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Leading Edge

Select
Targeting Resistance
Wrestling with cancer can be frustrating. Despite the progress in developing therapies that can effectively control tumor
growth, the devil almost always strikes back with resistance.
Even for the recent excitement in using immunotherapy to
achieve unprecedented success in some cancer patients,
resistance has been seen in clinical settings and is under
active investigation (Restifo et al., 2016).
How do we tackle resistance to cancer therapy? One effective approach is to nail the culpritpinpointing the cell population intrinsically insensitive to the treatment and targeting
their vulnerability. Recent work from Tessa L. Holyoake and
her team successfully applied this strategy on chronic
myeloid leukemia (CML; Abraham et al., 2016). CML is characterized by the aberrant activation of ABL1 tyrosine kinase
due to chromosome translocation, and tyrosine kinase inhibitors (TKIs) have been the standard treatment with clinical
efficacy. However, patients with CML eventually relapse
because the survival of leukemic stem cells (LSCs) does
not rely on the elevated kinase activity and therefore cannot
be eradicated by TKIs. Through integrated analyses, the
team exposed the essential role of p53 and c-MYC on the
CML network and an addictive dependency of LSCs on these
two signaling hubs. They further showed that a combinatory
treatment targeting p53 and c-MYC could effectively kill
LSCs, raising the hope of using this approach for treating
CML patients relapsing from TKIs (Abraham et al., 2016).
Leukemia is not the only type of cancer in which targeting
intrinsic resistance from a specific population is starting to
show promise. Indeed, by developing a mouse model with
knockin reporters, Tannishtha Reya and colleagues were
able to identify high expression of the stem cell determinant
Musashi (Msi) as a marker for populations in pancreatic cancer with strong tumor-initiating capacity and conferring drug
resistance. Inhibiting Msi significantly changed the trajectory
of disease progression and almost doubled the survival time
in mouse models. Moreover, simultaneously inhibiting two of

its potential direct targets was effective in killing tumor cells


resistant to gemcitabine, a FDA-approved chemotherapy
drug for treating pancreatic cancer. To further establish Msi
as a valuable target for drug development, the authors
went on to develop antisense oligonucleotides specifically
targeting MSI1, which successfully inhibited tumor growth
in patient-derived xenograft (PDX) models (Fox et al., 2016).
While drug resistance can be an intrinsic feature of a particular population within the tumor before the drug is even
applied, it can also arise as an adaptive response to the treatment itself. Aiming to better understand this adaptation and to
explore therapeutic opportunities, Scott Lowe and his team
employed a systematic approach to screen for factors that
could sensitize KRAS mutant lung cancer cells to trametinib,
an FDA-approved drug targeting the downstream effector
signaling of mutant KRAS. They found that activation of the
FGFR pathway underlies the resistance of tumor cells to trametinib. Consistently, inhibiting FGFR1 with either shRNAs or
ponatinib that has FGFR1 as one of its targets achieved synthetic lethality with trametinib in treating KRAS mutant lung
cancer cells. Despite the profound efficacy in tumor suppression, this combinatory approach showed minimal, if any,
toxicities in various in vivo models, including PDX, strongly
supporting its therapeutic potential (Manchado et al., 2016).
Strategies narrowing down specific components in the
signaling pathways conferring intrinsic or adaptive resistance
represent a major direction of effort for targeting resistance to
therapy. However, its not the only way. As reported by Cerezo
et al., rather than targeting a particular oncogenic driver in the
context of melanoma resistant to BRAF inhibitors, using compounds to induce ER stress was proven effective in eliminating
resistant cancer cells by promoting apoptosis and autophagy.
Remarkably, this approach did not seem to affect normal melanocytes or fibroblasts, indicating a rather attractive therapeutic window for future development (Cerezo et al., 2016).
By definition, resistance is hard to eradicate. Nonetheless,
in realizing what we have achieved along the way in this
marathon of finding a cure for cancer, we have every reason
to believe that we are heading in the right direction.
REFERENCES
Abraham, S.A., Hopcroft, L.E., Carrick, E., Drotar, M.E., Dunn, K.,
Williamson, A.J., Korfi, K., Baquero, P., Park, L.E., Scott, M.T., et al. (2016).
Nature 534, 341346.
Cerezo, M., Lehraiki, A., Millet, A., Rouaud, F., Plaisant, M., Jaune, E.,
Botton, T., Ronco, C., Abbe, P., Amdouni, H., et al. (2016). Cancer Cell 29,
805819.
Fox, R.G., Lytle, N.K., Jaquish, D.V., Park, F.D., Ito, T., Bajaj, J.,
Koechlein, C.S., Zimdahl, B., Yano, M., Kopp, J.L., et al. (2016). Nature 534,
407411.
Manchado, E., Weissmueller, S., Morris, J.P., Chen, C.C., Wullenkord, R.,
Lujambio, A., de Stanchina, E., Poirier, J.T., Gainor, J.F., Corcoran, R.B.,
et al. (2016). Nature 534, 647651.
Restifo, N.P., Smyth, M.J., and Snyder, A. (2016). Nat. Rev. Cancer 16,
121126.

Expression of the stem cell gene Musashi (red) in human pancreatic


cancer (cancer cells: green). Image courtesy of Dawn Jaquish.

Jiaying Tan
Cell 166, July 28, 2016 2016 Published by Elsevier Inc. 523

Leading Edge

Conversations
Brain Exploration, Off the Beaten Path
Model organisms, such as rodents, monkeys, or Drosophila, have driven much of recent research
in neuroscience. However, studies in other, more unusual systems have broadened the types
of questions that are being asked and have revealed the diverse ways in which species tackle common problems. Cell editor Mirna Kvajo talked with Nachum Ulanovsky, Gilles Laurent, and Anthony
Leonardo about their research and how studying bats, reptiles, and dragonflies informs big questions in neuroscience. An annotated excerpt of the conversation appears below, and the full conversation is available with the article online.

Anthony Leonardo
Janelia Research Campus

Nachum Ulanovsky
Weizmann Institute of
Science

Mirna Kvajo: It seems that a lot if not most of research in


neuroscience is being done in a couple of model organisms;
we hear about mice, we hear about rats, the Drosophila, and
then also monkeys. Most of these organisms are used to
address a broad spectrum of questions, and something that is
happening now is a raised awareness about how many of
these questions we can actually ask [in these traditional
systems]. And it seems that theres a surge of interest in
alternative model organisms.
The three of you are using something that people could call
alternative organisms, right? Youre working on bats, the
dragonfly, reptiles. Just to start off, I wanted to understand
what are your reasons for picking these organisms? What
kind of questions are you asking, and are some of these
questions such that cant be asked in other types of
organisms?

Many of the tools that we use


now actually come from the
study of unusual systems.
GFP, the channelrodopsins,
and CRISPR/Cas9.

Gilles Laurent
MPI for Brain Research

Anthony Leonardo: Ill speak up first. We study prediction


in dragonflies and how they anticipate where prey is going
and use this to construct a flight path. That process requires
internal models of how the body works and how the prey
moves. And the reason weve been studying prediction there
is largely because the problem the animal solves is very clear
when you look at the behavior . In the case of our system,
this sort of prey capture is not unlike reaching out your arm to
grab something, so its really a ubiquitous behavior and you
could study it in any one of a number of systems. But because
of how these animals do the behavior: its easy to elicit and
they do it with a certain amount of complexity, and they have
to solve it with certain accuracy, all those things conspire to
make the system much easier to understand than in other
places. Thats been the reason for me: its not that its the only
place to study it, but we think its the cleanest, clearest place,
and then we can take what weve learned there and apply it to
other systems.
Nachum Ulanovsky: Maybe I could continue on that. I think
this idea some call Kroghs Principle. The notion that for every
problem or every question in biology there are some organisms
that are particularly useful to address it. It could be because
their behavior is very precise. In our case of the bats, we are
studying place cells, grid cells, head-direction cellsthe
spatial system. The one reason is indeed that, on one hand, the
bat is a mammal, so the anatomy of its hippocampal system is
very similar to rodents, so we have that constraint. On the other
Cell 166, July 28, 2016 2016 Published by Elsevier Inc. 525

(L to R) Mirna Kvajo, Nachum Ulanovsky, Gilles Laurent, and Anthony Leonardo

hand, there are certain questions that are difficult to ask in


rodents, but theyre more approachable in bats. For example,
the representation of 3D space or representation of very large
spaces because they fly long distances .
There is another component to that, or another reason to
study non-standard animals, and this is the comparative
approach. Contrast and compare, so that what we find [in bats]
is that a lot of the things are very similar [to rats]; we find place
cells, grid cells, head-direction cells. But there are certain
things that are very different, so, for example, the theta
oscillations are very prominent in the [rat] hippocampal system,
and we dont see that in the bat. This means that those theories
of grid cells that rely in an obligatory manner on a perfect

The problem in the funding


situation is us; its driven by us,
and this has reached a point
that to me is quite dramatic.
526 Cell 166, July 28, 2016

oscillation, this argues against them. This comparative


approach is very powerful and used to be prevalent in
neuroscience, and unfortunately it disappeared. But I agree
with you, I feel it has a little bit of a comeback recently.
Gilles Laurent: I think that Nachum and Anthony have
summarized things really well. Forty, thirty years ago people
used what they called model systems, and it was a common
thing that youd go to Neuroscience [Society for Neuroscience
meeting] and people would work on Tritonia and crabs and
bats and barn owls and so on, and little by little all this has
disappeared, and as you guys were saying now, youre trying
to force all these things back onto one species and for
sometimes good reasons, but not always. I think all of
us agree on the danger of this trend, which means that,
practically, a population of scientists able to tackle interesting
problems on a variety of species, to take into account the
diversity of the animal world, of evolution, of comparisons and
their value its going to disappear as a culture, and thats
really dangerous.
MK: Im curious, Im sure that all of you must have
challenges, and you must be envious of people who are using
well-standardized and well-understood models which have a
lot of tools, and especially now in this age of tool making, you
must feel like, OK, I wish I could do this in my model. How do

You see a behavior outdoors


and start asking, How is that
implemented? And you can
eventually bring it to a
laboratory setting and do a
controlled experiment.
you think about this? Do you think that, for your particular
models, or just in general, theres going to be an age of tools,
or are you adjusting your questions to what you can ask?
NU: I think there will be an age of tools for sure. I think that,
often, people driven to these exotic or unusual systems are
inherently tool builders to some extent. When you pick one of
these unusual organisms, youre picking it because its a
question-driven enterprise and that leads very naturally to
saying, What is the tool that I need to answer this question,
and can I develop it here? And certainly in our work, we are
very inspired by our colleagues in genetic systems and wed
like to try to develop versions of those tools that we can
apply even for our very localized problems, so theyre not of
ubiquitous use but they solve our problems. So, I think that will
come as its needed; theres no fundamental impediments.
Its a question of time, effort, and funding, but it can certainly
be done.
GL: I was going to say that the three of us dont work in the
purely grant-driven American system. I think that the funding
issue is a fundamental one now. Those, the few of us who work
on unusual systems, tend to work in systems that allow the
funding and provide the funding to do that, and its becoming
less and less possible. And when you talk to your colleagues,
they say, Well the funding situation doesnt allow it. The
problem in the funding situation is us; its driven by us, and this
has reached a point that to me is quite dramatic. We dont even
have the confidence in pushing for that diversity.
NU: On one hand I agree, and on the other hand I also have
colleagues in the U.S. who study unusual animals. And those of
them who ask good questions, its clear that they can get
funding. I dont think its as tough . Its tough maybe, but its
possible, for sure. But, to address your question about tools, so
yeah, when you study an unusual animal, you have to develop
tools almost by definition because nobody will do it for you.
Sometimes this is an unusual toollike in our case for the bats,
we want to study them freely flying, so we have to develop
methods to record wirelessly from single neurons in flight, etc.
So, these are the kinds of tools we have to develop that dont
exist elsewhere in the world. But often times when were talking
about tools in neuroscience nowadays, its molecular tools, and

we need genomes and all these things. I think . with the advent
of genome editing, it might become a bit less of an issue.
AL: I started working on an unusual non-genetic system in an
era right when genetic systems were really exploding, and it
was clear that it was tactically not the wisest decision in terms
of certain aspects. And the thing that always sort of struck me is
that the genetic access to these sorts of weirdo systems is only
going to get easier over time, whereas the computations the
animals do and the behaviors they do are fixed. So its not that
mice and flies are going to evolve new behaviors suddenly that
youre going to be able to study in them. So there is a real
reason and a utility in saying, OK, this organism is solving this
computation, and this is a good place to study it, and were
going to work on it at the level of tools we have now, and
gradually more tools will become available and well gain
deeper levels of understanding it. As opposed to forcing that
problem into a genetic system where its very hard to study and
you make progress very slowly, even with the elegance of the
tools there.
GL: You could also turn this around by saying that many of
the tools that we use now actually come from the study of
unusual systems like bioluminescence in jellyfishyou get GFP
and the channelrhodopsins and CRISPR/Cas9. That doesnt
come from directed research at the beginning; its really
curiosity driven. If we lose this, we lose a lot of these
advantages.
AL: Yeah, I think on that same token, it is interesting to notice
that a lot of the problems being studied at a deep mechanistic
level in our genetic systems are problems that were described
at the level of algorithms and principles in other systems
things that we used to study in Hoverflies and locusts and all
these sort of exotic creatures that are being tapped. They
provided essentially the foundation on which these more
mechanistic studies can be built in other systems. And that
really arises from the ubiquity of evolution and the fact that
these principles do transcend the system, and almost by
definition you should be able to look at these things in different
places, and the breadth and depth can combine effectively.
I want to add yet another component, another advantage of
maintaining this diversity and studying non-standard species:
the natural behaviors. I mean, you cannot study a laboratory rat
or laboratory mouse in the wild. You can study a wild rat or wild
mice, which in some of their behaviors are quite different than
the ones in the laboratory. Whereas these non-standard
organisms, they are literally wild animalsliterally, we capture
them from the wild. You can study them also outdoors, so
weve been studying the bats, GPS tracking them outdoors,
looking at their navigation, etc. I think this really opens your
thinking to asking different questions. You see a behavior
outdoors and start asking, How is that implemented? And
you can eventually bring it to a laboratory setting and do a
controlled experiment, but even being able to study the animal
out in the wild is something that you typically cannot do. Or
even if you can, its not done by most people on standard
laboratory animals.

Cell 166, July 28, 2016 527

Leading Edge

Voices
Big Questions in Evolution
Evolution of Cell Types and Tissues

From Chemistry to Communities

From Whether to How

Detlev Arendt

Nicole King

Sean B. Carroll

European Molecular Biology Laboratory

University of California, Berkeley, HHMI

University of WisconsinMadison, HHMI

Evolution refers to the historic unfolding


of organismal life on Earth, which already
intrigued Greek philosophers. It cannot be
studied directlyjust as the plot of a
theatre play cannot be inferred from
watching the last second. Instead; we
study the fossil record; also, synthetic
biology has increasing power to mimic
key steps in evolution. The comparative
approach, however, remains most informative. It infers ancient conditions from
the comparison of extant species. Classically, evolutionary biologists compare
tissues and organsduring development
and in adults. More recently, comparative
genomics allow tracking the increase in
protein complexity in divergent lineages.
Now, a new level of comparison has
emerged, linking organ, tissue, and
protein evolutionthe cell type. Cells are
the basic building blocks of life; once we
understand their evolutionary diversification into types, this will solve the secrets
of multicellular life. A combination of
whole-organism single-cell transcriptomics, proteomics, expression atlases, and
CRISPR-Cas9-based functional studies
in various organisms opens up exciting
new questions: What is the cell type
complement in one species as compared
to others? How is it specified and maintained? What are the cell type-specific
molecular machines, or modules, and
how did these diversify in evolution?
Answering such questions will allow us to
examine even more complex structurestissues and organs, ultimately learning
about their evolution as an emergent
property of their constituting cell types.

Evolution is our family story. It holds the


keys to explaining our ancestry, our
connections with other life forms, and our
eventual, unavoidable extinction. One of
the most exciting frontiers in evolutionary
biology concerns how it all beganthe
origin of life. From our modern vantage,
over 3 billion years after prebiotic
chemistry gave rise to life, how can we
reconstruct the first major evolutionary
transition? The most meaningful
advances have begun with an explicit
model that can be tested experimentally
using techniques from chemistry,
physics, and biology. These approaches
have uncovered plausible prebiotic
chemical pathways leading to ribonucleotides, lipids, and amino acids. The gap
between simple macromolecules and
cells is huge, making this one of the most
compelling intellectual playgrounds for
budding evolutionary biologists. Fast forward to today, as we slowly come to
terms with the outcomes of our 3+ billion
year evolutionary history. Far from existing in isolation, evolution has led nearly all
archaea, bacteria, and eukaryotes to form
obligate associations with communities of
other organisms. Eukaryotic genomes
have evolved through rampant gene exchange with bacteria, our metabolic
pathways are interconnected with those
of our resident bacteria, and this is to say
nothing about pathogens. Understanding
our deep history will require new
approaches that take into account coevolution writ large, the dynamic,
complex, and evolvable interactions
among communities of organisms.

After the debut of Darwins On the Origin


of Species (1859), the big question for
naturalists was whether the mechanism
he (and Alfred Wallace) proposednatural selectioncould explain the exquisite
adaptation of organisms to their environment, and the formation of new species.
More than 150 years later, evolutionary
biologists are still focused on these two
principal evolutionary phenomena. But
the questions have shifted from whether
to howhow do new adaptations arise,
and how does speciation occur?
Empowered by molecular genetics,
biochemistry, and developmental
biology, the focus has expanded from the
organismal scale to the molecular: How is
genetic variation generated and maintained? How do new protein activities,
protein complexes, or physical traits
evolve? Evolutionary biology is the midst
of what Dean and Thornton have dubbed
The Functional Synthesis (Nature
Reviews Genetics 8, 675688) that seeks
a functional understanding of the
mutational paths to new phenotypes.
Biologists are dissecting some of the very
same phenomena that gripped Darwin
and his contemporaries including mimicry
in butterflies, the radiation of Galapagos
finches, the diversity of vertebrate limbs,
and yes, even the giant horns of dung
beetles, as well as experimenting on
model organisms such as bacteria, yeast,
and fruit flies. They are revealing the kinds
of mutations that are necessary and
permissible to make the endless forms
that have been and are being evolved.

528 Cell 166, July 28, 2016 2016 Published by Elsevier Inc.

Developmental Biases in Evolution

Evolution with Foresight

Predictive Evolutionary Genomics

Patricia Wittkopp

Eugene Koonin

Leonid Kruglyak

University of Michigan

National Institutes of Health

UCLA, HHMI

Contemporary evolutionary biology has


been built upon a rich foundation of
theoretical models providing hypotheses
for how and why biodiversity came to be.
When most of these models were developed, little was known about the mechanisms of inheritance that connect one
generation to the next nor about how this
hereditary material directs the development of diverse lifeforms. With the basic
mechanisms of genetics and development now known, evolutionary biologists
have been able to ask how genetic
changes have altered development to
produce diverse phenotypes. These
studies have shown that changes in gene
expression as well as gene function have
contributed to phenotypic evolution and
have also revealed genetic connections
among traits that can influence the evolution of disparate traits. These observations motivate two current big questions
in evolutionary biology: How do existing
genetic and developmental systems
influence the origin of phenotypic
variation and thus shape evolutionary
change? and How can our knowledge
of genetic and molecular mechanism be
integrated into evolutionary theories to
produce more complete models of
evolution? Addressing these questions
will not only help uncover evolutionary
changes that occurred in the past but will
also improve our ability predict evolutionary changes most likely to occur in the
future. The potential to predict paths of
evolutionary change is especially exciting,
as it could be used to help combat
cancer, control disease outbreaks, and
develop bioengineering solutions to the
diverse challenges we face in our
changing world.

For me, perhaps, the most pressing


question in evolutionary biology is: are
evolutionary mechanisms evolvable?
More precisely, is it possible to demonstrate that certain molecular mechanisms
have evolved under specific selective
pressure for increased evolvability or
simply increased rate of a particular type
of evolutionary change? Traditionally,
existence of such dedicated mechanisms
and devices for evolution had been
anathema to theorists because evolution
has no foresight. Yet, I believe that
several such mechanisms have already
been discovered inadvertently, not at all
as a result of focused efforts. One of these
has become famous for its utility in
genome engineering: the CRISPR-Cas
systems of bacterial and archaeal adaptive immunity. CRISPR-Cas is an elaborate molecular machine for directed
change of microbial genomes that makes
the organism immune to a specific
pathogen. Clearly, this is an evolved
mechanism of (quasi)Lamarckian microevolution. The CRISPR-Cas example
shows that evolution has both memory of
past events and some foresight as new
encounters with the same pathogen are
predicted. Another strong case in point
are the gene transfer agents, defective
bacteriophages that, instead of the phage
genome, package random segments of
microbial DNA and transfer them by infecting other microbes. I believe that the
discovery of these dedicated, evolved
mechanisms of genome evolution refutes
the simplistic no foresight view and
calls for an amended evolutionary theoretical framework.

The millions of species on Earth span a


stunning range of phenotypic diversity:
Darwins endless forms most beautiful.
We know, in broad outline, that this
diversity arises from differences in
genomes. With todays DNA sequencing
technologies, it is relatively straightforward to sequence genomes and identify
the differences between them. Indeed,
this has been accomplished for thousands of species, and the list is growing
rapidly. Lagging a long way behind is our
ability to pinpoint which specific genome
sequences underlie each species unique
phenotypic features. To sharpen the
focus on this problem, its worth posing
two related questions. First, given a
genome sequence, what organism will it
produce? This question is readily
answered by the developmental program
of each species (together with the initial
conditions of the starting cell), but we
cannot answer it in a very specific way.
What collection of data, coupled with
what predictive algorithms, would allow
us to tell whether a genome encodes a
mouse, an elephant, or a whale? Second,
given the features of an organism, can we
design a genome that encodes it? What
genome sequence would specify a
Tyrannosaurus rex, a Pterosaur, or a
creature that never existed but whose
existence isnt prohibited by any laws of
biology? Both of the questions appear
well-posed and answerable in principle,
and the difficulty of answering them in
practice highlights how far we still have to
go in our understanding of evolution.

Cell 166, July 28, 2016 529

Leading Edge

Previews
In Praise of Descriptive Science:
A Breath of Fresh AIRE
Mark M. Davis1,*
1Howard

Hughes Medical Institute, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford,
CA 94305, USA
*Correspondence: mmdavis@stanford.edu
http://dx.doi.org/10.1016/j.cell.2016.07.018

Meyer et al. find that subjects lacking the AIRE gene, critical for self-tolerance in T lymphocytes,
show a broad range of autoantibody specificities, which can have extremely high affinities. The
data also suggest that some of these autoantibodies can, surprisingly, prevent some types of autoimmunity, particularly type I diabetes.
In molecular biology, there has been a
persistent bias against the value of
descriptive biology. This might go back
to a remark attributed to Ernest Rutherford that There are two kinds of science;
physics and stamp collecting. Of course,
the real power in any scientific area
comes with a precise knowledge of
mechanisms, but it shouldnt be forgotten
that careful observation and discovering
new phenomena is the starting point of
every field. A case in point is the paper
in this issue of Cell by Meyer et al.
(2016), who take advantage of new technologies that are allowing us to get precise data about the human immune system to discover some remarkable and
unexpected properties of human beings
with a particular immune deficiency.
They start, innocently enough, with a
straightforward enquiry into the nature of
the autoantibodies in subjects that are
deficient in what is known as the AIRE
gene, which was originally identified in
APS1an autoimmune syndrome characterized by autoantibodies, impaired
endocrine function, and chronic Candida
infections (Nagamine et al., 1997;
Finnish-German APECED Consortium,
1997). Later work in mice has shown
that Aire has a specific role in stimulating
the expression of tissue-specific genes
in the thymus that wouldnt normally be
expressed in that organ and that this
helps ensure T cell tolerance to self-antigens (Mathis and Benoist, 2009). There
is also evidence that Aire has a role in
ensuring T cell tolerance in peripheral immune organs such as the spleen and
lymph nodes (Gardner et al., 2013). Tolerance is induced at least in some cases by

clonal deletion of self-specific T cells (Anderson et al., 2005) but also might take the
form of inhibiting activation (Davis, 2015).
In some mouse strains, Aire deficiency
leads to severe autoimmunity and early
death, but in other strains and in human
beings, the effects are more subtle. In
this study, the authors gathered specimens and data on 81 patients. Then they
analyzed the autoantibodies in their
serum for specificity. Remarkably, they
found that each patient expresses on
average approximately 100 different
specificities, such that, all together, they
were able to identify over 3,700 antibody
specificities, showing that there was an
almost random pattern of targets. However, there were some specificities that
were shared, particularly anti-cytokine
antibodies to cytokines in the type I interferon group. Even more remarkable is
that, when they characterized some of
these autoantibodies with respect to
their affinity, they found that many were
astonishing high, with one having a KD of
10 14M and others in the picomolar range
(10 12M), much higher than the nanomolar affinities that one gets with a standard
immunization. They further note that patients with this syndrome seem resistant
to many autoimmune diseases (multiple
sclerosis, lupus, and others) but a fraction
do develop type I diabetes. While there is
a growing literature correlating anti-cytokine antibodies with a susceptibility to
particular infectious diseases (Kisand
et al., 2010), they postulated that such autoantibodies might, in some cases, have a
protective effect. In particular, a-interferons have been implicated in type I diabetes in mice, and so they looked to see

530 Cell 166, July 28, 2016 2016 Published by Elsevier Inc.

whether the expression of autoantibodies


to these cytokines correlated with this
type of autoimmunity in their cohort.
Indeed, they found that, while all the patients in their substudy had antibodies to
this family of cytokines, those expressed
by the 8 APS1 subjects with type I diabetes did not neutralize, whereas the
13 patients who did not have diabetes
did. While not a proof, this is a seriously
smoking gun suggesting a critical role
for these cytokines in this particular
(and major) autoimmune disease. It is
also interesting that, while a-interferon is
prominent in anti-viral responses and
has been used therapeutically, APS1 patients do not seem to be particularly prone
to viral infections, indicating that there is
enough redundancy in other parts of the
immune system to carry the load.
So how to summarize this study?
Figure 1 attempts to do this by showing
that the lack of AIRE activity in the thymus
and in the periphery in patients with
the APS1 syndrome leads to a failure of
T cell tolerance in those T cells that are
specific for the many self-antigens that
AIRE is responsible for expressing. Just
how many this represents is evident in
the many different autoantibody specificities in this cohort.
But why would these autoantibodies
have such high affinities? A key factor in
stimulating a given B cell to mutate its
immunoglobulin genes from micromolar
to nanomolar affinities in the course of
an immune response cells are follicular
helper T cells (TFH). The authors suggest
that the lack of T cell tolerance in general
and TFHs in particular could divert B cells
originally having other specificities onto

human mutations can uncover new phenomena worthy of further study. This not
only opens up new vistas regarding our
understanding of immune function and
dysfunction but also shows how work
on the human immune system can not
only inform translational work but add to
our understanding of basic principles as
well.
REFERENCES
Anderson, M.S., Venanzi, E.S., Chen, Z., Berzins,
S.P., Benoist, C., and Mathis, D. (2005). Immunity
23, 227239.
Davis, M.M. (2015). Immunity 43, 833835.
Finnish-German APECED Consortium. (1997). Nat.
Genet. 17, 399403.

Figure 1. A Simplified View of T-B Lymphocyte Interactions in the Presence or Absence of


the AIRE Genes Influence on T Cell Tolerance
Normally, follicular helper CD4+ T cells emerge from the thymus with those expressing self-specific T cell
receptors either purged or suppressed (upper part of the figure). They are then able to stimulate B cells
specific for the same antigen to mutate their antibody genes to achieve higher affinities after a bolus of
immunizing antigen or an infection. In AIRE-deficient subjects, it is suggested that the absence of T cell
tolerance allows multiple T-B interactions of this sort due to the continuous presence of self-antigen,
resulting in the very high affinities seen in Meyer et al. (2016). Here, arrows denote increases in antibody
affinity.

this self-reactive path. But an additional


wrinkle could come from recent work by
Goodnow and colleagues (Sabouri et al.,
2014), who have found that, in addition
to TFHs boosting the affinities of antibodies for foreign antigens,, autoantibody-producing B cells can be selected
to reduce their affinity for self. If this
reversal of affinity was dependent on
input from T cells enforcing self-specific

tolerance (either regulatory T cells or


perhaps TFH cells that had escaped clonal
deletion in the thymus), and if those cells
were absent due to the lack of Aire, then
the ubiquitous presence of self-antigens
coupled with a selection for high affinity
antibodies in germinal centers might
result in the amazing affinities seen in
this system. In any event, these results
show how carefully analyzing particular

Gardner, J.M., Metzger, T.C., McMahon, E.J., AuYeung, B.B., Krawisz, A.K., Lu, W., Price, J.D., Johannes, K.P., Satpathy, A.T., Murphy, K.M., et al.
(2013). Immunity 39, 560572.
Kisand, K., Be Wolff, A.S., Podkrajsek, K.T.,
Tserel, L., Link, M., Kisand, K.V., Ersvaer, E., Perheentupa, J., Erichsen, M.M., Bratanic, N., et al.
(2010). J. Exp. Med. 207, 299308.
Mathis, D., and Benoist, C. (2009). Annu. Rev. Immunol. 27, 287312.
Meyer, S., Woodward, M., Hertel, C., Vlaicu, P.,
Haque, Y., Karner, J., Macagno, A., Onuoha,
S.C., Fishman, D., Peterson, H., et al. (2016). Cell
166, this issue, 582595.
Nagamine, K., Peterson, P., Scott, H.S., Kudoh, J.,
Minoshima, S., Heino, M., Krohn, K.J., Lalioti,
M.D., Mullis, P.E., Antonarakis, S.E., et al. (1997).
Nat. Genet. 17, 393398.
Sabouri, Z., Schofield, P., Horikawa, K., Spierings,
E., Kipling, D., Randall, K.L., Langley, D., Roome,
B., Vazquez-Lombardi, R., Rouet, R., et al.
(2014). Proc. Natl. Acad. Sci. USA 111, E2567
E2575.

Cell 166, July 28, 2016 531

Leading Edge

Previews
Broadening Horizons:
New Antibodies Against Influenza
Katherine J.L. Jackson1 and Scott D. Boyd1,*
1Department of Pathology, Stanford University, CA 94305, USA
*Correspondence: sboyd1@stanford.edu
http://dx.doi.org/10.1016/j.cell.2016.07.023

Seasonal influenza vaccine formulation efforts struggle to keep up with viral antigenic variation.
Two studies now report engineered or naturally occurring human antibodies targeting the influenza
hemagglutinin (HA) stem, with exceptional neutralizing breadth (Joyce et al., 2016; Kallewaard et al.,
2016). Antibodies with similar structural features are elicited in multiple subjects, suggesting that
modified vaccine regimens could provide broad protection.
To paraphrase Jane Austen, it is a truth
universally acknowledged that an individual in possession of a good broadly
neutralizing antibody response to a virus
must be in want of characterization. The
human immune response to influenza
has been no exception. The tools of
monoclonal antibody (mAb) characterization from single B cells, and tracking of the
clonal evolution of B cell populations by
high-throughput antibody sequencing,
are providing an increasingly high-resolution map of human immunity to a range of
pathogens and vaccines (Andrews et al.,
2015; Liao et al., 2013). Influenza is a significant global health challenge with millions of infections and up to half a million
deaths globally each year, despite efforts
to increase vaccination in at-risk populations. Influenza virus is a challenging
target for antibodies because of antigenic
drift and shift, genetic processes that produce ongoing and occasionally abrupt
antigenic changes. Antibodies elicited
by seasonal influenza vaccination tend
to target the rapidly mutating globular
head of hemagglutinin (HA), facilitating
viral escape from responses raised to
other viral strains (Krammer and Palese,
2013). Broadly neutralizing antibodies
(bnAbs) directed against the structurally
conserved HA stem could provide more
enduring protection, and examples of
these have been isolated (Krammer and
Palese, 2013). These antibodies have revealed some stereotyped features, such
as IGHV1-69 usage in multiple subjects
(Corti et al., 2010). These responses in vivo
in humans, however, seem to arise from
low-frequency clones, are present at
lower titers than head-specific antibodies,

and have neutralization breadth usually


limited to homologous subtypes. bnAbs
with neutralizing breadth covering both
influenza A group 1 and group 2 subtypes
would be far more valuable, if they could
be reliably stimulated by new vaccine regimens. Such antibodies have only rarely
been observed in responses to vaccination or infection (Corti et al., 2011). In this
issue, Joyce et al. (2016) and Kallewaard
et al. (2016) apply distinct approaches to
obtain bnAbs with breadth against both
influenza A group 1 and group 2 subtypes.
Joyce et al. (2016) study stereotyped or
convergent antibody responses across
multiple human donors after H5 hemagglutinin primed and repeatedly boosted
influenza vaccination, and they define
new multi-donor bnAb classes (Figure 1).
Kallewaard et al. (2016) engineer and
improve a single-subject-derived mAb
to obtain unprecedented neutralization
breadth. The identification of convergent
classes of antibodies capable of neutralizing both influenza A group 1 and group
2 subtypes builds upon a growing body
of literature showing that the diverse antibody-mediated responses of different humans can converge on similar solutions to
complex antigen targeting (Jackson et al.,
2014; Truck et al., 2015). The bnAb developed by Kallewaard et al. (2016) shows
structural similarities to one of the bnAb
classes identified by Joyce et al. (2016).
Kallewaards engineered MEDI8852
bnAb reacts to representatives of all influenza A subtypes from the last 80 years and
is derived from a naturally occurring antibody whose heavy chain uses IGHV6-1,
IGHD3-3, and IGHJ3 gene segments
with low levels of somatic hypermutation.

532 Cell 166, July 28, 2016 2016 Elsevier Inc.

This bnAb interacts with a highly


conserved epitope in the hydrophobic
groove in the fusion domain plus a large
portion of the fusion peptide (Kallewaard
et al., 2016). Similarly, one of the convergent antibody classes isolated from three
subjects by Joyce et al. (2016) also utilizes
IGHV6-1 and IGHD3-3, binds the HA
stem but avoids the conserved HA1 glycans of both group 1 and group 2 subtypes, and uses a somatically mutated
CDR H3 residue to insert into the Trp21
pocket in the hydrophobic groove of
HA2. Two additional convergent bnAb
classes identified by Joyce et al. (2016),
both using IGHV1-18, bind the HA fusion
peptide-helix A region suggesting this region is a recurrent target for bnAbs.
These two studies contribute to
ongoing efforts to understand the threedimensional structural features and the
underlying primary antibody sequences
used to recognize viruses that can alter
many, but not all, of their epitopes. They
add to a growing list of such structurally
conserved vulnerable targets for neutralization in influenza, akin to those for HIV
(Liao et al., 2013), and demonstrate that
shared antibody structural motifs for binding viral epitopes are formed in different
individuals, despite the vast diversity of
antibody repertoires. Analogous convergent antibodies contribute to human antibody responses to many, if not all, pathogens (Jackson et al., 2014; Truck et al.,
2015), but not all convergent antibodies
are neutralizing or otherwise protective.
Structurally constrained pathogen epitopes required for survival may be under
selection so that they are not highly antigenic and may only be accessible to

peutic drug candidates that could be


particularly helpful in populations with
compromised immune function, such as
the very elderly.
The combination of structural analysis
and extensive sequence characterization
of antibody repertoires, married with functional testing of mAbs, has greatly empowered the analysis of human B cell responses to real pathogens and vaccines.
These new studies underscore the value
of taking cues from the structural problem
solving of the immune system, when
designing new candidate therapeutics,
and they add to the evidence that there
are predictable features of human adaptive immune responses that can be accessed by properly designed vaccine regimens to achieve greater efficacy.

REFERENCES
Andrews, S.F., Huang, Y., Kaur, K., Popova, L.I.,
Ho, I.Y., Pauli, N.T., Henry Dunand, C.J., Taylor,
W.M., Lim, S., Huang, M., et al. (2015). Sci. Transl.
Med. 7, 316ra192.

Figure 1. Convergent Broadly Neutralizing Antibodies for Influenza


After exposure to influenza vaccines or viral infections, each individual produces a mixture of different
antibodies specific for a variety of epitopes on the hemagglutinin protein (indicated by antibody color and
epitope color on the hemagglutinin trimer; epitopes are colored on only one of the HA trimers for clarity).
Antibodies that bind to the highly variable head region are most often strain-specific, while antibodies that
bind the more conserved stalk region have the potential to neutralize a wider variety of strains. Rare stalkbinding antibodies that neutralize influenza strains spanning the major antigenic group 1 and group 2
categories have been previously reported. Joyce et al. (2016) now identify several categories of such
broadly neutralizing antibodies (indicated with circles) that also show structural convergence between
different donors (i.e., very similar antibodies are elicited in different individuals, despite the huge diversity
of antibody repertoires). Kallewaard et al. (2016) describe an engineered antibody with particularly
extensive neutralizing breadth, which shares structural similarity with one of the antibody classes in Joyce
et al. (2016) and binds to a similar epitope.

antibodies that are difficult for humans to


elicit, for example, because they are autoreactive or polyreactive and trigger
poorly defined human B cell tolerance
mechanisms (Liao et al., 2013).
It is less clear how much of any individuals actual serum antibody response to
influenza vaccination is composed of
such bnAbs or whether optimized vaccination regimens could elicit them at
high enough titers to be functionally protective. Ongoing antibody proteomic
research with improved mass spectrometry methods may help to address these
key questions, particularly if applied in
clinical trials in which protection from
infection can be measured (Wine et al.,
2013). What do these studies teach us
about immunogen design or selection?

The pragmatic goal of better vaccine efficacy against divergent viral strains could
be served equally well by eliciting rare antibodies with extreme breadth or a handful
of antibody lineages with complementary
partial breadth. The success of each scenario could depend on the titers reached
and the durability of the response. Evaluating the feasibility of these goals will
likely require testing vaccine regimens using heterologous antigens that are more
divergent than those in current vaccine
formulations and assessing antigenic
combinations, the ordering of antigen
administration, and the effects of adjuvants, while analyzing the kinds of antibodies that are stimulated in each case.
Of course, very potent bnAbs are potential passive immunoprotective or thera-

Corti, D., Suguitan, A.L., Jr., Pinna, D., Silacci, C.,


Fernandez-Rodriguez, B.M., Vanzetta, F., Santos,
C., Luke, C.J., Torres-Velez, F.J., Temperton,
N.J., et al. (2010). J. Clin. Invest. 120, 16631673.
Corti, D., Voss, J., Gamblin, S.J., Codoni, G., Macagno, A., Jarrossay, D., Vachieri, S.G., Pinna, D.,
Minola, A., Vanzetta, F., et al. (2011). Science
333, 850856.
Jackson, K.J., Liu, Y., Roskin, K.M., Glanville, J.,
Hoh, R.A., Seo, K., Marshall, E.L., Gurley, T.C.,
Moody, M.A., Haynes, B.F., et al. (2014). Cell
Host Microbe 16, 105114.
Joyce, M.G., Wheatley, A.K., Thomas, P.V.,
Chuang, G., Soto, C., Bailer, R.T., Druz, A., Georgiev, I.S., Gillespie, R.A., Kanekiyo, M., et al.
(2016). Cell 166, this issue, 609623.
Kallewaard, N.L., Corti, D., Collins, P.J., Neu, U.,
McAuliffe, J.M., Benjamin, E., Wachter-Rosati, L.,
Palmer-Hill, F.J., Yuan, A.Q., Walker, P.A., et al.
(2016). Cell 166, this issue, 596608.
Krammer, F., and Palese, P. (2013). Curr. Opin. Virol. 3, 521530.
Liao, H.X., Lynch, R., Zhou, T., Gao, F., Alam, S.M.,
Boyd, S.D., Fire, A.Z., Roskin, K.M., Schramm,
C.A., Zhang, Z., et al.; NISC Comparative
Sequencing Program (2013). Nature 496, 469476.
Truck, J., Ramasamy, M.N., Galson, J.D., Rance,
R., Parkhill, J., Lunter, G., Pollard, A.J., and Kelly,
D.F. (2015). J. Immunol. 194, 252261.
Wine, Y., Boutz, D.R., Lavinder, J.J., Miklos, A.E.,
Hughes, R.A., Hoi, K.H., Jung, S.T., Horton, A.P.,
Murrin, E.M., Ellington, A.D., et al. (2013). Proc.
Natl. Acad. Sci. USA 110, 29932998.

Cell 166, July 28, 2016 533

Leading Edge

Previews
Baby Nuclear Pores Grow Up Faster All the Time
C. Patrick Lusk1,*
1Yale School of Medicine, New Haven, CT 06510, USA
*Correspondence: patrick.lusk@yale.edu
http://dx.doi.org/10.1016/j.cell.2016.07.011

Annulate lamellae (AL) are stacked ER-derived membranes containing nuclear pore complex-like
structures whose fate and function have remained a mystery. During the short interphase of early
embryonic cells, AL are rapidly delivered into the nuclear envelope through fenestrations, highlighting the remarkable dynamics of the nuclear envelope.
The confinement of the genome within the
nucleus suggests that it, like most organelles, is a physically distinct cellular entity.
However, the two nuclear membranes
that comprise the nuclear envelope (NE)
are made up of one lipid bilayer that is
contiguous with the endoplasmic reticulum (ER) (Figure 1). The morphological
and biochemical identity of the NE is
classically defined by the presence of nuclear pore complexes (NPCs), which are
enormous 100 MD transport channels
composed of nucleoporin (nup) proteins.
Importantly, NPCs do not disrupt the continuity of the lipid bilayer between the NE
and ER but effectively seal NE pores
by controlling the passage of soluble
and membrane-bound macromolecules
into and out of the nucleus. Interestingly,
the morphological distinction between
NE and ER is blurred in some cell types,
as extra-nuclear NPC-like structures
occur in stacked ER cisternae (Cordes
et al., 1996). These annulate lamellae
(AL) were considered repositories of
excess nups, but this function was not
formally established. Moreover, it is challenging to contemplate how (or whether)
AL could be incorporated into the NE
without breaking the NE seal that would
also impose a topological barrier to such
an event. In this issue of Cell, Hampoelz
et al. (2016) show how rapidly dividing
cells in the Drosophila embryo overcome
this barrier by visualizing an elegant
mechanism of AL incorporation into the
NE. In addition to providing a solution to
the long-standing question of the function and fate of AL, they also introduce
a mechanism of membrane remodeling
that is challenging our conventional view
of a static interphase NE.
A common feature of early embryonic
divisions is their rapidity, necessitating

constant NE breakdown and reformation


cycles. While Drosophila cells do not fully
break down their NEs, NPC disassembly
and NE fenestration allow spindle microtubules access to the chromosomes. During
the ensuing 10 min interphase (consider
that a typical mammalian cell interphase
lasts several hours!), the NE almost triples
in surface area while maintaining a constant NPC density. These observations
suggest an intimate link between rapid
NE expansion and de novo NPC assembly
(DAngelo et al., 2006), but what are the
sources of membrane and NPCs? As the
embryonic Drosophila cells are chock-full
of AL that disappear during interphase,
a likely possibility is that they provide
the raw materials for NE expansion.
Indeed, by taking advantage of sophisticated time-lapse fluorescence microscopy coupled with correlative light EM
and slice and view focused ion beam
scanning EM, Hampoelz et al. convincingly demonstrate that AL are incorporated into the NE through large fenestrations (in some cases spanning several
micrometers) in the NE that likely persist
from the preceding mitosis (Figure 1A).
The molecular mechanism of incorporation likely involves a growth in nuclear volume that might mechanically stretch the
NE, further dilating these fenestrations
and providing an entryway for the AL to
come into direct physical contact with
intranuclear factors like chromatin. Undoubtedly, membrane remodeling proteins that help shape the ER contribute
to these processes as well through mechanisms that will await future study.
Interestingly, a key indicator of the
capacity of the embryonic nuclei to undergo the dramatic NE remodeling necessary for AL incorporation is the marked
mobility of NPCs, a bellwether of a

534 Cell 166, July 28, 2016 2016 Elsevier Inc.

uniquely dynamic NE-ER system. Consistent with this idea, as the embryonic cells
progressively immobilize NPCs by the
increased expression of intranuclear scaffolds like the lamins and lamin-binding
proteins, they lose the capacity to incorporate AL into the NE. Thus, AL incorporation may be halted as cell fate and NE
composition become cemented. Alternatively, AL incorporation might be slowed
because of a requirement to remodel a
rigid intranuclear lamin scaffold, a likely
necessity for NE dynamics in highly differentiated cells with more established nuclear architecture (King and Lusk, 2016).
A model in which the AL incorporate
into the NE makes the prediction that the
NE and AL together comprise a compartment that is distinct from the rest of the
ER. Remarkably, and consistent with this
idea, the permeability barrier of the NE
to macromolecules remains intact despite
the presence of the NE fenestrations, suggesting that the AL effectively seal the
NE like a lid that would be part of a
larger, yet-to-be defined NE-AL system
(Figure 1). A major challenge for the future
is to understand how membranes competent for AL formation either remain connected with the NE during mitotic NE
breakdown or establish connections by
generating holes in a sealed NE. A likely
possibility for the former scenario is that,
during NE breakdown, some membranes
(perhaps those that associate with the
mitotic spindle) retain a biochemical or
morphological signature of the NE that
establishes competence for NE reformation and NPC assembly. The future
identification of this molecular signature
might represent the minimal fundamental
component that triggers NPC assembly
and provides the ultimate differentiator
of the NE and ER.

tion of NPC assembly upon AL incorporation into the NE (Figure 1). This NPC
maturation must rely on positional
cues that could reflect binding to chromatin (Franz et al., 2007; Rasala et al.,
2006) or to components of the nuclear
basket known to be required for NPC assembly but missing from AL (Vollmer
et al., 2015). Thus, one exciting possibility is that the maturation mechanism
reflects conformational changes to the
NPC scaffold that expose otherwise
hidden anchor points for the remaining
nups. One wonders whether this process
might be reversed during NPC disassembly or dynamically controlled to alter
the transport properties of the NPC in
response to environmental (or other) inputs. In either case, understanding the
molecular basis behind these putative
conformational changes will likely be a
watershed in our understanding of NPC
assembly and function.

REFERENCES
Cordes, V.C., Reidenbach, S., and Franke, W.W.
(1996). Cell Tissue Res. 284, 177191.

Figure 1. Insertion of Annulate Lamellae into the Nuclear Envelope


(A) Annulate lamellae (AL) contain NPC skeletons lacking the full complement of nucleoporins (nups) that
are embedded in stacked ER membranes. They are directly connected to the NE around large fenestrations. The NPC skeletons have nups capable of establishing a diffusion barrier to large macromolecules
and are unlikely to mediate active nuclear transport of nuclear transport receptor (NTR) cargo complexes.
(B) During rapid NE expansion in interphase, the AL are incorporated into the NE as NPC skeletons mature
to complete NPC assembly. INM and ONM, inner and outer nuclear membrane, respectively.

What can AL teach us about the


mechanism of NPC assembly? As the
core scaffold components of the NPC
assemble into AL within minutes, it is clear
that building an NPC skeleton is a surprisingly efficient and rapid process; studying
AL formation could thus provide an
experimental system to determine the
mechanisms underlying these events. An
advantage of such a system is that it
might harmonize reported differences in
the biochemical (Doucet et al., 2010;
Vollmer et al., 2015) and kinetic (Dultz
and Ellenberg, 2010; Dultz et al., 2008) requirements of NPC assembly observed
either at the end of mitosis or during interphase. For example, these differences
might simply reflect layers of regulation
present at these distinct phases of the
cell cycle (Wandke and Kutay, 2013) or

in unique cell types with distinct nuclear


organizations that would be absent from
a minimal AL assembly system.
To ultimately determine whether there
are fundamental mechanistic differences
in NPC assembly throughout the cell
cycle or into AL will require a deeper
understanding of the biochemical and
structural intermediates that define the
steps in each of these putative pathways. Perhaps most importantly, it needs
to be definitively established whether
these modes of assembly require a
membrane fusion step to insert NPCs
into double membranes (Wandke and
Kutay, 2013). Just as critically, as the
NPC skeletons in AL lack critical asymmetric components of the NPC and
several central channel nups, mechanisms must exist to trigger the comple-

DAngelo, M.A., Anderson, D.J., Richard, E., and


Hetzer, M.W. (2006). Science 312, 440443.
Doucet, C.M., Talamas, J.A., and Hetzer, M.W.
(2010). Cell 141, 10301041.
Dultz, E., and Ellenberg, J. (2010). J. Cell Biol. 191,
1522.
Dultz, E., Zanin, E., Wurzenberger, C., Braun, M.,
Rabut, G., Sironi, L., and Ellenberg, J. (2008).
J. Cell Biol. 180, 857865.
Franz, C., Walczak, R., Yavuz, S., Santarella, R.,
Gentzel, M., Askjaer, P., Galy, V., Hetzer, M.,
Mattaj, I.W., and Antonin, W. (2007). EMBO Rep.
8, 165172.
Hampoelz, B., Mackmull, M.-T., Machado, P.,
Ronchi, P., Bui, K.H., Schieber, N., SantarellaMellwig, R., Necakov, A., Andres-Pons, A., Philippe, J.M., et al. (2016). Cell 166, this issue,
664678.
King, M.C., and Lusk, C.P. (2016). Curr. Opin. Cell
Biol. 41, 917.
Rasala, B.A., Orjalo, A.V., Shen, Z., Briggs, S., and
Forbes, D.J. (2006). Proc. Natl. Acad. Sci. USA
103, 1780117806.
Vollmer, B., Lorenz, M., Moreno-Andres, D., Bodenhofer, M., De Magistris, P., Astrinidis, S.A.,
Schooley, A., Flotenmeyer, M., Leptihn, S., and
Antonin, W. (2015). Dev. Cell 33, 717728.
Wandke, C., and Kutay, U. (2013). Cell 152, 1222
1225.

Cell 166, July 28, 2016 535

Leading Edge

Previews
A Biomarker Harvest
from One Thousand Cancer Cell Lines
Yu-Han Huang1 and Christopher R. Vakoc1,*
1Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
*Correspondence: vakoc@cshl.edu
http://dx.doi.org/10.1016/j.cell.2016.07.010

Identifying molecular biomarkers that predict cancer drug efficacy is crucial for the advancement of
precision medicine. In this issue of Cell, Iorio et al. nominate hundreds of potential genetic and
epigenetic biomarkers through high-throughput drug screening in 1,000 molecularly annotated
cancer cell lines.
Developing personalized therapies that
exploit the unique molecular abnormalities in a patients tumor is a central objective of modern cancer research. Genome
sequencing initiatives, such as The
Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium
(ICGC), have defined the complex genetic
landscapes of the most common human
malignancies; however, only a small
percentage of cancer mutations is
considered to be actionable with existing
therapies (Garraway and Lander, 2013;
Stratton et al., 2009). While new targets
and drugs are clearly needed, a major
obstacle in implementing precision therapies is our incomplete understanding of
the relationship between tumor genotype
and drug sensitivity. To address this
issue, many investigators have turned to
large-scale chemical screens in genetically annotated human cancer cell lines
as a means of nominating predictive biomarkers (Figure 1). While cancer cell lines
in culture are imperfect models of human
tumors, they tend to remain addicted to
the oncogenes that initiated tumor formation and hence are well-validated tools for
studying oncogene-targeted therapies
(Sharma et al., 2010). In this issue, Iorio
et al. present one of the largest attempts
to date at mining predictive correlates of
drug sensitivity using a panel of 1,000
annotated cancer cell lines treated with
265 compounds (Iorio. et al., 2016).
The strategy taken by the authors is the
following: (1) perform a deep genetic and
epigenetic analysis of each cell line,
focusing on the features that match the
recurrent alterations found in human tumors, (2) measure the sensitivity of each
cell line to 265 different compounds/

drugs, which includes approved and


investigational agents, and (3) perform
computational analyses to search for genetic/epigenetic alterations that correlate
with resistance and sensitivity to each
drug. Several important observations
have been made during the implementation of this screening platform. First, the
large panel of cell lines used in this study
capture much of the gene mutations,
DNA copy number alterations, and epigenetic changes found in primary human tumors. Furthermore, the authors use machine learning to investigate which data
type (genomic alterations, DNA methylation, or gene expression) is the best
predictor of drug sensitivity. When performing a pan-cancer analysis, gene
expression is the superior predictor of
sensitivity, whereas within a specific cancer, drug sensitivity is best explained by
genomic analysis. Importantly, many of
the pharmacogenomic relationships identified in Iorio et al. could be validated when
evaluating prior analyses of the Cancer
Cell Line Encyclopedia (CCLE) (Barretina
et al., 2012; Seashore-Ludlow et al.,
2015), thus alleviating concerns about
the reproducibility of results from independent drug screening platforms
(Haibe-Kains et al., 2013). Taken together,
these findings provide significant insight
into our assessment of human cancer
cell lines and drug sensitivity profiling as
tools for therapeutic investigation.
The output of the analysis in Iorio et al.
is a stunning number of genotype-drug
sensitivity associations. In total, 688 statistically significant interactions have
been identified between individual genetic/epigenetic events and specific cancer drugs, with 262 of these associations

536 Cell 166, July 28, 2016 2016 Elsevier Inc.

being classified as large effectthat is,


reaching a comparable strength of association as observed between clinically
validated kinase inhibitors and their
genetically altered kinase target (imatinib/BCR-ABL, vemurafinib/BRAFV600E).
We list here just a few of these novel associations: sensitivity to the anti-androgen
bicalutamide in squamous cell lung cancer lines has been found to be associated
with inactivating mutations of the chromatin modifier MLL2. In stomach cancer
cell lines, truncating mutations of the transcriptional corepressor BCOR are associated with sensitivity to LY317615, an inhibitor of protein kinase C b. Sensitivity
to the BRD2/BRD3/BRD4 bromodomain
inhibitor JQ1 in breast cancer lines is
linked to the mutational status of the
RNA polymerase II subunit POLR2B. The
functional relationship between these
non-oncogene drug targets and these
specific genetic alterations is presumably
indirect and can be contrasted with the
classical targeted therapy paradigm of
direct oncoprotein inhibition (e.g., imatinib/BCR-ABL) (Luo et al., 2009). Hence,
this study may have exposed a vast array
of synthetic-lethality genetic interactions
for future mechanistic characterization
(Kaelin, 2005).
When Iorio et al. is considered together
with other large-scale compound
screening initiatives (Figure 1), it becomes
apparent that an explosion of new
biomarker-guided therapeutic opportunities is emerging, which now await validation in pre-clinical models and/or in
cancer patients. Unfortunately, the historical experience of pharma and academia
in developing drugs against non-oncogene drug targets, in particular those

Figure 1. The Major Cancer Cell Line Drug Screening Initiatives


The NCI-60 project was initiated in the 1980s and has to date tested >100,000 compounds in 59 cancer cell lines. More recently, the scale of cancer cell line drug
screens has expanded dramatically (e.g., Cancer Cell Line Encyclopedia [CCLE]/Cancer Therapeutics Response Portal [CTRP], and Genomics of Drug Sensitivity
in Cancer [GDSC]) and now includes a detailed genetic analysis of each cell line.

discovered in cell lines, would lead us to


anticipate a low probability of success
during clinical translation. However, the
analysis presented in Iorio et al. may
have addressed the critical issue that
has undermined the prior pursuits of
non-oncogene targets: the lack of predictive genetic biomarkers. A key question
going forward will be whether the inherent
limitation of cancer cell lines as tumor
models will obfuscate the pharmacogenomic relationships identified in this
study, as validation experiments proceed
into more physiological tumor models.
Despite these concerns, the remarkable
scale of this cell line screening effort
places us in a strong position of having
hundreds of potential hypotheses to be
explored. Hence, even a low rate of validation could still amount to a major
advance in the development of targeted
cancer therapies. In this regard, a largescale drug efficacy evaluation in genetically annotated patient-derived xenograft
(PDX) tumor models would be a justified
follow-up venture to this work.
While the clinical significance of the
findings in Iorio et al. remains to be deter-

mined, the utility of this cell line resource


for basic cancer research is unambiguous. NCI-60 was initiated in the 1980s as
the first compound screening initiative in
cancer lines and contributed fundamental
insights to our understanding of drug
mechanisms of action (Chabner, 2016).
The deepening genomic, epigenomic,
and, ultimately, metabolomic characterization of cell lines is allowing investigators to pinpoint cancer-sustaining molecular mechanisms with unprecedented
depth, rigor, and speed. As such efforts
have expanded in recent years, it is now
common practice in many research labs,
including our own, to use publicly available cancer cell line resources to guide
the experimental evaluation of any new
gene or small molecule for its relevance
to cancer biology.

ACKNOWLEDGMENTS
C.R.V. is supported by the Leukemia and Lymphoma Society, the Burroughs-Wellcome Fund,
the Pershing Square Sohn Cancer Research Alliance, the Starr Cancer Consortium, and the NIH/
NCI grant RO1 CA174793.

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Cell 166, July 28, 2016 537

Leading Edge

Review
The Genetics of Transcription Factor
DNA Binding Variation
Bart Deplancke,1,* Daniel Alpern,1 and Vincent Gardeux1
1Laboratory

of Systems Biology and Genetics, Institute of Bioengineering, Ecole Polytechnique Federale de Lausanne and Swiss Institute of
Bioinformatics, 1015 Lausanne, Switzerland
*Correspondence: bart.deplancke@epfl.ch
http://dx.doi.org/10.1016/j.cell.2016.07.012

Most complex trait-associated variants are located in non-coding regulatory regions of the
genome, where they have been shown to disrupt transcription factor (TF)-DNA binding motifs. Variable TF-DNA interactions are therefore increasingly considered as key drivers of phenotypic variation. However, recent genome-wide studies revealed that the majority of variable TF-DNA binding
events are not driven by sequence alterations in the motif of the studied TF. This observation implies
that the molecular mechanisms underlying TF-DNA binding variation and, by extrapolation, interindividual phenotypic variation are more complex than originally anticipated. Here, we summarize
the findings that led to this important paradigm shift and review proposed mechanisms for local,
proximal, or distal genetic variation-driven variable TF-DNA binding. In addition, we discuss the
biomedical implications of these findings for our ability to dissect the molecular role(s) of noncoding genetic variants in complex traits, including disease susceptibility.
Introduction
Analysis of genomic variation in humans (Auton et al., 2015) as
well as in model species such as the mouse (Keane et al.,
2011; Yalcin et al., 2011) and fruit fly (Huang et al., 2014; Massouras et al., 2012) is providing unprecedented opportunities to
understand the genetic basis of complex traits, including disease
susceptibility. An important insight that emerged from genomewide association studies (GWAS) is that the vast majority of
significantly associated genetic variants is located in non-coding
regions and may thus impact gene regulation. For example, of
465 unique trait/disease-associated single nucleotide polymorphisms (SNPs) derived from 151 GWAS studies, only 12% are
located in protein-coding regions, while 40% fall within introns
and another 40% in intergenic regions (Hindorff et al., 2009). In
addition, genome-wide profiling of accessible chromatin regions
using DNase I hypersensitivity (DHS) mapping revealed that
almost 60% of non-coding GWAS SNPs and other variants are
located within DHS sites, with another 20% being in complete
linkage disequilibrium (LD) with variants that lie in a proximate
DHS site (Maurano et al., 2012). Since DHS sites reflect the occupancy of DNA binding proteins such as transcription factors
(TFs), these data indicate that GWAS loci may alter the binding
of TFs and, as such, induce variation in gene expression and
ultimately in complex organismal phenotypes. In this Review,
we summarize the findings that led to this increasingly accepted
notion of the importance of variation in TF-DNA binding in mediating phenotypic diversity. In addition, we strive to clarify why, for
the majority of studied traits or diseases, establishing a mechanistic link between regulatory and phenotypic variation is still
very challenging.
For this purpose, we explore the molecular mechanisms mediating TF-DNA binding variation and address a major question in
538 Cell 166, July 28, 2016 2016 Elsevier Inc.

the fieldnamely, why the majority of variable TF-DNA binding


events appear to be driven by mechanisms other than nucleotide
variation in the cognate motifs. We thereby focus on human interindividual, molecular variation and restrict this Review to discussing mechanisms underlying variable TF-DNA binding. Consequently, we will only briefly cover the functional consequences
of this variation or other modes of regulatory variation, which
have been extensively detailed elsewhere both for humans and
model organisms (Albert and Kruglyak, 2015; Lehner, 2013;
Lowe and Reddy, 2015; Mackay et al., 2009; Pai et al., 2015).
A Brief Historical Perspective on Variable TF-DNA
Interactions as Key Drivers of Inter-individual,
Phenotypic Diversity
The discovery of regulatory sequences (or operators) in bacteria by Jacob and Monod initiated the debate of whether variation
in regulator-operator interactions could drive phenotypic diversity (Jacob and Monod, 1961). It was proposed that this variation could arise either through mutations in the regulator itself or
through mutations in the operator that would alter or abolish its
specific affinity for the repressor (i.e., regulator) (Jacob and
Monod, 1961). This fundamental prediction proved to be accurate across species, and multiple examples have since been
revealed that support both scenarios (Barrera et al., 2016; Hoekstra and Coyne, 2007; Lynch and Wagner, 2008; Wray, 2007).
The first concrete evidence supporting the importance of such
non-coding or regulatory variation for human traits or diseases
started to emerge in the early 1980s, when the molecular mechanisms underlying thalassemias were investigated. These heritable blood disorders, characterized by an abnormal form of
hemoglobin, made it intuitive to explore the globin gene locus
for disease-causing genetic variants. Numerous variants were

detected, including several polymorphisms in the b globin gene


(HBB) promoter that correlated with reduced HBB expression
(Orkin et al., 1982; Poncz et al., 1982). For example, a single
nucleotide substitution (C to G) at position 87 of HBBs transcription start site (Orkin et al., 1982) was hypothesized to affect
the recruitment of a transcriptional activator. However, it was
only 11 years later, when the erythroid Kruppel-like factor
(KLF1) was cloned, that the affected site (CA(C/G)CC) was
matched with a TF (Miller and Bieker, 1993) (Table 1). This
groundbreaking example (as well as several others listed in
Table 1) support the idea of TF-DNA interactions being key
drivers of phenotypic variation. However, the fact that the underlying molecular mechanisms were uncovered for these diseases
is still more the exception than the rule.
Assessing the Impact of Genetic Variation on TF-DNA
Binding: A Complex Affair
The ability to elucidate the molecular mechanisms underlying
thalassemia, haemophila B, or malaria resistance was made
possible because several critical pieces of information were
available that are often missing in other genotype-phenotype
relationship studies: (1) knowledge of the affected gene, which
facilitated the identification of the causal mutation(s); (2) availability of DNA binding specificity data for the implicated TF;
and (3) relatively straightforward imputation of the effect of the
causal mutation(s) on TF-DNA binding. Below, we will discuss
each of these three items in more detail and explain why they
collectively complicate studies that investigate the impact of genetic variation on molecular or organismal variation.
Identification of Causal Mutation(s) and Affected
Gene(s)
In contrast to cases like the thalassemia mutations discussed
above, GWAS studies identify genetic variants linked to particular traits, but not necessarily those actually causing the disease
(Manolio, 2013). In addition, such studies do generally have little
prior knowledge regarding which genes will be uncovered.
Therefore, by simply matching a GWAS SNP with a TF binding
site, one risks wrongly inferring that a TF must affect the expression of the gene that is most proximal to a particular binding site.
However, the actual culprit could be another genetically linked
but unprobed variant such as an indel or a rare SNP that impacts
a different binding site and thus a distinct TF and/or target gene.
This potential misidentification is why significant efforts are
currently undertaken to fine-map complex traits using statistical
arguments (Figure 1; see also the Imputing DNA binding variation section) and/or integrative genomic approaches to identify
causal variants and their target genes at nucleotide-level resolution. A striking recent example involves variants that have been
consistently associated with an elevated body mass index in
both children and adults (Dina et al., 2007; Frayling et al.,
2007). These variants are located in the first and second introns
of a large (>250 kb) gene named fatso, or FTO, because its deletion causes a fused toes phenotype in mouse (Peters et al.,
1999). Given its association with obesity, it was subsequently rephrased as fat mass and obesity-associated gene and was
widely mechanistically studied for its role in energy homeostasis
(Fischer et al., 2009). However, recent studies revealed that the
focal variants are, in fact, located in a regulatory element that

controls the expression of the TF-coding genes IRX3 and IRX5


more than 1 Mbp away (Claussnitzer et al., 2015; Smemo
et al., 2014). Thus, these variants appear to have little impact
on the FTO gene, even though they are positioned within its introns. Rather, one variant disrupts the binding site of ARID5B
(AA(T/C)ATT), resulting in elevated IRX3 and IRX5 expression.
This, in turn, increases the formation of white fat cells, possibly
leading to excessive fat accumulation (Claussnitzer et al.,
2015). Significant efforts involving a battery of advanced computational (Claussnitzer et al., 2014) and experimental approaches
were required to study the molecular function of the FTO
variants and their relationship with body mass index. This
complexity illustrates why the number of mechanistically wellstudied relationships between regulatory and phenotypic variation is still relatively low. It also explains why the majority of
such studies focused on gene proximal variants (Table 1), especially prior to 2005, when the importance of chromosome conformation in gene regulation was still less established.
Incomplete TF Motif Catalog
In the early 1990s, DNA binding variation of the TFs CEBPa and
HNF4A, as well as GATA1, was linked to, respectively, haemophilia B and malaria resistance (Table 1). The identification of
these TFs is intuitive since they were among the relatively few
TFs whose DNA binding properties had been described at the
time when these genetic studies were carried out (Faisst and
Meyer, 1992). Several other studies have since established regulatory variant-phenotype relationships on the basis of GATA1
(Table 1), illustrating how such studies tend to be restricted to
investigating phenotypes that involve well-characterized TFs.
While the development of several large-scale in vitro DNA
binding characterization technologies, such as protein-binding
microarrays (PBM) (Berger et al., 2006), bacterial one-hybrid
(B1H) screening (Meng et al., 2005), and high-throughput (HT)SELEX (Jolma et al., 2010), has enabled a significant expansion
of the TF motif catalog, it is worth noting that at least one-third of
human TFs remains uncharacterized (Box 1). In other words,
several hundred TFs are still devoid of DNA binding specificity
models such as the most routinely used position weight matrix
(PWM) (Stormo and Zhao, 2010) (Figure 1). This lack of information severely limits the ability to analyze the effects of genetic
variation on TF-DNA binding, as a large fraction of human TFs
can simply not be taken into account in such studies.
This problem becomes even larger when considering that
many TFs do not bind DNA as single entities but, rather, in the
form of obligate heterodimers such as TFs containing bZIP,
bHLH, MADS box, or Rel DNA binding domains. Since the focus
of DNA binding specificity determination studies has largely
been on single protein-DNA interactions, DNA binding motifs
for such heterodimers are underrepresented in current regulatory lexicons. Moreover, many TFs also participate in facultative
heterodimers since they can bind to DNA both in monomeric or
dimeric context. It is difficult to know how many of such heterodimers routinely form in cells, but predictions range from 3,000
(Ravasi et al., 2010) to >25,000 (Jolma et al., 2015). Interestingly,
these cooperative TF pairs often show distinct binding site
preferences compared to the respective, individual TFs, as the
heterodimer core motif typically consists of closely packed individual motifs that overlap at their flanks. Consequently, individual
Cell 166, July 28, 2016 539

Table 1. Examples Linking Variable TF-DNA Binding to Phenotypic Variation Arranged by Date of Characterization
Causal Variant Position
Relative to TSS

Phenotype

Affected Gene

Hereditary persistance of fetal


haemoglobin

HBG

175 bp

Haemophilia B Leyden

F9

20 bp; 10 bp;

6 bp

Affected Binding
Site

TFBS
Outcome

GATA1; TAL1

Gain

(Martin et al., 1989;


Wienert et al., 2015)

HNF4a; C/EBPa;
OC1/OC2

Loss

(Reijnen et al., 1992;


Crossley and Brownlee,
1990; Funnell et al., 2013)

Reference(s)

Haemophilia B Brandenburg

F9

26 bp

AR

Loss

(Crossley et al., 1992)

Delta-thalassemia

HBD

77 bp

GATA1

Loss

(Matsuda et al., 1992)

Duffy blood antigen/chemokine


receptor expression

DARC

46 bp

GATA1

Loss

(Tournamille et al., 1995)

Familial combined hyperlipidemia

LPL

39 bp

OCT1

Loss

(Yang et al., 1995)

Bernard-Soulier syndrome

GP1BB

133 bp

GATA1

Loss

(Ludlow et al., 1996)

Osteoporosis

COLlAl

Sp1

Gain

(Grant et al., 1996)

Maturity-onset diabetes of the


young

HNF1A

58 bp

HNF4A

Loss

(Gragnoli et al., 1997)

Asthma

IL10

509 bp

YY1

Gain

(Hobbs et al., 1998)

Pyruvate kinase deficiency

PKLR

72 bp

GATA1

Loss

(Manco et al., 2000)

Congenital erythropoietic porphyria

UROS

70 bp;

GATA1; CP2

Loss

(Solis et al., 2001)

Psoriasis

SLC9A3R1

237 bp

RUNX1

Loss

(Helms et al., 2003)

Systemic lupus erythematosus

FASLG

844 bp

CEBPB

Loss

(Wu et al., 2003)

Esophageal cancer

COX-2

1195 bp

c-MYB

Gain

(Zhang et al., 2005)

Treacher Collins syndrome

TCOF1

346 bp

YY1

Loss

(Masotti et al., 2005)

Alpha-thalassemia

HBA

13 bp

GATA1

Gain

(De Gobbi et al., 2006)

Holoprosencephaly

SHH

460 kb

SIX3

Loss

(Jeong et al., 2008)

Various cancers

TERT

187 bp

ETS2

Loss

(Xu et al., 2008)

Nonsyndromic cleft lip

IRF6

14 kb

AP2

Loss

(Rahimov et al., 2008)

Pierre Robin syndrome

SOX9

1.44 Mb

MSX1

Loss

(Benko et al., 2009)

Prostate cancer

MYC

200 kb

FOXA1

Gain

(Jia et al., 2009)

Colorectal cancer

MYC

300 kb

TCF7L2

Gain

(Tuupanen et al., 2009)

Asthma and autoimmune diseases

ZPBP2;
GSDMB;
ORMDL3

5 kb; +44 kb; +54 kb

CTCF

Loss

(Verlaan et al., 2009)

Myocardial infarction

SORT1

44 kb

CEBPA

Loss

(Musunuru et al., 2010)

Beta-thalassemia

HBB

71 bp

GATA1

Loss

(Al Zadjali et al., 2011)

Coagulant factor VII deficiency

F7

60 bp

HNF4A

Loss

(Zheng et al., 2011)

Osteoarthritis

GDF5

41 bp

YY1

Loss

(Dodd et al., 2013)

Breast cancer

CCND1

127 kb;

ELK4; GATA3

Loss; Gain

(French et al., 2013)

Melanoma, various cancers

TERT

+2bp;

ETS2

Gain

(Horn et al., 2013)


(Huang et al., 2013)

Increased cancer susceptibility

KITLG

+20 kb

Hirschsprung disease

SOX10

Insulin resistance

PPARG2

Type 2 diabetes and proinsulindecrease

ARAP1

Melanoma

SDHD

25 bp;

Pancreatic agenesis

PTF1A

25 kb

Acute lymphoblastic leukemia

TAL1

7.5 kb

Obesity and Type 2 diabetes

IRX3; IRX5

0.5 Mb;

Colorectal cancer

FASLG

1377 bp;

540 Cell 166, July 28, 2016

+2 kb

90 bp

76 kb

66 bp;

88 bp

P53

Loss

(Zeron-Medina et al., 2013)

30 kb

AP2; SOX10

Loss

(Lecerf et al., 2014)

6 kb

PRRX1

Loss

(Claussnitzer et al., 2014)

PAX6/PAX4

Loss

(Kulzer et al., 2014)

EHF, ELF1 & ETS1

Loss

(Weinhold et al., 2014)

FOXA2, PDX1

Loss

(Weedon et al., 2014)

MYB

Gain

(Mansour et al., 2014)

ARID5B

Loss

(Claussnitzer et al., 2015)

SP1; STAT1

Loss

(Wang et al., 2016)

+418 bp
7 bp;

4 bp

1.2 Mb
670 bp

Figure 1. A Methodological Workflow for Identifying Regulatory Variants


Sequence-based, computational methodologies that evaluate the impact of potential regulatory variants on TF-DNA binding and downstream regulatory processes are schematically presented. For every putative variant (SNV, as in this example, or indel), a reference and alternate (containing the variant) sequence of
pre-defined length (illustrated by the distinct shades of gray) is extracted. The chosen length defines the sequence environment and varies according to the
type of model that is used. The middle yellow panel shows the common workflow, where both sequences are scored (SALT and SREF) according to a specific model
representation to obtain a differential score (DS) that may indicate a change in DNA binding or more generally in chromatin state. As shown, DS supports a model
in which the variant impacts a gene regulatory process. The bottom part of the figure illustrates the two main strategies that are employed for modeling the
regulatory effect of a variant. The choice of the strategy depends on the posed question: does the variant impact (1) the binding of a TF (left) or (2) the local
chromatin landscape (right)? In the first scenario, computational methods are used that depend on the availability of a comprehensive catalog of TF binding
sequences or motifs (Box 1). The de novo motif discovery part schematizes the procedure that is required to obtain such a catalog, illustrating the use of
sequence over-representation strategies that are applied on both in vivo (ChIP-seq, DHS-seq, etc.) and in vitro (e.g., PBM, HT-SELEX, or B1H) derived datasets.
These strategies then produce TF motifs that can be represented either in regular expression format or using PWM- or HMM-based models. In this example, the
linear HMM model is a generic representation of the PWM motif, with each node (state) of the HMM representing the position of a base in the motif. Additionally, a
second HMM model is depicted, which inherently takes a variable space within the motif into account, for accurate representation of more complex binding
scenarios (e.g., TF dimers). To answer the second question (lower-right), computational methods mainly rely on machine learning models that are trained on a
wide variety of features such as a k-mer vocabulary built on regulatory versus background sequences or additional (epi)genomic datasets. These more elaborate
models can also be used to score the two input sequences. The pipeline then evaluates the regulatory nature of the variant by directly assessing the differential
score DS or by calculating a p value based on the distribution of the scores. Of note, this pipeline can be applied multiple times on different variants, after which
the results can be aggregated and compared to prioritize variants.

Cell 166, July 28, 2016 541

Box 1. How Many Human TFs Have Assigned Motifs?


While seemingly straightforward, it turns out to be difficult to precisely enumerate the number of TFs with defined motifs. There is no consensus on
the number of TF-coding genes in the human genome. A comprehensive, manual curation primarily based on the presence of sequence-specific
DNA binding domains revealed 1,391 high-confidence genes, with another 216 listed as plausible (Vaquerizas et al., 2009). However, this list
may not be exhaustive as a protein microarray-based survey of DNA binding capacity revealed that hundreds of proteins among 3,000 that
were not annotated as TFs were able to bind to DNA in a site-specific manner (Hu et al., 2009). Thus, additional experimental efforts will be required
to derive a more precise estimate of the number of TFs that the human genome encodes. Therefore, we need to simplify the question to how many of
the 1,400 high-confidence TFs have annotated motifs. A very recent, expansive analysis involving almost 1,000 high-quality ChIP-seq and 542 HTSELEX datasets produced binding site models for 601 human TFs that are retrievable from the HOCOMOCO database (Kulakovskiy et al., 2016).
Why only 40% of human TFs feature experimentally derived motifs despite the development of powerful DNA binding characterization technologies such as PBM (Berger et al., 2006), bacterial one-hybrid (B1H) (Meng et al., 2005), or HT-SELEX (Jolma et al., 2010) is unclear but may largely
be due to technical limitations, including loss of DNA binding properties or weak in vitro expression of full-length proteins. This is why most in vitro
DNA binding assays rely on analysis of the DNA binding domains (DBDs) of TFs, as these are easier to work with in terms of cloning and expression
while exhibiting DNA binding properties that appear largely comparable to the respective full-length protein versions (Jolma et al., 2013).
The largest TF families that still resist a comprehensive characterization are the high-mobility group (HMG) TFs and C2H2 zinc finger proteins (Jolma
et al., 2013), of which the human genome encodes more than 700 (Weirauch and Hughes, 2011). Progress is being made though, as illustrated by a
recent study that combined PBMs and B1H assays to probe thousands of individual C2H2 zinc finger domains with the aim of inferring a specific
DNA recognition code (Najafabadi et al., 2015). The resulting motifs proved to be highly diverse in terms of nucleotide composition and exhibited
extensive degeneracy, which means that these motifs can be represented by many different sequences and that small internal perturbations in these
motifs tend to have little impact on DNA binding. Consequently, there is still ample room for alternative approaches or technologies that will enable
the further expansion or fine-tuning of the current catalog of human TF PWMs, also named the human regulatory lexicon.
Nevertheless, it may not be necessary to gather experimental data for all TFs, given that many have nearly identical DNA binding properties because
their DBDs are highly similar. Indeed, TFs (independent of organism) whose DBDs share >87.5% of their amino acids were found to bind to motifs
that were almost indistinguishable from one another (Weirauch et al., 2014). Applying this principle to human TFs adds another 200 inferred motifs to
the current catalog, which can be found in the Cis-BP database (Weirauch et al., 2014). In sum, the DNA binding properties of a significant fraction of
human TFs remain uncharacterized without even taking into account heterodimer or higher-order complex formation (see main text).

TFs may still be able to bind to this core motif, albeit with much
lower affinity. This may, in part, explain the observed discrepancy between in vivo DNA occupancy levels and in-vitro-derived
DNA binding affinities (Biggin, 2011), since these in vivo binding
events may reflect binding by interacting TF pairs and not individual TFs. It is therefore clear that a large portion of motifs
remain to be characterized, emphasizing the need for new technologies or efforts to close this gap.
Imputing DNA Binding Variation
It has often proven difficult to infer whether a specific polymorphism will significantly change TF-DNA binding and act as a
regulatory variant, even if the PWM model of the TF is available.
This complication stems from difficulties in capturing the
DNA binding complexity of a TF in a robust binding model either
to confidently detect a genuine binding site within a given
sequence or to accurately infer the impact of a variant on detected motifs.
The Accuracy of Binding Models and Robustness of Motif
Detection. The majority of motif detection methodologies rely
on PWM representation since PWMs perform relatively well
with respect to capturing the overall binding affinity. This is
because PWMs can be modeled as a numerical matrix, which
enables the scoring of a given sequence according to its similarity to a motif (Figure 1). Nevertheless, it is important to acknowledge that this model also has several limitations, which may
impede the discovery of the correct binding patterns. For
example, PWM models assume that the nucleotide binding energies are independent (Stormo and Zhao, 2010), which proved
not to be generally valid (Bulyk et al., 2002; Jolma et al., 2013;
Maerkl and Quake, 2009; Nutiu et al., 2011), and are also suboptimal to represent the binding of TF dimers, since many of these
542 Cell 166, July 28, 2016

bind to two sequences that are separated by a spacer with variable length. These caveats have spurred the development of
different models for representing TF motifs, such as hidden Markov models (HMMs) (Gelfond et al., 2009; Zhao et al., 2005) and
more advanced machine learning models, stimulated by the
increasing availability of multiple layers of genomic, transcriptomic, and epigenomic information. Among these are support
vector machine (SVM) or neural network (NN) approaches that
are trained on datasets containing both known regulatory and
random sequences, with the goal of recognizing and scoring
new putative regulatory sequences (Gao and Ruan, 2015)
(Figure 1 and Table S1). Such representations have many advantages over conventional models because they are highly flexible.
In addition, they are not limited to the DNA sequence recognized
by the TF and can incorporate additional features that are also
important to model TF-DNA binding. These features include
the 3D structural conformation of DNA and its steric characteristics (Levo and Segal, 2014; Rohs et al., 2009; Zhou et al., 2015),
the chemical properties used to model TF amino acid-nucleotide
contacts at the atomic level (Bauer et al., 2010; MaienscheinCline et al., 2012), protein concentration (Djordjevic et al.,
2003; Wang and Batmanov, 2015) that allows for a more accurate estimation of DNA occupancy and thus intrinsic DNA binding affinity (Biggin, 2011; Simicevic et al., 2013), and, finally,
the nucleotide composition of motif-neighboring sequences.
Indeed, recent work revealed that the sequence environment
of a genuine binding site tends to be distinct from that of unbound sequences. In particular, it was shown to exhibit specific
sequence features such as high GC content (White et al., 2013)
or a higher similarity to the core motif (Dror et al., 2015) that
may guide TFs to their cognate binding sites. These findings

have important consequences in terms of predicting DNA


binding events, since motif-scanning tools typically penalize for
local nucleotide composition biases. Instead, a better practice
may now involve rewarding motifs that are surrounded by established DNA binding-promoting features, such as a high GC fraction or lower-scoring and thus weaker homotypic (i.e., similar)
motifs. Together, these studies illustrate that the formulation of
DNA binding models and computational detection of genuine
binding sites is far from trivial and that further efforts aimed at
integrating a wide range of genomic datasets will be required
to increase the robustness of motif definition and mapping
approaches.
The Complexity of Correctly Inferring the Effect of Motif Variation on TF-DNA Binding. Genetic variants that change a TF motif
often affect the binding ability of a TF to that site because of an
altered DNA binding affinity (Table 1 and Figure 1). Initial efforts
to computationally predict relevant regulatory variants simply
revolved around the consideration of all SNPs that overlap with
TF binding sites (Ameur et al., 2009; Chorley et al., 2008; Ponomarenko et al., 2001). However, given the degenerate nature of
binding motifs (i.e., binding is not binary but is variable depending on different sequences), these kinds of analyses tend not to
provide good sensitivity. A more refined approach in this regard
is to analyze the difference in DNA binding affinity (for example,
scored using a PWM) between two alleles, i.e., the reference and
the alternate impacted by the variant (Figure 1 and Table S1). The
greater this difference, the greater the predicted impact of the
variant on binding of the respective TF and thus also the greater
the likelihood of it being causal.
More recent machine learning methods no longer depend on
the use of a strict motif database and directly infer regulatory
effects from k-mer vocabularies trained on ChIP-seq or other
experimental data. These vocabularies consist of all possible
DNA sequences of length k that collectively capture specific
sequence properties of certain regulatory elements such as
cell-type-specific enhancers. This methodological development
stems from the general appreciation in the field that the motif
alone cannot accurately predict differential DNA binding and
thus should be complemented (or even replaced) with information on the sequence environment around the focal variant, as
well as on other DNA or chromatin features that enhance the
models overall predictive power (as already covered in the previous section). Indeed, it is now well accepted that only a minority
of motif-disrupting variants effectively result in altered DNA binding of the respective TF (Heinz et al., 2013; Kilpinen et al., 2013;
Maurano et al., 2015; Spivakov et al., 2012). One possible explanation is based on the finding that, across the genome, TF motifs
appear to occur in clusters with some built-in redundancy (Gotea
et al., 2010), in line with the observation that the sequence
environment of relevant TF binding sites tends to have a certain
similarity to the core motif (Dror et al., 2015). These clustered
sites may buffer genetic perturbations that affect one of the
motifs. Indeed, the greater the number of such homotypic motifs,
the greater the buffering effect (Kilpinen et al., 2013). Given
the pervasive nature of this buffering phenomenon (Maurano
et al., 2015), the failure to take such neighboring homotypic
motifs into account may result in false TF-DNA binding event
predictions.

