
relevant PLZF target in hematopoietic cells. PML, also identified
from its involvement in chromosomal translocations with the
RARA gene in APL, is required for HSC maintenance and inhibits
mTORC1 (Bernardi et al., 2006; Ito et al., 2008); thus illustrating
a striking commonality of function between the RARA partners
of APL.
We have identified a role for Plzf-mediated Redd1 expression
in inhibiting mTORC1 activation in SPCs. As mTORC1 activity
has a significant impact on the response of SPCs to GDNF, the
tight regulation of this pathway is critical. Redd1 is also induced
in response to cellular stress and in particular, by hypoxia
(Brugarolas et al., 2004; Shoshani et al., 2002). Interestingly,
the HSC niche has been suggested to exist in a hypoxic state
(Kubota et al., 2008; Parmar et al., 2007) that, if the SPC niche
had similar characteristics, could be responsible for Redd1
induction. However, SPCs reside close to the vasculature
in vivo (Yoshida et al., 2007) and Redd1 expression is maintained
when SPCs are cultured at atmospheric oxygen levels. Thus
Redd1 expression in this case depends on a Plzf tonic transcrip-
tional activity and its induction is seemingly independent of
hypoxia.
As mTORC1 inhibition enhances SPC function even in a WT
setting, our data also have important therapeutic implications.
It is tempting to speculate that rapamycin could be used to
enhance SPC activity in humans; potentially allowing improved
SPC culture from testicular biopsies and generation of therapeu-
tically relevant MASCs (Figure 6E). Furthermore, our findings
provide a novel and straightforward explanation for the require-
ment of Plzf in germline maintenance.
EXPERIMENTAL PROCEDURES
Mouse Maintenance and Manipulation
Plzf / mice are previously described (Costoya et al., 2004). Mice were treated
with rapamycin (LC Laboratories) at a daily dose of 4 mg/kg as described
(Yilmaz et al., 2006). Transgenic mice expressing EGFP from the b-actin
promoter were from Jackson Laboratories. Testis transplant assays were
performed as described (Seandel et al., 2007) with 50 3 103 unfractionated
or 5 3 103 avneg Thy-1low c-Kitneg testis cells injected per recipient testis.
Teratoma formation assays in NOD/SCID mice (Jackson Laboratories) were
performed as described (Seandel et al., 2007).
Antibody Generation
Monoclonal anti-PLZF antibody (clone 9E12) was raised in Armenian hamster
against a pool of keyhole limpet hemocyanin (KLH)-conjugated peptides at the
Memorial Sloan-Kettering Cancer Center (MSKCC) antibody facility and reacts
to a peptide within the hinge domain of mouse Plzf (RSKEGPGTPTRRSVIT
SARE). Antibody was directly conjugated to Alexa 488 or Alexa 647 for use
in flow cytometry (MSKCC antibody facility).
Flow Cytometry
Single cell suspensions were prepared from testis as described (Ogawa et al.,
1997) and resuspended in phosphate-buffered saline (PBS) containing 2%
fetal bovine serum (FBS) for subsequent staining and analysis (Filipponi
et al., 2007). For a detailed description of flow cytometry methods and anti-
bodies used, refer to the Extended Experimental Procedures.
Cell Culture and Treatment
SPC fractions from testis were plated directly onto mitotically inactivated MEF
feeder cells. Medium for SPC culture was as previously described (Seandel
et al., 2007) supplemented with 10 ng/ml IGF-I (Peprotech). MASCs and
mouse ESCs (v6.5) were cultured in embryonic stem cell medium on MEF
feeders (Seandel et al., 2007). Knockdown of Redd1 expression in SPCs by
shRNA was performed using pLKO.1 lentiviral vectors (Open Biosystems).
A detailed description of methods is included in the Extended Experimental
Procedures.
Immunofluorescence, Immunohistochemistry, and Western Blot
Tissue and cultured cells were fixed in 4% paraformaldehyde before
processing for immunofluorescence (IF) and immunohistochemistry (IHC) as
described (Costoya et al., 2004; Filipponi et al., 2007). For western blot anal-
ysis, cells were lysed in RIPA buffer and processed as described (Costoya
et al., 2008). Detailed methods are included in the Extended Experimental
Procedures.
Chromatin Immunoprecipitation (ChIP)
SPCs were trypsinized and resuspended in SPC medium then incubated in
tissue culture dishes for 30 min to remove contaminating (adherent) MEFs.
Preparation and immunoprecipitation of chromatin was performed using
a SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology)
according to manufacturers instructions and monoclonal anti-PLZF antibody
(Calbiochem). DNA samples were analyzed by quantitative PCR using a
QuantiTect SYBR Green PCR kit (QIAGEN) and Light Cycler (Roche). Primer
sequences for the Redd1 promoter are included in the Extended Experimental
Procedures.
Luciferase Assay
REDD1 promoter constructs are described (Lin et al., 2005) and were cloned
from U2OS genomic DNA into the pGL3-enhancer vector (Promega). Lucif-
erase assays were performed with 293HEK cells as described (Barna et al.,
2002). Please refer to Extended Experimental Procedures for details.
Quantitative Reverse Transcription Polymerase Chain Reaction
(qRT-PCR)
RNA was harvested using Trizol reagent (Invitrogen) then used for first strand
cDNA synthesis and subsequent quantitative PCR analysis as described
(Filipponi et al., 2007). For primer sequences and detailed methodology please
see Extended Experimental Procedures.
Statistical Analysis
Results from cell growth assays, testis transplants, and flow cytometry anal-
ysis were assessed for statistical significance by a standard two-tailed t test.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures and
six figures and can be found with this article online at doi:10.1016/j.cell.
2010.06.041.
ACKNOWLEDGMENTS
We thank current and past members of the Pandolfi lab and in particular Taka-
hiro Maeda and Rosa Bernardi for helpful discussion and advice. We would
also like to thank Antonella Papa for generating luciferase reporters, Sharmila
Fagoonee for experimental help, Leif Ellisen for Redd1 promoter constructs,
and the flow cytometry facilities of BIDMC and MSKCC for expert support.
M.S. is a Stanley and Fiona Druckenmiller Fellow of the New York Stem Cell
Foundation. This work was supported by NIH grants to P.P.P.
Received: October 5, 2009
Revised: April 1, 2010
Accepted: May 28, 2010
Published: August 5, 2010
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Cell 142, 468–479, August 6, 2010 ª2010 Elsevier Inc. 479
Myc-Nick: A Cytoplasmic Cleavage
Product of Myc that Promotes a-Tubulin
Acetylation and Cell Differentiation
Maralice Conacci-Sorrell,1 Celine Ngouenet,1 and Robert N. Eisenman1,*
1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
*Correspondence: eisenman@fhcrc.org
DOI 10.1016/j.cell.2010.06.037
SUMMARY
The Myc oncoprotein family comprises transcription
factors that control multiple cellular functions and
are widely involved in oncogenesis. Here we report
the identification of Myc-nick, a cytoplasmic form of
Myc generated by calpain-dependent proteolysis at
lysine 298 of full-length Myc. Myc-nick retains
conserved Myc box regions but lacks nuclear locali-
zation signals and the bHLHZ domain essential for
heterodimerization with Max and DNA binding. Myc-
nick induces a-tubulin acetylation and altered cell
morphology by recruiting histone acetyltransferase
GCN5 to microtubules. During muscle differentiation,
while the levels of full-length Myc diminish, Myc-
nick and acetylated a-tubulin levels are increased.
Ectopic expression of Myc-nick accelerates myo-
blast fusion, triggers the expression of myogenic
markers, and permits Myc-deficient fibroblasts to
transdifferentiate in response to MyoD. We propose
that the cleavage of Myc by calpain abrogates the
transcriptional inhibition of differentiation by full-
length Myc and generates Myc-nick, a driver of cyto-
plasmic reorganization and differentiation.
INTRODUCTION
The Myc family (c-Myc, N-Myc, and L-Myc) of basic-helix-loop-
helix-zipper (bHLHZ) transcription factors controls the expres-
sion of a large number of target genes and noncoding RNA
loci. These Myc targets mediate the physiological effects of
Myc on cell proliferation, metabolism, apoptosis, growth, and
differentiation (Eilers and Eisenman, 2008). To promote tran-
scriptional activation at target genes, Myc forms heterodimers
with its partner Max and recruits chromatin-modifying com-
plexes to E-box-containing promoters. Myc is also involved in
transcriptional repression through the inhibition of the transcrip-
tional activator Miz1 (Kleine-Kohlbrecher et al., 2006). Aberrant
elevation of Myc levels has been shown to contribute to the
genesis of many types of human tumors (Hanahan and Wein-
berg, 2000).
480 Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc.
Myc family proteins contain highly conserved regions termed
Myc boxes (MB) that are essential for Myc’s biological activities
(see Figure 1E). A major determinant of Myc transcriptional
function is MBII, which is the site of recruitment of coactivator
complexes containing histone acetyltransferases (HATs) such
as GCN5 (McMahon et al., 2000) and TIP60 (Frank et al.,
2003). MBI functions as a phosphorylation-dependent binding
site for the ubiquitin ligase Fbw7 (Welcker et al., 2004), whereas
MBII is one of the binding sites for the ligase SKP2 (Kim et al.,
2003; von der Lehr et al., 2003). Fbw7 and Skp2 both contribute
to the rapid degradation of Myc protein (t1/2
20 min). The C
terminus of Myc harbors nuclear localization signals and the
bHLHZ motif that mediates dimerization with Max and DNA
binding.
Several variant forms of Myc protein have been previously
identified. All of them are nuclear-localized, low-abundance
proteins generated by alternative translation initiation. A weak
CUG translational initiation site, upstream and in-frame of the
predominant AUG codon, produces an N-terminally extended
form of c-Myc called c-Myc1 (Hann et al., 1988). Another Myc
protein variant is MycS, generated by internal translational initia-
tions at two AUG codons located
100 amino acids from the
normal N terminus (Spotts et al., 1997). MycS lacks MBI but
contains MBII and retains much of full-length Myc’s biological
activity (Xiao et al., 1998).
As expected, given their broad role as transcriptional regula-
tors, Myc family proteins are predominantly localized to the
cell nucleus during proliferation. Surprisingly, however, there
have been multiple reports of cytoplasmically localized Myc,
mostly in differentiated cells. For example, N-Myc localiza-
tion was shown to change from nuclear to cytoplasmic in
differentiating neurons of the neural crest, retinal ganglion
cells, neurons of spinal ganglia (Wakamatsu et al., 1993, 1997),
and Purkinje cells (Okano et al., 1999; Wakamatsu et al.,
1993). Cytoplasmic Myc was also reported in tumors with
diverse origins (Bai et al., 1994; Calcagno et al., 2009; Pietilai-
nen et al., 1995). These studies relied on immunostaining pro-
tocols and the form of the Myc protein involved was not char-
acterized.
Interestingly, association of Myc with several cytoplasmic
proteins has been reported. The best characterized is the
interaction of c-Myc with tubulins (Alexandrova et al., 1995)
(Koch et al., 2007; Niklinski et al., 2000). Myc has also been
reported to interact with other proteins that are predominantly
 A B IB: anti Myc N262 C IB: anti Myc 9E10
IB: anti Myc 143
c-Myc c-Myc c-Myc