More generally, epigenomic properties such as nucleosome


location (Soufi et al., 2015) or density (Barozzi et al., 2014) or
DNA methylation (Domcke et al., 2015) may impact the ability
of a TF to bind to a certain DNA sequence. Upon screening
1,300 human TFs for their ability to bind to one of 150 distinct
CpG-containing motifs, 47 were found to bind to DNA with
several exhibiting methylation-specific DNA specificities (Hu
et al., 2013). This is consistent with an earlier study demonstrating that the TF Kaiso is capable of binding not only to an
unmethylated motif, TCCTGCNA, but also to a methylated,
clearly distinct palindromic motif, TCTmCGmCGAGA, with
even greater affinity (Raghav et al., 2012). How frequently such
methylation-dependent changes in DNA binding occur and the
extent to which other DNA modifications affect DNA binding
specificities is still a matter of debate. Nevertheless, it is clear
that it adds another complexity in linking DNA variation to variable TF binding.
Ongoing computational studies are attempting to take these
complexities into account by implementing big data analyses
that are creating extended machine learning models that rely on
multilayered information of different types of genomic data,
including TF motifs, DNase hypersensitivity sites (DHS), chromatin marks, etc. (see, for example, Table S1). As such, they
can recognize regulatory regions based not only on pure
sequence information, but also on the chromatin state of the
DNA both at the variant locus as well as at neighboring regions.
Once correctly trained, these approaches can be very precise
and predict causal variants and their effects at distinct molecular
levels (Alipanahi et al., 2015; Zhou and Troyanskaya, 2015)
(Figure 1).
However, it is important to emphasize that their performance
depends not only on the diversity of input data, but also on the
correct selection of relevant features. For example, it has been
repeatedly shown that it is crucial to gather data that are specific
to the variant-linked trait or disease in terms of cell type, differentiation stage, tissue, or species since regulatory activity is variable and context dependent (Consortium, 2012; Maurano
et al., 2015). Another limitation of these extended representation
models that may dampen their widespread implementation is
their inherent black box nature. Indeed, most of the binding
patterns that were unveiled by these techniques are difficult to
interpret, especially when no visual representation is provided.
However, despite these caveats, advanced models have the
potential of uncovering completely novel and potentially unexpected cross-mechanisms that more standard methodologies
may fail to grasp.
TF-DNA Binding Is Itself a Complex, Molecular Trait
We are currently limited in our ability to predict TF binding as well
as in our understanding of how genetic variation impacts on this
process. Nevertheless, there is general consensus that differential, regulatory control by TFs is a major driver of phenotypic variation. A key aspect of this regulatory variation is variable TF-DNA
binding. It is in this regard intriguing that only a minority of variable TF binding events are driven by nucleotide changes in the
motifs of the studied TFs. For example, upon assessing binding
variation of the TF NFKB in ten distinct human lymphoblastoid
cell lines (LCLs), only 79 out of >1,100 variable TF-DNA binding
Cell 166, July 28, 2016 543

Figure 2. Distinct Modes of Genetic Variation-Mediated Changes in TF-DNA Binding


(A) Only a minority of variable TF-DNA binding events are caused by DNA variants disrupting the cognate TF recognition motif.
(BD) The majority of variably binding events are motif variation independent, signifying that a variant located either proximally (<200 bp, B and C) or distally (D) to the
focal motif affects the binding of the respective TF. Proximal variants can affect local cooperative DNA binding (B), which involves physical protein-protein interactions that require overlapping or very closely located (a few bp) motifs, or collaborative DNA binding (C), which reflects TF interdependencies needed, for
example, to compete with nucleosomes and thus to access DNA. In contrast, distal variants (D) may alter chromatin state or conformation (e.g., DNA loops), which
could affect the stability of interactions with DNA and between TFs.

events had a SNP directly located in the NFKB motif and induced
a binding difference that was consistent with its perceived
impact on motif quality (i.e., reduced binding was linked to a
SNP that lowered the PWM binding score and vice versa)
(Kasowski et al., 2010). One of the possible reasons that were
listed (next to LD or putative epigenomic variation) involved
trans-effects. However, ChIP-seq analyses of >20 TFs revealed
extensive, allele-specific DNA binding (in a constant trans
environment), effectively refuting this hypothesis (Reddy et al.,
2012). Subsequent studies in human LCLs and in cells or
tissues derived from distinct mouse strains observed a similar
pattern (Heinz et al., 2013; Kilpinen et al., 2013; Soccio et al.,
2015; Stefflova et al., 2013), collectively emphasizing the importance of cis-regulatory variation. Importantly, only a minority
of differential allelic occupancy events involved nucleotide
changes in the respective motifs (Reddy et al., 2012). However,
this does not mean that variation in the motifs of other TFs
should also be dispensed as a possible molecular mechanism for these observationsquite the contrary, in fact, as we
will clarify in greater detail in the next paragraphs (see also
Figure 2).
If a particular genetic variant does not affect the motif of the
studied TF, what then causes the respective TF to exhibit differential DNA binding? It appears that an important fraction (at least
7.5% according to our own estimate [Kilpinen et al., 2013]) of
variable TF-DNA binding events can be explained by alterations
of proximal motifs (Reddy et al., 2012). Thus, at some genomic
sites, TFs appear to be dependent on the proximal presence of
other TFs to bind to DNA. Qualitative motif analysis combined
with prior knowledge about the biological process in which the
focal TF is operational lends credibility to this notion. For
example, in mouse white adipose tissue, PPARg binding sites
that vary between strains and do not harbor an altered PPARg
motif were analyzed for enriched, polymorphic motifs. The topscoring motifs corresponded to the TFs CEBPa and glucocorticoid receptor (Soccio et al., 2015) that exhibit extensive
co- localization with PPARg in mature white fat cells (Siersbk
et al., 2014). Similarly, differential PU.1 binding correlated with
544 Cell 166, July 28, 2016

alterations in the motifs for the TFs CEBP and AP-1, which
modulate macrophage activity (Heinz et al., 2013). However,
this correlation appears to differ according to macrophage subtype. Indeed, a follow-up study in mouse microglia revealed that
other TF motifs correlate better with variable PU.1 DNA binding,
emphasizing the importance of cellular context in determining
this type of TF interactions (Gosselin et al., 2014). Together,
these studies strongly support the notion of pervasive DNA binding whose occurrence is dependent on the presence of other
TFs. Since it is well appreciated that regulatory regions tend to
harbor binding sites for multiple TFs, this notion may not be
entirely surprising. Nevertheless, it is worthwhile in the current
context of genetic variation to briefly revisit this mode of DNA
binding, which is interchangeably called cooperative or collaborative DNA binding (Gosselin et al., 2014; Mirny, 2010; Slattery
et al., 2014; Waszak et al., 2015). We would thereby like to argue
that, for the sake of discussion and molecular understanding, it
might be valuable to differentiate between these two terms
(Figure 2).
Local, Cooperative TF-DNA Binding
In the context of protein-DNA interactions, cooperativity was
initially used in describing the assembly of E. coli lambda repressors on DNA (Ptashne et al., 1980). Binding of a lambda
dimer on a first operator site facilitates binding of another
lambda dimer on the second operator site, given that physical
interactions between the first and second dimer increase the
affinity of the latter for DNA, which explains why cooperative
DNA binding is evoked to define this process. Consequently,
the term cooperativity may be especially suited for DNA binding
processes that involve TFs whose physical interactions at the
protein level may increase the affinity of the entire complex to
specific sites in the genome. For example, binding of the winged
HTH DNA binding domain-containing TF IRF4 is cooperatively
enhanced by the TF PU.1 (Escalante et al., 2002). This is
because binding of the two TFs contorts the DNA in a peculiar
S shape, placing the TFs in an optimal position for electrostatic
and hydrophobic interactions and thus stabilizing the entire
complex (Escalante et al., 2002). Consequently, individual

nucleotide alterations in one of the two binding sites may alter


the extent of cooperativity between two heterodimerizing TFs,
as has recently been quantified for the PPARg-RXRa heterodimer (Isakova et al., 2016). This, in turn, illuminates why the
disruption of either of the two TF motifs tends to affect binding
of the respective heterodimer.
Proximal, Collaborative TF-DNA Binding
For TFs to physically interact on DNA and thus for cooperative
DNA binding to occur, one would intuitively expect that the
respective motifs would be located very close to one another
or would even overlap. However, DNA binding relationships exist
between TFs whose motifs are separated tens, hundreds, or
even thousands of base pairs from one another. For example,
upon examining which TFs (based on motif matches) associated
with NFKB binding enrichment (based on ChIP-seq data), EBF1
and STAT1 were among the most correlated TFs (Karczewski
et al., 2011). Interestingly, this covariation signal of EBF1 and
STAT1 motifs within variable binding regions of the TF NFKB remained significant up to 500 bp from the NFKB binding peak center, suggesting that DNA binding dependencies between these
TFs were maintained over a relatively long distance. It is now
increasingly appreciated that many such dependencies do not
require direct contacts but instead reflect a relatively well-understood phenomenon termed collaborative DNA binding in which
two or more TFs compete with a nucleosome to access DNA
(Biggin, 2011; Mirny, 2010; Spitz and Furlong, 2012) (Figure 2).
Given that the intrinsic affinity of a nucleosome for DNA is
much greater than that of a TF alone (Polach and Widom,
1996), it may often require two or more collaborating TFs to
displace the nucleosome. In this scenario, TFs would be mutually
dependent, and this is indeed what is observed. For example,
HNF4A and CEBPa functioning in mouse liver exhibit a mutual
dependency, given that loss of HNF4A affected CEBPa DNA
binding and vice versa, whereas the absence of HNF4A did not
impact on the DNA binding dependency between CEBPa
and FOXA1 (Stefflova et al., 2013). A similar DNA binding interdependency was found between PU.1 and CEBPa in primary
macrophages (Heinz et al., 2013). It is worth noting that such
nucleosome-mediated, collaborative DNA binding could still be
regarded as a form of indirect, cooperative DNA binding, as
modeling has revealed an analogy between this process and
the one involving cooperative binding of oxygen to hemoglobin
(Mirny, 2010). Nevertheless, to avoid confusion, it may be best
to continue to define this process as collaborative DNA binding.
Interestingly, several of these collaborating TFs have previously been defined as pioneer TFs that are uniquely able to access and open silent or compacted chromatin (Iwafuchi-Doi
and Zaret, 2014). The fact that, at a wide range of loci, they are
nevertheless dependent on other TFs to access DNA constitutes
in this regard an intriguing paradox. For example, FOXA1 is
defined as an archetypical pioneer TF (Mancini and West,
2015), given its ability to open closed chromatin by binding to
DNA with its core DNA binding domain and to core histones
with a binding motif that is located in its C terminus (Cirillo
et al., 2002). However, its DNA binding interdependency with
CEBPa or potentially other TFs suggests that FOXA1s pioneering ability may often not be sufficient to allow DNA binding,
implying that FOXA1 requires the cumulative contribution of

other TFs to displace nucleosomes and successfully unlock


chromatin. This model may be consistent with the dispensability
of FOXA1 and the related TF FOXA2 in maintaining the chromatin
state in liver cells (Li et al., 2012). Similarly, PU.1 is also recognized as a pioneer factor for its ability to promote nucleosome
depletion (Barozzi et al., 2014; Heinz et al., 2010), yet it depends
on CEBP TFs to bind DNA at many genomic sites. What emerges
is that, at some loci, these TFs may act as true pioneer TFs,
whereas at others, they may require collaborations with other
TFs to open chromatin.
Consequently, it is of interest to better understand what distinguishes genomic sites with pioneer activity from collaborative
ones. An interesting observation in this regard is that regions
with high PU.1 occupancy in primary macrophages had, in general, similar motif scores to those with lower PU.1 binding, but
the two types of regions differed in nucleosome organization
(Barozzi et al., 2014). Specifically, the latter regions were surrounded by two nucleosomes (in contrast to sites with high
PU.1 binding) and showed enrichment for the NFKB motif, suggesting that, at those regions, PU.1 and NFKB need to collaborate to outcompete nucleosomes and thus to achieve high
DNA occupancy. As such, TFs may have locus-dependent TF interdependencies reflecting both nucleosome structure and the
presence of distinct TF motif clusters, consistent with the dependency of PU.1 on NFKB at some sites or either OCT2, BLIMP1,
or STAT2 at others in human LCLs (Kilpinen et al., 2013). This
view is also consistent with the flexible binding site grammar
that is typically observed in enhancers in that the position of individual TF motifs within enhancers tends to be of secondary
importance to their simple presence (Arnosti and Kulkarni,
2005). In other words, since collaborative DNA binding does
not require physical contacts, the spacing and orientation of motifs can be flexible with respect to preserving enhancer activity,
as long as the motifs are intact. This also implies that, at collaborative genomic sites, TFs should in principle bind to DNA in
seemingly joint fashion since loss of one TF-DNA interaction
(either in cis [e.g., because of a DNA mutation] or in trans
[because of TF dysfunction]) would reduce the binding capacity
of all other TFs at this locus. Such collective DNA binding
behavior has indeed been observed for TFs mediating heart
development in Drosophila melanogaster (Junion et al., 2012),
and evidence for simultaneous, collaborative TF-DNA binding
is also available for mammals (Adam et al., 2015; Siersbk
et al., 2014; Tijssen et al., 2011). A final illustration of this important notion is the dependency of the pioneer TF NRF1 (Sherwood
et al., 2014) on other TFs to keep a specific set of its target sites
from being methylated, which otherwise would block NRF1 DNA
binding to these sites (Domcke et al., 2015). This example again
illuminates how sequence context may affect the ability of a TF to
bind independently to DNA, even if this TF may normally act as a
pioneer factor. In sum, based on the currently available data,
care needs to be taken when classifying TFs into specific categories without considering sequence and chromatin context.
Variable Chromatin Modules Mediate Long-Range
TF-DNA Binding Interdependence
The previous sections highlighted that proximal variants can
affect DNA binding through cooperative or collaborative mechanisms. However, many of the variants that drive TF-DNA binding
Cell 166, July 28, 2016 545

variation are located beyond the sequence span that is required


for the formation of local TF-TF interactions or for competition
with local nucleosomes, respectively. One possibility is that proteins overcome this distance restraint by inducing DNA looping
through physical interactions. Even though this is an energetically costly process (Saiz and Vilar, 2006), both short- and
long-range looping have now been extensively documented
(de Wit et al., 2013; Gheldof et al., 2010; Lieberman-Aiden
et al., 2009; Rao et al., 2014; Saiz and Vilar, 2006) and may
play an important yet poorly understood role in mediating longdistance TF-DNA binding interdependencies.
It is therefore valuable to explore approaches to study the molecular origin of both short- and especially long-range TF-DNA
binding variation. One such approach is the identification of genetic polymorphisms that significantly correlate with changes in
DNA occupancy. Genomic regions in which such variants are
located are interchangeably termed TF or binding quantitative
trait loci (tfQTLs or bQTLs), as their detection suggests that a
polymorphism within this locus causally affects the ability of a
TF to bind to DNA. One study adopting this approach aimed to
identify variants that affect DNA binding of the insulator protein
CTCF by profiling its binding landscape in human LCLs using
ChIP-seq, after which tfQTLs were explored within a 50 kb region
centered around the CTCF binding region (Ding et al., 2014).
Only a minority of detected tfQTLs overlapped the CTCF motif,
even when the local LD structure was taken into account. A
similar picture emerged from a comparable study on PU.1
DNA binding variation also in human LCLs, since PU.1 tfQTLs exhibited a bimodal log-normal distribution in terms of their distance to the PU.1 binding region (Waszak et al., 2015). The first
mode represented tfQTLs that were located close to or at the
PU.1 binding site and, consistent with the CTCF study, encompassed only a minority of the significantly associated variants.
The second mode featured tfQTLs that were located distally to
the PU.1 binding region with a median distance between 20
and 30 kb. Together, these findings suggest that many variable
CTCF or PU.1 binding events are driven by long-distance mechanisms, which renders TF-DNA binding a complex molecular
trait by itself. Consequently, and even though the effect size of
distal tfQTLs tends to be inferior to that of proximal ones (Waszak
et al., 2015), it will be valuable to decipher how these distal variants affect TF-DNA binding, given that they constitute the majority of DNA binding QTLs.
In the LCLs, PU.1 binding variation often correlated with variation in active chromatin marks such as H3K4me1 or H3K27ac
not only locally, but often over extended distances (Waszak
et al., 2015). That is, high PU.1 DNA occupancy coincided with
both high proximal and distal H3K4me1 and H3K27ac enrichment and vice versa. Such regions with a high level of molecular
coordination between TF and chromatin marks have recently
been termed variable chromatin modules (VCMs; Waszak
et al., 2015; Figure 3A). Each VCM is thus composed of molecular phenotypes (e.g., the level of DNA occupancy by a TF or
enrichment for a specific chromatin mark) that are highly coordinated, often over multiple kbp of DNA. More than 14,000 distinct
VCMs were discovered in human LCLs, covering about 5% of
the genome (Waszak et al., 2015). The majority of these were totem VCMsso named because they were composed of
546 Cell 166, July 28, 2016

stacked or overlapping molecular phenotypes that did not correlate with other neighboring molecular phenotypes. Thus, a totem
VCM represents local chromatin state variation (Figure 3A). The
remaining multi-VCMs are more interesting since, while a
minority, they typically cover two or more distinct regulatory elementshence the term multiand capture the majority of all
detected molecular phenotypes (Figure 3A). The origin of a
multi- VCM is less intuitive than that of a totem-VCM. Its structure suggests, however, a higher-order chromatin organization
that is reminiscent of the modular genomic structure that has
been uncovered in the form of topologically associating domains
(TADs) (Dixon et al., 2012; Nora et al., 2012).
These TADs constitute distinct, three-dimensional genomic
structures in which sequences are more likely to interact with
one another than with those located outside the respective
TAD. However, VCMs and TADs constitute different molecular
entities because VCMs tend to be embedded within TADs and
thus tend to be smaller (Waszak et al., 2015) (Figure 3B). In addition, TADs are relatively stable across cell types and during
development and are even conserved across species (Dixon
et al., 2012; Vietri Rudan et al., 2015), whereas VCMs are by definition variable. As such, multi-VCMs correspond conceptually
better to sub-TADs, which are more fine-grained (sub-Mb),
genomic topologies that have been shown to be dynamic across
cellular differentiation (Dixon et al., 2015; Phillips-Cremins et al.,
2013) and to even differ between individual cells (Giorgetti et al.,
2014). In addition, sub-TADs have been suggested to define cisregulatory networks (Berlivet et al., 2013), with their internal
conformational dynamics being directly related to embedded
transcriptional activity (Giorgetti et al., 2014; Tang et al., 2015).
In parallel, the vast majority of gene-associated multi-VCMs exhibited a molecular activity state that significantly correlated with
the transcriptional activity of the included gene(s) (Waszak et al.,
2015) (Figure 3B). Moreover, the more regulatory elements
encompassed in a VCM, the more likely it was to associate
with variable gene expression. Together, the conceptual similarities between sub-TADs and multi-VCMs suggest that the latter
also reflect fine-grained configurations of interacting regulatory
elements with one or a few target genes whose collective, molecular activity is highly coordinated. As such, VCMs may
provide substantial insights into the structural and thus modular
organization of the chromatin landscape, including TF-DNA
interactions.
Which mechanisms lie at the origin of multi-VCMs? Since the
long-range molecular coordination that typifies multi-VCMs has
been observed at the allelic level (Kasowski et al., 2013; Kilpinen
et al., 2013; McVicker et al., 2013) and since recent chromatin
interaction analysis by paired-end tag sequencing (ChIA-PET)
data has also provided evidence for allele-specific chromatin topologies (Tang et al., 2015), it is reasonable to assume that the
observed molecular variation is largely driven by genetic factors.
Moreover, most of the molecular variation within each VCM
could be captured by a single, quantitative phenotype (Waszak
et al., 2015), which suggests that the activity state of a VCM
can be attributed to relatively few but strong causal variants.
QTL mapping using the activity state of each VCM as input
yielded vcmQTLs that were highly enriched in TF-occupied regions (Waszak et al., 2015) (Figure 3A). Together with previous

Figure 3. Variable Chromatin Modules


(A) Correlated TF (e.g., PU.1 or RNA polymerase II [PolII]) binding and chromatin mark (e.g., H3K27Ac, H3K4me1, H3K4me3) enrichment analyses across individuals allows the mapping of variable chromatin modules (VCMs) (shown in light green in the upper panel and in network format in the panel below). VCMs
thus embody variable regions with highly coordinated, molecular phenotypes.
(B) The majority of VCMs have a totem structure of stacked molecular phenotypes that do not correlate with other neighboring molecular phenotypes and, as
such, reflect local chromatin state variation. Multi-VCMs encompass sub-Mb regions involving distinct regulatory elements whose activity is highly coordinated
and driven by a single or a few highly penetrating variants (vcmQTL) with enrichment in TF-bound regions.
(C) VCMs constitute functional entities of higher-order chromatin organization embedded within topologically associating domains (TADs) and provide a molecular rationale as to how TF-DNA binding can be affected by distal genetic variation.

Cell 166, July 28, 2016 547

observations that TF-DNA binding perturbations are initiating


drivers of downstream changes in chromatin state and gene
expression (Kasowski et al., 2013; Kilpinen et al., 2013; McVicker
et al., 2013) (Table 1), these findings support a model in which the
alteration of one or a few TF binding events affects all molecular
phenotypes in the respective VCM, including other embedded
TF-DNA interactions (Figure 3B). Thus, the ability of a TF to
bind to a VCM-associated genomic region appears to be a function of the respective VCMs activity state, which itself seems
determined by one or few key TFs. As such, VCMs provide a conceptual framework to rationalize how distal genetic variation can
affect TF-DNA binding.
Defining these key TFs remains a work in progress, since only
few TFs reached significant enrichment in terms of their overlap
with vcmQTLs (Waszak et al., 2015). This suggests that each
VCM may have its own set of activity-determining TFs. Interestingly, distinct pairs of these same TFs were also enriched at pairs
of regulatory elements that belonged to the same VCM (Waszak
et al., 2015), suggesting that the functional interactions between
these TFs (or among themselves) may be instrumental for forming VCMs. Together, the presented findings support a scenario
in which the activity of each VCM is driven by a set of cell- and
chromatin-context-specific TFs. This would be consistent with
TF-DNA binding being highly dependent on proximal sequence
environment and chromatin organization, which may differ
from one VCM to the next. In addition, it would be compatible
with the multiple enhancer variant hypothesis (Corradin
et al., 2014), which dictates that linked variants in distinct regulatory elements often jointly contribute to gene expression variation. The VCM landscape may, as such, also be compatible with
the LD structure of the genome.
What is the molecular nature of VCM-embedded TF-TF interactions? Based on the conceptual similarity between VCMs and
sub-TADs and on how canonical enhancer-promoter interactions are established (Ciabrelli and Cavalli, 2015), it is conceivable that they are mediated by either direct physical contacts
or by indirect protein-protein interactions involving more generic
factors such as mediator, CTCF, and cohesins (Dekker and
Mirny, 2016). The latter proteins may function to stabilize the
interactions both with DNA and between TFs such that
distal DNA binding interdependencies arise. However, other
interaction-independent mechanisms could also underwrite
such interdependencies, including long-range, transcription-,
or repression-coupled chromatin remodeling processes (Hathaway et al., 2012; Smolle and Workman, 2013). Further experimentation will be required to elucidate the involvement and
contributions of key individual TFs or TF pairs in VCM formation.
From Causal Variant to Complex Phenotype
While the identification and characterization of a trait- or disease-associated variant that causally disrupts TF-DNA binding
is difficult, elucidating how it impacts on other potentially downstream molecular and biological processes may be equally if not
more challenging (Edwards et al., 2013). One intuitive strategy to
expand on the relatively few cases so far in which a causal relationship between molecular and phenotypic variation was established (Table 1) is the integration of other genetic or molecular
data to infer the functional consequences of the focal variant.
548 Cell 166, July 28, 2016

For example, distinct QTL datasets can be used to determine


whether the variant impacts not only TF-DNA binding, but also
the chromatin landscape, gene expression, or even other molecular phenotypes (Pai et al., 2015). The most common molecular
QTL analysis, involving the identification of variants that associate with gene expression changes (i.e., eQTLs), is highly informative in this regard. For six distinct human populations, the
most significant eQTLs were consistently found to overlap TF
binding sites (Auton et al., 2015), thus providing direct insights
into the identity of genes whose expression may be affected
by variable TF-DNA binding.
However, other layers of molecular phenotypesmore associated with regulatory functions and therefore often defined as
regulatory QTLscan also be associated with genotypes. These
include: (1) DNase I sensitivity (ds)QTLs that are strongly enriched in predicted TF binding sites in addition to being major determinants of gene expression variation (Degner et al., 2012);
(2) chromatin (c)QTLs or histone marks (hm)QTLs that are largely
concordant with TF-DNA binding and transcription (Grubert
et al., 2015; Waszak et al., 2015), and (3) methylation (m)QTLs
that also often exhibit a functional link with the other regulatory
QTLs (Banovich et al., 2014; Domcke et al., 2015; GutierrezArcelus et al., 2013; Heyn et al., 2013; McClay et al., 2015). Since
regulatory QTLs as well as eQTLs were found to be enriched in
complex trait or disease susceptibility variants (Albert and Kruglyak, 2015; Grubert et al., 2015; Nicolae et al., 2010; Waszak
et al., 2015), their joint analysis may reveal how specific perturbations triggered by causal genetic variants in a certain condition
or environment may first spread through transcriptional and
other molecular networks before affecting the cellular, tissue,
and finally organismal networks (Lehner, 2013; Mackay et al.,
2009).
An intriguing observation that emerged from such analyses is
that many regulatory QTLs do not overlap eQTLs, even if they
overlap other types of regulatory QTLs (Degner et al., 2012; Grubert et al., 2015; Waszak et al., 2015). This is consistent with the
well-established finding that many changes in TF-DNA binding
have no measurable effect on gene expression (Cusanovich
et al., 2014; Farnham, 2009). Thus, regulatory QTL analyses suffer from the same limitations as complex trait or disease susceptibility GWAS studies, i.e., difficulties in uncovering leading
causal variants among LD blocks or in reaching statistical significance without a high number of samples (Veyrieras et al., 2008).
Approaches that link chromatin organization to transcriptional
function such as ChIA-PET (Dowen et al., 2014; Tang et al.,
2015) or VCM mapping (Waszak et al., 2015) may in this regard
prove valuable, as they can provide a structural framework for interpreting regulatory variation. Indeed, as regulatory variants
tend to impact different layers of molecular phenotypes, it is
intrinsically valuable to know how these layers are coordinated
across distinct genomic domains. For example, many VCMs
were identified that consisted of active chromatin marks as
well as TF binding sites, even though an important portion of
such VCMs did not vary along with the expression of neighboring
genes. These VCMs, termed island VCMs (Waszak et al.,
2015), thus represent coordinated changes in TF binding and
chromatin state without measurable impact on gene expression.
Accordingly, QTLs for such island VCMs tend to overlap with

tfQTL and cQTLs, but not with eQTLs. There are several complementary hypotheses that could explain the existence of such
island VCMs, including (1) futile regulatory activity without
transcriptional consequences (Cusanovich et al., 2014; Farnham, 2009; Wasserman and Sandelin, 2004); (2) regulatory
redundancy, which prevents a gene-specific regulatory network
from collapsing even if one node or edge is impacted (Pai et al.,
2015), consistent with the shadow enhancer concept (Hong
et al., 2008); (3) regulatory regions that are not transcriptionally
operational, at least in the studied condition/cellular environment, which implies that the activity of these regions is tissue
specific. Indeed, if, in a hypothetical study, a complex trait-associated regulatory variant would be linked to an island VCM, it
might indicate that an incorrect system or context is being
studied, as its disconnection with gene expression is unlikely
to yield a cellular or organismal phenotype. This reasoning is
consistent with the observation across several studies that
GWAS variants tend to be most enriched for eQTLs in tissues
that are relevant to the phenotype (Emilsson et al., 2008; Nica
et al., 2010; Torres et al., 2014). However, in most cases, the
causal variants are obviously unknown a priori. To identify
them, it may prove valuable to, similar to eQTLs, map VCMs in
as many distinct cell types/tissues as possible. The resulting
set of VCMs may then provide guidance to both variant identification and characterization. Indeed, the most interesting candidates among the set of associated GWAS variants would be
those that impact not only on the chromatin topology (e.g.,
vcmQTLs) and state of the respective locus (e.g., cQTLs or
tfQTLs), but also on expression of the embedded gene. Once
identified, it should be relatively straightforward to detangle the
underlying molecular mechanisms since the coordinated, molecular phenotypes that make up the focal VCM should provide
clear insights into the flow of regulatory information, i.e., from
causal nucleotide over gene to ultimately cellular or organismal
phenotype.

DNA in machine learning approaches. In addition, it is increasingly appreciated that the chromatin context needs to be accounted for when searching for causal, regulatory variants and
that, in general, the use of cell types or systems that are most
relevant for the studied trait or disease will yield the best results.
It is also important to recognize that only a small fraction of all
variable TF-DNA binding events is actually driven by variation
within the motif of the studied TF. Thus, similar to gene expression, TF-DNA binding is a complex molecular trait by itself, which
has profound implications for our understanding of how regulatory variation arises.
Well-established concepts in the gene regulation field provide
an intuitive molecular foundation for local or proximal variantdriven DNA binding variation. Specifically, the former involves
cooperative DNA binding that is mediated by direct, physical
interactions between TFs, while the latter appears to be driven
by collaborative DNA binding that is likely reflective of sequenceor chromatin-context conditioned TF interdependencies to
displace nucleosomes and open chromatin. However, the mechanisms that underlie distal variant-driven DNA binding changes
are much less well understood (Figure 2). The identification of
3C-, ChIA-PET-, or VCM-based chromatin entities that link
structural information to transcriptional function is important in
this regard since they offer a molecular rationale to explain these
prevalent, long-range DNA binding dependencies. Sustained
efforts will therefore be required to unravel the modular structure
of the variable (epi)genome across a wide range of cells or
tissues. Thus, although many challenges remain, exciting progress is being made in elucidating the genetic basis of TF-DNA
binding variation that will undoubtedly improve our ability to
achieve a nucleotide-level understanding of the molecular mechanisms underlying many complex traits, including disease
susceptibility.

Conclusions
The fundamental discovery that most complex trait-associated
variants are located in non-coding, putatively regulatory regions
of the genome has focused the spotlight on TF-DNA interactions
as important mediators of phenotypic variation. Yet, to date,
relatively few examples are available in which a clear mechanistic relationship between TF-DNA binding variation and phenotypic variation was established (Table 1). To clarify why this is
such a challenging task, we focused in this Review on elucidating
how the impact of genetic variation on TF-DNA binding can be
assessed and why, contrary to expectations, this is itself already
inherently complex. There are several current limitations that will
have to be addressed to improve our ability to identify and interpret regulatory variation, including the need for new experimental or computational approaches that will enable us to
expand the TF motif catalog, to better predict genuine TF binding
sites, and to evaluate how motif variation affects TF-DNA binding. Promising research avenues in this regard include the development of new technologies to characterize monomeric and
higher complex TF-DNA binding properties and the incorporation of additional DNA binding features such as the sequence
environment and the conformational and chemical nature of

Supplemental Information includes one table and is available with this article
online at http://dx.doi.org/10.1016/j.cell.2016.07.012.

SUPPLEMENTAL INFORMATION

ACKNOWLEDGMENTS
We thank Richard Benton (University of Lausanne), Sebastian Waszak (EMBL),
Alina Isakova, Antonio Meireles-Filho, Petra Schwalie, and other members of
the Deplancke Laboratory, as well as the anonymous reviewers for useful comments on the manuscript. We also would like to acknowledge scientific discussions with all members of the Effect of sequence variation on chromatin
structure and transcription Sinergia Consortium (i.e., the Reymond and Hernandez Laboratories [UNIL] and the Dermitzakis Laboratory [University of
Geneva]). This work was supported by the Swiss National Science Foundation
grant CRSI33_130326, by SystemsX.ch (AgingX, 51RTP0_151019), and by
institutional support from the Swiss Federal Institute of Technology in Lausanne (EPFL).
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Leading Edge

Review
Mitochondria and Cancer
Sejal Vyas,1 Elma Zaganjor,1 and Marcia C. Haigis1,*
1Department

of Cell Biology, Ludwig Center at Harvard, Harvard Medical School, Boston, MA 02115, USA
*Correspondence: marcia_haigis@hms.harvard.edu
http://dx.doi.org/10.1016/j.cell.2016.07.002

Mitochondria are bioenergetic, biosynthetic, and signaling organelles that are integral in stress
sensing to allow for cellular adaptation to the environment. Therefore, it is not surprising that mitochondria are important mediators of tumorigenesis, as this process requires flexibility to adapt to
cellular and environmental alterations in addition to cancer treatments. Multiple aspects of mitochondrial biology beyond bioenergetics support transformation, including mitochondrial biogenesis and turnover, fission and fusion dynamics, cell death susceptibility, oxidative stress regulation,
metabolism, and signaling. Thus, understanding mechanisms of mitochondrial function during
tumorigenesis will be critical for the next generation of cancer therapeutics.
Introduction
Historical Perspective
Louis Pasteur identified the importance of oxygen consumption
in 1861, finding that yeast divided more in the presence of oxygen and that oxygen inhibited fermentation, an observation
known as the Pasteur effect. The discovery of mitochondria
in the 1890s, described cytologically by both Richard Altmann
and Carl Benda, began to shed light on this observation, and in
1913, the biochemist Otto Warburg linked cellular respiration
to grana derived from guinea pig liver extracts (Ernster and
Schatz, 1981). Warburg stated that the granules functioned to
enhance the activity of iron-containing enzymes and involved a
transfer to oxygen (Ernster and Schatz, 1981). In the following
decades, many scientists elucidated the machinery that drives
mitochondrial respiration, including tricarboxylic acid (TCA)
cycle and fatty acid b-oxidation enzymes in the mitochondrial
matrix that generate electron donors to fuel respiration and electron transport chain (ETC) complexes and ATP synthase in the inner mitochondrial membrane (IMM) that carry out oxidative
phosphorylation (Ernster and Schatz, 1981). This biochemical
understanding of mitochondrial oxidative phosphorylation gave
mechanistic insight into the Pasteur effect, which could be reconstituted by adding purified, respiring liver mitochondria to
glycolytic tumor supernatants and observing inhibited fermentation (Aisenberg et al., 1957). The ability of mitochondria to inhibit
a glycolytic system suggested an active and direct role for mitochondria in regulating oxidative versus glycolytic metabolism
(Aisenberg et al., 1957).
Warburgs seminal discovery that cancer cells undergo aerobic glycolysis, which refers to the fermentation of glucose to
lactate in the presence of oxygen as opposed to the complete
oxidation of glucose to fuel mitochondrial respiration, brought
attention to the role of mitochondria in tumorigenesis (Warburg,
1956). While the Warburg effect is an undisputed feature of
many (but not all) cancer cells, Warburgs reasoning that it
stemmed from damaged mitochondrial respiration caused immediate controversy (Weinhouse, 1956). We now understand
that while damaged mitochondria drive the Warburg effect in

some cases, many cancer cells that display Warburg metabolism possess intact mitochondrial respiration, with some cancer subtypes actually depending on mitochondrial respiration.
Decades of studies on mitochondrial respiration in cancer have
set the framework for a new frontier focused on additional functions of mitochondria in cancer, which have identified pleiotropic
roles of mitochondria in tumorigenesis.
A major function of mitochondria is ATP production, hence its
nickname powerhouse of the cell. However, mitochondria
perform many roles beyond energy production, including the
generation of reactive oxygen species (ROS), redox molecules
and metabolites, regulation of cell signaling and cell death, and
biosynthetic metabolism. These multifaceted functions of mitochondria in normal physiology make them important cellular
stress sensors, and allow for cellular adaptation to the environment. Mitochondria similarly impart considerable flexibility for tumor cell growth and survival in otherwise harsh environments,
such as during nutrient depletion, hypoxia, and cancer treatments, and are therefore key players in tumorigenesis.
There is no simple canon for the role of mitochondria in cancer
development. Instead, mitochondrial functions in cancer vary
depending upon genetic, environmental, and tissue-of-origin
differences between tumors. It is clear that the biology of mitochondria in cancer is central to our understanding of cancer
biology, as many classical cancer hallmarks result in altered
mitochondrial function. This review will summarize functions of
mitochondria biology that contribute to tumorigenesis, which
include mitochondrial biogenesis and turnover, fission and
fusion dynamics, cell death, oxidative stress, metabolism and
bioenergetics, signaling, and mtDNA (Figures 1 and 2).
Mitochondrial Biogenesis and Turnover
Mitochondrial mass is dictated by two opposing pathways,
biogenesis and turnover, and has emerged as both a positive
and negative regulator of tumorigenesis. The role of mitochondrial biogenesis in cancer is regulated by many factors, including
metabolic state, tumor heterogeneity, tissue type, microenvironment, and tumor stage. Additionally, mitophagy, the selective
Cell 166, July 28, 2016 2016 Elsevier Inc. 555

Figure 1. Mitochondria and Cancer


The role of mitochondrial metabolism, bioenergetics, mtDNA, oxidative stress regulation, fission and fusion dynamics, cell death regulation, biogenesis, turnover,
and signaling in tumorigenesis.

autophagic pathway for mitochondrial turnover, maintains a


healthy mitochondrial population. Importantly, regulation of
both mitochondrial biogenesis and mitophagy are central to
key oncogenic signaling pathways.
Transcriptional and Signaling Networks Regulating
Biogenesis
Mitochondrial biogenesis is regulated by transcriptional programs that coordinate induction of both mitochondrial- and nu556 Cell 166, July 28, 2016

clear-localized genes that encode mitochondrial proteins. The


transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1a) is a central regulator of mitochondrial biogenesis through interactions with
multiple transcription factors (Tan et al., 2016). PGC-1a levels
often reveal tumor reliance on mitochondrial mass, with high
PGC-1a expression resulting in a dependence on mitochondrial
respiration (Tan et al., 2016). In contrast, PGC-1a acts as a tumor

Figure 2. Mitochondria and Stages of


Tumorigenesis
Mitochondrial biology supports tumorigenesis at
multiple stages. Mutations in mitochondrial enzymes generate oncometabolites that result in
tumor initiation. Oxidative stress and mitochondrial signalling can also support tumor initiation.
Mitochondrial metabolic reprogramming, oxidative stress, and signaling can promote tumor
growth and survival. Mitochondria additionally
regulate redox homeostasis and susceptibility to
cell death via alterations in morphology to promote
cell survival. Alterations in mitochondrial mass via
regulation of biogenesis and mitophagy also
contribute to survival depending on cancer type.
Mitochondrial metabolic reprogramming, biogenesis, and redox homeostasis and dynamics also
contribute to metastatic potential of cancer cells.

suppressor in some cancer types, with overexpression resulting


in induction of apoptosis (Tan et al., 2016). Additionally, PGC-1a
is downregulated in hypoxia inducible factor-1 alpha (HIF-1a)activated renal cell carcinomas, reinforcing a switch to glycolytic
metabolism in low oxygen conditions (LaGory et al., 2015; Zhang
et al., 2007). Therefore, it is important to identify factors that
contribute to the dichotomous effect of PGC-1a on tumor
viability, as this has the potential to identify specific susceptibilities for cancer subtypes.
PGC-1a-dependent mitochondrial biogenesis may also support anchorage-independent cancer cell growth, a key step in
metastasis. Proteomic analysis identified upregulation of mitochondrial proteins involved in metabolism and biogenesis upon
low-attachment culture conditions (Lamb et al., 2014). Additionally, increased mitochondrial mass co-enriched with tumor-initiating activity in patient-derived breast cancer lines, which could
be blocked by PGC-1a inhibition (De Luca et al., 2015). These
findings remain relevant in vivo, as circulating tumor cells
(CTCs) developed from primary orthotopic breast tumors show
increased mitochondrial biogenesis and respiration, with PGC1a silencing decreasing CTCs and metastasis (LeBleu et al.,
2014). Thus, PGC-1a-dependent mitochondrial biogenesis
may contribute to tumor metastatic potential.
A key activator of mitochondrial biogenesis in cancer is c-Myc,
a transcription factor that globally regulates cell cycle, growth,
metabolism, and apoptosis. Over 400 mitochondrial genes are
identified as c-Myc targets, and initial studies demonstrated
that gain/loss of Myc increases/reduces mitochondrial mass,
respectively (Li et al., 2005). In normal physiology, c-Myc couples mitochondrial biogenesis with cell-cycle progression.
However, elevated mitochondrial biogenesis due to oncogenic
c-Myc increases cellular biosynthetic and respiratory capacity

by upregulating mitochondrial metabolism to support rapid proliferation,


complementing c-Mycs effects on stimulating cell-cycle progression and glycolytic metabolism to coordinate rapid cell
growth (Figure 3).
Another effector of mitochondrial
biogenesis is the mammalian target of
rapamycin (mTOR) signaling pathway,
which is critical for cellular growth and energy homeostasis
and is misregulated in many diseases including cancer. mTOR
regulates mitochondrial biogenesis both transcriptionally via
PGC-1a/Yin Yang 1 (YY1) activation, resulting in mitochondrial
gene expression, and translationally via repression of inhibitory
4E-binding proteins (4E-BPs) that downregulate translation of
nuclear-encoded mitochondrial proteins (Morita et al., 2015)
(Figure 3).
The transcriptional networks regulating biogenesis impact
therapeutic outcomes by providing cancer cells with metabolic
flexibility to adapt to targeted treatments and tumor microenvironments. In B-Raf or N-Ras mutant melanomas, resistance to
MEK inhibitors was partially due to a switch to oxidative metabolism mediated by PGC-1a upregulation and was overcome
by mTORC1/2 inhibition, which repressed PGC-1a expression
(Gopal et al., 2014; Haq et al., 2013). Likewise, in a mouse model
of K-Ras mutant pancreatic ductal adenocarcinoma, cells that
survive oncogene ablation have increased PGC-1a expression
and mitochondrial function, and the reliance on mitochondrial
respiration resulted in sensitivity to oxidative phosphorylation inhibitors (Viale et al., 2014). Cancer cells can adapt their mitochondrial function according to the specific stress. For example,
c-Myc upregulation and glycolytic gene expression enables
resistance to metformin, a complex I inhibitor, in pancreatic cancer cells, which actively utilize mitochondrial respiration due to
PGC-1a expression (Sancho et al., 2015). Similarly, c-Mycdependent mitochondrial biogenesis is normally opposed by
the HIF-1a signaling pathway, but this balance is altered during
oncogenic c-Myc-driven transformation (Dang et al., 2008).
Therefore, an important consideration in cancer therapeutics
will be addressing routes of bioenergetic plasticity provided by
mitochondria.
Cell 166, July 28, 2016 557

Figure 3. Effects of Classical Oncogenic and Tumor Suppressive Pathways on Mitochondrial Biology
Key mechanisms of mitochondrial regulation by c-MYC, K-RAS, PI3K, and p53 signaling pathways. Through transcriptional regulation, c-Myc induces mitochondrial biogenesis and metabolism in addition to its stimulation of cell-cycle progression and glycolysis. c-Myc promotes mitochondrial fusion and respiration,
which can result in increased ROS production and oxidative signaling. Hyperactive PI3K signaling through either PI3K mutation or loss/mutation of the PTEN
tumor suppressor results in mTOR activation, which is additionally regulated by nutrient availability, to regulate cell growth. mTOR promotes mitochondrial
biogenesis both transcriptionally and translationally. Low nutrient conditions that result in a high AMP/ATP ratio activate AMPK, which opposes the mTOR
pathway. During chronic nutrient deprivation, AMPK can also promote mitochondrial biogenesis to allow for metabolic flexibility. Loss of p53 promotes survival
not only via transcriptional regulation of cell death programs, but also through direct interactions with Bcl-2 proteins at the mitochondria. p53 can also induce
mitochondrial respiration to promote tumorigenesis by allowing for metabolic flexibility. Oncogenic K-Ras mutations result in a coordinated program of mitochondrial regulation, reprogramming mitochondrial metabolism through multiple mechanisms as well as promoting mitochondrial fission and mitophagy.

Mitophagy
Clearance of damaged mitochondria via mitophagy is critical for
cellular fitness since dysfunctional mitochondria can impair ETC
function and increase oxidative stress. A major trigger for
mitophagy is via the PTEN-induced putative kinase 1 (PINK1)/
Parkin pathway. This pathway is activated upon mitochondrial
membrane depolarization, a signal of mitochondrial dysfunction
that results from multiple causes including lack of reducing
equivalents, hypoxia, and impaired electron transport. An alternate pathway for mitophagy induction is through the HIF-1a
target genes Bcl-2 and adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) and BNIP3-like (BNIP3L/NIX), which inhibit mitochondrial respiration during hypoxic conditions that could result
in excessive ROS.
558 Cell 166, July 28, 2016

Is mitophagy beneficial or harmful to cancers? Similar to autophagy, which is shown to be both pro- and anti-tumorgenic
based on context, the function of mitophagy in transformation
likely depends on tumor stage (Mancias and Kimmelman,
2016). Mitophagy-deficient Parkin null mice develop spontaneous hepatic tumors, and Parkin loss increases tumorigenesis
in multiple cancer models (Matsuda et al., 2015). Additionally,
BNIP3 and NIX are identified as tumor suppressors in multiple
cancer models (Chourasia et al., 2015). Thus, in certain stages
of tumorigenesis, decreased mitophagy may allow for a permissive threshold of dysfunctional mitochondria to persist, generating increased tumor-promoting ROS or other tumorigenic
mitochondrial signals. In contrast, established tumors may
require mitophagy for stress adaptation and survival. Supporting

this concept, BNIP3 is induced in patient glioblastoma samples


in response to hypoxia caused by anti-angiogenic therapy and
combinatorial angiogenesis and autophagy inhibition had a
potent anti-tumor effect in xenograft glioma models (Hu et al.,
2012). Additionally, oncogenic K-Ras-driven transformation upregulates mitophagy for the clearance of dysfunctional mitochondria, and the accumulation of dysfunctional mitochondria
switches adenoma tumor fate to benign oncocytomas instead
of carcinomas (Guo et al., 2013).
Fission and Fusion Dynamics
Mitochondria are extremely dynamic, and the balance of fission
and fusion dictates their morphology. A critical step in mitochondrial membrane fission is dynamin-related protein-1
(Drp1) recruitment to mitochondria and interaction with its outer
mitochondria membrane (OMM) receptors, where it causes
membrane constriction fueled by GTPase activity. Drp1 mitochondrial translocation and activity is regulated by phosphorylation mediated by multiple kinases that respond to distinct
cell-cycle and stress conditions (Mishra and Chan, 2016). The
mitofusins, Mfn1 and Mfn2, along with optic atrophy-1 (Opa1)
mediate mitochondrial fusion. Mitochondria exist as either
fused, tubular networks or as fragmented granules depending
on cellular state, with mitochondrial metabolism, respiration,
and oxidative stress regulating fission/fusion machinery (Mishra
and Chan, 2016). Mitochondrial morphology also affects susceptibility to mitophagy and apoptosis (Kasahara and Scorrano,
2014).
Multiple studies have demonstrated an imbalance of fission
and fusion activities in cancer, with elevated fission activity
and/or decreased fusion resulting in a fragmented mitochondrial
network (Senft and Ronai, 2016). Importantly, restoration of
fused mitochondrial networks in these studies, through either
Drp1 knockdown/ inhibition or Mfn2 overexpression, impaired
cancer cell growth, suggesting that mitochondrial network remodeling is important in tumorigenesis. Increased Drp1 expression is associated with a migratory phenotype in multiple cancer
types, further highlighting the role of mitochondrial dynamics in
metastasis (Senft and Ronai, 2016).
Altered mitochondrial dynamics are a key feature of K-Rasdependent cellular transformation, with oncogenic K-Ras
stimulating mitochondrial fragmentation via ERK1/2-mediated
phosphorylation of Drp1 (Kashatus et al., 2015; Serasinghe
et al., 2015). Knockdown or inhibition of Drp1 renders cells
resistant to oncogenic K-Ras-mediated transformation and impairs tumor growth (Kashatus et al., 2015). Additionally, remodeling of the mitochondrial network upon oncogenic K-Ras
expression affects mitochondrial function, decreasing membrane potential and increasing ROS generation (Serasinghe
et al., 2015). Thus, K-Ras-mediated mitochondrial network remodeling creates a state of upregulated tumorigenic stimuli
to support cellular transformation. c-Myc also affects mitochondrial dynamics by altering the expression of multiple
fission and fusion proteins (Graves et al., 2012). However, the
net effect causes mitochondrial fusion (von Eyss et al., 2015),
and further studies are needed to understand the differential
effects of oncogenic signaling pathways on mitochondrial
dynamics.

Cell Death
A hallmark of cancers is their ability to evade cell death, a phenomenon tightly linked to mitochondria. The pro-apoptotic
Bcl-2 family members Bax and Bak are recruited to the OMM
and oligomerize to mediate mitochondrial outer membrane permeabilization (MOMP), resulting in pore formation and cytochrome c release from mitochondria into the cytosol to activate
caspases, the executors of programmed cell death. During
normal physiology, anti-apoptotic family members such as
Bcl-2 and Bcl-XL bind and inhibit Bax/Bak. Tumor cells escape
apoptosis by downregulating pro-apoptotic Bcl-2 genes and/or
upregulating anti-apoptotic Bcl-2 genes, achieved through multiple mechanisms reviewed elsewhere (Lopez and Tait, 2015).
The balance of pro- and anti-apoptotic proteins affects a cancer
cells susceptibility to apoptotic stimuli and may predict how a
tumor will respond to chemotherapy (Sarosiek et al., 2013).
Mitochondrial shape also dictates apoptotic susceptibility, as
Drp1 loss delays cytochrome c release and apoptotic induction,
although follow-up work indicated that fission was not required
for Bax/Bak-mediated apoptosis (Martinou and Youle, 2011).
Instead, a GTPase-independent function of Drp1 in membrane
remodeling and hemifusion results in Bax oligomerization and
subsequent MOMP, indicating that Drp1 can promote apoptosis
independent of fission (Martinou and Youle, 2011). The
importance of mitochondrial shape in apoptosis is further demonstrated by Mfn-1-loss induced mitochondrial hyperfragmentation, causing resistance to apoptotic stimuli due to the loss of
Bax interaction with mitochondrial membranes. In this study,
Drp1 inhibition rescued sensitivity to apoptotic stimuli by
restoring a balanced mitochondrial network (Renault et al.,
2015). Additionally, Mfn1 is a target of the MEK/ERK signaling
pathwayphosphorylated Mfn1 inhibits mitochondria fusion
and interacts with Bak to stimulate its oligomerization and
subsequent MOMP (Pyakurel et al., 2015). Therefore, while fission
and fusion do not necessarily regulate apoptosis per se, a balance
of these activities appears to generate a mitochondrial shape that
supports interactions with pro-apoptotic Bcl2 proteins.
Oxidative Stress
ROS, in the form of superoxide and hydroxyl free radicals, and
hydrogen peroxide, are produced from physiological metabolic
reactions. Mitochondria are major contributors to cellular ROS
and have multiple antioxidant pathways to neutralize ROS
including superoxide dismutase (SOD2), glutathione, thioredoxin, and peroxiredoxins. The early observation that cancer
cells have high ROS levels led to an overly simple hypothesis
that inhibiting ROS could be a successful therapeutic strategy.
However, a more complex picture is emerging, in which ROS
stimulates signaling and proliferation, and the concomitant
upregulation of antioxidant pathways prevents ROS-mediated
cytotoxicity and may even enhance tumor survival (Shadel and
Horvath, 2015; Sullivan and Chandel, 2014).
Multiple physiological reactions, including electron transport
by the ETC and NAD(P)H oxidases result in ROS production,
and these are often exacerbated during tumorigenesis by
oncogenic signaling, ETC mutations, and hypoxic microenvironments. High levels of ROS contribute to the oxidation of macromolecules, such as lipids, proteins, and DNA, and can contribute
Cell 166, July 28, 2016 559

to genomic instability to promote transformation. However,


modest elevations of ROS observed in many tumors can regulate
cell signaling via cysteine oxidation. Indeed, H2O2 inactivates
the tumor suppressor PTEN by oxidizing active site cysteine
residues, causing the formation of a disulfide bond, which
prevents PTEN from inactivating the PI3K pathway (Sullivan
and Chandel, 2014). Since ROS can inactivate protein tyrosine
phosphatases through oxidation of cysteine residues, ROS
may have many yet to be discovered effects on diverse,
mitogen-activated pathways that are normally inhibited by phosphatases (Sullivan and Chandel, 2014). ROS-mediated regulation of oncogenic signaling also affects metastasisoxidation
of cysteines in Src increased its oncogenic ability, promoting tumor cell migration and metastasis in multiple tumor types, and
these phenotypes were blocked by a ROS scavenger (Porporato
et al., 2014).
In response to elevated ROS, many tumors upregulate protective antioxidant pathways. For example, oncogenic K-Ras, B-raf,
and c-Myc actively inhibit ROS through regulation of nuclear factor (erythroid-derived 2)-like 2 (NRF2), a transcriptional regulator
of the antioxidant response, to promote tumorigenesis (DeNicola
et al., 2011). Similarly, a study in melanoma found that circulating
tumor cells had higher levels of NADPH than primary tumor sites,
presumably to combat the increased ROS caused by the stress
of metastasis (Piskounova et al., 2015). In this system, antioxidants promoted distant metastasis, while folate pathway inhibition prevented metastasis due to decreased NADPH production
but had no effect on the primary tumor. Similarly, antioxidant
treatment increased the number of metastasis in a mouse model
of malignant melanoma, causing increased invasiveness dependent on glutathione synthesis cells (Le Gal et al., 2015). Thus,
successful tumors maintain ROS levels within a window that
stimulates proliferation without causing cytotoxicity. The balance of ROS production and antioxidant expression is critical
for maintaining this tumor-promoting ROS level.
The requirement for upregulated antioxidant pathways may be
an Achilles heel for tumor cells: combination therapy using glutathione and thioredoxin pathway inhibitors has promising results
in vitro and in vivo in breast cancer models (Harris et al., 2015).
Targeting other aspects of mitochondrial metabolism that
contribute to redox regulation has also been proven to be a
successful anti-cancer strategy. For example, inhibition of glutamate dehydrogenase 1 (GDH1) increased ROS by reducing
levels of fumarate, an activator of antioxidant glutathione peroxidase 1 (GPx), to slow cancer growth (Jin et al., 2015).
Metabolism
One hallmark of tumors is metabolic reprogramming, which supports macromolecule synthesis, bioenergetic demand, and
cellular survival (Pavlova and Thompson, 2016). Mitochondria
are hubs for metabolic reactions and drive this reprogramming
through multiple mechanisms.
Alterations in Glucose Utilization
Many tumors divert glycolytic intermediates into the pentose
phosphate pathway, serine biosynthesis, and lipid biosynthesis,
as opposed to complete oxidation by mitochondrial respiration.
In some tumors, this is achieved by limiting pyruvate utilization
by mitochondria. The availability of pyruvate for mitochondrial
560 Cell 166, July 28, 2016

oxidation is regulated by pyruvate kinase (PKM), which catalyzes


the final step of glycolysis to generate pyruvate. Cancers specifically upregulate the PKM2 isoform, which has low activity, allowing upstream glycolytic intermediates to accumulate and
be used for anabolic processes (Christofk et al., 2008). Additionally, the mitochondrial pyruvate carrier (MPC1 and MPC2) is
either lost or downregulated in a number of cancers (Schell
et al., 2014). MPC re-expression had a profound effect on
reducing tumor growth in xenograft models, suggesting that
the expression of this fuel gatekeeper is an important determinant of growth (Yang et al., 2014). Furthermore, MPC loss stimulates compensatory pathways that maintain fuel oxidation by
the TCA cycle, including upregulation of glutaminolysis and the
use of fatty acids and branched chain amino acids, demonstrating mitochondrial metabolic flexibility (Schell et al., 2014;
Vacanti et al., 2014; Yang et al., 2014). Thus, mitochondria
remain functional during aerobic glycolysis, and mitochondrialdependent metabolic reprogramming can support bioenergetic
homeostasis during Warburg metabolism.
Reprogramming of Amino Acid Metabolism
Glutamine can be a substrate for TCA cycle oxidation and a
starting material for macromolecule synthesis (DeBerardinis
et al., 2007). The amide nitrogen on glutamine is used in nucleotide and amino acid synthesis, and glutamine-derived carbons
are used in glutathione, amino acid, and lipid synthesis. Catabolism of glutamine, termed glutaminolysis, is elevated in many
glutamine-addicted tumors and is often driven by c-Myc upregulation of glutaminase (GLS), which converts glutamine to glutamate and ammonia (Stine et al., 2015). Glutamate is oxidized
to a-ketoglutarate (a-KG) by GDH, providing an entry point into
the TCA cycle. This process is inhibited by the mitochondriallocalized sirtuin, SIRT4, a tumor suppressor in multiple cancer
models. SIRT4 expression in B cell lymphoma cells downregulates glutamine uptake and inhibits growth, whereas SIRT4
loss in an Em-myc B cell lymphoma model increases glutamine
consumption and accelerates tumorigenesis (Jeong et al.,
2014). In addition, transaminases utilize glutamate nitrogen to
couple a-KG production to synthesis of non-essential amino
acids, and tumor cells can utilize this pathway to support biosynthesis and redox homeostasis. For example, oncogenic K-Ras
reprograms glutamine metabolism by transcriptional downregulation of GDH1 and upregulation of GOT1, the aspartate
transaminase, to produce cytosolic oxaloacetate, which can ultimately lead to an increase in NADPH/NADP+ ratio through conversion to pyruvate (Son et al., 2013). Similarly, transaminases,
but not GDH1, are upregulated in 3D cultures of proliferating
mammary epithelial cells compared to quiescent cells, suggesting that this pathway is important during cancer cell proliferation
to support biosynthesis (Coloff et al., 2016).
In tumor cells with dysfunctional mitochondria due to ETC or
TCA cycle enzyme mutations, a fraction of glutamine-derived
a-KG undergoes reductive carboxylation to support biosynthesis and redox homeostasis (Mullen et al., 2011). This pathway
is dependent on the NADP+/NADPH-utilizing isocitrate dehydrogenase isoforms IDH1 (cytosolic) and IDH2 (mitochondrial),
catalyzing the reverse reaction of isocitrate production from
a-KG. Reductive carboxylation can also support anchorage independent tumor growth in spheroid cultures by mitigating

mitochondrial ROS in a coordinated cycle in which cytosolic


reductive carboxylation by IDH1 supports mitochondrial oxidative metabolism and NADPH production by IDH2 (Jiang et al.,
2016).
As nutrients are oxidized to produce biosynthetic precursors,
electrons are removed from carbon. Therefore, electron acceptors can quickly become limiting in highly proliferating cells.
This observation was highlighted in a series of studies demonstrating that beyond ATP production, mitochondrial respiration
is required to replenish electron-accepting cofactors NAD+ and
FAD (Birsoy et al., 2015; Sullivan et al., 2015). Interestingly,
when mitochondrial respiration is impaired, rather than ATP,
the electron acceptors are most limiting for de novo synthesis
of aspartate, a key amino acid required for protein and nucleotide synthesis.
Aside from coordinating fuel oxidation, mitochondria contribute to tumor progression through nucleotide synthesis via
one-carbon (1C) metabolism. The mitochondrial folate synthesis pathway consists of serine hydroxylmethyltransferase 2
(SHMT2) and methylenetetrahydrofolate dehydrogenase 2
(MTHFD2). A meta-analysis of gene expression profiles identified MTHFD2 as overexpressed in many human tumors and
further studies revealed its importance in survival of cancer
cells (Nilsson et al., 2014). Unlike the cytosolic arm of folate
metabolism that primarily uses serine, the mitochondrial arm
also uses glycine as a carbon source, a potential vulnerability
for cancers that upregulate this pathway. Metabolic profiling
of NCI-60 lines revealed high correlation of proliferation with
glycine consumption along with the increase in SHMT2,
MTHFD2, and MTHFD1L (Jain et al., 2012). While the cytosolic
pathway can compensate for loss of mitochondrial 1C metabolism, cells become dependent on extracellular serine and
glycine for growth and are thus susceptible to inhibition of serine
catabolism, highlighting the importance of mitochondrial 1C
metabolism in supporting tumorigenesis during nutrient deprivation (Ducker et al., 2016). For example, SHMT2 is expressed in
ischemic tumor zones, providing proliferative advantage under
hypoxic conditions (Kim et al., 2015). Additionally SHMT2 regulation of serine metabolism also contributes to NADPH production and detoxification of ROS under hypoxia, a function important for survival of Myc-driven cancers (Ye et al., 2014).
Lipid Metabolism
Unlike other fuels, lipid utilization in cancer is less defined at the
molecular level. Cancer-specific alterations of lipid metabolism
seem to be unique to tumor type, allowing for some cancers to
upregulate fatty acid oxidation (FAO) while others are more
dependent on lipid synthesis. Upregulation of lipogenesis is
postulated to be a common feature across most tumors, in
part to produce membranes for proliferation (Currie et al.,
2013). Inhibition of ATP-citrate lyase (ACLY), which converts
mitochondrial-derived citrate to acetyl-CoA in the cytoplasm to
support lipogenesis, impairs tumorigenesis in multiple models
(Currie et al., 2013). In contrast, certain cancer types including
lymphomas and leukemias rely primarily on FAO for ATP production (Carracedo et al., 2013). Additionally, FAO may be a
preferred fuel choice for cancers undergoing stress as it is a
crucial survival mechanism for breast cancer cells undergoing
loss of attachment to the extracellular matrix (Carracedo et al.,

2013). However, mechanisms that upregulate FAO in cancers


remain poorly understood. In one example, tumor cell upregulation of the brain-specific isoform of carnitine palmitoyltransferase (Cpt-1c), required for mitochondrial FA import, resulted in
increased FAO and ATP production and resistance to metabolic
stress (Carracedo et al., 2013). Moreover, increased FAO may
confer benefits beyond ATP generation such as maintaining
redox homeostasis (Carracedo et al., 2013). Finally, production
of acetyl-CoA from oxidized fatty acids could be used for epigenetic remodeling of chromatin, subsequently causing lasting
changes in metabolism.
Studying Cancer Metabolism In Vivo
Recent work has highlighted the importance of studying cancer
metabolism in models comparable to the in vivo disease. For
example, while glutamine fuels TCA cycle anaplerosis in vitro,
this is not necessarily true of all tumors in vivo. Studies
comparing the fate of labeled glucose and glutamine in mouse
models of K-Ras-driven non-small-cell lung cancer showed minimal contribution of glutamine to TCA cycle intermediates (Davidson et al., 2016). Additionally, studies in glioblastoma cells
showed that glutamine-dependent anaplerosis was not required
for growth, with cells secreting glutamate even under glutamine
starvation conditions (Tardito et al., 2015). In this study, glutamine synthase (GS) expression sustained growth and purine
nucleotide biosynthesis during glutamine starvation. Furthermore, primary patient-derived glioma stem-like cells grew independently of glutamine supplementation. These studies highlight
the importance of understanding in vivo metabolic requirements
of tumor cells when designing therapeutic strategies.
Mitochondrial Signaling
Mitochondrial biology and tumorigenic signaling intersect at
multiple levels. First, classical oncogenic signaling pathways
alter mitochondrial functions to support tumorigenesis. Second,
direct signals from mitochondria affect cellular physiology and
tumorigenesis. Finally, mutations in mitochondrial enzymes can
result in oncometabolite production, a novel set of mitochondrial
signaling molecules that function in tumor initiation.
Classical Oncogenic and Tumor Suppressive Pathways
Regulate Mitochondrial Biology
The resurgence of mitochondrial research has led to the discovery that established tumor suppressors and oncogenes directly
regulate mitochondrial biology. Several hallmark cancer signaling pathways that alter mitochondrial biology to promote
transformation are described herein (Figure 3).
In addition to promoting mitochondrial biogenesis, numerous
studies have linked c-Myc with mitochondrial metabolism in cancer. The importance of mitochondrial metabolism in c-Myc
driven growth was demonstrated in a functional screen of
Myc-responsive cDNAs to rescue cell growth of c-Myc-null cells.
The screen identified SHMT2, the first reaction in mitochondrial
1C metabolism, as the only target that could partially rescue
growth (Nikiforov et al., 2002). While the tumorigenic contribution
of increased mitochondrial biogenesis and metabolism in oncogenic c-Myc-driven cancers is difficult to separate from its global
upregulation of transcription, suppression of glutaminolysis can
inhibit proliferation of c-Myc driven lymphoma cells (Jeong et al.,
2014; Le et al., 2012).
Cell 166, July 28, 2016 561

An important signaling pathway in hypoxic tumor microenvironments is mediated by HIF-1a, which upregulates glycolytic
metabolism in low oxygen conditions and inhibits mitochondrial
respiration (Mucaj et al., 2012). Mitochondrial-derived ROS also
regulate the HIF-1a pathway via inhibition of prolyl hydroxylases (PHDs), negative regulators of HIF signaling. SIRT3, a
mitochondrial deacetylase, is an important regulator of this
pathway by maintaining redox homeostasis via deacetylation
and activation of mitochondrial SOD2 and IDH2 and indirectly
through transcriptional upregulation of antioxidant pathways
(Bause and Haigis, 2013). SIRT3-dependent reduction in mitochondrial ROS results in HIF-1a degradation, limiting glycolysis
and the Warburg effect in tumors (Bell et al., 2011; Finley et al.,
2011).
In addition to the pleiotropic effects of oncogenic K-Ras
signaling on proliferation, apoptosis, and metabolism, oncogenic K-Ras results in a coordinated program of mitochondrial
regulation that supports transformation (Pylayeva-Gupta et al.,
2011). Multiple K-Ras-dependent mechanisms can downregulate mitochondrial respiration including upregulation of mitochondrial fission (Kashatus et al., 2015; Serasinghe et al.,
2015), transcriptional downregulation of complex I (Wang et al.,
2015), and ERK-phosphorylation-dependent mitochondrial
translocation of phosphoglycerate kinase I (PGK1) (Li et al.,
2016). Oncogenic K-Ras also promotes upregulation of mitophagy to preserve mitochondrial function under starvation conditions. Autophagy inhibition in cancers with active K-Ras results
in a decline in mitochondrial respiration, TCA metabolite, and
energy levels during starvation; thus, this pathway may be
important for tumor cell survival in nutrient-depleted microenvironments (Guo et al., 2011).
The PI3K/Akt signaling pathway stimulates cell growth and is
often activated in cancer either through oncogenic mutations
in signaling kinases or loss/mutation of the PTEN tumor suppressor, a key phosphatase that shuts off this pathway (Papa et al.,
2014). Although PI3K signaling induces cell growth and upregulates glycolysis, metabolic adaptation via a switch to mitochondrial oxidative phosphorylation can mediate resistance to PI3K
inhibitors, undermining the effectiveness of PI3K-specific targeted therapy (Ghosh et al., 2015). A major downstream effector
of active PI3K/Akt signaling is mTOR, which participates in
mTORC1 and mTORC2 signaling complexes to couple nutrient
and growth-factor sensing to cellular growth through regulation
of translation, anabolic metabolism, and autophagy (Dibble
and Cantley, 2015). In addition to regulating mitochondrial
biogenesis, mTORC1 stimulates multiple mitochondrial metabolic pathways. The transcriptional repression of SIRT4 downstream of mTORC1 activity results in GDH activation to
upregulate glutaminolysis (Csibi et al., 2013). mTORC1 also induces the mitochondrial folate pathway to promote de novo
purine synthesis via activation of the transcription factor ATF4
to result in upregulation of MTHFD2 expression (Ben-Sahra
et al., 2016).
The AMP-regulated kinase (AMPK) signaling network is activated during low energy conditions, directly inhibiting multiple
targets including mTORC1 to restore energy homeostasis.
AMPK is a critical downstream target of the liver kinase B1
(LKB1) tumor suppressor, which is mutated in the inherited
562 Cell 166, July 28, 2016

cancer disorder Peutz-Jeghers syndrome and mediates many


LKB1 tumor suppressive functions (Faubert et al., 2015). However, AMPK loss does not fully recapitulate LKB1 loss and
AMPK has both pro- and anti-tumorigenic effects, which
appear dependent on the presence of other oncogenic drivers
as well as tumor stage (Faubert et al., 2015). While AMPK
loss can uncouple proliferation from energy sensing to allow
for unhindered proliferation with oncogenic growth signaling,
AMPK functions in metabolic adaptation and mitochondrial
homeostasis can be beneficial in established tumors. For
example, AMPK promotes mitophagy through phosphorylation
of ULK kinases and is required for cell survival during starvation (Faubert et al., 2015). Additionally, AMPK activation in
response to ETC dysfunction results in mitochondrial fragmentation through direct phosphorylation of mitochondrial
fission factor, an OMM receptor for Drp1 (Toyama et al.,
2016). Finally, sustained energy deprivation can result in
AMPK-mediated upregulation of mitochondrial biogenesis via
PGC-1aallowing the cell further metabolic plasticity (Faubert
et al., 2015).
p53 is a commonly mutated tumor suppressor and has been
extensively studied due to its transcriptional regulation of cellcycle and apoptotic genes. It is now appreciated that p53 also
has functions in the regulation of cellular metabolism via transcriptional activation of metabolic genes (Berkers et al., 2013).
p53 limits glycolysis and drives transcription of genes required
for ETC assembly and maintenance (Berkers et al., 2013). However, more recent work has suggested an alternate side to p53s
role in tumorigenesis, with its ability to allow for adaptation to
metabolic stress resulting in pro-survival effects in tumor cells.
These pro-survival effects are partially accomplished through
upregulation of mitochondrial FAO and respiration, allowing cancer cells to adapt to starvation conditions (Jiang et al., 2015). In
addition to transcriptional regulation of mitochondrial activity,
p53 also directly functions at the mitochondria to induce
apoptosis in response to stress via interactions with Bcl-2 family
members (Vaseva and Moll, 2009). Tumor-derived p53 mutations no longer interact with Bcl-2 and do not trigger mitochondrial outer membrane permeabilization (Vaseva and Moll,
2009). Thus, in addition to effects on transcriptional activity,
p53 mutations can also promote cancer survival through direct
mitochondrial functions.
Mitochondrial Retrograde Signals
Mitochondria are important stress sensors, and retrograde
signaling from the mitochondria allows the cell to adapt to its
environment. Metabolites generated by mitochondrial metabolic
pathways, including the TCA cycle, b-oxidation, and the ETC,
affect both nuclear gene transcription via chromatin modification
as well as cytosolic signaling pathways. For example, the
TCA cycle intermediate a-KG is a cosubstrate for many enzymes
in the cytoplasm and nucleus including the PHD family
and the 10-11-translocation methylcytosine dioxygenase (TET)
and Jumunji-C histone demethylase (JHDM) families of chromatin-modifying enzymes. In the case of chromatin regulation, glutamine-derived a-KG contributes to TET-dependent
demethylation reactions (Carey et al., 2015). Additional mitochondrial regulation of chromatin occurs through histone
acetylation. ACLY-dependent production of acetyl-CoA from

mitochondrial-derived citrate is used by histone acetyl transferases (HATs), and oncogenic signaling pathways modify histone
acetylation patterns in a ACLY-dependent manner (Lee et al.,
2014; Wellen et al., 2009). In addition to chromatin modification,
acetyl-CoA generated from mitochondrial-derived citrate is used
for the acetylation of many cytosolic and mitochondrial proteins
to modulate protein activity. Thus, mitochondrial-derived metabolites can effect signaling pathways, nuclear transcription, and
chromatin modification.
In addition to signaling molecules, readouts of mitochondrial
integrity including Dcm and MOMP also function as important
signals, enabling the cell to respond to unhealthy/dysfunctional
mitochondria. Since the membrane potential generated by
healthy mitochondria is required for protein import into the mitochondrial matrix and intermembrane space via the TIM22 and
TIM23 translocator complexes, loss of membrane potential impairs import. If the defect in protein import is severe, the cell
can initiate mitophagy to clear these unhealthy mitochondria
as discussed above. Additionally, ATP generation by the ETC
is an important signaling output with diminished ETC activity
increasing the AMP/ATP ratio to activate AMPK signaling. ETC
dysfunction can also result in decreased NAD+ levels, a co-substrate for both the sirtuin and poly(ADP-ribose) protein families,
which have many functions in tumorigenesis (German and Haigis, 2015; Vyas and Chang, 2014). Finally, ROS regulates cytosolic signaling networks to promote tumorigenesis (as discussed
above).
Mitochondrial Oncometabolites
Dominant mutations in mitochondrial enzymes led to the exciting
identification of mitochondrial-derived signaling molecules,
termed oncometabolites. Mutant versions of cytoplasmic and
mitochondrial IDH isoforms, found in a striking 20% of acute
myeloid leukemias and 70% of glioblastomas, reduce a-KG to
generate the oncometabolite (R)-2-hydroxyglutarate ([R]-2-HG)
(Dang et al., 2009; Ward et al., 2010). In addition, loss of function
of TCA cycle enzymes succinate dehydrogenase (SDH) and
fumarate hydratase (FH), underlying the inherited cancer predispositions hereditary paraganglioma syndrome and hereditary
leiomyomatosis and renal-cell cancer syndrome, respectively,
result in the accumulation of metabolic intermediates succinate
and fumarate, which function as oncometabolites when in
excess.
A major mode of action of these oncometabolites is owed to
their structural similarity to a-KG, allowing them to act as
competitive inhibitors of a-KG-dependent enzymes including
the TET and JHDM families of chromatin-modifying enzymes
and the PHD family (Nowicki and Gottlieb, 2015). Inhibition of
TET activity leads to hypermethylation of CpG islands, found
near gene promoters, which results in gene silencing (Nowicki
and Gottlieb, 2015). Additionally, repressive histone methylation
marks on H3K9 and H3K27 are observed in IDH1 and IDH2
mutant gliomas due to JHDM inhibition (Lu et al., 2012). Therefore, through the production of oncometabolites, mitochondria
exert strong influence on chromatin structure to promote
tumor initiation. Both succinate and fumarate accumulation stabilize HIF-1a via PHD inhibition, reinforcing the Warburg effect
(MacKenzie et al., 2007; Nowicki and Gottlieb, 2015). In contrast,
(R)-2-HG activates PHD enzymes, diminishing HIF-1a levels,

which resulted in the enhancement of proliferation of astrocytes


(Koivunen et al., 2012). (R)-2-HG alone reversibly recapitulates
the effects of IDH mutation on leukemogenesis while its enantiomer (S)-2-HG had no effect even though it more potently
inhibited TET2 and PHDs, suggesting that differential requirements for HIF-1a depending on cell type can influence neoplasia
(Losman et al., 2013).
FH deficiency also supports tumorigenesis independently of
a-KG/HIF-1a. The high level of fumarate accumulation in FHdeficient tumors/cells results in increased protein succinylation
through the covalent modification of fumarate on cysteines.
Cysteine succinylation inhibits Kelch-like ECH-associated protein 1 (KEAP1), a negative regulator of Nrf2, to result in upregulation of antioxidant pathways (Adam et al., 2011). Additionally,
accumulated fumarate can bind to glutathione to generate succinylated glutathione, an alternate glutathione reductase substrate that decreases NADPH and increases ROS levels (Sullivan
et al., 2013). Thus, FH deficiency can alter redox homeostasis to
promote tumorigenesis.
mtDNA Mutations
The presence of a separate mitochondrial genome adds to the
unique and complex biology of this organelle, as mutations in
mtDNA impact tumorigenesis. Mitochondria contain multiple
copies of a circular 16kB genome that encodes for 13 ETC subunits, mitochondrial rRNAs, and tRNAs. In addition to distinct
mtDNA haplotypes that exist among different human populations, many germline and somatic mtDNA mutations associated
with cancer risk have been identified (van Gisbergen et al.,
2015). Although the functional consequence of many of these
polymorphisms/mutations is not well understood, some mutations occur in ETC genes and can result in increased oxidative
stress due to ETC dysfunction to promote tumorigenesis. Differences in mtDNA copy number are implicated in tumorigenesis, although both low and high copy numbers have been
associated with various cancers, similar to the varying associations between mitochondrial biogenesis and tumorigenesis
(Reznik et al. 2016). Since mitochondria contain multiple copies
of the mtDNA genome, cells are either homoplasmic or heteroplasmic regarding their mtDNA composition, with mutant
copies of the genome spreading through the mitochondrial
network through fission and fusion cycles. In this way, dominant mtDNA mutations become established in a clonal cell
population. mtDNA mutations and haplotypes associated with
various cancer types are reviewed elsewhere (van Gisbergen
et al., 2015).
Concluding Remarks
Mitochondria are complex organelles that influence cancer initiation, growth, survival, and metastasis, and many facets of mitochondrial biology beyond energy production actively contribute
to tumorigenesis. These include mitochondrial mass, dynamics,
cell death regulation, redox homeostasis, metabolic regulation,
and signaling. The interplay between these aspects of mitochondrial biology results in coordinated programs of mitochondrial
regulation of cellular physiology and highlights the pleiotropic
functions of mitochondria in cancer. Additionally, similar to
the transforming discoveries of oncogenic mutations in growth
Cell 166, July 28, 2016 563

factor signaling pathways, mutations in mitochondrial metabolic


enzymes are an exciting new frontier in cancer biology.
The flexibility that mitochondria bestow tumor cells, including
alterations in fuel utilization, bioenergetics, cell death susceptibility, and oxidative stress, allows for survival in the face of
adverse environmental conditions such as starvation and during
chemotherapeutic and targeted cancer treatments. Therefore,
in order to effectively treat cancer, the escape routes to therapeutic interventions provided by mitochondria must also be
consideredfuture studies into combination therapies that
remove this flexibility will be important to advance cancer
treatments.
ACKNOWLEDGMENTS
We apologize for all the primary literature and work we could not cite due to
space limitations. This review is not meant to be a comprehensive summary
of all the work done in the field of mitochondrial functions in cancer. We thank
Jonathan Coloff, Lydia Finley, Karina Gonzalez, Jessica Spinelli, and Alison
Ringel for discussion on the manuscript. S.V. is supported by a postdoctoral
fellowship from the American Cancer Society (127097-PF-14-255-01-TBE).
E.Z. is supported by a postdoctoral fellowship from the American Heart Association (15POST25560077). M.C.H. is supported by the Ludwig Center at Harvard, the Paul F. Glenn Foundation, and the National Institute of Diabetes and
Digestive and Kidney Diseases (1R01DK103295-01A1).
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Article

Mitotic Checkpoint Regulators Control Insulin


Signaling and Metabolic Homeostasis
Graphical Abstract

Authors
Eunhee Choi, Xiangli Zhang, Chao Xing,
Hongtao Yu

Correspondence
hongtao.yu@utsouthwestern.edu

In Brief
Mitotic checkpoint proteins that prevent
chromosomes from incorrectly
segregating have another unexpected
job: they regulate insulin signaling. This
role draws a link between chromosome
stability and nutrient metabolism.

Highlights
d

p31/ mice show glucose intolerance and insulin resistance


Low-level aneuploidy does not underlie metabolic defects in
p31/ mice
The insulin receptor (IR) directly binds to MAD2 through a
canonical MIM
p31comet blocks premature clathrin-mediated endocytosis of
unstimulated IR

Choi et al., 2016, Cell 166, 567581


July 28, 2016 2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.cell.2016.05.074

Article
Mitotic Checkpoint Regulators Control
Insulin Signaling and Metabolic Homeostasis
Eunhee Choi,1 Xiangli Zhang,2 Chao Xing,2 and Hongtao Yu1,*
1Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road,
Dallas, TX 75390, USA
2Bioinformatics Core, Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center,
6001 Forest Park Road, Dallas, TX 75390, USA
*Correspondence: hongtao.yu@utsouthwestern.edu
http://dx.doi.org/10.1016/j.cell.2016.05.074

SUMMARY

Insulin signaling regulates many facets of animal


physiology. Its dysregulation causes diabetes and
other metabolic disorders. The spindle checkpoint
proteins MAD2 and BUBR1 prevent precocious chromosome segregation and suppress aneuploidy. The
MAD2 inhibitory protein p31comet promotes checkpoint inactivation and timely chromosome segregation. Here, we show that whole-body p31comet
knockout mice die soon after birth and have reduced
hepatic glycogen. Liver-specific ablation of p31comet
causes insulin resistance, hyperinsulinemia, glucose
intolerance, and hyperglycemia and diminishes the
plasma membrane localization of the insulin receptor (IR) in hepatocytes. MAD2 directly binds to IR
and facilitates BUBR1-dependent recruitment of
the clathrin adaptor AP2 to IR. p31comet blocks the
MAD2-BUBR1 interaction and prevents spontaneous clathrin-mediated IR endocytosis. BUBR1
deficiency enhances insulin sensitivity in mice.
BUBR1 depletion in hepatocytes or the expression
of MAD2-binding-deficient IR suppresses the metabolic phenotypes of p31comet ablation. Our findings
establish a major IR regulatory mechanism and
link guardians of chromosome stability to nutrient
metabolism.
INTRODUCTION
Insulin regulates systemic metabolic homeostasis in animals, as
well as cell survival, growth, and proliferation in multiple tissues
and organs. The main framework of insulin signaling has been
established for decades (White, 2003). At the plasma membrane
(PM), insulin binds and activates the insulin receptor (IR), a receptor tyrosine kinase that consists of a dimer of two disulfidelinked chains, IRa and IRb. The activated IR phosphorylates itself
and insulin receptor substrate 1/2 (IRS1/2), creating phosphotyrosine (pY)-containing docking motifs for downstream effectors and adaptors. These proteins activate the PI3K-AKT and
MAP kinase pathways to promote cellular glucose uptake,

glycogen and protein synthesis, and cell growth and survival


(Boucher et al., 2014). The activated IR can be internalized
through clathrin-mediated endocytosis (Goh and Sorkin, 2013),
thus terminating signaling. Dysregulation of insulin signaling
has been linked to human diseases, including diabetes and cancer (Boucher et al., 2014; Pollak, 2012).
In response to kinetochores not properly attached to spindle
microtubules, the spindle checkpoint inhibits the anaphasepromoting complex/cyclosome (APC/C) and delays anaphase
onset, thereby suppressing chromosome segregation errors
(Jia et al., 2013; London and Biggins, 2014; Musacchio, 2015).
The checkpoint proteins MAD2 and BUBR1 can simultaneously
bind to CDC20, converting it from an APC/C activator to a
subunit of an APC/C-inhibitory complex called the mitotic checkpoint complex (MCC) (Izawa and Pines, 2015). MAD2 is an unusual protein with multiple folded conformers, including the
latent open MAD2 (O-MAD2) and the active closed MAD2
(C-MAD2) (Luo and Yu, 2008; Mapelli and Musacchio, 2007).
On checkpoint activation, unattached kinetochores convert
O-MAD2 to CDC20-bound C-MAD2. C-MAD2-CDC20 then associates with BUBR1 to form MCC (Kulukian et al., 2009), in
which BUBR1 contacts both C-MAD2 and CDC20 (Chao et al.,
2012). During checkpoint inactivation, p31comet (also known as
MAD2L1BP) specifically binds to C-MAD2 and disrupts the
C-MAD2-BUBR1 interaction, thus promoting MCC disassembly
and timely anaphase onset (Hagan et al., 2011; Jia et al., 2011;
Xia et al., 2004; Yang et al., 2007). The intricate interactions
among p31comet, MAD2, and BUBR1 are crucial for proper chromosome segregation and genomic stability.
In this study, we discover the role of the p31comet-MAD2BUBR1 module of mitotic regulators in insulin signaling, thus
linking a critical chromosome segregation network to a major
pathway regulating metabolic homeostasis.
RESULTS
p31comet Ablation in Mice Causes Neonatal Lethality and
Liver Glycogen Shortage
Mad2 overexpression in mice promotes aneuploidy and tumorigenesis through hyperactivation of the spindle checkpoint (Sotillo et al., 2007). To test whether inactivation of p31comet might
similarly cause hyperactivation of the checkpoint and promote
tumorigenesis, we generated mice with floxed alleles of p31comet
(p31F/F) and crossed them with CAG-Cre transgenic mice to
Cell 166, 567581, July 28, 2016 2016 Elsevier Inc. 567

Figure 1. Whole-Body and Liver-Specific


Ablation of p31comet Reveals Its Role in
Metabolism
(A) Wild-type (WT), p31+/, and p31/ littermates at
E18.5 and birth. Arrows indicate milk spots.
(B) Survival curves of WT, p31+/, and p31/
neonates.
(C) H&E staining and periodic acid-Schiff (PAS)
staining (magenta) of livers from WT and p31/
newborns. Scale bar, 100 mm.
(D) Liver glycogen levels of WT and p31/ newborns.
(E) Fed blood glucose levels of WT, liver-p31/,
and liver-Insr/ mice. Mean SEM. *p < 0.05,
**p < 0.01, ***p < 0.0001 versus WT; mean
SEM (EI).
(F) Serum insulin concentrations of WT, liverp31/, and liver-Insr/ mice.
(G) Liver glycogen levels of WT, liver-p31/, and
liver-Insr/ mice.
(H) Glucose tolerance test in 2-month-old male
mice. WT, n = 21; liver-p31/, n = 16; liver-Insr/,
n = 9.
(I) Insulin tolerance test in 2-month-old male mice.
WT, n = 16; liver-p31/, n = 9; liver-Insr/, n = 12.
See also Figures S1 and S2 and Table S1.

delete p31comet in the whole body (Figures S1AS1C). Homozygous p31comet knockout (p31/) mice were born in the expected
Mendelian ratio. Heterozygous p31+/ mice did not show
568 Cell 166, 567581, July 28, 2016

discernible differences from wild-type


(WT) littermates (Figures 1A and 1B).
p31/ newborns and embryos at E18.5,
however, showed mild growth retardation
and died within 5 hr after birth, in spite of
suckling activity. Thus, instead of being
susceptible to tumorigenesis, p31/
mice exhibited neonatal lethality.
The p31comet protein was expectedly
absent in p31/ mouse embryonic fibroblasts (MEFs) (Figure S1D). The p31comet
protein level in p31+/ MEFs was about
65% of that in WT, and these p31+/ cells
did not show discernable abnormalities.
The p31/ MEFs proliferated more
slowly than did WT MEFs (Figure S1E)
and had elevated apoptosis, decreased
S-phase population, and increased
G2/M population (Figure S1F). Consistent
with established mitotic roles of p31comet,
p31/ MEFs exhibited a mitotic delay
and abnormal mitotic features, including
lagging chromosomes (Figures S1G
S1I). Consequently, p31/ MEFs displayed increased polyploidy and aneuploidy (Figures S1J and S1K). These
results validate the functions of p31comet
in mitotic regulation and in suppressing
chromosome instability.
The mitotic and aneuploidy phenotypes of p31/ MEFs were
similar to those reported for MEFs with a hypomorphic allele of
Cdc20 (Malureanu et al., 2010). Mice with the same hypomorphic

Cdc20 allele were, however, viable (Malureanu et al., 2010).


Similarly, partial inactivation of other mitotic regulators, including
BUBR1, induces aneuploidy in mice without compromising
their viability (Baker et al., 2004). Therefore, mitotic defects and
aneuploidy alone in mice do not necessarily lead to neonatal
lethality.
We investigated the defects underlying the neonatal lethality
of p31/ mice. The p31+/ mice did not show differences
discernible from WT littermates. Despite mild growth retardation, p31/ embryos and newborns did not show gross
morphological and developmental defects. The lung and respiratory muscle in p31/ animals were normal, and there was
evidence of lung inflation. p31/ mice also showed normal
liver development (Figure S2A). However, unlike hepatocytes
from WT and p31+/ mice, hepatocytes from p31/ E18.5 embryos and newborns had reduced cytoplasmic vacuolation
(Figures 1C and S2A), suggestive of defects in hepatic
glycogen storage.
Indeed, liver sections of p31/ newborn mice showed
dramatically reduced periodic acid Schiff (PAS) staining, which
detects polysaccharides, including glycogen (Figure 1C). As a
control, the skeletal muscle in the hind limbs of p31/ animals
exhibited normal PAS staining (Figure S2B). Direct biochemical
measurement confirmed that the p31/ liver had significantly
lower glycogen content (Figure 1D). Glycogen storage in the
liver is crucial for energy homeostasis of newborn mice, and
hypoglycemia is a major cause of neonatal lethality (Girard and
Pegorier, 1998). We suspect that the insufficient energy to
breathe and inability to transition from placenta to nursing, as
consequences of decreased glycogen stores in the liver, are
causal factors of neonatal lethality in p31/ animals. These phenotypes of p31/ mice closely resemble those of insulin receptor (IR) knockout (Insr/) mice (Joshi et al., 1996), although we
did not observe muscle hypotrophy in p31/ mice, as seen
with Insr/ mice.
Liver-Specific p31comet Ablation Causes Metabolic
Disorders in Mice
Liver-specific insulin receptor knockout (liver-Insr/) mice survive to adulthood and develop severe hepatic insulin resistance
and metabolic syndromes (Biddinger et al., 2008; Michael et al.,
2000), indicating that insulin resistance in the liver is a major
cause of metabolic disorders. We generated liver-specific
p31comet knockout mice (liver-p31/) by crossing p31F/F mice
with Albumin-Cre mice. Liver-p31/ mice were born in the expected Mendelian ratio, were indistinguishable from WT littermates, and survived to adulthood. Histological analysis did not
reveal hepatic developmental defects in liver-p31/ embryos
and mice (Figures S2C and S2D). The hepatic glycogen level of
liver-p31/ E18.5 embryos was reduced, but to much less
extent than p31/ embryos, allowing liver-p31/ mice to survive. Clearly, p31comet has functions in non-liver tissues that
help to maintain hepatic glycogen levels. Liver-Insr/ mice
showed normal hepatic architecture, but had scattered focal
dysplasia in the periportal area (Figure S2D), as reported previously (Michael et al., 2000).
2-month-old liver-p31/ mice showed hyperglycemia and
hyperinsulinemia in the fed state, although their hyperinsulinemia

was less severe compared to liver-Insr/ mice (Figures 1E and


1F; Table S1). Despite having elevated serum insulin levels, liverp31/ mice had a decreased level of hepatic glycogen (Figures
1G and S2D). Again, the hepatic glycogen shortage in liverp31/ mice was less severe than that in liver-Insr/ mice. The
hepatic triglyceride levels of liver-p31/ mice were decreased
by about 50%, whereas their serum triglyceride levels were
moderately increased (Table S1). These results indicate that specific ablation of p31comet in the liver can promote systemic
changes in glucose and lipid metabolism.
Liver-Insr/ mice showed severe glucose and insulin intolerance (Michael et al., 2000) (Figures 1H and 1I). 2-month-old male
liver-p31/ mice also developed glucose and insulin intolerance, albeit to lesser degrees. These data indicate that loss
of p31comet in the liver suffices to produce defects in insulin
modulation of glucose metabolism. The whole-body p31+/
mice had normal glucose metabolism (Figure S2E), indicating
that p31comet is not haploinsufficient.
Liver-Insr/ mice progressively developed liver dysfunction,
but surprisingly did not show severe glucose/insulin intolerance
at an older age (Michael et al., 2000). Indeed, the blood glucose
levels of 6-month-old liver-Insr/ and liver-p31/ mice became
normal (Figure S2F). Similar phenotypes of whole-body and liverspecific p31comet and Insr knockout mice implicate p31comet in
insulin signaling.
p31comet Regulates Insulin Signaling
In the liver, an important downstream event of insulin signaling is
the stimulation of glycogen synthesis by activating glycogen
synthase (GS) (Boucher et al., 2014). As expected, insulin treatment stimulated the GS activity in WT hepatocytes, and this
stimulation was absent in liver-Insr/ hepatocytes (Figure 2A).
Strikingly, insulin-dependent GS activation was abolished in
liver-p31/ hepatocytes, consistent with the hepatic glycogen
shortage in p31/ mice. Thus, p31comet is required for a crucial
branch of insulin signaling in hepatocytes.
To determine at which step p31comet regulated IR signaling,
we monitored the status of IR autophosphorylation, activating
phosphorylation of AKT (pT308), and inhibitory phosphorylation
of GSK3b (pS9) in whole-liver lysates (Figure 2B). WT, liverp31/, and liver-Insr/ mice were fasted overnight and injected with insulin via inferior vena cava. Liver lysates were
prepared from these mice and subjected to quantitative immunoblotting. The p31comet protein was reduced by about 80% in
liver-p31/ whole-liver lysates (Figure 2B). As hepatocytes
make up about 85% of total cells in the liver, the residual
p31comet in liver-p31/ animals was likely from non-parenchymal cells. IR autophosphorylation and AKT pT308 were
greatly reduced in both liver-Insr/ and liver-p31/ animals.
GSK3b pS9 was also reduced in both groups, but to a lesser
degree, implicating the existence of IR-independent mechanisms for this phosphorylation. Consistent with the in vivo findings, freshly isolated liver-p31/ primary hepatocytes showed
weakened and delayed IR autophosphorylation and AKT pT308
at multiple time points and over a wide range of insulin concentrations (Figures 2C, 2D, S3A, and S3B). Therefore, p31comet is
required for insulin signaling, at the step or upstream of IR
autophosphorylation.
Cell 166, 567581, July 28, 2016 569

Figure 2. p31comet Ablation Causes Insulin Resistance, whereas Bub1b Insufficiency Enhances Insulin Sensitivity

(A) Glycogen synthase (GS) activity of WT, liver-p31/, and liver-Insr/ hepatocytes treated without () or with (+) insulin (Ins). Mean SD (n = 4 independent
experiments).
(B) Immunoblots of whole-liver lysates of WT, liver-p31/, and liver-Insr/ mice treated without () or with (+) insulin (Ins). Each lane contains lysate from an
individual mouse. The relative band intensities are quantified and shown below.
(C and D) Insulin signaling in primary WT and liver-p31/ hepatocytes treated with 10 nM insulin for the indicated times (C) or increasing concentrations of insulin
for 5 min (D). Cell lysates were blotted with the indicated antibodies.

(legend continued on next page)

570 Cell 166, 567581, July 28, 2016

Aneuploidy Alone Is Insufficient to Promote Insulin


Resistance
The status of aneuploidy in normal hepatocytes is still controversial, and it is formally possible that the insulin signaling
defects of liver-p31/ mice are a secondary consequence of
severe aneuploidy in hepatocytes. To assess the extent of hepatic aneuploidy in these mice, we isolated live hepatocytes
from WT and liver-p31/ mice, sorted tetraploid cells, amplified and sequenced genomic DNA from single cells, and
analyzed the whole-genome copy number variation. We
analyzed hepatocytes from mice harboring hypomorphic alleles
of BUBR1 (Bub1bH/H) as a control (Baker et al., 2004). Consistent with a previous study (Knouse et al., 2014), none of the
15 WT hepatocytes were aneuploid, whereas about 20%
Bub1bH/H (2 out of 10) were aneuploid (Figures 2E and S3C).
Only 1 out of 10 p31/ hepatocytes was aneuploid. Thus,
the aneuploidy incidence of p31/ hepatocytes was surprisingly low.
We tested whether p31comet restoration rescued the metabolic and insulin signaling defects of liver-p31/ mice. Adeno-associated virus (AAV)-mediated expression of p31comet,
but not of GFP, in the liver rescued the hyperglycemia and
glucose/insulin intolerance phenotypes and insulin signaling
defects in liver-p31/ mice (Figures 2F2H and S3D). We performed single-cell sequencing to assess the extent of aneuploidy in hepatocytes from liver-p31/ mice injected with
AAV-GFP or AAV-p31. Only 1 of 40 hepatocytes in the AAVGFP group was aneuploid, and 2 of 39 hepatocytes in the
AAV-p31 group were aneuploid (Figures 2I and S3E). Thus,
the extent of aneuploidy in liver-p31/ hepatocytes is very
low (about 5%). p31comet restoration rescues the metabolic defects of p31comet ablation without eliminating aneuploidy,
arguing against aneuploidy as the underlying reason for the
observed defects.
Next, we analyzed glucose metabolism and insulin signaling in
Bub1bH/H mice. The aneuploidy incidence in Bub1bH/H hepatocytes was higher than that in liver-p31/ hepatocytes (Figures
2E, S3C, and S3E) (Knouse et al., 2014). Strikingly, Bub1bH/H
mice exhibited a reduced blood glucose level in the fed state
(Figure 2J) and increased glucose tolerance (Figure 2K) and insulin sensitivity (Figure 2L). In contrast to liver-p31/ hepatocytes,
IR autophosphorylation and AKT phosphorylation in response to
insulin were more robust in Bub1bH/H hepatocytes (Figures S3F
S3I). Thus, another aneuploid mouse model (Bub1bH/H) exhibited opposite insulin signaling phenotypes. These findings
argue against aneuploidy as the underlying cause of defective insulin signaling in p31/ animals, and are more consistent with a
karyotype-independent regulatory function of p31comet in insulin
signaling.

p31comet Prevents Unscheduled Clathrin-Mediated


Endocytosis of IR
The requirement for p31comet in IR autophosphorylation indicates
that p31comet acts upstream in the pathway, possibly at the level
of IR itself. On insulin stimulation, the activated IR at the PM can
be internalized by clathrin-dependent or clathrin-independent
pathways (Goh and Sorkin, 2013). We generated human
HepG2 hepatocellular carcinoma cell lines stably expressing
the C-terminal GFP-tagged IR and examined the localization of
IR-GFP after transfection with control or p31comet small interfering RNAs (siRNAs) (Figure 3A). In control cells, IR localized
to PM in the absence of insulin and was internalized on insulin
treatment and co-localized with RAB7, a late endosome marker.
In contrast, IR-GFP in p31comet-depleted cells had much weaker
PM localization and was enriched in intracellular compartments
(ICs), even without insulin treatment. A large fraction of these IRpositive ICs were positive for RAB7. The GTPase dynamin is
essential for clathrin-mediated endocytosis (McMahon and Boucrot, 2011). Addition of dynasore (Macia et al., 2006), a chemical
inhibitor of dynamin, blocked the aberrant IR internalization in
p31comet-depleted HepG2 cells (Figures 3B and 3C). Co-depletion of the clathrin heavy chain (CLTC) also restored IR at PM
in p31comet-depleted cells. Thus, p31comet suppresses unscheduled clathrin-dependent IR internalization in the basal state in
HepG2 cells.
Next, we analyzed the localization of the endogenous IR
and regulation of IR by insulin in the liver of WT, liver-p31/,
and liver-Insr/ mice in vivo (Figure 3D). IR localized to
the PM of hepatocytes in WT liver and underwent insulin-triggered internalization. The PM staining of IR was absent in
liver-Insr/ hepatocytes, although we could detect residual
IR signals in non-parenchymal cells. Even without insulin
injection, the PM IR signal in the liver of liver-p31/ animals
was already weak, whereas the IR signal in RAB7-positive
ICs was strong. AAV-mediated p31comet expression in liverp31/ mice restored the PM IR signal in the liver (Figure 3E).
These results establish a role of p31comet in suppressing IR
internalization in vivo.
We monitored the kinetics of insulin endocytosis in WT and
liver-p31/ hepatocytes. At several time points, the intensities
of internalized, fluorescently labeled insulin in liver-p31/ hepatocytes were much weaker than those in WT hepatocytes (Figures 3F and 3G). In contrast, the internalization of transferrin,
another client of clathrin-mediated endocytosis, was normal in
liver-p31/ hepatocytes (Figures 3F and 3H). These results indicate that p31comet specifically regulates insulin endocytosis, but
not all forms of clathrin-mediated endocytosis. Both defective insulin endocytosis and insulin-triggered IR autophosphorylation
in p31comet-deficient cells can be attributed to the decreased

(E) Segmentation plots of euploid (WT) and aneuploid hepatocytes from liver-p31/ and Bub1bH/H mice.
(FH) Fed blood glucose levels (F), glucose tolerance test (G), and insulin tolerance test (H) of WT and liver-p31/ mice injected with AAV-GFP or AAV-p31. WT
(AAV-GFP), n = 10; liver-p31/ (AAV-GFP), n = 8; liver-p31/ (AAV-p31), n = 7; mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus liver-p31/ (AAV-GFP);
py < 0.05, yyp < 0.01 versus WT (AAV-GFP).
(I) Segmentation plots of aneuploid cells from liver-p31/ mice injected with AAV-GFP or AAV-p31.
(JL) Fed blood glucose levels (J), glucose tolerance test (K), and insulin tolerance test (L) of WT and Bub1bH/H mice. For GTT and ITT, at least 12 mice in each
group were analyzed. Mean SEM. *p < 0.05, **p < 0.01.
See also Figure S3.

Cell 166, 567581, July 28, 2016 571

Figure 3. p31comet Suppresses Spontaneous IR Endocytosis in the Absence of Insulin


(A) HepG2 cells stably expressing IR-WT-GFP were transfected with the indicated siRNAs, serum starved, and stained with anti-GFP (IR; green) and anti-RAB7
(red) antibodies and DAPI (blue). The boxed region was magnified and shown on the right. Scale bars, 10 mm.

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572 Cell 166, 567581, July 28, 2016

level of functional IR at the PM, due to the premature internalization of IR prior to insulin stimulation.
MAD2 Directly Binds to a Canonical MIM Motif in IR
We wondered whether p31comet function in regulating IR internalization involved MAD2. MAD2 had been reported as an IR-binding protein in yeast two-hybrid and proteomic screens (Hutchins
et al., 2010; ONeill et al., 1997). MAD2 binds to MAD1 and
CDC20 through the MAD2-interacting motif (MIM) with the
consensus of (K/R)ccXcX3-4P (c, a hydrophobic residue;
X, any residue; Figure 4A). The C-terminal cytoplasmic tail of IR
contained a putative MIM (Figure 4A). A peptide containing this
motif (IRMIM-WT) efficiently bound to purified recombinant
MAD2 WT and a monomeric mutant R133A (Figure 4B), but did
not interact with MAD2 DC, a truncation mutant that could not
form C-MAD2. A mutant IR peptide (IRMIM-4A) did not interact
with MAD2. Immunoprecipitation with a C-MAD2-specific antibody (Fava et al., 2011) confirmed that IR-bound MAD2 formed
C-MAD2 (Figure 4C). IRMIM-WT bound to C-MAD2R133A with a
dissociation constant of 380 nM (Figure 4D). Thus, IR contains
a functional MIM.
MAD2 WT, but not DC, interacted with the cytoplasmic
domain of IRb (IRb-C) (Figure 4E), indicating that the MIM is
not masked in the intact cytoplasmic domain of IR and is available for MAD2 binding. MAD2 binding did not affect the kinase
activity of IR, and vice versa. Furthermore, IR-WT-MYC interacted with endogenous MAD2 in 293FT cells, whereas IR-4AMYC did not (Figure 4F). Depletion of p31comet enhanced the
IR-MAD2 interaction, suggesting that p31comet might promote
IR-C-MAD2 disassembly. Endogenous MAD2 and IR interacted
with each other in HepG2 cells (Figure 4G) and in whole-liver lysates of WT and liver-p31/ mice (Figure 4H). Unlike in 293FT
cells depleted of p31comet, in liver lysates of liver-p31/ mice
we did not observe a significant increase of the IR-MAD2 interaction, suggesting that p31comet might not actively promote IRMAD2 disassembly in hepatocytes.
A previous report showed that insulin-stimulated IR activation
reduced the IR-MAD2 interaction in IR-overexpressing CHO
cells (ONeill et al., 1997). In contrast, we found that the IRMAD2 interaction in vitro and in human cells was not regulated
by insulin or IR autophosphorylation (Figures 4E4G). Thus, IR
binds to MAD2 through a canonical MIM in vitro and in vivo.
This interaction is constitutive and is not regulated by insulin.
p31comet Regulates IR Endocytosis by
Counteracting MAD2
We generated HepG2 cells stably expressing IR-4A-GFP and
examined the subcellular localization of this MAD2-binding-defi-

cient mutant. IR-WT localized to the PM in control-depleted


cells, but localized to intracellular compartments in p31cometdepleted cells (Figures 5A and 5B). In contrast, IR-4A retained
its PM localization, even in p31comet-depleted cells. Similar results were obtained in p31/ MEFs (Figures 5C and 5D). Expression of p31comet WT, but not of the MAD2-binding-deficient
Q83A/F191A (QF) mutant (Yang et al., 2007), restored IR at the
PM of p31comet-depleted cells (Figures 5E and 5F). Adenovirus-mediated expression of IR-4A, but not of IR-WT or GFP,
in the liver of liver-p31/ mice restored IR at the PM in hepatocytes (Figure 5G). These results establish the importance of IRMAD2 and MAD2-p31comet interactions in IR endocytosis and
indicate that p31comet regulates insulin signaling through suppressing MAD2.
p31comet Blocks MAD2-BUBR1-Dependent AP2
Recruitment
Adaptor protein 2 (AP2) is crucial for clathrin-mediated endocytosis and interacts with both clathrin and the cargo (McMahon
and Boucrot, 2011). AP2 is a heterotetramer of the a, b2, m2,
and s2 subunits. BUBR1 has been reported to interact with
AP2-b2 (AP2B1) (Cayrol et al., 2002). Through its aC dimerization
helix, C-MAD2 can directly bind to BUBR1 (Chao et al., 2012).
MAD2 might bridge an interaction between IR and BUBR1AP2, thus promoting clathrin-mediated endocytosis of IR.
Consistent with this hypothesis, the aberrant IR endocytosis in
p31comet-depleted HepG2 cells required clathrin, AP2, MAD2,
and BUBR1 (Figures 6A, 6B, and S4AS4C). Depletion of
MAD2 or BUBR1 partially blocked IR endocytosis induced by insulin. Thus, MAD2 and BUBR1 are required for untimely IR internalization in cells with a compromised p31comet function and for
insulin-triggered IR endocytosis.
The recombinant BUBR1 N-terminal domain (BUBR1N) associated with IR-bound MAD2 WT (Figure 6C). It bound much less efficiently to MAD2 R133E/Q134A (RQ), which was monomeric and
contained mutations of two critical residues in aC. p31comet diminished the BUBR1N-MAD2 interaction (Figure 6C), without substantially affecting the IR-MAD2 interaction. p31comet bound
weakly to MAD2 RQ and did not further reduce the residual binding between BUBR1N and MAD2 RQ. Thus, p31comet inhibits the
interaction between BUBR1 and IR-bound C-MAD2 in vitro. In
human cells, MYC-BUBR1 interacted with IR and AP2B1 at a
basal level (Figure 6D). These interactions were enhanced when
clathrin-mediated endocytosis of IR was blocked with dynasore.
Depletion of p31comet further enhanced these interactions,
consistent with the role of p31comet in suppressing IR endocytosis.
In keeping with these protein-binding data, BUBR1, p31comet,
and MAD2 could be detected on PM in HepG2 cells with total

(B) HepG2 cells stably expressing IR-WT-GFP were transfected with the indicated siRNAs, serum starved, treated without or with 80 mM dynasore (Dyn), and
stained with indicated antibodies. Scale bars, 10 mm.
(C) Quantification of the ratios of PM and IC IR-GFP signals of cells in (B) (mean SD; *p < 0.0001).
(D) Liver sections of WT, liver-p31/, and liver-Insr/ mice injected with PBS or insulin (+Ins) were stained with anti-IR (red) and anti-RAB7 (green) antibodies and
DAPI (blue). Scale bars, 10 mm. Asterisks indicate sinusoids.
(E) Liver sections of WT and liver-p31/ mice injected with AAV-GFP or AAV-p31 were stained with anti-IR (red) antibodies and DAPI (blue). Scale bars, 10 mm.
(F) Representative images of endocytosis assays with Cy3-insulin and Alexa 568-transferrin in WT and liver-p31/ hepatocytes at 20 min. The insulin and
transferrin signals are shown in red. The DAPI signals are shown in blue. Scale bar, 10 mm.
(G and H) Endocytosis of insulin (G) and transferrin (H) in WT and liver-p31/ hepatocytes. The intensities of internalized fluorescent signals at the indicated times
were quantified (mean SD; n = 3 independent experiments with >70 cells analyzed at each time point).

Cell 166, 567581, July 28, 2016 573

Figure 4. MAD2 Binds to a Canonical MIM in the C-Terminal Tail of IR


(A) Sequence alignment of the C-terminal tail of IR proteins and human CDC20 and MAD1, with the conserved residues in the MAD2-interacting motif (MIM)
boxed. The MIM consensus is shown on top. Sequences of IRMIM-WT and IRMIM-4A peptides are also shown.
(B) Beads coupled to IRMIM-WT or IRMIM-4A were incubated with the indicated MAD2 proteins. Input and proteins bound to beads were analyzed by SDS-PAGE
and stained with Coomassie (CBB).
(C) Beads coupled to a C-MAD2-specific antibody were incubated with the indicated MAD2 proteins in the presence or absence of IRMIM-WT. Input and beadsbound proteins were detected with the anti-MAD2 antibody.
(D) ITC analysis of binding between MAD2 and IRMIM-WT, with the Kd and binding stoichiometry (N) indicated.

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574 Cell 166, 567581, July 28, 2016

internal reflection fluorescence (TIRF) microscopy (Figure S4D),


and the PM co-localization of IR and BUBR1 was increased by
dynasore treatment with or without insulin stimulation (Figures
S4E and S4F). We then measured the co-localization efficiency
of IR and AP2B1 on the cell surface in control and p31cometdepleted cells (Figures 6EEG). In control cells, IR had a diffusive
PM staining, with no clear enrichment in the AP2B1 puncta (Figure 6E). The punctate staining pattern of AP2B1 became more
pronounced in p31comet-depleted cells, and IR efficiently colocalized with AP2B1 in these surface puncta (Figures 6E and
6F). Co-depletion of BUBR1 or MAD2 in p31comet-depleted cells
reduced the co-localization of IR and AP2B1 (Figures 6F and 6G).
Therefore, p31comet attenuates clathrin-mediated endocytosis of
IR through suppressing MAD2-BUBR1-dependent recruitment
of AP2.
The p31comet-MAD2-BUBR1 Module Regulates Insulin
Signaling
We tested whether the p31comet-MAD2-BUBR1 module indeed
regulated insulin signaling. Insulin-triggered IR and AKT phosphorylation was markedly reduced in liver-p31/ hepatocytes,
indicative of defective insulin signaling (Figures 7A and S5A).
Depletion of BUBR1 or MAD2 from liver-p31/ hepatocytes
significantly restored insulin signaling. Co-depletion of MAD2,
BUBR1, AP2B1, or CLTC similarly restored insulin signaling in
p31comet-depleted HepG2 cells (Figures S5B and S5C). These
results indicate that p31comet-MAD2-BUBR1 controls insulin
signaling through regulating IR endocytosis.
Next, we generated liver-specific p31comet and BUBR1 doubleknockout (liver-p31/-;Bub1b/) mice and tested if BUBR1 ablation rescued the metabolic defects of liver-p31/ mice. Similar to
Bub1bH/H mice, liver-p31/;Bub1b/ mice were normal at birth,
but only attained 40% of normal weight at 3 weeks. Strikingly,
liver-p31/;Bub1b/ mice showed hypoglycemia in the fed state
(Figure 7B), increased glucose tolerance (Figure 7C), and insulin
hypersensitivity (Figure 7D), phenotypes similar to those in
Bub1bH/H mice and opposite of those in liver-p31/ mice.
The cell and nuclear sizes of hepatocytes in liver-p31/;
Bub1b/ mice were larger than those of hepatocytes in the
single-knockout animals (Figures S5D and S5E). The percentage
of hepatocytes with 8N or greater DNA content was higher in
liver-p31/;Bub1b/ mice (Figure S5F). This increased polyploidization is likely due to the complete loss of Bub1b in the
liver. Despite this striking ploidy change, liver-p31/;Bub1b/
mice retained the insulin and glucose hypersensitivity seen in
Bub1bH/H mice. These results do not support a causal link between polyploidization and insulin resistance. We could not
perform single-cell sequencing of p31/;Bub1b/ hepatocytes
to ascertain their aneuploidy status, as live-cell sorting produced
no intact cells.

Because IR-4A does not undergo unscheduled endocytosis


caused by p31comet inactivation (Figure 5), we hypothesized
that the expression of IR-4A might suppress the metabolic phenotypes of liver-p31/ mice. Expression of IR-4A-GFP, but not of
IR-WT-GFP, rescued the insulin signaling defects in HepG2 cells
depleted of p31comet (Figures S6A and S6B) and in primary hepatocytes isolated from liver-p31/ mice (Figures S6C and S6D).
Adenovirus-mediated expression of IR-4A, but not of IR-WT or
GFP, restored insulin-stimulated IR autophosphorylation and
AKT phosphorylation in the liver of liver-p31/ mice (Figure 7E).
Expression of IR-4A also significantly decreased fed blood
glucose levels (Figure 7F) and restored glucose tolerance (Figure 7G) and insulin sensitivity (Figure 7H) in liver-p31/ mice.
These results indicate that p31comet regulates insulin signaling
through suppressing the functions of the IR-MAD2 interaction
in vivo.
DISCUSSION
Combining mouse genetics, cell biological and biochemical
methods, and single-cell genomics, we have discovered a critical role for the p31comet-MAD2-BUBR1 module of mitotic regulators in insulin signaling through regulating IR endocytosis
(Figure 7I).
In the mouse, p31comet deficiency diminishes IR at the PM
prior to insulin stimulation and causes defective insulin signaling
and metabolic syndrome. The insulin signaling defects caused
by p31comet ablation are not limited to the liver, as whole-body
p31/ mice exhibit neonatal lethality, whereas liver-specific
p31/ mice are viable. Indeed, p31/ MEFs exhibited defective
insulin-induced adipogenesis (Figures S7A and S7B). Insulin
signaling, GLUT4 translocation, and insulin-stimulated glucose
uptake were defective in the poorly differentiated p31/ adipocytes (Figures S7CS7E).
While we cannot rule out mitotic defects and the low extent of
aneuploidy as factors contributing to the metabolic defects in
p31comet-deficient mice, two complementary lines of evidence
argue against aneuploidy as the determining factor. The first
line of evidence involves the use of BUBR1-deficient (Bub1bH/H)
mice. These mice harbor aneuploidy in hepatocytes, but are
more sensitive to insulin, a phenotype opposite of that of liverp31/ mice. Furthermore, liver-specific ablation of BUBR1
rescues the metabolic defects of liver-p31/ mice, despite
increasing polyploidization in hepatocytes. These findings support our conclusion that BUBR1 facilitates IR endocytosis and
attenuates insulin signaling. It also indicates that aneuploidy or
polyploidy alone is insufficient to produce insulin signaling defects caused by p31comet ablation.
The second line of evidence comes from genetic suppression experiments with transgene expression in the liver. The

(E) GST pull-down assays with recombinant GST-IRb-C and MAD2. Input and beads-bound proteins were blotted with the indicated antibodies. The relative
intensities of pY and MAD2 (mean SEM; n = 3 independent experiments) are shown below.
(F) 293FT cells were co-transfected with IR-WT-MYC or IR-4A-MYC constructs and the indicated siRNAs, serum starved, and treated without () or with (+)
100 nM insulin (Ins) for 20 min. The total cell lysate (TCL) and anti-MYC immunoprecipitate (IP) were blotted with the indicated antibodies.
(G) HepG2 cells were transfected with siLuc or siMAD2, serum starved, and treated without () or with (+) 100 nM insulin (Ins) for 20 min. TCL, anti-MAD2 IP, and
IgG IP were blotted with the indicated antibodies.
(H) Total liver lysates from WT, liver-p31/ and liver-Insr/ mice, and anti-MAD2 and IgG IP from these lysates were blotted with the indicated antibodies.

Cell 166, 567581, July 28, 2016 575

Figure 5. p31comet Suppresses Spontaneous IR Endocytosis through Counteracting the IR-MAD2 Interaction
(A) HepG2 cells stably expressing IR-WT-GFP or IR-4A-GFP were transfected with siLuc or si-p31, serum starved, and stained with anti-GFP (IR; green) antibody
and DAPI (blue). Scale bars, 10 mm.
(B) Quantification of the ratios of PM and IC IR-GFP signal intensities in (A) (mean SD; ****p < 0.0001).
(C) WT and p31/ MEFs were infected with IR-WT-GFP or IR-4A-GFP retroviruses, serum starved, treated without or with dynasore (Dyn), and stained with antiGFP (IR; green) antibody and DAPI (blue). Scale bar, 10 mm.
(D) Quantification of the ratios of PM and IC IR-GFP signal intensities in (C) (mean SD; ****p < 0.0001).
(E) IR-GFP-expressing HepG2 cells were co-transfected with the indicated siRNAs and plasmids and stained with anti-GFP (IR; green) and anti-MYC (p31comet;
red) antibodies and DAPI (blue). Scale bar, 10 mm.
(F) Quantification of the ratios of PM and IC IR-GFP signal intensities in (E) (mean SD; ****p < 0.0001).
(G) Liver sections of WT and liver-p31/ mice injected with Ad-GFP, Ad-IR-WT, or Ad-IR-4A were stained with anti-IR (red) antibody and DAPI (blue). Scale
bar, 10 mm.

576 Cell 166, 567581, July 28, 2016

Figure 6. p31comet Prevents MAD2- and BUBR1-Dependent IR Endocytosis through Blocking AP2 Recruitment
(A) IR-GFP-expressing HepG2 cells were transfected with the indicated siRNAs, treated without or with 100 nM insulin (Ins) for 5 min, and stained with DAPI (blue)
and anti-GFP (IR; green) and anti-RAB7 (red) antibodies. Scale bars, 10 mm.

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Cell 166, 567581, July 28, 2016 577

aneuploidy incidence in p31/ hepatocytes is surprisingly low.


Restoring p31comet expression using viral vectors does not eliminate this low-level aneuploidy, but rescues metabolic and insulin
signaling defects in liver-p31/ mice and hepatocytes. Expression of IR-4A similarly rescues the phenotypes of liver-p31/
mice and hepatocytes. These results indicate that the insulin
signaling defects caused by p31comet ablation are not simply
an indirect consequence of aneuploidy. Rather, they strongly
support our conclusion that p31comet and MAD2 directly regulate
insulin signaling through a physical interaction with IR.
We propose that O-MAD2 can bind to IR without the need
for MAD1-mediated conformational activation (Figure 7I). IRbound MAD2 then recruits AP2 to IR through BUBR1 and promotes IR endocytosis. p31comet prevents IR-bound MAD2
from binding BUBR1 and blocks AP2 recruitment to IR, thereby
inhibiting clathrin-mediated endocytosis of IR. Insulin-triggered IR autophosphorylation events have been shown to be
a requirement for insulin-stimulated IR endocytosis (Goh
and Sorkin, 2013). MAD2 and BUBR1 are also required for
insulin-stimulated IR endocytosis in cells without genetic
inactivation of p31comet. Thus, insulin stimulation might suppress p31comet-mediated blockade of BUBR1-AP2 association
with IR. Future experiments are needed to test this intriguing
possibility.
The p31comet-MAD2-BUBR1 module specifically regulates the
endocytosis of cell-surface receptors that contain the MIM. The
MIM is highly conserved in all vertebrate IR proteins (Figure 4A),
suggesting that the metabolic function of the p31comet-MAD2BUBR1 module might have been acquired in vertebrates. The insulin-like growth factor 1 receptor (IGF1R) does not contain the
MIM and does not bind to MAD2 (ONeill et al., 1997). On the
other hand, IR might not be the only client cell-surface protein
regulated by MAD2. For example, MAD2 can bind to another
membrane protein, ADAM17, a metalloprotease with myriad
functions in cancer biology (Nelson et al., 1999). We have identified a functional MIM in the cytoplasmic tail of ADAM17 (Figures
S7FS7H). Additional studies are required to establish the functional significance of this interaction and to systematically identify cell-surface proteins that interact with MAD2.
BUBR1 insufficiency in mice causes aging-related disorders
(Baker et al., 2004), and, conversely, BUBR1 overexpression extends lifespan and delays aging in mice (Baker et al., 2013). An
evolutionarily conserved function of IR/IGF1R signaling in
longevity and aging has been widely documented (Bluher
et al., 2003; Kimura et al., 1997). We have now linked BUBR1
to insulin signaling. Future experiments are needed to test

whether changes in insulin signaling contribute to the aging phenotypes of mice with altered BUBR1 expression.
Liver-specific ablation of p31comet in mice produces metabolic
disorders reminiscent of type 2 diabetes, including insulin resistance. The underlying mechanisms of insulin resistance in type 2
diabetes are complex and not fully understood (Samuel and
Shulman, 2012). Our findings presented herein implicate premature IR internalization prior to insulin binding as a potential mechanism underlying insulin resistance. It will be interesting to
examine IR levels or localization in liver biopsies of human
type 2 diabetes patients.
Our results establish the role of mitotic checkpoint regulators
in insulin signaling and metabolic homeostasis. Our study provides a striking example of how an entire branch of key regulators in one cellular process can be repurposed to control
another. By virtue of its ability to link signaling pathways originating from kinetochores and the plasma membrane, the
p31comet-MAD2-BUBR1 module may offer a potential conduit
for extracellular hormones to regulate chromosome segregation
and karyotypes.
EXPERIMENTAL PROCEDURES
Generation and Phenotypic Analyses of p31/ and Liver-p31/ Mice
All animal experiments were performed in accordance with institutional guidelines and with approval from the Institutional Animal Care and Use Committee
of UT Southwestern Medical Center.
The strategy of targeting p31comet (Mad2l1bp) is described in Figure S1. The
p31F/F mice were crossed with transgenic mice expressing Cre recombinase
under the Actin promoter to generate the p31/ mice and were mated with
transgenic mice expressing Cre recombinase under the Albumin promoter
to generate liver-p31/ mice. See the Supplemental Experimental Procedures
for information about the mouse crosses and husbandry and generation of
liver-p31/;Bub1b/ mice.
Tissue histology and immunohistochemistry were performed by an oncampus core facility. Hepatic glycogen content was measured with the
Glycogen Assay Kit (Sigma). Glucose and insulin tolerance tests, metabolic
profiling, glycogen synthase activity assay, and in vivo insulin signaling assay
were performed with established protocols. Prism was used for the generation
of all curves and graphs and for statistical analyses. See the Supplemental
Experimental Procedures for details.
Cell Culture, Transfection, and Viral Infection
Mouse embryonic fibroblasts (MEFs) and primary hepatocytes were isolated
and cultured following standard protocols. 293FT and HepG2 cells were
cultured in DMEM, supplemented with fetal bovine serum. Plasmid
transfections into 293FT cells and HepG2 were performed with polyethylenimine (PEI; Sigma) and Lipofectamine 2000 (Invitrogen), respectively. siRNA
transfections were performed with Lipofectamine RNAiMAX (Invitrogen).

(B) Quantification of the ratios of PM and IC IR-GFP signal intensities in (A) (mean SD; ****p < 0.0001).
(C) The indicated proteins were incubated for 1 hr and added to beads coupled to the IRMIM-WT peptide. Proteins bound to beads were blotted with the indicated
antibodies. The relative BUBR1 intensities (mean SEM; n = 3 independent experiments) are shown below.
(D) 293T cells were transfected with plasmids encoding MYC-BUBR1, AP2B1, and IR and treated with or without dynasore (Dyn). The total cell lysates (TCL), antiMYC IP, and IgG IP were blotted with the indicated antibodies. The relative intensities of IRb and AP2B1 (mean SEM; n = 3 independent experiments) are shown
in the figure.
(E) HepG2 cells stably expressing IR-WT-GFP were transfected with the indicated siRNAs and stained with anti-GFP (IR; green) and anti-AP2B1 (red) antibodies.
Two boxed regions (1 and 2) were magnified and shown. Scale bar, 10 mm.
(F) Quantification of the Manders coefficients of IR and AP2B1 co-localization in (E) (mean SEM; siLuc, n = 16; si-p31, n = 26; si-p31/siBUBR1, n = 12; si-p31/
siMAD2, n = 11; **p < 0.005).
(G) Western blot analysis of cell lysates in (E) and (F).
See also Figure S4.

578 Cell 166, 567581, July 28, 2016

Figure 7. The p31comet-MAD2-BUBR1 Module Controls Insulin Signaling


(A) Hepatocytes from WT and liver-p31/ mice were transfected with siRNAs, serum starved, and then treated with 10 nM insulin (Ins) for 5 min. Cell lysates were
blotted with the indicated antibodies.
(BD) Fed blood glucose levels (B), glucose tolerance test (C), and insulin tolerance test (D) of WT, liver-p31/, Bub1bH/H, and liver-p31/;Bub1b/ mice. At least
eight mice in each group were analyzed. Mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus WT; py < 0.05, yyp < 0.01, yyyp < 0.001,
yyyyp < 0.0001 versus liver-p31/;Bub1b/.

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Cell 166, 567581, July 28, 2016 579

Recombinant adenoviruses and adeno-associated viruses were generated at


Agilent Technologies and Vector Biolabs, respectively, and were introduced to
mice through tail-vein injection. See the Supplemental Experimental Procedures for adipocyte differentiation and glucose uptake assay, generation of
stable cell lines, list of siRNAs, and other details.

SUPPLEMENTAL INFORMATION

Single-Cell Whole-Genome Sequencing


Hepatocytes were isolated from WT, liver-p31/, and Bub1bH/H mice and
sorted into single cells with flow cytometry. GenomePlex Single Cell Whole
Genome Amplification (WGA4; Sigma) was performed. Library generation
and sequencing were performed by an on-campus facility. Sequencing reads
were aligned against the reference genome. Copy numbers were estimated
with a bin size of 500 kb. The average gene copy number of each chromosome
was calculated, and log2 (copy number/average autosome copy number) was
used to classify aneuploidy, with cutoff set at 0.15. See the Supplemental
Experimental Procedures for details.

AUTHOR CONTRIBUTIONS

Immunoprecipitation and Immunoblots


Cleared cell lysates were incubated with antibody-conjugated beads. The proteins bound to beads were eluted with SDS sample buffer and analyzed by
SDS-PAGE and quantitative western blotting. Membranes were scanned
with the Odyssey Infrared Imaging System (LI-COR). See the Supplemental
Experimental Procedures for details and list of antibodies.
Immunofluorescence, Live-Cell Imaging, and Metaphase Spreads
Immunofluorescence microscopy was performed on cells grown on coverslips
or on liver sections following standard fixation and staining procedures.
Cells were imaged on various microscopes with the appropriate objectives.
Live-cell imaging was performed on MEFs expressing H2B-mRFP. Metaphase
spreads were prepared for MEFs, and images were captured with a Zeiss
Axioscope upright microscope. Identical exposure times and magnifications
were used for all comparative analyses. Images were analyzed with Image J.
See the Supplemental Experimental Procedures for details and for the endocytosis assay.
Flow Cytometry
MEFs pulsed with bromodeoxyuridine (BrdU) were fixed and stained with fluorescein isothiocyanate (FITC)-anti-BrdU antibody (BD Bioscience) and propidium iodide. Hepatocytes were fixed and stained with propidium iodide. Cells
were analyzed with FACSCalibur or FACS Aria II SORP (BD Bioscience). Data
were processed with FlowJo. See the Supplemental Experimental Procedures
for details.
Protein Binding Assays
Recombinant MAD2 proteins were purified as previously described (Luo et al.,
2004). BUBR1N (residues 1370) and mouse p31comet were purified with
affinity and conventional chromatography. IR peptides were chemically
synthesized. Glutathione S-transferase (GST)-IR (residues 10111382) was
purchased from Promega. The isothermal titration calorimetry (ITC) assay of
IR-WT binding to MAD2 was performed as previously described (Xia et al.,
2004). Peptide- or protein-bound beads were used to pull down prey proteins
in line with standard procedures. See the Supplemental Experimental Procedures for details.

Supplemental Information includes Supplemental Experimental Procedures,


seven figures, and one table and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2016.05.074.

E.C. designed and performed all the experiments, analyzed the data, and
wrote the paper. X.Z. and C.X. analyzed copy number variation in hepatocytes.
H.Y. supervised the project, provided suggestions, and edited the paper.
ACKNOWLEDGMENTS
We thank Xuemin Zhang for assistance with generating the p31F/F mice, Jan
van Deursen for providing the Bub1bH/H mice and other reagents, Mayuko
Hara for performing ITC, Bing Li for preparing BUBR1N, Min Kim for advice
on adenovirus production, Ralph DeBerardinis for providing 293FT and
HepG2 cells, Vanessa Schmid and Rachel Bruce for single-cell sequencing,
John Shelton and James Richardson for advice on histological analysis, and
Xuelian Luo for advice on protein purification. We are grateful to anonymous
reviewers for suggesting the genetic suppression experiments. This work is
supported, in part, by grants from the Clayton Foundation (to H.Y.) and the
NIH (UL1TR001105 to C.X.). H.Y. is an HHMI Investigator.
Received: March 23, 2015
Revised: December 9, 2015
Accepted: May 24, 2016
Published: June 30, 2016
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Cell 166, 567581, July 28, 2016 581

Article

AIRE-Deficient Patients Harbor Unique High-Affinity


Disease-Ameliorating Autoantibodies
Graphical Abstract

Authors
Steffen Meyer, Martin Woodward,
Christina Hertel, ..., Part Peterson,
Kai Kisand, Adrian Hayday

Correspondence
kai.kisand@ut.ee (K.K.),
adrian.hayday@kcl.ac.uk (A.H.)

In Brief
Self-reactive antibodies specific for type I
interferons are associated with protection
against type I diabetes in patients with an
autoimmune syndrome caused by
mutations in AIRE.

Highlights
d

Each AIRE-deficient patient has a private repertoire of


autoantibody reactivities
Loss of B cell tolerance occurs during T cell-dependent
somatic hypermutation
Patient autoantibodies have unprecedented affinities for
conformational epitopes
Patient autoantibodies can display disease-ameliorating
properties in vivo

Meyer et al., 2016, Cell 166, 582595


July 28, 2016 2016 The Authors. Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.cell.2016.06.024

Article
AIRE-Deficient Patients Harbor Unique
High-Affinity Disease-Ameliorating Autoantibodies
Steffen Meyer,1,11 Martin Woodward,2,11 Christina Hertel,1,11 Philip Vlaicu,1,11 Yasmin Haque,2,11 Jaanika Karner,3,11
Annalisa Macagno,4 Shimobi C. Onuoha,4 Dmytro Fishman,5,6 Hedi Peterson,5,6 Kaja Metskula,7 Raivo Uibo,7 Kirsi Jantti,8
Kati Hokynar,8 Anette S.B. Wolff,9 APECED patient collaborative, Kai Krohn,8 Annamari Ranki,10 Part Peterson,3
Kai Kisand,3,* and Adrian Hayday2,*
1ImmunoQure

AG, Konigsallee 90, 2012 Dusseldorf, Germany


Gorer Department of Immunobiology, Kings College, London SE19RT, UK
3Molecular Pathology, Institute of Biomedicine and Translational Medicine, University of Tartu, Ravila 19, Tartu 50411, Estonia
4ImmunoQure Research AG, Wagistrasse 14, 8952 Schlieren, Switzerland
5Institute of Computer Science, University of Tartu, Liivi 2, Tartu 50409, Estonia
6Quretec Ltd., U
likooli 6A, Tartu 51003, Estonia
7Department of Immunology, Institute of Biomedicine and Translational Medicine, University of Tartu, Ravila 19, Tartu 50411, Estonia
8Clinical Research Institute HUCH Ltd., Haartmaninkatu 8, 00290 Helsinki, Finland
9Department of Clinical Science, University of Bergen, Laboratory Building, 8th floor, 5021 Bergen, Norway
10Department of Dermatology, Allergology and Venereology, Institute of Clinical Medicine, University of Helsinki, Skin and Allergy Hospital,
Helsinki University Central Hospital, Meilahdentie 2, 00250 Helsinki, Finland
11Co-first author
*Correspondence: kai.kisand@ut.ee (K.K.), adrian.hayday@kcl.ac.uk (A.H.)
http://dx.doi.org/10.1016/j.cell.2016.06.024
2Peter

SUMMARY

APS1/APECED patients are defined by defects in the


autoimmune regulator (AIRE) that mediates central
T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/
APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins.
Thereby, autoantibodies from 81 patients collectively
detected many thousands of human proteins. The
loss of B cell tolerance seemingly occurred during
antibody affinity maturation, an obligatorily T celldependent step. Consistent with this, many APS1/
APECED patients harbored extremely high-affinity,
neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically
active in vitro and in vivo, and those neutralizing
type I interferons (IFNs) showed a striking inverse
correlation with type I diabetes, not shown by other
anti-cytokine antibodies. Thus, naturally occurring
human autoantibodies may actively limit disease
and be of therapeutic utility.
INTRODUCTION
T lymphocyte tolerance is essential for limiting autoimmune disease. Tolerance occurs centrally when developing thymocytes
with strongly self-reactive T cell receptors (TCRs) are deleted
following engagement of self-antigen-derived peptides presented by major histocompatibility complex (MHC) antigens.
The expression of thousands of tissue-specific self-antigens

(TSAs) by medullary thymic epithelial cells (mTEC) is directly promoted by AIRE, a poorly understood transcriptional regulator
(Mathis and Benoist, 2009; Klein et al., 2014). Reflecting its
importance, AIRE deficiency is defined by the APS1/APECED
syndrome for which autoimmune polyendocrinopathy and
chronic mucocutaneous candidiasis are pathognomonic (Nagamine et al., 1997).
There are also several mechanisms of peripheral T cell tolerance, including requirements for co-stimulatory signals for the
activation of naive T cells; the expression of molecular brakes
(e.g., CTLA-4, PD-1) by activated T cells; and the suppression of
effector T cells in trans by FOXP3-expressing T-regulatory
(T-reg) cells. Reflecting its importance, FOXP3 deficiency is
defined by early-onset, life-threatening autoimmunity (Bennett
et al., 2001; Wildin et al., 2001).
Central and peripheral tolerance mechanisms have likewise
been hypothesized to shape the B cell compartment. Thus,
self-reactive B cells developing in the bone marrow may be
censored by clonal deletion, clonal anergy, or B cell receptor
(BCR) editing in which secondary gene rearrangements replace
the initial BCR with a new specificity (Goodnow et al., 2010; Pillai
et al., 2011; Ubelhart and Jumaa, 2015). Peripheral B cell tolerance is less well characterized, although some checkpoints
have been inferred. For example, immature transitional B cells
recently emigrated from the bone marrow contain many autoreactive and polyreactive cells, whereas there are relatively few
among mature naive B cells, strongly suggesting that tolerance
is imposed as transitional B cells differentiate into naive B cells
(Wardemann et al., 2003).
Interestingly, this B cell checkpoint is T cell dependent, as reflected by its impairment in patients with T-reg deficiencies
(Kinnunen et al., 2013). Likewise, CD40L and MHC class II
deficiencies that each impair T-B interactions also display
more autoreactive B cells (Meffre and Wardemann, 2008). These

582 Cell 166, 582595, July 28, 2016 2016 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

(legend on next page)

Cell 166, 582595, July 28, 2016 583

considerations raise the possibility that B cell tolerance is largely


governed by the state of T cell tolerance.
Certainly, any autoreactive B cell that might progress through
to the naive B cell compartment of a healthy individual should
lack cognate autoreactive T cells to help it mature. Likewise,
T cell help is required in the germinal center (GC) reaction in
which B cells undergo somatic hyper-mutation (SHM) of the
immunoglobulin (Ig) variable (V) region genes, thereby driving
T cell-dependent selective expansion of clones with increased
antigen affinity (Brink, 2014). The question that then arises is
whether major defects in central T cell tolerance provoke wideranging losses of B cell tolerance at either or both of these
stages.
An approach to assessing this is to examine B cell reactivities
in AIRE-deficient APS1/APECED patients whose under-expression of TSAs in the thymus is predicted to lead to increased
numbers of peripheral autoreactive T cells. Thus, there are reports of APS1/APECED patients carrying autoantibodies against
twenty-five TSAs, with prevalence ranging from 6% to 69%
(Kisand and Peterson, 2015). Their specificities include steroidogenic enzymes, consistent with the patients polyendocrinopathies (Krohn et al., 1992; Uibo et al., 1994; Winqvist et al.,
1993). In addition, most patients display autoreactivities toward
type I IFNs and T helper (Th)-17-related cytokines, antibodies to
which limit resistance to Candida infection (Kisand et al., 2010;
Meager et al., 2006; Puel et al., 2010).
These findings notwithstanding, there has been no large-scale
analysis of the scope and nature of autoantibodies in APS1/
APECED patients, thereby resolving how T cell tolerance impacts upon B cell tolerance in humans. By analyzing 81 APS1/
APECED patients, we found that each was much more likely
than a healthy relative or an unrelated control to harbor strong
serum reactivities toward 100 human proteins. About 10 of
those, including type I IFNs and interleukin-22 (IL22), were
recognized by almost all patients, whereas others were mostly
private specificities. Hence, 81 patients collectively harbored
antibodies toward >3,700 human proteins.
Focusing on antibodies to type I IFNs, IL22, and IL17, we
found unexpectedly that most were reactive to conformational
determinants and included highly mutated antibodies of subpicomolar affinity. Because their gemline counterparts were
not self-reactive, B cell autoreactivity was most probably
driven by self-reactive T cells in the GC reaction. The autoantibodies commonly neutralized their targets in vivo, and APS1/
APECED patients with signature type 1 diabetes (T1D)-associated antibodies (e.g., anti-GAD65) commonly failed to develop
T1D so long as they harbored powerfully neutralizing IFNa-specific antibodies. Thus, autoantibodies naturally arising in subjects with defective central T cell tolerance may be disease
ameliorating.

RESULTS
High-Titer Autoreactivities in APS1/APECED
Sera from 81 APS1/APECED patients from discrete Finnish, Norwegian, Slovenian, and Sardinian cohorts were directed against
a ProtoArray displaying 9000 immobilized recombinant human
proteins or protein fragments. Because some patients were
sampled longitudinally, 97 sera were assayed in total. Control
sera were from healthy first-degree relatives (n = 9) and healthy
unrelated volunteers (n = 12) across the same age range. Data
readouts for the binding of individual sera were normalized by
applying robust linear modeling (Sboner et al., 2009), whereafter
each signal was assigned a Z score denoting the number of standard deviations (SD) above or below the mean of the combined
healthy relatives and controls.
Most patients and the combined controls displayed Z scores
of 12 for 200 proteins (Figure 1A). However, when the convention was employed of defining Z R 3 as bona fide positives, the
patients segregated from the two control cohorts, considered
either jointly or separately. Thus, each control serum displayed
reactivities of Z R 3 toward an average of %20 proteins, with
most recognizing < 10 (Figures 1A, 1B, and S1A). Given that
there was inter-individual variation, the 21 control sera collectively displayed Z R 3 reactivities toward 406 distinct proteins,
i.e., 5% of those displayed on the array (Figure 1B). For only
2 proteins was Z R 4, and for none was Z R 5 (Figures 1A and
1B). Hence, as expected, the control cohorts largely lacked
high-titer serum autoreactivities.
Conversely, most patients at any one time displayed Z R 3
autoreactivities toward R 80 proteins (Figures 1A, 1B, and
S1A). These data were re-analyzed with stringent procedures
to minimize false-positives, including exclusion of any signals
that might have arisen from cross-sample print contamination. With this achieved, the patients private autoantibody
repertoires collectively detected 3,731 distinct targets (Figure 1B). Furthermore, almost all patients displayed Z R 4
scores for at least 10 proteins (mean of 30), collectively
recognizing > 1,500 proteins, and > 50% of patients displayed
Z R 5 scores for R 10 proteins (mean of > 12), collectively
recognizing 636 proteins (Figures 1A and 1B). Hence, high-level
reactivity toward multiple self-proteins was a disease-defining
property. This was further illustrated by the qualitative difference in Z score distribution curves for patients versus controls,
which cannot simply be explained by there being 5-fold more
patient sera (Figure 1C). Thus, whereas sampling greater
numbers would likely have increased the protein species detected by control cohorts at Z R 4, it would not bridge the
1,000-fold gap between two proteins detected by 21 control
sera versus > 1,500 proteins detected by 97 patient sera
(Figure 1B).

Figure 1. Immune Response Profiling of APS1/APECED


(A) Distributions of hits between patients and controls at different Z scores.
(B) Z scores for all samples against all protein features and mean hits for each group calculated for Z R 3, Z R 4, and Z R 5. The number of distinct proteins
targeted in each group (P, n = 97; C, n = 21) at Z scores denoted. The complexity factor was calculated by dividing the number of distinct proteins by average
number of hits per patient.
(C) The max Z score distribution of all proteins in patient and control groups.
(D) Fraction of patients recognizing each of 3,731 proteins at Z R 3. Red dots depict 126 proteins shared between patients and controls.

584 Cell 166, 582595, July 28, 2016

In sum, 81 different patients collectively displayed strong reactivities to >40% of human proteins arrayed. For most proteins
(blue dots 133731, Figure 1D), reactivities were spread across
the cohort, reflecting high inter-patient variation, whereas 12
proteins (blue dots 112, Figure 1D), including several type I
IFNs, were recognized by > 60% of patients, as reported
(Meager et al., 2006). However, the public specificities were
not enriched among the 126 reactivities shared between patients
and controls at z > 3 (red dots, Figure 1D), emphasizing that their
common autoantigenicity is unique to the patients. Patient autoreactivity frequencies were largely comparable across geographical locations, albeit somewhat less in Norway and Slovenia, and
age ranges (Figures S1B and S1C). Indeed, most anti-IFN autoantibodies of APECED patients were reported to increase early in
life and remain stable thereafter (Meager et al., 2006; Wolff et al.,
2013).
The collective targets of patient antibodies included intracellular, trans-membrane, and secreted proteins. Because many
proteins displayed on the ProtoArray may be denatured, there
may be false-negatives that underestimate patient reactivities
to conformational determinants. Although a detailed analysis of
the types of proteins targeted will be presented, it is evident
that the proteins most commonly detected by patient sera
included numerous cytokines, particularly type I IFNs, for which
reason this study focuses on the nature of those autoreactivities.
Strong, Selective Anti-Cytokine Reactivities
Human type I IFN genes include 13 IFNa genes, 1 IFNb gene, and
1 IFNu gene. There is also a type II IFNg gene and three type III
IFNl genes. IFNg is largely limited to lymphocytes, whereas
type I and type III IFNs are broadly expressed, with their functional uniqueness and/or redundancy unresolved (Ivashkiv and
Donlin, 2014). As assessed by ProtoArray, patient sera showed
significantly stronger reactivities than controls toward all IFNa
subtypes, albeit the reactivities to some (e.g., a1/13, a5, and
a14) were higher than those to others (e.g., a2, a16, and a21)
(Figure 2A). The differential between patients versus controls
was emphasized by luciferase-based immunoprecipitation
(LIPS) in which many target proteins were recognized in their
native conformations (Figure 2B). Many patients showed strong
reactivities to IFNu but rarely toward IFNb (Figure 2B) and never
toward IFNk and IFN, two phylogenetically distant type I IFNs
(data not shown). By contrast, patient sera harbored reactivities
significantly above controls toward IL1a, IL5, IL6, IL17A, IL17F,
IL20, IL22, IL28A (IFNl2), IL28B (IFNl3), and IL29 (IFNl1) (Figure 2C). Whereas reactivities toward some targets (e.g., IL17F,
IL22) were common to most patients, reactivities toward others
(e.g., IL20, IL28, IL6) were not (Table S1), and with the exception
of IL5, patient sera mostly did not detect either Th2 cytokines
(e.g., IL4 and IL13) or IL21, a Tfh (T follicular helper) cell cytokine
that drives high-affinity antibody maturation. There were also no
reactivities toward G-CSF and GM-CSF (Table S1), which drive
the development of myeloid cells associated with the patients
inflammatory endocrinopathies.
Cytokine reactivities were largely validated by ELISA, which
confirmed that IFNg was only rarely and weakly recognized by
patient sera (Figure 2D; Table S1) and that there was no reactivity
toward TNFa (data not shown). By contrast, ELISA revealed au-

toantibodies toward IL32a and IL32g, two poorly characterized


proinflammatory cytokines (Figure 2D; Table S1). In sum, 81
APS1/APECED patient sera collectively displayed strong reactivities to a very selective subset of human cytokines.
Very High-Affinity Human Antibodies
To understand the nature of patient serum reactivities, nine
IFNa-specific monoclonal antibodies (mAbs) were derived by
limit-dilution cloning from memory B cells of four patients. Two
were characterized in detail (26B9 and 19D11), whereas a
more limited analysis of the others strongly argued that the properties of 26B9 and 19D11 were generally representative of
patients cytokine-specific antibodies. First, their VH and Vk
sequences were highly mutated relative to their germline
counterparts, with non-conservative replacements enriched
in complementarity-determining regions (CDRs), as expected
(white; Figure 3A). The antibodies bore no obvious resemblance
to each other in V-gene segment or CDR3 usage. Conversely, a
third anti-IFNa antibody, 50E11, shared with 19D11 the same VH
(IGHV1-69) and junctional (IGHJ4) segments and a very similar
light chain (IGKV3-11 versus V3-20) (Figure S2A). However, there
were very different template-independent nucleotide insertions
in the VH CDR3s of 19D11 and 50E11, and the somatic mutation
patterns were different: whereas 19D11 and 26B9 showed high
mutation frequencies in VH CDR2 and Vk CDR1, 50E11 did not
(Figures 3A and S2A).
The recombinant antibodies 26B9 and 19D11 harvested from
transfected CHO cells were immobilized on surface plasmon
resonance (SPR) chips over which were run recombinant human
IFNa2b, IFNa4, IFNa14, and IFNu, the latter being recognized by
26B9 but not by 19D11 (Figure 3B). These experiments revealed
very slow off-rates reflecting extremely high affinities of the antibodies for their targets, ranging from KD = 3.28e 14M for 26B9
toward IFNa14 to KD = 2.09e 11M for 26B9 toward IFNa2b (Figures 3B and 3C). Sub-picomolar/near-femtomolar dissociation
constants were likewise shown by 19D11 (Figures 3B and 3C).
Thus, APS1/APECED patients harbor some of the strongest
affinity antibodies described.
18-mer peptides spanning IFNa2b and IFNu were used to
map linear epitopes recognized by 26B9 and 19D11. However,
no specific reactivities were detected (data not shown), consistent with the antibodies binding conformational determinants
shared by several type I IFNs. Also, the antibodies reacted poorly
or not at all to mouse IFNs (Table S2).
To investigate the origins of the high-affinity, conformationspecific antibodies, germline counterparts for 19D11, 26B9,
and 50E11, albeit with the same CDR3-VDJ sequences, were expressed and tested by LIPS against recombinant human IFNa2b,
IFNa8, and IFNa14. There was no measurable interaction with
any target (Figure 3D), although the antibodies quality was
evident from their comparable detection by anti-human IgG (Figure S2B). These data argue that the strong autoreactivity toward
IFNs developed de novo during affinity maturation, rather than
being an intrinsic property of the germline repertoire that is
enhanced by affinity maturation.
The high affinities of 26B9 and 19D11 were not unique. Thus, a
patient-derived IgGk mAb (20A10) specific for IL20 (which is
not a target detected by most patients; Figure 2C; Table S1)
Cell 166, 582595, July 28, 2016 585

Figure 2. Serology of APS1/APECED to IFNs and Other Cytokines


Seroreactivity of APS1/APECED patients (blue) and contols (red) toward selected interferons and cytokines as measured in ProtoArray (A), LIPS (B and C), and
ELISA (D).

586 Cell 166, 582595, July 28, 2016

(legend on next page)

Cell 166, 582595, July 28, 2016 587

displayed a KD of 9.1e 14M, (Table S3). Likewise one IgGk mAb


(17E3) and one IgGl mAb (24D3), each specific for IL17F, displayed dissociation constants of <10 pM, and one IgGk antibody
(30G1) and one IgGl antibody (35G11) specific for IL22 displayed dissociation constants of 37 pM and 39 pM, respectively.
As a comparison, a CHO cell-expressed form of fezakinumab, a
humanized anti-IL22 mAb tested in the clinic, displayed a KD of
54 pM (Table S3). The only exception to this pattern was 2C2,
an IgGl mAb specific for IL32g (for which no human antibody
has been reported), which displayed nanomolar dissociation
(Table S3).
Similar to IFNa antibodies, most cytokine-specific antibodies
did not detect linear peptides from relevant target proteins,
strongly suggestive of complex conformational determinants
(data not shown). The one exception was 20A10, which bound
to an IL20 peptide and within which key amino acids were identified by mutagenesis (Figures S2C and S2D).
The antibody sequences of IL17F-reactive 17E3 and 9A2 and
of IL22-reactive 30G1 and 35G11 displayed myriad non-conservative mutations enriched in the CDRs. Again their germline
counterparts did not detect the respective targets (Figures 3D,
S2E, and S2F). Moreover, neither patient-derived antibodies
nor their germline counterparts showed any general autoreactivity (judged by immunofluorescent staining of tissue sections or
HEp-2 cells) or reactivity to Candida albicans, thus arguing
against candida infection being the trigger for autoantibody generation (data not shown).
The highly mutated CDRs of all studied antibodies suggested
that they derived from GC reactions that partially rely on Tfh cells.
Aberrant generation and/or activation of Tfh cells has been
described in several autoimmune diseases (Ueno et al., 2015),
but when four pediatric and four adult APS1/APECED were
compared to controls, we found no differences in the percentages of circulating CXCR5+ Tfh cells, or their activation state,
as judged by ICOS (inducible costimulator) and CCR7 levels
(Figure S3).
Biologically Active Human Antibodies
To test the biological activities of 19D11 and 26B9, HEK293 cells
transfected with type I IFN-stimulated response elements (ISRE)
fused to firefly luciferase were treated with recombinant forms
of each of 12 IFNa subtypes and IFNu in the presence or
absence of increasing concentrations of 19D11 or 26B9.
Following treatment, firefly luciferase values were normalized
to those of co-transfected Renilla luciferase, so as to control
for variations in transfection efficiency. Both antibodies strongly
inhibited the IFN-dependent response, with median IC50 values
of 2.83 ng/ml for 26B9 and 0.9 ng/ml for 19D11 (Figure 4A; Table
S4). By comparison, median IC50 values of 76.24 ng/ml and

10.86 ng/ml, respectively, were displayed by in-house-generated recombinant sifalimumab and rontalizumab, two anti-IFN
mAbs used in clinical trials for systemic lupus erythematosus patients (Table S4).
Predictably, the antibodies varied in their inhibition of IFNstimulated responses. Thus, 26B9 neutralized IFNu, but not
IFNa16, and only poorly inhibited IFNa8 (Figure 4A; Table S4).
Likewise, in the same assay, rontalizumab failed to efficiently
neutralize IFNa6, IFNa7 and IFNa10, whereas sifalimumab
neutralized several IFNa subtypes only weakly. By contrast,
19D11 neutralized all 12 IFNa subtypes tested (Table S4).
Patient-derived IFN-specific mAbs were also assessed for
their capacity to inhibit STAT1 phosphorylation in cells treated
with each of 12 IFNa subtypes, IFNu, IFNb, or IFNg (Figures
4B, 4C, and 4D). As predicted from the luciferase assay,
19D11 inhibited STAT1 phosphorylation levels (normalized to
total STAT1 or tubulin) driven by all IFNa subtypes but did not
affect responses to IFNu, IFNb, or IFNg. By contrast, 25C3, an
additional patient-derived mAb (Table S2), was highly selective
for discrete IFNa subtypes, whereas other antibodies tested,
including 26B9, showed neutralization profiles between those
of 19D11 and 25C3 (Figures 4B4D). Only 26B9 and 31B4
neutralized IFNu, and none neutralized IFNb or IFNg. By comparison, sifalimumab, rontalizumab, and AGS-009 (another
IFNa-targeting mAb in clinical development) showed variable
and less uniform inhibition of STAT1 phosphorylation induced
by different IFNa subtypes (Figure S4A).
The striking biological activities of patient mAbs were not
limited to those specific for IFNs in that potent functional target
neutralization was shown by mAbs targeting IL17F, IL22,
IL32g, and IL20, respectively (Figure S4B).
Biologically Active Human Antibodies In Vivo
We next asked whether patient autoantibodies could functionally
neutralize targets in vivo. To test this, mice were treated intraperitoneally (i.p.) with a single aliquot of antibodies 26B9, 19D11, or
sifalimumab, and their ears inoculated intradermally (i.d.) on
days 1, 3, 6, and 8 with recombinant human IFNa5 or IFNa14
(Figure 5A) and IFNu (data not shown). Relative to repeated inoculation with vehicle/PBS, the cytokines induced ear swelling, reflecting an inflammatory response that includes rapid TNFa and
IFNg induction (Figures S5A and S5B). This ear swelling was
significantly inhibited by single injections of antibodies (Figure 5B). Again, neutralization varied toward the effector IFNa
subtype: 26B9 and 19D11, but not sifalimumab, largely ablated
the IFNa5 response, whereas all three partially yet significantly
limited swelling induced by IFNa14 (Figure 5B).
Specific, antibody-mediated neutralization in vivo was likewise seen when the same assay was applied to human IL17F

Figure 3. Affinity of Patient-Derived mAbs


(A) Amino acid sequences of 26B9 and 19D11 anti-IFN antibodies aligned with closest corresponding germline IgVH, DH, JH, VL, and JL sequences. Identities
highlighted in blue; conservative mutations in yellow; non-conservative in white; CDRs underlined in red.
(B) Plasmon resonance data: antibodies 19D11 and 26B9 were immobilized on Biacore chips; different concentrations of recombinant human IFNa2b, IFNa4,
IFNa14, and IFNu were passed over; response units were recorded; and dissociation constants (KD) calculated.
(C) Scatter chart of KD values derived from (B).
(D) Binding determined by LIPS of APS1/APECED-derived mAbs and of germline counterparts to IFNa2, IFNa8, IFNa14 (19D11, 50E11, and 26B9), IL22 (35G11
and 30G1), and IL20 (20A10 and 2A11). Binding to immobilized IL17F (17E3 and 9A2) was determined by ELISA.

588 Cell 166, 582595, July 28, 2016

Figure 4. In Vitro Neutralization

(A) IC50 analysis of APS1/APECED-derived anti-IFN


mAbs 19D11 and 26B9 in HEK293T MSR cells
transfected with ISRE dual-luciferase reporter
constructs and treated with IFNa subtypes shown.
Error bars correspond to SEM of multiple measurements.
(BD) IFN-induced STAT1 tyrosine phosphorylation
detected by western blot and normalized to total
STAT1 or to tubulin levels as loading controls.
Vertical lines in (B) and (C) denote cropped lanes.

or IL32g (Figures 6A and 6B). For IL17 neutralization, the data are
clearly consistent with the known capacity of APS1/APECED
patients antibodies to neutralize Th17-family cytokines (Kisand
et al., 2010; Puel et al., 2010), thereby predisposing to Candida
infection.
Additionally, the detection of mouse IL22 by antibody 30G1
offered an opportunity to measure its bio-activity toward endogenous IL22, a primary effector of imiquimod (IMQ)-induced
dermatitis used to model psoriasis (van der Fits et al., 2009).
IMQ-induced pathology measured by modified PASI scoring
was significantly ameliorated by 30G1 relative to IgG control,
particularly following an initial inflammatory response (Figures
6C and S6). Again, 30G1 was at least as effective as an inhouse-expressed anti-IL22 antibody, fezakinumab (see above)
(Figure 6C). Collectively these data establish the capacity of
patient anti-cytokine antibodies to limit pathologies induced by
their targets in vivo.

Clinical Correlates of Neutralizing


Antibodies
Given the results from animal models, it
was appropriate to consider the potential
impact of APS1/APECED antibodies in
the patients themselves. Because circulating IFNa levels are extremely low in
human peripheral blood, even following
vaccination (Sobolev et al., 2016), circulating IFN levels do not offer robust biomarkers of anti-IFNa antibodies. Neither
does measurement of interferon-stimulated genes (ISGs) because many, e.g.,
CXCL10, can be upregulated by type II
IFNs (Welcher et al., 2015). By contrast,
antibody activities may be reliably reflected
in discrete pathologies, as in the correlation of anti-IL22 with candidiasis.
In this regard, many datasets, particularly in mouse models, suggest that type I
IFN contributes to type 1 diabetes (T1D)
(Carrero et al., 2013; Downes et al., 2010;
Foulis et al., 1987; Huang et al., 1995; Li
et al., 2008). Although APECED/APS1
patients by definition suffer from polyendocrinopathy, T1D affects only 10%
20% of patients and manifests primarily
in adulthood (Husebye et al., 2009;
Kisand and Peterson, 2015). This is despite the fact that
radioimmunoassays have revealed that many APS1/APECED
patients carry GAD65-reactive autoantibodies, a clinically
applied biomarker for likely onset of T1D (Ziegler et al., 2013).
Consistent with this, ProtoArray and LIPS data showed that
many patients carried GAD65- and/or GAD67-reactive antibodies, but among them relatively few presented with T1D (red
dots, Figures 7A and 7B). Collectively, these many observations
suggest that patients at risk of T1D, as judged by anti-GAD65/
GAD67, might fail to develop T1D if they harbored powerfully
neutralizing anti-IFNa antibodies. Indeed, we reported a seemingly exceptional APS1/APECED patient, completely lacking
IFNa-neutralizing antibodies and presenting with T1D (Kisand
et al., 2008).
To investigate this, the 8 patients presenting with T1D
(red dots, Figure 7B; mean age SD, 48 11 years) were
compared with an available cohort of 13 patients without
Cell 166, 582595, July 28, 2016 589

Figure 5. Biological Activity of IFN mAbs


(A) Experimental timeline: mAb administered i.p. at
day 0; human IFNa administered i.d. on days 1, 3,
6, and 8. Ear thickness measured on all days (prior
to cytokine injection) except for day 5.
(B) I.p.-administered IFN mAbs reduced IFNa-induced ear inflammation.
Significance calculated by two-way ANOVA, with
*p % 0.05, **p % 0.01, ***p % 0.001, and ****p %
0.0001. Error bars denote SEM.