Vector
Sparse Dense Cultures
Cell density Cell density
+ - + c-Myc
62
62 62 Myc
Myc Myc 49
49 49
Myc-nick 38
Myc-nick 38
38 28
17
14
Tubulin Tubulin
Tubulin
Total lysates
Sin3A Sin3A
50 5 .5 50 50 5 .5 X 105 Cells 50 5 .5 50 5 .5 X 105 Cells

Nuclear Cytoplasmic Nuclear Cytoplasmic

D IB: anti Myc N262 E

Nuclear Cytoplasmic Myc-nick
Myc antibodies
2

N262 ....................................... +
26

26
G

G
A

IP:
Ig

Ig
H
N

H
N

Myc
143......................................... +
274................................ +
Myc-nick 9E10 -
MycS
C19 -

Max

F
Sparse Dense
c-Myc
DAPI

20µM

Ab: N262 9E10

Figure 1. Identification of Myc-Nick in the Cytoplasm of Cells Grown at High Density

(A) Total cell lysates of Rat1 myc null fibroblasts infected with c-Myc or empty retroviral vectors were prepared for western blot by adding boiling sample buffer.
(B and C) Nuclear and cytoplasmic fractions of HFF cells expressing c-Myc were prepared 48 hr after plating at the indicated increasing densities.
(D) Immunoprecipitation of HA-c-Myc (N-terminal tag) with anti-N262, anti-HA, and normal IgG from nuclear and cytoplasmic fractions of HFFs. Note that Max is
only coimmunoprecipitated along with nuclear c-Myc.
(E) Schematic representation of antibody mapping.
(F) HFF cells infected with c-myc-expressing retrovirus were cultured for 4 days after reaching confluency (middle and right panels) and compared with a
subconfluent culture (left panel) by immunofluorescence using N262 and 9E10 antibodies.
See also Figure S1.

cytoplasmic, such as cdr2 (Okano et al., 1999) and AMY-1 (Taira RESULTS
et al., 1998). However the nature of the cytoplasmic Myc protein
and its potential function remains an enigma. Here we report the Myc-Nick Is a Truncated Form of Myc Localized
identification of Myc-nick, a cytoplasmically localized cleavage Predominantly in the Cytoplasm
product of Myc, and provide evidence for its role in cytoskeletal While studying regulation of c-Myc degradation, we noticed an
organization and cell differentiation. inverse correlation between the levels of full-length c-Myc and

Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc. 481

a cytoplasmic 42 kDa protein in anti-Myc immunoblots derived
from confluent fibroblast cultures (Figures 1A and 1B). As
described below, this protein, which we have named Myc-nick,
is a cytoplasmic cleavage product of full-length c-Myc gener-
ated at high cell density (Figure 1B). Myc-nick is recognized by
three antibodies against the N-terminal two-thirds of c-Myc
(anti-Myc N262, 274, 143; Figures 1A and 1B and Figure S1A
available online) but not by anti- C-terminal antibodies (anti-
Myc 9E10, C19; Figure 1C). Furthermore, an anti-HA antibody
immunoprecipitates Myc-nick from cytoplasmic extracts of cells
expressing N-terminally HA-tagged c-Myc (Figure 1D). In addi-
tion, cytoplasmic Myc bearing N-terminal but not C-terminal
epitopes is detected by imunofluorescence in confluent cultures
(see below, Figure 1F). Together, these results indicate that
Myc-nick is a truncated protein lacking the C-terminal portion
of c-Myc while preserving an intact N terminus comprising
Myc boxes I–III (Figure 1E). This makes Myc-nick distinct from
any other previously identified form of Myc (see Introduction).
We have detected endogenously expressed Myc-nick in the
cytoplasm of a large number of cell lines including human
foreskin fibroblasts (HFFs), Wi38, L cells, HCT116, SW480, HeLa
(Figure S1B), C2C12 (Figure 7D), 293T, A431, Rat1, U2OS, ES
cells, and mouse neurospheres (not shown), in addition to mouse
tissues such as muscle, brain, and cerebellum (Figure 7A and
Figure S7A). We also observed Myc cytoplasmic localization
by immunofluorescence of confluent cultures of HFFs (Figure 1F
and Figure S5A) and Rat1 myc null cells (not shown) expressing
c-myc. Therefore a protein with the size and properties of Myc-
nick is very widely expressed. In some settings we observe
relatively low and variable amounts of Myc-nick in the nucleus
(e.g., Figure 2A).
Myc-Nick Is Generated by Proteolytic Cleavage
of c-Myc in the Cytoplasm
We considered the possibility that Myc-nick is generated in the
cytoplasm because the nuclear export inhibitor Leptomycin B
had no effect on the production or cytoplasmic localization of
Myc-nick (Figure 2A). To determine whether a cytoplasmic
activity could convert full-length c-Myc into Myc-nick, we incu-
bated in vitro-translated [35S]-methionine-labeled c-Myc or puri-
fied full-length recombinant c-Myc with nuclear or cytoplasmic
extracts from Rat1 myc null cells. Only cytoplasmic extracts
(CE) were capable of producing a protein (Figures 2B–2D) having
the same apparent molecular weight as Myc-nick that was
recognized by antibodies against the N terminus but not C
terminus of c-Myc (Figure S2A). Increasing the incubation time
with CE augmented Myc-nick production and decreased the
amounts of full-length c-Myc input (Figure 2C). In agreement
with our observations made in vivo, CE of dense cultures were
more efficient in cleaving c-Myc than CE of sparse cultures
(Figure S2B). In experiments designed to characterize the cyto-
plasmic activity responsible for formation of Myc-nick, we found
that inhibitors of transcription or translation had no effect on
Myc-nick formation (Figure 2E) whereas heating or adding
protease inhibitors to the CE blocked Myc-nick, consistent
with proteolysis (Figure 2F). Whereas MG132, an inhibitor of the
proteasome (and other cysteine proteases), blocked Myc nick
formation, specific proteasome inhibitors such as Epoxomycin
482 Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc.
and Lactacystein failed to block the formation of Myc-nick
in vitro and in vivo (Figures 2G and 2H). In addition, inhibition
of either the Fbw7 or Skp2 degradation pathways, known to be
responsible for proteasomal Myc turnover, had no effect on
Myc-nick formation (data not shown). Moreover, incubation of
Myc with 20S and 26S proteasomes failed to produce Myc-
nick (not shown). These results indicate that full-length c-Myc
is converted into Myc-nick by a cytoplasmic protease that is
independent of the proteasome.
Myc Is Cleaved by a Calcium-Activated Calpain
to Produce Myc-Nick
In a systematic search for the proteases mediating cytoplasmic
cleavage of Myc, we ruled out both caspases and lysosomal
proteases on the basis of inhibitors and cleavage conditions
(Figure S2C). However, we found that all calpain inhibitors tested
including calpeptin, calpain inhibitor XII (Figure 3A), and calpain
inhibitor VI (Figure 3B) blocked the formation of Myc-nick in vitro
and in vivo. Moreover, MG132, although well known as a protea-
some inhibitor, has also been shown to inhibit calpains (Mailhes
et al., 2002). Calpains had been linked to Myc stability earlier, but
the antibodies used in those studies would not have detected
Myc-nick (Gonen et al., 1997; Small et al., 2002). Calpains
comprise a large family of calcium-dependent cytoplasmic
cysteine proteases that function at neutral pH and are primarily
associated with partial protein cleavage rather than complete
protein degradation. The most well studied members of this
family are mcalpain and mcalpain. These ubiquitously expressed
catalytic subunits form functional heterodimers with a calpain
regulatory subunit (calpain r) to bind calcium. To determine
whether Myc-cleavage is regulated by calcium-activated cal-
pains, we employed siRNA to knock down calpain r in cells
expressing c-Myc either endogenously or under control of
a retroviral vector. In both settings a partial silencing of calpain
r correlated with decreased Myc-nick and increased full-length
c-Myc (Figure 3C). Knockdown of m or mcalpains alone did not
block the formation of Myc-nick, most likely due to the presence
of other redundant calpains (not shown). Because calpain
activity is calcium dependent we next examined the effects on
Myc-nick of modulating calcium levels. Treatment with the
calcium chelators Bapta or EGTA reduced the ability of CE to
cleave c-Myc (Figures 3D and 3E). Incubation of either IVT
c-Myc (Figure 3F) or recombinant c-Myc (Figure 3G) with purified
m or mcalpain (together with calpain r) produced Myc-nick.
These results demonstrate that Myc-nick is directly generated
by calcium-dependant calpain cleavage of full-length c-Myc.
Lysine 298 Is the Primary Calpain Cleavage Site in c-Myc
To map the calpain cleavage region on c-Myc, we used a series
of internal deletion mutants lacking 60 residue segments (c-Myc
DA-DG) (Tworkowski et al., 2002) (Figure 3H) and determined
whether any failed to generate a shorter c-Myc protein. Only
the deletion of residues 252–315 (DE) resulted in loss of a Myc-
nick-like product (Figure 3I). This region contains a high scoring
PEST domain, often present in unstable proteins and frequently
associated with calpain cleavage sites. We further narrowed our
search to residues 270–315, based on the ability of antibody 274
to detect Myc-nick (Figure S1A and Figure 3H). Although there is
 A B C - - - - - - + MG
IB: N262 - - - - - + - NE
- - - + CE 10' 1 2 4 16 16 16 hours
Nuclear Cytoplasmic - + - - NE + + + + + - + CE
0 10 30 0 10 30 ng LB
Myc Myc Myc
Myc-nick Myc-nick Myc-nick