DISCUSSION

T1D but with strong GAD65 reactivity (relative luciferase


units > 5) (blue dots, Figure 7B; mean age SD, 31 12
years). Consistent with T1D developing in adult APS1/APECED
patients, GAD65 reactivities mostly arose post-adolescence,
and hence the patient cohorts comprised 20 adults and one
8 year old.
As expected, all 21 patients harbored antibodies to IFNa
and IFNu (see Figure 2B), but when tested for IFNa and IFNu
neutralization, the antibodies showed a striking segregation
with clinical status (Figures 7C, 7D, and S7A): patients without
T1D collectively neutralized all IFNa subtypes, whereas those
with T1D showed only low or negligible neutralization. Particularly strong differences were seen vis-a-vis IFNa1, IFNa2,
IFNa5, IFNa8, IFNa14, and IFNa17 neutralization (Figure 7C).
The two subgroups of the 21 patients also showed statistically
significant differences in neutralizing IFNu, but the difference
was weaker than for IFNa (Figure S7A). Interestingly, the two
GAD65-reactive non-diabetics who displayed relatively low
IFNa neutralization were young adults who may be en route to
developing T1D.
In a small subcohort of GAD65/67-reactive patients for whom
longitudinal samples were available, the three T1D patients
(red bars) again showed lower IFNa neutralization relative to
the two patients without T1D. Moreover, one patient was able
to neutralize IFNa4 in 1978 but by 2012 could no longer do so
and presented with T1D (Figure S7B).
Such striking correlations with T1D (Figure 7D) were not
evident for any other naturally arising anti-cytokine antibodies,
supporting the view that IFNa may contribute critically to the natural progression of T1D. Moreover, although the data do not
prove that active anti-IFN antibodies underpin selective protection from T1D, they provide a firm foundation for exploring the
potentials of APS1/APECED-derived autoantibodies to ameliorate other major diseases that are rarely if ever present in
APS1/APECED patients.
590 Cell 166, 582595, July 28, 2016

This analysis of the impact of AIRE deficiency on human B cells has revealed
a signature pattern of humoral autoreactivity with general implications for our
understanding of autoimmunity. First,
the autoantibodies studied were mostly
extremely high affinity and specific for
native conformational epitopes. These
properties were shared by antibodies
specific for cytokines targeted by most patients (e.g., IFNa,
IL17, IL22) and by antibodies specific for IL20 to which few
patients displayed reactivity. Because such properties are
very rare among antibodies raised by immunization, when B
cells are primed de novo to antigen for short periods of time,
it seems inappropriate to continue to model one type of mAb
on the other.
Second, essentially all 81 APS1/APECED patients studied
showed strong reactivities toward a common set of 1015 proteins, coupled with patient-specific reactivity profiles toward
8090 additional proteins. This limited frequency (< 1% of proteins displayed on the array) is consistent with a recent report
that B cell tolerance was not globally disrupted in 51 APS1/
APECED patients sampled (Landegren et al., 2016). Nonetheless, the patient-to-patient variation in reactivity profiles meant
that the 97 sera analyzed in our study collectively harbored antibodies toward over 3,500 proteins.
The patient-to-patient variation argues that B cell autoimmunity resulting from AIRE deficiency is not simply an amplification
of sporadic, low-level autoreactivities seen in healthy controls
but has distinct origins. By this perspective, defects in central
T cell tolerance may underpin other autoimmune and autoinflammatory pathologies attributed to high-affinity autoantibodies.
Whereas this contrasts with the widely held view that autoimmune diseases mostly reflect peripheral tolerance defects, it
aligns with data that central tolerance defects contribute to the
NOD mouse model of T1D (Geng et al., 1998; Zucchelli et al.,
2005). Moreover, wherever autoantibodies reflect central T cell
tolerance defects, donor-to-donor variation is to be expected,
as individuals will generate distinct TCR repertoires via quasirandom gene rearrangements, will be exposed to different physiologic and environmental triggers that promote the selective
outgrowth of autoreactive T cell clones, and will differ in immune
response modifier genes (e.g., HLA) that regulate the magnitude
of antigen-specific responses.

Autoantibodies to some non-tissue-restricted antigens, including multiple type I IFNa subtypes, are displayed by almost
all patients, sometimes early post-partum (Wolff et al., 2013).
Most likely, the immunogenicity of these proteins arises by
mechanisms distinct from those shaping patient-specific autoantibody repertoires. Possibly the public autoantibodies arise
from a direct impact of AIRE deficiency on B cell tolerance,
for example, via the dysregulation of AIRE-expressing thymic
B cells that resemble GC B cells by several criteria (Yamano
et al., 2015). Arguing against this, however, autoantibodies to
type I IFNs, Th17 cytokines, and additional self-proteins are
found in thymoma patients with AIRE-sufficient B cells (Kisand
et al., 2011; Meager et al., 1997; Wolff et al., 2014). This likewise
argues against autoantibodies to type I IFNs and Th17 cytokines
originating from defects in lymph node AIRE+ cells termed
eTACs (Gardner et al., 2008). Although studies in mice have
suggested tolerizing roles of eTACs, the functions of their rare
human counterparts are unknown (Poliani et al., 2010).
AIRE deficiency may, however, act indirectly on thymic B cells,
for example by hyperactivity of functionally competent thymic gd
cells (Ribot et al., 2009) that may likewise be dysregulated in thymoma. Such cells may create an intra-thymic milieu favoring
priming rather than tolerance of thymic B cells toward proteins
highly expressed in the thymus (Dudakov et al., 2012; Meager
et al., 2006).
Notwithstanding this possibility, our findings suggest that
high-affinity autoantibodies in APS1/APECED patients prob-

Figure 6. In Vivo Activity of Cytokine-Reactive mAbs


(A) mAb administered i.p. at day 0, and human
IL17F administered i.d. on days 1, 3, 6, and 8. Ear
thickness measured on all days (prior to cytokine
injection) except day 5.
(B) As in (A), but with human IL32g administered i.d.
(C) anti-IL22-specific mAb injected i.p. into 9-week
mice prior to and during IMQ treatment. Efficacy
measured by Psoriasis Area and Severity Index
(PASI).
Significance calculated by two-way ANOVA, with
*p % 0.05, **p % 0.01, ***p % 0.001, and ****p %
0.0001. Error bars denote SEM.

ably reflect dysregulated GC reactions,


wherein autoreactive T cells, e.g., Tfh
cells, that were not tolerized in
the thymus promote the competitive
outgrowth and affinity maturation of
B cells that were initially primed to exogenous antigen(s) but whose mutated
IgGs bind to self-proteins. Consistent
with this, autoantibodies targeting thyroid-stimulating hormone receptor in
Graves disease cross-react to Yersinia
enterocolitica antigens (Brink, 2014;
Hargreaves et al., 2013), and activated
peripheral blood Tfh cells correlate positively with serum autoantibodies and disease activity/severity in
multiple autoimmune diseases (Ueno et al., 2015). Although our
analysis of four adult and four pediatric APS1/APECED patients
revealed no alterations in Tfh cell numbers relative to agematched healthy controls, this did not exclude Tfh cells being
enriched in autoreactive specificities. Moreover, no patients displayed neutralizing autoantibodies to IL21, a major mediator of
Tfh cells in the GC.
This etiology of APS1/APECED B cell autoimmunity is strikingly similar to proposed origins of highly mutated anti-desmoglein-3 antibodies in autoimmune pemphigus (Di Zenzo et al.,
2012) and of anti-GM-CSF antibodies pathognomonic in pulmonary alveolar proteinosis (Piccoli et al., 2015). In those
studies, as in this, the closest germline counterparts (unmutated common ancestors [UCAs]) showed no reactivity toward
the targets of the affinity-matured autoantibodies. By contrast,
germline versions of antiviral antibodies showed only slightly
reduced binding to target viral antigens (Corti et al., 2011,
2013). Moreover, it is not the case that UCAs intrinsically lack
autoreactivity, as germline counterparts of some autoantibodies with few replacement mutations showed autoantigen
reactivity in pemphigus patients (Cho et al., 2014). The underlying defect(s) in T cell tolerance that dysregulate affinity maturation in pemphigus, pulmonary alveolar proteinosis, and other
organ-specific autoimmune diseases may be limited to few
antigens, by contrast to broad-spectrum defects in APS1/
APECED.
Cell 166, 582595, July 28, 2016 591

That almost all APS1/APECED-derived mAbs were biologically active in vivo against a range of cytokine targets has
profound implications for patients. Clearly, immune-effector responses may be reduced, as in the association of anti-IL22
with susceptibility to Candidiasis (Kisand et al., 2010). Likewise,
gut barrier integrity may be compromised, leading to increased
levels of anti-commensal antibodies (Hetemaki et al., 2016).
Conversely, despite the common neutralization of IFNa and
IFNu, APS1/APECED patients do not show severe viral infections, as were recently reported for a child genetically impaired
in type I IFN (Ciancanelli et al., 2015). Possibly preserved
IFNb function mediates anti-viral protection in APS1/APECED
patients.
On the other hand, some autoantibodies may target key
mediators of immunopathologies, thereby ameliorating disease.
Thus, a unique correlation was observed between antibodymediated neutralization of IFNa and failure to develop T1D,
providing a novel strand of support for animal studies arguing
that targeting type I IFNs could be effective in T1D. The concept
that naturally arising autoantibodies may be beneficial is not
widely considered, despite its underpinning the widespread
592 Cell 166, 582595, July 28, 2016

Figure 7. Clinical Correlation of T1D and IFN


Neutralization
(A and B) Seroreactivity to GAD67 and GAD65
measured by ProtoArray and LIPS in APS1/
APECED patients with (red) or without (blue) T1D.
(C) IFNa-neutralizing titers in patients with T1D
(n = 8) and anti-GAD65 seropositive patients
without T1D (n = 13). y axis shows inhibitory concentration IC50 reflecting serum dilutions at which
IFN activity was reduced 50%.
(D) Heatmap of seroreactivity toward GAD67,
GAD65, and IFNa analyzed by ProtoArray and
LIPS combined with neutralization capacity in
patients with and without T1D.
Significance calculated by Mann Whitney using
GraphPad Prism v.6, with *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001. Error bars
denote SEM. Significance values in (D) compare
T1D+ and T1D groups for each parameter.

use of therapeutic mAbs. In this regard,


it is striking that despite their severe flaws
in central T cell tolerance, APS1/APECED
patients do not present with systemic
sclerosis, Sjogrens syndrome, MS, or
SLE. These pathologies are considered
to involve interplays of IL17/Th17 and
type I IFNstwo main targets of APS1/
APECED autoantibodies (Ambrosi et al.,
2012). Likewise, Th17-driven psoriasis
was diagnosed in only two of our patients,
each of whom lacked autoantibodies
to IL17A, IL17F, and IL22 (our unpublished data). Furthermore, atopy/allergy
is seemingly rare among APS1/APECED
patients, although whether anti-IL5 antibodies underpin this requires more study.
For now, the data presented by this study strongly suggest that
antibodies recovered from APS1/APECED patients include ones
with profound therapeutic and diagnostic potential.
EXPERIMENTAL PROCEDURES
More details are available in the Supplemental Experimental Procedures.
Human Samples
Eighty-one APS1/APECED patients were diagnosed by mutational analysis of
AIRE and by autoantibodies to type I IFNs. All provided informed consent, and
many were analyzed previously (Kisand et al., 2011; Kluger et al., 2015; Meloni
et al., 2012; Wolff et al., 2007). Approvals by local ethics committees are
described in the Supplemental Experimental Procedures. Ages at serum sampling were 473 years; mean = 31.9. For protoarray there were 12 agematched controls and 9 healthy first-degree relatives, and there were
additional healthy controls for LIPS and ELISA.
Immune Response Profiling by ProtoArray
Sera of patients, healthy relatives, and controls were tested against > 9,000 human proteins displayed on the Human Protein Microarray v5.1 (ThermoFisher
Scientific). Preprocessing methods were applied to account for technical variability. First, corresponding local background intensity was subtracted,

whereafter values were log-transformed and subjected to robust linear normalization (Sboner et al., 2009). Z scores were calculated as the number of standard deviations of the signal from the mean of the corresponding controls
and healthy relatives; Z R 3 was considered positive. After scoring, stringent
quality assessment was undertaken, including high correlation coefficients of
duplicate spots of printed proteins (average r = 0.92), reactivity toward known
autoantibody targets, and perfect correlation of signals for proteins spotted in
different locations. Printing contaminants were identified as proteins showing
high correlation coefficients with known APECED antibody targets and were
further verified by cross-reference to another protoarray (5.0) used for 23 patients and 7 controls. Thus, 31 suspect false-positives were identified and
excluded from further consideration.
Antibody Isolation and Cloning
Cloning, production, and purification of human mAbs were performed as
described (patent application WO2013/098419). In brief, memory B cells
(CD22+, IgD , IgM , CD3 , CD8 , and CD54 ) were flow-sorted (MoFlo)
from patient PBMC, incubated transiently with EBV-containing B95-8 supernatant (SN) for 3.5 hr at 37 C, and then incubated in Transferrin- and CpGsupplemented IMDM at 37 C, 5% CO2, at 10 cells/well in 96-well plates
coated with irradiated PBMC feeders. Short-term, oligoclonal B cell culture
SN were analyzed for IgG and antigen-specific antibodies detected by
ELISA and/or LIPS. Positive wells were harvested, cells single-cell-sorted
into reverse transcriptase (RT) buffer (Life Technologies), and RT-PCR
performed using Superscript III (Life Technologies) and random hexamers.
IgG VH, Vl, and Vk regions were amplified from cDNA by two-step nested
PCR reaction using Advantage 2 cDNA polymerase (Clontech) and primer
mixes specific for germline families (VBASE database). Nested primers
attached restriction sites for V-region cloning into expression vectors
providing IgG1, Ig-k, or Ig-l constant regions. Recombinant antibodies were
produced in HEK293T cells and antigen specificity analyzed by ELISA. Corresponding closest germline region sequences were identified using the
VBASE2 database (Retter et al., 2005). CDRs were identified by IMGT definitions (Lefranc, 2003).
Complete Ig-VH and VL regions described in US7741449 (Sifalimumab),
US7087726 B2 (Rontalizumab), US8361463 (ACO-1), and US20070258982
A1 (Fezakinumab) were ordered as CHO-codon-optimized synthetic constructs (GenScript) and expressed as above.
mAb Characterization In Vitro
EC50 binding of mAbs was determined by ELISA. Neutralizing capacities of
type I IFN-specific mAbs were studied using phospho-STAT1 quantification
in immunoblot and ISRE-luciferase reporter assay. IL17F, IL22, IL20, and
IL32 neutralization assays were performed on respective responsive cell lines.
mAB affinities were measured with a Biacore T200 (GE Healthcare). Epitope
mapping used overlapping 18-mer peptides.
mAb Characterization In Vivo
C57BL/6J (WT; from Charles River) mice were administered i.p. with mAbs
(day 0) and inoculated i.d. on days 1, 3, 6, and 8 with cognate human cytokines,
IFNa2a, IFNa2b, IFNa4, IFNa14, IL17F, and IL32g, and their ear thicknesses
measured with a micrometer. For IL22 mAbs cross-reactive to mouse, bioactivity was assessed in imiquimod-treated mice.
SUPPLEMENTAL INFORMATION

AUTHOR CONTRIBUTIONS
S.M., A.M., and S.C.O. cloned monoclonal antibodies from patient samples,
and K.J. and K.H. assisted. S.M., P.V., and A.M. characterized antibodies
in vitro; M.W. and Y.H. did so in vivo. C.H. analyzed ProtoArray data and wrote
and edited the paper. J.K. assayed neutralization by sera and Tfh subsets and
performed LIPS. D.F. and H.P. analyzed ProtoArray data. K.M. and R.U.
screened sera for T1D autoantibodies and tested germline antibody specificities. K. Krohn and A.R. developed the clinical database, sampled Finnish
patients, and employed ELISA. A.S.B.W. sampled Norwegian patients,
contributed to the clinical database, and assayed antibodies. APECED patient
collaborative contributed to the clinical database and sampled respective patients. P.P., K. Kisand, and A.H. supervised research, reviewed data, and
wrote and edited the paper.
ACKNOWLEDGMENTS
We are indebted to patients; to the Finnish APECED and Addison patients
association; and to attending physicians and carers. We thank M. Rothe,
P. Adler, A. Remm, M. Pihlap, M. Karlsberg, A. Tallqvist, M. Tuukkanen,
L. Prassmayer, M. Wordehoff, A. Peters, R. Repke, B. Mathis, and particularly
E. Stuart and K. Henco for critical insight and support. We thank staff of the
Biological Services Unit at Kings College London. Funding was by the
following: ImmunoQure AG, the Wellcome Trust, and CRUK (to A.H.) and
Estonian Research Council grant IUT2-2 and European Union Project 20142020.4.01.15-0012 (to J.K., P.P., and K. Kisand). P.P., K. Krohn, K. Kisand,
A.R., and A.H. are cofounders and shareholders of ImmunoQure AG, and
A.M., C.H., P.V., S.C.O., and S.M. were/are employees of ImmunoQure AG.
Received: January 27, 2016
Revised: April 24, 2016
Accepted: June 10, 2016
Published: July 14 2016
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Cell 166, 582595, July 28, 2016 595

Article

Structure and Function Analysis of an Antibody


Recognizing All Influenza A Subtypes
Graphical Abstract

Authors
Nicole L. Kallewaard, Davide Corti,
Patrick J. Collins, ..., Qing Zhu,
Steven J. Gamblin, John J. Skehel

Correspondence
zhuq@medimmune.com (Q.Z.),
john.skehel@crick.ac.uk (J.J.S.)

In Brief
Identification of a human monoclonal
antibody that reacts effectively with all
influenza A hemagglutinin subtypes
paves the way for developing
immunotherapy for people infected with
the flu virus.

Highlights
d

Binding to all influenza A subtypes neutralizing seasonal and


pandemic strains
Utilizes a rare VH (VH6-1) and carries a low level of somatic
mutations
Highly conserved epitope encompassing fusion peptide and
hydrophobic groove
Superior therapeutic window compared to oseltamivir in
animals

Kallewaard et al., 2016, Cell 166, 596608


July 28, 2016 2016 The Authors. Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.cell.2016.05.073

Accession Numbers
5JW5
5JW4
5JW3
KX398429
KX398468

Article
Structure and Function Analysis of an Antibody
Recognizing All Influenza A Subtypes
Nicole L. Kallewaard,1,8 Davide Corti,2,8 Patrick J. Collins,3,8 Ursula Neu,3,8 Josephine M. McAuliffe,1 Ebony Benjamin,1
Leslie Wachter-Rosati,1 Frances J. Palmer-Hill,1 Andy Q. Yuan,4 Philip A. Walker,5 Matthias K. Vorlaender,3 Siro Bianchi,2
Barbara Guarino,2 Anna De Marco,2 Fabrizia Vanzetta,2 Gloria Agatic,2 Mathilde Foglierini,6 Debora Pinna,6
Blanca Fernandez-Rodriguez,6 Alexander Fruehwirth,6 Chiara Silacci,6 Roksana W. Ogrodowicz,5 Stephen R. Martin,5
Federica Sallusto,6 JoAnn A. Suzich,1 Antonio Lanzavecchia,6,7,9 Qing Zhu,1,9,* Steven J. Gamblin,3,9
and John J. Skehel3,9,*
1Department

of Infectious Disease and Vaccines, MedImmune LLC, One MedImmune Way, Gaithersburg, MD 20878, USA
BioMed SA, Via Mirasole 1, 6500 Bellinzona, Switzerland
3Mill Hill Laboratory, The Francis Crick Institute, London NW7 1AA, UK
4Department of Antibody Discovery and Protein Engineering, MedImmune LLC, One MedImmune Way, Gaithersburg, MD 20878, USA
5Structural Biology Science Technology Platform, Mill Hill Laboratory, Francis Crick Institute, London NW7 1AA, UK
6Institute for Research in Biomedicine, Universita
` della Svizzera italiana, 6500 Bellinzona, Switzerland
7Institute for Microbiology, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland
8Co-first author
9Co-senior author
*Correspondence: zhuq@medimmune.com (Q.Z.), john.skehel@crick.ac.uk (J.J.S.)
http://dx.doi.org/10.1016/j.cell.2016.05.073
2Humabs

SUMMARY

Influenza virus remains a threat because of its ability


to evade vaccine-induced immune responses due
to antigenic drift. Here, we describe the isolation,
evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin
(HA) subtypes. MEDI8852 uses the heavy-chain
VH6-1 gene and has higher potency and breadth
when compared to other anti-stem antibodies.
MEDI8852 is effective in mice and ferrets with a
therapeutic window superior to that of oseltamivir.
Crystallographic analysis of Fab alone or in complex
with H5 or H7 HA proteins reveals that MEDI8852
binds through a coordinated movement of CDRs
to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large
portion of the fusion peptide, distinguishing it from
other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency
of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected
humans.
INTRODUCTION
Influenza virus infection remains a serious threat to global
health and the world economy. Annual epidemics result in a
high number of hospitalizations, with an estimated 35 million
cases of severe disease and 250,000500,000 deaths globally,
and higher mortality rates are possible during pandemics
(Wright et al., 2007). Given the emergence of anti-viral drug-

resistance, short treatment windows for antivirals and the


lack of cross-protective vaccines, there is an unmet medical
need for new therapeutic options that can effectively treat influenza infection.
There are three types of influenza viruses, A, B, and C
causing disease in humans, and influenza A and B are responsible for frequent seasonal epidemics. However, influenza A
infections account for the majority of hospitalizations and
are the only type to cause pandemics (Wright et al., 2007). Influenza A is subtyped by its two major surface proteins, hemagglutinin (HA) and neuraminidase (NA). HA is the main target of
neutralizing antibodies that are induced by infection or vaccination. The globular HA head domain mediates binding to the
sialic acid receptor, while the HA stem mediates the subsequent fusion between the viral and cellular membranes that is
triggered in endosomes by the low pH (Skehel and Wiley,
2000). Genetically, there are 16 influenza A subtypes of HA,
which form two structurally and antigenically distinct groups
(Nobusawa et al., 1991; Russell et al., 2004). In addition, two
new HA analogs recovered from bats, H17 and H18, have
been included in this classification (Tong et al., 2012, 2013).
Currently, H1 and H3 HA subtypes are associated with human
disease and viruses containing H5, H7, H9, and H10 HAs are
associated with sporadic human infections due to direct transmission from avian species.
The majority of influenza virus neutralizing antibodies elicited
by vaccination or infection bind to the globular head of HA and
recognize homologous strains within a given subtype (Russell
et al., 2008). These antibodies neutralize virus infectivity by
blocking sialic acid receptor binding either directly (Knossow
and Skehel, 2006; Schmidt et al., 2013) by interacting with the
receptor binding site at the tip of the molecule or indirectly, by
projecting over the binding site thereby rendering it inaccessible
(Fleury et al., 1999; Xiong et al., 2015). These antibodies are
involved in the selection of viruses with variant HAs in the

596 Cell 166, 596608, July 28, 2016 2016 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

process of antigenic drift, necessitating the annual re-development of influenza vaccines.


In the past 8 years, several laboratories have described a new
class of influenza-neutralizing antibodies that target conserved
sites in the HA stem that showed different levels of cross-reactivity toward group 1 (Corti et al., 2010; Sui et al., 2009; Throsby
et al., 2008; Wrammert et al., 2011), group 2 (Dunand et al., 2015;
Ekiert et al., 2011; Friesen et al., 2014; Tan et al., 2014) and
groups 1 and 2 viruses (Corti et al., 2011; Dreyfus et al., 2012; Nakamura et al., 2013; Wu et al., 2015). Anti-stem antibodies are
less potent at direct viral neutralization as compared to antihead antibodies, but were shown to induce potent antibodydependent cellular cytotoxicity (ADCC) of infected cells in vitro
and in vivo (Corti et al., 2011; Dilillo et al., 2016; DiLillo et al.,
2014), while anti-head antibodies were not or less effective at
mediating ADCC. In general, the human antibody response
to the HA stem region is more frequent against group 1 as
compared to group 2 HAs and is dominated by VH1-69 antibodies (Pappas et al., 2014; Sui et al., 2009; Wrammert et al.,
2011). Although subdominant, the group 1 stem response was
shown to be recalled after heterologous boosts by the new
pandemic H1N1 virus in 2009 (Corti et al., 2011; Wrammert
et al., 2011). The antibody response to the HA stem region of
group 2 HAs is less frequent, possibly due to the presence of a
conserved glycan bound to N38 in HA1 that may shield the access to the most conserved sites in the HA stem and to the
lack of exposure to heterologous group 2 viruses (i.e., H7) or to
new pandemic H3N2 viruses. Finally, antibodies capable of reacting with the HA stem region of both group 1 and 2 subtypes
are extremely rare and usually do not show complete coverage
of all subtypes. It has been hypothesized that such broadly
cross-reactive antibodies might have potential as therapeutic
agents and studies on their mechanism of action, epitope specificity, and ontogeny could also inform the design of cross-protective influenza virus vaccines (Corti and Lanzavecchia, 2013;
Yewdell, 2013).
A problem related to the development of anti-stem antibodies as immunotherapeutics is their variable neutralizing
potency against viruses belonging to different subtypes and
the existence of natural escape mutants. In view of the limitations of group 1 and group 2 antibodies isolated so far, we
searched for an antibody capable of potently neutralizing
group 1 and 2 influenza A viruses within a narrow range of
antibody concentrations. In this study, we isolated and optimized an antibody, named MEDI8852, that exhibited unprecedented breadth and potency, being able to neutralize a diverse
panel of representative viruses spanning >80 years of antigenic evolution. Unlike other broadly neutralizing stem-reactive antibodies, MEDI8852 is unique in that it uses a rare
VH (VH6-1) and carries a low level of somatic mutations. Crystallographic analysis of the Fab alone or in complex with H5
and H7 HA proteins reveals that MEDI8852 binds a highly
conserved epitope on H5 and H7 that is markedly different
from other structurally characterized stem-reactive neutralizing antibodies. The characterization of this unique epitope
and the breadth and potency of neutralization exhibited by
MEDI8852 support its development for immunotherapy in
influenza virus-infected humans.

RESULTS
Isolation, Genetic Description, and Optimization of
MEDI8852
Four broadly reactive antibodies were isolated from the memory
B cells of a selected donor based on influenza A HA protein
cross-reactivity as previously reported (Traggiai et al., 2004;
Pappas et al., 2014). These antibodies (FY1, FY5, FY6, and
FY18) belong to the same lineage carrying VH6-1*01/D3-3*01/
JH3*02 and VK1-39*01/JK1*01 gene segments (Figure 1A). We
reconstructed the genealogical trees of this lineage and produced the unmutated common ancestor (UCA), the four clonally
derived antibodies, and three antibodies representing the evolutionary branching points (BP) of the lineage (Figure 1B). Purified
antibodies were tested for neutralizing activity against multiple
viruses of different group 1 and 2 subtypes (Figure 1C). Interestingly, the UCA antibody exhibited neutralizing activity against
group 1 viruses, but not group 2 viruses, albeit with lower
potency as compared to some of the mutated antibodies. Of
note, the first BP (BP1) gained neutralization activity toward early
group 2 H3N2 viruses (HK/68 and VC/75). Two antibodies (i.e.,
FY1 and FY5) of this lineage acquired neutralization activity
against group 2 viruses through two independent pathways of
somatic mutations. The same analysis was extended to the lineage of FI6, a previously described antibody cross-neutralizing
group 1 and group 2 viruses (Corti et al., 2011). Isolation of five
additional antibodies from this lineage allowed the reconstruction of a complex genealogy tree (Figure S1). Similarly to what
was observed for the FY1 lineage, the FI6-UCA antibody exhibited neutralizing activity against group 1 viruses only and
evolved through two independent pathways of somatic mutations that led to the group 1-specific FI370 and FI6038 antibodies
and to the group 1 and 2 cross-reactive antibodies FI6, FI2013
and FI4013. Taken together, these findings suggest that in
both lineages, the UCA was initially selected by a group 1 virus
and developed to a branching point characterized by crossreactivity toward a limited number of group 2 viruses. From
this point, the final antibody may have been selected further for
binding to group 1 only (e.g., FY6 and FI370) or group 2 HAs
(e.g., FY1 and FI6). These results are consistent with a model
in which the development of cross-reactive group 1 and 2 antibodies is started by group 1 HAs and then further selected
through boosts by group 2 HAs.
The FY1 antibody was chosen as the lead, based on its potency, breadth, and low somatic mutations for further in vitro
optimization through parsimonious mutagenesis of the complementarity determining regions (CDRs) combined with reversion
of unnecessary somatic mutations in the frameworks. The optimization focusing on affinity binding resulted in a 14-fold and
5-fold improved Fab affinity to H3 HA and H1 HA proteins determined by surface plasmon resonance, respectively (Table S2).
The resulting antibody was named MEDI8852 (VH and VL sequences shown in Figure 1A) and was compared side by side
with the parental FY1 antibody for binding and neutralization of
a large panel of influenza A viruses. MEDI8852 showed higher
binding activity as compared to FY1 against the group 1 HA proteins of H1, H2, H5, H6, and H9 subtypes and group 2 HA proteins of H3 and H7 subtypes, with a mean half-maximal effective
Cell 166, 596608, July 28, 2016 597

FY5

I
.
.
.
.
.
.
.
.

113

107

102

T I F GV N I D A F D I WGQG TMV T V S S
.V. . . . . . . . . . . . . . . . . . . . .
.V. . . . . . . . . . . . . . . . . . . . .
.V. . . . . . . . . . . . . . .K. . . . .
.V. . . . . . . . .V. . . . . . . . . . .
. V . . L . . . . Y . . . . . . AK . . . . .
.V. . . . . . . . . . . .L. .K. . . . .
. V . . . . V . . . .M. . . . . . . . . . .
. V . . . . V . . . .M. . . . . . . . . . .

107

100

96

90

85

80

75

70

100
100A
100B
100C
100D
100E
100F
100G
100H

95

90

85

80

82A
82B
82C

75

70

65

60
65

60

10-1
C

FY5

FY6

FY
6
FY
18

25 (13)

100

17 (6)

FY
5

18 (8)

FY18

FY18

101

FY
1

17 (18)

9 (6)

BP3

15 (12)

BP3

BP2

2 (2)

6 (3)

3 (2)

BP

15 (11)

FY1

BP2

BP

5 (1)

S S L QP E D F A T Y Y CQQS R T F GQG T K V E
. . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . .V. . . . . . . . . . . . . . . . . .
.N. . . . . . . . . . . .L. . . . . . . . . . .
. TF . A . . V . . . . . . . . . . . . . . . . . .
. . . . . . .V. . . . . .L. . . . .H. . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .

102

BP

BP1

2 (1)

I
.
.
.
.
.
.
.
.

UCA
2 (2)

UCA
BP1

55

50

55

50

45

40

35

30

T CR A SQS I S S Y L NWYQQK PGK A P K L L I Y A A S S L QSGV P S R F SGS GSG T D F T L T


. . . . . . .L. . . .H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
S. . . . . .L. . . .H. . . . . . . . . . . . . . . . . .T. . . . . . . . . . . . . . . . . . . . .
S. . . . . . L . . . .H. . . . . . .Q. . . . . . . . . .T. . . . . . . . . . . . . . . . . . . . .
S . . T . . . LR . . . H . . . . . . . . . . . . . . . . S . T . . . . . . . . . . . . . . . . . . . . .
S . . . - . . L . . . . H . . . . . . . QP . . . . . . . . T T . . . . . . . . . . . . . . . . . . . . .
S . . . . . RLN . . . H . . . . T . . Q . . . . . . . . T . T . . . . . SP . . . . . . . . . . . . . .
. . .T. . .L. . . .H. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
G. . . . . . . . . . . . . . . . . . .
. . . T . . . L . . . TH . . . . . . . . . . . . . L . . . . . R
RG

4 (2)

FY1

52A
52B

30

25

15
15

10

5
5

20

I
.
.
.
.
.
.
.
.

SGD S V S S N S A AWNW I RQS P S RG L EWL GR T Y Y R S KWY ND Y A V S V K S R I T I N P D T S K NQ F S L Q L N S V T P E D T A V Y Y C A RGGH


. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .E. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .E. . . . . . . . . . . . . . . .V. . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .E. . . . . .V. . . . . . . . .V. . . . . . . . . . .S. . . . . . . . . .
. . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DF L . R . . . . . . . . . N . EV . . R . T . . . . D . . . L . . . . . . . .
. . .R. . . . . .V. . . . . . . . . . . . . . . . . . . . . . . . .Y. . .E. . . . . .V.D. . . . . . .V. . . . . . . . . . .S. I . . . . . . . .
. . . T . . . . R . T . . . M . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . . V V . . . . . . . . . V . . . . . T . . . D . SG . . F . . . . . .
. . . . . . . . N . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . . . . . . . V . . . . . . . . . . . H . K. . . . . . . . . F . . V . S . .
. . . . . . . YN . V . . . . . . . . . . . . . . . . . . . . . . G . . . . . . E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . .

IC50 ( g/ml)

I QMT QS P S S L S A S VGDR V T
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . I .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . .

45

25

D
UCA
.
BP1
.
BP2
.
BP3
.
FY18
.
FY5
.
FY6
.
FY1
MEDI8852 .

40

VL

35
35A
35B

I
.
.
.
.
.
.
.
.

20

UCA
QVQ L QQSGPG L V K P SQ T L S L T C A
BP1
. . . . . . . . . . . . . . . . . . . . . . .
BP2
. . . . . . . . . . . . . . . . . . . . . . .
BP3
. . . . . . . . . . . . . . . . . . . . . . .
FY18
. . . . . . . . . . . . . . . . . . . . . . .
FY5
. . . . . . . . . . . . . . . . . . . . . . .
FY6
. . . . . . . . . . . . . . . . . . . . . .V
FY1
. . . . .E. . . . . . . . . . . . . . . . .
MEDI8852 . . . . . . . . . . . . . . . . . . . . . . .

10

VH

I
.
.
.
.
.
.
.
.

K
.
.
.
.
.
.
.
.

Group 1

Group 2

H1N1 WSN/33
H1N1 PR/34
H1N1 FM/47
H1N1 BJ/95
H1N1 SZ/95
H1N1 NC/99
H1N1 SI/2006
H1N1 SD/2007
H1N1 CA/2009
H1N1 BR/2010
H2N2 JP/57
H5N1 VT/2004
H6N2 AB/85
H9N2 HK/97

H3N2 HK/68
H3N2 VC/75
H3N2 SG/93
H3N2 WH/95
H3N2 SY/97
H3N2 PA/99
H3N2 CA/2004
H3N2 WI/2005
H3N2 PT/2009
H3N2 VC/2011
H7N3 BC/2004

FY6

Figure 1. Developmental Pathway of the MEDI8852 Lineage


(A) Alignment of VH and VL amino acid sequences of four mutated antibodies with their UCA and branchpoint (BP) configurations and MEDI8852. Amino acid
substitutions are highlighted in red. Residue positions are according to Kabat numbering. Dots indicate identical residues. Boxes indicate CDR borders according
to IMGT (solid line) and Kabat (dashed line).
(B) Genealogy trees of VH (left) and VL (right) nucleotide sequences generated using dnaml. The number of mutations is indicated on the branches with amino acid
substitutions in parentheses.
(C) Neutralization of influenza A viruses. IC50 values were determined against a panel of 25 influenza A isolates. Values above 50 mg/ml were scored as negative
(dashed line). Average IC50 values were obtained from at least two independent experiments. Full viral strains designations are listed in Table S1.
See also Figure S1.

concentration (EC50) of 0.064 mg/ml versus 0.124 mg/ml for


MEDI8852 and FY1, respectively (Figure 2B; Table S2). In addition, we investigated the binding of FY1 and MEDI8852 to the
remaining HAs including the 1918 H1N1 pandemic strain and
two recently identified HA analogs recovered from bats (H17
and H18) (Tong et al., 2012, 2013), by flow cytometric analysis
of cell-surface expressed HAs (Figure 2C). Of note, MEDI8852
bound to all HAs and gained reactivity against H12 HA over the
parental FY1 antibody.
To examine if the higher potency and breadth of binding
activity of MEDI8852 as compared to FY1 translated into potent
and broad antiviral activity, we measured neutralizing activity
of both antibody variants in MDCK cells against a diverse
panel of seasonal H1N1 and H3N2 viruses and emerging,
potentially epidemic viruses, isolated over a period spanning
>80 years (19332014). All seasonal influenza viruses tested
were neutralized by FY1 and MEDI8852 with median IC50 values
of 1.33 mg/ml and 0.51 mg/ml, respectively, resulting in nearly
a 3-fold increase in overall potency (Figure 2D). However,
both antibodies exhibited comparable activity in neutralizing
group 1 and group 2 viruses with similar IC50 values (1.03 and
2.02 mg/ml for FY1 and 0.34 and 0.61 mg/ml for MEDI8852
against 18 H1N1 and 18 H3N2 viruses, respectively) (Figure 2D).
598 Cell 166, 596608, July 28, 2016

The increase in overall activity of MEDI8852 compared to FY1


was also apparent when tested against 13 non-seasonal influenza viruses including H5 and H7 viruses isolated from recent
human infections. MEDI8852 neutralized these viruses having
an overall median IC50 of 1.21 mg/ml (range 4.050.41 mg/ml)
versus FY1 with a median IC50 of 3.59 mg/ml (range 11.05
0.76 mg/ml) (Figure 2E). These results indicate that the optimization of MEDI8852 resulted in a 3-fold increase in the potency of
neutralization and the ability to bind to all HA subtypes of influenza A viruses.
To extend the evaluation, we directly compared the in vitro
neutralization activity and breadth of MEDI8852 with the previously published cross-group neutralizing mAbs FI6v3, CR9114,
and 39.29 (Corti et al., 2011; Dreyfus et al., 2012; Nakamura
et al., 2013), using a diverse panel of seasonal and non-seasonal influenza strains from group 1 and group 2 (Figure 2F).
As reported previously, these antibodies neutralized group 1
and group 2 viruses although they exhibited distinct differences
in both potency and breadth. Among all the antibodies tested,
MEDI8852 is the only one that demonstrated neutralizing activity against all the viruses tested with a median IC50 0.99 mg/ml
(range 8.750.09 mg/ml). CR9114 failed to neutralize the human
H2N2 A/Japan/57 virus. Both FI6v3 and 39.29 were unable to

H8

H4 H14 H3
H12

H10
H7

H9

IC50( g/ml)

H2
H11

H6 H1

0.1

102

101

100

FY1

10-2

H5

10-1

10-1

H15

H16
H13

Group 1
H1
H2
H5
H6
H9
Group 2
H3
H7

EC50( g/ml)

Group 1

100

MEDI8852

H1N1
WSN/33
PR/34
FM/47
NJ/76
KW/86
TX/91
SZ/95
BJ/95
NC/99
SD/2007
SI/2006
CA/2009
BR/2010
HK/2010
NH/2010
NY/2012
WA/2012
BO/2013

H3N2
HK/68
VC/75
LA/87
SG/93
WH/95
SY/97
PA/99
CA/2004
WI/2005
PT/2009
VC/2011
BR/2011
NY/2012
TX/2012
AM/2013
SW/2013
NC/2014
PU/2014

IC50 ( g/ml)

101
100

10-1

CR9114

39.29

FI6v3

10

MEDI8852

104
103
102
101

FY1 MEDI8852

102

10-2

MFI

Group 2

FY1 MEDI8852

Mock
H1 SC/18
H8 ON/68
H11 ME/74
H12 AL/76
H13 MA/77
H16 SW/99
H17 GU/09
H18 PU/10
H4 CZ/56
H10 GE/49
H14 AS/82
H15 AU/79

102

IC50 ( g/ml)

H2N2 JP/57
H2N3 MO/2006
H5N1 HK/2003
H5N1 VT/2004
H6N2 AB/85
H6N1 HK/97
H9N2 HK/97
H9N2 HK/99
H3N2v MN/2010
H3N2v IN/2011
H7N7 NT/2003
H7N3 BC/2004
H7N9 AN/2013

101

100

10-1

H1N1 PR/34
H1N1 SD/2007
H1N1 SI/2006
H1N1 CA/2009
H1N1 HK/2010
H1N1 WA/2012
H1N1 BO/2013
H2N2 JP/57
H2N3 MO/2006
H5N1 HK/2003
H5N1 VT/2004
H6N2 AB/85
H6N1 HK/97
H9N2 HK/97
H9N2 HK/99

FY1 MEDI8852

Figure 2. MEDI8852 Binds to All Influenza A


HA Subtypes and Exhibits Neutralization
of Influenza A Seasonal and Non-seasonal
Viral Strains
(A) Phylogenetic tree of influenza A HAs. Group 1
and group 2 colored in red and blue are further
subdivided into 3 clades (H8, H9, and H12;
H1, H2, H5, and H6; H11, H13, and H16) and 2
clades (H3, H4, and H14; H7, H10, and H15),
respectively.
(B) ELISA binding average EC50 values of FY1 and
MEDI8852 to purified recombinant HA proteins.
(C) Binding of FY1 and MEDI8852 to surfaceexpressed HA proteins as determined by flow
cytometry. Shown are MFI values.
(D and E) FY1 and MEDI8852 neutralization IC50
values were determined against a panel of 36
seasonal influenza A isolates (D) and 13 nonseasonal influenza viruses (E).
(F) Neutralization average IC50 values of MEDI8852,
39.29, FI6v3, and CR9114 were determined from at
least two independent experiments using a panel of
24 seasonal and non-seasonal influenza viruses
and plotted as a single symbol. Full viral strains
designations are listed in Tables S1 and S2.

H3N2 HK/68
H3N2 WI/2005
H3N2 PT/2009
H3N2 BR/2011
H3N2 NY/2012
H3N2 PU/2014
H3N2 SW/2013
H7N7 NT/2003
H7N9 AN/2013

neutralize the contemporary human isolate H7N9 A/Anhui/


2013, and 39.29 was incapable of neutralizing the A/
Netherlands/2003 H7N7 virus, both H9N2 viruses, and a
contemporary H3N2 virus, A/Palau/2014, at the highest concentration tested (50 mg/ml). In addition to better overall
breadth of neutralization, MEDI8852 exhibits equal or greater
neutralization potency than the other cross-reactive monoclonal antibodies with a median IC50 of 0.99 mg/ml, compared
to 2.13, 7.57, and 1.76 mg/ml for CR9114, 39.29, and FI6v3,
respectively, when the non-neutralized viruses are excluded
from the analysis.
MEDI8852 Mechanisms of Antiviral Activity
The cross-subtype neutralizing antibodies reported to date
inhibit HA-mediated membrane fusion activity in vitro (Corti
et al., 2011; Dreyfus et al., 2012; Nakamura et al., 2013). Activation of fusion requires cleavage of the precursor, HA0, and exposure of the cleaved HA to the low pH of endosomes. In assays
of these two processes, we have shown that MEDI8852 inhibits
the host cell protease cleavage of both H1 (group 1) and H3
(group 2) HA0 that would prevent membrane fusion (Figure 3A),
and MEDI8852 binding to cleaved HA also prevents its low
pH-induced conformational change, which is required for membrane fusion by stabilizing the pre-fusion conformation (Figures
3B and 3C).

In addition, we have shown that


MEDI8852 mediates the lysis of infected cells by human primary NK cells
(ADCC), the antibody-dependent cellular
phagocytosis (ADCP) of MDCK cells
expressing H1 or H3 HAs by human
monocyte-derived macrophages and
the complement-dependent cytotoxicity
(CDC) of influenza-infected MDCK cells in the presence of complement (Figures 3D, 3E, 3F, and S2).
Prophylactic and Therapeutic Efficacy of MEDI8852 in
Mice and Ferrets
We evaluated the antiviral activity of MEDI8852 in mice challenged with a lethal dose of three different influenza A viruses,
A/California/7/2009 H1N1 (CA/2009 H1), A/Wilson Smith N/33
H1N1 (WSN/33 H1), and a reassortant A/Hong Kong/8/68
H3N1 (rHK/68 H3). A dose-ranging study was conducted in
which MEDI8852 was administered at the time of virus challenge. Mice (100%) receiving MEDI8852 at 3 or 1 mg/kg survived
challenge with CA/2009 H1, while 60% and 20% of mice survived that received 0.3 and 0.1 mg/kg of MEDI8852, respectively
(Figure 4A). Consistent with the survival data, lung viral titers
were significantly reduced compared to control antibody-treated
animals when MEDI8852 was administered at 3 mg/kg (Figure 4B). In addition, we observed that the two highest doses
of MEDI8852 significantly protected lung function in mice
compared to the control antibody as measured by pulse oximetry (Figure S3A).
To evaluate the therapeutic utility of MEDI8852, we administered MEDI8852 at different time points following infection with
WSN/33 H1 or rHK/68 H3 virus. At a dose of 10 mg/kg, survival
rates of 90%100% were achieved even when treatment was
Cell 166, 596608, July 28, 2016 599

Isotype Control
0 5 10 20 40

MEDI8852
5 10 20 40

Fusion Inhibition (%)

100

MEDI8852
H1N1
H3N2
Control Ab
H1N1
H3N2

40

MEDI8852
Control Ab

Figure 3. MEDI8852s Antiviral Mechanisms


of Action

(A) HA cleavage inhibition assay of uncleaved HA0


recombinant proteins of A/New Caledonia/20/99
(H1N1) or A/Hong Kong/8/68 (H3N2), pre-treated
60
H3
20
with MEDI8852 or a non-relevant isotype control
40
10
antibody, MPE8v3, after digestion with TPCKB
HA + MEDI8852
20
trypsin for 0, 5, 10, 20, or 40 min.
7.38 7.00 6.74 6.49 6.21 5.92 5.54
0
(B) Inhibition of low pH-activated conformational
0
HA
-1
0
1
2
3
4
5
change in HA showing SDS PAGE gels of H5
10-3 10-2 10-1 100 101 102 103
10 10 10 10 10 10 10
HA with and without MEDI8852, incubated at
Ab (ng/ml)
F
Ab (ng/mL)
Fab E
60
20
decreasing pH values and neutralized after
MEDI8852
MEDI8852
Control Ab
digestion with TPCK- trypsin.
Control Ab
40
15
(C) Fusion inhibition assay using MEDI8852 (solid)
HA alone
or MPE8v3 (open) incubated with A/Puerto Rico/8/
20
7.38 7.00 6.78 6.52 6.21 5.87 5.51
10
34 (H1N1) virus (red) or A/Aichi/2/68 (H3N2) virus
HA
0
(blue) and human red blood cells and exposed
5
to low pH to induce viral fusion. Percent fusion
-20
inhibition was calculated based on the amount of
0
hemoglobin present in the supernatant.
1
2
3
4
5
10-2 10-1 100 101 102 103 104
10 10 10 10 10
(D) ADCC activity on A549 cells infected with
Ab (ng/mL)
Ab (ng/ml)
A/Puerto Rico/8/34 (H1N1) and incubated with
MEDI8852 (red) or MPE8v3 (black) antibody in
the presence of human NK cells, antibody-dependent killing was measured in quadruplicate by LDH release.
(E) ADCP activity on MDCK cells expressing H1 HA from A/South Dakota/06/2007 that were labeled CFSE and incubated with MEDI8852 (red), or an irrelevant
control, R347 (black) antibody in the presence of violet-labeled human macrophages in duplicate. Percent phagocytosis was determined by the amount of total
macrophages that were labeled with violet and CFSE.
(F) CDC activity on MDCK cells infected with A/Puerto Rico/8/34 (H1N1) and incubated with a serial dilution of MEDI8852 (red) or MPE8v3 (black) antibody in the
presence of rabbit complement. Antibody-dependent killing was measured in triplicate by LDH release. Error bars represent two times the SD at each antibody
concentration.
See also Figure S2.
30

ADCC (%)

80

CDC (%)

ADCP (%)

H1

delayed until day 4 post infection with WSN/33 H1, or day 3 post
infection with rHK/68 H3 (Figures 4C and 4E). Significant survival
benefits were also seen with 1 and 3 mg/kg doses when administered on days 1, 2, or 3 post infection, albeit lower survival rates
than the 10 mg/kg (Table S3). MEDI8852 treatment of 10 mg/kg
at all times post infection resulted in significantly decreased viral
titers, compared to control antibody treated and untreated
animals, with a clear trend for greater reductions with earlier
treatment (Figures 4D and 4F).
To further investigate MEDI8852s therapeutic potential, we
determined the therapeutic window for treating ferrets infected
with the highly pathogenic avian influenza virus, A/Vietnam/
1204/2004 H5N1 (VT/2004 H5N1). In these studies, ferrets
were infected intranasally with 1 LD90 of VT/2004 and then
treated with a single intravenous (i.v.) dose of 25 mg/kg of
MEDI8852 at 1, 2, or 3 days post infection. We also used as a
comparator, the anti-influenza drug oseltamivir at 25 mg/kg
twice a day (b.i.d.) (Figure 4G). As expected, all control animals
showed signs of infection including fever peaking from days 1
3 post infection and 100% mortality by day 7 post infection (Figures 4G and 4H). In comparison, ferrets treated with MEDI8852
or oseltamivir on day 1 post infection were completely protected.
When treatment was delayed until 2 or 3 days post infection,
MEDI8852 provided complete protection with 100% survival
while oseltamivir only partially protected animals with survival
rates of 71% and 29%, respectively. In addition, MEDI8852
treatment resulted in a period of fever reduction following
administration, which was not observed in oseltamivir or control
antibody-treated animals (Figure 4H). Similar efficacy and therapeutic window results were seen when MEDI8852 and oseltami600 Cell 166, 596608, July 28, 2016

vir treatments were compared in a lethal murine model (Figures


S3B and S3C).
The Structures of Complexes Formed between
MEDI8852 Fab and H5, Group 1, and H7, Group 2, HAs
To provide insight into the structural basis for MEDI8852 breadth
and potency, we have determined the structures of the
MEDI8852 Fab fragment at 1.9 A and of its complexes with H5
and H7 HA proteins at 3.7 A and 3.75 A resolution, respectively
(Figures 5A and S4A). The structures of the HA proteins in both
complexes are similar to those of the apo structures determined
before (Russell et al., 2004; Xiong et al., 2015). MEDI8852 makes
similar contacts with both H5 and H7 HAs, by binding in a very
similar orientation to both HAs (Figures 5B5D), each Fab interacting with just one protomer of the HA trimer. Overall, the interactions bury 1,750 and 1,646 A2 from solvent for the H5 and H7
complexes, respectively, consistent with their high binding affinity (Table S2). MEDI8852 contacts the fusion domain of HA and
interacts with three regions of HA2, a central hydrophobic
groove, the fusion peptide and helix A, and with specific residues
in the HA1 component of the fusion domain (Figure 6). The location of MEDI8852 in the complex with the HA proteins is consistent with the in vitro assays showing that MEDI8852 stabilized
the pre-fusion conformation inhibiting fusion as well as blocking
the proteolytic cleavage of the HA precursor on the neighboring
subunit (Figure 3).
Although the structures of the complexes were determined at
intermediate resolution, the interfaces between the HAs and
MEDI8852 Fab are well-ordered and the electron density maps
in these areas are among the clearest of the overall complexes

40
20
0
0

WSN/33 H1
Survival (%)

C 100
80
40
20
2

4 6 8 10 12 14
Days Post Infection

rHK/68 H3
Survival (%)

100
80
40

80
60
40

MEDI8852 F
10 mpk

MEDI8852 Oseltamivir
25 mpk 25 mpk (2x)
Day 1
Day 2
Day 3
*
Ctr. mAb
(Day 1)
2 4 6 8 10 12
Days Post Infection

7
6
5
4

Ctl. mAb
Naive

Day 1
Day 2
Day 3
Day 4

Figure 4. MEDI8852 Provides Dose-Dependent Protection from Lethal Influenza


Infection in Mice and Ferrets Even When
Treatment Was Delayed

D 10

MEDI8852
10 mpk
Day 1
Day 2
Day 3
Day 4

4 6 8 10 12 14
Days Post Infection

20
0
0

Ctl. mAb
Naive

Ctl. mAb
Naive

20

100

A/VT/2004 H5N1
Survival (%)

*
*
*

60

0
0

*
*
*
*

60

0
0

4 6 8 10 12 14
Days Post Infection

Log TCID50 /g

60

Log TCID50 /g

80

8
Log TCID50 /g

MEDI8852 B
Day 0 Tx
3 mpk
*
1 mpk
*
0.3 mpk
*
0.1 mpk
0.03 mpk

Temperature (C) Temperature (C)

CA/2009 H1
Survival (%)

A 100

9
8
7
6
5
4
3

*
*

9
8
7
6
5
4
3

41
40
39
38
37
36

(MEDI8852)

41
40
39
38
37
36

2
3
(Oseltamivir)

1
2
3
4
Days Post Infection

(unbiased omit electron density maps are shown in Figures S4B


and S4C). We can, therefore, have confidence in our description
of the inter-molecular contacts: hydrogen bonds described in
the text should be regarded as potential interactions, however,
given the limitations of defining the exact geometry of these interactions. The principal contact areas involve three CDRs,
CDRH3, CDRH2, and CDRL1, with minor interactions with
CDRH1 and CDRL3 (Figure 6A). CDRH3 makes extensive contacts with the bottom of a hydrophobic groove between helix A
of HA2 and the fusion domain component of HA1 (Figures 6B
and 6C). Phe100A(CDRH3) inserts into this groove made by HA2
residues Ile45, Val48, Thr49, and Val52 of helix A, Trp21 of the
fusion peptide, and Thr309 of HA1. Val100C(CDRH3) binds in a
lower position in the same groove and interacts with the main
chain of HA2 residues 1921 of the fusion peptide as well as
with the side chains of Trp21 and of HA1 His8 (Figure 6B).
Asn100D(CDRH3) also makes hydrophobic contact with Val18 in

(A) Kaplan-Meier survival curves.


(B) Lung viral titers on day 5 post infection determined by TCID50 assay after mice were treated with
MEDI8852 at 3, 1, 0.3, 0.1, and 0.03 mg/kg (single
i.p. dose) and then infected with CA/2009 H1.
(C) Kaplan-Meier survival curves.
(D) Lung viral titers on day 5 post infection in mice
infected with WSN/33 H1 virus, on study day 0,
then treated with MEDI8852 or irrelevant control
antibody, R347 (single i.p. dose) at 10 mg/kg at
various days post infection.
(E) Kaplan-Meier survival curves.
(F) Lung viral titers on day 5 post infection in mice
infected with rHK/68 H3 virus, on study day 0, then
treated with MEDI8852 at 10 mg/kg or R347 (single
i.p. dose) at various days post infection.
(G) Kaplan-Meier survival curves of ferrets infected with 1LD90 of A/Vietnam/1203/2004 H5N1
virus on study day 0. Treatment with MEDI8852 at
25 mg/kg (closed symbols solid line), oseltamivir
at 25 mg/kg (open symbol dashed line), or R347
(open symbol solid line) was initiated at the indicated day post infection.
(H) Temperature of ferrets treated with MEDI8852,
or oseltamivir at various days post infection. Dotted
line designates the average normal temperature of a
ferret at 38.5 C. Error bars represent the SE of the
mean for each determination. *For murine studies,
significance was determined compared to control
antibody treatment with p < 0.005 for survival (logrank test) and p < 0.05 for lung viral titers (Students
t test); for ferret survival studies, significance was
determined by comparing to oseltamivir on the
indicated initiation day with p < 0.05 for survival (logrank test).
See also Figure S3 and Table S3.

the fusion peptide (Figure 6C). The


CDRH2 loop interacts, through VH germline-encoded residues, with HA2 residues
1519 of the fusion peptide (Figure 6C). In particular, HA2 Val18
is almost completely protected from solvent by Tyr56(CDRH2) and
Arg52B(CDRH2). There is also van der Waals interaction between
the Ca of HA2 Gly16 and Tyr52(CDRH2). Tyr52(CDRH2) is positioned
within hydrogen bonding distance of Gly16. Arg50(CDRH2) forms a
salt bridge with HA2 Asp19. Arg50(CDRH2) also contributes to a
polar patch on the antibody surface that includes Arg96(CDRL3)
and Asp58(CDRH2) and Asp100F(CDRH3). Finally, CDRL1 interacts
with the N-terminal region of helix A of HA2 (Figure 6D).
Ser30(CDRL1) and Ser31(CDRL1) are in hydrogen bonding distance
of HA2 Gln42, while Tyr32(CDRL1) and Leu29(CDRL1) make hydrophobic contacts with the aliphatic moiety of HA2 Lys38 of
helix A. The side chain of Tyr32(CDRL1) stacks against HA2
Gln42 of helix A and its hydroxyl group is in hydrogen bonding
distance of the main chain of HA2 Asp19 of the fusion peptide.
The epitope recognized by MEDI8852 is highly conserved between both HA proteins, consistent with the antibodys broad
Cell 166, 596608, July 28, 2016 601

Figure 5. MEDI8852 Binds to a Unique Site


within the H5 and H7 HA Proteins
(A) Overview of MEDI8852 in complex with H5
hemagglutinin. One HA protomer and the cognate
MEDI8852 Fab fragment are highlighted in color,
the other two copies in the trimer are colored gray.
The HA1 polypeptide is colored blue, the HA2
polypeptide is colored red, with the fusion peptide
at the N terminus of HA2 highlighted in yellow. The
heavy chain of the MEDI8852 Fab is colored orange, the light chain is colored green.
(B) Overlay of MEDI8852 bound to group 1 (H5) and
group 2 (H7) HAs. The antibodies are shown in
cartoon representation together with Helices A
and B of the HA. The components are colored
according to (A) and the view orientation is
approximately that shown by the black arrow in (A).
(C and D) H5 (C) and H7 (D) HAs are shown in
surface representation. Only the HA residues in the
MEDI8852 binding epitope that differ between H5
and H7 are labeled. The CDR loops of MEDI8852
that are in contact with HA are shown in cartoon
and stick representation and colored by element.
See also Figures S4, S7, and Table S4.

activity against group 1 and group 2 influenza viruses (Figures


5C, 5D, and S5). However, the membrane proximal fusion domains of both group 1 and group 2 HAs have a few distinct structural differences such as glycosylation status of HA1 position 38,
which could potentially affect the binding of some antibodies
(Corti et al., 2011; Ekiert et al., 2009; Sui et al., 2009). In the H7
complex with MEDI8852, the bulky carbohydrate chain attached
to HA1 Asn38 changes its orientation to allow antibody binding,
as observed before in the FI6 Fab-H3 HA complex (Corti et al.,
2011). Another notable feature of the MEDI8852 complex with
H7 HA is the involvement of HA2 Tyr38 in an aromatic stacking
interaction with Tyr32(CDRL1). Interestingly, the tyrosine at position 38 is not conserved across all HA subtypes only in H7,
H10, and H15. The arginine in H6, H9, H11, and H12 could
engage in a similar stacking interaction although the leucine,
lysine, and glutamine found in the remaining HA proteins could
not interact in the same way. Thus, the high and similar affinities
with which MEDI8852 reacts with these HAs, suggests that the
differences in glycosylation at HA1 38 and the side chain at
HA2 38 make a minor contribution to the overall energetics of
binding (Figure 2B; Table S2).
Conformational Rearrangements in MEDI8852 on
Complex Formation
The availability of a high-resolution structure of MEDI8852 Fab
and of well-ordered interfaces in the structures of the complexes
formed with H5 and H7 HAs enables us to analyze conformational changes in the Fab upon HA binding, particularly in the
602 Cell 166, 596608, July 28, 2016

CDRH3 and CDRL1 loops. The loop


formed by residues 97100F of CDRH3
undergoes a largely rigid-body rotation, pivoted around Gly96(CDRH3) and
Ala100G(CDRH3) (Figure 7A) to facilitate
interactions with HA. As a consequence,
the side chain of Phe100A(CDRH3) moves by 5 A, to insert
into the hydrophobic groove of the epitope, near HA2 48. In addition, residues 2732(CDRL1) are restructured, with an average
displacement of 10 A between apo and bound forms. The reorientation of the side chains of Tyr32(CDRL1) and Leu29(CDRL1) enables them to interact with HA2 Tyr38 in the H7 complex.
Ser30(CDRL1) and Ser31(CDRL1) in the complex form a helical
structure that places them in hydrogen bonding distance with
Gln42 of HA2.
There are five mutations found in the vitro optimization of
FY1 to MEDI8852 that are not in direct contact with HA, but
are contained within CDR loops (Figures 6E and 6F). The
mutated residues are seen to stabilize the conformations that
the loop regions adopt in complex with HA while the parental residues (FY1) do not appear to be able to make similar stabilizing
interactions (Figure S6). Thus, the optimization process of FY1
to MEDI8852 results in the selection of amino acid substitutions
that stabilize the induced fit conformation that the CDR loops
adopt in complex with HA.
Comparison of the Epitope of MEDI8852 with Those of
Other Broadly Neutralizing Antibodies
We have compared the mode of interaction of MEDI8852 with
other broadly neutralizing antibodies that recognize the membrane proximal fusion domain of HA. The cross-group neutralizing antibodies 39.29, FI6v3, and CR9114 (Corti et al., 2011;
Dreyfus et al., 2012; Nakamura et al., 2013), as well as the group
1-neutralizing antibodies F10 and CR6261 (Ekiert et al., 2009; Sui

Figure 6. Binding Epitope of MEDI8852 on H5 HA


(A) HA is shown in surface representation and residues that are contacted by MEDI8852 are highlighted in color (blue for HA1, red for HA2 and yellow for fusion
peptide residues). Secondary structure elements of HA are shown in cartoon representation. The hydrophobic groove on HA is outlined in gray. The CDR loops of
MEDI8852 that are in contact with HA are shown in cartoon representation and colored orange and green for the heavy and light chains, respectively. The colored
boxes indicate the three parts of the binding epitope that are shown in more detail in (B), (C), and (D).
(B) Interactions of MEDI8852 with the hydrophobic groove of H5 HA. HA is drawn in surface representation, with the main chain shown in cartoon representation
and amino acids that are in contact with MEDI8852 shown in stick representation. W21, which adopts different rotamers in group1 and group 2 influenza viruses,
is colored magenta. MEDI8852 is also shown in cartoon representation, with contact residues shown in stick representation. Hydrogen bonds and salt bridges are
indicated by dashed lines.
(C) Interactions of MEDI8852 with the fusion peptide of H5 HA. Shown in the same style as in (B).
(D) Interactions of MEDI8852 with the base of helix A of H5 HA. Shown in the same style as in (B).

(legend continued on next page)

Cell 166, 596608, July 28, 2016 603

et al., 2009), all recognize helix A of HA2 and the adjacent hydrophobic groove (Figure 7B, blue box). In contrast, the group
2-neutralizing antibodies CR8020 and CR8043 (Ekiert et al.,
2011; Friesen et al., 2014) recognize a different region of the
fusion peptide and a small b sheet below it in the fusion domain
(Figure 7B, red box). The epitope of MEDI8852, uniquely among
the cross-group neutralizing antibodies reported to date, represents a combination of both regions. A structural comparison of
HA-bound MEDI8852 overlapped with the antibodies CR8020
and CR9114 is shown in Figures S7A and S7B, which reveals
that the overall orientation of MEDI8852 is such that it sits slightly
higher on the HA than the CR8020 antibody and lower than
CR9114. The nearest paratope residue of MEDI8852 is 20
from the membrane proximal end of the HA.
MEDI8852 and 39.29 antibodies bind similarly to residues in
the hydrophobic groove and adjacent helix A in the fusion
domain. Both antibodies contain the four amino acid sequence
ValPheGlyVal/Ile in their otherwise dissimilar CDRH3 loops.
These tetrapeptides superpose in the complexes with an allatom root-mean-square deviation (RMSD) of 0.7 A, indicating
that they interact with their cognate HAs in a similar way (Figure 7C). However, other contacts made by these antibodies
with HA are quite different between MEDI8852 and 39.29, reflecting the fact that the two antibodies are not particularly similar
in sequence and are derived from different germline sequences
(VH6-1*01 and VK 1-39*01 for MEDI8852 versus VH3-30*01
and VK3-15*01 for 39.29). MEDI8852 contacts the base of helix
A and the fusion peptide with its CDRL1 and CDRH2 loops,
respectively (Figure 6). By contrast, contacts made by the heavy
chain of 39.29 Fab mainly involve CDRH3. 39.29 appears to bury
helix A using all three light chain CDR loops. As a consequence,
the 39.29-HA interaction buries a total of 2,287 A2, while
MEDI8852 achieves similar affinity with a smaller buried surface
of 1,646 A2.
MEDI8852 is the second cross-group neutralizing antibody for
which structures of complexes with both group1 and group 2
HAs have been reported. The first was FI6v3, which, although
it recognizes both group 1 and group 2 HAs, has higher in vivo
neutralizing activity against group 1 viruses (Corti et al., 2011).
The cross-group binding of FI6v3 has been attributed to its
long and flexible HCDR3, which can accommodate the differences in conformation and environment of HA2 Trp21 observed
between group 1 and group 2 viruses. There is a much more significant rearrangement between FI6v3 bound to H1 HA (group 1)
versus H3 HA (group 2) than there is between MEDI8852 in its
complexes with H5 HA (group 1) and H7 HA (group 2) which overlap very closely (Figures 5B and S7C). MEDI8852, therefore,
binds in a very similar way to HAs of both groups. This greater
structural conservation of the binding interface is likely responsible for its broader neutralizing ability.

DISCUSSION
There is an unmet medical need for effective treatments against
severe influenza. The potential of broadly infectivity-neutralizing
antibodies used therapeutically to address this need has provided a stimulus for their isolation and characterization. Among
the antibodies considered to date, the anti-HA human monoclonal antibody MEDI8852 has demonstrated significant breadth
of its infectivity-neutralizing capacity. MEDI8852 reacts with HAs
of all influenza antigenic subtypes, potently neutralizes diverse
virus strains with numerous HA subtypes, and can block infection and lethality caused by influenza viruses when administered
up to 4 days after challenge with the virus in mice and up to
3 days post challenge in ferrets with the highly pathogenic
H5N1 virus. This potential ability to overcome the unpredictable
characteristics of influenza, namely the antigenic shift, that results in disease during pandemic periods, and the antigenic drift,
that occurs with the emergence of antigenically novel viruses, is
a major advantage for a candidate anti-influenza therapeutic
antibody.
The mechanisms of MEDI8852-mediated neutralization of
infection involve processes at the beginning and the end of the
infection cycle. Binding of the antibody to HAs on the infecting
virus inhibits HA-mediated membrane fusion that is required
for the initiation of infection. At the end of infection, antibody
binding to precursor HA0 can block its cleavage and prevent
the formation and spread of newly made infectious virus. Additionally, binding of MEDI8852 to HAs displayed on the surfaces
of infected cells results in their recognition and lysis by other
components of the immune system: NK cells, macrophages,
and complement. These multiple mechanisms exhibited by
MEDI8852 presumably combine to ensure the observed effectiveness of antibody treatments in infected mice and ferrets.
The epitope recognized by MEDI8852 is consistent with the
ways it blocks HA function in membrane fusion and with the
locations of epitopes that have been described previously for
influenza group-specific cross-reactive antibodies and for
more broadly reactive antibodies. However, the regions of HA
that interact with MEDI8852 are a combination of those previously assigned to group 1 specificity (primarily a hydrophobic
groove and the adjacent helix A of HA) (Ekiert et al., 2009; Sui
et al., 2009) or group 2 specificity (a separate part of the fusion
peptide, near its N terminus) (Ekiert et al., 2011; Friesen et al.,
2014). The structural characterization of MEDI8852 bound and
unbound structures also highlight the coordinated movement
of the CDRH3 and the CDRL1 to insert into the hydrophobic
grove of the HA, as well as the rearrangement of the orientation
of the glycan attached to Asn38 of the H7 virus to allow antibody
binding. Importantly, structures of the complexes formed by
MEDI8852 with H5 and H7 HAs indicate that the locations and

(E) Location of mutations found during affinity maturation of FY1 to MEDI8852. The variable domains of MEDI8852 are shown in cartoon representation, viewed
from the direction of HA. Regions of the heavy and light chains in contact with HA are colored orange and green, respectively. Interacting sidechains are shown in
stick representation. Residues that differ between the parental and affinity-maturated antibody are shown in sphere representation.
(F) Sequences of MEDI8852 variable region framework and CDR residues. CDRs (according to Kabat) are highlighted in orange and green for the heavy and light
chains respectively. Residues in contact with HA are colored red and residues changed during affinity maturation from FY1 are colored cyan, with corresponding
residues of FY1 indicated.
See also Figure S6.

604 Cell 166, 596608, July 28, 2016

Figure 7. MEDI8852 Binds to a Unique Site within the H5 and H7 HA Proteins through CDR-H3 and CDR-L1 Conformational Rearrangements
upon Complex Formation
(A) Conformational rearrangements in MEDI8852 on complex formation. Conformational change of the CDRH3 and CDR-L1 loops upon HA engagement. The apo
structure of MEDI8852 is shown in blue, the bound structure is shown in orange and green for the heavy and light chains, respectively. The beginning and end of
the moving regions are indicated with black ovals. HA (H7) is shown as a gray surface. The apo structure does not make interactions with HA and does not fit into
its surface featuresthe conformational change is necessary for productive HA engagement.
(B) Epitopes of different broadly neutralizing antibodies on the HA surface. Residues of HA that are in contact with the heavy chain are colored orange, residues
that are in contact with the light chain are colored green, and residues that are in contact with both chains are colored yellow. The blue box encases the part of the
MEDI8852 epitope (helix A and hydrophobic groove) that can also be found in other broadly neutralizing antibodies as well as group 1 specific ones. The red box
encases the part of the MEDI8852 epitope that can also be found in group 2 specific antibodies (middle of fusion peptide).
(C) Comparison of the structures of the conserved CDRH3 tetra-peptide in the complexes between MEDI8852 and H7 HA (left panel) and 39.29 and H3 HA (right
panel). In both cases the tetra-peptide is shown in stick representation with other loops of the antibody shown as coil, colored as in panel A. The HAs are shown in
surface representation.
See also Figure S7.

Cell 166, 596608, July 28, 2016 605

orientations of the bound antibodies are very similar, and this


structurally conserved ability to interact with both regions of
HA presumably results in effective cross-reactivity.
The comparison of the structures of the complexes formed by
MEDI8852 with H5 and H7 HAs with the previously reported
complex formed between the cross-reactive monoclonal antibody 39.29 and H3 HA (Nakamura et al., 2013) indicated that
the two antibodies had the amino acid sequences, V-F-G-VMEDI8852 and V-F-G-I- 39.29, in their HCDR3 loops that occupied equivalent positions in the complexes. Conceivably, the
structure of this shared component of the antibodies might be
used in the preparation of immunogens or to select candidate
molecules on the basis of their affinity for the tetra-peptides.
Of note, a recent paper described the development of a computationally designed protein binding to Helix A in the stem region
of group 1 HAs that showed in vitro and in vivo antiviral efficacy
(Koday et al., 2016).
The reconstruction of the developmental pathway of MEDI8852,
as well as of FI6, suggests that the generation of such broadly
reactive antibodies may require a stepwise stimulation by group
1 HAs, followed by the selection of mutated variants by group 2
HAs. Indeed, the MEDI8852 donor was born in the 1950s
and it is possible that this lineage was primed by H2N2 and
further matured through multiple H3N2 exposures. This hypothesis is further strengthened by the observation that the UCA
mAb neutralized with high potency the strain H2N2 JP/57 (i.e.,
IC50 = 0.6 mg/ml). The UCA and mutated antibodies of the
MEDI8852 and FI6 lineages represent useful tools to design
stem-based immunogens that can be used in a heterologous
prime-boost mode to prime the group 1 reactive naive B
cells and selectively expand those that also cross-react with
group 2 HAs.
Based on the results reported, MEDI8852 is currently being
evaluated for safety and efficacy in adults with uncomplicated
influenza infection in an outpatient setting (https://.clinicaltrials.
gov: NCT02603952) prior to conducting studies in patients hospitalized with influenza caused by type A strains.
EXPERIMENTAL PROCEDURES

In Vitro Fusion and HA Cleavage Assays


Antibody-mediated fusion inhibition was tested using a low pH-induced red
blood cell fusion model adapted from protocol described in Wang et al.
(2010). The ability of MEDI8852 to inhibit the low pH-activated conformational
change in trypsin-digested H5 HA was analyzed by SDS/PAGE. The ability of
antibody to block the HA0 cleavage by TPCK-treated trypsin was measured by
western blot analysis (Supplemental Experimental Procedures).
Measurement of Fc-Effector Function
ADCC activity was measured with the LDH release assay using primary human
NK cells as effector cells and H1N1 or H3N2 influenza virus infected A549 cells
as a target. ADCP activity was measured by flow cytometry using fluorescently
labeled monocyte-derived macrophages and H1 or H3 HA-expressing MDCK
as target cells. CDC activity was measured with the LDH release assay using
rabbit complement on influenza H1N1-infected MDCK cells (Supplemental
Experimental Procedures).
Therapeutic Efficacy Studies in Mice and Ferrets
All animal studies were approved and conducted in accordance with
MedImmunes Institutional Animal Care and Use Committee (murine studies)
and Southern Research Institutes Institutional Animal Care and Use Committee (ferret studies) and performed in Association for the Assessment and
Accreditation of Laboratory Animal Care (AAALAC)-certified facilities.
MEDI8852 or R347 control mAb was administered as a single intraperitoneal
(i.p.) dose at various days post infection, depending on the virus strain. For
oseltamivir comparison studies, mice were administered 25 mg/kg oseltamivir
by mouth (PO) b.i.d. for 5 days, or a single 10 mg/kg dose i.v. of MEDI8852 with
vehicle PO b.i.d. for 5 days. Viral loads in the lungs were measured by TCID50
assay on day 5 post infection. Five- to six-month-old ferrets were challenged
intranasally with A/Vietnam/1203/04 (H5N1) virus and treated with a single
25 mg/kg i.v. dose of MEDI8852 (or R347 control) or oseltamivir at 25 mg/kg
BID for 5 days initiated at different days post infection. Bio-metric data systems chip was used for temperature monitoring. (Supplemental Experimental
Procedures).
HA-MEDI8852 Complex Preparation, Crystallization, and Structure
Determination
H5 and H7 HAs were purified from the virus membrane and mixed with purified
MEDI8852 antibody Fab fragments and incubated overnight at 4 C for complex formation. Complexes were further purified by size-exclusion chromatography and concentrated for crystallization. Crystals were frozen by direct
immersion in liquid nitrogen and diffraction datasets were collected at 100 K
at the IO2 and IO4 beamlines at the diamond light source (Harwell). Structures
were solved by molecular replacement and refined using standard protocols. Macromolecular structures have been deposited under the accession
numbers PDB: 5JW5 (apo MEDI8852), PDB: 5JW4 (H5 complex), and PDB:
5JW3 (H7 complex). Crystallographic statistics are summarized in Table S4
(Supplemental Experimental Procedures).

Monoclonal Antibody Isolation and Ex Vivo Affinity Maturation


Monoclonal antibodies were isolated from memory B cells, as previously
described (Pappas et al., 2014; Traggiai et al., 2004) from blood donors who
had given written informed consent, following approval by the Cantonal Ethical
Committee of Cantone Ticino, Switzerland. FY1 antibody was further modified
to revert the non-germline framework amino acid changes and its affinity was
improved through parsimonious mutagenesis of CDRs (Supplemental Experimental Procedures).

ACCESSION NUMBERS

Recombinant HA Protein and Binding Assays


Recombinant HA proteins were expressed and purified as previously
described (Benjamin et al., 2014). The binding of antibodies to HAs was
measured by ELISA or by staining HA transfected cells using flow cytometry
(Supplemental Experimental Procedures).

SUPPLEMENTAL INFORMATION

Viruses and Microneutralization Assay


Wild-type influenza strains and cold-adapted (ca) live-attenuated influenza
vaccine viruses (complete viral strain designations shown in Table S1) were
propagated in embryonated chicken eggs, titered, and used to infect MDCK
cells to determine neutralizing activity as described in the Supplemental
Experimental Procedures.

606 Cell 166, 596608, July 28, 2016

The accession number for the coordinates and structure factors reported in
this paper is PDB: 5JW5 (apo MEDI8852, 5JW4 (H5 complex), and 5JW3
(H7 complex). The accession number for the sequences for all of the antibodies reported in this paper is GenBank: KX398429-KX398468.

Supplemental Information includes Supplemental Experimental Procedures,


seven figures, and four tables and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2016.05.073.
AUTHOR CONTRIBUTIONS
B.F.-R. carried out PCR of immunoglobulin sequences from B cells. G.A.
produced and purified antibodies. M.F. analyzed immunoglobulin genetic

elements, produced figures, and carried out bioinformatic analysis. D.P. and
C.S. carried out donors selection and screenings for the identification of
cross-reactive antibodies. F.V. and A.F. carried out cloning HAs and testing
antibodies for binding in cytofluorimetry. S.B. performed biochemical and
cellular assays to test the fusion and HA maturation inhibiting activities of isolated antibodies. B.G. and A.D.M. carried out ADCC and CDC studies. F.S.
wrote the paper. A.L. and D.C. directed the B cell isolation studies, analyzed
data, and wrote the paper. J.M.M. and E.B. carried out in vivo studies. L.W.-R.
carried out ADCP studies. F.J.P.-H. carried out the ELISA binding studies.
N.L.K., Q.Z., L.W.-R., and F.J.P.-H. carried out the neutralization studies.
A.Q.Y. lead the antibody optimization. J.A.S. edited the paper and provided
supervision. N.L.K. and Q.Z. directed antibody optimization, in vitro and in vivo
characterization, analyzed the data, and wrote the paper. P.J.C., U.N.,
P.A.W., M.K.V., R.W.O., S.R.M., S.J.G., and J.J.S. designed and performed
structural research, contributed new reagents and analytical tools, analyzed
data, and wrote the paper.
CONFLICTS OF INTEREST
A.L. is the scientific founder of Humabs BioMed SA. A.L. holds shares in Humabs BioMed. B.G., A.D.M., G.A., F.V., and D.C. are employees of Humabs
Biomed. This work was funded by MedImmune, LLC, a wholly owned subsidiary of AstraZeneca Pharmaceuticals. N.L.K., J.M.M., E.B., L.W.-R., F.J.P.-H.,
A.Q.Y., J.A.S., and Q.Z. were employed by MedImmune, LLC when work was
executed and may currently hold AstraZeneca stock or stock options.

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Huang, M., Qu, X., Huang, Y., Salgado-Ferrer, M., et al. (2015). Preexisting human antibodies neutralize recently emerged H7N9 influenza strains. J. Clin.
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Throsby, M., Goudsmit, J., and Wilson, I.A. (2009). Antibody recognition of a
highly conserved influenza virus epitope. Science 324, 246251.
Ekiert, D.C., Friesen, R.H.E., Bhabha, G., Kwaks, T., Jongeneelen, M., Yu, W.,
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conserved neutralizing epitope on group 2 influenza A viruses. Science 333,
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M. (1999). A complex of influenza hemagglutinin with a neutralizing antibody
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ACKNOWLEDGMENTS

Knossow, M., and Skehel, J.J. (2006). Variation and infectivity neutralization in
influenza. Immunology 119, 17.

We thank Jose Martinez and the MedImmune Laboratory Animal Resource


staff for in vivo study assistance; Robert Woods for additional affinity characterization; and Sandrina Phipps, Arnita Barnes, and Kannaki Senthil for generating HA reagents. This work was supported by the European Research
Council (grant 670955 BROADimmune) and the Swiss National Science Foundation (grant 160279). A.L. is supported by the Helmut Horten Foundation. We
thank the staff at the Diamond Light Source Synchrotron for assistance and
beam-line access under Diamond Light Source Proposal 9826. This work
was funded by the Francis Crick Institute, London. U.N. was also funded by
a Marie Curie Actions Intra-European Fellowship (grant 629829).

Koday, M.T., Nelson, J., Chevalier, A., Koday, M., Kalinoski, H., Stewart, L.,
Carter, L., Nieusma, T., Lee, P.S., Ward, A.B., et al. (2016). A Computationally
Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection
from Influenza Independent of a Host Immune Response. PLoS Pathog. 12,
e1005409.

Received: February 22, 2016


Revised: April 4, 2016
Accepted: May 25, 2016
Published: July 21, 2016
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608 Cell 166, 596608, July 28, 2016

Article

Vaccine-Induced Antibodies that Neutralize Group 1


and Group 2 Influenza A Viruses
Graphical Abstract

Authors
M. Gordon Joyce, Adam K. Wheatley,
Paul V. Thomas, ..., Peter D. Kwong,
John R. Mascola, Adrian B. McDermott

Correspondence
pdkwong@nih.gov (P.D.K.),
jmascola@nih.gov (J.R.M.),
adrian.mcdermott@nih.gov (A.B.M.)

In Brief
Quantifying B cells capable of producing
broadly neutralizing antibodies against
influenza serves as a metric to guide the
development of a universal influenza
vaccine.

Highlights
d

Isolation of group 1 and group 2 influenza A-neutralizing


antibodies from H5N1 vaccinees
Discovery of three classes of broadly neutralizing antibodies
directed to the HA stem
Delineation of sequence signatures specific for broadly
neutralizing antibodies
Antibody quantification by NGS to guide the development of
a universal vaccine

Joyce et al., 2016, Cell 166, 609623


July 28, 2016 Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.cell.2016.06.043

Accession Numbers
5K9J
5K9K
5K9O
5K9Q
5KAN
5KAQ
KX386124KX387227

Article
Vaccine-Induced Antibodies that Neutralize
Group 1 and Group 2 Influenza A Viruses
M. Gordon Joyce,1,8 Adam K. Wheatley,1,8 Paul V. Thomas,1,8 Gwo-Yu Chuang,1,8 Cinque Soto,1,8 Robert T. Bailer,1
Aliaksandr Druz,1 Ivelin S. Georgiev,1,2,3 Rebecca A. Gillespie,1 Masaru Kanekiyo,1 Wing-Pui Kong,1 Kwanyee Leung,1
Sandeep N. Narpala,1 Madhu S. Prabhakaran,1 Eun Sung Yang,1 Baoshan Zhang,1 Yi Zhang,1 Mangaiarkarasi Asokan,1
Jeffrey C. Boyington,1 Tatsiana Bylund,1 Sam Darko,1 Christopher R. Lees,1 Amy Ransier,1 Chen-Hsiang Shen,1
Lingshu Wang,1 James R. Whittle,1 Xueling Wu,1 Hadi M. Yassine,1 Celia Santos,1,4 Yumiko Matsuoka,4
Yaroslav Tsybovsky,5 Ulrich Baxa,5 NISC Comparative Sequencing Program,6 James C. Mullikin,6 Kanta Subbarao,4
Daniel C. Douek,1 Barney S. Graham,1 Richard A. Koup,1 Julie E. Ledgerwood,1 Mario Roederer,1 Lawrence Shapiro,1,7
Peter D. Kwong,1,* John R. Mascola,1,* and Adrian B. McDermott1,*
1Vaccine

Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
of Pathology, Microbiology, and Immunology and Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville,
TN 37232, USA
3Department of Electrical Engineering and Computer Science, Vanderbilt University, Nashville, TN 37232, USA
4Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda,
MD 20892, USA
5Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Frederick National Laboratory for
Cancer Research, Frederick, MD 21702, USA
6NIH Intramural Sequencing Center (NISC), National Human Genome Research Institute, National Institutes of Health, Bethesda,
MD 20892, USA
7Department of Biochemistry & Molecular Biophysics and Department of Systems Biology, Columbia University, New York, NY 10027, USA
8Co-first author
*Correspondence: pdkwong@nih.gov (P.D.K.), jmascola@nih.gov (J.R.M.), adrian.mcdermott@nih.gov (A.B.M.)
http://dx.doi.org/10.1016/j.cell.2016.06.043
2Department

SUMMARY

Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/
MIV-prime-boost influenza vaccine trial, we sorted
hemagglutinin cross-reactive memory B cells and
identified three antibody classes, each capable of
neutralizing diverse subtypes of group 1 and group
2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs
to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences
from at least one multidonor class, andin half the
subjectsmultidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast,
these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency
of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide
universal influenza A immunization strategies.
INTRODUCTION
Influenza A viruses can be categorized into two phylogenetic
groups (group 1 and group 2), each containing diverse subtypes

(Figure 1A). Currently, group 1 influenza viruses from the H1 subtype (1918 and 2009 H1N1 pandemics), and the group 2 H3 subtype (1968 H3N2 pandemic), co-circulate and cause seasonal infections in over 10% of the human population each year. Other
subtypes have emerged or threaten to re-emerge including the
group 1 H2 subtype, endemic in humans from 19571968, the
group 1 H5 subtype, which includes lethal avian strains (Subbarao et al., 1998), and the group 1 H6 and H9 and the group 2
H7 and H10 subtypes, which have been associated with human
infections and fatalities in recent years (Butt et al., 2005; Morens
et al., 2013). Frequent zoonotic cross-overs that may cause pandemics of unpredictable frequency and severity highlight the
need for a universal influenza vaccine that is capable of eliciting
protection against divergent influenza A viruses.
Potential approaches to a universal influenza vaccine involve
the elicitation of neutralizing antibodies that recognize the
influenza hemagglutinin (HA) from multiple subtypes. One means
to accomplish this involves ontogeny-based strategies, which
seek to identify antibodies of reproducible classes and to induce
similar antibodies by vaccination (Jardine et al., 2015; Lingwood
et al., 2012; Pappas et al., 2014). We consider antibodies to be of
the same class when they recognize the same region, employ the
same structural mode of recognition, and develop through
similar recombination and maturation pathways (Kwong and
Mascola, 2012). Reproducible classes, which are observed in
multiple individuals, represent immunological solutions to the
challenge of broad influenza A neutralization that might be available to the general human population.
The influenza A-neutralizing stem-directed antibodies that utilize the HV1-69 germline gene are one such multidonor class
(Ekiert et al., 2009; Kashyap et al., 2010; Sui et al., 2009; Throsby
Cell 166, 609623, July 28, 2016 Published by Elsevier Inc. 609

B
H12
H8

H7
H15

H10
H1
H4
H14
H18

H2

H17

H3

H5
H
5

10 5

10 4

01
16
31
36
54
56

10 4
10 3
10 2

10 3

10
27
29

10 2
10 2

59

10 1

10 1

10 1
H5N1

Group 1

Subject 16

Subject 31

0.08%

0.22%

0.07%

Subject 36

Subject 54

Subject 56

10 3

H3-A/Perth/16/2009

H11

Subject 01

Selection of subjects displaying


varying levels of neutralization against
group 1 and group 2 strains
Neutralization (recipricol ID5 0 titre)

H9
H6

H13 H16

H3N2

H7N7

Group 2 D

0.2

Subject 01

Subject 16

H5-A/Indonesia/5/2005

Subject 31

E
Number of
genetic
Similarities

Antibody name
(subject.lineage.clone)

235

136

HV4-34

HV1-18

HV3-23

Subject 54

Subject 36

Subject 56

HV6-1

HV1-18

HV6-1
HV
1-18

94

0.13%

0.29%

0.11%

102

13
HV 4-30

HV1-18

65
HV6-1

HV3-64D

Neutralization breadth

Frequentist
probability

Number of
clones per
subject per
genetic cluster

HV
gene

HD
gene

HJ
gene

CDR H3
HV
length* maturation

LV
gene

LJ
Gene

CDR L3
length*

Binding
competition

Group 1

Group 2

31.g.01
54.f.01
56.a.09

4.9E-12

1
1
30

HV6-1
HV6-1
HV6-1

HD3-3
HD3-3
HD3-3

HJ4/5
HJ4/5
HJ4/5

16
16
16

8%
6%
4%

KV3-20
KV3-20
KV3-20

KJ2
KJ3
KJ2

9
9
9

n.d.
+++
+++

n.d.
H1, H2, H5
H1, H5

n.d.
H3, H7
H3, H7

01.k.01
31.b.09

3.6E-6

1
26

HV1-18
HV1-18

HD3-9
HD3-9

HJ4
HJ4

15
15

9%
5%

KV2-30
KV2-30

KJ5
KJ2

9
9

+++
+++

H1, H5
H1, H5

H3
H3, H7

16.g.07
54.a.84

6.9E-5

8
92

HV1-18
HV1-18

HD2-15
HD2-15

HJ2
HJ2

21
21

11%
11%

KV1-12
KV3-11

KJ2
KJ1

9
9

+++
+++

H1, H5, H9
H1, H5, H9

H3, H7
H3, H7
H3, H7

16.a.26

93

HV1-18

HD2-2

HJ5

21

8%

KV1-39

KJ2

+++

H1, H5, H9

54.a.39

92

HV1-18

HD2-2

HJ5

21

6%

KV3-11

KJ1

+++

H1, H9

H3, H7

31.a.83
56.h.01

0.004

104
2

HV3-23
HV3-23

HD3-9
HD3-9

HJ6
HJ6

24
28

8%
8%

KV3-15
KV2-29

KJ2
KJ4

9
9

+++
+++

H1, H2, H5, H9


H1, H2, H9

H3, H7
None

01.s.01
31.f.01

0.007

1
1

HV1-69
HV1-69

HD5-18
HD3-22

HJ4
HJ4

15
15

10%
7%

KV4-1
KV3-20

KJ3
KJ2

9
9

n.b.
+++

None
H1, H2, H5, H9

None
None

31.f.01
56.ND.11

5.1E-5

1
1

HV1-69
HV1-69

HD3-22
HD3-22

HJ4
HJ6

15
15

7%
6%

KV3-20
KV3-20

KJ2
n.r.

9
n.r.

+++
n.d.

H1, H2, H5, H9


n.d.

None
n.d.

54.ND.03
56.g.01

0.001

1
1

HV1-69
HV1-69

HD3-22
HD3-22

HJ6
HJ6

13
13

8%
6%

n.d.
KV3-20

n.d.
KJ2

n.d.
9

n.d.
n.b.

n.d.
None

n.d.
None

HV7-4-1

HD3-9

HJ4

21

1%

KV3-15

KJ1

+++

H1, H5

H3

HV3-49

HD3-9

HJ4

21

9%

KV2-30

KJ1

n.d.

n.d.

n.d.

1
1

HV3-23
HV4-4

HD6-13
HD6-13

HJ4
HJ4

15
15

0%
3%

KV4-1
KV2-40

KJ1
KJ2

9
7

n.b.
n.d.

None
n.d.

None
n.d.

54.e.01
56.k.01

6.9E-5

0.001

01.i.01
56.i.01

0.032

n.a.

31.d.01

n/a

HV3-30

HD3-9

HJ3

21

4%

KV4-1

KJ2

+++

H1, H2, H5, H9

None

n.a.

01.a.44

n/a

53

HV4-34

HD2-8

HJ6

24

12%

KV1-9

KJ2

+++

H1, H2, H5

H3, H7

*IMGT CDR 3 lengths used; n.d.: not determined; : crystal structure in complex with HA determined; n.b.: no binding to HA; n.r.: not recovered; None: no pseudovirus neutralization observed; n.a.: not applicable

Figure 1. H5N1 Vaccine Recipients Have Cross-Reactive B Cells that Utilize the Same Genetic Elements and Neutralize Group 1 and Group 2
Influenza A Viruses
(A) Phylogenetic tree depicting influenza A subtypes, generated using HA sequences (one per subtype except H1, H3, H5, and H7) with program MEGA6. Scale
bar indicates distance per fractional nucleotide change.
(B) Neutralization by serum from 63 vaccinees, sampled 2 weeks after final H5N1 immunization and assessed against vaccine strain (A/Indonesia/5/2005) and
heterologous group 2 strains (H3N2: A/Hong Kong/1-4-MA21-1/1968; H7N7: A/Netherlands/219/2003). Ten subjects were selected for flow cytometric (FACS)
characterization, as highlighted in key. Dotted line indicates the limit of detection.
(C) FACS analysis of PBMC samples isolated from H5N1-vaccine recipients 2 weeks after final vaccination and co-stained with HA probes H5 (A/Indonesia/5/
2005) and H3 (A/Perth/16/2009). Sizable populations of H5-H3 cross-reactive memory B cells observed in six of ten subjects (Figure S2).

(legend continued on next page)

610 Cell 166, 609623, July 28, 2016

et al., 2008). In terms of reproducibility, the HV1-69-derived antibodies have the additional advantage of utilizing heavy chainonly recognition, and prior studies have shown their vaccineinduced elicitation (Khurana et al., 2013; Ledgerwood et al.,
2011, 2013; Sui et al., 2009; Wheatley et al., 2015; Whittle
et al., 2014). However, HV1-69-derived antibodies generally do
not neutralize both group 1 and group 2 strains of influenza A,
and, only a single HV1-69-derived antibody has been identified
(CR9114) capable of neutralizing both group 1 and group 2
strains of influenza A (Dreyfus et al., 2012). Other broadly neutralizing antibodies have been identified, such as FI6v3 and 39.29,
both of which derive from the HV3-30 germline gene; however,
co-crystal structures with HA reveal different modes of recognition (Corti et al., 2011; Nakamura et al., 2013), and FI6v3 and
39.29 are thus not members of the same class. Indeed, a reproducible antibody class capable of neutralizing both group 1 and
group 2 influenza A viruses has not been observed in multiple
donors.
We previously showed that subjects enrolled in the phase I
clinical trial, VRC 310who received an A/Indonesia/05/2005
monovalent inactivated virus (MIV) vaccine primed by an H5
DNA plasmid vaccine (Ledgerwood et al., 2011, 2013) (Table
S1A)showed transient expansion of H1- and H5-cross-reactive memory B cells specific to the HA stem (Wheatley et al.,
2015; Whittle et al., 2014). To determine whether these memory
B cells might encode multidonor class antibodies capable of
neutralizing group 1 and group 2 influenza A virus, we sorted
memory B cells that reacted with both H5 (group 1) and H3
(group 2) HAs. Immunoglobulin transcripts from post-vaccination cross-reactive memory B cells were sequenced, and the
encoded antibodies were synthesized and characterized. Specifically, we assessed breadth and potency of influenza A virus
neutralization, determined representative crystal structures in
complex with HA, analyzed sequence convergence based on
V(D)J-gene recombination and somatic hypermutation (SHM),
and tested sequence signatures for their ability to identify group 1
and group 2 neutralizing antibodies. Our findings reveal reproducible immunological pathways to achieve broadly reactive
antibodies and support a B cell ontogeny-based approach to obtaining a universal influenza A vaccine.
RESULTS
Identification of Memory B Cells Cross-Reactive with
Group 1 and Group 2 Influenza A HAs
We studied ten subjects from the VRC 310 H5N1 vaccine trial
who displayed a range of vaccine-elicited serum H5N1 neutralization activity, as well as varied but detectable responses against
group 2 strains A/Hong Kong/1-4-MA21-1/1968 (H3N2) or
A/Netherlands/219/2003 (H7N7) (Figures 1B and S1; Tables S1B
and S2). We used recombinant group 1-specific (H5) and group
2-specific (H3) HA probesmodified to prevent sialic acid binding

(HADSA) (Wheatley et al., 2015; Whittle et al., 2014)to co-stain


and sort peripheral blood mononuclear cells (PBMCs) isolated
2 weeks post H5N1 MIV boost (Figures 1C and S2). We recovered
sequences of memory B cell immunoglobulin gene transcripts
from six of the ten studied subjects (Figure 1D).
The sequence repertoire of each subject was generally dominated by clonally related transcripts comprising a small number
of clonal expansions. Transcripts derived from diverse HV genes
including HV1-18, HV3-23, HV3-64D, HV4-30, HV4-34, and
HV6-1 (Figures 1D and 1E). Notably, transcripts from HV1-69,
which often predominate group 1-specific stem-reactive antibodies (Lingwood et al., 2012; Pappas et al., 2014; Wheatley
et al., 2015), comprised only 2.5% of this set of group 1 (H5+)group 2 (H3+) double-positive memory B cells.
Group 1 and Group 2 Neutralizing Antibodies from
Different Vaccinees Are Genetically Similar
Immunoglobulin sequences recovered from H5+ and H3+ crossreactive memory B cells (Table S3) showed surprising similarity.
Notably, many immunoglobulin sequences from different donors
derived from the same genetic elements (Figure 1E). To analyze
commonalities of immunoglobulin transcripts between subjects,
we considered the following seven genetic elements: inferred
heavy variable (HV), heavy diversity (HD), heavy joining (HJ)
genes, third-heavy chain complementarity-determining region
(CDR H3) length for heavy chain-gene transcript, inferred light
variable (LV) and light joining (LJ) genes, and CDR L3 length for
corresponding light chain transcripts (Figure 1E). Frequentist
analysis indicated the presence of four or more of the same genetic elements in separate lineages to be statistically significant
(p % 0.001) (Figure 1E), and representative antibodies were
cloned and expressed from such lineages. Of note, our antibody
nomenclature specifies donor, lineage, and clone; e.g., antibody
56.a.09 (Figure 1E, third row) is named for subject (56), lineage
within this subject (a), and clone within this lineage (09). Most
of the expressed antibodies bound HA, and all antibodies that
bound HA competed with the antigen-binding fragment (Fab)
of the stem-directed antibodies CR9114 (Dreyfus et al., 2012)
or F10 (Sui et al., 2009) (Figure 1E), and negative stain-electron
microscopy (EM) indicated binding to the HA stem (Figure S3).
Most of these antibodies neutralized viruses from both group 1
and group 2, including subtypes H1, H3, H5, and H7, with select
antibodies also demonstrating neutralization of viruses from
subtypes H2, H9, and H10 (Figures 1E and S4; Table S4).
Notably, in both pseudovirus and microneutralization assays
of influenza A viruses, the breadth and potency for several of
the newly identified antibodies were comparable to those of
antibody CR9114 (Dreyfus et al., 2012).
To understand the immunological basis of these multidonor
humoral responses against influenza A, we analyzed the recombination, SHM, and structural constraints, which drove the generation and development of these antibodies.

(D) Clonal diversity of H5-H3 cross-reactive B cells. The HV repertoire from each subject is shown as a pie chart; with each slice representing a unique HV clone or
clonally related family. Total number of HV sequences recovered per subject is indicated by the number at the center of each pie chart.
(E) Genetic and functional characteristics of selected antibodies recovered from H5-H3 cross-reactive B cells. Structurally characterized antibodies
indicated by .
See also Figures S1, S2, S3, and S4 and Tables S1, S2, S3, and S4.

Cell 166, 609623, July 28, 2016 611

A Multidonor Class of Broadly Neutralizing Antibodies


with HV6-1+HD3-3 Germline Genes
Three memory B cell lineages, from subjects 31, 54, and 56,
shared heavy chain sequences derived from recombination of
HV6-1, HD3-3, and HJ4 or HJ5 to yield highly similar amino
acid sequences in the CDR H3 (Figure 1E, top row; Table S3).
In each case, the heavy chain was paired with a light chain
sequence from KV3-20, KJ2, or KJ3 resulting in a CDR L3 of
nine amino acids. Similar affinity maturation patterns were
observed: a Val100bIleHC alteration of an HD-gene-encoded
section of the CDR H3 was completely conserved in all three lineages (Figure 2A) (for clarity, each residue number is followed by
a subscript denoting parent molecule: HC, heavy chain; LC, light
chain; HA1, HA2, or HA, either HA subunit or HA in general Kabat
numbering is used for antibodies, thus, residue 100b refers to the
second insertion b after residue 100 [Kabat et al., 1991]).
Notably, these antibodies displayed neutralization breadth and
potency that rivaled that of CR9114 and exceeded that of the
group-specific CR6261 or CR8020 or of the head-directed antibody CH65 (Ekiert et al., 2009, 2011; Whittle et al., 2011) (Figures
2B and S4; Table S4).
To provide insight into the structural basis for the similarity
between these HV6-1+HD3-3 antibodies, we determined the
crystal structure of the antigen-binding fragment (Fab) for antibody, 56.a.09, alone, and in complex with A/Hong Kong/1-4MA21-1/1968 (H3N2) HA at 3.3 A resolution (Figures 2C and
S5; Tables S5 and S6). Unexpectedly, the crystallized HA was
not trimeric, with the asymmetric unit for the crystallized FabH3 complex comprising an HA head of one protomer interacting
with the HA stem of an adjacent protomer in a head-to-stem
dimeric arrangement (Figures S5B and S5C). Despite this nontrimeric arrangement, the Ca-root-mean-square deviation
(RMSD) between the 56.a.09-bound HA and the ligand-free HA
was <1 A, and, for clarity, we thus depict the 56.a.09 bound complex as a typical HA trimer (Figure 2C).
Antibody 56.a.09 recognized a conserved region on the HA
stem in a manner that avoided glycans at residues Asn21HA1
(conserved on group 1 viruses) and Asn38HA1 (conserved on
group 2 viruses), providing a structural explanation for its
extraordinary group 1 and group 2 neutralization breadth. Antibody 56.a.09 bound primarily with its heavy chain (934 A2 buried
surface area [BSA] versus 386 A2 BSA for the light chain). Heavy
chain binding involved the HD3-3-encoded CDR H3 (Figure 2C)
with Phe100HC and Gly100aHC contributing 240 A2 of BSA
and the SHM-altered Val100bIleHC inserting directly into the
Trp21HA2 pocket (contributing over 100 A2 of interactive surface).
In addition, Met98HC interacted with a conserved aromatic residue present on all light chains, helping to orient the CDR H3
(Figure 2D). Heavy chain binding also involved the HV6-1 germline-encoded CDR H2, which uniquely encodes a nine-amino
acid CDR H2, contributed 182 A2 of BSA, was unmutated in contact residues in all three subjects (Figure S6), and interacted with
the conserved fusion peptide (Figure 2E). With respect to light
chain, the largely unmutated KV3-20-derived V genes (4%6%
SHM) observed in lineages from these three subjects interacted
with the HA stem through both CDR L1 and CDR L3 (Figure 2F;
Table S6), with Tyr33LC contributing the largest BSA among all
light chain residues (73.1 A2).
612 Cell 166, 609623, July 28, 2016

HV6-1+HD3-3-derived antibodies were thus found in three independent donors, shared genetic elements in both the heavy
and light chain, displayed convergent affinity maturation, and
appeared to share the same mode of recognition (structurefunction analysis of recognition interface and SHM are provided
in Figure S7). Furthermore, we tested for functional complementation: swapping of heavy and light chains of the three HV61+HD3-3-derived antibodies resulted in six functional antibodies
from nine possible pairings (all three pairings with the heavy
chain of antibody 31.g.01 failed to express) (Table S7). Overall,
these results indicated the HV6-1+HD3-3-derived antibodies
form a multidonor class. Structural analysis indicated numerous
light chains to be compatible with binding, and 99% of the human population (Abecasis et al., 2012) possess alleles of the
HV6-1 and HD3-3 genes compatible with the class elicitation
and recognition described here (Figures 2D2G).
A Second Multidonor Class of Broadly Neutralizing
Antibodies with HV1-18+HD3-9 Germline Genes
Two memory B cell lineages from subjects 1 and 31 shared
immunoglobulin heavy chain sequence derived from recombination of HV1-18 with HD3-9 and HJ4 to yield highly similar amino
acid sequences in a CDR H3 of 15 amino acids (Figures 1E and
S6). Notably an Arg96HC residue was encoded by N-nucleotide
addition in both cases (Figure 3A). In each donor, the heavy chain
was paired with a light chain derived from KV2-30. Encoded immunoglobulins were expressed and shown to neutralize primarily group 1 strains of influenza A, although a few group 2 strains
were neutralized (Figure S4; Table S4). Overall neutralization
from these HV1-18+HD3-9 antibodies appeared more similar
to the group 1-specific antibody CR6261 than to the very broad
CR9114 or HV6-1-derived antibodies; nevertheless, neutralization breadth encompassed 50% of influenza A subtypes that
commonly infect humans (Figure 3B).
We determined the crystal structure of Fab 31.b.09 in complex
with A/California/04/2009 (pH1N1) HA (Figure 3C; Tables S5 and
S6). Similar to the 56.a.09-H3 complex structure, the crystallized
hemagglutinin in the Fab 31.b.09 complex was not a trimer, but a
molecular dimer (Figure S5). Despite this unexpected nontrimeric arrangement, the Ca-RMSD between the 31.b.09bound HA and the ligand-free HA in the stem region was 0.6 A,
and for clarity, we depict the 31.b.09 bound complex in a more
typical trimeric arrangement (Figure 3C). The HV1-18+HD3-9derived antibody 31.b.09 bound an epitope that overlapped
the HV6-1+HD3-3 class epitope, but with antibody rotated
105 (mostly involving a rotation perpendicular to the trimer
axis) (Figure 3C). Antibody 31.b.09 bound with both heavy and
light chains (343 A2 BSA for heavy chain and 540 A2 BSA for
the light chain). Heavy chain interactions were generated
through CDR H2 and H3 loops. In the CDR H2 (127 A2 BSA),
the HV1-18 germline-encoded Tyr53HC recognized the fusion
peptide of HA2 and Asn56HC recognized helix A of HA2, while
the CDR H3 (216 A2 BSA) was positioned over the fusion peptide-helix A interface with Ile99HC and Leu100HC inserting into
the hydrophobic groove between these two conserved elements
(Figures 3C3E). Light chain interactions involved CDR L1 and
L3, which recognized helix A (Figure 3F). We tested functional
complementation: swapping of heavy and light chains between

A
HV6-1+HD3-3

92

94

96

98

100

100b

100d

100f

102

56.a.09
footprint

Phe
100

C
CDR H3
A/Hong Kong/1-4MA21-1/1968 H3 HA0

Helix A

Met 98
Trp 21HA2

Ile 100b
56.a.09
light chain

CDR L1

Fusion
peptide
CDR H2

CDR L3

90

Thr 57

N-terminusLC

56.a.09
heavy chain

Asp 92LC

H3 HA

E
Phe
100

Tyr
50LC
Ser
53

CDR H3
Met
98

Trp 21HA2
Ile
100b

Trp
21HA2

Arg
52b

CDR L1
Tyr
33

Fusion
peptide

Helix A

Arg
50
CDR L3

CDR H2
Lys
55

Tyr
56

Asp 92 LC

Thr
57

Gln 95LC

Ile
100d

Longer HD motif: 100 x X


Compatible HD genes:
HD3-3 with compatible alleles
HD3-3*01 and HD3-3*02
Shorter HD motif: x
Compatible HD genes:
HD1-1, HD1-14, HD1-20, HD2-2,
HD2-8, HD2-15, HD3-3, HD3-9,
HD3-10, and HD6-13

HV motif: 9 residue CDR H2


Compatible HV genes:
HV6-1 with
compatible alleles
HV6-1*01 and HV6-1*02

LV motif: Y at residue 33
Compatible LV genes:
30 genes compatible
Key:
= F,Y or W
x = G, A or S
= I, V, L, or M
X = any residue

Figure 2. A Multidonor HV6-1+HD3-3 Class of Broadly Neutralizing Antibodies


(A) Immunoglobulin heavy chains utilizing germline genes HV6-1, HD3-3, and HJ4 or HJ5. Germline HV, HD, and HJ gene-encoded nucleotide and amino acid
residues are shown in black, with junction-encoded residues in light blue and residues that have undergone SHM in red. Nucleotides removed by exonuclease
trimming indicated with a line through the letters. Conserved HD3-3-encoded residues (IFG) highlighted by a black box; recurrent SHM-derived Ile100bHC
highlighted by a red box. HA contacts indicated with open circles (B) denoting antibody main-chain-only contacts, open circles with rays () denoting antibody
side chain-only contacts, and filled circles (C) denoting both main-chain and side-chain contacts.
(B) Neutralization breadth-potency curve for HV6-1+HD3-3 antibodies, with breadth shown as percentage of pseudoviruses neutralized at each IC50 cutoff, and
virus panel comprising 15 strains that includes influenza A subtypes known to infect humans (H1, H2, H3, H5, H7, H9).
(C) Co-crystal structure of Fab 56.a.09 in complex with an H3 HA monomer (A/Hong Kong/1-4-MA21-1/1968). Fab heavy and light chains colored purple and
cyan, respectively, and depicted in surface representation, while the H3 HA is depicted in ribbon and shown as a trimer (Figure S5 shows HA crystal packing).
Inset: interacting CDR loops of the 56.a.09 Fab are shown in ribbon and sticks and colored as in (A) with the antibody footprint outlined. Note that labels for heavychain residues do not explicitly show HC.
(D) A five-amino acid motif within the CDR H3 inserts into the conserved Trp21 pocket of H3.
(E) HV6-1encoded CDR H2 depicted with HA fusion peptide; HV6-1 is the only HV gene that encodes a nine-residue CDR H2.
(F) Light chain interactions that contribute to the antibody-binding surface; these are not specific to KV3-20.
(G) Analysis of antibody gene compatibility, highlighting additional CDR H3 residues that may be compatible with HA binding, e.g., 100HC could be a F, Y, or W
residue (U), 101HC could be a G, A, or S residue (x).
See also Figures S3, S4, S5, S6, and S7 and Tables S3, S4, S5, S6, and S7.

Cell 166, 609623, July 28, 2016 613

HV1-18+HD3-9

92

94

96

98

100

31.b.09
footprint

A/California/04/2009
H1 HA0

CDR L1

31.b.09
light chain

CDR L3

Ile 27e

Trp 21HA2

90

Leu 100
31.b.09
CDR H3

Helix A

His 98

31.b.09
heavy chain

Fusion
peptide
CDR H2

Helix A
Trp 21HA2

Trp 21HA2

CDR H3

Helix A
Fusion
peptide

Leu
100

CDR L1
Ile 27e

CDR L3

CDR H2

Fusion
peptide

His 93
Tyr
53

His 98

Asn
56

Trp 21HA2
Trp 94
Helix A

HD Motif: Leu 100


Compatible HD genes:
All 34 D genes compatible

CDR H2 motif: Trp at residue 50


Tyr at residue 53, Asn at 56
Compatible HV genes:
HV1-18
HV1-68
HV7-34-1
HV7-81

LV motif: CDR L1=16 aa


Compatible LV genes:
KV2-4
KV2D-18
KV2-18
KV2D-24
KV2-24
KV2D-26
KV2-28
KV2D-28
KV2-29
KV2D-29
KV2-30
KV2D-30

Figure 3. A Multidonor HV1-18+HD3-9 Class of Broadly Neutralizing Antibodies


(A) Immunoglobulin heavy chains utilizing germline genes HV1-18, HD3-9, and HJ4, with sequences annotated as described in Figure 2A.
(B) Neutralization breadth-potency curve for HV1-18+HD3-9 antibodies on a panel of influenza A viruses that includes subtypes known to infect humans.
(C) Co-crystal structure of Fab 31.b.09 in complex with an H1 trimer (A/California/04/2009). Fab heavy and light chains colored dark green and light green, respectively,
and depicted in surface representation, while the H1 HA is depicted in ribbon and colored blue, green, and white. Inset: interacting CDR loops of the 31.b.09 Fab are
shown in ribbon and sticks and colored as in (A) with the antibody footprint outlined. Note that labels for heavy-chain residues do not explicitly show HC.
(D) A conserved motif within the CDR H3 inserts into the highly conserved Trp21 pocket of HA while also interacting with the fusion peptide.
(E) The HV1-18-encoded CDR H2 also interacts with the opposing side of the fusion peptide.
(F) Light chain interactions from CDR L3 and CDR L1 also contribute to the antibody binding surface area.
(G) Analysis of antibody gene compatibility.
See also Figures S3, S4, S5, S6, and S7 and Tables S3, S4, S5, S6, and S7.

two antibodies of the putative class resulted in functional antibodies for all four of the possible pairings (Table S7). Overall,
the results indicated the HV1-18+HD3-9-derived antibodies
614 Cell 166, 609623, July 28, 2016

form a multidonor class. The light chain had a 16-amino acid


CDR L1, which could be encoded by 12 other LV genes, and residue Ile27eLC, which forms a critical contact, was a product of

SHM. The multiple alternative D gene alleles that could be used


to generate CDR H3s compatible with recognition by this multidonor class, in combination with a large number of compatible
light chains, led to a calculated distribution of potential HV1-18
combinations in the human population of close to 100% (Abecasis et al., 2012) (Figure 3G).
A Third Multidonor Class of Broadly Neutralizing
Antibodies with HV1-18 Germline Gene and Q-x-x-V
Motif
Multiple B cell lineages in two subjects (16 and 54) produced
distinctive HV1-18-derived immunoglobulins sharing five genetic
elements and having a CDR H3 of 21 amino acids derived from
recombination with either HD2-2 or HD2-15 genes (Figure 4A;
Table S3). This set of immunoglobulins all shared an SHMderived Thr54HC, a Gln98HC encoded by P-nucleotide addition,
and a germline HD-encoded aliphatic residue at position100aHC
(Q-x-x-V motif). Neutralization breadth and potency for this set of
immunoglobulins were similar to that of antibody CR9114 (Figure S4; Table S4), neutralizing all common human-infecting subtypes of influenza A except H2 and exceeding substantially the
breadth of group 1-specific or group 2-specific stem antibodies
or the head-directed antibody CH65 (Figure 4B). To understand
the basis of their recognition, we crystallized representative
antibodies with HA. Co-crystal structures of representative
HV1-18+HD2-2 (16.a.26) and HV1-18+HD2-15 (16.g.07) Fabs
in complex with the A/Hong Kong/1-4-MA21-1/1968 (H3N2)
HA revealed highly similar epitopes (Figures 4C4H; Tables S5
and S6). Antibody binding occurred primarily through heavy
chain recognition of the conserved HA stem. With 16.a.26, the
heavy chain contributed 514 A2 of BSA while the light chain
contributed 283 A2 of BSA, and with 16.g.07, the heavy chain
contributed 646 A2 of BSA while the light chain contributed
329 A2 of BSA.
Heavy chain interactions were generated primarily through
CDR H2 (150 A2 BSA for 16.a.26 and 200 A2 BSA for
16.g.07) and CDR H3 (300 A2 BSA for 16.a.26 and 450 A2
BSA for 16.g.07) with the HV1-18 germline-encoded Tyr53HC
and the SHM-derived Thr54HC recognizing the N-terminal region
of HA1 and the hydrophobic groove between helix A and the
fusion peptide of HA2 (Figures 4D and 4G; Table S6). The CDR
H3s of these antibodies also bound the conserved hydrophobic
groove next to helix A and adjacent to Trp21HA2. The conserved
Val100aHC inserted into a pocket present on both group 1 and
group 2 HAs, just above Trp21HA2 and proximal to Ile48HA2.
Despite differences between residues encoded by HD2-2
(16.a.26) and HD2-15 (16.g.07), the CDR H3s from both antibodies were oriented perpendicular to the Fab axis and interacted similarly with HA. The conserved Gln98HC interacted
with Gln42HA2 by occupying a germline-encoded pocket unique
to HV1-18-encoded antibodies, which was formed by framework
residues Gly33HC and Ser52HC (Figures 4E and 4H). The location
of Gln98HC within the framework pocket likely stabilized the
perpendicular orientation of the CDR H3 relative to the antibody-framework regions and may allow for CDR H3-motifs
derived from diverse D genes to bind to this conserved hydrophobic groove. In this regard, we note that sequences from subject 01 with an HV1-18 germline gene and a Gln98-x-x-Val100a

motif in a 17-amino acid CDR H3 were able to neutralize H3


and H5 strains of influenza. Although light chains of this class
contribute approximately one-third of the total buried surface
area, analysis of antibodies of this class revealed light chain sequences to derive from diverse LV genes, with only KV3-11 appearing twice. The recently reported HV1-18-derived group 1
and group 2 neutralizing antibody CT149 uses a Gln98HC-x-xVal100aHC motif with a 19-amino acid CDR H3 to bind the HA
stem (Wu et al., 2015) and recognized HA in a manner highly
similar to both 16.a.26 and 16.g.07. We tested the functional
complementation for antibodies 16.a.26, 16.g.07, 54.a.39,
54.a.84, and CT149 from donors 16, 54, and SH-K1 (the source
of antibody CT149). Swapping of heavy and light chains between
these five antibodies resulted in ten functional antibodies from
the 25 possible pairings (Table S7). Functionality correlated
strongly with HV gene identity and CDR H3 length (Table S7),
suggesting a requirement for specific heavy-light chain pairings.
Despite this requirement, the HV1-18 antibodies with Thr54HC
and CDR H3 98Q-x-x-V motif appeared to form a multidonor
class. Thus, the recombination of HV1-18 with many alternative
D gene segments yields a Q-x-x-V motif in the CDR H3, which
may comprise a common solution for neutralization of group 1
and group 2 influenza A viruses. The many alternative HD gene
alleles that can be used to generate CDR H3s for this multidonor
class in combination with no obvious light chain bias led to a
calculated distribution of potential HV1-18 combinations in the
human population of close to 100% (Abecasis et al., 2012) (Figures 4I and 4J).
Multidonor and Lineage-Unique Antibodies that
Neutralize Group 1 and Group 2 Viruses Recognize
Similar Epitopes
For the six subjects analyzed, the most highly expanded lineage
was lineage a from subject 31, from which we sequenced 104
clones (Figure 1E; Table S2). Clone 83 of this lineage (antibody
31.a.83) derived from HV3-23, HD3-9, and HJ6 and displayed
the highest neutralization breadth of the antibodies we
sequenced and expressed, neutralizing all of the common influenza A subtypes that infect humans (Figure 5A). Structural characterization of antibody 31.a.83 in complex with A/Hong Kong/14-MA21-1/1968 (H3N2) HA (Table S5) revealed binding to the
conserved HA stem, primarily through CDR H3 residues, many
of which were somatically hypermutated (Figures 5B5D; Table
S6). Antibody 31.a.83 utilized hydrophobic residues to contact
an epitope adjacent to helix A and involving the Trp21HA2 pocket
with its CDR H3 parallel to helix A in a manner that avoided the
conserved N-glycan at Asn38HA1 present in group 2 HAs.
A second lineage, h from subject 56 (lineage 56.h), also
derived from the same germline genes as antibody 31.a.83 (Figures 5B and S6). However, critical CDR H3 contact residues of
antibody 31.a.83 were not conserved and a four-amino acid shift
relative to the V-gene was observed (Figure 5B). Moreover, the
representative antibody we expressed and analyzed from this
second lineage, antibody 56.h.01, neutralized H1, H2, and H9
subtypes, but not H3, H5, or H7 subtypes (Figures 1E and S4;
Table S4). Together, these observations indicated lineages
31.a and 56.h to have different modes of recognition, providing
an example of influenza A-targeting antibodies that derived
Cell 166, 609623, July 28, 2016 615

HV1-18 Q-x-x-V

92

94

96

98

99

100a

100c 100d

100f

100h

100j

101

103

92

94

96

98

99

100a

100c 100d

100f

100h

100j

101

103

A/Hong Kong/1-4MA21-1/1968 H3 HA0

16.a.26
heavy chain

16.a.26
footprint

Leu 100c

16.a.26
light chain

CDR H3

NAG 38HA1

Gly 33

Val 100a
90

170

Trp 21HA2

Gln 98
Ser 52

CDR H3

Gln 98

Tyr 53

Thr 54

Gln 42HA2

16.a.26
heavy chain

Helix A
Trp 21HA2

A/Hong Kong/1-4MA21-1/1968 H3 HA0

16.g.07
footprint

16.g.07
light chain

16.g.07
heavy chain

Pro
100c

CDR H3

Val 100a

Gly 33

NAG 38HA1
90

Gln 98
170

Trp 21HA2
CDR H3

Gln 98

Tyr 53

Thr 54

Ser 52

Gln 42 HA2

16.g.07
heavy chain

Helix A
Trp 21HA2

HD Motif: Val 100a


Compatible HD genes:
All 34 D genes compatible

HV Contact Residue Motif : Tyr 53


Compatible HV genes:
HV1-18, HV1-45, HV1-68,
HV1-NL1, HV7-34-1, and HV7-81
HV Structural Motif:
Gly33 and Ser52/Thr52
Compatible HV genes:
HV1-18 with compatible alleles
HV1-18*01, HV1-18*02,
HV1-18*03, and HV1-18*04

Figure 4. A Multidonor HV1-18 Class of Broadly Neutralizing Antibodies with Q-x-x-V Motif
(A) Immunoglobulin heavy chains utilizing germline genes HV1-18, HD2-2 or HD2-15, and HJ5 or HJ2, with sequences annotated as described in Figure 2A.
(B) Neutralization breadth-potency curve for HV1-18 (Q-x-x-V) class antibodies on a panel of influenza A viruses that includes subtypes known to infect
humans.
(C) Co-crystal structure of Fab 16.a.26 (HV1-18, HD2-2, HJ5) in complex with H3-HK68. Fab heavy and light chains colored orange and lavender respectively and
depicted in surface representation while HA is depicted in ribbon and shown as a trimer. Note that labels for heavy chain residues do not explicitly show HC.
(D) The 16.a.26 CDR H3 is depicted with junction-encoded and mutated residues colored as in (A) and germline-encoded residues in orange with the antibody
footprint on the HA outlined in black.
(E) Antibody heavy chain depicted in surface representation with CDR H3 loop in ribbon. 16.a.26 Gln98HC occupies a turn in the CDR H3, which interacts with the
conserved residue Gln42HA2.
(F) Co-crystal structure of Fab 16.g.07 (HV1-18, HD2-15, and HJ2) in complex with A/Hong Kong/1-4-MA21-1/1968 (H3N2) HA depicted as in (C).
(G) The 16.g.07 CDR H3 depicted as in (D) with the antibody footprint on the HA outlined in black.

(legend continued on next page)

616 Cell 166, 609623, July 28, 2016

92

94

96

98

A/Hong Kong/1/1968
H3 HA0

100c

100

100f

100h

31.a.83
footprint

31.a.83
footprint

31.a.83
light chain

Arg 31LC

31.a.83
light chain Ile 100

Tyr 91LC

Ile 100
70

Leu 100c

Leu 100c
Trp 21HA2
CDR H2

Met 100d

31.a.83
heavy chain

Lineage-unique group 1-2


neutralizing antibody epitopes

FI6v3
HV3-30
15% HV SHM
22aa CDR H3

39.29
HV3-30
10% HV SHM
18aa CDR H3

(Corti et al., 2011)

(Nakamura et al., 2013)

31.a.83
HV3-23
8% HV SHM
24aa CDR H3

Fusion
peptide

CDR H2

Multidonor group 1-2


neutralizing antibody epitopes

56.a.09
HV6-1
4% HV SHM
16aa CDR H3

31.b.09
HV1-18
4% HV SHM
15aa CDR H3

16.a.26
HV1-18
8% HV SHM
21aa CDR H3

16.g.07
HV1-18
11% HV SHM
21aa CDR H3

CT149
HV1-18
12% HV SHM
19aa CDR H3

CR9114
HV1-69
14% HV SHM
14aa CDR H3

(Wu et al., 2015)

(Dreyfus et al., 2012)

Figure 5. A Conserved Site of Group 1 and Group 2 Influenza A Virus Vulnerability Targeted by Multidonor and Lineage-Unique Antibodies
(A) Neutralization breadth-potency curve for HV3-23-derived antibodies on a panel of influenza A viruses that includes subtypes known to infect humans.
(B) Immunoglobulin heavy chains utilizing germline genes HV3-23, HD3-9, and HJ6, with sequences annotated as described in Figure 2A.
(C) Co-crystal structure of Fab 31.a.83 in complex with A/Hong Kong/1-4-MA21-1/1968 (H3N2) HA.
(D) Epitope for antibody 31.a.83 (outlined in black) with heavy chain depicted in yellow and junction-encoded residues (highlighted in blue), mutated residues (in
red), and germline-encoded residues (in gold) with light chain depicted in tan, HA protomer1 in gray, and HA protomer2 in dark gray. Note that labels for heavychain residues do not explicitly show HC.
(E and F) Antibodies capable of neutralizing group 1 and group 2 influenza A viruses recognize overlapping epitopes within the HA stem (colored red). The HA
surface is colored blue for structures determined previously (FI6v3 PDB: 3ZTN; 39.29 PDB: 4KVN; CT149 PDB: 4R8W; CR9114 PDB: 4FQI), and gray for
structures determined in the current study.
See also Figures S3, S4, S5, and S6 and Tables S3, S4, S5, S6, and S7.

from the same heavy chain-VDJ genes, but did not use the same
mode of recognition nor share convergent development.
Thus, antibody lineages may be multidonor (common or public), meaning that they are observed in different individuals and
share the same genetic elements and mode of recognition (Henry Dunand and Wilson, 2015; Zhou et al., 2013), or unique (uncommon or private), meaning that they have only been observed
a single time. We observed no substantial difference between

epitopes of multidonor and unique antibodies capable of neutralizing group 1 and group 2 influenza A viruses (Figures 5E and 5F),
nor did we observe segregation in antibody approach to HA used
by multidonor or unique antibodies (Figures S3E and S3F). Antibodies from multidonor lineages did, however, have lower SHM
(averaging 8% for multidonor versus 11% for the unique). The
lower SHM suggested that multidonor antibodies may undergo
more parsimonious development. We note that unique lineages

(H) The 16.g.07 CDR H3 is depicted as in (E) with Gln98HC contacting Gln42HA2.
(I) Analysis of D-gene compatibility.
(J) Analysis of V-gene compatibility.
See also Figures S3, S4, S5, S6, and S7 and Tables S3, S4, S5, S6, and S7.

Cell 166, 609623, July 28, 2016 617

Sequence
signature*

Class

PostVRC310
HeavyLight
(515,594#)

DeKosky et al.
Heavy-Light
partial
sequences
(3,019,679)

Pre-TIV
Jiang et al.
Heavy-only
(759,337)

Post-TIV
Jiang et al.
Heavy-only
(3,045,513)

Healthy
normal
donors
Heavy-only
(1,239,173)

HV6-1
+HD3-3

VH6-1 + D3-3
98MIFGI
CDR H3 = 16

21
(0.004%)
3

0
(0%)
0

0
(0%)
0

13
(0.0004%)
1

0
(0%)
0

HV1-18
+HD3-9

VH1-18
96RxxILTG
CDR H3 = 15

16
(0.003%)
3

17
(0.0006%)
1

0
(0%)
0

123
(0.004%)
3 (2)

64
(0.0052%)
2 (1)

VH1-18
53Y54T 98QxxV
CDR H3 = 17-21

309
(0.06%)
14

0
(0%)
0

0
(0%)
0 (1)

242
(0.008%)
3

2
(0.00016%)
1

*Class signature used for transcript identification


#515,594 B cells were sorted of which 645
yielded sequences used in frequency calculations

HD gene family

Subject number
16

> 30 sequences
2

5 67

HV gene family
HV6-1+HD3-3
HV1-18+HD3-9
HV1-18 (Q-x-x-V)

31

10-30 sequences

HV6-1+HD3-3.1.SRP015957H+56.a.09 L
+F10 competition
HV6-1+HD3-3.2.SRP015957H+54.f.01 L
+F10 competition
HV6-1+HD3-3.2.SRP015957H+56.a.09 L
+F10 competition

54

< 10 sequences

HV1-18+HD3-9

HV1-18 Q-x-x-V

HV1-18+HD3-9.1.SRP047462H+31.b.09 L
+F10 competition
HV1-18+HD3-9.1.SRP047462
+F10 competition

HV1-18+QxxV.1. SRP047462
HV1-18+QxxV.2. SRP047462
HV1-18+QxxV.3. SRP047462

No. of multidonor class sequences


(Sequence frequency)
No. of unique lineages

HV-HD repertoire of cross-reactive antibodies by subject

6
5
4
3
2
1

HA-stem binding of sequence signature-identified antibodies


HV6-1+HD3-3

56

1
36

Clustering of VRC 310 and sequence signature-identified antibodies


based on neutralization data

HV6-1+HD3-3
HV1-18+HD3-9
HV1-18 (Q-x-x-V)

Neutralization (recipricol IC50 titer (g/ml))

HV1-18
(Q-x-x-V)

Prevalence of multidonor antibodies against influenza A

Neutralization of sequence signature-identified antibodies


100

HV6-1+
HD3-3

HV1-18+
HD3-9

Control antibodies

Limit of detection (>50 g/ml)


Median IC50

10

0.1

0.01

0.001

0.0001
0.296 0.274 50.0 50.0

0.050 0.015 0.55

50.0 50.0

H
V6
-1
+H
H
D
V6
3-1
3.
+H
2.
H
SR
D
V1
3P0
-1
15
8+ 3.1
.S
95
H
R
D
7
P0
39.
15 H _5
1.
SR 957 4.f.
01
H
P0
H_
V1
L
56
47
-1
.a
46
8+
.0
2
H
9L
D
3- H -31
9.
.b
1.
SR .09
L
P0
47
46
2
C
R9
11
4
FI
6V
3
C
R6
26
1
C
R8
02
0
C
H6
5

Group 1 Strains
H1N1-A/South Carolina/1/1918
H1N1-A/Puerto Rico/8/1934
H1N1-A/New Caledonia/20/1999
H1N1-A/Califonia/04/2009
H2N2-A/Singapore/1/1957
H2N2-A/Canada/720/2005
H5N1-A/Vietnam/1203/2004
H5N1-A/Indonesia/05/2005
H9N2-A/Hong Kong/1073/1999
Group 2 Strains
H3N2-A/Hong Kong/1/1968
H3N2-A/Beijing/353/1989
H3N2-A/Perth/16/2009
H7N7-A/Netherlands/219/2003
H7N9-A/AnHui/1/2013
Influenza B
INF B-Brisbane/60/2008

0.2

Figure 6. Sequence Signatures of Multidonor Antibody Lineages


(A) Multidonor class antibodies capable of neutralizing group 1 and group 2 influenza A viruses: sequence signature, number, and frequency of signatureidentified class members. The number of singleton transcripts from 454-derived NGS data is reported in italicized font; these were omitted from transcript and
lineage quantifications as described in the Supplemental Experimental Procedures. Additional sequences MEDI8852 (Kallewaard et al., 2016) and SFV005-2G02
(Li et al., 2012) have been identified, which correspond to the HV6-1+HD3-3 and HV1-18+HD3-9 classes, respectively.
(B) HV-HD germline-gene origin plots. One box shown per subject with antibody frequencies plotted as circles at their HV (horizontal), HD (vertical) coordinates.
The size of the plotted circle corresponds to the number of antibody sequences as shown in key at left. Multidonor antibodies in different subjects connected by
lines colored according to each multidonor class.
(C) Binding and competition MSD-ECLIA for antibodies identified in (DeKosky et al., 2015; Jiang et al., 2013) databases; competition with stem antibody F10
highlighted in red.
(D) Neutralization of influenza pseudoviruses. Median IC50 for each antibody is indicated by a horizontal line with value (mg/ml) shown at the base of the graph.
(E) Clustering of antibodies based on their neutralization fingerprints on a 15-virus panel.
See also Figure S6 and Tables S3, S4, and S7.

accounted for the majority of cross-reactive B cells in four of the


six analyzed VRC 310 donors (1, 31, 36, and 56); in light of the
positive functional characteristics of several of the unique lineages (e.g., 31.a.83), it seems likely that these antibodies would
contribute to a protective response. As the number of donors
with sequenced cross-reactive memory B cells increases, we
618 Cell 166, 609623, July 28, 2016

would expect some of the antibodies described here as


unique to be observed in other donors.
Sequence Signatures for Multidonor Antibody Classes
We analyzed the multidonor antibodies identified here for
class-specific sequence signatures (Figure 6A) as a means to

quantify class transcripts and to identify potential class members in other subjects. Notably, despite a sequence signature
requiring residue 54HC to be altered by affinity maturation, the
HV1-18 (Q-x-x-V) class accounted for over half of the H5+ and
H3+ cross-reactive B cells we sequenced (Figure 6A) and could
be found in half the analyzed vaccinees (Figure 6B).
For other subjects, we searched published human antibody
datasets, both those with paired heavy-light sequences
(DeKosky et al., 2015), as well as those with heavy chain-only sequences (Figure 6A; Table S1C). Searches with the HV61+HD3-3 signature did not yield sequence matches in paired
heavy-light sequences, but in the published heavy chain-only
datasets, we found 13 matches, which appeared to derive
from a single lineage (Figure S6). We chose both consensus as
well as the sequence closest to consensus to synthesize and
reconstitute with light chains of HV6-1+HD3-3 class antibodies
from subjects 54 and 56. One of these reconstituted antibodies
did not express, but the other three did and bound H1 HA in a
manner that could be competed with antibody F10 (Figure 6C).
We tested two of these antibodies and both neutralized group
1 and group 2 influenza A strains (Figure 6D; Table S3), and
the neutralization signatures (Georgiev et al., 2013) of the synthesized antibodies clustered in a dendrogram with known HV61+HD3-3 class antibodies (Figure 6E).
With the HV1-18+HD3-9 signature, we found 17 matches in
published paired heavy-light chain sequences, which appeared
to derive from a single lineage (Figure 6A). We synthesized
consensus sequences and reconstituted published heavy and
light chains as well as the published heavy chain and light chain
of this class from subject 31. Both of these reconstituted antibodies bound H1 hemagglutinin in a manner that could be
competed with antibody F10 (Figure 6C), neutralized group 1
influenza A strains (Figure 6D; Table S4), and clustered in a
neutralization dendrogram with known HV1-18+HD3-9 class
antibodies (Figure 6E). The neutralization breadth of these antibodies was lower than those isolated from VRC 310 subjects,
likely due to the use of germline sequences to complete the
CDR L1 and CDR L2 regions of this antibody (the somatic mutation of 27ELC to Ile is required for optimal recognition)
(Figure 3F).
With the HV1-18 (Q-x-x-V) signature, searches did not yield
sequence matches in paired heavy-light sequences, but in
published heavy chain-only datasets (Table S1C), we found
244 matches, which appeared to derive from 4 lineages (Figures 6A and S6). We synthesized four sequences (consensus
or closest NGS read) and reconstituted with the five light
chains used previously in swapping experiments (Table S7).
None of these reconstituted antibodies bound a set of HAs
(Figure 6C) or neutralized any of the 15 viruses in our neutralization panel. Analysis of the tested heavy chains indicated that
their CDR H3 length matched that of CT149 in three of four
cases, but was below the 78% identity threshold that correlated with function in heavy-light complementation of this class
(Table S7).
Altogether, the results indicate sequence signatures with sufficient specificity to identify other functional class members by
sequence alone could be obtained for two multidonor classes:
HV6-1+HD3-3 and HV1-18+HD3-9. The sequence signature

for the third class, HV1-18 (Q-x-x-V), was complicated by incompatibility of some heavy-light pairs from this class; nonetheless,
sequence searches for this third class did place an upper limit on
the prevalence of this multidonor antibody class in the searched
databases.
Vaccine Induction of Multidonor Broadly Neutralizing
Antibodies
In the VRC 310 trial, we observed a significant expansion
(p = 0.0284) of H5+H3+ memory B cells following H5 DNA
prime-MIV boost, ranging from an increase of 1.2- to 10.6-fold
(Figure 7A). Notably, subjects with the largest increases and
the highest frequencies of multidonor antibodies had the largest
percentages of antibodies belonging to the three multidonor
classes identified here (Figure 7B). Our initial observation of a
high number of transcripts with multiple genetic commonalities
may be explained by the preferential expansion of multidonor
class transcripts; indeed, the fold-increase in cross-reactive B
cells by subject correlated with the percentage of antibody sequences with inter-subject genetic commonalities (Figure 7C).
Importantly, the frequency of cross-reactive memory B cells
post-VRC 310 vaccination correlated with the sequence signature-identified prevalence of multidonor class antibodies
(p = 0.0045) (Figure S6D). Moreover, while we did not see significant correlation between fold increase in overall sera titer versus
an increase in cross-reactive memory B cells, we did observe a
significant correlation in titers with the H1N1 virus, A/Singapore/
8/1986, which we previously found to be especially sensitive to
neutralization by stem-directed antibodies (Lingwood et al.,
2012) (Figure 7D).
To quantify the frequency of multidonor class antibodies in
unvaccinated subjects, we examined NGS-determined memory
B cell transcripts from healthy normal donors (Figure 6A). To
compare frequencies from VRC 310-vaccinated versus unvaccinated subjects, we equated the total number of sorted memory
B cells with the total number of transcripts and observed a substantially higher transcript frequency (and to a lesser degree,
a higher lineage frequency) for multidonor class sequences on
H5N1 vaccination in the VRC 310 trial (Figure 7E).
We also examined published antibody sequences from subjects immunized with the 2009 or 2010 seasonal influenza
vaccine (Jiang et al., 2013) (Figures 6A, 7F, and S6). Overall, transcripts matching the HV1-18+HD3-9 signature were 103 more
prevalent prior to vaccination than the other multidonor transcripts; however, this class appeared not to expand on either
seasonal or VRC 310 vaccination. By contrast, transcripts
matching HV6-1+HD3-3 and HV1-18 (Q-x-x-V) signatures appeared to be present at low frequency prior to vaccination, to increase on seasonal vaccination, and to increase up to 1,000-fold
on VRC 310 vaccination (Figure 7F).
DISCUSSION
The vaccine induction of broadly neutralizing antibodies against
influenza A virus has been a long standing immunological goal.
While the human immune system can generate antibodies
capable of neutralizing group 1 and group 2 strains of influenza
(Corti et al., 2011; Dreyfus et al., 2012; Nakamura et al., 2013;
Cell 166, 609623, July 28, 2016 619

Figure 7. Vaccine Induction of Antibodies Capable of Neutralizing Group 1 and Group 2 Influenza A Viruses
(A) Frequencies of H5-H3 cross-reactive memory B cells pre- and post-H5N1 vaccination. Subjects for whom pre-immunization samples were no longer available
are indicated with open symbols; subject name and fold increase shown for others.
(B) Frequency of multidonor class sequences by donor and multidonor class.
(C) Fold increase in cross-reactive B cells relative to prevalence of heavy chain sequences with three (out of a possible four) of the same heavy chain genetic
elements in at least one sequence in any of the six analyzed subjects (Pearson correlation with the total number of sequences provided in Figure 1D).
(D) Fold increase in cross-reactive B cells relative to the fold increase in sera neutralization titer for all tested influenza A strains (shown in black) (see Figures 1 and
S1), or the single H1N1-A/Singapore/8/1986 strain (shown in red).
(E) Bar graphs of transcript frequencies (left) and lineage frequencies (right). Left: frequency of multidonor-class transcripts by dataset. Right: frequency of
multidonor class lineages for each dataset.
(F) Transcript frequency versus dataset and goal. Stars depict upper-bound estimates, and circles depict frequencies confirmed by neutralization.
(G and H) Multidonor antibodies displayed in ribbon with class-conserved contact residues shown in stick. Antibody epitopes shown in purple (HV6-1+HD3-3),
green (HV1-18+HD3-9), and orange (HV1-18 with Q-x-x-V) with black outlines. Glycans shown in surface representation and colored by conservation: conserved
(light green) or variable (dark green).
See also Figures S3 and S6 and Tables S3 and S4.

620 Cell 166, 609623, July 28, 2016

Wu et al., 2015), clear evidence of their induction by vaccination


had not been reported. In this study, we used a sequence-based
structural approach to identify three multidonor classes of antibodies capable of neutralizing group 1 and group 2 influenza A
viruses in VRC 310-vaccinated subjects. One multidonor class
utilized HV6-1+HD3-3 germline genes (Figure 2); a second class
utilized HV1-18+HD3-9 germline genes and a 15-amino acid
CDR H3 (Figure 3); and a third class utilized HV1-18 germline
gene and a Gln98HC-x-x-Val100aHC motif (Figure 4). For each
class, we delineated sequence signatures (Figure 6). Despite
the lack of serological indicators of vaccine-induced improvement in cross neutralization with VRC 310-vaccinated subjects
(Figure S1), we observed a significant vaccine-induced expansion of cross-reactive memory B cells (Figure 7A) and a clear increase in the frequency of transcripts for two of the multidonor
antibody classes (Figure 7F). These findings reveal the vaccine
induction of broadly neutralizing influenza A antibodies.
Stereotypic antibody signatures have been reported for some
bacterial polysaccharide antigens (Adderson et al., 1993), CD4induced, V1V2-directed, and VRC01 classes of HIV-1-neutralizing antibodies (Dosenovic et al., 2015; Gorman et al., 2016;
Huang et al., 2004; Jardine et al., 2015; Zhou et al., 2013), and
both stem- and head-directed influenza neutralizing antibodies
(Pappas et al., 2014; Schmidt et al., 2015). Thus, despite the potential repertoire of human immunoglobulins being large (Boyd
et al., 2009) and SHM further increasing diversity to the point
where highly similar modes of antigen recognition might be expected very infrequently, our findings reveal that multidonor classes can be induced by vaccination in humans (Figures 7 and S7).
The coexistence of multiple vaccine-induced pathways to
generate influenza group 1 and group 2 neutralizing antibodies
is encouraging for efforts aimed at achieving analogous responses in genetically diverse human populations. The stemdirected antibodies induced here potently neutralize in pseudotype assays (Figure S4), but less potently in live influenza A virus
assays (Table S4). While determination of the in vivo concentrations of stem antibodies required for protective efficacy may
require passive infusion trials in humans, it seems likely that a
protective response will require higher titers of group 1 and
group 2 neutralizing antibodies than achieved in the VRC 310
trial. Our NGS-based measurements indicated VRC 310 vaccination to boost the frequency of transcripts from two multidonor
classes substantially toward the target goal, and we even
observed increases after seasonal vaccination (Figure 7F). In
this regard, sequence signature-based quantification may provide a suitably sensitive technology to detect transcript frequencies of group 1 and group 2 neutralizing antibodies, to
measure their expression in appropriate memory B cell subsets
and long-lived plasma cells, and to assess their durability.
Appropriate SHM is an additional aspect, which we investigated
by analyzing the recognition of germline-reverted versions of
each of the three multidonor classes as well as the effect of
mutational analysis of critical contacts (Figure S7). Further
studies aimed at increasing the prevalence of group 1 and group
2 neutralizing influenza antibody lineagesguided by the
sequence signatures identified heremay provide a means to
achieve the protective efficacy required of a universal influenza
A vaccine. In this regard, it is helpful to know that 100- to

1,000-fold increases in the transcript frequencies for two multidonor classes of influenza A neutralizing antibodies could be
achieved through immunization with a divergent influenza (Figures 6A and 7F), likely by enhancing immune focus to the HA
stem (Figures 7G and 7H), an approach utilized by stem-only
or headless immunogens (Impagliazzo et al., 2015; Yassine
et al., 2015) and by chimeric HA immunogens (Krammer et al.,
2015). It will be fascinating to evaluate how these immunogens,
in various vectored, subunit, and prime-boost combinations, will
fare at further inducing, maintaining, or expanding the multidonor
broadly neutralizing antibodies identified here.
EXPERIMENTAL PROCEDURES
Ethics Statement and VRC 310 Study Design
The VRC 310 study protocol and associated procedures were approved by the
National Institute of Allergy and Infectious Diseases (NIAID) Institutional Review Board. All participants provided written informed consent in accordance
with the Declaration of Helsinki. The VRC 310 study (ClinicalTrials.gov identifier
NCT01086657) (Ledgerwood et al., 2011, 2013) was conducted by the Vaccine
Research Center, NIH. See Table S1 and the Supplemental Experimental Procedures for details.
Expression of HA Probes, Flow Cytometry, Cell Sorting, and
Sequencing
HA constructs consisting of the extracellular domain of HA modified to ablate
sialic acid binding and C-terminally fused to (1) a T4-fibritin trimerization motif,
(2) a biotinylatable AviTag sequence, and (3) a hexahistidine affinity tag were
synthesized (Genscript), cloned into a CMV-expression plasmid, and expressed as previously described (Whittle et al., 2014). Cryopreserved PBMC
samples were stained and sorted on a fluorescence-activated cell sorting
(FACS) Aria II using fluorescently labeled recombinant H1 (A/New Caledonia/
20/1999), H5 (A/Indonesia/05/2005), or H3 (A/Perth/16/2009) probes; single
memory B cells binding to both H5 and H3 probes were sorted, and
sequencing of immunoglobulin genes by multiplex PCR was performed as previously described (Whittle et al., 2014). See the Supplemental Experimental
Procedures for details.
Production of Pseudotyped Lentiviral Vectors and Influenza A
Viruses and Measurement of Antibody Neutralizing Activity
Influenza HA pseudotyped lentiviral vectors expressing a luciferase reporter
gene were produced and used to infect 293A cells. All influenza viruses
used in the microneutralization assays were expanded in Madin-Darby canine
kidney epithelial (MDCK) cells in the presence of Tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma) and titrated in MDCK cells.
See the Supplemental Experimental Procedures for details.
Structural Analysis and Sequence Bioinformatics
Both negative stain-EM and X-ray crystallography were used to characterize
antibodies from the VRC 310 trial and their complexes with HA. An Antibodyomics1.0 pipeline, modified to analyze both 454 and Illumina output,
was used to analyze B cell transcripts for the presence of sequence signatures specific to multidonor antibodies. Frequentist probabilities were used
to determine likelihoods of sequence convergence and germline prevalence
in the human population. See the Supplemental Experimental Procedures for
details.
ACCESSION NUMBERS
The accession numbers for the coordinates and structure factors reported in
this paper are Protein Data Bank (PDB): 5K9J, 5K9K, 5K9O, 5K9Q, 5KAN,
and 5KAQ. The accession numbers for the sequences for all of the antibodies
reported in this paper are GenBank: KX386124KX387227. The accession
numbers for the NGS data reported in this paper are Short Reads Archive:
SRP026397 and SRP073039.

Cell 166, 609623, July 28, 2016 621

SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and seven tables and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2016.06.043.
AUTHOR CONTRIBUTIONS
M.G.J., A.K.W., P.V.T., G.-Y.C., C.S., P.D.K., J.R.M., and A.B.M. conceived,
designed, and coordinated the study. M.G.J., A.K.W., P.V.T., G.-Y.C., C.S.,
L.S., P.D.K., J.R.M., and A.B.M wrote and revised the manuscript and figures.
B.S.G. and J.E.L. carried out VRC 310 trial and provided subject samples.
R.A.K. and M.R. provided support for B cell analysis and sorting. J.C.B.,
M.K., and J.R.W. designed HA constructs. M.G.J., A.K.W., P.V.T., R.T.B.,
A.D., R.A.G., M.K., W.-P.K., K.L., S.N.N., M.S.P., E.S.Y., B.Z., Y.Z., M.A.,
S.D., C.R.L., A.R., L.W., X.W., H.M.Y., C.S., Y.M., Y.T., U.B., and NISC CSP
performed experiments. M.G.J., G.Y.-C., C.S., C.-H.S., and P.D.K. carried
out bioinformatics analysis. M.G.J., A.K.W., P.V.T., G.-Y.C., C.S., R.T.B.,
I.S.G., M.K., W.-P.K., K.L., T.B., S.D., Y.T., U.B., J.C.M., K.S., D.C.D., L.S.,
P.D.K., J.R.M., and A.B.M. analyzed data. All authors read and approved the
manuscript. Detailed author contributions are provided in Supplemental
Information.
ACKNOWLEDGMENTS
We thank D. Ambrosak and R. Nguyen for assistance with flow cytometry;
J. Chrzas, J. Gonczy, U. Chinte, and staff at Southeast Regional Collaborative Access Team (SER-CAT) for help with X-ray diffraction data collection;
G. Georgiou and S.R. Quake for human antibody sequences; J. Stuckey
for assistance with figures; and members of the Structural Biology Section,
Structural Bioinformatics Core Section and Virology Laboratory of the Vaccine Research Center for helpful comments. Support for this work was provided by the Intramural Research Program of the Vaccine Research Center
and the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH. This work was supported in part with federal funds
from the Frederick National Laboratory for Cancer Research, NIH, under
contract HHSN261200800001E. Use of insertion device 22 (SER-CAT) at
the Advanced Photon Source was supported by the U.S. Department of
Energy, Basic Energy Sciences, Office of Science, under contract W-31109-Eng-38.
Received: January 22, 2016
Revised: April 12, 2016
Accepted: June 23, 2016
Published: July 21, 2016
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Cell 166, 609623, July 28, 2016 623

Article

Hexokinase Is an Innate Immune Receptor for the


Detection of Bacterial Peptidoglycan
Graphical Abstract

Authors
Andrea J. Wolf, Christopher N. Reyes,
Wenbin Liang, ..., K. Mark Coggeshall,
Moshe Arditi, David M. Underhill

Correspondence
david.underhill@csmc.edu

In Brief
The metabolic enzyme hexokinase
unexpectedly acts as a pattern
recognition receptor that recognizes
bacterial peptidoglycan and triggers
activation of inflammasomes.

Highlights
d

Peptidoglycan-derived N-acetylglucosamine activates the


NLRP3 inflammasome
N-acetylglucosamine in the cytosol is detected by
hexokinase
Hexokinase release from mitochondrial outer membranes
triggers NLRP3 activation
Metabolic conditions affecting hexokinase activity trigger
inflammasome formation

Wolf et al., 2016, Cell 166, 624636


July 28, 2016 2016 Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.cell.2016.05.076

Article
Hexokinase Is an Innate Immune Receptor for the
Detection of Bacterial Peptidoglycan
Andrea J. Wolf,1,2 Christopher N. Reyes,1 Wenbin Liang,3,6 Courtney Becker,1 Kenichi Shimada,2,4 Matthew L. Wheeler,1,2
Hee Cheol Cho,3,7 Narcis I. Popescu,5 K. Mark Coggeshall,5 Moshe Arditi,2,4 and David M. Underhill1,2,*
1F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles,
CA 90048, USA
2Division of Immunology, Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
3Cedars-Sinai Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
4Division of Pediatric Infectious Diseases, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
5Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
6Present address: University of Ottawa Heart Institute and Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa,
ON K1Y 4W7, Canada
7Present address: Departments of Biomedical Engineering and Pediatrics, Emory University, Atlanta, GA 30322, USA
*Correspondence: david.underhill@csmc.edu
http://dx.doi.org/10.1016/j.cell.2016.05.076

SUMMARY

Degradation of Gram-positive bacterial cell wall


peptidoglycan in macrophage and dendritic cell
phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates
processing and secretion of interleukin (IL)-1b and
IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl
dipeptide component in the cytosol by NOD2, we
report here that NLRP3 inflammasome activation is
caused by release of N-acetylglucosamine that is
detected in the cytosol by the glycolytic enzyme
hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria
outer membranes, and we found that this is sufficient
to activate the NLRP3 inflammasome. In addition,
we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and
localization induce inflammasome activation. While
previous studies have demonstrated that signaling
by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic
enzyme can act as a pattern recognition receptor.
INTRODUCTION
Macrophages and dendritic cells play essential roles in initiating inflammation by releasing cytokines and chemokines in
response to pathogen-associated molecular patterns (PAMPs)
detected by innate immune receptors. Surface receptors, such
as surface Toll-like receptors (TLRs) and C-type lectin receptors,
detect extracellular PAMPs (Kumar et al., 2011). In addition, microbes internalized by phagocytes are enzymatically degraded,
releasing small molecules that are screened for potential danger
by a panel of intracellular innate immune receptors, such as
intracellular TLRs and Nod-like receptors. We and others have

found that degradation of Staphylococcus aureus in phagosomes is a key factor in determining the types and amounts
of inflammatory cytokines produced following phagocytosis (Ip
et al., 2010; Wolf et al., 2011; Muller et al., 2015). In particular,
we noted that production of interleukin (IL)-1b and IL-18 required
the degradation of S. aureus cell wall peptidoglycan (PGN) and
that this response is suppressed when the organism modifies
its PGN to become resistant to degradation (Shimada et al.,
2010).
IL-1b and IL-18 play essential roles in controlling bacterial infections, in part, by recruiting neutrophils to sites of infection
and polarizing T cell responses. Unlike many other cytokines,
IL-1b and IL-18 are transcribed as pro-cytokines in the cytosol.
Signaling to multiprotein complexes known as inflammasomes
activates caspase-1 to process and secrete the cytokines (Lamkanfi and Dixit, 2014; Martinon et al., 2004). While there are
several varieties of inflammasomes, the one responsible for responding to PGN is defined by the presence of NOD-like receptor family, pyrin domain-containing 3 (NLRP3). The mechanism
by which NLRP3 is activated by PGN is not known. It is generally
thought that all of the immunomodulatory activity of the S. aureus
PGN comes from the degradative release of muramyl dipeptide
(MDP), which is detected by cytosolic NOD2 receptor. However,
we observed that NLRP3 inflammasome activation in response
to S. aureus PGN was not affected by the loss of NOD2 (Shimada
et al., 2010). Thus, the fragment of PGN that must be generated
through degradation to activate the inflammasome and how it is
sensed have not been established.
Diverse particulate stimuli that activate the NLRP3 inflammasome have been identified, including crystals such as silica,
alum, asbestos, uric acid, and cholesterol (Dostert et al., 2008;
Duewell et al., 2010; Hornung et al., 2008; Martinon et al.,
2006). Like PGN particles, phagocytosis of these crystals is a
necessary step in the process leading to inflammasome activation. For crystalline particles, which are non-degradable and
non-microbial, it has been suggested that disruption of the
phagosomal compartment leads to NLRP3 inflammasome activation (Hornung et al., 2008). However, we have previously
observed that phagosomes containing PGN remain intact, and

624 Cell 166, 624636, July 28, 2016 2016 Published by Elsevier Inc.

Nlrp3-/-

0.8

3.0

0.6

2.0

0.5

0.7

2.5

Sup

***

0.3

***

0.2

dT

0.1
SA
PGN

pd
A:

U
T
AT
P

0.5

Strep
PGN

IL-1 p45

Lys

***

***

Tubulin

BS
PGN

D
UT
PGN
ATP
Nig

IL-1 (ng/ml)

4
3
2
1
0
5

34

64

77

K+ (mM)

92

121 150

S. aureus
ATP
16
14
12
10
8
6
4
2
0

E
UT
% LDH release

C
6

IL-1 p17

***

0.4

1.5
1.0

IL-1 (ng/ml)

WT

0.9

3.5

IL-1 (ng/ml)

***

1.0

4.0

T
AT
P
SA
St PG
re N
p
BS -PG
-P N
G
N

64 92 150
K+ (mM)

45
40
35
30
25
20
15
10
5
0

ATP

20

40
Time (h)

PGN

60

80

Figure 1. PGN Activates the NLRP3 Inflammasome Independent of Potassium Efflux and Cell Death
(A) LPS-primed BMDMs from wild-type and Nlrp3 / mice were untreated (UT) or stimulated with 5 mM ATP for 2 hr or pdA:dT or PGN from DoatA S. aureus (SA),
Streptomyces (Strep), or B. subtilis (BS) at 2040 mg/ml, and IL-1b was assayed in the supernatant after 6 hr.
(B) Immunoblot of mature IL-1b in supernatants (Sup) or pro-IL-1b in cell lysates (Lys) of wild-type macrophages stimulated as in (A).
(C and D) LPS-primed BMDMs were stimulated in the presence of increasing concentrations of extracellular KCl with (C) 20 mg/ml PGN 6 hr, 5 mM ATP 2 hr,
10 mg/ml nigericin (Nig) 2 hr, or (D), S. aureus (DoatA) 6 hr.
(E) LPS-primed BMDMs were stimulated with PGN (20 mg/ml) or ATP (5 mM), and release of lactate dehydrogenase (LDH) into supernatants was measured at
indicated times and shown as percentage of maximum at each time point.
Error bars indicate SD. ***p < 0.001.
See also Figure S1.

this, together with the observation that lysosomal degradation is


necessary, suggests the existence of an alternative mechanism
for specifically sensing PGN degradation products.
In this study, we have identified N-acetylglucosamine (NAG), a
sugar subunit of the backbone of PGN, as an activator of the
NLRP3 inflammasome. Anthrax bacteria specifically de-acetylate NAG in PGN, and we show that this PGN becomes a poor
activator of IL-1b secretion in vitro and in vivo. Mechanistically,
we observed that purified NAG and NAG released upon degradation of PGN in phagosomes are detected via inhibition of the
glycolytic enzyme hexokinase, resulting in its dissociation from
the mitochondrial outer membrane. Using a peptide that competes with hexokinase for binding to mitochondria and induces
its dissociation from the outer membrane, we observed that
hexokinase dissociation alone is sufficient to induce NLRP3
inflammasome activation. These conclusions are further sup-

ported by the observation that specific metabolic perturbations


that affect hexokinase function also induce inflammasome activation. Together, the data suggest a model in which hexokinase
effectively acts as a pattern recognition receptor, alerting the
cell to degradation of bacterial PGN in phagosomes and activating an inflammatory response via disruption of the glycolytic
pathway and mitochondrial function.
RESULTS
PGN-Induced NLRP3 Inflammasome Activation Is
Independent of Potassium Efflux and Pyroptosis
We and others have shown that phagocytosis of Gram-positive
PGN by bone marrow-derived macrophages (BMDMs) stimulates secretion of IL-1b via the NLRP3 inflammasome (Figures
1A and 1B) (Martinon et al., 2004; Shimada et al., 2010), a
Cell 166, 624636, July 28, 2016 625

process that, as we have previously shown, requires degradation (Shimada et al., 2010). The diminished IL-1b production by
Nlrp3 / BMDMs in response to PGN are not a consequence
of differential phagocytosis or lysosomal enzyme activity, which
are equivalent in wild-type and Nlrp3 / BMDMs (Figures S1A
S1C). In the process of evaluating inflammasome activation in
response to PGN, we observed some behaviors inconsistent
with current models of mechanisms of activation. First, it has
been suggested that efflux of cytosolic potassium is essential
for NLRP3 inflammasome activation (Munoz-Planillo et al.,
2013). While we confirmed that IL-1b secretion triggered by
ATP or nigericin in lipopolysaccharide (LPS)-primed macrophages is strongly inhibited by extracellular potassium, we found
that PGN-induced IL-1b secretion is not affected by extracellular
potassium (Figure 1C). We also observed no effect of extracellular potassium on IL-1b secretion in response to whole DoatA
S. aureus (Figure 1D), a strain that makes a PGN that is
highly sensitive to phagosomal degradation and that we have
previously shown to be a strong activator of the NLRP3 inflammasome (Shimada et al., 2010). Second, unlike many inflammasome activators, PGN-induced caspase-1 activation does not
result in pyroptosis, as measured by the release of lactate dehydrogenase (LDH) (Figure 1E), annexin V staining (Figure S1D),
or propidium iodide uptake (Figure S1E). We only observe
background levels of cell death over 3 days in macrophages
stimulated with PGN (Figure 1E). Compared to classic NLRP3
activators like ATP and nigericin, PGN-induced inflammasome
activation occurs over a longer time period; for example, in Figure 1A, PGN induces much less IL-1b over 6 hr than ATP triggers
in 2 hr. Given these unique features of PGN-induced activation,
we set out to determine how PGN is sensed by macrophages.
NAG Is the Minimal Inflammasome-Activating
Component of PGN
PGN, which makes up as much as 80% of the dry weight of typical
Gram-positive bacteria, is a polysaccharide of repeating units of
N-acetylmuramic acid (NAM; MurNAc) and NAG (GlcNAc) crosslinked by short amino acid side chains (Figure 2A).
MDP, the NOD2-activating fragment of PGN, has been
suggested to stimulate IL-1b release under certain conditions
(Faustin et al., 2007; Ferwerda et al., 2008; Hsu et al., 2008;
Marina-Garca et al., 2008; Martinon et al., 2004; Pan et al.,
2007). However, when we treated macrophages with soluble
MDP or lipofectamine-complexed MDP (to deliver it to the
cytosol), they did not mimic the response to PGN that we saw
(Figure 2B). Furthermore, as noted earlier, we have previously
shown that PGN-induced IL-1b secretion is not blocked in macrophages lacking NOD2 (Shimada et al., 2010). Together, the
data suggest that some other lysosomal degradation product
of PGN must be detected.
Therefore, we examined the inflammasome-activating capacity of other potential PGN degradation products. We observed
that the NAG sugar subunit from the backbone of PGN becomes
a potent activator of IL-1b processing and secretion in LPSprimed macrophages when it is complexed with lipofectamine
to deliver it to the cytosol (Figure 2C). We found that delivery
of NAG to the cytosol is important to its function, since soluble NAG only induced IL-1b when added to culture medium
626 Cell 166, 624636, July 28, 2016

at high concentrations (Figure S2A). The IL-1b detected in


response to lipofectamine-complexed NAG was confirmed to
be cleaved IL-1b p17 by immunoblot (Figure 2D), and inflammasome assembly was detected by NLRP3 and caspase-1 p10
proximity ligation (Figure 2E). In contrast, when we exposed
LPS-primed cells to lipofectamine-complexed NAM (the other
sugar subunit of the PGN backbone) or other sugars like glucosamine (GAM), glucose, or sucrose, they triggered little or no IL-1b
secretion (Figure 2F). Lipofectamine-complexed NAG alone was
unable to induce tumor necrosis factor a (TNF-a) in un-primed
cells (Figure 2G), indicating that NAG does not reproduce the
priming activity of PGN. In addition to mouse macrophages,
PGN and NAG activate IL-1b secretion in mouse dendritic cells,
as well as human macrophages and dendritic cells primed
with LPS (Figures S2BS2D). Like PGN, NAG inflammasome
activation is dependent on the NLRP3 inflammasome (Figure 2H),
independent of potassium efflux (Figure 2I), and does not induce
pyroptosis (Figure 2J).
Acetylation of PGN Is Necessary for Its InflammasomeActivating Potential
We reasoned that, if NAG is the critical component of PGN
involved in NLRP3 inflammasome activation, PGN without
NAG should not stimulate the inflammasome. PGN produced
by Bacillus anthracis (anthrax) is unusual in that as much as
88% of its NAG is de-acetylated to GAM due to the expression
of PGN NAG deacetylase activity (Zipperle et al., 1984). Thus,
anthrax PGN contains little NAG, although it can be chemically
re-acetylated in vitro (Zipperle et al., 1984). Native NAG-deficient
anthrax PGN induces little or no inflammasome activation, while
re-acetylated anthrax PGN strongly induces activation from
LPS-primed (Figure 3A) or PAM3CSK4-primed (Figure S3A) macrophages. Native anthrax PGN is internalized by macrophages
slightly less efficiently than the re-acetylated PGN in vitro (Figures 3B and 3C), although this difference cannot account for
the profound lack of IL-1b secretion. The re-acetylated anthrax
PGN-induced IL-1b was completely NLRP3 dependent (Figure 3D). The level of IL-1b produced by the re-acetylated
PGN-stimulated macrophages is sufficient to induce potent
inflammatory immune responses in vivo, since we observed
that neutrophil recruitment to the peritoneum of mice injected
with these macrophages was largely, although not entirely,
NLRP3 dependent (Figure S3B).
Re-acetylated anthrax PGN was significantly more inflammatory upon direct intraperitoneal injection into mice than native
anthrax PGN, as measured by neutrophil infiltration (Figure 3E).
PGN induces inflammation via a wide variety of mediators
(e.g., cytokines, chemokines, and complement). We observed
that the IL-1b receptor antagonist anakinra partially inhibited
re-acetylated anthrax PGN-induced neutrophil infiltration (Figure 3F), indicating that IL-1b production plays an important role
in the overall response.
PGN and NAG Inhibit Hexokinase and Induce Its
Dissociation from Mitochondria
In order to determine how NAG activates the NLRP3 inflammasome, we began by investigating how PGN and NAG relate to
mechanisms previously implicated in NLRP3-induced IL-1b

B
NAM

IL-1 (ng/ml)

L-Ala
D-Ala

D--Glu
L-Lys

(Gly)5

L-Lys
D--Glu

D-Ala

L-Ala

NAG

NAM

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1.4

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Sup

0.2

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Blue = DAPI

po

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300

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12

Red = NLRP3/p10 PLA+


Nlrp3-/0.6

WT

700
TNF (ng/ml)

800

IL-1 (ng/ml)

IL-1 (pg/ml)

L-NAG

Lipo

L-NAG

10

dT

2 6 8 20 2 6 8 20 2 6 8 20 2 6 8 20 h

L-

K+ (mM)

A:

34 64 77 92 121 150

pd

0
5

AG

20

0.2

po

0.4

30

Li

0.6

40

0.8

50

1.0

LDH % of Max

IL-1 (ng/ml)

60

L-NAG

1.2

Figure 2. NAG Is the NLRP3 Inflammasome-Activating Component of PGN


(A) Schematic diagram of PGN structure.
(B) LPS-primed BMDMs were stimulated with lipofectamine-complexed MDP (L-MDP), soluble MDP (sMDP, 10 mg/ml), pdA:dT, or lipofectamine alone (Lipo) for
6 hr. UT, untreated.
(C) Cells were stimulated for 6 hr with lipofectamine complexes containing increasing amounts of NAG.
(D) IL-1b processing was assessed by immunoblot as in (C). Sup, supernatant; Lys, lysate.
(E) Proximity ligation assay (for association of NLRP3 with caspase-1) of LPS-primed BMDMs treated for 3 hr with lipofectamine alone (Lipo) or complexed with
NAG (L-NAG) (experiment was performed 23).
(F) Cells were stimulated for 6 hr with different sugars complexed with lipofectamine (NAM, N-acetylmuramic acid; GAM, glucosamine; Gluc, glucose; and Suc,
Sucrose).