D E F
- - + - - - Boiled CE
mg/µl CHX mg/µl ActD - - - - - + PI
yc
W

.01 .1 .5 .01 .1 .5
- + + - + + CE
M
M

- + + CE - + + + + + + + CE

Myc Myc Myc

Myc-nick Myc-nick Myc-nick

Silver IB: 143 1 2 3 4 5 6 7 8

G H IB: N262
µM MG132 mM Lacta mM Epoxo MG132 (80nM) Lacta (200nM) Epoxo (400nM)

0 1 10 100 0 0.1 1 10 0 0.1 1 10 - + - + - + - + - + - +

Myc
Myc-nick

N C N C N C
in vitro
in vivo

Figure 2. Myc-Nick Is a Product of c-Myc Cytoplasmic Cleavage Independent of the Proteasome

(A) CRM1-dependant nuclear export is not involved in the formation or localization of Myc-nick. Rat1 myc null cells infected with c-Myc were treated with
leptomycin B (LB) for 4 hr before harvesting.
(B) c-Myc gives rise to Myc-nick in vitro when incubated with cytoplasmic extracts. Radiolabeled IVT c-Myc was incubated for 2 hr with cytoplasmic (CE) or
nuclear extracts (NE) from Rat1 c-myc null cells.
(C) Timecourse of in vitro cleavage of c-Myc.
(D) Recombinant c-Myc is cleaved in the presence of CE. One microgram of recombinant c-Myc was incubated with 30 mg of CE and processed for western blot.
(E) IVT c-Myc was incubated with CE for 4 hr in the presence of of Actinomycin D (ActD) or cyclohexamide (CHX).
(F) The cleavage of c-Myc is inhibited by protease inhibitors and by heat inactivation. CE was boiled prior to incubation with IVT c-Myc and the protease inhibitor
(PI) was added to the incubation mixture.
(G and H) The cleavage of c-Myc is inhibited by MG132, but not by Lactacystein or Epoxomycin. (G) IVT c-Myc was incubated with CE, in the presence of
increasing amounts of MG132, Lactacystein, and Epoxomycin for 1 hr. (H) Rat1 myc null cells expressing c-Myc were incubated with MG132, Lactacystein,
or Epoxomycin for 2 hr prior to harvesting. Nuclear (N) and cytoplasmic (C) fractions are shown.
See also Figure S2.

no universal consensus for a calpain cleavage site, a comparison
of 106 sites present in 49 known calpain substrates indicated
a preference for calpain cleavage after K or R, and to lesser
extent Y, especially when these amino acids are flanked by P,
V, and L (Figure 3J) (Tompa et al., 2004). We noted a region local-
ized C-terminal to the PEST domain containing the sequence
PLVLKRC (Figure 3H, marked in red). This region is evolutionarily
conserved in c-Myc and N-Myc but not L-Myc (Figure S3A),
consistent with the fact that c-Myc and N-Myc, but not L-Myc,
are cleaved (Figures S3B and S3C). To determine whether this
region functions as a calpain cleavage site, we synthesized
a 10 amino acid peptide corresponding to the putative c-Myc
calpain cleavage region (291–300) and to another nearby region
of c-Myc (236–245) (Figure 3H, in red and blue, respectively) and
asked whether they could act as competitive inhibitors of Myc-
nick formation in vitro. Addition of increasing amounts of the
peptide containing the putative calpain cleavage site blocked
the formation of Myc-nick in vitro whereas the control peptide

Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc. 483

A B C Figure 3. Myc-Nick Is Generated by Calpain
μM Calpeptin μM inhibitor XII IB: N262 IB: N262 Cleavage of Full-Length Myc
0 0 0.1 1 10 100 0 1 10 100 N C HCT116 HFF-Myc
- + - + Reg siRNA
(A) IVT c-Myc was incubated with CE for 1 hr in the
- + + + + + + + + + CE - + - + inhibitor VI
Myc Myc Myc presence of the calpain inhibitors calpeptin and
Myc-nick Myc-nick Myc-nick calpain inhibitor XII.
Tubulin Calpain r (B) Dense cultures of HFF cells infected with a
Sin 3A Tubulin c-Myc retroviral vector were incubated with cal-
pain inhibitor VI for 2 hr prior to harvesting.
D (C) siRNA for calpain regulatory subunit (Reg)
E F μ m Calpain
0 1 1 1 ng Calpain
G IB: 143 reduces the formation of Myc-nick in HCT116
- + - Bapta 0 0 .1 1 10 100 μM EGTA - - + - - Calpeptin + + Purified c-Myc and HFF-Myc cells.
- + + CE - + + + + + CE - + - - - CE - + μCalpain (D) Rat1 myc null cells were incubated for 2 hr in the
Myc Myc Myc
presence of the calcium chelant Bapta, and then
Myc
Myc-nick Myc-nick Myc-nick cytoplasmic extracts were prepared and incubated
Myc-nick
with IVT c-Myc (as in 2B).
(E) IVT c-Myc was incubated with CE for 1 hr in the
presence of increasing amounts of EGTA.
(F) IVT c-Myc was incubated with purified recombi-
H nant mcalpain or mcalpain and r subunit for 1 hr
1 63 126 189 252 315 378 439 on ice.
MBI MBII MBIII NLS BHLH LZ

A B C D E F G (G) One hundred nanograms of purified mcalpain

and r subunit was incubated with 2 mg of recombi-
274 antibody
SPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLK RCHVSTHQHNYAAPPST nant c-Myc for 30 min on ice.
* ** * (H) Schematic representation of c-Myc protein
PEST domain
indicating A-G deletion regions (deletion end-
points indicated above), putative calpain cleavage
I K region in red.
PHSPLVLKRC SPEPLVLHEE
(aa 291-300) (aa 236-245)
(I) c-Myc and the deletion mutants DA-DG were
c

ΔG
c- c ΔC

M D
M A
M B

M E
M F
c- My

J
c- yc Δ
c- c Δ

c- Δ
c- c Δ
c- yc Δ

0 .1 1 10 μg expressed in 293T cells, and 48 hr later the pres-

c- yc

0 .1 1 10
c-

yc

yc
y
y

y
M
M

M
A-

- + + + - + + + CE
ence of a Myc-nick-like protein in cytoplasmic
H

P3 P2 P1 extracts was determined.