(legend continued on next page)

Cell 166, 624636, July 28, 2016 627

secretion. As noted earlier, they do not require potassium efflux


or induce pyroptosis. However, PGN (Figure 4A) and NAG (Figure S4A) both trigger the appearance of mtDNA in the cytosol.
DNA release from the mitochondria has been associated with
NLRP3 activation in response to other stimuli (Shimada et al.,
2012; Zhong et al., 2016; Zhou et al., 2011), and while its exact
relationship to PGN-induced NLRP3 inflammasome regulation
is not clear, the observations prompted us to investigate further
how PGN-derived NAG could affect mitochondria.
Early biochemical studies attempting to identify the cytosolic
enzyme responsible for phosphorylation of glucose, the first
step in glycolysis, noted that NAG could competitively inhibit
the process (Spiro, 1958). The enzyme inhibited by NAG was
later identified as hexokinase (Wilson et al., 2011). Using purified
mouse macrophage mitochondria and recombinant human
hexokinase, we confirmed that NAG is a dose-dependent inhibitor of hexokinase enzymatic activity (Figures 4B and 4D), while
other breakdown products of PGN, including NAM and MDP,
do not inhibit hexokinase (Figures 4B and 4C).
As a competitive inhibitor, NAG, by definition, competes with
glucose for binding to the active site of the enzyme. Binding of
glucose to hexokinase has been directly characterized by solving
the crystal structure of hexokinase bound to glucose, and NAG
binding to the active site has been modeled (Aleshin et al.,
1998a, 1998b; Madej et al., 2014) (MMDB: 43169). Consistent
with these structural studies and with previous enzymatic studies
and our current data (Figure 4B), we have further observed NAG
directly binding to hexokinase by protein thermal shift assay (Figure S4B). Even though NAG binds to hexokinase, the enzyme
cannot phosphorylate NAG (Figure S4C), which is consistent
with its role as a competitive inhibitor. Acetylation of NAG is
important for its role as a hexokinase inhibitor, because de-acetylated NAG, i.e., GAM, is phosphorylated by hexokinase (Figure S4C). Because GAM is phosphorylated by hexokinase,
though less efficiently, it is not perceived as an inhibitor, suggesting why GAM does not activate the NLRP3 inflammasome (Figure 2F), and anthrax PGN, which is naturally de-acetylated, is
less inflammatory than re-acetylated anthrax PGN (Figure 3). To
further examine the role of acetylation, we solubilized native
and re-acetylated anthrax PGN, as well as S. aureus PGN, with
macrophage lysosomal extracts and observed that degradation
products derived from PGNs rich in NAG, re-acetylated anthrax
and S. aureus PGN, partially inhibited hexokinase activity (Figure S4D), while native anthrax PGN with low NAG content did not.
NAG inhibition of hexokinase was of particular interest,
because the enzyme (hexokinase I and/or II, depending on the
cell type) associates with the mitochondrial outer membrane
through an interaction with the voltage-dependent anion channel
(VDAC). This interaction with the VDAC is involved in regulation of glycolysis, mitochondrial stability, ROS production, and
permeability transition pore formation (Pastorino and Hoek,

2008). Previous investigators had noted that the knockdown of


the VDAC somehow blocks NLRP3 inflammasome activation,
but how this related to microbial sensing or whether this has
any relationship to cellular metabolism has not been clear
(Zhou et al., 2011). Previous studies on cellular metabolism
and regulation of apoptosis have observed that the association
of hexokinase with the VDAC is closely regulated by signaling
and feedback inhibition (Pastorino and Hoek, 2008). We hypothesized that the release of NAG after phagocytosis and degradation of PGN could affect mitochondria by influencing hexokinase
association with mitochondrial VDAC.
To test this hypothesis, we measured the ability of PGN
and NAG to induce release of hexokinase from mitochondria.
We observed that macrophages stimulated with PGN or NAG
showed elevated cytosolic levels of hexokinase by immunoblot
(Figures 4E and 4F). The increased hexokinase in the cytosol is
not a result of generalized loss of mitochondrial integrity; we
did not detect increased cytosolic levels of other mitochondria
proteins, including Tom20 or cytochrome c (Figures 4E and
4F), and mitochondrial membrane potential was not affected
(Figures S4E and S4F). Thus, while acute activation of the
NLRP3 inflammasome by some stimuli such as ATP may cause
severe cell damage and mitochondrial disruption, PGN and NAG
appear to induce a physiologically tolerable level of hexokinase
release that does not involve degradation of mitochondria or
collapse of total cellular mitochondrial function.
To more quantitatively measure hexokinase dissociation from
mitochondria, we measured hexokinase release into the cytosol
by ELISA (Figure 4G) and enzyme activity (Figure 4H) and
observed increases in response to PGN and NAG. We observed
hexokinase dissociation from mitochondria after phagocytosis
of a variety of Gram-positive bacteria (Figure S4G). Overall
expression of hexokinase was not affected (Figure S4H). Hexokinase release in response to PGN is upstream of NLRP3, since
we observed normal hexokinase release into the cytosol in
NLRP3-deficient macrophages (Figure S4I). To more directly
evaluate the effect of NAG on hexokinase, we microinjected
sugars directly into the cytosol of primary BMDMs expressing
GFP-tagged hexokinase, which localizes to mitochondria (Figure 4I). While injection of NAM had no effect on hexokinase localization, injection of NAG induced dissociation of hexokinase
from mitochondria within minutes, confirming that free NAG in
the cytosol is sufficient to trigger hexokinase release. Mitochondria remain intact, as observed by the expression of DsRed
tagged with a mitochondrial localization sequence.
Hexokinase Dissociation from Mitochondria Is Sufficient
to Trigger NLRP3 Inflammasome Activation and IL-1b
Production
We predicted that, if hexokinase inhibition and release from
the mitochondria constitute an initiating step in NLRP3

(G) Unprimed BMDMs were treated with lipofectamine-complexed sugars as in (F), and TNF-a was measured in the supernatant after 6 hr.
(H) LPS-primed BMDMs from wild-type (WT) and Nlrp3 / mice were stimulated as described above.
(I) LPS-primed BMDMs were stimulated with lipofectamine-complexed NAG in the presence of increasing concentrations of extracellular KCl for 6 hr.
(J) LPS-primed BMDMs, stimulated as described above, were assessed for LDH release at indicated times.
Error bars indicate SD. ***p < 0.001, Students t test.
See also Figure S2.

628 Cell 166, 624636, July 28, 2016

Figure 3. Acetylation of NAG Is Necessary for Inflammasome Activation by PGN


(A) LPS-primed BMDMs were treated with the increasing doses of native (AxPGN) or re-acetylated (Ac-AxPGN) anthrax PGN (20160 mg/ml) for 6 hr, and IL-1b in
the supernatant was measured by ELISA. UT, untreated.
(B) TRITC-labeled native or re-acetylated anthrax PGN (40 mg/ml) was incubated with LPS-primed BMDMs for 1 hr, and internalization was confirmed by
fluorescence microscopy.
(C) TRITC-labeled native or re-acetylated anthrax PGN internalization by BMDMs was measured by flow cytometry after 6 hr.
(D) LPS-primed BMDMs from wild-type (WT) and Nlrp3 / mice were stimulated with 80 mg/ml AxPGN for 6 hr, 80 mg/ml Ac-AxPGN for 6 hr, 5 mM ATP for 2 hr, or
pdA:dT for 6 hr, and IL-1b was measured by ELISA.
(E) Mice were injected i.p. with PBS (n = 8) or 10 mg of AxPGN (n = 7) or Ac-AxPGN (n = 7). After 4 hr, cells in the peritoneal lavage were harvested and analyzed by
flow cytometry. Total cells (left panel) and neutrophils (middle and right panels) were increased in response to re-acetylation of AxPGN. ns, not significant.
(F) Mice were injected i.p. with 10 mg Ac-AxPGN without (Ctl) (n = 5) or with 25 mg/kg anakinra (n = 5) and assayed as in (E) (experiment was performed 13).
Error bars indicate SD. **p % 0.01; ***p < 0.001, one-way ANOVA and Newman-Keuls multiple comparison test (E) and unpaired Students t test (F).
See also Figure S3.

Cell 166, 624636, July 28, 2016 629

20
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HK Activity (U/ml)

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HK Activity (U/ml)

**

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100

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Fold Induction

5.0
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% Actiivity

T
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hr

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CytoC

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Specificity
Ctl

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N
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po
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AG

HK activity
(% of Total Cell Activity)

Before
*

Antibody
Specificity
Ctl

cytosol

NAG

***

Tubulin

I
3h

hr
HK2

H
20
18
16
14
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10
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CytoC
cytosol

Lipo

HK2 (ng/ml)

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NAG (mM)

**

5
***

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3

***

**

2
1
0

1 2 3 1 2 3 1 2 3 1 2 3 h
UT
PGN L-NAG pdA:dT

After

HK2
GFP
Mito
DsRed

Before

NAM

After

HK2
GFP
Mito
DsRed

Figure 4. Hexokinase Is the Receptor that Detects NAG for Inflammasome Activation
(A) LPS-primed BMDMs were stimulated with ATP (5 mM) for 2 hr or PGN (40 mg/ml) as indicated, and the presence of mtDNA in the cytosolic fraction was
measured by RT-PCR.
(B and C) Hexokinase (HK) activity in purified mouse macrophage mitochondria was assessed in the presence of increasing concentrations of (B) NAG, NAM,
sucrose, or (C) MDP (25 mM). UT, untreated.
(D) NAG inhibits the activity of purified human hexokinase.
(E and F) Hexokinase in the cytosol fraction of LPS-primed BMDMs stimulated for the indicated times with PGN (40 mg/ml, from S. aureus) or lipofectaminecomplexed NAG was detected by immunoblot. Clotrimazole (CTM) treatment was used as a positive control for hexokinase release from mitochondria, and
mitochondrial markers Tom20 and cytochrome c (CytoC) were included to control for mitochondrial integrity. Control lysates were included (Antibody Specificity
Ctl) to confirm antibody staining for each marker (non-continuous lane from the same gel). Lipo, lipofectamine.
(G) LPS-primed BMDMs were stimulated as indicated PGN (40 mg/ml), and hexokinase 2 in the cytosol was determined by ELISA.
(H) LPS-primed BMDMs were stimulated as indicated, and cytosolic hexokinase enzyme activity was measured.
(I) LPS-primed BMDMs expressing hexokinase 2 fused to GFP (HK2-GFP) and DsRed targeted to mitochondria (Mito-DsRed) were microinjected with NAG or
NAM as indicated. Association of hexokinase with mitochondria was visualized before and 1 min after injection (n = 10 cells assessed for each sugar).
Error bars indicate SD. *p % 0.05; **p % 0.01; ***p % 0.001, Students t test.
See also Figure S4.

630 Cell 166, 624636, July 28, 2016

Figure 5. Hexokinase Dissociation from Mitochondria Is Sufficient to Activate the NLRP3 Inflammasome
(A) LPS-primed BMDMs were treated with cell-permeable hexokinase dissociation peptide (HKVBD) or scrambled control peptides (Ctl) fused to TAT peptide
(20 mM) for the indicated times, and the amount of hexokinase in the cytosolic fraction was determined by immunoblot. Control lysate was included on each gel
(Antibody Specificy Ctl) to confirm antibody staining for each marker (non-continuous lane from the same gel). UT, untreated.
(B) LPS-primed BMDMs expressing HK2-GFP and mitochondria-localized DsRed (Mito-DsRed) were imaged following treatment with HKVBD and Ctl peptides
fused to TAT (20 mM) to assess hexokinase redistribution.
(C and D) LPS-primed BMDMs were treated with HKVBD or control peptides fused to cell-permeable antennapedia peptide; IL-1b (C) and IL-18 (D) were
measured by ELISA after 2 hr.
(E) LPS-primed BMDMs from wild-type and Nlrp3 / mice were stimulated with 5 mM ATP, pdA:dT, HKVBD or control peptides fused to antennapedia peptide,
and IL-1b was measured in the supernatant after 2 hr.
(F and G) LPS-primed BMDMs were treated with HKVBD or control peptides fused to TAT peptide; (F) cleaved IL-1b and caspase-1 were detected by immunoblot at
2 hr, and (G) inflammasome assembly was observed by NLRP3 and caspase-1 p10 proximity ligation (PLA, red). Nuclei were stained with DAPI (blue). Sup, supernatant.

(legend continued on next page)

Cell 166, 624636, July 28, 2016 631

inflammasome activation by PGN, then forcing hexokinase to


dissociate from the VDAC would be sufficient to trigger IL-1b
secretion. Previous metabolic studies have characterized the
binding site between hexokinase II and the VDAC and have
shown that a peptide derived from hexokinase II (HKVBD) can
be used to block binding and cause dissociation of the enzyme
from the VDAC (Chiara et al., 2008; Majewski et al., 2004; Pastorino et al., 2002). When we treated LPS-primed macrophages
with a cell-permeable version of this peptide, hexokinase rapidly
dissociated from mitochondria, as measured by immunoblot
of the cytosol fraction (Figure 5A). We also directly observed
HKVBD-peptide-induced dissociation of GFP-tagged hexokinase 2 from macrophage mitochondria by microscopy within minutes of exposure to the peptide (Figure 5B). When we treated
LPS-primed BMDMs with the hexokinase dissociation peptide,
we observed dose-dependent release of mature IL-1b by ELISA
(Figure 5C; Figure S5A), as well as IL-18 (Figure 5D). IL-1b processing and secretion in response to the HKVBD peptide were
faster than PGN (Figures S5B and S5C) and NLRP3 dependent
(Figure 5E). Inflammasome activation was confirmed by observation of cleaved IL-1b p17 and caspase-1 p10 in the supernatant of treated BMDMs (Figure 5F). Proximity ligation assay
revealed direct association of NLRP3 and caspase-1, evidence
of initial inflammasome assembly, within minutes of exposure
to the peptide (Figure 5G). To determine whether hexokinase
dissociation from the VDAC on mitochondrial membranes is sufficient to activate inflammatory responses in vivo, we injected
mice intraperitoneally with cell-permeable control or HKVBD
peptides. The HKVBD peptide was sufficient to induce inflammation and recruitment of inflammatory cells (Figures 5H and
S5D), and this response was reduced in mice deficient in caspase-1 and -11 (Figure 5I).
While NAG inhibits hexokinase activity and, therefore, triggers
hexokinase dissociation from mitochondria, HKVBD peptide induces hexokinase dissociation but does not inhibit hexokinase
activity (Figure S5E). Therefore, we conclude that hexokinase
dissociation, rather than inhibition, is the important upstream
step in inflammasome activation. HKVBD peptide can be toxic
to the cells during extended exposure, but during the short exposure in which IL-1b is induced, we observed only a small amount
of increased cell death (Figure S5F). Consistent with PGN
and NAG, we observed an increase in cytosolic mtDNA when
we stimulated cells with HKVBD peptide (Figure S5G). Since
HKVBD-peptide-induced hexokinase release is sufficient to activate the NLRP3 inflammasome, and since PGN degradation
leads to hexokinase release, we conclude that this mechanism is sufficient to explain how PGN activates the NLRP3
inflammasome.
Metabolic Conditions that Result in Hexokinase
Inhibition Lead to Inflammasome Activation
Glycolysis is regulated, in part, by feedback inhibition of hexokinase by its enzymatic product glucose-6-phosophate (G6P).

High levels of G6P trigger the release of hexokinase from mitochondria and, thus, reduce the rate of further G6P production
(Gerber et al., 1974; Pastorino et al., 2002). Therefore, we predicted that excess G6P would activate the NLRP3 inflammasome. Indeed, when we treated primed BMDMs and bone
marrow-derived dendritic cells (BMDCs) (data not shown) with
lipofectamine-complexed G6P, we observed IL-1b secretion
by ELISA (Figure 6A) and production of cleaved IL-1b p17 and
caspase-1 p10 in the supernatant (Figure 6B). This activation
was NLRP3 dependent (Figure S6A). Consistent with its role as
a hexokinase inhibitor, we observed increased hexokinase in
the cytosol following G6P treatment (Figure 6C). 2-deoxyglucose
(2-DG) is a glycolytic inhibitor that is commonly used in studies
of cell metabolism. It competes with glucose in the glycolytic
pathway. When we treated primed BMDMs with 2-DG, we
observed dose-dependent induction of IL-1b by ELISA (Figure 6D), as well as cleaved IL-1b p17 and caspase-1 p10 in the
supernatant (Figure 6E), as has recently been reported by others
(Nomura et al., 2015). However, 2-DG does not function as an
inhibitor of hexokinase like NAG or G6P. Instead, 2-DG is metabolized by hexokinase to 2-deoxyglucose-6-phosphate (2-DG6P)
(Figure S4C), which cannot be utilized by downstream glycolytic
enzymes (Wick et al., 1957). The result is a buildup of 2-DG6P
that inhibits hexokinase like G6P but is sensitive to the presence
of glucose, hexokinases preferred substrate. Thus, while 2-DG
can trigger inflammasome activation in primed macrophages in
media containing glucose, it is more effective in the absence of
glucose (Figure 6F) and is completely dependent on NLRP3 (Figure S6B). Consistent with our previous observations, 2-DG treatment leads to an increase in hexokinase in the cytosol of BMDMs
(Figure 6G). Lastly, we treated primed cells with citrate, a natural
intermediate in the tricarboxylic acid (TCA) pathway that inhibits
phosphofructokinase when it accumulates. Buildup of citrate
thus backs up the glycolytic pathway and naturally elevates
cytosolic G6P levels (Berg et al., 2002). As expected, treating
primed cells with citrate triggered NLRP3-dependent IL-1b
production and caspase-1 cleavage (Figures 6H and S6C). While
these metabolic stresses can cause cell death under certain
conditions, we observed IL-1b production under conditions
that do not cause substantial cell death (Figure S6D). These
data suggest an intriguing relationship between cellular metabolism and inflammatory signaling.
DISCUSSION
While the original evolutionary role of phagocytosis was to eat
and degrade other microbes to obtain nutrients, this study
suggests that mammalian phagocytes have adapted the cells
metabolic machinery for utilizing these nutrients to detect the
presence of microbial-derived sugars and metabolic perturbation as danger signals. In this study, we have shown that the
NAG subunit of the sugar backbone of bacterial PGN induces
inflammasome activation by inhibiting hexokinase, the first

(H and I) Indicated mice were injected i.p. with 500 ml of PBS (n = 6), 240 mM HKVBD (n = 7), or control peptide fused to TAT peptide (n = 6); peritoneal cavities were
lavaged after 4 hr; total cells were counted; and neutrophil content was determined by flow cytometry (both experiments were each done 13). ns, not significant.
Error bars indicate SD. *p % 0.05; **p % 0.01; ***p % 0.001, Students t test (CE and H) and one-way ANOVA and Newman-Keuls multiple comparison test (G).
See also Figure S5.

632 Cell 166, 624636, July 28, 2016

H
G

Figure 6. Metabolic Perturbations Affecting Hexokinase Activate the NLRP3 Inflammasome


(A and B) LPS-primed BMDMs were treated with increasing concentrations of lipofectamine-complexed glucose-6-phosphate (G6P) for 6 hr. IL-1b was measured
in the supernatant by ELISA (A), and cleaved IL-1b and caspase-1 were detected by immunoblot (B). UT, untreated; Lipo, lipofectamine.
(C) Hexokinase was detected in the cytosolic fraction following treatment with lipofectamine-complexed G6P for the indicated times. Control lysate was included
on each gel (Antibody Specificity Ctl) to confirm antibody staining for each marker (non-continuous lane from the same gel). CTM, clotrimazole.
(D and E) LPS-primed BMDMs were treated with increasing concentrations of 2-deoxyglucose (2-DG) for 6 hr. IL-1b was measured in the supernatant (Sup) by
ELISA (D), and cleaved IL-1b and caspase-1 were detected by immunoblot (E).
(F) LPS-primed BMDMs were treated with 2-DG in the presence or absence of glucose for 6 hr, and IL-1b was measured in the supernatant by ELISA.
(G) Hexokinase was measured in the cytosolic fraction following treatment with 2-DG in the absence of glucose.
(H) LPS-primed wild-type or Nlrp3 / BMDMs were treated with increasing concentrations of sodium citrate for 6 hr, and IL-1b was measured in the supernatant
by ELISA.
Error bars indicate SD. *p % 0.05; **p % 0.01; ***p % 0.001, Students t test.
See also Figure S6.

Cell 166, 624636, July 28, 2016 633

step in glycolysis. The inhibition of hexokinase results in hexokinase dissociation from the mitochondria, which, as we have
observed, is sufficient to initiate an NLRP3 inflammasomeactivating cascade in the cell. This model is supported by the
observation that several metabolic perturbations that inhibit
hexokinase function, such as treatment with glucose-6-phosphate, 2-deoxyglucose, or citrate, all lead to inflammasome
activation. How exactly hexokinase release from the mitochondrial outer membrane promotes NLRP3 inflammasome activation remains to be understood.
Mitochondrial dynamics have been broadly implicated in regulation of the NLRP3 inflammasome, including studies demonstrating a role for mitochondrial movement along microtubules
(Misawa et al., 2013), regulation of mitochondrial fission and
growth (Park et al., 2015), mitophagy (Zhong et al., 2016), and
release of mtDNA into the cytosol (Shimada et al., 2012; Zhong
et al., 2016) in modulating inflammasome activation. NAG inhibition of hexokinase was particularly interesting, because hexokinases I and II, the primary isoforms that regulate glycolysis, are
known to associate with the VDAC in the mitochondrial outer
membrane (John et al., 2011; Pastorino and Hoek, 2008; Pastorino et al., 2002; Rasola et al., 2010). The VDAC is known to regulate mitochondrial ROS (reactive oxygen species) production
(da-Silva et al., 2004), is a suggested component of the mitochondrial permeability transition pore that can release large molecules (including mtDNA) into the cytosol (Rasola et al., 2010;
Tomasello et al., 2009), and is localized to regions enriched for
cardiolipin (Sun et al., 2012) an NLRP3 activator. The interaction
of hexokinase with the VDAC on the outer membrane of mitochondria provides hexokinase with preferential access to newly
produced ATP transported from the matrix by the VDAC (Pastorino and Hoek, 2008). Hexokinase inhibition and dissociation from
the mitochondria constitute an essential step in regulation of the
rate of glycolysis (da-Silva et al., 2004; Pastorino and Hoek,
2008). Excess glucose-6-phosphate generated by hexokinase
leads to feedback inhibition of hexokinase and its dissociation
from mitochondria, slowing glycolysis. In addition, the interaction of hexokinase with the VDAC protects cells from mitochondrial ROS production (da-Silva et al., 2004) and suppresses
pro-apoptotic interactions between the VDAC and Bcl-family
members (Bax, Bid, etc.), which promotes sustained opening
of the mitochondrial permeability transition pore (Chiara et al.,
2008; Majewski et al., 2004; Pastorino and Hoek, 2008; Pastorino et al., 2002; Rasola et al., 2010). Each of these processes
have been previously implicated in NLRP3 inflammasome regulation, but how they relate to microbial sensing has not been
understood (Shimada et al., 2012; Zhou et al., 2011).
At first thought, NAG would seem to be a poor candidate to be
a PAMP detected by the innate immune system, since it is not
unique to bacteria. However, free NAG is not generally found in
the cytosol of mammalian cells and is primarily generated only
in small amounts following degradation of glycosylated proteins.
In biosynthetic pathways, uridine diphosphate (UDP)-NAG is
synthesized directly from glycolytic intermediates and utilized
in glycosylation processes without existing as free NAG. In
contrast, during degradation of particulate PGN in phagosomes,
unusually large amounts of NAG can be expected to become
available. The presence of a transporter that moves NAG from
634 Cell 166, 624636, July 28, 2016

lysosomes into the cytosol has been biochemically documented


(Jonas and Jobe, 1990; Jonas et al., 1989), although the molecular identity of the transporter is not yet known. We interpret the
data to suggest that macrophages have adapted to use their
ancient and essential metabolic glycolysis pathway to directly
sense unusually high levels of bacteria-derived NAG, generated
only upon phagocytosis of bacteria. Interestingly, unlike many
strong NLRP3 inflammasome activators, PGN does not stimulate pyroptosis. It is possible that this resistance to cell damage
is a result of the lower level of prolonged inflammasome activation stimulated by PGN, as compared to strong acute NLRP3
activators such as ATP. This is consistent with the idea that,
from the perspective of mounting an effective host defense,
macrophage cell death would seem to be an inappropriate
response to detection of PGN.
Though many studies over the years have suggested that
innate immune signaling has important effects on metabolism,
these studies have not implicated metabolic enzymes themselves as sensors of non-self (Haneklaus and ONeill, 2015;
Wen et al., 2012). Our findings suggest a novel area for crosstalk
between metabolism and innate immune signaling. The observation that modulation of a metabolic process can directly induce
inflammation may have profound implications for diseases as
wide ranging as diabetes, obesity, atherosclerosis, or inflammatory bowel disease, which have been linked to inflammasome
activation (Wen et al., 2012).
EXPERIMENTAL PROCEDURES
Mice
C57BL/6 mice were purchased from Jackson Laboratory. Nlrp3 / mice
(Mariathasan et al., 2006) were obtained from Dr. K. Fitzgerald (University of
Massachusetts), and Casp1 / mice also deficient in caspase-11 (Kuida
et al., 1995) were obtained from R.A. Flavell (Yale University). Mice were
housed in specific pathogen-free conditions in the Cedars-Sinai animal facility,
and all animal experiments were conducted according to Cedars-Sinai Medical Center Institutional Animal Care and Use Committee guidelines.
Cell Preparation and Stimulation/Lipofectamine Complexing
BMDMs and dendritic cells were grown as described previously (Wolf et al.,
2011), using 10% L-cell conditioned media or 50 ng/ml recombinant human
macrophage colony-stimulating factor (M-CSF) or murine granulocyte-macrophage (GM)-CSF, respectively. Cells were plated at 1 3 105 in a 96-well plate
and primed with LPS (50100 ng/ml) for 34 hr, followed by treatment with
PGN (2040 mg/ml) for 6 hr or ATP (5 mM) or nigericin (10 mM) for 2 hr. Lipofectamine-complexed stimuli were prepared by mixing pdA:dT (1 mg/ml) or sugars
(1 M) prepared in Opti-MEM with 24 ml lipofectamine (Invitrogen) per 100 ml for
30 min at room temperature. Cells were stimulated with 10 ml of the mix per
well. All sugar solutions were adjusted to a pH 7.4 prior to mixing with lipofectamine. Infection of cells with DoatA S. aureus and other bacteria was
done as described previously (Shimada et al., 2010; Wolf et al., 2011). Cell supernatants were analyzed by ELISA for IL-1b and TNFa (BioLegend) and IL-18
(MBL International).
Anthrax PGN
PGN from Bacillus anthracis Sterne stain was purified at the University of Oklahoma Health Sciences Center, as previously described (Iyer and Coggeshall,
2011; Langer et al., 2008). Anthrax PGN was re-acetylated according to previously published methods (Vollmer and Tomasz, 2000). AxPGN and Ac-AxPGN
were labeled with TRITC (Biotium) or Alexa Fluor 647 in PBS for 1 hr at 37 C
and washed to remove unreacted fluorophore. Labeled PGN was added to
BMDMs and spun down briefly, and cells were allowed to internalize PGN

for 1 hr and washed. Cells were either imaged or treated with PBS + proteinase
K (1 U/ml) to remove unbound particles and analyzed by flow cytometry to
determine the degree of phagocytosis.

support came from the Janis and William Wetsman Family Chair in Inflammatory Bowel Disease at Cedars-Sinai Medical Center (D.M.U.). Thanks to the
laboratory of Dr. Robin Shaw for use of their FemtoJet microinjection system.

Intraperitoneal Injections of PGN and HKVBD


C57BL/6 mice (n = 7 per group) were injected intraperitoneally (i.p.) with 500 ml
sterile PBS alone or containing 10 mg/ml AxPGN or Ac-AxPGN or 240 mM TATfused HKVBD or scramble peptide. For adoptive transfer experiments, wildtype and Nlrp3 / BMDMs were primed with 100 ng/ml PAM3CSK4 for 4 hr
and then given 80 mg/ml Ac-AxPGN for 1 hr. Cells were washed 33 with
PBS and counted, and 1 3 105 cells in 500 ml sterile PBS were injected i.p.
Mice were rested for 4 hr before being euthanized, and the peritoneal cavity
was lavaged 23 with 5 ml of cold sterile PBS + 2 mM EDTA. Cells were
counted; stained to assess cell types using antibodies against CD11b,
CD11c, Gr-1, CD3, and CD19 (BioLegend); and analyzed by flow cytometry
using FlowJo software.

Received: October 23, 2015


Revised: March 11, 2016
Accepted: May 25, 2016
Published: June 30, 2016

Cell Fractionation and Hexokinase Detection


Cells were grown in six-well plates and stimulated as described earlier.
Mitochondria and cytosol were separated using protocols previously described
(Pastorino et al., 2002), with some modification. Cells were lifted in 600 ml of
cell disruption buffer (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA,
250 mM sucrose, Roche cOmplete Protease Inhibitor) and incubated on ice for
5 min. The cells were disrupted by passing 30 times through a 22-gauge needle.
In some cases, cytosol was isolated by adding 50 mg/ml digitonin to the disruption buffer and incubating with rocking for 10 min. Lysates were then centrifuged
at 10,000 3 g for 10 min, and the supernatant was designated cytosol. For hexokinase assays, the pellet containing mitochondria was washed and resuspended
in mitochondria suspension media (20 mM HEPES, 1.5 mM MgCl2, 250 mM
sucrose). Cytosolic hexokinase was measured by either immunoblot or mouse
hexokinase 2 ELISA (Novateinbio) on equivalent amounts of protein lysate.
Proximity Ligation Assay
BMDMs were plated on glass coverslips and primed with LPS for 4 hr. Cells
were stimulated for designated times with the indicated stimuli, fixed with
4% paraformaldehyde, permeablized using 0.1% Triton X-100, and stained
with anti-NLRP3 (Cryo-2) (AdipoGen) and anti-caspase-1 p10 (Santa Cruz
Biotechnology). The Duolink In Situ PLA Kit was used according to the manufacturers instructions (Olink Biosciences).
Statistical Analysis
All experiments were conducted with triplicate measurements a minimum of
three times unless otherwise stated in the figure legends. Experiments were
analyzed using GraphPad Prism software or Microsoft Excel. The Grubbs
test was used to identify and exclude a single outlier (Figure 3E).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures
and six figures and can be found with this article online at http://dx.doi.org/
10.1016/j.cell.2016.05.076.
An audio PaperClip is available at http://dx.doi.org/10.1016/j.cell.2016.05.
076#mmc3.
AUTHOR CONTRIBUTIONS
Studies were designed by A.J.W. and D.M.U. and performed by A.J.W.,
C.N.R., C.B., M.L.W., and K.S. Microinjection experiments were performed
by A.J.W. and W.L. Anthrax PGN was prepared by K.M.C. and N.I.P. Further
advice and conceptual development were provided H.C.C. and M.A. The
manuscript was prepared by A.J.W. and D.M.U.
ACKNOWLEDGMENTS
This study was funded by grants from the NIH (GM085796 to D.M.U.,
T32AI089553 to A.J.W., AI067995 to M.A., and AI062629 to K.M.C.). Further

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636 Cell 166, 624636, July 28, 2016

Article

Amyloid-like Self-Assembly of a Cellular


Compartment
Graphical Abstract

Authors
Elvan Boke, Martine Ruer, Martin Wuhr, ...,
David Drechsel, Anthony A. Hyman,
Timothy J. Mitchison

Correspondence
elvan_boke@hms.harvard.edu

In Brief
Amyloid-like self-assembly of a specific
protein drives formation of a cellular
compartment in oocytes.

Highlights
d

The organelle content of the Balbiani body is held together


by an Xvelo matrix
Xvelo forms amyloid-like networks in vitro, which can recruit
RNA and mitochondria
Prion-like domain of Xvelo dictates specificity in amyloid
assembly
Amyloid-like polymerization is conserved among vertebrate
Balbiani body organizers

Boke et al., 2016, Cell 166, 637650


July 28, 2016 2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.cell.2016.06.051

Article
Amyloid-like Self-Assembly
of a Cellular Compartment
Elvan Boke,1,* Martine Ruer,2 Martin Wuhr,1,3 Margaret Coughlin,1 Regis Lemaitre,2 Steven P. Gygi,3 Simon Alberti,2
David Drechsel,2 Anthony A. Hyman,2 and Timothy J. Mitchison1
1Department

of Systems Biology, Harvard Medical School, Boston, MA 02115, USA


Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
3Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
*Correspondence: elvan_boke@hms.harvard.edu
http://dx.doi.org/10.1016/j.cell.2016.06.051
2Max

SUMMARY

Most vertebrate oocytes contain a Balbiani body, a


large, non-membrane-bound compartment packed
with RNA, mitochondria, and other organelles. Little
is known about this compartment, though it specifies
germline identity in many non-mammalian vertebrates. We show Xvelo, a disordered protein with
an N-terminal prion-like domain, is an abundant constituent of Xenopus Balbiani bodies. Disruption of the
prion-like domain of Xvelo, or substitution with a
prion-like domain from an unrelated protein, interferes with its incorporation into Balbiani bodies
in vivo. Recombinant Xvelo forms amyloid-like networks in vitro. Amyloid-like assemblies of Xvelo recruit both RNA and mitochondria in binding assays.
We propose that Xenopus Balbiani bodies form by
amyloid-like assembly of Xvelo, accompanied by
co-recruitment of mitochondria and RNA. Prion-like
domains are found in germ plasm organizing proteins in other species, suggesting that Balbiani
body formation by amyloid-like assembly could be
a conserved mechanism that helps oocytes function
as long-lived germ cells.
INTRODUCTION
Female germ cells, oocytes, are highly specialized cells. In many
species, oocytes are long-lived and lie dormant for months or
years before they are activated prior to fertilization (Li and Albertini, 2013). They ensure the continuity of the species by providing
the female genome and mitochondria, along with most of the
housekeeping machinery and nutrients the early embryo will
need after fertilization. Oocytes have a unique subcellular organization, with a large nucleus, called the germinal vesicle, and
a large cytoplasm. In many species, the cytoplasm of the early
oocytes contains a highly specialized compartment called the
Balbiani body. It is non-membrane bound and densely packed
with mitochondria, RNA, ER, and Golgi. The Balbiani body assembles early in oocyte formation (Lei and Spradling, 2016), disappears in late-stage oocytes in mammals (Pepling et al., 2007),
and forms dispersed isles at the vegetal pole in late-stage oo-

cytes of Xenopus and zebrafish (Bontems et al., 2009; Kloc


et al., 2004).
How, or why, the Balbiani body forms is largely mysterious
(Gupta et al., 2010; Heim et al., 2014; Kloc and Etkin, 1995;
Kloc et al., 1993; Marlow and Mullins, 2008). In some species,
including frogs and fish, but not newts and mammals, one function of the Balbiani body is to serve as an embryonic determinant
that specifies germline identity by forming germ plasm (Lesch
and Page, 2012; Richardson and Lehmann, 2010). Germ plasm,
a special part of oocyte cytoplasm, protects specific maternal
RNAs from degradation and is believed to host healthy mitochondria during development to pass on to future generations
(Houston and King, 2000; Kogo et al., 2011). Germline specification is the only known function of the Balbiani body, but it is
notable that oocytes from species that use inductive processes
to specify their germline, including mammals, still contain a Balbiani body, whose ultrastructure is similar to those of organisms
that use germ plasm (Hertig, 1968). We do not know what functions the Balbiani body serves in all vertebrates. We speculate
their most conserved function is to protect the quality of mitochondria and other organelles during long periods of oocyte
dormancy, which can extend for decades in humans.
An elegant maternal effect screen in zebrafish identified only
one gene, bucky ball, required for Balbiani body formation
(Dosch et al., 2004). In buc mutants, the Balbiani body did not
form, and oocyte polarity was disrupted. Overexpression of
Bucky ball protein stimulated numerous Balbiani bodies and
ectopic germ cell formation (Bontems et al., 2009; Heim et al.,
2014; Marlow and Mullins, 2008). Bucky ball is proposed to be
a structural organizer of zebrafish Balbiani bodies, but the
biochemical basis of this proposed function has been unclear.
Xenopus laevis provides complementary advantages for analysis of Balbiani bodies because oocytes are abundant and
accessible, easy to manipulate due to their large size, and
amenable to live imaging. Xenopus oocytes are classified in six
different stages according to Dumont, 1972. Stage I oocytes
contain a clearly visible Balbiani body (Figure 1A). The Balbiani
body gradually disappears during oocyte development to give
rise to dispersed germ plasm islands at the vegetal pole of the
stage VI oocytes during oocyte maturation (Kloc et al., 2004).
Here, we report that the Balbiani body forms via amyloid-like
self-assembly of Xvelo, the Xenopus homolog of Bucky ball. Amyloids and amyloid-like proteins have largely been studied in the
context of neurodegenerative diseases, such as Alzheimers
Cell 166, 637650, July 28, 2016 2016 Elsevier Inc. 637

B
2 M NaCl

Balbiani
body
50 m

Bb

95C

N
50 m

Time (min)

10 m

2 m

0.5 m

50 m

Figure 1. A Balbiani Body Is a Non-Membrane-Bound Compartment Packed with Membranous Organelles


(A) Phase contrast image of a stage I Xenopus laevis oocyte. Bb, Balbiani body; N, nucleus or germinal vesicle.
(B) Balbiani body immobilized in perfusion chambers. 2 M NaCl (first panel) or 95 C 50 mM HEPES, 100 mM KCl (pH 7.6) buffer (second panel) was perfused into
the chambers.
(C) Thin-section electron microscope (EM) images of isolated Balbiani bodies from stage I Xenopus oocytes. Mitochondria (dark spots), RNP particles (green
arrow head), and Golgi stacks (yellow arrow head) are clearly visible.
(D) Stage I oocytes were incubated in 10 mM Thioflavin T in 13 MMR for 10 min and washed twice with 13 MMR.
See also Figure S1, Table S1, and Movie S1.

disease, amyotrophic lateral sclerosis, or prion diseases (Haass


and Selkoe, 2007; Koo et al., 1999; Polymenidou and Cleveland,
2011). Several examples of amyloid-like aggregation mechanisms with normal biological functions have recently emerged
(Berchowitz et al., 2015; Fowler et al., 2006; Hou et al., 2011;
Li et al., 2012). Both pathological and physiological amyloidforming proteins contain low-complexity regions, which are
intrinsically disordered but can undergo conformational conversions into an amyloid-like fibrillar state (Kato et al., 2012). These
regions are also found in yeast prion proteins, a class of infectious proteins that generate heritable phenotypic diversity in
clonal population of yeast cells (Alberti et al., 2009; Halfmann
et al., 2012; Wickner et al., 2013). Protein regions with sequence
compositions similar to yeast prions are called prion-like domains (PLDs) (Alberti et al., 2009; Si et al., 2003).
RESULTS
A Balbiani Body Has a Rigid Structure that Stains with
Thioflavin T
We began our studies on Balbiani bodies by manually dissecting
them from Xenopus laevis stage I oocytes, which are transparent
and 50300 mm in diameter (Figure 1A). Stage I oocytes were
placed in a glass-bottom dish together with a cytoskeleton stabilizing buffer and mechanically disrupted using tweezers, leaving the Balbiani body intact. The Balbiani body preserved its circular shape and did not disintegrate under the considerable
shear forces applied during isolation (Movie S1). Balbiani bodies
also retained their structure under harsh conditions, including
638 Cell 166, 637650, July 28, 2016

high salt (2 M NaCl) (Figure 1B, first panel) and high temperature
(up to 95 C) (Figure 1B, second panel). Confirming previous work
on intact oocytes, thin-section electron microscopy of the isolated Balbiani bodies revealed densely packed mitochondria,
ER, and Golgi stacks as well as compact fibrillar elements that
others have shown to be made of ribonucleoprotein (RNP) (Figure 1C) (al-Mukhtar and Webb, 1971; Balinsky and Devis,
1963; Billett and Adam, 1976).
To probe the molecular properties of the Balbiani body, we
introduced a number of small molecules into the oocytes. Thioflavin T (ThT), a dye that stains the b sheet-rich structures of amyloid (Alberti et al., 2010; Nilsson, 2004), strongly accumulated in
the Balbiani body (Figure 1D), suggesting it is rich in b sheet
structures, which is a hallmark of amyloids.
Xvelo Is Highly Concentrated in Balbiani Bodies
To analyze the composition of Balbiani bodies, we used quantitative mass spectrometry (McAlister et al., 2014; Wuhr et al.,
2014). This revealed that the most enriched Balbiani body proteins that are not part of organelles are Velo1 (commonly known
as Xvelo) and fetal hemoglobin subunits, Hba1 and Hbg1 (Figures S1AS1C). We focused on Xvelo because it is homologous
to zebrafish Bucky ball, which plays a crucial role in Balbiani
body organization (Bontems et al., 2009). Computational analysis of Xvelo sequence predicted an intrinsically disordered protein with no known domains, apart from a C-terminal positively
charged region that could bind RNA.
Quantitative western blotting using a peptide antibody against
the C terminus of Xvelo (Figure S2A) provided an estimate of

Xvelo

Mitochondria

Overlay

50 m

2 m
20 m

0.25 s

30 min

60 min

Xvelo-GFP

Prebleach

100

Intensity (A.U.)

Xvelo - WT

80
60
40
20
0
0

20
40
Time (min)

60

Figure 2. Xvelo Forms a Stable Matrix


(A) mRNA encoding for Xvelo-GFP was microinjected into stage I oocytes. MitoTracker Deep Red was used to label mitochondria. Oocytes were imaged live with
a laser scanning confocal microscope with a 403 water-immersion objective.
(B) Magnification of the Balbiani body in (A).
(C) Internal rearrangement of fluorescent Xvelo-GFP particles after half bleach over time.
(D) The fluorescent recovery of the half-bleached Xvelo-GFP in the Balbiani body in (C) and two other biological replicates is shown by quantification of fluorescence in bleached region over time. Fluorescent intensity changes in the bleached region per pixel over time were plotted after it was normalized for photobleaching by using an unbleached neighboring area and background subtraction.
See also Figure S2.

Xvelo concentration in oocytes of 8 mM (Figures S2B and S2C).


If all the Xvelo was concentrated in the Balbiani body, and
assuming an average late stage I oocyte diameter is 250 mm
and Balbiani body diameter is 60 mm, this corresponds to
560 mM in the Balbiani body. Compared with the published
concentrations of proteins in a frog egg, where the most concentrated proteins were alpha-1-antitrypsin (Serpina1) and actin
(Actg1) at 17.6 and 14.3 mM, respectively (Wuhr et al., 2014),
one can appreciate the exceptionally high concentration of Xvelo
in Balbiani bodies. This high local concentration of Xvelo,
together with its homology to the zebrafish protein Bucky ball,
makes it a likely candidate for a structural organizer of the Balbiani body.
Xvelo Forms a Stable Matrix in Balbiani Bodies
To investigate Xvelo function in Xenopus, we began by injecting
mRNAs encoding for Xvelo tagged with GFP (Xvelo-GFP) into the
oocyte cytoplasm. Xvelo-GFP mainly localized to the Balbiani
body with little soluble protein present in the cytoplasm (Figure 2A). More detailed analysis at higher magnification showed
that Xvelo-GFP filled the gaps between the mitochondria in the
Balbiani body (Figure 2B). These results suggest that Xvelo

may form a matrix in which mitochondria and other organelles


are embedded.
To test whether Xvelo was part of a stable matrix, we used
FRAP (fluorescence recovery after photobleaching) to probe its
dynamics. We again injected mRNA encoding for Xvelo-GFP,
and after overnight incubation, we imaged the oocytes. After
photobleaching Xvelo-GFP, maximum recovery of 20% was
seen after 1 hr (Figures 2C and 2D). We conclude that Xvelo matrix is highly stable, with slow turnover, consistent with a role as a
structural matrix.
Xvelos Association with the Balbiani Body Requires Its
Prion-like Domain
We next investigated how the Xvelo matrix is formed and held
together. Despite the lack of any conventional domains, Xvelo
has a PLD at its N terminus, which is detected by several prion
detection algorithms (Figure 3A; Figure S3A) (Alberti et al.,
2009; Lancaster et al., 2014; Toombs et al., 2012). It also has a
lysine/ arginine rich region at its C terminus that might act as
an RNA binding domain (Figure 3A). To assess the behavior of
these different parts of Xvelo in vivo, we performed a structurefunction analysis by dividing the protein into four fragments
Cell 166, 637650, July 28, 2016 639

A
NH2
1
WT

82

398

F4

K/R rich region


...KIKEQDKPPKKKGALK
SSKRKQTRT...

4D

NQPRPYFYAQP...GNPDDPDDSVAL

7D

NQPRPDDDAQP..GNPDDPDDSVAL

Fragment 3

Fragment 4

Xvelo-woF1

GFP

Fragment 2

779 aa
COOH

598
F3

F2

Prion-like domain
82
21
58
NQPRPYFYAQP...GNPYFPYYSVAL

Fragment 1

150

F1

Xvelo-WT

F1-7D

Xvelo-4D

F1-4D

Xvelo-7D

F1-WT

F1-7D

F1-WT

F1-4D

50 m

GFP

Ccy/CBb

0.8

50 m

t = 0.1 s

t = 10 min

0.2
0

GFP

Xvelo-WT

Zoom

F1-4D

F1- 4D

50 m

F1-7D

F1-7D
Fluorescence Recovery (% of initial)

0.4

F1-WT

F1-WT

Prebleach

0.6

100
80

WT
F1 - 4D
F1 - 7D

60
40
20
0
0

100 200 300 400 500 600


Time (s)

Figure 3. Xvelo Self-Assembly Is Dependent on Its Prion-like Domain


(A) Diagram of the known structural elements of Xvelo. Prion-like domain, mutants (4D and 7D) and the fragments of Xvelo (F1F4) are marked in the figure.
(B) mRNAs encoding for Xvelo fragments shown in (A) and Xvelo without fragment 1 (Xvelo-woF1) are in vitro synthesized and micro-injected into stage I oocytes.
Oocytes were imaged after overnight incubation in oocyte culture medium (OCM).

(legend continued on next page)

640 Cell 166, 637650, July 28, 2016

(see Supplemental Experimental Procedures for details; Data


S1A).
We in vitro synthesized mRNAs encoding for the four Xvelo
fragments tagged with GFP and injected them into oocytes at
equal concentrations. Each fragment of Xvelo localized differently (Figure 3B). The C-terminal F4 fragment, which carries
the positively charged region, localized to nucleoli. Nucleoli are
RNA-rich, thus this localization may reflect predicted RNA binding activity of the F4 fragment (Figure 3B). The N-terminal F1
fragment, which carries the PLD, localized to the Balbiani
body. Its localization pattern was indistinguishable from the
full-length protein (Figures 2A and 3B). An additional fragment
lacking the F1 fragment but retaining the rest of the Xvelo protein
(Xvelo-woF1) did not localize to the Balbiani body (Figure 3B).
Thus, we conclude that the N terminus, which contains the
PLD, is the key region that targets Xvelo to the Balbiani body.
We next designed mutants that would disrupt the propensity
of Xvelo for amyloid-like self-assembly. Charged amino acids
are strongly disfavored in PLDs, as they interfere with the formation of a hydrophobic nucleus (Alberti et al., 2009; Lopez de la
Paz and Serrano, 2004; Serio et al., 2000). Thus, to create defective PLD mutants of Xvelo, we mutated either four or seven aromatic residues in its PLD to a negatively charged amino acid
(aspartate) (Figure 3A). We call these 4D and 7D mutants,
respectively (Figure 3A). The mutants no longer scored positive
in prion detection algorithms (Figures S3B and S3C). We injected
mRNAs encoding for wild-type or mutant versions of Xvelo
tagged with GFP into the oocytes and imaged after overnight incubation. We used mRNAs encoding for both fragment 1 (the
PLD) and full-length Xvelo to observe the effects of the mutants
in the oocytes in case the full-length protein interferes with the
assembly pattern of the mutants. Fragment 1 proteins carrying
the 4D and 7D mutations still exhibited a partial localization to
the Balbiani body, but they also showed a diffuse signal in the
cytoplasm, which was not observed for the wild-type protein
(Figures 3C and 3D). The effect was much stronger for the 7D
mutant, which was barely enriched in the Balbiani body (Figures
3C and 3D). Full-length proteins behaved similar to their F1 counterparts (Figures 3D and S3D). Thus, we conclude that the PLD of
Xvelo is essential and sufficient for Balbiani body localization,
and the two conserved motifs enriched for aromatic residues
provide an essential structural role in Xvelo targeting.
To investigate whether the mutations in the Xvelo PLD change
the association dynamics of the GFP-tagged constructs with the
endogenous Xvelo matrix, we performed FRAP on wild-type and
mutants. Fragment 1 had a slow turnover rate, similar to the fulllength protein (Figures 2C, 2D, 3E, 3F, S3E, and S3F). However,

the mutants recovered from photobleaching significantly faster


than wild-type; the 100% recovery time was 10 min for 4D and
3 min for 7D mutants after photobleaching (Figures 3E and 3F).
(This should be compared to the wild-type, which recovers to
20% after 1 hr in Figures 2C and 2D.) The recovery times of
the full-length mutants were similar to the F1 fragments (Figure S3F). These results suggest that the PLD drives the association of Xvelo with the pre-assembled Xvelo in the matrix.
If the mutants change the association dynamics of Xvelo with
the matrix, they might impose a dominant-negative effect (i.e.,
inhibit the assembly of wild-type Xvelo into the matrix). To test
this, we co-injected mRNAs encoding for full-length Xvelo-WTmCherry and GFP-tagged F1 wild-type and mutants at equal
concentrations and imaged by live confocal microscopy the
next day. Wild-type Xvelo co-localized with the F1 fragment in
the Balbiani body, and there was little signal in the cytoplasm
(Figure 3G). However, in the presence of the 4D mutant, the
wild-type Xvelo also formed discrete small aggregates in the
cytoplasm (Figure 3G). The 7D mutant also increased the amount
of diffusely localized wild-type Xvelo, but it did not cause the
punctate localization pattern seen with the 4D mutant (Figure 3G). The difference between the mutants may be due to
the fact that the 4D mutant binds more strongly to wild-type
Xvelo because of its remaining hydrophobic motif. The same
patterns were observed when the experiments were repeated
with full-length mutants (Figure S3G). Thus, we conclude that
mutant PLDs can partially inhibit the self-assembly of wild-type
Xvelo, most likely by binding to it and blocking growth into larger
assemblies.
Recombinant Xvelo Forms Micron-Scale Networks
In Vitro
To test whether Xvelo can form amyloid-like fibers on its own, we
analyzed recombinant Xvelo-GFP in vitro. Xvelo-GFP did not express at all in bacteria (data not shown) but expressed well in baculovirus-infected insect Sf9 cells. Purification of Xvelo-GFP
from insect cells was challenging due to its strong tendency to
aggregate, but we found that addition of 300 mM arginine to
Xvelo-GFP solubilized it in an otherwise physiological buffer (Figure S4A). The guanidino fragment of arginine may function
similar to guanidinium ion as a solubilizing agent (England and
Haran, 2011; Tsumoto et al., 2004).
We purified soluble Xvelo-GFP in 300 mM arginine and then
diluted into 30 mM arginine, which induced self-assembly (Figure 4A, first panel). Xvelo-GFP self-assembled first into small,
then large micron-scale networks over time (Figure 4A, first
panel). To test if network assembly required the PLD of Xvelo,

(C) mRNAs encoding for wild-type and PLD mutants of fragment 1-GFP were microinjected into oocytes. Oocytes were incubated overnight and imaged.
(D) Ratio of GFP concentration in the oocyte cytoplasm (Ccy) to the Balbiani body (CBb) in oocytes injected with mRNAs encoding for indicated proteins. Relative
concentrations were calculated by using oocyte or the Balbiani body volume from z stacks and the fluorescent intensity of GFP. Mean values and SEs of 10
oocytes are plotted.
(E) Internal rearrangement of fluorescent wild-type or mutant F1-GFP particles after photobleaching over time.
(F) The fluorescent recovery of photobleached wild-type or mutant F1-GFP in Balbiani bodies in (E) and two other biological replicates for each are shown by
quantification of fluorescence in bleached region over time normalized by an unbleached neighboring region.
(G) mRNAs encoding for full-length Xvelo-mCherry wild-type and GFP-tagged fragments (F1-WT-GFP, F1-4D-GFP, and F1-7D-GFP) were in vitro synthesized.
F1-WT-GFP or mutants were mixed with equal amounts of full-length Xvelo-mCherry mRNA and microinjected into the oocytes. After overnight incubation, the
oocytes were imaged by scanning confocal microscopy.
See also Figure S3.

Cell 166, 637650, July 28, 2016 641

Xvelo-WT

0 hour

1 hour

2 hours

3 hours

24 hours

98
62
49
38
28

Total Network Mass (A.U.)

Xvelo-7D-GFP

C
F1-WT-GFP

kDa
198

Xvelo-4D-GFP

Xvelo-WT-GFP

Xvelo-7D

Xvelo-4D

F1-WT

40 m

150

100
Xvelo-WT
F1-WT
Xvelo-4D
Xvelo-7D

50

0
0

24

Time (h)

Figure 4. Xvelo Forms Micron-Scale Networks In Vitro


(A) 15 mM of recombinant Xvelo-GFP, F1-WT-GFP, and full-length mutants (Xvelo-4D-GFP and Xvelo-7D-GFP) were diluted into a low-arginine buffer (30 mM) to
promote their self-assembly. The reaction mixtures were incubated at 25 C for the indicated time intervals and squashed under a coverslip to be imaged by
spinning-disc confocal microscopy.
(B) Coomassie-stained gels depicting recombinant Xvelo-GFP, Xvelo-4D-GFP, Xvelo-7D-GFP, and F1-WT-GFP.
(C) Quantification of networks in (A). 20 images were taken and stitched together, a threshold was applied, and the network intensities were measured. The
integrated intensity of networks per sample at each time point (total network mass) is plotted. Means and SEs of three biological replicates are shown.
See also Figure S4.

we expressed and purified the F1 fragment containing the PLD.


F1-WT-GFP also formed large networks over time, with striking
similarity to networks formed by full-length Xvelo, showing that
network assembly is driven by the PLD. We confirmed the critical
role of the PLD by showing that two full-length PLD mutants,
Xvelo-4D-GFP and Xvelo-7D-GFP, did not form networks (Figure 4A, lower panels) (Figure 4B).
To quantify Xvelo self-assembly in vitro, we tested Xvelo concentrations similar to its physiological concentration (Figure S2C). To balance between the aggregation propensity of
Xvelo at high concentrations and the measured physiological
concentration of 8 mM in oocytes if it was uniformly distributed,
642 Cell 166, 637650, July 28, 2016

we chose a stock concentration of 15 mM for each recombinant


protein so that after 10-fold dilution, the Xvelo concentration in
the mixture was 1.5 mM. We quantified total network mass by
summing the fluorescence intensity of networks in an area corresponding to a total of 20 images (Figures 4B and S4B). This analysis further confirmed that the F1 and full-length Xvelo-GFP have
indistinguishable kinetics of network formation while the PLD
mutants could not form any networks (Figure 4C). Xvelo-4DGFP formed small precipitates after dilution out of high arginine,
but these precipitates remained in solution without forming networks after overnight incubation (Figure 4A, third panel). Xvelo7D-GFP was completely soluble with no precipitates upon

Control

1 % SDS

0.1h
12h
24h

1000

Xvelo-WT

900

ThT (A.F.U)

800

40 m

700
600
500
400
300

F1 - WT

200

ThT

Blank

Mot3

Xvelo-7D

Xvelo-4D

F1- WT

Xvelo

Overlay

Arbitrary units (A.U)

E
EB1

Xvelo-7D

Xvelo-4D

F1 - WT

Xvelo-WT

Xvelo-WT

100

-OC

mCherry

500
400
300
200
100
0

Arbitrary units (A.U)

20 m

Ponceau

200

400
300
200
100
0

600

Egg extract, boiled

Oocyte st I, boiled

Egg extract, boiled

Oocyte stage I

Xvelo-WT

100nm

Xvelo-4D

400

SDS - PAGE

SDD - AGE

Egg extract

200

Distance (m)

Oocyte st I, boiled

600

ThT

10 m

400

500

-Xvelo

Ponceau

Figure 5. Xvelo Shows Amyloid-like Features In Vivo and In Vitro


(A) SDS was added to Xvelo and F1-WT-GFP networks to a final 1% concentration, and the reactions were incubated at room temperature for 15 min. The
resulting mixtures were squashed under a coverslip and imaged by a spinning-disc confocal microscope.
(B) A final concentration of 5 mM of Thioflavin T was added to the wild-type, F1, and mutant network reactions at the indicated time points. Yeast prion Mot3 was
used as a positive control, whereas blank was only buffer and ThT. ThT fluorescence was measured (a.u.) by a fluorescence plate reader.
(C) 1 mg RFP-tagged wild-type, F1, and mutant recombinant proteins were dot-blotted on a nitrocellulose membrane and assayed for reactivity with a-amyloid
fibril OC. EB1-RFP was used as a negative control.
(D) Negative stain electron microscopy images of the untagged Xvelo-WT and Xvelo-4D self-assembly reactions (scale bars, 100 nm).

(legend continued on next page)

Cell 166, 637650, July 28, 2016 643

dilution, indicating its complete inability to self-assemble. We


also found that the assembly kinetics of Xvelo networks were
dependent on Xvelo concentration (Figure S4C). We conclude
that Xvelo can form networks on its own in vitro and that the
Xvelo PLD is essential and sufficient for the formation of these
networks.

yloid-like networks in stage I oocytes and in vitro. Although Xvelo


is present in the eggs at a detectable, albeit a much lower, concentration of 80 nM (Figures 5F and S2D), it does not form
SDS-resistant assemblies in the eggs (Figure 5F). Thus, the amyloid-like characteristics of Xvelo are transient and regulated
during development.

Xvelo Networks Exhibit Amyloid-like Properties In Vitro


and In Vivo
We next tested whether the Xvelo networks we see in vitro have
amyloid-like properties. For this purpose we tested a number of
different criteria. First we showed that both Xvelo-WT and F1GFP networks were resistant to SDS treatment (Figure 5A). We
noticed a partial solubilization of the Xvelo network by SDS (Figures 5A and S5A), suggesting the presence of a more stable fiber
backbone, which is decorated with more loosely associated
Xvelo that can be released by treatment with SDS. Next we
monitored acquisition of Thioflavin T (ThT) fluorescence, using
RFP-tagged Xvelo, fragment 1 and mutants Xvelo-4D and
Xvelo-7D, and the yeast Mot3 prion as a rapidly aggregating positive control (Alberti et al., 2009). F1 and full-length Xvelo networks quickly acquired ThT fluorescence in a PLD-dependent
manner (Figure 5B). We supported these findings by showing
that full-length and F1 networks were recognized by an anti-amyloid fibril antibody, whereas the mutants or an unrelated protein
(EB1-RFP) were not (Figure 5C). Finally, negative-stain transmission electron microscopy (TEM) revealed that Xvelo-WT forms fibers reminiscent of amyloids in vitro in a PLD-dependent manner
(Figure 5D).
To confirm that the Xvelo matrix behaves like an amyloid
in vivo, we examined whether Xvelo staining coincides with the
strong staining of the Balbiani body with ThT. For this purpose,
we injected oocytes with mRNA encoding for Xvelo-mCherry,
incubated the oocytes with ThT, and imaged them by live
confocal microscopy. Xvelo and ThT signals overlapped strongly
(Figure 5E). Considering Xvelo is the only enriched protein in the
Balbiani body with a PLD and that the endogenous Xvelo concentration exceeds 500 mM in the Balbiani body, the majority
of the ThT signal likely comes from endogenous Xvelo.
Next, we used semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) to check the solubility of Xvelo. SDD-AGE
allows the resolution of a wide size range of SDS-resistant aggregates (Alberti et al., 2010; Bagriantsev et al., 2006). We collected
stage I oocytes and used egg extracts as a comparison. Indeed,
Xvelo formed SDS-resistant aggregates in vivo detected by the
SDD-AGE gel. Moreover, it did not form any detectable SDSresistant aggregates in the mature egg (Figure 5F).
Therefore, because Xvelo forms SDS-resistant, filamentous
assemblies that bind ThT in a manner that depends on the presence of its prion-like domain, we conclude that Xvelo forms am-

Prion-like Domain Specificity for Targeting to the


Balbiani Body
To examine whether other proteins with prion-like domains
might also be involved in organizing the Balbiani body, we first
looked in our mass spectrometry list for other proteins with a
PLD. However, apart from the common contaminant yolk protein, vitellogenin, none of the other enriched proteins in the Balbiani body were predicted to contain a prion-like domain.
To investigate whether Xvelo is unique in its ability to form a
stable matrix in the Balbiani body, we selected five RNA binding
proteins with prion-like domains, namely CPEB3, Dazap1, FUS,
hnRNPA1, and Tia1, as well as the aggregation-prone mutant of
FUS, FUS-156E, and injected mRNAs encoding for GFP-tagged
versions of these proteins into the oocytes. Apart from CPEB3,
all these proteins are present naturally in Xenopus eggs and oocytes (Wuhr et al., 2014). Among these proteins, hnRNPA1,
CPEB3, FUS, and FUS156E did not localize to the Balbiani
body (Figure 6A). Tia1 and Dazap1 localized to the Balbiani
body (as well as the cytoplasm and nucleus, respectively). However, fast turnover rates of both Tia1 and Dazap1 after photobleaching strongly suggest that they do not incorporate into a
stable matrix (Figures S6A and S6B). Both Tia1 and Dazap1
are implicated in translational repression by binding to 30 UTRs
of mRNAs (Dixon et al., 2003; Steger, 2001), and thus, their localization pattern can be explained by their binding to the repressed
mRNAs in the Balbiani bodies upon overexpression.
Our experiments suggest that the PLD of Xvelo has specific
features to target to and form a stable matrix in the Balbiani
body. Part of the evidence that Xvelo structurally organizes Balbiani bodies is its sequence homology to zebrafish Bucky ball,
whose genetics pointed to such a function (Bontems et al.,
2009). To test if the two proteins exhibit similar sub-cellular dynamics, we expressed Bucky ball in Xenopus oocytes as a GFP
fusion and looked at the characteristics of incorporation of
Bucky ball into Xenopus Balbiani bodies. Bucky ball targeted
to the Balbiani body, co-localizing with Xvelo (Figure 6B). Bucky
ball turnover time after photobleaching was still a little faster
than Xvelo, but of a similar order of magnitude (Figures 6C
and 6D). Thus, we conclude that sequence features conserved
in the PLDs of Bucky ball and Xvelo are required for targeting to
the Balbiani bodies in oocytes. This experiment also provides
strong evidence that Bucky ball and Xvelo are functional
homologs.

(E) Stage I oocytes were injected with mRNA coding for Xvelo-mCherry and incubated overnight. The oocytes were incubated in 10 mM ThT, washed twice, and
imaged by confocal microscopy. Bottom: zoomed in images. Line scans showing the co-localization of Xvelo-mCherry and ThT stain from five Balbiani bodies
were plotted. Each color represents the line scan of a different Balbiani body. We speculate that the outer rim Xvelo-mCherry signal belongs to the newly
translated Xvelo-mCherry protein that has just started to form a new, immature matrix and does not yet stain with ThT.
(F) SDD-AGE detects SDS-resistant Xvelo aggregates in vivo. Equal amounts of cytoplasmic extracts of stage I oocytes and mature eggs were loaded onto SDSPAGE. A five times more amount of egg extracts was loaded for SDD-AGE gels to make Xvelo concentrations comparable between the oocyte and egg extract
lanes. Xvelo was detected by an anti-Xvelo antibody.
See also Figure S5.

644 Cell 166, 637650, July 28, 2016

hnRNPA1

Tia1

Dazap1

FUS

FUS-156E

DIC

GFP

CPEB3

50 m

GFP

GFP

Xvelo-mCherry

Merge

Xvelo-mCherry

FUS(PLD)Xvelo

FUS

Buc(PLD)Xvelo

Buc

FUS/Xvelo

Buc /Xvelo

Buc

Prebleach

t = 0.1 s

t = 15min

D
Fluorescence Recovery (% of initial)

50 m

100

Buc
Buc(PLD)Xvelo
FUS(PLD)Xvelo
Xvelo - WT

80
60
40
20
0
0

200

400
600
Time (s)

800

1000

Figure 6. Xvelo Has Unique Properties for Forming a Stable Matrix


(A) mRNAs encoding for GFP-tagged hnRNPA1, CPEB3, Tia1, Dazap1, FUS, and FUS156E were in vitro synthesized and microinjected into the oocytes. After
overnight incubation, the oocytes were imaged by laser scanning confocal microscopy.

(legend continued on next page)

Cell 166, 637650, July 28, 2016 645

To further investigate the specificity of the Xvelo PLD for targeting to the Balbiani body, we swapped the PLD of Xvelo with
the PLD of FUS, an unrelated prion-like RNA binding protein,
and with the PLD of Bucky ball. The resulting chimeric proteins
were named FUS(PLD)Xvelo and Buc(PLD)Xvelo, respectively
(Figure S6C). Buc(PLD)Xvelo localized to the Balbiani body,
with a FRAP time in between Xvelo-WT and Bucky ball-WT (Figures 6C and 6D). FUS(PLD)Xvelo localized to the cytoplasm and
weakly to the Balbiani body (Figure 6B). The Balbiani body-localized protein recovered quickly after photobleaching, with a halflife of 1 min, much faster than that observed for Buc(PLD)Xvelo
(2 hr) (Figures 6C and 6D). Taken together, these data provide
strong evidence that the prion-like domains of functional homologs Xvelo and Bucky ball have unique features that target and
form a stable matrix in the Balbiani body.
We next examined the specificity of PLD interactions in vitro
with an aggregation assay that compared the assembly properties of Xvelo and FUS. We repeated previous reports showing
that FUS-WT forms liquid droplets in vitro, whereas the aggregation prone mutant FUS-156E forms aggregates (Patel et al.,
2015). We mixed Xvelo-RFP with either FUS-GFP or FUS156E-GFP in vitro in a high-arginine, high-salt buffer and then
diluted out the arginine and salt to initiate aggregation. XveloRFP networks and FUS-WT-GFP droplets or FUS-156E-GFP aggregates formed in the vicinity of each other but clearly were
separate and did not interact with one another (Figure S6D).
Thus, we conclude that PLDs do not aggregate with one another
randomly, even when they are in close proximity at high
concentration.
Xvelo Binds RNA and Clusters Mitochondria
A key biological function of Xvelo during Balbiani body formation
is likely to be the binding and concentration of organelles and
RNA through its amyloid-like self-assembly. We looked for direct
evidence that Xvelo can form a network that is sufficient to bind
and concentrate mitochondria and RNA. To test whether XveloGFP networks can bind RNA, we used a non-specific control
mRNA coding for mCherry and an RNA that is enriched in Balbiani bodies, the Xenopus homolog of nanos, xcat-2 (Zhou and
King, 1996). Both of the RNAs bound to the networks (only
mCherry-RNA results are shown). In contrast, the F1 fragment
that lacks the C-terminal motif did not bind to either of the
RNAs. Thus, Xvelo networks can sequester RNA in a manner
that requires a putative RNA-binding domain at the C terminus
of Xvelo (Figure 7A), but apparently without any strong RNA
sequence preference in vitro.
Germ cells receive a pool of organelles from cyst cells to form
Balbiani bodies and become oocytes in mice (Lei and Spradling,
2016). In early-stage oocyte development, these organelles are

clustered into Balbiani bodies by an unknown mechanism. We


asked whether Xvelo can cluster organelles on its own in a
cell-free system and thus stimulate aspects of Balbiani body
reconstruction in vitro. For this, we used an established cellfree system, cytoplasmic extracts from Xenopus eggs with intact
actin, which contain abundant organelles and RNA, like the germ
cell environment prior to Balbiani body formation (Field et al.,
2014; Lei and Spradling, 2016). We labeled the mitochondria
with MitoTracker and added Xvelo-GFP to the Xenopus egg extracts to 1.5 mM final concentration. Xvelo-GFP self-assembled
as expected, and induced co-clustering of mitochondria (Figure 7B). Mitochondrial clustering activity depended on the PLD
of Xvelo (Figure 7B, second panel). When we treated these clustered mitochondria with 2 M KCl, mitochondria were still stably
bound to, and entrapped by, the Xvelo network (Figure 7B, third
panel). We used line-scans to quantify co-clustering of Xvelo assemblies and mitochondria over five frames, in an area extending
2 mm2 (Figure 7C). Note the similar line-scans for Xvelo-WT and
mitochondria, demonstrating co-clustering, and the lack of mitochondrial clustering with the 4D mutant, which does not selfassemble. Although fragment 1 can still form networks similar
to full-length networks in egg extracts, these networks cannot
recruit mitochondria (Figure S7A). This allows us to speculate
that entrapment of mitochondria is not based on the geometrical
properties of Xvelo networks.
As a control, we introduced FUS-WT and FUS-156E to egg extracts. Both FUS-WT and FUS-156E behaved as expected in the
egg extracts; WT formed droplets, and 156E formed aggregates.
Strikingly, FUS structures were avoided by mitochondria in the
egg extracts, as opposed to the clustering we see by Xvelo (Figure 7D). Thus, we conclude that mitochondrial clustering in egg
extracts is a specific property of Xvelo that requires its amyloid-like self-assembly.
DISCUSSION
Here, we show that Xvelo, a highly enriched protein in Xenopus
Balbiani bodies, forms a mitochondria-embedding, SDS-resistant matrix in vivo pervading the entire volume of the Balbiani
body in early-stage oocytes. Xvelo has a prion-like domain in
its N terminus, which is sufficient and necessary to target and
incorporate into the Balbiani body. Pure protein experiments
further support our in vivo data that Xvelo has amyloid-like properties. Our key functional experiment, namely clustering of mitochondria by Xvelo in egg extracts, is a reconstruction of aspects
of Balbiani body formation. The fact that Xvelo can cluster mitochondria but the prion-like domain mutant does not strongly suggests that this clustering is dependent on its prion-like domain.
Thus, these data suggest that the organelle-rich Balbiani body

(B) mRNAs encoding for Xvelo-mCherry and GFP-tagged Bucky ball (Buc), FUS, and the PLD-swap versions of Xvelo, in which the PLD of Xvelo was replaced
either by the PLD of Bucky ball (BucPLDXvelo) or the PLD of FUS (FUSPLDXvelo), were injected into the oocytes at equal concentrations and imaged after
overnight incubation.
(C) Internal rearrangement of fluorescent Bucky ball-GFP (Buc), and PLD-swap versions of Xvelo, Buc(PLD)Xvelo-GFP and FUS(PLD)Xvelo-GFP, after photobleaching over time.
(D) The fluorescent recovery of photobleached constructs in Balbiani bodies in (C), as well as Xvelo-WT and two other biological replicates for each, is shown by
quantification of fluorescence in a bleached region over time normalized by an unbleached neighboring region.
See also Figure S6.

646 Cell 166, 637650, July 28, 2016

RNA (546-14-UTP)

GFP

Mitochondria

Zoom/Merge

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+ 2M KCl
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Figure 7. Xvelo-GFP Aggregates Bind to RNA and Cluster Mitochondria


(A) Labeled RNAs, Xenopus nanos homolog xcat-2, and an mRNA encoding for mCherry protein were prepared using the MEGAscript SP6 kit with ChromaTide
Alexa Fluor 546-14-UTP. RNAs were added to pre-assembled networks and imaged with a spinning-disc confocal microscope.
(B) Recombinant Xvelo-WT-GFP or the prion-like domain mutant, Xvelo-4D-GFP, was added to Xenopus egg extracts with intact actin. Xvelo-GFP fills the gaps
between mitochondria (arrows, compare to Figure 2B). Mitochondria were labeled with Mitotracker Deep Red. Images were taken with a spinning-disc confocal
microscope. Bottom: the extracts were diluted with KCl so that the final KCl concentration was 2 M.

(legend continued on next page)

Cell 166, 637650, July 28, 2016 647

is organized by a functional amyloid into a dense matrix that sequesters mitochondria and other organelles.
The Balbiani body changes its properties during development:
it is a stable structure in the early oocyte, and it either disappears
in mammals (Hertig and Adams, 1967; Pepling et al., 2007) or
dissociates into small dispersed isles, called germ plasm, in
the mature oocytes of germ-plasm-containing species. This suggests that its formation and dispersal are regulated. We did not
detect any SDS-resistant Xvelo aggregates in egg extracts (Figure 5F), suggesting Xvelo does not form amyloid-like structures
in the egg. How this transformation occurs remains unclear. Our
preliminary data suggest that Xvelo is extensively phosphorylated in the egg, and most of these sites are not phosphorylated
in the oocyte. Regulation by phosphorylation could be a mechanism determining the dispersal of the Balbiani body, perhaps by
kinases or phosphatases that are activated at fertilization. Understanding the regulation of the physical state of Xvelo as the
oocyte matures will be important to elucidate the fate of the organelles residing in the Balbiani body.
Balbiani bodies are found in most vertebrates; mouse Balbiani
bodies were identified only recently, but Balbiani bodies in humans were observed decades ago, although they are almost untouched in the literature (Hertig and Adams, 1967; Pepling et al.,
2007). Could proteins similar to Xvelo be required for the formation of Balbiani-like structures in other organisms? In zebrafish,
bucky ball was identified in a maternal effect mutant screen as
the only gene that is essential for Balbiani body formation (Dosch
et al., 2004). Although the sequence similarity between Xvelo and
Bucky ball proteins are poor (Data S1B), these proteins have long
patches of intrinsically disordered regions, and score positive in
prion detection programs (Data S1C). Oskar is a key protein
required to organize Drosophila pole plasm (Ephrussi et al.,
1991), but no homologs of Oskar have been identified in vertebrates. We found that Oskar also is a disordered protein with a
predicted PLD (Data S1D). This suggests that amyloid-like selfassembly of a disordered protein could be an evolutionary
conserved mechanism for Balbiani body formation. Strikingly,
a recent paper has also linked the formation of large amyloidlike aggregates to gametogenesis in yeast, suggesting amyloid-like mechanisms may be involved in germline specification
across kingdoms (Berchowitz et al., 2015).
Despite the low sequence conservation of Xvelo and Bucky
ball (Data S1B), key residues in their PLDs are conserved, suggesting that these residues are structurally important and underlie the observed specificity of assembly. Xvelo does not interact
with other proteins with PLDs, such as FUS, upon self-assembly
in vitro. This is in contrast to the promiscuous behavior of many
disease-causing amyloids, which often show cross-seeding interactions and promote mutual nucleation events. We attribute
this to the fact that Xvelo self-assembly does not involve an intermediate liquid-like state. We speculate that fast assembly into an
inert amyloid-like structure prevents aberrant interactions with

other prion-like proteins, thus reducing the likelihood of a disease condition. We propose that rapid assembly kinetics and
high specificity are important driving forces underlying the evolution of functional amyloids.
What are the potential advantages of using an amyloid-like
mechanism to form the Balbiani body? One could imagine packing away germline components in amyloid-like structures is protective. The tightly packed structures could prevent other proteins from diffusing into them, such as regulators, thus keeping
the organelles in a dormant state. It could act to slow down the
diffusion of small toxic molecules generated by mitochondria,
which could be damaging. It also provides a novel way to organize the cytoplasm, forming a rigid, giant body, in which the organelles are clustered together into one place and kept there for
many years. Future studies are likely to provide mechanistic
insight into the central question of how the germline of an organism provides young cytoplasm with its complement of organelles
in every generation while the somatic cells age and die.
EXPERIMENTAL PROCEDURES
Detailed methods are available in Supplemental Experimental Procedures.
Oocyte Handling and Injections
All experiments using Xenopus and zebrafish were done with approval of the
Harvard Medical School (HMS) animal care review board. Ovaries were surgically removed from adult female Xenopus laevis frogs and treated with 2 mg/ml
collagenase 1A (Sigma) in 13 MMR by gentle rocking, until most of the oocytes
were clearly dissociated. Oocytes were later injected with mRNAs encoding
for indicated proteins by using a FemtoJet express microinjector (Eppendorf).
Xvelo Protein Purification from Insect Cells
Recombinant versions of MBP-Xvelo-GFP, -RFP, and no-fluorescent tag (for
negative-stain electron microscopy studies) were expressed in Sf9 insect cells
using the baculovirus expression system. Insect cells were harvested in lysis
buffer (50 mM HEPES [pH 7.6], 100 mM KCl, and 1 M arginine). The MBP
(maltose binding protein) tag was captured using dextrin Sepharose resin
and cleaved off using HRV 3C protease (MPI-CBG, in-house) by incubation
overnight on ice.
Microscopy
Differential interference contrast (DIC) and phase contrast microscopy for microinjections, perfusion chambers, and Balbiani body isolations were performed using a standard wide-field epifluorescence Nikon inverted microscope equipped with a Hamamatsu Orca CCD camera and 43, 103, and
203 dry objectives. Live confocal microscopy with oocytes was performed using Nikon A1R Laser Scanning confocal equipped with 103 dry and 403 water-immersion objectives. Spinning disc confocal images were taken with a Nikon Ti inverted microscope with Yokagawa CSU-X1 spinning disk confocal
with Spectral Applied Research Aurora Borealis modification, equipped with
203 dry, 403, and 603 oil-immersion objectives.
Semi-denaturing Detergent-Agarose Gel Electrophoresis
Stage I oocyte and egg extracts were prepared according to Field et al. (2014)
with intact actin. SDD-AGE was adapted from Alberti et al. (2009). The protein
concentrations of the lysates were adjusted and protein samples were mixed
with 43 sample buffer (80 mM Tris, 40 mM acetic acid, 2 mM EDTA, 20% [v/v]

(C) Line scans of Xvelo-WT-GFP and Xvelo-4D-GFP and mitochondria in Xenopus egg extracts. Five images were stitched together to have an area spanning
larger than 2 mm2. Each color represents a different field.
(D) Recombinant FUS-WT-GFP or the aggregation prone mutant, FUS-G156E-GFP, was added to Xenopus egg extracts with intact actin. Arrows point to the
exclusion zones of mitochondria in the presence of FUS structures.
See also Figure S7.