V,L,F L,V RKY
V L K Myc (J) Amino acid preference for calpain cleavage
Myc-nick region according to Tompa et al. (2004). P1–P3
IB: 143+274
indicate the position of preferred residues in rela-
tion to the cleavage site, bold letters indicate the
c-Myc calpain cleavage site.
(K) IVT c-Myc was incubated with CE in the pres-
L M N IB: 143+274
ence of a peptide that contains the potential
- + - + Δ291-300 calpain cleavage site (amino acids 291–300 in
98 0

WT L295A K298A R299A

K2 -30
K2 A
A

+ - + -
W or

Wt c-Myc red) or a nearby sequence (amino acids 236–245

91

97
ct
T
Δ2

0 2 4 0 2 4 0 2 4 0 2 4 h incubation
Ve

- - + + CE
in blue).
Myc Myc Myc
Myc-nick (L) WT and D291–300 IVT c-Myc were incubated
Myc-nick Myc-nick
with CE for 1 hr (as in 2B).
(M) IVT WT, L295A, K298A, and K299A c-Myc were
incubated with CE for the indicated time points.
(N) Cleavage products produced from the indi-
cated c-Myc mutants in 293T cells.
See also Figure S3.

had no effect (Figure 3K). Next, we generated a deletion mutant Myc that become more pronounced in the cleavage-deficient
of Myc lacking residues 291–300 and found it to be resistant to mutants.
cleavage by CE (Figure 3L). When this mutant was ectopically
expressed in 293T cells the cleavage was also reduced in Expression of Myc-Nick Alters Cell Morphology
comparison to the wild-type (WT) c-Myc (Figure 3N), indicating and Increases Acetylation of a-Tubulin
that this is the major calpain cleavage site within c-Myc. Next, To study Myc-nick function, we generated a truncated form of
we made point mutations in the calpain cleavage region (labeled Myc containing amino acids 1–298 (referred to as Myc-nick*).
with asterisks in Figure 3H) and assessed their cleavage. We We found that ectopically expressed Myc-nick* is localized
found that the K298A mutation reduced the cleavage of c-Myc predominantly to the cytoplasm and migrates on SDS-PAGE
in vitro (Figure 3M) and in vivo when ectopically expressed in with the same apparent molecular weight as Myc-nick generated
293T cells (Figure 3N). Although K298 appears to be the major by cleavage of full-length c-Myc (Figure 4A). Myc-nick is
calpain cleavage site, a K298A mutant was still cleaved in vivo degraded at a comparable rate to full-length c-Myc with a half-
most likely because when this residue is mutated the cleavage life of about 30 min (data not shown). Because Myc-nick
is shifted to R299 (and perhaps L297, V296). We also identified contains the MBI phosphodegron, with its GSK3b phosphoryla-
weaker calpain cleavage sites localized in the C terminus of tion and Fbw7-binding sites, we would expect Myc-nick to be

484 Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc.

 A B
IB: N262
vector Myc Myc-nick*
- - + - MG132
Myc
C
Myc-nick
Myc
N
α-tubulin
Myc-nick
1 2 3 4
DAPI/α
Vector Myc
Figure 4. The Expression of Myc-Nick* Promotes Changes in Cell Morphology
(A) Myc-nick* (1–298) is cytoplasmic and has the same apparent molecular weight as Myc-nick derived from full-length c-Myc (compare lanes 2 and 4, upper
panel). Rat1 myc null cells expressing empty vector, full-length c-Myc, and Myc-nick were fractionated into nuclear (N) and cytoplasmic (C) fractionations.
(B) Myc-nick*-expressing cells extend protrusions at the wound edge. A confluent monolayer of Rat1 myc null cells expressing either vector or Myc-nick was
scratched using a 100 ml tip and phase contrast images were taken at 12 hr.
(C) Rat1 myc null cells expressing empty vector, c-Myc, and Myc-nick at 14 days after selection.
See also Figure S4.
degraded through similar proteasomal pathways as full-length
Myc. Indeed, blocking proteasome activity by pharmacological
inhibitors or by silencing E3 ligases and components of the pro-
teasome induced the stabilization of both full-length Myc and
Myc-nick (data not shown). Calpain inhibitors are incapable of
inducing accumulation of Myc-nick, indicating that calpains are
unlikely to play a major role in Myc-nick turnover in vivo.
Whereas overexpression of full-length c-Myc in Rat1a myc
null fibroblasts is associated with increased proliferation
and apoptosis, the ectopic expression of Myc-nick* had no
detectable effect on either cell doubling time or survival (data
not shown). However, Myc-nick*-expressing cells displayed
dramatic morphological changes—they appeared spindle-like
with long protrusions that occasionally formed intercellular con-
tacts (Figure 4C, Figure S4, Figure S5C). This morphology was
specific for Myc-nick* expression and could not be produced
by expressing the C-terminal 100 residues of c-Myc (not shown).
Introducing a scratch wound across a confluent cell monolayer
showed that whereas control cells migrated into the wound by
extending lamellipodia, Myc-nick*-expressing cells aligned
parallel to each other and extended long protrusions into the
wound (Figure 4B).
The morphological changes induced by Myc-nick* are
suggestive of altered cell-cell contacts and/or major cytoskeletal
reorganization. The elongated cellular protrusions present in
Myc-nick-expressing cells resemble specialized structures
formed by stable microtubules. Because stable microtubules
display increased acetylation of a-tubulin on lysine 40 (Hubbert
et al., 2002), we stained Myc-nick*-expressing cells with anti-
Vector Myc-nick * Myc-nick *
Vector Myc-nick *
60μM
Myc-nick* Myc-nick*
bodies against a-tubulin and acetylated a-tubulin. Figure 5A
shows that although acetylated a-tubulin immunostaining
in vector and c-Myc-expressing cells is low, Myc-nick* cells
display intense staining of elongated structures (Figures 5A
and 5F). This was confirmed by immunoblotting for acetylated
a-tubulin in Rat1 myc null or 293T cells expressing Myc-nick*
(Figure 5C, Figure S6B). Importantly neither the levels of total
cellular acetylated lysine (Figure S6A) nor the levels of tyrosi-
nated tubulin (not shown) were affected. To ask whether Myc-
nick* expression increased microtubule stability, we treated cells
expressing Myc-nick*, c-Myc, or vector with nocodozole for
15 min. This treatment disrupted unstable microtubules with
a half-life of about 10 min but not stable microtubules with
a half-life >2 hr. Only Myc-nick*-expressing cells possessed
microtubules that survived nocodozole treatment and these
stained strongly with antiacetylated a-tubulin (Figure 5B).
Myc-Nick Interacts with a- and b-Tubulins
We observed partial cytoplasmic colocalization between Myc
and a-tubulin in several cell types expressing c-Myc or Myc-
nick (Figure 5E; Figures S5A–S5C). Using 293T cells expressing
GFP-tubulin and Myc-nick we performed immunoprecipitation
with anti-GFP and immunobloted with anti-Myc. Myc-nick was
coimmunoprecipitated with GFP-tubulin but not GFP-EB1,
a microtubule-binding protein (Figure S5D). In addition, immuno-
precipitation of either c-Myc or endogenous a-tubulin from cyto-
plasmic extracts indicated that Myc-nick interacts with a-tubulin
in vivo (Figure 5D). To examine in vitro interactions we incubated
35
[S]-methionine-labeled IVT c-Myc with purified brain tubulins
Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc. 485
 A Figure 5. Myc-Nick* Cells Display In-
Vector c-Myc Myc-nick* creased Levels of Acetylated a-Tubulin
and Microtubule Stabilization
α−tubulin/DAPI

(A) Immunofluorescence for a-tubulin (upper

panels) and acetylated a-tubulin (lower panels) of
Rat1 myc null cells infected with empty vector,
c-Myc, or Myc-nick*.
(B) As for (A) but incubated in the presence of
nocodazole for 15 min prior to fixation.
Vector c-Myc Myc-nick* (C) Immunoblotting of Rat1 cell extracts using anti-
acetyl. α−tub/DAPI

bodies against the indicated proteins.

(D) NE or CE of Rat1 myc null cells express-
ing c-Myc were immunoprecipitated with anti
a-tubulin or normal mouse IgG and immunoblotted
for Myc (top panel), or immunoprecipitated with
60μM anti-c-Myc N262 antibody, and immunoblotted
with anti-a-tubulin.
B (E) Immunofluorescence for Myc and acetylated
Vector c-Myc Myc-nick*
a-tubulin in A431 lung epithelial cells cell trans-
fected with Myc-nick.
α−tubulin

(F) Detail of Myc-nick-expressing cell stained for

10μM Nocodazole 15'

acetylated a-tubulin.
See also Figure S5.

Vector c-Myc Myc-nick*

recruits an acetyltransferase to microtu-
acetyl. α−tub

bules. We observed marked acetylation

of a-tubulin in cytoplasmic extracts incu-
bated in the presence of either recombi-
nant c-Myc (Figure 6A) or IVT c-Myc
60μM (Figure 6B and Figure S6C). Importantly,
Myc proteins are known to associate
C
yc k*
k*

Myc-nick*
M nic
ic

D with the acetyltransferases GCN5 and

-n

E F
M c
y
-
ro

yc
M
Pu

N C N C N C
c-

acetyl α-tub
TIP60 (Frank et al., 2003; Sterner and
IB: Myc

Myc
Berger, 2000) via TRRAP that interacts
α-tubulin
Myc-nick
*
acetyl α-tub with MBII (residues 106–143) (McMahon
Input α-tub IgG
γ-tubulin IP et al., 1998). We therefore tested a Myc-
nick deletion mutant lacking Myc box II
IB: α-tub

actin
HDAC 3
HDAC 6
for 1 hr, then immunoprecipitated a, b, g, bIII and acetylated
a-tubulin and exposed the gel to detect radioactive Myc. Full-
length c-Myc was coimmunoprecipitated mostly with b-tubulin
(Figure S5E, middle panel). We also incubated IVT c-Myc with
CE for 1 hr to produce Myc-nick and then performed immuno-
precipitation for tubulins as above. The results showed that
Myc-nick interacts with a- and b-tubulin (Figure S5E, upper
panel). The binding of Myc-nick to tubulin is consistent with the
previous report of an N-terminal region of Myc capable of asso-
ciating with tubulin (Alexandrova et al., 1995).
Myc-Nick Directly Regulates a-Tubulin Acetylation
through GCN5
Because Myc-nick lacks the Myc C-terminal dimerization and
DNA-binding domains, we surmised that the increase in acety-
lated a-tubulin induced by Myc-nick* expression is independent
of Myc transcriptional activity. One possibility is that Myc-nick
N C N C
α-tubulin Merge
(DMBII) and found that its ability to pro-
Input IP:Myc
mote a-tubulin acetylation was reduced
10μM
(Figures 6C and 6D). In addition, the
acetyl. α−tub/DAPI
DMBII Myc-nick* mutant failed to induce
the cell morphological changes that we
had detected with WT Myc-nick* whereas a comparably sized
deletion in a region adjacent to MBII was similar to WT (Figure 6E).
The dependence on MBII for tubulin acetylation and altered
cell morphology suggests involvement of the acetyltranferases
known to bind this region. We detected substantial amounts of
GCN5 and TRRAP in the cytoplasm (Figure S6D), whereas
Tip60 was predominantly nuclear (not shown). To test GCN50 s
involvement in a-tubulin acetylation we performed siRNA-medi-
ated knockdown of TRRAP and GCN5 in Myc-nick*-expressing
cells and found that decreasing either one of these proteins
reduced the levels of acetylated a-tubulin (Figure 6F) and
reverted the changes in cell morphology induced by Myc-nick
to that of vector-infected cells (Figure 6G). Moreover, GCN5
coimmunoprecipated with a-tubulin and Myc in cytoplasmic
extracts of 293T cells transfected with GCN5 (Figure 6H).
Both ectopic expression of GCN5 in 293T cells (Figures 6D
and 6I) and addition of full-length recombinant GCN5 to

486 Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc.

 I
BI
*
ni ick
A C E

M
*Δ
c- r

-n
M c
o
y
ct

yc

ck
M
+ + + + CE Vector Myc-nick*

ve
- - 0.1 1 μg c-Myc acetyl α-tub

in vitro CE
- + + + GTP
acetyl α-tub. α-tubulin

Myc IVT

I
BI
B D

N *
G ick

M
Myc-nick* Δ106-143

*Δ
ni 5
M r
-n
o
+ + - - vector IVT

ct
yc

ck
Myc-nick* Δ145-160

C
(ΔMBII)

ve
- - + + c-Myc IVT
acetyl α-tub

in vivo 293T cells

acetyl α-tub
α-tubulin
α-tubulin Myc-nick
GCN5

F G
G P
TR l
ro

5
A
N
R
nt

si RNA
C
co

Control si RNA GCN5 si RNA TRRAP si RNA

TRRAP

GCN5

acetyl α-tub

α-tubulin 60μM

Myc-nick

HDAC6

H J L - + + + + + - microtubules 5μg
- - - - + + + GCN5 0.1μg
r
5

GCN5
to
N
c

+ - + + - + - c-Myc 0.5μg
C
Ve

- - - + + + c-Myc 0.2μg
G

GCN5
10% input
acetyl α-tub
Myc *
30μg CE

1.0 1.6 1.9 1.8 2.4 2.7 fold change Tubulin

IP: α-tubulin
IB: GCN5 p300
IP: Myc - - - + + + c-Myc 0.2μg GCN5 catalyctic
IB: GCN5 acetyl α-tub Ponceau domain
1.0 1.1 1.1 2.4 2.6 2.5 fold change

acetyl α-tub
I K
GCN5
G r
5
o
N
ct
C
Ve

- - - + + c-Myc
acetyl α-tub α-tubulin
5μg microtubules

acetyl α-tub
1.3 2.7 1.0 3.1 4.2 fold change β-tubulin
GCN5 + p300 1 2 3 4 5 6 7
- - + + c-Myc
α-tubulin acetyl α-tub
0.9 1.0 1.2 1.1 fold change

Figure 6. c-Myc and the HAT GCN5 Promote a-Tubulin Acetylation

(A) CE was incubated with recombinant c-Myc and c-Myc dilution buffer or (B) with c-Myc IVT or vector IVT for 1 hr at 37 C. The samples were immunoblotted as
indicated.
(C) CE was incubated with IVT vector, c-Myc, Myc-nick, and Myc-nick DMBII (D106–143) for 30 min at 37 C, then processed for immunoblotting.
(D) 293T cells were transfected with empty vector, GCN5, Myc-nick, and Myc-nick DMBII and processed for immunoblotting after 48 hr.
(E) Rat1 myc null cells were infected as indicated and photographed 10 days after selection.
(F and G) Rat1 myc null cells expressing Myc-nick were transfected with 100 nM of control, TRRAP, or GCN5 siRNA and 76 hr later processed for immunoblotting
(F) or photographed (G).
(H) CE of 293T cells transfected with empty or GCN5 vectors were immunoprecipitated with anti-a-tubulin or anti-Myc (143+274) and immunoblotted for GCN5.
(I) 293T cells were transfected with control or GCN5-expressing vectors and 48 hr later processed for immunoblotting.
(J–L) GCN5 induces acetylation of a-tubulin. (J) CE were incubated with 200 ng of Myc, 100 ng or 300 ng of GCN5 (upper panel), or 100 ng or 500 ng of p300 (lower
panel). (K) Assembled microtubules were incubated with 100 ng or 500 ng of recombinant full-length GCN5 (upper panel) or p300 (lower panel) in the presence or
absence of 200 ng of purified c-Myc. (L) Purified assembled microtubules were incubated with recombinant c-Myc and GCN5-catalytic domain. The asterisk
indicates a nonspecific bacterial band copurified with GCN5-catalytic domain.
See also Figure S6.

cytoplasmic extracts (Figure 6J, upper panel) resulted in nant GCN5 also induced the acetylation of a-tubulin present
increased levels of acetylated a-tubulin. The addition of Myc in assembled microtubules (Figure 6K) and synergized with
further increased the levels of a-tubulin acetylation induced by c-Myc to promote further acetylation (Figure 6K, compare
GCN5 in cytoplasmic extracts (Figure 6J). Full-length recombi- lanes 1 to 4 and 2 to 5). The GCN5 catalytic domain alone was

Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc. 487

 A B GM DM
C D GM DM
1 2 3 8 16 weeks S D D Density primary myoblasts S M D D Density

α T58P α N262
Myc

primary mouse myoblasts

Myc 1.8
Myc

Calpain activity
1.6
Myc-nick 1.4
Myc-nick
Myc-nick TroponinC 1.2
1.0 Myc
Calpain3 0.8 Myc-nick
Tropomyosin 0.6
acetyl α-tub 0.4 Tropomyosin
mouse hindlimb muscles
0.2 Tubulin
Tubulin
GM DM C2C12

E F
c-Myc c-Myc/DAPI acetyl α-tub /DAPI acetyl α-tub /DAPI G
Rhabdomyosarcoma
HFF RHJT RD RH1
S D S D S D S D

20μM 60μM alveolar embryonal

C2C12 DM GM DM Subtype
ni r

H J L M
o
*
ct
ck
ve

Vector Myc-nick Vector GM DM

human myoblasts (pectoral)

Troponin C
- + - + Myc-nick*
(rectus abdominus)

Desmin
TroponinC
human myoblast

Myogenin
Myc-nick* Tropomyosin
acetyl α-tub
Myogenin
α-tubulin
C2C12

C2C12
Caveolin 3
I
r

K
BI
to

ΔM *

I
ck
c
or

ve
ni
*
ct
ck

Myc-nick*
RD Rhabdomyosarcoma

Troponin C
ve

ni

Desmin
(rectus abdominus)

Troponin C Tropomyosin
human myoblast

acetyl α-tub acetyl α-tub

Myc-nick*
Myc
MyoD
α-tubulin nick*
nick* ΔMBII
α-tubulin 60μM α-tubulin
N
O myc -/- myc +/+
TroponinC /DAPI