648 Cell 166, 637650, July 28, 2016

glycerol, 3% [w/v] SDS, and bromophenol blue) and incubated at room temperature for 15 min before loading onto a 1.8% agarose gel containing 13
TAE and 0.1% SDS. The gel was run in running buffer (13 TAE, 0.1% SDS)
at 90 V, followed by wet transfer to nitrocellulose membranes (Amersham Biosciences). Xvelo protein was detected by an anti-Xvelo antibody.

Bontems, F., Stein, A., Marlow, F., Lyautey, J., Gupta, T., Mullins, M.C., and
Dosch, R. (2009). Bucky ball organizes germ plasm assembly in zebrafish.
Curr. Biol. 19, 414422.

SUPPLEMENTAL INFORMATION

Dosch, R., Wagner, D.S., Mintzer, K.A., Runke, G., Wiemelt, A.P., and Mullins,
M.C. (2004). Maternal control of vertebrate development before the midblastula transition: mutants from the zebrafish I. Dev. Cell 6, 771780.

Supplemental Information includes Supplemental Experimental Procedures,


seven figures, one table, one movie, and one data set and can be found with
this article online at http://dx.doi.org/10.1016/j.cell.2016.06.051.
AUTHOR CONTRIBUTIONS
E.B. and T.J.M. conceived the project together. E.B. designed the experiments
with A.A.H., S.A., and T.J.M. All experiments were performed by E.B., except
for mass spectrometry analysis (M.W. and S.P.G.) and electron microscopy
(M.C. and M.R.). Protein expression and purification were performed by
M.R., D.D., and R.L. The manuscript was written by E.B. with input from
A.A.H., S.A., and T.J.M.
ACKNOWLEDGMENTS
We thank members of the T.J.M. and A.A.H. labs, especially Avinash Patel for
helpful discussions, Andrei Pozniakovski for cloning, and Christine Field for
help in making extracts. We would like to thank Doris Richter and Sonja
Kroschwald for technical assistance. We are grateful to the Protein Expression, Electron Microscopy and Image Processing facilities of the MPI-CBG
for their support. We would also like to thank the Nikon Imaging Center at
Harvard Medical School for microscopy support. Proteomic analysis was supported by NIH grant R01-GM103785 (PI Marc W. Kirschner). M.W. was supported by the Charles A. King Trust Postdoctoral Fellowship. This work was
supported by the MaxSynBio consortium, which is jointly funded by the
Federal Ministry of Education and Research of Germany and the Max Planck
Society (to A.A.H.) and NIH grant GM39565 (to T.J.M.).
Received: January 20, 2016
Revised: May 6, 2016
Accepted: June 29, 2016
Published: July 28, 2016
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Article

Compositional Control of Phase-Separated Cellular


Bodies
Graphical Abstract

Authors
Salman F. Banani, Allyson M. Rice,
William B. Peeples, Yuan Lin,
Saumya Jain, Roy Parker,
Michael K. Rosen

Correspondence
michael.rosen@utsouthwestern.edu

In Brief
What are the general principles that
define the composition of phaseseparated cellular bodies?

Highlights
d

Cellular bodies are organized by scaffolds and recruit clients


Clients bind to free sites in the scaffolds, and binding scales
with client valency
Relative scaffold stoichiometries control client recruitment in
switch-like fashion
Cells can control these parameters and thus regulate cellular
body composition

Banani et al., 2016, Cell 166, 651663


July 28, 2016 2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.cell.2016.06.010

Article
Compositional Control
of Phase-Separated Cellular Bodies
Salman F. Banani,1 Allyson M. Rice,1 William B. Peeples,1 Yuan Lin,1 Saumya Jain,2 Roy Parker,2 and Michael K. Rosen1,*
1Department

of Biophysics and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390, USA
of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80309, USA
*Correspondence: michael.rosen@utsouthwestern.edu
http://dx.doi.org/10.1016/j.cell.2016.06.010
2Department

SUMMARY

Cellular bodies such as P bodies and PML nuclear


bodies (PML NBs) appear to be phase-separated liquids organized by multivalent interactions among
proteins and RNA molecules. Although many components of various cellular bodies are known, general
principles that define body composition are lacking.
We modeled cellular bodies using several engineered multivalent proteins and RNA. In vitro and in
cells, these scaffold molecules form phase-separated liquids that concentrate low valency client proteins. Clients partition differently depending on the
ratio of scaffolds, with a sharp switch across the
phase diagram diagonal. Composition can switch
rapidly through changes in scaffold concentration
or valency. Natural PML NBs and P bodies show
analogous partitioning behavior, suggesting how
their compositions could be controlled by levels of
PML SUMOylation or cellular mRNA concentration,
respectively. The data suggest a conceptual framework for considering the composition and control
thereof of cellular bodies assembled through heterotypic multivalent interactions.
INTRODUCTION
Eukaryotic cells compartmentalize biological processes to
achieve spatial and temporal control over biochemical reactions.
Compartmentalization has long been studied in the context of
membrane-bound organelles, where mechanisms of biogenesis
and transport of molecules into and out of the organelle are well
understood. Cells also contain numerous membrane-less organelles, collectively referred to as cellular bodies (Mao et al., 2011).
These structures, which include P granules, P bodies, nucleoli,
and promyelocytic leukemia nuclear bodies (PML NBs), are
micron-sized assemblies of proteins and often RNA found in
the cytoplasm and nucleoplasm of eukaryotic cells. They appear
to be functionally important, as inferred from their conservation
among evolutionarily distant species (Handwerger et al., 2005)
and their tendency to concentrate functionally related groups
of molecules (Mao et al., 2011; Mohamad and Boden, 2010).
Ultrastructural analysis of cellular bodies suggests that they
are porous structures with densities comparable to those of

the nucleo- or cytoplasm (Handwerger et al., 2005). Analysis in


live cells has revealed that, macroscopically, the bodies persist
for hours to days. Yet, they are highly dynamic at the molecular
level, turning over their contents within seconds to minutes
(Dundr et al., 2004; Weidtkamp-Peters et al., 2008). Recent
work has demonstrated that bodies exhibit liquid-like properties
(Brangwynne et al., 2009; Brangwynne et al., 2011; Chen et al.,
2008; Kroschwald et al., 2015; Patel et al., 2015; Wang et al.,
2014). These and other behaviors suggest that cellular bodies
are condensed phases that form through liquid-liquid phase
separation of the nucleo- or cytoplasm (Hyman et al., 2014).
Cellular bodies are often enriched in multivalent molecules (Li
et al., 2012)proteins that harbor multiple modular domains or
stretches of low-complexity amino acid sequence with repeated
interaction motifs (Decker et al., 2007; Han et al., 2012; Kato
et al., 2012; Reijns et al., 2008) or charged elements (ElbaumGarfinkle et al., 2015; Nott et al., 2015); RNA species that contain
multiple protein-binding elements; or combinations thereof.
Interactions between multivalent macromolecules can drive
polymerization-driven phase separation (Banjade et al., 2015;
Fromm et al., 2014; Li et al., 2012; Mitrea et al., 2016; Nott
et al., 2015), resulting in the formation of a condensed, droplet
phase suspended in the bulk solution phase. It has been suggested that this fundamental macromolecular behavior may be
an important principle governing the organization of cellular
bodies (Fromm et al., 2014; Li et al., 2012; Mitrea et al., 2016;
Nott et al., 2015). Indeed, expressing engineered multivalent proteins or ectopically tethering high copy numbers of body components (high local valency) in cells is sufficient to form dynamic,
membrane-less puncta that resemble bona fide cellular bodies
(Chung et al., 2011; Kaiser et al., 2008; Li et al., 2012).
Cellular bodies typically contain tens to hundreds of types of
molecules (Buchan and Parker, 2009; Fong et al., 2013). Where
characterized in detail, only a small number of these components
appear to be essential for the structural integrity of the body
(Clemson et al., 2009; Hanazawa et al., 2011; Ishov et al.,
1999). We refer here to such molecules as scaffolds. In contrast,
the remaining majority of components are dispensable for body
assembly and often reside in the bodies only under certain conditions (Dellaire et al., 2006; Grousl et al., 2009). These molecules, which we refer to here as clients, often contain elements
that specifically bind to elements in the scaffolds, often via low
valency interacting elements of the same class as those in the
scaffolds (e.g., Chalupnkova et al., 2008; Lin et al., 2006). For
example, P bodies assemble in part via scaffolding interactions
between RNA binding proteins and RNA but also recruit several
Cell 166, 651663, July 28, 2016 2016 Elsevier Inc. 651

RNA binding proteins that are not important for P body assembly (Buchan and Parker, 2009). Within cellular bodies, clients
diffuse much more rapidly than scaffolds (Dundr et al., 2004;
Weidtkamp-Peters et al., 2008), suggesting that client-scaffold
interactions are more transient than the interactions among
scaffolds.
Compositional regulation is a general property of many cellular
bodies and may be crucial to their function. Cellular body compositions change during the phases of the cell cycle or in
response to stresses (Dellaire et al., 2006; Grousl et al., 2009).
Despite their importance, the fundamental principles governing
cellular body composition have been experimentally difficult to
elucidate, owing to the complex nature of both scaffolds and
clients and the diversity of species that reside within bodies.
However, simplified model systems composed of few types
of molecules, each with well-defined interaction elements, can
help isolate key molecular parameters and thus have the potential to reveal generalizable concepts.
Here, we describe the biochemical and cellular behavior of
three different sets of engineered molecules as simplified but
representative multivalent scaffolds and low valency clients,
which form model cellular bodies. Clients were differentially recruited into the bodies based on the relative stoichiometries of
the scaffolds. Changes in client recruitment occurred sharply
and on cellular timescales as the scaffold stoichiometries or valencies changed. Client partitioning also depended on client
valency. These findings lead to a simple mass action model
that predicts many features of the observed client partitioning
behavior and suggests how cellular body compositions could
be regulated in cells. Behaviors analogous to those of the model
systems were observed in PML NBs in mammalian nuclei and P
bodies in yeast cytoplasm. Thus, although natural cellular bodies
are complex, their compositions may be governed by simple underlying rules and could be altered based on parameters that are
easily tunable through cellular and evolutionary mechanisms.
RESULTS
Scaffold Stoichiometries Dictate Client Recruitment
We began by studying three independent pairs of interacting
multivalent scaffolds in vitro. These systems consisted of (1) a
protein with ten repeats of human SUMO3 (polySUMO) and a
protein with ten repeats of the SUMO Interaction Motif (SIM)
from PIASx (polySIM); (2) a protein with four repeats of the second SH3 domain from Nck (polySH3) and a protein containing
four repeats of a Proline-Rich Motif (PRM) from Abl1 (polyPRM)
(Li et al., 2012); and (3) the PTB protein (contains four RNA recognition motifs [RRMs]) and an RNA with five repeats of the RRM
recognition element UCUCU (polyUCUCU) (Li et al., 2012).
Each of these pairs phase separated when mixed together, but
not when individual components were alone in solution (Li
et al., 2012; Figure S1A; and data not shown).
To model client recruitment into the bodies, we engineered a
series of fluorescently labeled, monovalent clients (containing
a single element that binds the scaffold) and characterized their
partitioning into droplets generated by their cognate scaffolds.
We mixed (1) GFP-SUMO and RFP-SIM (or GFP-SIM) with
polySUMO/polySIM (Figure 1A); (2) GFP-PRM and RFP-SH3
652 Cell 166, 651663, July 28, 2016

with polySH3/polyPRM (Figure 1B); and (3) GFP-RRM and


UCUCU-AlexaFluor647 (AF647) with PTB/polyUCUCU (Figure 1C). Partition coefficients (PCs) for the clients, defined as
the ratio of concentrations in the droplet to the bulk phases,
ranged from 1 to 10 across the phase diagram (Figures S1B
and S1D). Client recruitment in all three systems was qualitatively
similar. Clients partitioned asymmetrically about the diagonal of
the phase diagram (the line of equal scaffold stoichiometry) or
near to it; each client was enriched only on the side where its
cognate scaffold was in stoichiometric excess in the solution.
For example, when polySIM was in excess (above the diagonal),
GFP-SUMO was enriched in the droplets (PC 3), but when
polySUMO was in excess (below the diagonal), GFP-SUMO
concentrated nearly equally in both phases (PC 1) (Figure S1B).
GFP-SIM showed an opposite pattern of enrichment (PC 3
when polySUMO was in excess; PC 1 when polySIM was in
excess). Recruitment preference transitioned sharply, in
switch-like fashion, as the diagonal was crossed. For the polySUMO/polySIM system, neither GFP alone nor clients mutated
at their binding sites were enriched in droplets on either side of
the diagonal (Figures S1B and S1C). Thus, binding to the scaffold
proteins is necessary and sufficient for enrichment into the
droplets (PC > 1).
Together, these data show that, regardless of the molecular
system, low valency clients partition asymmetrically into droplets formed by heterotypic scaffold interactions, with a sharp
switch in client recruitment preference across the diagonal.
Valency of Client Affects Client Recruitment
Since the clients of a given cellular body can differ in their valencies, we examined how client valency affected partitioning. We
fused to GFP 2 or 3 tandem repeats of SUMO or SIM and
measured the PC for these clients across the polySUMO/
polySIM phase diagram (Figure 2). Like their monovalent counterparts, the di- and trivalent clients partitioned into the droplets
predominantly on one side of the phase diagram, transitioning
sharply in their PCs across the diagonal. However, both the
di- and trivalent clients had larger magnitudes of maximum
partitioning than their monovalent counterparts, a feature that
increased with valency: max PC was 19 and 37 for GFP(SUMO)2 and GFP-(SUMO)3, respectively, and 21 and 61 for
GFP-(SIM)2 and GFP-(SIM)3, respectively. In all cases, maximum
partitioning occurred just past the diagonal, substantially
enhancing the sharpness of the switch between client preferences. The increased partitioning was likely due to higher
apparent affinity of the di- and tri-valent clients for the scaffold.
Indeed, isothermal titration calorimetry (ITC) experiments verified that apparent affinity of the clients to cognate sites increases
with increasing valency (Figure S2 and Table S1).
These data demonstrate that, in addition to position on the
phase diagram, client valency can strongly influence client partitioning and thus droplet composition.
Mass Action Explains Switch-like Partitioning of Low
Valency Clients
We sought to understand the origin of the switch-like nature of
client partitioning. Our data suggest that partitioning depends
strictly on SUMO-SIM interactions between clients and scaffolds

A
GFP-SUMO

SIM Module
Concentration (M)

Figure 1. Phase Diagram Position Dictates


Client Recruitment

polySUMO + polySIM
RFP-SIM

Merge

90
80
70
60
50
50

60

70

80

90

50

60

70

80

90

50

SUMO Module Concentration (M)

60

70

80

90

Scale: 100 m

Solutions of multivalent scaffolds plus the indicated clients were imaged for client fluorescence.
AF, Alexa fluorophore.
(A) GFP-SUMO (green) and RFP-SIM (magenta)
(100 nM each) were mixed with the indicated
module concentrations of polySUMO and polySIM.
(B) GFP-PRM (green) and RFP-SH3 (magenta)
(200 nM each) were mixed with the indicated
module concentrations of polyPRM and polySH3.
(C) UCUCU-AF647 (green) and RFP-RRM
(magenta) (200 nM each) were mixed with the
indicated module concentrations of polyUCUCU
and PTB.
See also Figure S1.

polyPRM + polySH3
GFP-PRM

RFP-SH3

Merge

RRM Module
Concentration (M)

SH3 Module
Concentration (M)

sites accessible to its cognate client.


Conversely, the scaffold that is stoichiometrically deficient will effectively be
350
saturated by scaffold-scaffold interac300
tions and be invisible to its cognate client
in either phase.
250
The scaffold composition of the droplet
and bulk phases varied in a smooth,
200
graded fashion across the phase dia200 250 300 350 400
200 250 300 350 400
200 250 300 350 400
gram, with PCs ranging from 30125
PRM Module Concentration (M)
Scale: 100 m
(Figures 3A and 3B). For most of the
phase diagram, the PC of the two scafC
folds was similar (within an 2-fold
polyUCUCU + PTB
range), such that the droplets were
UCUCU-AF647
RFP-RRM
Merge
essentially concentrated counterparts of
70
the bulk solution (Figure 3C). Thus, at
each point in the phase diagram, the
60
scaffold in excess has a higher concentration of free sites in the droplet than in
50
the bulk (Figure 3D) and consequently
40
concentrates its cognate client into the
droplets. The scaffold that is stoichiomet30
rically deficient has few free sites in either
20 30 40 50 60
20 30 40 50 60
20 30 40 50 60
phase, and its cognate client remains
UCUCU Module Concentration (M)
Scale: 100 m
uniformly distributed. Since the stoichiometric relationship between the two
scaffolds switches abruptly in both
(Figures S1B and S1C). We reasoned that client partitioning phases near the diagonal, the capacity of the droplets to recruit
should be governed by the relative concentrations of available one client over the other also switches abruptly.
scaffold binding sites in droplets versus the bulk.
We modeled client partitioning by mass action (Figure 3E). We
The apparent dissociation constant for polySUMO/polySIM allowed clients to equilibrate between two simulated phases
(Kd % 1 nM, based on ITC measurements with (SUMO)5/ while binding to free sites at concentrations computed from
(SIM)5) is much less than the scaffold concentrations in either our experiments (Figure 3C, Table S1, and Supplemental Inforphase (Figure 3C), suggesting that most scaffold sites are occu- mation). This simple mass action model suffices to recapitulate
pied. Moreover, the apparent client-scaffold dissociation con- the key qualitative features of observed client partitioning (Figstants (Kd = 7010,000 nM, estimated from ITC measurements ure 3F): (1) selective partitioning of clients, restricted to only
with (SUMO)m + (SIM)m, for m = 1, 2 or 3) (Figure S2 and Table one side of the diagonal; (2) a sharp change in partitioning as
S1) are much weaker than the apparent polySUMO/polySIM af- the diagonal is crossed; and (3) the dependence of partitioning
finity. Thus, clients should be poor competitors of scaffold-scaf- on the apparent client-scaffold affinity. As described in the Supfold interactions. This analysis suggests that, in either phase, plemental Information, the model also predicts less intuitive feaonly the scaffold that is in stoichiometric excess will have free tures of the data, including non-monotonic partitioning, as well
400

Cell 166, 651663, July 28, 2016 653

polySUMO + polySIM
GFP-(SUMO)2

75
60
45
30
15
0
90

75
60
45
30
15
0
90

S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)

70

90

le
odu M)
50
OM

SUM tration (
cen
Con
60

75
60
45
30
15
0
90

S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)

70

80

90

Co

le
odu M)
50
OM

SUM tration (
cen
Con
60

70

80

90

75
60
45
30
15
0
90

S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)

le
odu M)
OM

SUM tration (
n
once

60

70

80

90

le
odu M)
50
OM

SUM tration (
cen
Con
60

GFP-(SIM)3
75
60
45
30
15
0
90

S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)

Co

Co
50

S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)

GFP-(SIM)2
Partition Coefficient

Partition Coefficient

GFP-(SIM)1

75
60
45
30
15
0
90

Co

Co
80

Partition Coefficient

S 80
nc IM 70
en M
60
tra od
tio ule 50
n
(
M
)

Co

GFP-(SUMO)3
Partition Coefficient

Partition Coefficient

Partition Coefficient

GFP-(SUMO)1

50

70

80

90

le
odu M)
OM

SUM tration (
n
once

60

50

70

80

90

le
odu M)
OM

SUM tration (
n
once

60

Figure 2. Client Valency Affects Partitioning


PCs (means of duplicate samples) of the indicated clients (100 nM) into droplets formed by the indicated module concentrations of polySUMO and polySIM.
See also Figure S2 and Table S1.

as dramatically high partitioning of one client and attenuated


partitioning of the other near the diagonal (Figure 3F; Figure 2,
trivalent clients; Figures S3 and S4).
Collectively, this analysis suggests that switch-like changes in
client partitioning fundamentally arise from the sharp inversion of
scaffold excess across the diagonal of the phase diagram.
Compositional States Interchange on Cellular
Timescales
Our data and analyses suggest how compositional states could
be controlled by mass action. We wondered whether transitions
between two compositional states were kinetically feasible on
cellular timescales. We equilibrated polySUMO/polySIM droplets at a point on the phase diagram where only one of the clients,
either GFP-SUMO or RFP-SIM, was preferentially enriched in the
droplets (but both were present in solution). We then abruptly
changed the concentration of the scaffold components to
move the system to a point across the diagonal where the reciprocal recruitment preference was expected (Figure 4A). The
droplets remained intact, spherical, and of relatively consistent
sizes throughout the experiment. Within 6 hr, all droplets
expelled the initially enriched client in exchange for the other
client (Figure 4B). Recruitment of the latter started at the outer
654 Cell 166, 651663, July 28, 2016

edges of the droplets and moved inward, and smaller droplets


exchanged clients more rapidly than larger droplets.
In fluorescence recovery after photobleaching (FRAP) experiments, clients diffused much more rapidly within droplets
than did scaffolds (i.e., for droplets 20 mm in diameter, RFPSIM and polySUMO had exponential recovery constants, t, of
1.3 min and 38 min, respectively; Figure S5 and Table S3).
Thus, scaffold rearrangements likely limit the rate of transitions
between compositional states. Scaling recovery times to droplets of 1 mm diameter, as often observed in cells, indicates that
compositions should interchange on a timescale of 6 s in
natural systems (Table S3).
We previously demonstrated how covalent modifications of
scaffolds could regulate the formation and dissolution of droplet
phases (Li et al., 2012). We likewise wondered whether covalent
modifications could also regulate droplet compositions. In cells,
SUMO modifications are dynamically added by the SUMO ligase
cascade and removed by SUMO proteases. We generated a
single component, fused (SUMO)9-(SIM)8 scaffold that could
be selectively cleaved by Ulp1, the yeast SUMO protease, to
produce (SUMO)7-(SIM)8, mimicking natural deSUMOylation
(see Experimental Procedures). Such (SUMO)m-(SIM)n (m s n)
fusions are essentially fixed on one side of the phase diagram

polySIM

Merge

90
80
70
60
50
50

60

70

80

90

50

60

70

80

90

50

60

70

SUMO Module Concentration (M)


polySUMO

polySIM
150
120
90
60
30
0
90

90

50

80
70
le
60
odu M)
OM

SUM tration (
n
ce
Con

90
80
70
le
60
odu M)
M
O

SUM tration (
n
ce
Con

50

Droplet

Free Sites

100

Clients

100

60
80

70
70

80
60

90 SUMO
50 SIM

Bulk
polySUMO
polySIM

60
80

70
60

80
50

Module Concentration (M)

90 SUMO
50 SIM

Partition Coefficient

102
polySUMO
polySIM

Droplet/Bulk Ratio

Module Concentration in Phase (M)

100
80
60
40
20
0
50
90

S 80
nc IM 70
en M
tra od 60
tio ule 50
n
(
M
)

Droplet

80

60

40

20
polySUMO
polySIM
0
50
90

60
80

70
70

80
60

Module Concentration (M)

80

60
100
40

20
102

90 SUMO
50 SIM

0
50
90

60
80

70
70

80
60

Kd, Client to Scaffold Affinity


(M Module)

3000
2500
2000
1500
1000
500
0
50
90

Bulk

Co

Co

Partition Coefficient

Partition Coefficient

150
120
90
60
30
0
90
S 80
nc IM 70
en M
tra od 60
tio ule 50
n
(
M
)

90

SUMO Client

80

Scale: 100 m

SIM Client

SIM Module
Concentration (M)

polySUMO

90 SUMO
50 SIM

Module Concentration (M)

Figure 3. A Mass Action Model Predicts Client Partitioning Behavior


(A) Imaging of polySUMO and polySIM (1% labeled with AF488 [green] and AF647 [magenta], respectively) fluorescence.
(B) PCs (means of duplicate samples) of polySUMO (left) and polySIM (right) calculated from imaging (see Experimental Procedures).
(C) Scaffold module concentrations (blue and yellow dots) in the droplet (top) and bulk (bottom) phases at the anti-diagonal data points from panel (B). To model
client partitioning, values of concentrations were smoothed and interpolated with a cubic spline to yield continuous curves from discrete data. The continuous,
interpolated values were used for subsequent calculations. Error bars represent SEM. Dotted line, phase diagram diagonal.
(D) Blue curve shows the ratio of free SUMO sites in the droplet phase to free SUMO sites in the bulk phase. Yellow curve shows the analogous ratio for free SIM
sites.
(E) Mass action model for the partitioning of a low valency client, L, that binds to free scaffold sites R1 and R2 in the droplet and bulk, respectively (see
Experimental Procedures).
(F) Predicted PC of clients as a function of affinity for scaffolds. Free site concentrations computed in (E) were used to parameterize the model (C) and predict
partitioning of client as a function of their apparent affinity (ranging from 102102 mM module) for the scaffolds (see Experimental Procedures).
See also Figures S3 and S4 and Table S2.

diagonal and have client recruitment preferences analogous


to the in trans systems (see Figure 5A). When mixed with
GFP-(SIM)2 and RFP-(SUMO)2, (SUMO)9-(SIM)8 droplets recruited the former but not the latter client (Figure 4C). Ulp1 cleavage, which shifted the scaffold to the other side of the phase
diagram diagonal, caused the droplets to expel GFP-(SIM)2
and recruit RFP-(SUMO)2. These data suggest that enzymatic

modifications of cellular body scaffolds, such as SUMOylation


and deSUMOylation, could robustly regulate body composition.
We conclude that droplets can transition, without compromising structural integrity, between substantially different compositional states on timescales accessible to cells. This can occur
with only subtle changes in the concentration or covalent modifications of their polymer scaffolds.
Cell 166, 651663, July 28, 2016 655

Figure 4. Droplets Interchange Composition on Cellular Timescales without Compromising Structural Integrity
(A) Schematic of experiment. After equilibration of 100 nM GFP-SUMO and 100 nM RFP-SIM with polySUMO and polySIM at module concentrations of 60 mM and
80 mM, respectively, concentrations of the polySUMO and polySIM were abruptly shifted to 80 mM and 60 mM, respectively, for trajectory 1 and vice versa for
trajectory 2.
(B) Time lapse imaging of droplets starting immediately after the abrupt change in concentrations of polySUMO and polySIM, showing merged, pseudocolored
fluorescence signals from GFP-SUMO (green) and RFP-SIM (magenta). Note that small droplets (white arrows, top) interconvert more quickly than larger droplets
(bottom).
(C) 6 mM of a (SUMO)9-(SIM)8 scaffold containing Ulp1 cleavage sites after only the two N-terminal SUMOs was equilibrated with 50 nM of GFP-(SIM)2 (green) and
RFP-(SUMO)2 (magenta). Time lapse imaging was started immediately after addition of 10 nM of Ulp1. Pseudocolored images showing merged fluorescent
signals from the two clients are shown.
See also Figure S5 and Table S3.

Engineered Cellular Puncta Selectively Concentrate


Low Valency Clients
We next asked whether the partitioning behavior observed
in vitro could also be observed in cells. For these experiments,
we used in cis [(SUMO)m-(SIM)n] scaffolds, which afforded tight
experimental control of the relative module concentrations independent of absolute concentrations. Both (SUMO)10-(SIM)5
and (SUMO)5-(SIM)10 phase separated in vitro at micromolar
concentrations. (SUMO)10-(SIM)5 droplets enriched GFP-SIM
(PC = 4.7), but not GFP-SUMO (PC = 1.3), and (SUMO)5(SIM)10 showed the reverse (PC = 2.8 for GFP-SUMO;
PC = 1.2 for GFP-SIM) (Figures 5A and 5B).
We then individually expressed RFP-(SUMO)10-(SIM)6 or RFP(SUMO)6-(SIM)10 in HeLa cells, where they each formed spherical, micron-sized puncta in the cytoplasm. In live cells, the
puncta occasionally contacted each other and coalesced into
larger structures, suggesting that they are phase-separated liquids (data not shown). When co-transfected with individual
YFP-tagged clients, RFP-(SUMO)10-(SIM)6 puncta only concentrated YFP-SIM. Reciprocally, RFP-(SUMO)6-(SIM)10 puncta
only concentrated YFP-SUMO. In both cases, neither YFP alone
656 Cell 166, 651663, July 28, 2016

nor clients with mutations at their binding sites were enriched


in cellular puncta (Figure S6A). We also obtained qualitatively
analogous data using co-expression of in trans polySUMO and
polySIM scaffolds, along with YFP-tagged clients (Figure S6B).
However, experimental uncertainties in the relative concentrations of the scaffold components made it difficult to assign cells
confidently to one side the diagonal.
Taken together, our data suggest that mass action-based
compositional control can be achieved as robustly in cells as
in vitro.
Scaffold Stoichiometries Control Client Recruitment
into Natural Cellular Bodies
We sought to determine whether natural cellular bodies could
exhibit compositional control analogous to our model systems.
We focused on two natural cellular bodies, PML NBs in mammalian
nuclei and P bodies in the yeast cytoplasm, systems in which the
interactions governing client recruitment were well characterized
and where their stoichiometries were experimentally perturbable.
PML NBs are micron-sized, membrane-less organelles in
mammalian nuclei that are involved in processes including

C
(SUMO)5-(SIM)10

Scaffold:

Scale:

RFP-(SUMO)6-(SIM)10

YFP-SIM

YFP-SUMO

YFP-SIM

Scale:

D
12

Scaffold
Client

10
8
6
4
2
0

GFP-SUMO

GFP-SIM

Scaffold
Client

10
8
6
4
2
0

GFP-SUMO

GFP-SIM

RFP-(SUMO)10-(SIM)6

RFP-(SUMO)6-(SIM)10

14
YFP-SUMO
YFP-SIM

12
10
8
6
4
2
0

10

12

Scaffold Partition Coefficient

14

Client Partition Coefficient

12

(SUMO)5-(SIM)10
Client Partition Coefficient

(SUMO)10-(SIM)5
Client Partition Coefficient

Client Partition Coefficient

RFP-(SUMO)10-(SIM)6
YFP-SUMO

Scaffold

Client:

GFP-SIM

Client

Scaffold
Client

GFP-SUMO

Scaffold

GFP-SIM

Scaffold

(SUMO)10-(SIM)5
GFP-SUMO

Client

Client:

Client

Scaffold:

14
YFP-SUMO
YFP-SIM

12
10
8
6
4
2
0

10

12

14

Scaffold Partition Coefficient

Figure 5. Cellular PolySUMO-PolySIM Puncta Selectively Recruit Low Valency Clients


(A) 60 nM of GFP-SUMO or GFP-SIM (green) was mixed with 12 mM of (SUMO)10-(SIM)5 (left) or (SUMO)5-(SIM)10 (right) (1% RFP-tagged; magenta), and the
resulting droplets were imaged for scaffold and client fluorescence.
(B) PCs for both scaffold (black bars) and clients (white bars) from experiment in (A). Graphs show averages from triplicate experiments. Error bars represent SEM.
Dotted line, PC = 1.
(C) Live cell fluorescence images of YFP-SUMO or YFP-SIM (green) co-transfected with RFP-(SUMO)10-(SIM)6 (left) or RFP-(SUMO)6-(SIM)10 (right) (magenta) into
HeLa cells.
(D) PCs of scaffolds and client components calculated from cells in the experiment. Each symbol represents the average PC into all puncta (typically 1-3) in a given
cell (12-35 cells per sample) when the indicated scaffold was co-transfected with YFP-SUMO (black circles) or YFP-SIM (white circles). Dotted line, PC = 1.
Red + sign, median PC.
See also Figure S6.

DNA damage repair, apoptosis, and anti-viral responses (Lallemand-Breitenbach and de The, 2010). The PML protein appears
to be the primary scaffold for these bodies (Ishov et al., 1999).
PML can self-assemble via elements within its Tripartite Motif
(TRIM) (Antolini et al., 2003; Huang et al., 2014) and also via binding of its conserved SIM element to SUMOs conjugated at up to
eight sites in the protein (Nisole et al., 2013; Shen et al., 2006).
Though not strictly required for body assembly (Brand et al.,
2010; Sahin et al., 2014), SUMO-SIM interactions likely
contribute substantially to body architecture, as deletion of the
SIM motif or perturbations to PML SUMOylation via mutagenesis, viral infection, or knockdown/overexpression of SUMO
ligases/proteases can cause changes in the size, number,
morphology, or dynamics of PML NBs (Best et al., 2002; Hattersley et al., 2011; He et al., 2015; Muller and Dejean, 1999; Shen
et al., 2006; Weidtkamp-Peters et al., 2008). SUMO-SIM interactions also appear to be critical for the recruitment of many PML
NB clients (e.g., Daxx and Sp100) (Lin et al., 2006; Van Damme
et al., 2010; Zhong et al., 2000).
We initially examined partitioning of GFP-tagged SUMO/SIM
clients into endogenous PML NBs in U2OS cells (Figure S7A).
Immunofluorescence imaging using an antibody against PML revealed numerous PML NBs in nearly all cell nuclei. For each

client, we measured the ratio of GFP fluorescence intensity


within the PML NBs to that in the bulk nucleoplasm (Intensity Ratio, IR = IntensityPML_NB/Intensitynucleoplasm, see Supplemental
Information). GFP-SIM was enriched in these bodies with a median IR of 2.9. In contrast, as previously reported for monovalent
SUMO clients (Ayaydin and Dasso, 2004), GFP-SUMO had little
enrichment in the PML NBs (median IR = 1.3). Increasing valency
increased the enrichment of the preferred client into PML NBs
(median IR = 8.1 for GFP-(SIM)3) but had no effect on the impartial client (median IR = 1.4 for GFP-(SUMO)3). Neither GFP alone
nor a client with mutated SIM sites were enriched (Figure S7B).
The selective, valency-dependent partitioning into PML NBs is
analogous to the behaviors of our polySUMO/polySIM model
system on the polySUMO-enriched side of the phase diagram diagonal (i.e., to that of (SUMO)10-(SIM)5, above the diagonal). Our
model predicts that PML NBs on the opposite side of the phase
diagram diagonal should exhibit inverted partitioning behavior
with respect to SUMO versus SIM clients. To create such
structures, we expressed either wild-type (WT) GFP-PML or a
PML mutant (GFP-PML(SUMO)) lacking some of the known
SUMOylation sites in PML/ mouse embryonic fibroblasts
(MEFs) (Figure 6A). The mutant protein is SUMOylated in cells,
but to a lesser degree than the wild-type protein (Figure S7F).
Cell 166, 651663, July 28, 2016 657

Scaffold:

RFP(SIM)3

GFP-PML(SUMO)

Client:

RFP(SUMO)3

Strain:

RFP(SIM)3

Xrn1-GFP
WT

lsm1

dcp2

Scaffold

Client:

GFP-PML
RFP(SUMO)3

Merge

Client

Intensity Ratio

Scale:
2 m

Scale:
10 m

10
8
6
4
2
0
1.88

3.71

4.08

Median

Client
Intensity Ratio

**
7
6
5
4
3
2
1
0

7
6
5
4
3
2
1
0
1.20

***

2.79

2.14

***

1.21

Median

***
***
***

Figure 6. Client Recruitment into Natural Cellular Bodies Is Affected by Scaffold Stoichiometries
(A) Images of RFP-SUMO or RFP-SIM (red) co-transfected with GFP-PML or GFP-PML(SUMO) (green) into PML/ MEFs (top); nuclear staining with Hoecsht
33342 (blue). Plots (bottom) show IRs from individual cells (black dots) and median values (red horizontal lines). Each symbol represents the average IR (see
Experimental Procedures) for all puncta in a given cell. 3244 cells were analyzed per sample, each on average containing 16 or 5 puncta per cell with GFP-PML or
GFP-PML(SUMO), respectively. Distributions were statistically compared using the Wilcoxon rank sum test followed by the Bonferonni correction for multiple
comparisons to determine significance. ***p < 0.001. Dotted line, IR = 1.
(B) Representative images of WT, lsm1D, or dcp2D yeast strains carrying Xrn1-GFP (green) in their genomes (top). Distributions of Xrn1-GFP IRs (bottom), where
each symbol represents IR corresponding to an individual P body. 13 P bodies per cell were analyzed from a set of 410 cells per sample. Analysis for
significance was performed as in (A). **p < 0.01.
See also Figure S7.

Both the WT and mutant scaffolds formed micron-sized puncta


in nuclei. Like natural PML NBs, GFP-PML puncta substantially
recruited RFP-(SIM)3 (median IR = 2.8), but not RFP-(SUMO)3
(median IR = 1.2). In reciprocal fashion, GFP-PML(SUMO) puncta
efficiently recruited RFP-(SUMO)3 (median IR = 2.1) but recruited
RFP-(SIM)3 poorly (median IR = 1.2). Neither scaffold could
recruit RFP alone nor clients with mutations at the SUMOor SIM-binding site (Figures S7C and S7D). Moreover, these results were independent of the SUMO paralog (SUMO1 versus
SUMO3) used to construct the client (Figure S7E).
These data suggest that decreasing the degree of PML
SUMOylation can shift the bodies to a region analogous to the
opposite side of the SUMO/SIM diagonal, where recruitment of
SUMO-containing clients is favored.
We next explored analogous compositional control in P
bodies, protein- and mRNA-rich cellular bodies in the cytoplasm
of eukaryotes that promote mRNA decay (Parker, 2012).
P bodies assemble through multivalent interactions of RNA
binding proteins composed of modular RNA binding domains
and self-associating disordered regions and mRNA molecules
(Decker and Parker, 2012). mRNAs that have exited translation
act as important P body scaffolds (Teixeira et al., 2005). We
658 Cell 166, 651663, July 28, 2016

thus asked whether modulating the levels of mRNA, thereby


affecting the relative stoichiometry of an important scaffold
component, could affect recruitment of clients into P bodies.
We used the lsm1D and dcp2D yeast strains, which are deficient
in mRNA decapping and therefore accumulate deadenylated
mRNAs that would otherwise be targets for degradation (Parker,
2012). We then measured the IR of the P body client Xrn1 (Jain
and Parker, 2013) fused to GFP (Xrn1-GFP), in the WT, lsm1D,
or dcp2D strains. Xrn1 is predicted to be recruited, at least in
part, by interactions with RNA. Compared to its recruitment
into P bodies in WT cells (median IR = 1.88), recruitment in the
lsm1D and dcp2D strains increased 2-fold (median IR = 3.71
and 3.71 and 4.08, respectively) (Figure 6B). This behavior was
qualitatively consistent with the behaviors of our engineered clients (Figure 1). The recruitment of the P body scaffold Edc3 (Jain
and Parker, 2013) also increased in the two deletion strains
concomitant with the increase in deadenylated mRNAs, consistent with the increased partitioning of scaffolds observed in
certain regimes of the phase diagram when the concentration
of the cognate scaffold increased (Figure 3B). Thus, these data
suggest that, like PML NBs, compositional control can be
achieved in P bodies by modulation of scaffold stoichiometries.

Figure 7. A Model for Compositional Control of Cellular Bodies

C
E

Collectively, these data indicate that the compositions of both


of these natural cellular bodies can be controlled by modulation
of stoichiometries of scaffold elements, analogous to the behaviors observed in our model systems. This behavior suggests
simple cellular mechanisms to rapidly and dramatically alter
the composition, and thus function, of cellular bodies in
response to stimuli.
DISCUSSION
Hierarchical Organization of Cellular Bodies
We propose a hierarchical model for the composition of cellular
bodies (Figure 7). The model has several key features. First, scaffolds self-associate by multivalent heterotypic interactions and
undergo assembly-driven phase separation (Li et al., 2012),
forming a condensed phase (Figure 7A)i.e., the cellular body.
Second, clients partition into bodies by interacting with scaffolds, often utilizing the same types of interacting elements as
those between scaffolds (Figure 7B). The typically lower valency
(and therefore lower apparent affinity) of clients minimizes their
competition with the higher affinity scaffold-scaffold interactions. As a result, clients are recruited by binding only to excess,
or free, scaffold sites. Thus, their recruitment will be governed by
the stoichiometric ratios of the scaffolds (Figure 7C). Third, since
the enrichment of excess sites switches sharply across the
phase diagram diagonal from one class of sites to the other,
bodies can change compositions in a switch-like manner as a
function of phase diagram position. Fourth, since scaffold and
client valencies can affect position on the phase diagram and
the degree of client partitioning, respectively, covalent modifications that change valency can be used to rapidly switch between
compositional states (Figure 7D).
We note that clients that bind regions of the scaffold that are
not involved in heterotypic assembly will be recruited but remain
relatively insensitive to changes in scaffold stoichiometries (Fig-

Multivalent scaffold molecules (high valency blue


and yellow molecules) assemble and phase separate to form the body (A). Many client molecules (low
valency blue and yellow molecules, with additional
domains) are enriched in the body through binding to
free cognate sites in the scaffold that is in stoichiometric excess (B). Client modules have a hatched
pattern to distinguish them from scaffold modules.
Stoichiometric excess of the scaffold modules can
be changed either through changes in the scaffold
concentrations (C) or through changes in
the scaffold valency (not shown). Since stoichiometric excess of the scaffolds in droplet (A) and bulk
(not shown) changes sharply across the phase diagram diagonal, the nature of the clients also switches
sharply across the diagonal. Higher valency promotes stronger recruitment of the clients (D). Molecules that bind to other regions of the scaffolds
(E, light blue trianguloids) will be recruited independently of the scaffold stoichiometry. Natural bodies
are composed of more complicated molecules, with
multiple types of interaction elements, but should
follow this same logic.

ure 7E). Moreover, molecules with otherwise appropriate physicochemical properties (e.g., complementary charge) may also
partition into droplets due to non-specific interactions (Li et al.,
2012). Clients containing multiple types of interaction elements,
some mirroring scaffold-scaffold interactions and others not,
could show complex behaviors that are essentially superpositions of these individual effects. This reasoning may explain
the recruitment of Xrn1-GFP into P bodies without perturbation
of mRNA content (i.e., on what may be the non-cognate side
of the phase diagram diagonal), as well as the enhanced
recruitment when cellular mRNA is increased, as observed in
Figure 6B.
Complexities of Natural Cellular Bodies
Although natural cellular bodies are appreciably more complicated than our engineered model systems, their compositions
may still be understood with simple extensions to the framework
we present here. First, cellular bodies may have multiple scaffolds held together by different types of multivalent interactions.
For example, RNA granules likely have multiple scaffolds with
contributions from both low-complexity sequence elements,
as well as RNA and RNA-binding domains. PML NBs likely
assemble by a combination of TRIM and SUMO-SIM interactions. Multiple types of scaffolds and scaffold interactions
may cooperate to synergistically promote polymerization and
phase separation, as suggested previously (Lin et al., 2015).
Moreover, clients may also possess multiple classes of low
valency elements that can each interact with scaffolds. Nevertheless, in the absence of cooperativity, one can think of the
different interaction motifs independently. For any given class,
the corresponding free sites in a scaffold will dictate partitioning
of clients that can bind to those sites. Indeed, perturbing one
type of interaction motif within PML NBs or P bodies had strong
effects on the recruitment of clients that bind to that motif
(Figure 6).
Cell 166, 651663, July 28, 2016 659

Second, for some systems, the distinction between scaffolds


and clients may be less stark than in our engineered systems
(see also Supplemental Information). As client valency approaches that of the scaffolds, this distinction breaks down,
and the client begins to compete with scaffold-scaffold interactions. For such clients, the change in partitioning across the
diagonal is likely to be less sharp, as we observe for scaffolds
(Figures 3A and 3B). Further investigations are needed to understand the distribution of scaffolds, clients, and such intermediate
molecules in the various known natural bodies.
Finally, several cellular bodies contain subcompartments
(condensed phases within the primary condensed phase) and
thus are not simple droplet/bulk systems (Brangwynne et al.,
2011; Feric et al., 2016; Jain et al., 2016; Wang et al., 2014).
Each subcompartment can have a unique composition organized by a distinct set of scaffold molecules. A client may bind
to free sites in any or all of the bodys subcompartments. Partition coefficients between any two sub-compartments or between a sub-compartment and the surrounding bulk will still
result from mass action driven by the corresponding free site
ratios.
Thus, despite the inherent complexities of natural cellular
bodies, they may still be understood through the lens of our simple model.
Biological Mechanisms of Regulating Body Composition
Biological processes could regulate the composition of
cellular bodies by acting on either scaffolds or clients on timescales ranging from physiologic to evolutionary. On the most
rapid timescales (seconds to minutes), covalent modifications
could change valencies of the scaffold components, shifting
the position of the system within the phase diagram. They
could also change valencies and affinities of the clients, influencing their degree of partitioning, as suggested here (Figure 2), and previously (Han et al., 2012; Kwon et al., 2013).
On slower timescales (hours to days), the scaffold concentrations could change via regulation of expression levels, or their
valencies could be changed by alternative splicing. On evolutionary timescales, changes in gene sequences could change
the affinity of scaffold components for each other or for clients, shifting composition and function in a more permanent
sense.
Some of these processes can be observed in PML NBs. For
example, the SUMOylation of PML is substantially decreased
during mitosis concomitant with loss of some SIM-containing
clients (Dellaire et al., 2006; Everett et al., 1999), and phosphorylation of the SIM in PML increases its affinity for SUMO (Cappadocia et al., 2015). Similarly, phosphorylation of the SIMs of PML
NB clients, including Daxx (Chang et al., 2011), increases their
interactions with the bodies.
Changes in Body Composition May Dictate Changes in
Function
Unlike macromolecular machines, cellular bodies continuously
rearrange the bonding interactions and organization of their constituent parts and thus are not stereochemically defined across
their lengths. Their functions, therefore, cannot be controlled
by allosteric transitions between conformational states, as
660 Cell 166, 651663, July 28, 2016

often occurs with macromolecules. Instead, transitions between


compositional states are likely to be key determinants of body
function. The differential partitioning of molecules in different regions of the phase diagram implies that it may be most appropriate to consider a given type of cellular body as a distribution
of entities (likely defined by a limited number of scaffolds) that
lie on a continuum of compositions subject to cellular control.
This idea was suggested previously for RNA-based bodies
based on the related compositions of P bodies, stress granules,
and RNA transport granules (Buchan and Parker, 2009). Similarly, in the case of PML NBs, a variety of structures in different
cell types and cell states have been characterized, unified by
their enrichment of the PML protein but varying in their composition of other components (Dellaire et al., 2006; Luciani et al.,
2006). Our data suggest that this behavior may be generally
applicable to many cellular bodies.
Since function is dictated by composition, this reasoning implies that cellular bodies may exhibit a continuum of functions,
rather than a limited set of discrete functions as seen for macromolecular machines in different conformations. Even though
cellular body function may be more continuous than discrete,
our data suggest that mechanisms could exist, as they do in canonical macromolecular machines, to mediate sharp switches
between different functional regimes. Moreover, we and others
(Molliex et al., 2015; Patel et al., 2015; Ramaswami et al.,
2013; Weber and Brangwynne, 2012) speculate that pathological states of cellular bodies may also lie on the same compositional and functional continuum. As such, manipulation or
depletion of certain scaffolds may be a promising approach to
mitigate the toxicities associated with these pathological granules. Indeed, in models of ALS, toxicities due to TDP-43 aggregation can be alleviated by removal of the Ataxin-2 scaffold
(Elden et al., 2010).
Implications for Cellular Body Function
Precise control of client partitioning could mediate colocalization
of reaction partners to accelerate reaction rates and increase reaction specificity. For example, polySUMOylation of cellular substrates was recently demonstrated to activate the ubiquitin E3
ligase RNF4, a process that could be driven or enhanced by
such compartmentalization (Rojas-Fernandez et al., 2014). Similarly, metabolic flux could be controlled by colocalization-mediated substrate channeling (Srere, 1987) or the colocalization of a
branch point enzyme and downstream molecules in a pathway
(Castellana et al., 2014). Compositional control may also help
regulate en masse reactions such as SUMOylation of many
cellular factors at PML NBs, which, analogous to DNA repair
foci (Psakhye and Jentsch, 2012), colocalize not only enzymes
of the SUMOylation cascade but also several SUMOylation
substrates (Van Damme et al., 2010). Partitioning into a cellular
body could also serve to sequester components away from their
cellular targets, as has been proposed in the regulation of Daxx
(Lallemand-Breitenbach and de The, 2010) and the priming of
RNA Polymerase II prior to transcription initiation (Kwon et al.,
2013). Indeed, strong depletion of clients from bulk solution
through dramatically high PC is consistent with behaviors we
observe in our mass action model (Figures 3F, S3E, and S3F
and Supplemental Information).

Conclusion
We demonstrate how cellular body assembly, when driven by
heterotypic polymerization and concomitant phase separation,
naturally leads to a simple and predictive model for compositional control of these structures. Our model suggests how
bodies could be switched sharply between distinct compositional (and thus functional) states on a range of biological timescales. Moreover, it suggests that superficially similar cellular
bodies composed of a given set of scaffolds may be markedly
different in their composition and function, depending on the
relative scaffold stoichiometries. Thus, a complete understanding of cellular bodies may require knowing relative scaffold
amounts in addition to scaffold identities.
Our studies thus provide a mechanistic framework for studying the biochemical and regulatory function of cellular bodies
owing to properties not attributable to any individual molecule
but rather to those intrinsic to the macroscopic structure itself.

Mass Action Model for Client Partitioning


Measured concentrations (from imaging) and affinities (from ITC, see Supplemental Information) of polySUMO and polySIM were used to calculate the free
sites concentrations in droplet and bulk phases. An equilibrium mass action
model was created to describe our systems as two compartments with unequal concentrations of receptors (free scaffold sites) and a permeable ligand
(client). The model was numerically solved using MATLAB and predicted PCs
were calculated as ratio of the total ligand concentration between the two
compartments (see Supplemental Information for details).
Cellular Experiments
For engineered polySUMO/polySIM and PML NB experiments, mammalian
cells (HeLa, U2OS, and PML/ MEFs) were cultured in DMEM supplemented
with 10% fetal bovine serum, 1% Penicillin-Streptomycin, and 1% GlutaMAX
in 5% CO2 at 37 C. Cells were transfected using Lipofectamine 2000 (Life
Technologies) and imaged 1824 hr after transfection. For P body experiments, WT and mutant yeast strains cells carrying Xrn1-GFP in the genome
were grown to log phase at 30 C in yeast minimal media supplemented with
a complete set of amino acids and 2% dextrose.

EXPERIMENTAL PROCEDURES

SUPPLEMENTAL INFORMATION

Genes, RNA, and Plasmids


polyPRM, polySH3, PTB, and the polyUCUCU RNA were described previously (Li et al., 2012). The RNA client UCUCU-AF647 (50 -UCUCUAAAAA-30 ;
30 -labeled with AF647), as well as (SUMO)5 and (SIM)5 as synthetic genes,
were purchased from Integrated DNA Technologies. Decavalent, fused, and
low valency SUMO/SIM constructs were constructed from (SUMO)5 and
(SIM)5 by PCR. To prevent conjugation and proteolysis, we mutated the C-terminal di-glycine motif in all SUMO proteins (see Supplemental Information).
The RRM client was constructed from the first RRM domain of PTB. The
mCherry, mEGFP, mVenus, and mCerulean (referred to as RFP, GFP, YFP,
and CFP, respectively) fusion proteins were produced by cloning into corresponding vectors (Clontech). Sequences of molecules used in this study are
listed in Table S4.

Supplemental Information includes Supplemental Experimental Procedures,


seven figures, and four tables and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2016.06.010.

Protein Expression, Purification, and Labeling


All purified proteins were expressed and purified similarly (see Supplemental
Information). Proteins were expressed in E. coli strain BL21 DE3T1R by induction with 1 mM IPTG. Proteins were purified with Ni-NTA Agarose Resin
(QIAGEN) or Amylose Resin (NEB), followed by ion exchange (Source 15Q
and/or Source 15S [GE Healthcare]) and size exclusion chromatographies using a Superdex 200 or Superdex 75 gel filtration columns (GE Healthcare). Proteins were labeled using maleimide-conjugated Alexa dyes (Life Technologies)
following the manufacturers protocol.
In Vitro Partitioning Assays
Scaffold molecules (1% Alexa-labeled) were mixed with GFP- or RFPtagged or Alexa-labeled clients in wells of chambered cover glass
(GraceBiolabs) or 384-well plates (Sigma) passivated with 30 mg/mL BSA
(Sigma). Mixtures were incubated for 2 to 4 hr for SH3/PRM and PTB/
RNA experiments and 2026 hr for SUMO/SIM experiments and imaged
at 203 magnification.
Image Acquisition and Analysis
Yeast cells were imaged using DeltaVision Elite microscope at 1003
magnification using a sCMOS camera. In all other experiments, imaging
was performed using spinning disk confocal microscopes equipped with
EMCCD cameras at 203 or 1003 magnification for in vitro or cellular experiments, respectively. Images were analyzed using ImageJ or MATLAB
(Mathworks) (see Supplemental Information). Fluorescence intensities
were calibrated to concentrations using standard solutions of purified client
molecules or corresponding fluorescent proteins, whose concentrations
were independently determined. When possible, care was taken to circumvent effects of the PSF in concentration determination (see Supplemental
Information).

AUTHOR CONTRIBUTIONS
Conceptualization, S.F.B. and M.K.R.; methodology, S.F.B. and M.K.R.; investigation, S.F.B., A.R., W.P., Y.L., S.J.; writing, S.F.B., A.R., R.P., M.K.R.; supervision, R.P. and M.K.R.
ACKNOWLEDGMENTS
We thank Louie Kerr, Kate Luby-Phelps, and Abhijit Bugde for assistance
with imaging; Chad Brautigam and Thomas Scheuermann for assistance
with ITC; Mark Kittisopikul for helpful discussions regarding image analysis,
numerical fitting, and statistical testing; Pier Paolo Scaglioni for providing
PML/ cells; Rama Ranganathan for critical reading of the manuscript;
and members of the Rosen lab for helpful discussions. This work was
supported by the Howard Hughes Medical Institute, the HCIA program
of HHMI, grants from the NIH (R01-GM56322) and Welch Foundation
(I1544), a Sara and Frank McKnight Graduate Fellowship (to S.F.B.), and
the NSF (1000196079; to A.M.R.).
Received: September 8, 2015
Revised: March 13, 2016
Accepted: June 1, 2016
Published: June 30, 2016
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Cell 166, 651663, July 28, 2016 663

Article

Pre-assembled Nuclear Pores Insert into the Nuclear


Envelope during Early Development
Graphical Abstract

Authors
Bernhard Hampoelz,
Marie-Therese Mackmull,
Pedro Machado, ..., Thomas Lecuit,
Yannick Schwab, Martin Beck

Correspondence
martin.beck@embl.de

In Brief
Rapidly growing embryos meet the
challenge of stocking the nuclear
envelope with nuclear pore complexes by
keeping a ready store of pores in stacked
membranous rings that feed into the
envelope without puncturing it.

Highlights
d

Annulate lamellae (AL) NPCs insert into the nuclear envelope


during interphase
AL-NPCs are pore scaffolds devoid of most transport
channel nucleoporins
NE-openings enable AL insertion, yet the permeability barrier
remains unperturbed
AL-NPC insertion operates only before gastrulation

Hampoelz et al., 2016, Cell 166, 664678


July 28, 2016 2016 The Author(s). Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.cell.2016.06.015

Article
Pre-assembled Nuclear Pores Insert
into the Nuclear Envelope
during Early Development
Bernhard Hampoelz,1 Marie-Therese Mackmull,1 Pedro Machado,2 Paolo Ronchi,2 Khanh Huy Bui,1,5 Nicole Schieber,4
Rachel Santarella-Mellwig,2 Aleksandar Necakov,1 Amparo Andres-Pons,1 Jean Marc Philippe,3 Thomas Lecuit,3
Yannick Schwab,2,4 and Martin Beck1,4,*
1European

Molecular Biology Laboratory, Structural and Computational Biology Unit, 69117 Heidelberg, Germany
Molecular Biology Laboratory, Electron Microscopy Core Facility, 69117 Heidelberg, Germany
3Aix-Marseille Universite
, CNRS, IBDM UMR 7288, 13009 Marseille, France
4European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, 69117 Heidelberg, Germany
5Present address: Department of Anatomy and Cell Biology, Groupe de Recherche Axe
sur la Structure des Proteines (GRASP), McGill
University, Montreal, Quebec H3A 0C7, Canada
*Correspondence: martin.beck@embl.de
http://dx.doi.org/10.1016/j.cell.2016.06.015
2European

SUMMARY

Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos,
NPCs reside not only within the NE, but also at
some endoplasmic reticulum (ER) membrane sheets,
termed annulate lamellae (AL). Although a role for AL
as NPC storage pools has been discussed, it remains
controversial whether and how they contribute to the
NPC density at the NE. Here, we show that AL insert
into the NE as the ER feeds rapid nuclear expansion
in Drosophila blastoderm embryos. We demonstrate
that NPCs within AL resemble pore scaffolds that
mature only upon insertion into the NE. We delineate
a topological model in which NE openings are critical
for AL uptake that nevertheless occurs without
compromising the permeability barrier of the NE.
We finally show that this unanticipated mode of
pore insertion is developmentally regulated and operates prior to gastrulation.
INTRODUCTION
In eukaryotes, the double membranous nuclear envelope (NE)
encloses the nucleoplasm and separates it from the cytoplasm.
The inner nuclear membrane (INM) provides contact with chromatin and the outer nuclear membrane (ONM) is continuous
with the endoplasmic reticulum (ER). The two bilayers are fused
at nuclear pore complexes (NPCs) that form aqueous channels
through which regulated transport of macromolecules occurs.
NPCs consist of multiple copies of 30 different nucleoporins
(Nups) that are organized into biochemically distinct sub-complexes (Figures S1A, S1A0 , and S1B). Two such modules, the inner ring complex (also called Nup93 complex) and the Y-complex (also called Nup107 complex) constitute the NPC scaffold
that is symmetric across the NE plane. FG-Nups (containing

phenylalanine-glycine rich intrinsically disordered protein domains) dock onto the scaffold. They constitute the permeability
barrier and interact with translocating cargo complexes. Some
of them (e.g., Nup214/88, Nup358 [RanBP2], and Nup153) introduce asymmetry by specifically binding to the cytoplasmic or nuclear face of the NPC, respectively (reviewed in Grossman et al.,
2012) (Figure S1B).
Obviously, the sheer size and compositional complexity of
NPCs renders its assembly and membrane insertion a very intricate task. Two distinct NPC assembly pathways that are temporally separated during the cell cycle have been described. First,
during interphase, NPCs are assembled de novo onto an enclosed NE (DAngelo et al., 2006). Interphase assembly occurs
ubiquitously throughout eukaryotes and strictly requires the
fusion of the INM and ONM by a mechanism that is only partially
understood (Doucet and Hetzer, 2010). Second, no membrane
fusion is required for NPC assembly at mitotic exit. This so-called
postmitotic assembly mode is restricted to eukaryotes that
disassemble their NPCs during mitosis into soluble sub-complexes after phosphorylation by mitotic kinases (Laurell et al.,
2011). In anaphase, de-phosphorylation of Nups is thought to
trigger the ordered re-assembly onto the separated chromatids
before or while membranes enclose daughter nuclei (Doucet
et al., 2010; Dultz and Ellenberg, 2010; Dultz et al., 2008). Both
insertion mechanisms rely on the stepwise recruitment of
pre-assembled sub-complexes. An insertion of pre-assembled
NPCs into the NE has (to the best of our knowledge) not yet
been described.
NPCs not only reside within the NE but are also found in
stacked cytoplasmic membranes termed annulate lamellae
(AL) that are a subdomain of the ER (Figure S1C) (Cordes
et al., 1996; Daigle et al., 2001). Based on two-dimensional
(2D) transmission electron micrographs these membrane stacks
have been perceived as parallel membrane sheets decorated
with NPCs (hereafter called AL-NPCs) that morphologically
appear similar to their counterparts on the nuclear envelope
(NE-NPCs) (Kessel, 1983). AL appear in some but not all transformed cell lines (Cordes et al., 1996; Daigle et al., 2001) and
are highly abundant in germ cells and early embryos throughout

664 Cell 166, 664678, July 28, 2016 2016 The Author(s). Published by Elsevier Inc.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Figure 1. AL-NPCs Insert into the Nuclear Envelope


(A and A0 ) Nuclear growth and NPC distribution during interphase in the Drosophila syncytium: Stills from a time-lapse movie recorded from a GFP::Nup107
expressing embryo imaged right after interphase onset (A) and 5 min later (A0 ). GFP::Nup107 localizes to the NE and to cytoplasmic foci (arrowheads) in (A), which
disappear at t + 5 min (A0 ).
(BB00 ) Cytoplasmic foci of Nup107 fluorescence localize to AL-NPCs. Correlative light and electron microscopy (CLEM) of a RFP::Nup107 expressing interphase
embryo. RFP::Nup107 is concentrated along the NE and at foci (boxed in B), that correspond to NPCs along ER membranes (arrowheads in B0 and B00 ).

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Cell 166, 664678, July 28, 2016 665

animal phyla, including Xenopus, Caenorhabditis elegans, sea


urchin, Drosophila, and also humans (Soupart and Strong,
1974). A role of AL as a storage compartment for maternally
deposited Nups that can be made available for meiosis and
the rapid cell cycles during early embryogenesis has been suggested (Lenart and Ellenberg, 2003; Longo and Anderson,
1968; Spindler and Hemleben, 1982) but not experimentally
proven. Despite these fundamental and long-standing pretensions the function of AL remains elusive and controversial, primarily for two reasons: (1) it has been difficult to conceive how
the insertion of parallel stacked membrane sheets containing
pre-assembled and possibly pre-oriented NPCs is topologically
possible; and (2) direct experimental evidence for a contribution
of AL-NPCs to the pool of NE-NPCs has never been obtained.
On the contrary a previous study in Drosophila embryos has detected large soluble pools of transport channel Nups and
concluded that NPC insertion likely proceeds from soluble cytosolic Nups (Onischenko et al., 2004).
Here, we address the function of AL in the physiological
context of the Drosophila blastoderm embryo that is rich in
AL, while it undergoes a series of 13 synchronized mitoses
in a syncytium (Figure S2A). Subsequently, the plasma membranes enclose the cortically aligned somatic nuclei in the
extended 14th interphase, forming the first epithelial cell layer
before the embryo initiates gastrulation (Schejter and Wieschaus, 1993). This occurs concomitantly with the broad
onset of transcriptional activity on the zygotic genome, a
major developmental transition present in all metazoan (Newport and Kirschner, 1982). In the syncytial blastoderm, cellcycle progression is very rapid, with interphase durations of
10 min during the early cell cycles. At least in mammalian
cells, de novo NPC interphase assembly has been described
to proceed with markedly slower kinetics (Dultz and Ellenberg,
2010). This led us to hypothesize that NPC assembly into a
closed NE in Drosophila embryos might occur by a different,
faster mechanism. By tracking NPCs in living embryos,
we demonstrate direct uptake of AL-NPCs into the NE, as
the ER feeds nuclear expansion. We derive a topological
model that explains how the INM becomes continuous with inserting membrane sheets from the ER. We conclude that AL
insertion to the NE is a previously unanticipated mode of

NPC insertion that relies on pre-assembled, yet immature


NPC scaffolds and operates prior to gastrulation.
RESULTS
Nuclear Pores Insert from the ER into the NE
To investigate whether AL-NPCs contribute to the pool of NENPCs, we conducted live-imaging experiments in Drosophila
blastoderm embryos before formation of the first somatic
cell layer (Figure S2A). During this stage of development, AL
are abundant and thus could potentially serve as a reservoir
for NE-NPCs. To track NPCs throughout early embryogenesis,
we expressed functional GFP or RFP fusions of Nup107
(Katsani et al., 2008) to image scaffold Nups and injected
sub-critical concentrations of the fluorescently labeled lectin
wheat germ agglutinin (WGA) to label FG-Nups. In interphase,
GFP::Nup107 localized to the NE and to prominent foci
throughout the cytoplasm (Figure 1A), similar to structures
that were previously characterized as AL (Cordes et al., 1996;
Daigle et al., 2001; Onischenko et al., 2004, 2005). As expected
these foci also stained positive for transport channel Nups (Figures 1D1D00 ) and always localized to membranes (Figures
S2CS2E00 ). To further confirm that these foci are morphologically identical to AL, we performed correlative light and electron
microscopy. RFP::Nup107 fluorescence was strongly enriched
along the NE and at AL (Figures 1B1B00 , S2B, and S2B0 ). We
conclude that fluorescence imaging in life embryos is wellsuited to study the spatiotemporal dynamics of annulate
lamellae.
Image quantification revealed a 2.5- to 3-fold increase in nuclear surface during interphases, indicating a considerable uptake of ER membranes within a few minutes (Figures 1A, 1A0 ,
and 1C). Thus, the surface area of the nucleus just before division
is more than twice as large as the combined surfaces of the two
daughter nuclei after division. This finding implies an excess of
nucleoporins with respect to the available nuclear surface after
mitosis. Both NE-NPCs and AL-NPCs disassemble during
mitosis (Cordes et al., 1996; Onischenko et al., 2005; Stafstrom
and Staehelin, 1984b). This leaves a fenestrated NE that, unlike
in vertebrates, stays throughout mitosis around the separating
sister chromatids and the mitotic spindle, except at centrosomes

(C) NPC density stays constant as nuclei grow. Quantification of normalized nuclear surface increase (blue curves) and the mean GFP::Nup107 fluorescence
intensity SD at the NE (red curves) during interphases. Values on both graphs are normalized to the earliest measured time point for each movie (n = 71 nuclei in
four embryos).
(DE) The Y-complex protein Nup107 and WGA-labeled transport Nups co-localize at the NE and at AL-NPCs that insert to the NE. Top view still (DD00 ) and
kymograph (E) from a time-lapse movie imaging a WGA-Alexa555 injected syncytial blastoderm embryo expressing GFP::Nup107 (see also Movie S2). Insertion is
captured in the kymograph (E) that spans the region of interest (ROI) boxed in (D).
(FG) Stills (FF00 ) and kymograph (G) of the blue shaded ROI from a time-lapse movie recording photo-converted EosFP::Seh-1 before (0 s, F) or after (6 s, F0 , and
145 s, F00 ) photo-conversion of AL-NPCs adjacent to the NE. NPC transfer from AL to the NE is documented by the lateral dispersion of the converted signal, which
starts after 100 s (G).
(H) AL-NPC number drops during early interphases. Quantification of the relative AL-NPC number inferred from GFP::Nup107 fluorescence for four embryos liveimaged over interphases. AL-NPCs were counted from 1 mm distant z sections spanning nuclear height in a constant field of view comprising 10 nuclei for each
embryo.
(I) GFP::Nup107 fluorescence intensity shifts from AL to the NE in the first 25% of interphases, when AL number drops the most (H). Fluorescence intensities were
integrated over consecutive confocal slices covering the entire nuclear height at the NE (NE-NPCs) and at AL (AL-NPCs). Cytoplasmic GFP::Nup107 was
determined from the mean fluorescence intensity. Analysis was done on two embryos (n = 13 and 11 nuclei, respectively). See Figure 1A for representative image;
error bars represent SD over multiple ROIs. All images in Figure 1 are acquired from embryos in cycles 1013 of the syncytial blastoderm stage.
See also Figure S1 and Movies S2 and S4.

666 Cell 166, 664678, July 28, 2016

Figure 2. AL-NPCs Resemble Pore Scaffolds


(A) Composition of NE-NPCs and AL-NPCs. Median intensity-based absolute quantification (iBAQ) scores of Nups detected in nuclei containing NE-NPCs,
microsomal membranes containing AL-NPCs and cytosol after fractionation of Drosophila embryos in the syncytial blastoderm stage (n = 3 biological replicates).
Nups are grouped into known subcomplexes and color-coded as represented in (E0 ).
(B) Western blot analysis of fractionated Drosophila syncytial blastoderm embryos. The Lamin Dm0 is exclusively nuclear (N), while a-tubulin is strongly enriched
in the cytoplasm (C), confirming fractionation quality. Detection with mAb414 recognizing a panel of FG-Nups reveals Nup358 predominantly in membranes (M)
and nuclei but absent from the cytosol. Other FG Nups are mostly soluble (see text). Amido black shows equal loading.

(legend continued on next page)

Cell 166, 664678, July 28, 2016 667

(Movie S1). Simultaneous with NE-NPC re-assembly at the


daughter nuclei in late mitosis, AL-NPCs appeared first at membranes at the spindle (Figures S2CS2C00 ; Movie S1), consistent
with the idea of excess nucleoporins at late mitosis/early
interphase.
To address if AL-NPCs contribute to the NE-NPC pool during
the following interphase, we first determined if NE-NPC density
decreases during nuclear surface expansion. We quantified the
mean fluorescence intensities of GFP::Nup107 at the NE and
found that it stayed almost constant (Figure 1C). As a consequence, NPCs have to insert constantly into the NE as its surface increases. Conversely, AL-NPCs were highly abundant in
early but not late interphase (Figures 1A and 1A0 ). We therefore
investigated their fate during interphase progression by
tracking AL-NPCs in living embryos and found that they insert
into the NE (Figures 1D1G and S2ES2E00 ; Movies S2 and
S3), along ER membranes (Figures S2ES2E00 ; Movie S3). To
directly confirm the transfer of Nups from the observed cytoplasmic foci to the NE, we imaged embryos expressing
photo-convertible Seh1-EosFP. After photo-conversion of a
fluorescent spot close to a nucleus, the signal remained locally
constrained for 100 s before it laterally resolved into the proximal NE over roughly the same time frame (Figures 1F1F00 and
1G; Movie S4), suggesting a critical event prior to lateral diffusion. We conclude that AL-NPCs insert into the NE during blastoderm interphases.
Previous EM-based morphometry suggested that the total
number of AL-NPCs stays constant in the syncytial blastoderm,
i.e., during the first 90 min of Drosophila embryogenesis (Onischenko et al., 2004). However, if AL-NPCs considerably
contribute to the pool of NE-NPCs by inserting into the NE while
it expands, their number should decrease at least temporarily on
much shorter timescales, namely during the 10 min of each
interphase. Indeed, we found that AL-NPCs diminished as interphases progressed (Figure 1H). This reduction was particularly
strong in the first half of interphase, when the rate of nuclear
growth and thus NPC insertion was highest (Figure 1C). To
estimate if the reduction of AL-NPC number reflects NPC redistribution from AL to the NE, we measured the respective
GFP::Nup107 fluorescence levels at both compartments in
three dimensions over time. For all quantified nuclei, the integrated NE fluorescence intensities of GFP::Nup107 increased
between 70% and 100% within the first quarter of interphase,
while inversely intensities at AL strongly decreased, mirroring
AL disappearance (Figure 1I). This supports a scenario in which
the pool of NE-NPCs is predominantly fed by integration of ALNPCs. Alternatively, AL could disassemble and soluble Nups
could add to pore formation at the growing NE from the cytoplasm. Yet this is unlikely since the GFP::Nup107 background
intensity in the cytoplasm remained constant as AL disassembled (Figure 1I). We conclude that our data rather support

a scenario in which pre-assembled NPCs insert from AL into


the NE.
There are, however, major impediments that challenge the
notion that intact NPC can insert into the NE: first, NPCs have
an inherent compositional directionality across the NE plane
(Figure S1B). If AL-NPCs were identical to NE-NPCs, they would
be assembled asymmetrically in the absence of a nuclear
compartment providing a directionality cue. They also had to
be inserted into the NE in the correct orientation. Second, the
integration of NPC-containing ER sheets into an intact NE poses
striking topological obstacles. In particular, how an AL membrane sheet can become continuous with the INM of a sealed,
intact NE is far from obvious. How is AL-NPC insertion thus
possible?
AL-NPCs Resemble Pore Scaffolds
The asymmetry of NPCs derives from sets of FG-Nups that are
found exclusively either at the nucleoplasmic or cytoplasmic
side of the NPC, in contrast to the symmetrically embedded
scaffold Nups of the inner ring and Y-complexes (Figures S1A
and S1B). We therefore explored if NPC composition was preserved in AL. We subjected blastoderm embryos to subcellular
fractionation and comparatively analyzed fractions enriched for
nuclei, microsomal membranes containing AL (devoid of the
NE) and soluble cytosolic proteins by quantitative mass spectrometry (Figures S3AS3D00 , S4A, and S4B). We found that
AL-NPCs contain the full set of NPC scaffold components,
namely all the members of the inner ring and Y-complexes
(Figures 2A and S4B). In contrast, their levels were low or undetectable in the cytosol, with the exception of Sec13, a known
member of the cytosolic coatomer complex (Fath et al., 2007)
(Figures 2A and S4B). These data further support the above-proposed scenario in which soluble pools of scaffold Nups cannot
significantly contribute to the maintenance of NE-NPC number
during interphase (see also Figure 1I).
The FG-Nups 358 and 98 displayed a subcellular distribution
that was similar to scaffold Nups (Figures 2A and S4B). Both
have been recently shown to critically contribute to NPC scaffold
formation (Fischer et al., 2015; Stuwe et al., 2015; von Appen
et al., 2015). Notably, the presence of Nup98 in AL-NPCs, a
protein that is the essential constituent of the NPC permeability
barrier (Hulsmann et al., 2012), suggests that NPCs are impermeable for larger molecules at all times. In contrast, the members of the Nup62/58/54 and Nup214/88 (the latter called Mbo
in flies) complexes as well as the nuclear basket components
Tpr (called Mtor in flies) and Nup153 were absent in AL-NPCs
(Figures 2A2D and S4B). Instead, the Nup214/88 and Nup62/
58/54 complexes were highly abundant in the cytosol (Figures
2A, 2B, and S4B), in agreement with previous biochemical results that identified certain FG-Nups to be predominantly soluble
and excluded from ER-membranes (Onischenko et al., 2004).

(CD00 ) Top views onto a fixed Drosophila syncytial blastoderm embryo in interphase. The nuclear basket components Nup153 (C and C0 ) and Mtor (D and D0 ) are
absent from AL-NPCs (arrowheads in C00 , D00 ), which stain positive for mAb414 (C and C00 ) or WGA (D and D00 ), both labeling FG-Nups (including Nup358).
(E and E0 ) AL-NPCs (E) are pore scaffolds made of transmembrane Nups, the inner ring, and Y-complex nucleoporins, extended by Nup358, which might or might
not be attached symmetrically across ER membranes. NE-NPCs (E0 ) recruit soluble Nups to construct the mature pore that is asymmetric across the NE.
See also Figures S3 and S4.

668 Cell 166, 664678, July 28, 2016

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Cell 166, 664678, July 28, 2016 669

One might thus surmise that NPC assembly is completed after


insertion into the NE by recruiting soluble Nups from the cytosol
in order to establish directionality and transport competence.
Indeed, the nuclear accumulation of nls::GFP was delayed as
compared to the burst phase of AL-NPC insertion (Figures S3E
and S3F). We conclude that AL-NPCs are pore scaffolds devoid
of most FG-Nups and all nuclear basket components. With the
exception of Nup358, Nups that asymmetrically distribute
across the NE-plane in NE-NPCs are absent from AL-NPCs (Figures 2E and 2E0 ).
Topology of AL Insertion
To address how AL-NPC insertion is topologically possible, we
sought to identify putative steps of AL insertion by ultrastructural
analysis. We first analyzed sections through staged blastoderm
embryos by transmission electron microscopy after high-pressure freezing and freeze substitution. AL were apparent in the
cytoplasm throughout the embryo as interconnected stacks of
membranes containing NPCs (Figures S5A, S6A, and S6B
S6B00 ). AL that were close to the NE and thus potentially could
engage in an insertion event, often appeared continuous with
the NE (Figures 3A, 3B, S5B00 , and S5C0 ). Strikingly, as evident
in multiple sections, these AL-NE fusion sites often were adjacent to apparent openings within the NE (Figures 3A and 3B).
At the edges of these openings, the INM and ONM were seen
to be fused in the electron micrographs, emphasizing that the
gaps are not sample preparation artifacts (Figures 3A and 3B).
We used correlative light and electron microscopy as described
above and recorded an electron tomogram at a site where a fluorescent spot of RFP::Nup107 close to the NE indicated a potentially ongoing insertion event (Figures 3C, 3C0 , and S5BS5B00 ).
Although the observed membrane topology in this region was
complex, it clearly showed the critical features: a patch of AL
engaged with the NE in direct proximity to NE openings. In
conclusion, the unanticipated discovery of NE openings offers
a topological explanation for how the INM becomes continuous
with AL (see below).
A limitation of the aforementioned analysis is that it resolves
membranes only when they are roughly aligned parallel to the
electron optical axis and thus manifest as a projection in the
electron micrographs. Because AL assume various orientations
with respect to the NE it is relatively unlikely to capture both in a
favorable two-dimensional projection. To better resolve inserting

AL sheets in 3D, we used the slice and view technique, in which a


low angle focused ion beam is used to mill away thin (510 nm)
layers of an embedded specimen alternating with image acquisition by focused ion beam-scanning electron microscopy (FIBSEM). The resulting volumes have an almost isotropic resolution
and can be virtually rotated to obtain slices in basically any
spatial direction. We first confirmed that the parallel AL-NPC
decorated ER sheets are indeed highly interconnected in three
dimensions and link to the NE close to openings of the nuclear
membrane (Figure S5C00 ). NE-openings were frequent and surprisingly large (Figures 3D and 3E). By tangentially slicing the
NE in volumes obtained by FIB-SEM, we could resolve inserting
AL-sheets as part of an ER compartment that enclosed large
parts of the respective NE-opening and contained NPCs (Figures
3F3F00 and 3G). We conclude that AL insertion typically occurs
in proximity to NE openings.
The three-dimensional data allowed us to deduce putative
topological intermediates of AL-insertion. Those involve establishing membrane connections from the adjacent sheet to the
nuclear membranes and in proximity to openings of the existing
NE. NE-openings could either form de novo or persist from the
previous mitosis (Figures 4A and 4A0 ). Importantly their existence suggests a model for AL uptake that elegantly resolves
the topological puzzle: NE openings link the INM to the inserting
sheet and convert the latter into NE (Figures 3F, 3G, and 4A).
Driven by nuclear expansion, both the adopted novel and
the underlying previously present NE sheet laterally slide
away from each other and augment nuclear surface (Figures
4A and 4B0 ). These topological intermediates can be viewed
as part of a spatiotemporal continuum of AL and NE membranes. This model would predict that redundant pieces of NE
should result from AL insertion (Figures 4A and 4B0 ). We indeed
could confirm the existence of NPC-decorated, redundant
NE membranes in both micrographs (Figures S5DS5F) as
well as nucleoplasmic GFP::Nup107 foci in live microscopy (Figure 6C). Such redundant NE could be resolved either by fission
or re-insertion in an equivalent way as AL insertion from the
cytoplasm.
The Permeability Barrier of the NE Is Maintained during
AL Insertion
The apparent NE openings suggest a compromised permeability barrier between the nucleoplasm and the cytosol, except

Figure 3. Topology of AL Insertion


(AB) NE-openings accompany AL insertions. (A and A0 ) Transmission electron micrographs (TEM) of two consecutive serial sections of a nucleus with closely
aligned AL-NPCs (arrowheads). Note the clear INM-ONM connection of the encircled opening (A), which is replaced by intact NE in (A0 ), while the boxed opening is
seen in both sections. (B) Electron micrograph of a nucleus where the NE is opened (boxed) and continuous with an ER stretch.
(C) Capture of an AL-insertion into the NE by correlative light and electron microscopy (CLEM) and tomography (C0 ). (C) RFP::Nup107 localizes to the NE and is
enriched at proximal AL-NPCs (arrowhead).
(C0 ) Single slice through a tomogram recorded at the region depicted by the yellow box in (C). Arrowheads point to a membrane connection of the inserting AL to
the NE, adjacent to a NE-opening.
(D) Multiple NE openings (arrowheads) are apparent in a volume containing a nucleus that was obtained by focused ion beam-scanning electron microscopy (FIBSEM; a single slice is shown superimposed with the NE that is isosurface-rendered in blue).
(E) Histogram of NE-opening diameters measured in TEMs as represented in (A, A0 , and B). Most NE-openings are in the range of 400800 nm.
(FG) FIB-SEM visualizes the continuity of AL membrane sheets with the NE. Slices through a FIB-SEM volume tangential to the nucleus (FF00 ) and isosurface
rendering of the same region (G). (FF00 ) Slices at slightly different angles. Note that cross-sectioned NE with NPCs in side view (yellow arrowheads in FF00 ) are
perpendicular to an AL membrane sheet that has a branched topology and contains multiple NPC in top view (white arrowheads in FF00 ).
See also Figures S5 and S6.

670 Cell 166, 664678, July 28, 2016

Figure 4. Model of AL-NPC Insertion


(AB0 ) Planar (A and A0 ) or three-dimensional (B
and B0 ) model for AL-NPC insertion, as inferred
from electron micrographs. Insertion involves the
establishment of membrane connections from the
inserting ER sheet containing AL-NPCs to the NE
and opening of the NE adjacent to the connected
ER sheet. NE openings could emerge de novo by
an unknown mechanism (A) or alternatively could
remain from the previous round of mitosis (A0 ). The
connected ER sheet (arrowhead in B) becomes
part of the NE while nuclear surface increases
by lateral dissipation within linked membrane
planes. Because pores limit lateral membrane
dissipation, NE-NPCs located in between the NE
opening and the AL-sheet connection predict
the formation of transient redundant NE stretches
(A and B0 ).
(C and C0 ) The permeability barrier of the NE
would be compromised if inserting ER sheets
fail to entirely surround (or sufficiently shield)
the NE opening against the cytoplasm (C). Alternatively no cytoplasmic influx through the
NE opening would occur in a concealed
compartment (C0 ).

that the NE openings would reside in a compartment that is


entirely surrounded by ER-membranes (Figures 4C and 4C0 ).
The electron microscopy data (Figures 3C0 and 3G) highlight
the topological complexity of AL insertion and indicate that
the same event might span a considerable fraction of the
nuclear surface area. As such, it is not ultimately possible
to conclude from the three-dimensional data whether or not
the NE remains topologically closed during AL insertion. We
therefore set out to experimentally test if the NE permeability
can be maintained despite AL fusion. We imaged GFP::
Nup107-expressing blastoderm embryos that were injected
with fluorescently labeled dextrans of different molecular
weight (Lenart and Ellenberg, 2006). Small dextrans of 10 and

25 kDa were not excluded by NPCs


and diffused into the nucleoplasm during
interphase (Figures 5A and 5A0 ). In
contrast, nuclei were impermeable to
155 kDa dextran (Figure 5A00 ), suggesting
an intact barrier. Importantly, 155 kDa
dextran did not even leak into the nucleoplasm as AL inserted to the NE (Figures
5B5B00 and 5E; Movie S5), demonstrating that insertion of AL does not
interfere with the permeability barrier of
the nuclear membranes.
To test if an NE opening of the observed
size would in principle cause dextran
influx, we artificially ruptured the nuclear
membranes by performing laser nanosurgery on the NE. Using GFP::Nup107,
we targeted the NE with a 950 nm Titan
Sapphire Laser and punctured the nuclear membranes (Figures 5C5C00 ; Movie
S6). Successful puncture was reflected by a strong mechanical
response of the entire nucleus apparent as NE folding and tumbling (Movie S6). Strikingly, 155 kDa-dextran accumulated in the
nucleoplasm of punctured nuclei within tens of seconds (Figures
5C0 and 5C00 ; Movie S6) with kinetics that did not depend on the
size of the punctured region (Figure 5C00 ). These experiments
conclusively demonstrate that the permeability barrier of the
NE was disrupted after laser-induced rupture while it was not
impaired when AL inserted (Figure 5E), despite comparable dimensions of the respective NE openings apparent in electron
micrographs (Figure 3E). These findings are in line with our
topological model and suggest that the NE-openings are entirely
surrounded by ER membrane sheets.
Cell 166, 664678, July 28, 2016 671

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672 Cell 166, 664678, July 28, 2016

NPC Organization and Insertion Mode Change during


Development
In mammalian cell lines, NPCs are stationary embedded within
the NE but mobile along ER membranes in AL (Daigle et al.,
2001). Our results demonstrate frequent AL insertions in
blastoderm embryos and indicate that AL-NPCs predominantly contribute to an increased NE-NPC number during
nuclear expansion. Lateral mobility of NPCs within the NE
could facilitate their re-distribution following AL insertion. We
thus performed FRAP experiments on GFP::Nup107 expressing
embryos to test whether AL-NPC became immobile upon
NE insertion. Strikingly, we observed fast recovery of GFP::
Nup107 all along the rim after photobleaching (Figures 6A
and 6A0 ). Together with our finding that NPC material laterally
dispersed after AL insertion to the NE (Figures 1F1F00 ), we
conclude that in the Drosophila syncytial blastoderm NPCs
are mobile within the NE. In contrast, GFP::Nup107 fluorescence did not recover after photo-bleaching of nuclei at the
onset of gastrulation, suggesting that dispersion of pores
within the envelope was abolished (Figures 6B and 6B0 ). The
impaired NE-NPC mobility in those nuclei was reflected by
distinct principles of pore organization along the NE when
compared to nuclei in younger embryos. GFP::Nup107 distributed uniformly along the NE of blastoderm embryos but
appeared clustered into distinct steady foci just before
embryos started to gastrulate (Figures 6C and 6D). The switch
in NPC mobility and organization coincides with the activation
of the zygotic genome (Figure 7A). Thus pore organization at
the NE could be controlled by zygotic genes or by transcription-associated changes in chromatin. Consistent with
both hypotheses, injection of the RNA polymerase inhibitor
a-amanitin prevented clustering of NPCs on later stage nuclei
(Figure 6E).
The nuclear lamina, a meshwork of intermediate filament
proteins that underlies the NE and projects into the nucleoplasm in metazoa is critical for NPC organization and mobility
within the NE (Daigle et al., 2001), but other NE proteins
could also be important for NPC mobility. Comparative analysis
of the proteomes of isolated nuclei from either blastoderm or

gastrulating embryos revealed a significant (p = 0.00024)


2.5-fold enrichment of a very prominent INM protein, lamin
B receptor (LBR), in older nuclei (data not shown). In mammalian cells LBR is recruited by the Y-complex member ELYS/
Mel28 to specific NE-subdomains and could thus directly
link to NPC distribution (Clever et al., 2012). Strikingly, LBR
was absent from the NE in syncytial embryos but became
localized to the rim of somatic nuclei during cellularization
in interphase 14 (Figures S7A and S7B). Notably, the protein
remained undetectable at the NE of nuclei from pole cells,
which are the posteriorly localized germ cell progenitors (Figures S7C and S7D). To address whether LBR is sufficient to
alter NPC organization at the NE, we ectopically expressed
the protein in WGA-injected syncytial blastoderm embryos.
In these embryos pores appeared clustered within the NE
and nuclei acquired an irregular morphology (Figure 6F).
Both phenotypes are reminiscent of wild-type nuclei at gastrulation onset. Strikingly, ectopic expression of LBR in the early
embryo also induced larger AL sizes (Figures 6F0 and 6G),
implying that LBR expression and NPC clustering counteracts
AL-insertion.
At last, we detected striking differences in the NPC insertion
mode between the different developmental stages. In contrast
to the early embryo (compare to Figure 1C), the mean fluorescent intensities of either GFP::Nup107 or fluorescently labeled
WGA at the nuclear rim strongly decreased as interphase 14
proceeded (Figure 6H), while nuclei significantly increase their
surface area (Fullilove and Jacobson, 1971). Interestingly, the
switch in NPC organization and insertion is concomitant with
the reported (Onischenko et al., 2004; Stafstrom and Staehelin,
1984a) and observed (Figures 6D and 6I) disappearance of ALNPCs from the cortical nuclei layer at early gastrulation. At the
same time AL remained abundant in pole cells (Figure 6I), suggesting that nuclei from the prospective soma and germline
have different NE organizations, compatible with our results
on differential LBR localization (Figure S7). Jointly, our data
suggest that AL insertion to the NE is an early developmental
program that is reduced or lost as the embryo matures
(Figure 7).

Figure 5. The Permeability Barrier of the NE Is Maintained during AL Insertion


(A) Kymographs from time-lapse movies recorded in syncytial blastoderm embryos expressing GFP::Nup107 injected with fluorescently labeled dextrans of
different molecular weight. Regions of interest (ROIs) for kymographs span entire nuclei as schematically depicted. The NE is permeable for 10 kDa (A) and 25 kDa
dextran (A0 ), but not for 155 kDa dextran (A00 ).
(BB00 ) The NE stays impermeable upon AL-insertion. Kymographs (scheme) of a time-lapse movie imaging an embryo expressing GFP::Nup107 (B and B0 )
injected with dextran-155 kDa-TRITC (B and B00 ). Dextran stays cytoplasmic upon insertion of GFP::Nup107-labeled AL-NPCs (arrow). Colored ROIs refer to the
graph in (E).
(CE) Laser puncture of the NE compromises its permeability barrier. (C) Top view still of a time-lapse movie imaging a GFP::Nup107 expressing syncytial embryo
injected with dextran-155 kDa-TRITC, where the NE of two nuclei was laser punctured simultaneously (indicated by the arrowhead and asterisk, respectively). (C0 )
Kymograph of the respective movie along the ROI (white box in C). Dextran leaks into the nucleoplasm of the punctured nucleus (arrowheads in C and C0 ), but not
in the neighboring control nucleus. (C00 ) Quantification of the mean dextran-TRITC fluorescence intensities SD for the respective ROIs color-coded in (C).
Dextran accumulates in the nucleoplasm of nuclei within 20 s after puncture with an initial kinetics that is independent of the size of the punctured region. (D)
Representative kymograph for GFP::Nup107 (D0 ) and dextran-155 kDa-TRITC (D00 ) after NE-puncture; colored ROIs refer to the graph in (E). (E) Dextran leaks into
the nucleoplasm upon NE puncture, but not when AL insert to the NE. Quantitation of dextran influx upon NE-puncture and AL insertion. GFP::Nup107 and
155 kDa-dextran-TRITC levels were inferred from their respective fluorescence intensities, determined from line scans on ROIs in kymographs as exemplified in
(B0 ), (B00 ), (D0 ), and (D00 ). Mean fluorescence intensities SD are plotted as a function of the distance from the NE for control nuclei (no insertion, n = 10 nuclei),
nuclei with AL-NPC insertions (n = 7) or punctured nuclei (n = 7). Experiments were aligned by the respective maximal GFP::Nup107 intensity value, delineating the
position of the NE.
See also Movies S5 and S6.

Cell 166, 664678, July 28, 2016 673

Figure 6. Developmental Regulation of NPC Organization and Insertion


(AD) Lateral mobility and organization of NPCs change during development. Representative top view stills (AD) and kymographs (A0 and B0 ) of time-lapse
movies imaging GFP::Nup107 in syncytial embryos (A, A0 , and C) or before gastrulation onset in interphase 14 (B, B0 , and D). GFP::Nup107 at the NE was photobleached (arrowheads in A0 and B0 ) at the depicted orange regions of interest (ROIs) (A and B) and recovered at syncytial blastoderm nuclei (A0 ) but not at nuclei
from interphase 14 embryos (B0 ). For both kymographs the respective ROIs are boxed in red in (A) and (B). (C and D) GFP::Nup107 distributes evenly along the NE
at the spherical nuclei of syncytial embryos (C) but appears clustered at the rim of the irregularly shaped nuclei at gastrulation onset (D).
(E) Pore clustering is zygotically induced. Top view still from a movie recording a GFP::Nup107 expressing embryo in interphase 14, injected with a-amanitin,
where nuclei stay round and pores fail to cluster due to inhibition of zygotic gene activation.
(FG) The zygotically induced gene LBR is sufficient to cluster NPCs and increase AL size. (F and F0 ) Top view stills from two syncytial blastoderm embryos where
LBR expression was maternally induced. WGA labeled NPCs appeared clustered along the NE (F), similar to wild-type embryos in interphase 14 (B and D) and
accumulate in larger AL-NPCs (F0 ) (arrowheads). (G) Histogram of AL-NPC sizes measured from images as shown in (C and F0 ). AL-NPCs are larger in blastoderm
embryos where LBR expression was ectopically induced (n = 282 AL), compared to control embryos (n = 282).
(H) The mode of NPC insertion is developmentally controlled. NPC density along the NE was inferred from the mean fluorescence intensities of GFP::Nup107 or
fluorescently labeled WGA, which were normalized and plotted as a function of interphase progression. NPC densities stayed almost constant in syncytial interphases but strongly decayed in interphase 14 (see also Figure 1C). Plotted are mean fluorescence intensities SD for 10 nuclei from each imaged syncytial
blastoderm (n = 5) or cellularizing (n = 4) embryo.
(I) During interphase 14, AL-NPCs were diminished from the somatic nuclear layer at the embryos cortex but not from pole cells.
See also Figure S7.

674 Cell 166, 664678, July 28, 2016

Figure 7. NPC Insertion in the Context of


Embryonic Development
(A) AL are abundant throughout the 120 min of
syncytial development but diminish from the
cortical layer in interphase 14, concomitant with
cellularization and the transcriptional activation at
zygotic induction.
(A0 ) NE-NPCs are laterally mobile and all along the
NE in the syncytial blastoderm, but immobilize and
cluster at the NE starting with cellularization.
(B) In each precedent interphase of the syncytial
blastoderm, AL number oscillates within the cortical nuclear layer on a timescale of 10 min, with
increasingly longer interphases in each cycle. Inverse to AL-NPC number, which decreases in each
interphase, NE-NPC number increases together
with nuclear surface expansion.
(C and D) AL-NPCs insertion to the NE occurs in the
range of 12 min. It involves an open NE and batch
insertion of AL-NPCs within a proximal ER sheet
(D). Lateral mobility allows NE-redistribution of inserted NPCs. Insertion of subsequent ER sheets
augments nuclear surface (C).

DISCUSSION
Collectively, the following scenario emerges from our data. AL
are abundant in early Drosophila embryos and predominantly
contribute to maintain the constant NE-NPC density in the expanding NE during interphase. The abundance of AL at the
cortical nuclei layer thereby oscillates together with the progression of the consecutive interphases until the start of global transcription when AL disappear and the mode of NPC insertion
changes (Figures 7A and 7B). During each onset of early interphases, AL-NPCs are assembled similarly to NE-NPCs but since
the combined nuclear surface of the two daughter nuclei is

smaller as compared to the parental nucleus, they remain in the cytoplasm. As interphases progress, AL-NPCs feed into
the pool of NE-NPCs alongside ER membranes that augment NE surface during
rapid nuclear expansion (Figure 7C). AL
insertion is enabled by NE openings that
might either persist from previous mitosis
or form de novo by an unknown mechanism (Figure 7D). Upon AL insertion, the
NE permeability barrier remains unperturbed, likely because the NE openings
are entirely surrounded by the ER
network. The inserting NPCs comprise
pre-assembled NPC scaffolds that recruit
the full set of Nups only subsequent to
insertion and only then establish transport
competence.
Why do the expanding nuclei of the syncytial blastoderm maintain a constant
number of NPCs per surface area despite
their transcriptional inactivity? One might
surmise that this is due to mechanical
properties but also temporal constraints. The insertion of NPCs
might be crucial to enable the massive influx of material into
the nucleoplasm during nuclear expansion (volume increase).
Indeed, the strained configuration of nuclei is reflected by their
strong mechanical response (NE tumbling) upon disruption of
the NE and permeability barrier after laser puncture. Second,
the batch transfer of entire NPC scaffolds as inherent parts of
membrane sheets overcomes the described kinetic constrains
of interphase assembly in mammalian cells, that are not compatible with the short interphases in the Drosophila syncytium (Dultz
and Ellenberg, 2010). Given the abundance of AL-NPCs and the
reported high insertion rate of NPCs into the NE of Xenopus
Cell 166, 664678, July 28, 2016 675

leavis oocytes (DAngelo et al., 2006) it appears likely that similar


mechanisms operate in vertebrates. It remains unclear how
sufficient amounts of AL are generated to globally feed nuclear
surface expansion over multiple cell cycles until the start of
transcription. However, Nups are maternally provided and AL
are abundant not only at the cortical layer of nuclei but also within
the interior of the embryo (Figures S6A and S6BS6B00 ). Therefore, a possibility that needs to be considered is that a source
of AL-NPCs already generated during oogenesis feeds nuclear
growth throughout the syncytial blastoderm.
In addition to their eminent role in transport, NE-NPCs organize the nuclear periphery by delineating zones of active
euchromatin as compared to transcriptionally repressed heterochromatin in between pores (Ptak et al., 2014). Crucial to
this is that NPCs are laterally immobile within the NE, which
was shown to depend on the nuclear lamina (Daigle et al.,
2001). Lamins are nuclear intermediate filament proteins
and come in two major types: B-type Lamins are ubiquitous,
while A-type Lamins are expressed exclusively when cells
differentiate. Both proteins engage in distinct meshworks and
also impact on NPC insertion rate (Lenz-Bohme et al., 1997;
Liu et al., 2000). Our work puts NPC organization and the
mode of pore insertion into a developmental context. We propose that in Drosophila AL insertion is innate to earliest
embryogenesis and diminishes when pores get laterally
restricted and cluster at the NE. There are no A-type lamins expressed at that stage, and specifically expressed INM proteins
could be crucial. Intriguingly, the formation of immobile pore
clusters coincides with the transcriptional upregulation of hundreds of genes at zygotic induction, a developmental transition
present in all metazoan that is accompanied by characteristic
changes in chromatin signatures (Rudolph et al., 2007; Vastenhouw et al., 2010). We reveal that the zygotically upregulated
INM protein LBR, a developmentally controlled INM tether of
peripheral heterochromatin (Solovei et al., 2013), is sufficient
to prematurely aggregate NPCs in blastoderm interphases,
when artificially expressed earlier in embryogenesis. This also
leads to larger AL likely because LBR counteracts AL insertion
for which lateral NPC mobility is required. Our data suggest a
zygotically induced regulation that links pore insertion and organization, NE composition and ultimately also chromatin organization at the nuclear periphery. All of these events eventually
contribute to the commitment of originally pluripotent somatic
nuclei into distinct lineages.
EXPERIMENTAL PROCEDURES
Detailed experimental procedures are available in the Supplemental
Information.
Embryo Injections, Live Imaging, and Immunostainings
Staged embryos were treated according to standard protocols and injected
with Alexa488 or Alexa555 conjugated WGA (100 mg/ml, Life Technologies),
a-amanitin (100 mg/ml, Sigma) or TRITC/FITC conjugated dextrans of
different molecular weight (Lenart and Ellenberg, 2006). Subsequently embryos were imaged on an inverted Zeiss LSM780 confocal microscope
equipped with a 633/1,4 NA oil immersion objective. For immunostainings,
embryos were fixed in 4% formaldehyde and processed according to standard protocols.

676 Cell 166, 664678, July 28, 2016

Sub-cellular Fractionation and Protein Identification by Mass


Spectrometry
Dechorionated embryos were lysed and nuclei were isolated by centrifugation at 5,000 rpm for 13 min and stripped from attached membranes
by centrifugation (45 min, 12,000 rpm) through a 1 M sucrose cushion.
Microsomal membranes were isolated by spinning the supernatant from
the nuclear precipitate at 40,000 rpm for 45 min. Samples were further
processed and analyzed by shotgun mass spectrometry as previously
described (Mackmull et al., 2015). Raw files for quantitative label free
analysis were analyzed using MaxQuant (Cox and Mann, 2008) and the
MS/MS spectra were searched against the Drosophila Swiss-Prot entries
using the Andromeda search engine (Cox et al., 2011). Protein differential expression was evaluated using the Limma package. Differences
in protein abundances were statistically determined using the Students
t test moderated by the empirical Bayes method. Significant regulated
proteins were defined by a cut-off of log2 fold change %1 or R1 and
p value % 0.01.
Transmission Electron Micrograph, FIB-SEM, and Correlative Light
and Electron Microscopy Imaging
Embryos were high pressure frozen, freeze-substituted and infiltrated with
resin. Blocks were subsequently trimmed for FIB-SEM or cut into 300-nm
sections for correlative light and electron microscopy (CLEM) analysis using
an ultramicrotome. For serial transmission electron microscopy (TEM), the
resin-embedded embryos were trimmed and consecutive 100 nm distant sections were obtained with a section thickness of 80 nm. TEM imaging was
carried out on a FEI Tecnai F30 equipped with Gatan US4000 CCD camera,
operated at 300 kV or a FEI Biotwin equipped with an Olympus Keen View
G2 camera operated at 120 kV, respectively. The fluorescence microscopy
(FM) imaging was carried out as previously described (Avinoam et al., 2015;
Kukulski et al., 2011). Tomography was performed in 1 increments at
4,7003 magnification on a FEI Tecnai F30 electron microscope. FIB-SEM
imaging was carried out on an Auriga 60 (Zeiss) using the Atlas3D software.
Datasets were acquired with 5 nm pixel size and 5 nm steps in z and aligned
using TrakEM (Fiji).
ACCESSION NUMBERS
The accession number for the mass spectrometry proteomics data reported in
this paper is ProteomeXchange Consortium: PXD004120.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and six movies and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2016.06.015.
AUTHOR CONTRIBUTIONS
B.H. conceived the project, designed and performed experiments, analyzed
data, and wrote the manuscript. M.T.M. designed and performed experiments and analyzed data. P.M., P.R., K.H.B., N.S., R.S.M., A.A.P., A.N.,
and J.M.P. performed experiments. T.L. designed experiments. Y.S. designed experiments and oversaw the project. M.B. conceived the project,
designed experiments, analyzed data, oversaw the project, and wrote the
manuscript.
ACKNOWLEDGMENTS
We thank Drs. Iain Mattaj and Peter Lenart for critical reading of the manuscript
and Dr. Yannick Azou-Gros for experimental assistance. We are grateful to our
colleagues Drs. V. Doye, N. Dostatni, A. Ephrussi, S. deRenzis, M. Mavrakis, A.
Akhtar, G. Krohne, I. Mattaj, and J. Ellenberg for reagents. Stocks obtained
from the Bloomington Drosophila Stock Center (NIH P40OD018537) were
used in this study. We gratefully acknowledge support by the European Molecular Biology Laboratory (EMBL) advanced light microscopy (ALMF), electron

microscopy (EMCF), and proteomic (PCF) core facilities and Petra Riedinger
for graphical design. We are grateful to Drs. P. Paul-Gilloteaux, X. Heiligenstein, and S. Mosalaganti for assistance and expertise in correlative light
and electron microscopy analysis. B.H. was supported by the Agence Nationale de la Recherche (ANR) (Programme Blanc) NeMo (to T.L) and the European Molecular Biology Organisation (EMBO). K.H.B. was supported by postdoctoral fellowships from the Swiss National Science Foundation (SNF),
EMBO, and Marie Curie Actions. M.B. acknowledges funding by EMBL and
the European Research Council (309271-NPCAtlas).
Received: October 25, 2015
Revised: April 15, 2016
Accepted: June 3, 2016
Published: July 7, 2016
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678 Cell 166, 664678, July 28, 2016

Article

Adjacent Codons Act in Concert to Modulate


Translation Efficiency in Yeast
Graphical Abstract

Authors
Caitlin E. Gamble, Christina E. Brule,
Kimberly M. Dean, Stanley Fields,
Elizabeth J. Grayhack

Correspondence
fields@uw.edu (S.F.),
elizabeth_grayhack@urmc.rochester.edu
(E.J.G.)

In Brief
Rather than protein synthesis relying
solely on readout of individual codons,
pairs of codons dictate translational
efficiency, suggesting unexpected
coupling between tRNA binding sites
within the ribosome.

Highlights
d

17 codon pairs in yeast mediate strong inhibition of


translation
Inhibition by codon pairs is distinct from dipeptide and
individual codon effects
Inhibitory pairs slow the ribosome on native mRNAs and
involve wobble decoding
Codon order is key to inhibition, implying distinct roles for
each position

Gamble et al., 2016, Cell 166, 679690


July 28, 2016 2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.cell.2016.05.070

Article
Adjacent Codons Act in Concert to
Modulate Translation Efficiency in Yeast
Caitlin E. Gamble,1,2,6 Christina E. Brule,3,4,6 Kimberly M. Dean,3,4,8 Stanley Fields,1,5,7,* and Elizabeth J. Grayhack3,4,7,*
1Departments

of Genome Sciences and Medicine, University of Washington, Seattle, WA 98195, USA


in Molecular and Cellular Biology, University of Washington, Seattle, WA 98195, USA
3Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA
4Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA
5Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA
6Co-first author
7Co-senior author
8Present address: BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, USA
*Correspondence: fields@uw.edu (S.F.), elizabeth_grayhack@urmc.rochester.edu (E.J.G.)
http://dx.doi.org/10.1016/j.cell.2016.05.070
2Program

SUMMARY

Translation elongation efficiency is largely thought of


as the sum of decoding efficiencies for individual codons. Here, we find that adjacent codon pairs modulate translation efficiency. Deploying an approach in
Saccharomyces cerevisiae that scored the expression of over 35,000 GFP variants in which three adjacent codons were randomized, we have identified 17
pairs of adjacent codons associated with reduced
expression. For many pairs, codon order is obligatory for inhibition, implying a more complex interaction than a simple additive effect. Inhibition mediated
by adjacent codons occurs during translation itself
as GFP expression is restored by increased tRNA
levels or by non-native tRNAs with exact-matching
anticodons. Inhibition operates in endogenous
genes, based on analysis of ribosome profiling
data. Our findings suggest translation efficiency is
modulated by an interplay between tRNAs at adjacent sites in the ribosome and that this concerted
effect needs to be considered in predicting the functional consequences of codon choice.
INTRODUCTION
Translation elongation shapes the proteome, influencing the
amount of protein produced per mRNA and folding of the
nascent protein (Gingold and Pilpel, 2011; Ingolia et al., 2009;
Thanaraj and Argos, 1996). During translation elongation, ribosomes coordinate interactions of codons in mRNA with the anticodons of cognate tRNAs, resulting in addition of an amino acid
to the growing polypeptide, followed by a three-base translocation of the mRNA. Synonymous codons specify addition of the
same amino acid to a growing polypeptide chain, but differ in
their relative use in the genome, the abundance of the tRNAs
that decode them, and their requirement for wobble (nonWatson-Crick) decoding interactions between the third base of
the codon and the first base of the anticodon.

Codon choice modulates translation efficiency (Gingold and


Pilpel, 2011), protein folding (Thanaraj and Argos, 1996; Zhang
et al., 2009), and mRNA decay (Presnyak et al., 2015). A set of
optimal codons, decoded by abundant tRNAs, is implicated
in high-translation efficiency in Saccharomyces cerevisiae and
Escherichia coli (Burgess-Brown et al., 2008; dos Reis et al.,
2004; Gingold and Pilpel, 2011; Pechmann and Frydman,
2013; Presnyak et al., 2015; Sharp and Li, 1987; Welch et al.,
2009). The importance of codon choice is underscored by the
finding that codon use differs substantially between genes expressed in proliferating human cells and in differentiated tissues
(Gingold et al., 2014). However, the parameters that modulate
elongation are not well understood.
While suboptimal codon use could reflect a lack of selective
pressure (Plotkin and Kudla, 2011), in some cases suboptimal
codon use is functionally important, for instance, in the expression of the Neurospora clock protein FRQ (Zhou et al., 2013)
and the cyanobacteria oscillator kaiABC (Xu et al., 2013). The
prevailing hypothesis for how suboptimal codons affect translation has been that their decoding by low-abundance tRNAs
slows ribosome progress. Variation in the decoding rates of individual codons has been detected in some studies (Curran and
Yarus, 1989; Gardin et al., 2014; Lareau et al., 2014; Pedersen,
1984; Srensen and Pedersen, 1991; Stadler and Fire, 2011),
but not in others (Ingolia et al., 2009; Pop et al., 2014; Subramaniam et al., 2014). Furthermore, it is not resolved how differences
in decoding impact translation efficiency, with different studies
suggesting that suboptimal codons affect mRNA decay (Presnyak et al., 2015), translation initiation rates (Chu et al., 2014),
or the recruitment of quality control systems (Letzring et al.,
2013).
Detection of codon-mediated effects is complicated by three
factors. First, changes in codon use affect mRNA sequence,
which also influences mRNA structure, protein and microRNA
binding sites, and splicing signals (Goodman et al., 2013; Kudla
et al., 2009; Weatheritt and Babu, 2013; Welch et al., 2009). Second, translation of particular amino acids or amino acid combinations, such as proline repeats, may affect both the rate and
efficiency of translation (Gutierrez et al., 2013; Lareau et al.,
2014). Third, codon-mediated effects almost certainly depend
on additional parameters beyond the single codon, including a
Cell 166, 679690, July 28, 2016 2016 Elsevier Inc. 679

codons location in the gene (Letzring et al., 2010; Pechmann


and Frydman, 2013; Tuller et al., 2010a, 2010b; Wolf and Grayhack, 2015) and sequence context (Boycheva et al., 2003; Fedorov et al., 2002; Moura et al., 2005).
Interactions between adjacent codons were first implicated in
translation efficiency by the biased use of codon context and
codon pairs in organisms in all three kingdoms (Fedorov et al.,
2002; Gutman and Hatfield, 1989). In human cells, recoding viral
genes with underused codon pairs reduces expression and
leads to attenuated viruses (Coleman et al., 2008). Moreover,
codon pairs and codon context affect the rate of translation elongation in the HisT leader peptide in Salmonella enterica (Chevance et al., 2014), while adjacent CGA codons inhibit translation
in S. cerevisiae more effectively than individual CGA codons
(Letzring et al., 2010). Thus, interactions between sites in the
ribosome may play important roles in regulating translation.
However, a major impediment to understanding translational
control mediated by codon choice has been the lack of an unambiguous method to identify codons or codon combinations that
reduce translation efficiency.
We reasoned that an analysis of extensive variation within a
small region could identify codon combinations that reduce
gene expression in the yeast S. cerevisiae. Large synthetic libraries of a reporter gene provide a robust tool for evaluating
the functional impacts of sequence variation (Goodman et al.,
2013; Kudla et al., 2009; Welch et al., 2009). We therefore
used fluorescence-activated cell sorting (FACS) and deep
sequencing to measure the expression of 35,811 GFP variants
in which three adjacent codons near the 50 end of the coding
sequence were randomized. We identified 17 codon pairs
associated with low expression and examined their effects on
translation. We have found that most of these codon pairs substantially reduce the rate of translation elongation on native yeast
mRNAs and dramatically reduce expression in only a single
orientation, consistent with inhibition by the codon pair and not
by the sum of individual codon effects. We conclude that the
rate of translation elongation is modulated by the concerted
effects of tRNA:codon interactions in two adjacent sites in the
ribosome.
RESULTS
Analysis of 35,811 Three-Codon Variants Reveals Codon
Pairs Linked to Reduced Expression
To identify codons or codon pairs that substantially inhibit yeast
translation, we randomized three adjacent codons at amino
acids 68 of a fusion protein encoding superfolder GFP in the
chromosomally integrated RNA-ID reporter (Dean and Grayhack, 2012). Codon-mediated translational control has been
recapitulated in this reporter, in which a bidirectional GAL1,10
promoter separately drives expression of GFP and RFP, with
normalization of GFP to red fluorescent protein (RFP) used to
reduce transcriptional noise. We created two libraries that randomized the three codons (Figure 1A): the (VNN)3 library encoded each codon by VNN (V = A, C, or G) to avoid insertion
of stop codons, and the (NNN)3 library encoded each codon by
NNN. This approach seemed likely to comprehensively define interactions between adjacent codons, since each codon pair, the
680 Cell 166, 679690, July 28, 2016

reverse of each codon pair, and the two individual codons would
be represented many times in different contexts.
To detect differences in GFP expression, we used fluorescence-activated cell sorting (FACS) to separate yeast cells into
three fluorescence bins. For the (NNN)3 library, we made and
separately sorted two independent yeast libraries. We estimated
that the assay detected expression levels, relative to a no-insert
GFP reference, from 75%100% in bin 1, from 25%75% in
bin 2 (median 43% of bin 1 median), and from 2.5%25% in bin 3
(median 6% of bin 1 median) (Figure 1A); GFP variants with stop
codons migrated into the background bin. Following FACS, we
sequenced the three-codon insertions from cells in each bin,
carried out quality filtering, and determined the relative distribution of sequences. We estimated mean expression (GFPSEQ) for
each sequence based on the sequences distribution across
bins and applying the median fluorescence of a bin to all reads
counts in that bin. GFPSEQ scores correlated across the three libraries (r = 0.91 to 0.93) (Figure S1A) and with mean GFP expression of 76 individual constructs measured by flow cytometry
(GFPFLOW) (r = 0.81), although binning limited the resolution
(Figure S1B).
We considered that amino acid sequences encoded by the insertions could affect GFP stability or expression, although most
of these effects should be mitigated by using superfolder GFP,
which has robust fluorescence even when fused to several insoluble proteins (Pedelacq et al., 2006). Thus, for downstream analysis, we included only the 35,811 unique DNA sequences
specifying one of 5,148 tripeptides that had at least one synonymous sequence above the mean of all GFPSEQ scores (which
left out 4.1% of tripeptides and 6.2% of DNA variants). For
each DNA sequence, the highest scoring sequence encoding a
synonymous peptide served as its synonymous reference. We
scored expression due to codon usage (syn-GFPSEQ) as the
GFPSEQ ratio of a given sequence and its synonymous reference.
As expected, most synonymous variants had similar expression (Figure 1B; Table S1), with a mean syn-GFPSEQ of 0.954.
However, 1,119 DNA sequences (low variants) had syn-GFPSEQ
ranging from 0.059 through 0.647 (three SDs or more below the
mean). Intermediate variants comprised 5,127 sequences from
0.648 through 0.953; high variants comprised 24,417 non-reference (as well as 5,148 reference) sequences with syn-GFPSEQ
greater than 0.953.
There were no examples in which the use of an individual
codon consistently reduced expression to a degree detectable
in our assay. The median syn-GFPSEQ for each set of variants
containing one or more copies of a given codon ranged from
0.97 to 1.00, but the use of broad expression bins limited our
ability to detect differences in GFPFLOW values between 75%
and 100% of the reference GFP. A subset of codons occurred
frequently in low variants (Table S2), suggesting that combined
use of particular codons may dramatically reduce expression.
To identify inhibitory codon pair candidates, we looked for
combinations of adjacent codons enriched in the low-variant
category. We found 293 six-base sequences (non-gapped
6-mers) enriched in the low variants at one or more of the four
possible starting positions of the nine-base insertions (permutation p value % 0.001; Table S3). Most six-base sequences were
enriched at a single position, as might be expected if they form

Figure 1. Identification of Six Base Sequences Linked to Low GFP Expression

C
1. Library of GFP variants

Library insertion

PGAL1,10

RFP

GFP

RFP

VNN VNN VNN


NNN NNN NNN
2. Fluorescence-activated cell sorting
Bin: bkgd
3 2 1

6-mer position

NNN-NNN-NNN
2
3
4
5
4

105
3
104
2

103

104
105
GFP
3. High throughput sequencing of bins

B
10000

High
Intermediate
Low

7500

500

5000

250

0
-log10(p-value)

DNA variants

750

2500

0.00

0
0.00

0.25

0.25

0.50

10

0.95

0.50
0.75
syn-GFPSEQ

Inhibitory
5

0.75

Arg-Pro

Optimal

1.00

(A) Schematic of the method to examine effects of


three randomized codons on superfolder GFP
expression, using the RNA-ID reporter. The FACS
sort of (NNN)3 library 1 is shown.
(B) Distribution of syn-GFPSEQ scores. Variants
were assigned to low- (magenta; n = 1,119), intermediate (gray; n = 5,127), and high- (gold;
n = 24,417, excluding high-expression synonymous references) expression categories.
(C) Significance of 6-mer enrichment in lowexpression variants by 6-mer position (14) in
the nine-base variable region (library insertion).
6-mers with at least one p value % 0.001 are
plotted based on hierarchical clustering of positional permutation p values. 57 6-mers are not
plotted due to missing values; this includes 6-mers
that form an in-frame stop codon. 6-mers with a
p value % 0.001 at both in-frame start positions
(1 and 4) are labeled (although CUG-AGG, CUGAUA*, and CUU-AGG are not plotted because they
form a stop codon at another position). Candidate
inhibitory pairs that remain enriched in a reduced
structure dataset are indicated with a star.
(D) Flow cytometry scatter plots from six individual
variants; label (GFP*100/RFP).
See also Tables S1, S2, S3, and S4.

CUU-CAG
CU U-CGG
GU A-CCG *
GU A-CGG
GUG-CGA *
GU A-CGA *
CG A-GCG *
CG A-CGG *
CG A-CGA *
CG A-CUG *
CG A-CCG *
CUG-GCG
CG A-AU A *
CUG-CCG *
CUG-CGA *
CU C-CCG *
CU U-CUG *
A U A-CGG *
A GG-CGA *
A U A-CGA *
CG A-CAU
A GG-CGG *
C U C-A U A *
CUG-CUG *

identified a reduced-structure subset of


variants as all those with a similar
CGA-CCG
AGA-CCA
degree of structure to the majority of
13.0 0.1
82.6 2.1
103
high expression variants, based on both
Arg-Ile
105
local and global structure predictions
(Figure S1C; Table S1; Supplemental
104
Experimental Procedures). We then evalCGA-AUA
AGA-AUU
uated whether each candidate pair re1 12.9 1.3
38.6 1.7
103
mained enriched among low-expression
105 Arg-Arg
variants present in the reduced-structure
subset. We found 20 of the 28 candidates
104
enriched at in-frame start positions (perAGG-CGG
AGA-AGA
mutation p value % 0.055) and revised
58.1 2.7 11 1.6 3.8
103
our candidate list to include only these
CUC-CGG
6-mer
103
104
105
(Figure 1C; Table S4). We conclude that
position
3
4
1
2
GFP
structure is unlikely to account for most
of the reduced expression by these 20
candidates.
part of a secondary structure or a recognition motif that also inExpression of GFP variants with a candidate inhibitory pair
cludes common sequences. However, 28 of these 6-mers was substantially reduced, with syn-GFPSEQ medians ranging
(0.75% of all possible 6-mers without a stop codon) were en- from 0.44 to 0.82 (Table S3). Candidate pairs were present in
riched at both in-frame start positions (permutation p value % 29% (n = 319) of all low-expression variants. We validated the
0.001 for each position) and comprised our initial list of inhibitory inhibitory effects of the 20 inhibitory codon pair candidates by
codon pair candidates (Figure 1C).
flow cytometry of individual constructs. For each pair, we asSince strong RNA secondary structure in the 50 end of an open sessed expression due to codon usage by comparing GFPFLOW
reading frame (ORF) can reduce translation efficiency in E. coli of two synonymous variants, one with the inhibitory codon pair
and S. cerevisiae (Goodman et al., 2013; Kudla et al., 2009; and the other with an optimized pair based on codon adaption
Shah et al., 2013; Tuller et al., 2010b), we investigated whether index (CAI), which scores codons based on their frequency of
enrichment of each 6-mer in low-expression variants is expli- use in highly expressed genes (Sharp and Li, 1987). All variants
cable primarily by formation of strong secondary structure. We with an inhibitory pair candidate had lower GFPFLOW, ranging
RFP

104

Cell 166, 679690, July 28, 2016 681

n=21

AGG-CGA* n=30

CUG-CUG
CUU-CUG
GUA-CCG*
GUA-CGA*
GUG-CGA*

0.00

0.4
0.2
0.0

D
Inhibitory pair
Single codon optimized
Insert
RFP PGAL GFP

0.25

0.50

0.75

Variant syn-GFP

1.0
0.8
0.6
0.4
0.2
0.0

1.00

SEQ

from 14%76% that of synonymous optimized variants (Figure 1D; Table S4).
Codon Pairs Mediate Frame-Dependent Inhibition in
Different Sequence Contexts
To assess the likelihood that inhibition is mediated by translation,
we examined the properties of the candidate pairs. If inhibition
were coupled to translation, then the enriched 6-base sequences would likely inhibit expression only when the two
codons were in-frame, but not when out-of-frame, where mechanisms decoupled from translation might explain enrichment.
For the 20 candidates, we compared the syn-GFPSEQ distribution of variants with these candidates at in-frame positions (the
six-base sequences starting at positions 1 and 4) (Figure 2A,
blue) to that of variants with the candidates at out-of-frame positions (the six-base sequences starting at positions 2 and 3)
682 Cell 166, 679690, July 28, 2016

Insert
RFP PGAL Rluc GFP
1.0
0.8
0.6
0.4
0.2
0.0

G
AAG CC
A- G
C CC
G
A- G
C
C
A

CUG-CGA*

0.6

(A) syn-GFPSEQ distribution of variants with each of


the 20 inhibitory codon pair candidates. Variants
with the indicated codon pair in-frame (blue) are
compared to variants with the codon pair out-offrame (the 6-mer at positions 2 and 3) (gray) and
variants with the same two codons in-frame,
but separated (purple). Boxplot shows median
centerline and edges marking the first and third
quartiles. Inhibitory pairs that depend on both
frame and adjacent positioning (corrected Wilcoxon p values % 0.006) are indicated with a star.
Pairs with Wilcoxon p values > 0.006 are shaded in
gray.
(B) The CGA-GCG pair is inhibitory in different
contexts. The GFPFLOW ratio from each of three
sets of variants is positioned above the corresponding variant in a syn-GFPSEQ boxplot of all
variants with the CGA-GCG codon pair (identical
to the blue CGA-GCG boxplot in 2A). The GFPFLOW
ratio (inhibitory/optimal) is a comparison of
GFPFLOW values from two synonymous variants,
one with an inhibitory codon pair and the other with
an optimized pair.
(C) Inhibitory pairs are effective in Renilla luciferase-GFP (light blue) or GLN4(1-99)-GFP (dark
blue). Here, the GFPFLOW ratio compares variants
with three copies of an inhibitory pair to synonymous variants with three copies of the optimized
pair.
(D) Each codon in the CUC-CCG and CGA-CCG
pairs contributes to inhibition. Schematics of the
respective reporters (Renilla luciferase-GFP reporters contain three copies of the pair) and the
GFPFLOW ratio from each of three sets of variants
(inhibitory codon pair, optimized 50 codon, and
optimized 30 codon) are shown.
See also Figure S2.

CUG-CCG*

1.0

GFPFLOW ratio
(Inhibitory/Optimal)

CUG-AUA*

GFP

0.8

C
G
-C
C
U
C G
-C
C
A

CUC-CCG*

Rluc

RFP PGAL GLN4 GFP

CUC-AUA

1.00

Insert

-C

CGA-GCG*

0.75

RFP PGAL

CGA-CUG*

0.50

CGA-CGG*

0.25

Variant syn-GFPSEQ

AG
G
CG -CG
A A
CG -AU
A A
CG -CC
A G
CG -CG
A A
CG -CG
A G
CG -CU
A G
CU -GC
C G
CU - C C
G G
CU -CC
G G
GU -CG
A A
GU -CG
G- A
CG
A

CGA-CGA*

0.27

0.00

CGA-CCG*

0.65
0.49

n = 30

CGA-AUA*

CGA-GCG
Arg-Ala

1.0
0.8
0.6
0.4
0.2
0.0

AUA-CGG*

GFPFLOW ratio
(Inhibitory/Optimal)

AUA-CGA*

n=29
n=25
n=36
n=47
n=18
n=11
n=17
n=21
n=27
n=30
n=5
n=27
n=17
n=15
n=22
n=14
n=17
n=25
n=16
n=29
n=38
n=18
n=16
n=21
n=15
n=26
n=30
n=9
n=6
n=12
n=3
n=7
n=15
n=9
n=11
n=22
n=17
n=11
n=30
n=22
n=12
n=25
n=15
n=16
n=25
n=14
n=12
n=27
n=15
n=18
n=25
n=27
n=20
n=36
n=20
n=21
n=30
n=26

GFPFLOW ratio
(Inhibitory/Optimal)

AGG-CGG*

Figure 2. Adjacent Codons Mediate FrameDependent Inhibition

Out-of-frame
In-frame
Separated

Codon pair:

GFPFLOW ratio
(Inhibitory/Optimal)

(Figure 2A, gray); 19 pairs had lower


syn-GFPSEQ scores when the codon
pairs were in-frame (corrected Wilcoxon
p values % 0.006; CUC-AUA not significant). Additionally, we compared synGFPSEQ distributions of variants with an inhibitory pair to that
of variants with these codons at non-adjacent, in-frame positions (separated) (Figure 2A, purple). If the codon pair, rather
than additive effects of single codons, mediates translation inhibition, then we would expect greater inhibition by adjacent codons than by non-adjacent codons. 17 of the 20 candidate pairs
had lower syn-GFPSEQ scores when the codons were adjacent
(corrected Wilcoxon p values % 0.006; CUC-AUA, CUG-CUG,
and CUU-CUG not significant). Thus, inhibition by these 17 pairs
is dependent on both frame and adjacent positioning of the two
codons.
Because the boxplots revealed a range of syn-GFPSEQ scores
from variants with the same inhibitory pair, we used GFPFLOW to
obtain higher resolution measurements of relative inhibition
by an inhibitory pair in different contexts, including in variants
with high syn-GFPSEQ scores. For three variants containing

or

IA wobble
UG wobble

Codon
Pair

R-R
R-R
I-R
I-R
R-I
R-P
R-R
R-R
R-L
R-A
L-P
L-I
L-P
L-R
V-P
V-R
V-R

AGG-CGA
AGG-CGG
AUA-CGA
AUA-CGG
CGA-AUA
CGA-CCG
CGA-CGA
CGA-CGG
CGA-CUG
CGA-GCG
CUC-CCG
CUG-AUA
CUG-CCG
CUG-CGA
GUA-CCG
GUA-CGA
GUG-CGA

CGA-CCG
105
Arg-Pro

Optimal
Pair

Vector
13.8 0.5
103
105 AGA-CCA
Arg-Pro

tP(CGG)*
87.6 2.4

Vector
33.1 1.6
AGA-GCU
Arg-Ala

tP(CGG)*
63.4 4.0
104
105

Vector
119.4 4.6
103

GFP

Vector

1.6

3 native tRNA

0.4
ND

ure S2B). Thus, each of these inhibitory


codon pairs mediates reduced expression either near the start of translation or
at internal coding sequence locations.
In two cases, we explicitly demonstrated that each codon in the pair
is necessary for inhibition. First, we
compared GFPFLOW of a variant with an
inhibitory pair (CUC-CCG) to GFPFLOW
of variants in which either the 50 or 30
codon was replaced with an optimized
codon (UUG-CCG, CUC-CCA). The
variant with the inhibitory pair showed dramatically lower
expression (14% of the optimized variants; Figure 2D). Second,
we tested three copies of the CGA-CCG pair at amino acid 100
and obtained similar results (Figure 2D). Thus, the inhibitory
codon pairs mediate reduced expression when present in-frame
in a coding sequence.

AG
GCG
A
CG
AAU
A
CG
ACC
G
CG
ACG
A
CG
ACG
G
CG
ACU
G
CG
AGC
G
CU
CCC
G
CU
GCC
G
CU
GCG
A
GU
ACG
A
GU
GCG
A

NA

(A) Wobble decoding is prevalent in the 17 inhibitory codon pairs.


(B) Flow cytometry scatter plots show GFPFLOW
from two sets of variants that contain an inhibitory
pair (top) or a synonymous optimized pair (bottom), in cells with either an empty vector or a
plasmid expressing the indicated tRNA; the nonnative exact matching tRNA is also indicated by a
star.
(C) The effect on the GFPFLOW ratio of expressing a
tRNA that decodes the 30 codon in an inhibitory
pair. Vector, blue; native tRNA, light purple; nonnative tRNA, dark purple. Error bars represent SD.
NA, not applicable; ND, not determined.
See also Figure S3.

3 non-native tRNA

0.8

NA

tA(UGC)
118.9 5.3
104
105

Figure 3. Codon Pair-Mediated Inhibition


Affects Translation and Is Suppressed by
Particular tRNAs

GFP

1.2

0.0

tA(UGC)
67.3 8.6

104
Vector
103 76.5 0.5
103

C
GFPFLOW ratio
(Inhibitory/Optimal)

CGA-GCG
Arg-Ala

Inhibitory
104
Pair
RFP

Amino
Acid

Vector
3 non-native tRNA (decodes Pro CCG)
3 native tRNA (decodes Ala GCG)

CGA-GCG (Figure 2B) and three containing CGA-CGG (Figure S2A), the ratio of inhibitory to optimal GFPFLOW scores was
always less than 0.66. Thus, the codon pair was inhibitory in
different contexts, although the magnitude of inhibition varied.
This variation could reflect effects from RNA structure or additional sequence context (nucleotide, codon, or amino acid).
Additionally, each three-codon insert introduces four codon
pairs (including the invariant codons at positions 5 and 9), all of
which could affect translation.
If inhibition by codon pairs is a general function of their translation by the ribosome, then the codon pairs should reduce
expression when positioned at diverse locations within the coding sequence. However, the magnitude by which codons affect
expression can depend on their location relative to the start of
translation; for example, CGA codon repeats are more inhibitory
near the start of the coding sequence (Letzring et al., 2010; Wolf
and Grayhack, 2015). Therefore, we tested whether inhibition occurs at internal locations by inserting three copies of an inhibitory
codon pair at amino acid 100 (between an N-terminal GLN4(1-99)
domain and GFP) and at amino acid 318 (between Renilla luciferase and GFP) (Letzring et al., 2010; Wolf and Grayhack,
2015); we carried out this test for the 12 pairs with the lowest
syn-GFPSEQ medians. In each case, GFPFLOW with the inhibitory
pairs was lower than with optimized pairs (from 20%67%; Figure 2C). We also showed that increasing the copy number of the
codon pairs results in greater inhibition (three pairs tested at
amino acid 6 and two pairs tested at amino acid 100) (Fig-

Wobble Decoding Is Central to Inhibition by Codon Pairs


The codon composition of the 17 pairs is consistent with the idea
that these pairs inhibit translation and that wobble decoding is
central to their effects. All ten codons in these pairs are implicated in poor translation by their infrequent use in highly expressed genes, as measured by CAI (Sharp and Li, 1987). The
Arg CGA codon, the only codon in yeast decoded via a purinepurine I,A wobble, is found in more than half of the candidate
pairs (Figure 3A). The other nine codons in the 17 pairs include
all three codons decoded exclusively by U,G wobble, as well
as six codons decoded by low-copy tRNAs (one or two gene
copies), three of which are also decoded by a second tRNA via
wobble interactions (Johansson et al., 2008) (Figure 3A). Wobble
interactions have been implicated in both slow decoding (Lareau
et al., 2014; Srensen and Pedersen, 1991; Stadler and Fire,
2011) and in inefficient translation (Letzring et al., 2010).
To determine if defects in decoding inhibitory codon pairs are
responsible for low expression, we evaluated the ability of overexpressed tRNAs to suppress the low GFPFLOW of variants with
inhibitory codon pairs. We initially examined suppression by
Cell 166, 679690, July 28, 2016 683

tRNAs that decode the 30 codon of inhibitory pairs, since the


30 codon is likely to occupy the ribosomal A site during the
inhibitory reaction. The expression defect for variants with 10
of 12 pairs tested was suppressed either by increasing the abundance of a native tRNA or by expressing a non-native tRNA that
enables decoding by Watson-Crick base pairing at all three
bases (exact matching) (Figures 3B and 3C; not for AGG-CGA
or CGA-CGG). Maximal suppression ranged from 1.8- to 7.7fold increases in GFPFLOW (relative to an empty vector), and suppression was only slightly augmented in the one case tested by
co-expression of two tRNAs, one for each codon (Figure S3A).
As shown below, for one of the pairs (CGA-CGG) in which
tRNA for the 30 codon did not suppress, the expression defect
was strongly suppressed by a non-native exact matching tRNA
that decodes the 50 codon (see below). Thus, for these 11
tRNA-suppressible pairs, inhibition is due to a translation defect.
Furthermore, since the expression defect for AGG-CGA was
alleviated by shifting the reading frame (Figure S3B), we infer
that inhibition in this case could also likely be a translational
defect.
To evaluate the role of wobble decoding in codon-mediated
inhibition, we compared the degree of suppression by 30 native
tRNAs to that of 30 exact matching, non-native tRNAs. The exact
matching tRNA was more effective at suppressing inhibition by
the eight tested pairs (substantially so for three pairs with a
30 Pro CCG codon [Figures 3B and 3C] and four pairs with a
30 Arg CGA codon, but marginally so for a pair with a 30 Leu
CUG codon [Figure 3C]). Because correcting wobble decoding
improved translation more effectively than did increased
amounts of the native tRNA, we conclude that I,A and U,G
wobble base pairings contribute to inhibition of translation by
codon pairs.
We also examined the GFP mRNA abundance from six variants with an inhibitory pair. The amount of GFP mRNA from
each of these variants with an inhibitory pair was reduced relative
to that from a synonymous optimized variant (Figure S3C), as
might be expected since many translational defects result in
mRNA degradation (Shoemaker and Green, 2012). For a variant
with CUC-CCG, expression of the non-native, exact matching

tRNAProCGG suppressed both mRNA and GFP defects (Figure S3D), illustrating a link between mRNA and translation efficiency for this variant with an inhibitory pair.
Inhibition by Codon Pairs Implicates Interactions
between Sites in the Ribosome
Since the codons had to be adjacent for low expression mediated by codon pairs, we considered that these pairs might act
in a concerted manner to mediate inhibition, with each codon
in the pair playing a unique role in the inhibitory effect and occupying a specific position in the ribosome. If inhibition occurs
when the 30 codon enters or occupies the ribosomal A site,
then the 50 codon in the pair would occupy the P site. In this
case, overproduction of a native tRNA that decodes the 50 codon
would not be expected to suppress inhibition by the codon pair.
In testing ten pairs (excluding CGA-CGA, which has identical
codons, and AGG-CGA, which is not tRNA suppressible), we
found that increased expression of a native tRNA corresponding
to the 50 codon had no significant effect on inhibition by eight
684 Cell 166, 679690, July 28, 2016

pairs, and only marginally suppressed inhibition by GUA-CGA


(Figure 4A). For CUC-CCG, overproduction of the single copy
native tRNALeu(GAG) substantially suppressed inhibition, but
overproduction of a different native tRNA (tRNALeu(UAG)) that
competes to decode the same codon by wobble interactions
increased inhibition (Figure 4A). Thus, reducing the use of
wobble decoding for the P site codon improved expression.
We demonstrated that charged tRNAArg(ICG) was increased
9-fold when the tRNA was expressed from a high copy 2m
plasmid (Figures 4A and 4B), even though this overproduction
resulted in no suppression of inhibitory pairs with a 50 CGA.
Furthermore, the use of an even higher copy leu2-d 2m plasmid
(Beggs, 1978) resulted in a 20-fold increase in charged
tRNAArg(ICG), but still no detectable suppression of these pairs
(Figures 4B and 4C). By contrast, the non-native exact matching

tRNAArgUCG suppressed expression defects (Figure 4A), and,
thus, translation of the 50 codons is also central to inhibition
by codon pairs. These results are consistent with the idea
that codon-anticodon interactions in the P site affect A-site
decoding.
If the position of each codon in the ribosome is critical for
codon pair-mediated inhibition, then the order of codons in an
inhibitory codon pair should be important for inhibition. Of the
17 pairs identified in this study, the CGA-CGA pair is composed
of identical codons and two sets of pairs are inhibitory with the
codons in either order (AUA-CGA, CGA-AUA and CUG-CGA,
CGA-CUG), leaving 12 inhibitory pairs with a single order of
codons. For each of these 12, we compared the GFP expression
of variants with the inhibitory pair to those with its reverse pair
(i.e., with the two codons in reverse order). In each case, variants
with the inhibitory pair tended to have lower syn-GFPSEQ scores
than variants with the reverse pair (corrected Wilcoxon p value %
0.006; Figure 4D). Similarly, two variants with the inhibitory pair
CUC-CCG had low GFPFLOW relative to a synonymous variant
with the optimized pair, whereas two variants with the reverse
pair, CCG-CUC, had high GFPFLOW (Figure 4E). Thus, the idea
that the 50 codon in an inhibitory pair has a role distinct from
the 30 codon is supported by failure of overproduced native
tRNAs that decode the 50 codon to suppress inhibition (eight
pairs), as well as by the dependence of GFP inhibition on the
order of codons (12 pairs). We conclude that inhibition by most
codon pairs is likely to involve interactions between tRNAs at
adjacent sites in the ribosome.
12 Inhibitory Codon Pairs Have Elevated Ribosome
Occupancies on Yeast Gene Transcripts
To assess the potential influence of inhibitory pairs on translation
of yeast genes, we examined the overall expression of genes
containing these pairs. The pairs occur 2,922 times in 1,868
genes (31.6% of the 5,917 yeast genes), including 28 occurrences in 659 genes (Engel et al., 2014). Consistent with the 17
inhibitory pairs having a negative impact on translation efficiency
and as expected from the low CAI of their individual codons,
these pairs predominantly occur in yeast genes with low to moderate expression (based on mRNA levels) (Figure 5A). Many
ORFs with an inhibitory pair tend to have reduced protein (Kulak
et al., 2014) per mRNA transcript (Presnyak et al., 2015)
compared to other ORFs within a similar CAI range (corrected

5 native tRNA

0.8
0.6
0.4

0.2
NA

0.0

NA

NA

0.4
0.2

Lane 1 2 3 4 5

Inhibitory pair
Reverse order pair
1.2

n=34
AGG-CGA* n=30

AUA-CGG* n=22
n=27
CGA-CCG* n=16
n=22
CGA-CGG* n=22
n=38

0.8

CGA-GCG* n=18
n=30

0.4

CUC-CCG* n=16
n=15

0.0

CUG-AUA* n=22

CU
C
CC -C
G- CG
C U -A
C - CU
AC
AC
U
UAC C
U- UC
CC -C
G- CG
CU
C

A
G

-C

UG

G
-C

UA

A
-C
G
CU

CG

CG

A-

CG

G
A-

CG

Inhibitory pair

AGG-CGG* n=40
n=36

CC
A-

CG

CG

A-

AU

0.0
CG

1/5x

0.6

A-

1x

Phe(GAA) Arg(ICG)

1x 1x 1/5x 1x

tR(ICG) 2 leu2-d

Vector

0.8

CG

2 leu2-d

vector
tR(ICG)
tR(ICG)

Deacyl.
vector
tR(ICG)
tR(ICG)

2 LEU2

GFPFLOW ratio
(Inhibitory/Optimal)

Figure 4. Inhibition Depends on Codon


Order and Pair Effect

5 non-native tRNA

1.0

GFPFLOW ratio
(Inhibitory/Optimal)

Vector

AG
GCG
A
CG
AAU
A
CG
ACC
G
CG
ACG
A
CG
ACG
G
CG
ACU
G
CG
AGC
G
CU
CCC
G
CU
GCC
G
CU
GCG
A
GU
ACG
A
GU
GCG
A

GFPFLOW ratio
(Inhibitory/Optimal)

Reverse order pair

(A) The effect on the GFPFLOW ratio of expressing a


tRNA that decodes the 50 codon of an inhibitory
pair. Vector, blue; native tRNA, light orange; nonnative tRNA, dark orange. Leu CUC is decoded by
two native tRNAs; the exact matching tRNA is
indicated by a star. Error bars represent SD. NA,
not applicable. CGA-CGA data are also shown in
Figure 3C.
(B) Charged tRNAArg(ICG) levels increase when
tRNAArg(ICG) is expressed from either a 2m or 2m
leu2-d vector, as measured with an acidic northern
blot probed for tRNAArg(ICG) and tRNAPhe(GAA).
Charged tRNA (black arrow) and uncharged tRNA
(gray arrow) are indicated.
(C) Effects of increasing native tRNAArg(ICG) by
expression from a 2m leu2-d vector on the
GFPFLOW ratio from each of eight sets of variants
(leu2-d vector, blue; tRNAArg(ICG), gray). Error bars
represent SD.
(D) syn-GFPSEQ distribution of variants with an
inhibitory pair (blue) compared to that of variants
with the same pair of codons in reverse order
(pink). Distributions are plotted for the 12 pairs for
which only a single order of the codons is present
in the list of inhibitory pairs. Boxplot edges mark
the first and third quartiles. Stars indicate a corrected Wilcoxon p value % 0.006. Blue boxplots
are identical to those in Figure 2A.
(E) Inhibition by the CUC-CCG pair depends on the
order of the codons. The GFPFLOW ratio for two
sets of variants, each with an inhibitory pair (blue)
and the reverse pair (pink), is shown. Error bars
represent SD.
See also Table S1.

n=22

CUG-CCG* n=28
n=30

To assess each codon pair for evidence


of reduced translation rates, we evaluGUA-CGA* n=18
ated the overall footprint count at codon
n=36
pairs relative to neighboring codon posiGUG-CGA* n=35
n=30
tions. For each pair, we located all of its
sites in yeast ORFs and aligned windows
0.00
0.25
0.50
0.75
1.00
of up to 100-codon positions, with the
Variant syn-GFPSEQ
pair at the center of each window. At
each of the aligned positions, we calcut test p value % 0.01; Figure 5B) or to ORFs with a reverse-order lated ribosome occupancy by summing footprint counts across
pair (corrected t test p value = 6.34 3 105 for group of 12 pairs; ORFs and normalizing to total counts from all positions. Based
Figure 5C).
on an even distribution of footprints, we would expect baseline
We proceeded to investigate whether translation elongation occupancy of about 0.01 at each position. Occupancies at those
slows at inhibitory codon pairs in yeast transcripts by exam- positions with the pair in ribosomal sites tended to be higher than
ining existing yeast ribosome profiling data, in which ribosome baseline occupancies at surrounding codon positions (Figlocations and the identities of codons in the ribosome are ure 6A). By combining occupancies from two positions (with
inferred from the sequences of ribosome-protected mRNA the pair in the ribosomal P, A sites and E, P sites), we obtained
fragments (footprints). Relative translation rates are inferred the pairs cumulative ribosome occupancy. We applied the
from the density of footprints, with positions of reduced same approach to calculate cumulative ribosome occupancies
translation speed yielding higher densities. Since cells treated for dipeptides and individual codons.
with cycloheximide were recently shown to have altered
Consistent with some previous reports (Artieri and Fraser,
footprint distributions (Hussmann et al., 2015), we evaluated 2014; Hussmann et al., 2015; Lareau et al., 2014), Pro-Pro sites
a yeast experiment carried out without cycloheximide (Jan had the highest cumulative occupancy of sites for a dipeptide
et al., 2014).
(0.04), and the codons CGA, CCG, and CGG had the highest
GUA-CCG* n=29
n=25

Cell 166, 679690, July 28, 2016 685

ORFs by mRNA Quartile


(Presnyak, 2015)

N.D.
n = 38

Yeast ORFs

Q4
n = 98

Inhibitory pairs
n = 1,868

Q3
n = 398

Q1
n = 621
68%

32%

Q2

n = 713

No inhibitory pairs
n = 4,049

C
Inhibitory pairs
Reverse order pairs

Inhibitory pairs
No inhibitory pairs

10

**
*

**
*

**
*

log2(protein/mRNA)

log2(protein/mRNA)

10

n = 708

n = 118
n = 92
n = 335
n = 304
n = 477
n = 517
n = 308
n = 570
n = 111
n = 390
n = 39
n = 279

n = 678

CAI bin: 0.425 0.450 0.475 0.500 0.525 0.550

**
*

Figure 5. Inhibitory Pairs Occur in Genes with Both Low Expression


and Translation Efficiency
(A) Proportion of S. cerevisiae ORFs with at least one of the 17 inhibitory pairs
(left) and the proportion of these ORFs present in each mRNA abundance
quartile (right), as based on steady state, total mRNA (Presnyak et al., 2015).
Q1 indicates the bottom 25% of S. cerevisiae transcript abundance.
(B) Estimated translation efficiency distribution (protein abundance [Kulak
et al., 2014] normalized to mRNA [Presnyak et al., 2015]) for ORFs with at least
one inhibitory pair (blue) or no inhibitory pair (gray) and grouped by CAI. CAI
bins, labeled by their lower CAI limit, are 0.025 in size. Stars indicate a corrected t test p value % 0.01 (*) or % 3.69 3 109 (***).
(C) Estimated translation efficiency distribution for ORFs with at least one of 12
inhibitory pairs for which the reverse-order pair was not in the inhibitory list
(blue) and ORFs with at least one of the reverse-order pairs (pink). ORFs with
both inhibitory and reverse-order pairs were excluded from the analysis. Stars
indicate a corrected t test p value % 6.34 3 105.

cumulative occupancies for individual codons (0.04 to 0.05).


Inhibitory pairs also had elevated cumulative occupancies. For
each inhibitory pair, we evaluated the significance of its cumulative ribosome occupancy by comparison to 10,000 permutations
of the footprint counts in each ORF and found that all 17 inhibitory pairs had higher occupancy than expected by chance (corrected permutation p value < 0.009), given ORF coverage and
footprint distributions. Twelve inhibitory pairs had cumulative occupancies in the top 0.6% of all codon pairs (more than three
SDs above the mean; Figure 6B). In particular, the four pairs
686 Cell 166, 679690, July 28, 2016

with the strongest inhibitory effects in the GFP assay were


among the five pairs with the highest occupancies (Figure 6B).
Thus, inhibitory pairs tended to be translated slowly, and pairs
with some of the highest occupancies also showed the greatest
inhibition of GFP expression.
To assess the pairs effects on ribosome occupancy, relative
to potential individual codon and dipeptide effects, we first
ranked synonymous codon pairs by each pairs cumulative
occupancy. Inhibitory codon pairs tended to have some of the
highest occupancies among codon pairs specifying a given
dipeptide (Figure 6C). We also carried out direct comparisons
between synonymous pairs using a Fishers exact test on the
footprint counts at each pair and its surrounding codon positions. We compared each inhibitory pair to two other pairs: a synonymous pair with the same 50 codon, but an optimized 30 codon,
and a synonymous pair with the same 30 codon, but an optimized
50 codon. For 12 inhibitory pairs, the proportion of footprints at
the inhibitory pair was higher than for each synonymous comparison (corrected Fishers exact p value % 6.79 3 108; Table S5).
In analyzing a separate ribosome profiling dataset by Lareau
et al. (2014), we found ten of these inhibitory pairs also reached
significance in synonymous comparisons (corrected Fishers
exact p value % 0.002). We conclude that ribosomes tend to
translate through 12 of 17 inhibitory codon pairs more slowly
than through either of the individual codons across matching
dipeptide sites.
We also evaluated the impact of codon order on ribosome
occupancy. For the 10 of 11 pairs that differed from synonymous pairs (excluding the CGA-CGA pair), the proportion of
footprints at the inhibitory pair was significantly higher than
for the reverse pair (corrected Fishers exact p value %
4.63 3 1032; Figure 6D). Thus, we conclude that slower translation of these inhibitory pairs is, in large part, due to codon pair
effects, rather than due to the simple result of sequential, individual codon effects.
DISCUSSION
We establish that codon pairs affect translation elongation and
translation efficiency in yeast in a manner distinct from the effects of their individual constituent codons. For 17 inhibitory
codon pairs, we show that it is the pair, rather than the sixbase sequence, the two individual codons, or the encoded
dipeptide, that is responsible for inhibition. GFP variants containing an inhibitory pair had significantly lower expression than variants in which the same six-base sequence was out of frame, the
two codons were present but separated, or one of the codons of
the pair was instead an optimal codon. We demonstrate that the
inhibition occurs during translation by suppressing it with overexpressed tRNA (11/12 pairs tested). Codon pair effects are
distinguished from individual codon effects by two findings reported here. First, the order of codons in the pair was required
for inhibition (for 12 of the 17 pairs). Second, translation rates
of many inhibitory pairs were slower (based on ribosome occupancies) than the rates of pairs encoding the same dipeptide
or of pairs with the reverse codon order. These findings implicate
interplay between adjacent ribosomal sites in codon pair-mediated inhibition.

0.05

CGA-GCG
Arg-Ala
n=35

CG

Cumulative ribosome occupancy


(P, A-site + E, P-site)

0.10

Ribosome occupancy

0.00
0.10
0.05

CUC-CCG
Leu-Pro
n=60

0.00
0.10
0.05

CGA-CUG
Arg-Leu
n=91

0.00
0.10
0.05

CUG-AUA
Leu-Ile
n=559

A-

G
CC

Mean

CG

G
A- G
A
CG CC
AU
C
AU
G
C
C
GA
-C

2 sd

3 sd

Inhibitory pair
Other codon pair

0.10

CG

CG

GA

A A
C
CG G
G
A- C-C
G
CC
U
U
U
A
A-C G G C CG
U
A
C
G
GCGG-C A
G
GU
CU -C
G
GA
CU
G
A A-C
CG
GG
G
AAU
UA G-C
-C
U
A
G
A
G
AG
AG
CU

0.05

0.00

0.00
0 +25 +50
-25
codon distance

-50

0.4

0.6
0.8
syn-GFPSEQ median

1.0

C
Inhibitory pair

Synonymous pair

Mean

UA*
GA

CU

Leu-Arg

A
CG G
AUAACG
AU

3 sd
Leu-Pro

0.05

2 sd

G*
CC
CUC CCG*

CUG

Leu-Ile

0.10

Ile-Arg

CG

CUG

0.00
*

UG

AC

CG

CG

AC

Arg-Pro

0.05

UA*
AA

CG

Arg-Leu

AG

CG

Arg-Ile

CG

0.10

Arg-Ala

CG

0.00

0.05

GA*
AC G*
CG ACG
G
CG
CG GA
AGGGGC
A

10 20 30

CG

C
GUA

Val-Pro

0.10

Ribosome occupancy

0.00

0.09
0.06
0.03
0.00
0.09
0.06
0.03
0.00
0.09
0.06
0.03
0.00
0.09
0.06
0.03
0.00

10 20 30
Rank
Reverse order pair

10 20 30

(A) Examples of ribosome occupancy for an


inhibitory pair and surrounding baseline positions.
At codon distance 0, the inhibitory pair is positioned in the P and A sites of the ribosome. Occupancy at each position is the sum of footprints
across aligned ORFs and normalized to total
footprints from all window positions.
(B) Median syn-GFPSEQ of variants with a given
pair versus cumulative ribosome occupancy for
two positions (with the pair in the P, A sites and E, P
sites). Horizontal lines represent the mean occupancy of all codon pairs and 2 or 3 SDs above the
mean (as indicated).
(C) Ranking of synonymous codon pairs by their
cumulative ribosome occupancy (at P, A and E,P
positions). Black dots below the bars indicate
synonymous pairs used in Fishers exact comparisons because they have a CAI-optimal codon
and a 50 or 30 codon identical to one of the inhibitory pairs.
(D) Ribosome occupancy by position in the ribosome for inhibitory pairs (blue) and pairs with the
reverse codon order (pink). Panels on the right
show two sets of pairs, for which both codon orders were identified as an inhibitory codon pair.
The black line indicates expected occupancy
(0.01), based on an even distribution of footprints.
Stars indicate inhibitory pairs with higher cumulative occupancy at the P, A-site and E, P-site positions compared to the reverse pair (one-sided
Fishers exact corrected p value % 4.63 3 1032).

Inhibitory pair
1, E site E, P site P, A site A site, +1

0.10

Arg-Arg

Cumulative ribosome occupancy (P,A-site + E,P-site)

0.15

Figure 6. Inhibitory Codon Pairs in Yeast


Gene Transcripts Have Elevated Ribosome
Occupancies

Val-Arg

suppressed inhibition in some cases


while overproduction of native wobble
0.00
decoding tRNA did not. Wobble decoding
A*
G
0.10
GC A*
has been implicated in both slow and
GU ACG
GU
0.05
inefficient decoding of individual codons
0.00
(Lareau et al., 2014; Letzring et al., 2010;
1 10 20 30
Srensen and Pedersen, 1991; Stadler
Rank
G CG CG UA CG G A GA GA UA GA UG
A
G
G
G
and Fire, 2011). Our findings are consisC
-C -A -C -C
CG CG CG CG -C G -C -A -C CC -CG -C
G- G- UA- GA- GA GA- UC UG UG UA- UA UG AUAGA UG GA
tent with a model in which wobble decodG
G
C
A C *C *C *C *C *C *G *G *G
A A
*C
*C
ing, rather than limited quantities of
tRNA, is central to codon pair-mediated
inhibition.
Codon-anticodon interactions at both the 50 and 30 codon play
The ribosome is a highly coordinated machine with communia major role in inhibition, as illustrated by three lines of evidence. cation between tRNAs in the A, P, and E sites mediated by
First, most inhibitory pairs (15/17) have a codon that relies on numerous protein and rRNA contacts (Demeshkina et al.,
wobble decoding, while synonymous pairs with codons that 2010). It is well established that tRNA:codon interactions at the
are decoded by the same tRNA species (but via Watson-Crick P site affect A site interactions, as during programmed framebase pairing) were not inhibitory. Moreover, in 12 of the 17 inhib- shifting (Atkins and Bjork, 2009) and in a post-peptide bond
itory pairs, the 30 codon is decoded by an abundant wobble de- quality control mechanism in E. coli (Zaher and Green, 2009).
coding tRNA (encoded by 3, 5, 6 and 10 tRNA gene copies, with However, it has not been appreciated that communication begene copy number strongly correlating with abundance [Tuller tween tRNAs at adjacent sites plays a general role in regulating
et al., 2010a]). Second, non-native exact matching tRNAs that the rate and efficiency of translation elongation. Concerted efdecode the 30 codons suppressed inhibition much more than fects of adjacent codons could occur at several steps in the elondid increased amounts of native, wobble decoding tRNAs (seven gation reaction, e.g., tRNA accommodation, formation of the
pairs). Third, exact matching tRNAs that decode the 50 codons hybrid state, translocation, or tRNA exit. Furthermore, inhibition
0.05

Cell 166, 679690, July 28, 2016 687

mediated by different pairs may work by distinct mechanisms,


since pairs differ with respect to their translation rate, dependence on codon order, requirement for wobble decoding, and
even in suppression by overproduction of a native exact matching 30 tRNA. However, we infer that acceptance of the 30 codon
into the A site is likely limiting for many of the identified pairs,
since overproduction of the native 30 tRNA frequently improved
GFP expression. Thus, inhibitory effects depend on a complex
interplay of the interactions between adjacent sites in the
ribosome, codon-anticodon interactions, and acceptance of a
codon into the A site.
That pairs of codons modulate translation efficiency may, in
part, explain why the effects of synonymous codons on translation efficiency have remained baffling (Plotkin and Kudla,
2011). Although several previous studies implicated codon
pairs in translation efficiency (Chevance et al., 2014; Coleman
et al., 2008; Gutman and Hatfield, 1989; Letzring et al., 2010),
most work has focused on the roles of individual codons (Plotkin and Kudla, 2011), with papers on codons outnumbering
papers on codon pairs or adjacent codons 175:1 (PubMed
citations of title and abstract). The prevailing model has been
that codons influence elongation efficiency primarily through
the small, additive effects of individual codons (Plotkin and Kudla, 2011) and indeed individual effects of some codons are
apparent (Hussmann et al., 2015; Lareau et al., 2014; Srensen
and Pedersen, 1991; Stadler and Fire, 2011). However, we
observed that the effects of an individual codon differed
considerably depending on which other codons it was paired
with. For example, eight CGA-NNN codon pairs had synGFPSEQ medians between 0.44 and 0.73, while the remaining
53 such pairs had medians >0.91. Moreover, the existence of
strong inhibitory pairs calls into question the idea that many,
individually small events sum to a substantial effect on expression. Instead, a few inhibitory codon pairs may act as discrete
regulatory signals and could be as strongly selected as miRNA
recognition sequences.
Inhibitory codon pairs in yeast, and potentially in other
organisms, may have broad effects on translation efficiency,
protein folding, and mRNA decay. Understanding the mechanisms by which inhibitory codon pairs impact translation
is essential to predict the functional implications of codon
composition.
EXPERIMENTAL PROCEDURES
Library Construction, FACS, and Flow Cytometry
Construction and transformation of (NNN)3 and (VNN)3 libraries of GFP variants in the RNA-ID reporter were performed as described previously (Dean
and Grayhack, 2012). Growth of each library, fluorescence-activated cell
sorting of 39.5 million cells from each library, and analysis of individual
variants were performed as described previously (Dean and Grayhack,
2012), with differences noted in the Supplemental Experimental Procedures.
Oligonucleotides and plasmids employed in this work are listed in Tables S6
and S7.
Sequencing of GFP Three-Codon Insertions
From genomic DNA samples, we amplified GFP library fragments through 25
PCR cycles, using primers specific to the flanking regions and containing a
FACS bin-specific index (Table S6). We then pooled the amplified fragments
and sequenced on an Illumina GAII sequencer with single-end reads. For qual-

688 Cell 166, 679690, July 28, 2016

ity control, we required each read to have accurately called six bases
(AACGCA) immediately downstream of our variable region and for each of
the nine variable base calls to have a score of Q30 or better. To compare
read counts across bins, we corrected for the number of cell sorting events
in a given bin. See additional filtering and scoring details in the Supplemental
Experimental Procedures.
Analysis of Ribosome Profiling Datasets
We analyzed a whole-cell ribosome profiling sample with no cycloheximide
treatment from Jan et al. (2014). A-site codon footprint tallies were provided
by Jeff Hussman as described in Hussmann et al. (2015). See additional details
in the Supplemental Experimental Procedures.
Acidic Northern Blot Analysis
Bulk RNA, prepared from 3 OD pellets, was resolved on 6.5% acrylamide
gels at pH 5 as described previously (Alexandrov et al., 2006).
Explanation of the Statistical Methods
To assess the significance of each 6-mer sequences enrichment in low GFP
variants, we tracked occurrences of the 6-mer in low variants across
100,000 permutations. Variants were assigned to one of ten pools based on
GC count, and we shuffled the expression categories within each pool. From
this analysis we derived p values for the frequency of each 6-mer in low variants, based on the probability of obtaining as many, or more, low-variant
counts by chance.
We also estimated the chance probabilities of footprint densities. For each
pair, we carried out 10,000 permutations, in which we shuffled the A-site
codon footprint counts within each ORF with the pair and recalculated footprint density at ribosomal site positions. To directly evaluate the significance
of differences between synonymous pairs, we performed one-sided Fishers
exact tests on 2 3 2 contingency tables with the footprint counts for each
pair at ribosome site positions and at the remainder of codon positions within
a 100-codon window. See additional details in the Supplemental Experimental
Procedures.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
three figures, and seven tables and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2016.05.070.
AUTHOR CONTRIBUTIONS
C.E.G., C.E.B, S.F., and E.J.G. wrote the manuscript. C.E.G. and C.E.B. acquired data and performed the computational and experimental analyses,
respectively; K.M.D. identified tRNA suppressible variants from a pilot screen;
and E.J.G. and S.F. supervised the work.
ACKNOWLEDGMENTS
We thank Eric Phizicky, Andrew Wolf, Scott Butler, Gloria Culver, Adam Geballe, David Mathews, David Morris, and Yi-Tao Yu for discussions and comments on the manuscript, Jeffrey Hussman for assistance with ribosome
profiling data, Josh Hatfield, Shannon Schmitt, Blake Bentley, and Erin Eidschun for assistance with experiments, XiaoJu Zhang and David Mathews
for initial help analyzing codon use in the yeast genome, the URMC Flow Cytometry Resource and NCCR (1S10RR029229901) for technical support. This
work was supported by an NSF grant (MCB-1329545) (to E.J.G.) and an NIH
grant (1P41 GM103533) (to S.F.). C.E.B. was also supported by an NIH T32
Training Grant (GM068411), and C.E.G. was supported by an NSF Fellowship.
S.F. is an investigator of the Howard Hughes Medical Institute.
Received: December 18, 2015
Revised: April 14, 2016
Accepted: May 19, 2016
Published: June 30, 2016

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