C MyoD MyoD C
M
M
M
M
M
M

-S
-S

G
G
D
G
G
D

TroponinC
Myogenin
60μM α-tubulin

P nick MyoD MyoD+nick

Phase

M
M

M
G

G
D

D
Rat1 myc -/-

TroponinC
Myogenin
20μM α-tubulin
MyoD MyoD+Myc-nick*
Rat1 myc+/+

Figure 7. Myc-Nick Accelerates Muscle Cell Differentiation

(A) Hindlimb muscles dissected from 1-, 2-, 3-, 8-, and 16-week-old mice were processed for immunoblotting using N-terminal anti-Myc sera (anti 143+274).
(B) Mouse primary myoblasts isolated from hindlimb muscles of 8-week-old mice were cultured as sparse cultures for 24 hr or as dense cultures in the presence of
either growth medium (GM) or differentiation medium (DM) for 3 days. Total cell extracts were immunoblotted for c-Myc (anti 143+274) and indicated proteins.
(C) Mouse primary myoblasts were cultured in GM or DM for 3 days, lysed in buffer A, and total calpain activity was measured using Suc-LLVY-AMC synthetic
substrate (n = 2; calpain activity in DM was compared with calpain activity in GM; set to 1 ± standard error of the mean [SEM]).
(D) C2C12 mouse myoblasts cultured at sparse (S), medium (M), or high densities (D). Dense cultures were harvested or switched to DM for 7 days. Total cell
extracts were immunoblotted for c-Myc using antibodies against total c-Myc (N262) or against phosphorylated T58 Myc, a signal for Myc degradation.
(E) C2C12 cells cultured in DM for 7 days and stained for endogenous c-Myc (anti-N262).
(F) C2C12 cells grown in the presence of GM or DM for 5 days and stained with anti-acetylated a-tubulin.
(G) Western blotting for Myc in rhabdomyosarcoma cell lines grown as dense or sparse cultures for 3 days.
(H and I) Human myoblasts expressing vector, or Myc-nick were cultured in DM and processed for immunoblotting after 4 days.
(J) Human myoblasts expressing vector or Myc-nick were cultured in DM and photographed after 2 days.

488 Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc.

sufficient to induce a-tubulin acetylation, but no synergy with
Myc was detected, most likely because association between
these two proteins occurs outside of GCN50 s active site (Fig-
ure 6L). As a further control, we tested p300, a HAT that binds
the C terminus of Myc (Faiola et al., 2005). p300 neither induced
tubulin acetylation nor did it synergize with Myc (Figures 6J and
6K, bottom panels). These data indicate that GCN5 can specifi-
cally acetylate tubulin and that Myc augments tubulin acetylation
by binding to tubulin and recruiting GCN5.
Myc-Nick Is Produced in Differentiating Cells
and Tissues
When examining the expression of Myc-nick in adult mouse
tissues, we found that brain, cerebellum, and skeletal muscle
express significantly higher levels of Myc-nick than any other
tissue (Figure 7A, Figure S7A). Interestingly, both neuronal and
muscle differentiation require major cytoskeletal rearrangements
that have been associated with increased microtubule stability
and elevated levels of acetylated a-tubulin. For example, it has
been demonstrated that the acetylation of a-tubulin by the
Elp3 acetyltransferase is required for proper cortical neuronal
migration and differentiation (Creppe et al., 2009). Increased
levels of acetylated a-tubulin are found during myogenic differ-
entiation (Gundersen et al., 1989). Importantly, inhibiting the
activity of the deacetylases HDAC6 and Sirt2 (known to deace-
tylate a-tubulin) generates augmented levels of acetylated
a-tubulin and promotes differentiation of myoblasts (Iezzi et al.,
2004). Furthermore, the presence of the primary cilium, a micro-
tubule-based antenna-like structure composed of acetylated
a-tubulin, is essential for cardiomyocyte differentiation (Clement
et al., 2009).
The extensive cytoskeletal changes that occur during muscle
differentiation are regulated by calcium and calpains (Dedieu
et al., 2004). The inhibition of calpain activity either by pharmaco-
logical inhibitors or by overexpression of Calpastatin (an endog-
enous inhibitor of calpains) blocks muscle cell differentiation
(Dedieu et al., 2004). In addition, both types of ubiquitous
calpains (m and m) were shown to regulate muscle cell differen-
tiation in vitro (Moyen et al., 2004). Moreover, mutations in the
calpain 3 gene (the muscle-specific calpain) cause limb girdle
muscle dystrophy 2A (LGMD2A) (Richard et al., 1995). Finally,
knocking out calpain r (the regulatory subunit shared by all
calcium-dependent calpains) is lethal due to impaired cardiovas-
cular development (Arthur et al., 2000).
To study the cleavage of Myc to Myc-nick during the process
of muscle differentiation, we employed human primary myo-
blasts purified from pectoral girdle, mouse myoblasts purified
from hindlimb muscles, and C2C12 cultured mouse myoblasts.
When stimulated to differentiate, these cells displayed low
levels of full-length c-Myc and high levels of Myc-nick when
compared to undifferentiated cycling cells (Figures 7B and 7D;
Figure S7B). Interestingly, Myc-nick levels were higher in a rhab-
domyosarcoma cell line from the alveolar subtype (Figure 7G),
a very aggressive tumor derived from partially differentiated
muscle cells.
The increased levels of Myc-nick in differentiated primary
mouse myoblasts correlate with an increase in calpain 3 levels
(Figure 7B; Figure S7B) and in total calpain activity (Figure 7C).
Similarly, when C2C12 cells were stimulated to differentiate,
they also displayed an increase in total calpain activity (Fig-
ure S7C) and in the ability to cleave Myc in vitro (Figure S7D).
Importantly, the levels of acetylated a-tubulin were also elevated
during the process of differentiation (Figure 7B; Figures S7B and
S7F). Acetylation of a-tubulin is accompanied by myoblast fusion
to form multinucleated myotubes (Figure 7F; Figure S7F). When
myoblasts are stimulated to differentiate they fuse into multinu-
cleated myotubes. In addition to displaying increased levels of
acetylated a-tubulin, these myotubes show decreased immu-
nostaining for nuclear Myc and increased cytoplasmic Myc
staining (Figure 7E). Conversely, undifferentiated satellite cells
in the same culture display nuclear staining for Myc (Figure S7E).
This is consistent with our results showing conversion of full-
length Myc into predominantly cytoplasmic Myc-nick.
In C2C12 cells, concomitant with increased Myc-nick abun-
dance, we observed a decrease in Myc-nick phosphorylation
at threonine 58 (T58) after the switch to differentiation conditions
(Figure 7D). Phospho-T58 mediates Fbw7-dependent degrada-
tion of Myc by the proteasome. Decreased phosphorylation at
this site is consistent with the notion that stabilization of Myc-
nick contributes to its elevated levels. Interestingly we found
that Myc-nick phosphorylation at T58 is also reduced in adult
muscle, brain, and cerebellum (Figure S7A and data not shown).
In summary, we found that during muscle differentiation there
is an increase in Myc-nick, concomitant with an elevation in cal-
pain activity and tubulin acetylation (Figures 7B–7D; Figure 7F;
Figures S7B–7D; Figure S7F).
Myc-Nick Accelerates Muscle Differentiation
We examined the effects of ectopic expression of Myc-nick* in
human primary myoblasts isolated from the pectoral girdle
(Figure 7H) and rectus abdominus (Figures 7I–7J), from the
human rhabdomyosarcoma cell line RD (Figure 7K), and in
C2C12 cells (Figures 7L and 7M). In all cells we found that
Myc-nick* expression accelerated muscle cell differentiation,
augmented a-tubulin acetylation, and elevated expression of
muscle-specific proteins (Figures 7H–7M).

(K) RD rhabdomyosarcoma cells expressing vector, Myc-nick, or Myc-nick DMBII (D106–143) were grown in DM for 4 days and processed for immunobloting.
(L) C2C12 cells expressing vector or Myc-nick were grown as dense cultures and stimulated to differentiate for 4 days and then photographed.
(M) C2C12 cells expressing vector or Myc-nick were grown to confluency and harvested or stimulated to differentiate for 3 days and then harvested. Total cell
lysates were immunoblotted with antibodies against the indicated proteins.
(N) Rat1 (myc+/+) cells expressing MyoD or MyoD + Myc-nick were cultured in DM for 3 days and photographed (lower panels) or stained for Troponin C and DAPI
(upper panels).
(O) Rat1 (myc+/+) or Rat1 myc null (myc/) cells expressing vector (lane C) or MyoD were cultured in GM, DM, or serum-free medium (lane S) for 4 days and
processed for immunobloting.
(P) Rat myc null (myc/) cells expressing Myc-nick, MyoD, or MyoD + Myc-nick were grown in GM or DM for 3 days and processed for immunoblotting.
See also Figure S7.

Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc. 489

 The expression of Myc-nick* in C2C12 myoblasts promoted
an increase in cell fusion not only in confluent cultures (Fig-
ure 7L; Figure S7H) but also in sparse cultures (Figure S7G).
The role of Myc-nick in promoting muscle cell differentiation is
partially dependent on MBII, as the differentiation of RD and
C2C12 cells by Myc-nick can be delayed but not inhibited by
the deletion of MBII (Figure 7K; Figure S7I).
To examine the requirement for Myc-nick production during
differentiation further, we first tested calpain inhibitor XII and
found that it reduces differentiation and tubulin acetylation
(Figure S7J). Next we employed c-Myc mutant (D291–300),
which is deficient in cleavage to Myc-nick. Similar to full-length
WT c-Myc, this mutant can induce proliferation, apoptosis,
fibrillarin expression, and phosphorylated H2AX in Rat1 myc
null cells (Figures S7L–S7M). However the D291–300 c-Myc
mutant significantly reduces C2C12 cell differentiation when
compared to c-Myc, even though the proteins are expressed
at equal levels. Whereas full-length c-Myc only blocks the
fusion of C2C12 myoblasts, the c-Myc mutant (D291–300) with
reduced ability to generate Myc-nick dramatically reduces the
expression of muscle markers in addition to blocking fusion
(Figures S7N–S7O).
Myc-Nick Renders Rat1 myc Null Cells Competent
to Differentiate
MyoD has been long known to induce transdifferentiation in
diverse cell types. We found that MyoD induces expression of
muscle-specific markers in Rat1 fibroblasts but not in Rat1
myc null fibroblasts (Figure 7O), indicating that myc is necessary
for the transdifferentiation process. Expressing Myc-nick
together with MyoD in myc+/+ Rat1 fibroblasts further elevated
the levels of muscle-specific markers and promoted cell fusion
compared to MyoD alone (Figure 7N). Importantly, we find that
in Rat1 myc null cells, the coexpression of Myc-nick (Figure S7K)
permitted transdifferentiation to muscle in response to MyoD
(Figure 7P). This experiment indicates that Myc-nick is sufficient
for MyoD-induced transdifferentiation, supporting the idea that
conversion of full-length Myc to Myc-nick is important in
MyoD-induced differentiation.
DISCUSSION
Recent studies have demonstrated that Myc directly regulates
transcriptional activation through the three RNA polymerases,
as well as transcriptional repression, and DNA replication (Eilers
and Eisenman, 2008). Here we identified and characterized Myc-
nick, a novel form of Myc performing transcription-independent
functions in the cytoplasm. One of these functions is to regulate
a-tubulin acetylation in cooperation with the HAT GCN5. More-
over, Myc-nick levels are elevated in differentiated muscle
tissues and Myc-nick overexpression accelerates muscle cell
differentiation.
Despite its widespread expression, Myc-nick has not been
previously characterized. There are several likely reasons for this.
First, the most commonly used antibodies against Myc (such as
9E10) recognize its C terminus and would not detect Myc-nick.
Second, the proportion of Myc-nick to Myc increases when
cells are grown as confluent cultures, conditions that are not
490 Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc.
commonly employed when studying Myc proteins. Third, most
studies use total protein or nuclear extracts and have not analyzed
the cytoplasmic pool of Myc. Fourth, Myc-nick may have been
mistaken for the similarly sized MycS protein (Hann et al., 1988),
a nuclear localized product of internal translation initiation.
Calpain Cleavage as a Posttranslational Functional
Switch
We found that Myc-nick is generated in the cytoplasm by
calpain-mediated cleavage of full-length Myc. Cytoplasmic
cleavage by calpains has been reported for over 100 proteins
including many cytoskeletal proteins, membrane receptors,
and transcription factors (Tompa et al., 2004). This number is
probably an underestimate because the lack of a clearly defined
consensus cleavage site for calpains makes the identification of
novel calpain substrates difficult. For most substrates the role of
calpain cleavage is not known. However, there are several note-
worthy examples of calpain cleavage operating as a functional
switch (Abe and Takeichi, 2007; Yousefi et al., 2006). Here we
have shown that the cleavage of Myc by calpain converts this
predominantly nuclear transcription factor into Myc-nick, a cyto-
solic factor that regulates a-tubulin acetylation. Based on our
findings and the examples in the literature, we surmise that the
partial proteolytic cleavage of proteins by calpains functions as
an irreversible posttranslational modification.
A Role for Myc-Nick in a-Tubulin Acetylation
We have shown that Myc-nick mediates the acetylation of
a-tubulin by forming a complex with microtubules and the HAT
GCN5. GCN5 has been long known to associate with nuclear
Myc through the highly conserved Myc box II, a region retained
in Myc-nick. Although tubulin acetylation was first described
20 years ago, little is known about the enzymes that catalyze
this reaction. Recently Elp3, a histone acetyltransferase, was
demonstrated to be critical in acetylation of a-tubulin in cortical
projection neurons, an event linked to neural differentiation and
migration (Creppe et al., 2009). We surmise that GCN5 repre-
sents another acetyltransferase targeting tubulin. Deacetylation
of a-tubulin is mediated by both HDAC6 and Sirt2 (Hubbert
et al., 2002; Matsuyama et al., 2002; North et al., 2003). However
the increase in acetylated a-tubulin by Myc-nick does not occur
through modulation of HDAC activity (Figures S6E and S6F).
A connection between a-tubulin acetylation and calpain acti-
vation was previously suggested by two independent studies.
First, calcium depletion, which inactivates calpains, was shown
to decrease the levels of acetylated a-tubulin in epithelial cells
(Ivanov et al., 2006). Second, the ectopic expression of calpain
6 causes microtubule stabilization, elevates the levels of acety-
lated a-tubulin, and impairs cytokinesis in HeLa cells (Tonami
et al., 2007). Our findings suggest that the induction of a-tubulin
acetylation by calpains could be mediated at least in part by
Myc-nick and GCN5.
The Role of Myc-Nick in Terminal Differentiation
The role of Myc family members in differentiation is complex.
Endogenous Myc is strongly downregulated during terminal
differentiation of many cell types. Moreover, ectopic expression
of Myc has been shown to block terminal differentiation. The
ability of Myc to negatively regulate differentiation is consistent
with its role in maintaining pluripotency in ES cells and in the
generation of induced pluripotent stem (iPS) cells (Cartwright
et al., 2005; Takahashi et al., 2007). However, Myc has also
been implicated in promoting both proliferation and differentia-
tion in specific cellular contexts such as in progenitor cells of
the skin (Gandarillas and Watt, 1997; Gebhardt et al., 2006), B
lymphocytes (Habib et al., 2007), and hematopoietic stem cells
(Wilson et al., 2004). Our data suggest that whereas full-length
Myc blocks differentiation at the transcriptional level, Myc-nick
may be involved in promoting differentiation through transcrip-
tion-independent mechanisms. In agreement with this hypoth-
esis there have been numerous reports of Myc antigenicity
localized predominantly in the cytoplasm of differentiated cells
(see Introduction). In addition, we show that ectopic expression
of Myc-nick accelerates muscle cell differentiation and renders
Rat1 myc null cells competent to differentiate into muscle
following introduction of MyoD.
A number of studies have shown a strong correlation between
muscle cell differentiation, calcium influx, and calpain activation
(Dedieu et al., 2004; Kumar et al., 1992). In addition, limb girdle
muscular dystrophy 2A (LGMD2A) is caused by mutations in
calpain 3 that affect its activity (Richard et al., 1995). During
the process of normal differentiation, myoblasts elongate and
fuse into syncytial myotubes. An early event during this process
is the remodeling of the microtubule cytoskeleton, involving
disassembly of the centrosome and the alignment of stable
microtubules into a parallel array along the long axis of the cell.
We observed an increase in the levels of acetylated a-tubulin
(an indicator of microtubule stabilization and cytoskeletal reorga-
nization) and an elevation in Myc-nick abundance during muscle
cell differentiation. Induction of a-tubulin acetylation and micro-
tubule stabilization are likely to be important events in terminal
differentiation especially when cells must establish and maintain
a new shape. Acetylation of a-tubulin was also demonstrated to
regulate cortical neuron differentiation and migration (Creppe
et al., 2009).
We propose a model for the regulation of Myc by calpains and
for the function of Myc-nick. During proliferation, full-length Myc
is rapidly synthesized and transported to the nucleus where it
transcriptionally activates growth- and proliferation-related
genes and represses genes involved in differentiation. External
cues that stimulate differentiation, such as cell-cell contact,
and calcium influx can lead to the activation of calpains. Acti-
vated calpains interact with and may retain Myc in the cytoplasm
where it is cleaved to produce Myc-nick. As the cleavage
removes the nuclear localization sequence (NLS) and the DNA-
and Max-binding domains, Myc-nick is predominantly cyto-
plasmic and transcriptionally inactive. Indeed we have shown
that Myc-nick, when expressed in cells devoid of full-length
Myc, does not stimulate proliferation, growth, or apoptosis—
the transcription-dependent functions of full-length Myc. In
such Myc-nick-expressing cells we instead observe morpholog-
ical changes and increased tubulin acetylation, which are unique
to Myc-nick. During muscle differentiation Myc-nick appears to
be stabilized by dephosphorylation of threonine 58 within the
Myc box I phosphodegron. We propose that one of the functions
carried out by Myc-nick in the cytoplasm involves binding tubulin
and recruiting GCN5 to mediate a-tubulin acetylation. Myc-nick
is also likely to have additional functions and partners in the cyto-
plasm as the deletion of MBII in Myc-nick, which should block
the interaction with GCN5, only partially reduced the ability of
Myc-nick to promote muscle differentiation. We predict that
the functions carried out by Myc-nick in the cytoplasm coop-
erate to promote cytoskeletal changes that can further drive
terminal differentiation. In our model, the cleavage of Myc by
calpains has two roles. First, it helps diminish nuclear Myc abun-
dance preventing newly synthesized Myc from entering the
nucleus, therefore eliminating the transcriptional blockade to
differentiation caused by Myc. Second, the production of Myc-
nick influences cytoskeletal organization and facilitates terminal
differentiation. We predict that Myc-nick will play a general role
linking myc to both nuclear functions and cytoplasmic organiza-
tion during differentiation of a wide variety of cell types.
EXPERIMENTAL PROCEDURES
Retroviral Infection and Transfection
For retroviral production, 293T cells were cotransfected with pBabe-puro
vector expressing Myc clones and amphotropic helper. Infected cells were
selected with 2 mg/ml puromycin for 4 days. Rat1 myc null cells were harvested
10–14 days after infection and C2C12 cells were stimulated to differentiate
after selection. For overexpression experiments, 293T (transfected with
Fugene-Roche) cells were harvested 3 days after transfection with a change
in culture medium 24 hr before harvesting. Sparse cultures (S) are about
20% confluent, medium cultures (M) are about 40%–60% confluent, and
dense cultures (D) are grown as confluent cultures for 2–4 days.
Total Cell Lysates and Nuclear and Cytoplasmic Fractionation
For total extracts, cells were lysed in either boiling sample buffer or in RIPA
buffer. For cellular fractionation, cells were lysed in buffer A on ice for 20 min,
centrifuged for 3 min, and the supernatant employed as the cytoplasmic frac-
tion. The pellet was resuspended in buffer B, rotated for 20 min, sonicated, and
centrifuged for 10 min. The supernatant was employed as the nuclear fraction.
Ten to twenty micrograms of total extracts or 10 mg of nuclear and 30 mg of cyto-
plasmic extracts were probed overnight with indicated antibodies. See
Extended Experimental Procedures for composition of buffers.
Immunofluorescence
Cells were grown on glass coverslips and fixed with 4% paraformaldehyde
for 20 min, permeabilized with 0.5% Triton X-100 for 5 min, and blocked
with Image IT FX signal enhancer (Invitrogen) for 30 min. Primary and
secondary antibodies were diluted in PBS at 1:200 and incubated for 1 hr.
In Vitro Cleavage of Myc
All cDNAs were cloned into pCS2+ and were transcribed from the SP6
promoter using the Promega wheat germ system, in the presence of cold
(Promega) or 35[S]-labeled methionine (Perkin Elmer) according to the manu-
facturer’s instructions. For the in vitro cleavage experiments, Myc (1 ml IVT
Myc or 1 mg recombinant Myc) was incubated with 30 mg of nuclear or cyto-
plasmic extracts in 20 ml of buffer G at 37 C and the samples were processed
for autoradiography or western blot as specified. Nuclear and cytoplasmic
fractions were dissolved in the same buffer and adjusted to identical salt
concentration for these experiments. For Myc cleavage using recombinant
calpains, 1 ml IVT c-Myc or 0.25 mg of purified c-Myc was incubated with the
indicated amounts of calpains in the presence of 20 ml buffer G. Total calpain
activity was measured using a Calbiochem kit.
In Vitro Tubulin Acetylation Assays
CE (30 mg) or purified assembled microtubules (1 mg) were incubated with 1 ml
of IVT c-Myc or IVT vector or with recombinant Myc for 1 hr in the presence of
Cell 142, 480–493, August 6, 2010 ª2010 Elsevier Inc. 491
acetylation buffer. Assembled microtubules were incubated at 30 C and CE
at 37 C.
See Extended Experimental Procedures for buffer composition, constructs,
siRNA sequence, pharmacological inhibitors, antibodies, cell lines, and culture
media.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures and
seven figures and can be found with this article online at doi:10.1016/j.cell.
2010.06.037.
ACKNOWLEDGMENTS
We are grateful to Nao Ikegaki, William Tansey, John Sedivy, and Peter Hurlin
for reagents essential for this work. We also thank Stephen Tapscott, Maura
Parker, Hector Rincon, Bill Carter, Linda Wordeman, and members of the
Eisenman lab for advice and discussion. M.C.-S. was a recipient of an
EMBO Long-Term Fellowship. This work was supported by NIH/NCI grant
CA20525 (R.N.E.).
Received: November 9, 2009
Revised: March 2, 2010
Accepted: May 18, 2010
Published: August 5, 2010
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 Erratum

SIRT1 Suppresses b-Amyloid Production

by Activating the a-Secretase Gene ADAM10
Gizem Donmez, Diana Wang, Dena E. Cohen, and Leonard Guarente*
*Correspondence: leng@mit.edu
DOI 10.1016/j.cell.2010.07.034

(Cell 142, 320–332; July 23, 2010)

As a result of an error during figure preparation, the model in Figure 6D was omitted. Additionally, in Figure S1B, Tau-pSer399 was
incorrectly typed. This has been corrected to Tau-pSer396 both in the figure and in the text. Corrected versions of both figures are
shown below.
These errors in no way affect the conclusions of the paper, and the authors apologize for any confusion caused by these errors.
The online version of the article has been corrected.

Figure 6. SIRT1 Deacetylates RARb

494 Cell 142, 494–495, August 6, 2010 ª2010 Elsevier Inc.

Figure S1. Related to Figures 1 and 2

Cell 142, 494–495, August 6, 2010 ª2010 Elsevier Inc. 495

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Michael R. Lieber1 and Thomas E. Wilson2
1
University of Southern California, Los Angeles, CA 90089, USA and 2University of Michigan, Ann Arbor, MI 48109, USA

496 Cell 142, August 6, 2010 ©2010 Elsevier Inc. DOI 10.1016/j.cell.2010.07.035 See online version for legend and references.
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CAMK
PDK

VRK3
STK32A (YANK1) STK32C (YANK3) MASTL

TTBK1

2)
TTBK2
(AD

(CLIK1L)

KK2
CI

FLJ3268(FUSED)
ma)

STK36
gamma 3)
CAB

MAST3

ULK4
gam

(CK1 gamma

AAK1 (BIKE)
(SGK494)

BMP2K

5 (NEK10)

ULK3
alph ) (PKA

AUR
(PKA beta CG

CSNK 2A2
VRK1

KA
VRK2

CSNK
ACA (PKA PRKA

FLJ25006

TLK2
LATS2
LATS1
DK

(AUR
TLK1
)

PKN3

PLK4
2A1 (CK2
HK4

1)
a)

MAST1
BCK

ULK1
CIT (CRIK)

AUR KB (AUR
4 (PD

ULK2

A)
(CK2 alpha
STK32B (YANK2)

AUR
KC
PDK

PRK PRKACB

(AUR B)
alpha 2)

PLK1

C)
NEK6
NEK7

1)
PKN2

ADRBK1 (GRK3, BARK2)

(GPRK7)(GRK2, BARK1)
PKN1

MAST2
MAST4

NEK2
A6 (RSK4)
SGK2

PLK3 (SNK
PLK2
I (PKC zeta)

NEK9
iota)

NEK8

(FNK )
GRK1 (RHOK)
RPS6K

ADRBK2

GRK6 (GPRK6)
PRKC (PKC

a)

)
gamm
Z

SGK3
PRKC

AKT3 SGK

2 (P70S6 1 (p70S6(RSk1)

GRK7

STK38 (NDR1)
AKT1 (PKB beta) (PKB

STK38L (NDR2)

NEK11
K beta) K)
RPS6K A5 (MSK1)
A4 (MSK2)
A1
delta )
PRKC E (PKC (PKC eta) D (PKC theta

RPS6K
)

alpha)

GRK5 (GPRK5)
GRK4 (GPRK4)
of a kinase binding assay

ROCK1
RPS6KB RPS6KB

RPS6K

ROCK2
RPS6K A2 (RSK3)
(PKC

A3 (RSK2)
(PKB
PRKC Q

NEK4
DMPK (DMPK1)
PRKC

AKT2

RPS6K
gamm on)

NEK3
a)
epsil
H
PRKC PRKC

beta) G (PKC

CDC42BPG (DMPK2)

NEK1
NEK5
)
alpha
PRKC B (PKC
A (PKC
PRKC

CDC42BPB (MRCK beta)

CDC42BPA (MRCK alpha)
Solutions for advancing your workflow from challenging
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