103

REVIEW / SYNTHSE

Tubulin synthesis and assembly in

differentiating neurons
Nicole B. Laferrire, Thomas H. MacRae, and David L. Brown

Abstract: Neurons are highly polarized cells that extend long processes, the axons and dendrites, to form contacts with target
cells. The formation and maintenance of this specialized morphology relies on the assembly of an organized microtubule
array that is the predominant component of the neuronal cytoskeleton. During this process there is an evolution in the
composition and dynamics of microtubules, resulting in stable microtubule bundles that provide structural support and
function in intracellular transport along the axon. In this essay we provide an overview of the mechanisms regulating the
synthesis and assembly of tubulin in differentiating neurons with particular attention to the roles of multiple tubulin isotypes,
posttranslational modifications of tubulin, and microtubule-associated proteins. We conclude that, ultimately, the
developmental regulation of microtubules in neurons may require the coordinated expression and posttranslational
modifications of tubulin and microtubule-associated proteins to provide biochemical forms that favour specific interactions,
each combination conferring distinctive dynamic and functional properties.
Key words: neuronal differentiation, microtubules, tubulin isotypes, tubulin posttranslational modifications,
microtubule-associated proteins.

Rsum : Les neurones sont des cellules trs polarises qui projettent de longs prolongements, les axones et les dendrites,
pour entrer en contact avec des cellules cibles. La formation et le maintien de cette morphologie spcialise ncessitent
lassemblage dun rseau organis de microtubules, principal constituant du cytosquelette neuronal. Au cours de ce
processus, la composition et la dynamique des microtubules voluent. Il en rsulte des fuseaux de microtubules stables qui
assurent le rle et le support structural du transport intracellulaire le long de laxone. Dans cet article, nous faisons une revue
des mcanismes rglant la synthse et lassemblage de la tubuline dans les neurones en diffrenciation, en sattardant
particulirement au rle des multiples isotypes de la tubuline, aux modifications post-traductionnelles de la tubuline et aux
protines associes aux microtubules. Nous concluons que la rgulation des microtubules au cours du dveloppement des
neurones ncessiterait la coordination de lexpression et des modifications post-traductionnelles de la tubuline et des
protines associes aux microtubules afin de gnrer les formes biochimiques favorisant des interactions spcifiques, chaque
combinaison confrant des proprits fonctionnelles et dynamiques distinctives.
Mots cls : diffrenciation neuronale, microtubules, tubuline, isotypes, modifications post-traductionnelles, protines
associes aux microtubules.
[Traduit par la rdaction]

Introduction
Neurons are highly specialized cells with a characteristic po-
larized morphology resulting from unique cytoskeletal arrange-
ments. A typical neuron consists of a cell body (perikaryon)
and extended neurites (one axon and up to several arborized
dendrites) that may be metres in length. Microtubules (Mts)
are a predominant component of the neuronal cytoskeleton,
they comprise 15 to 20% of total cell protein in brain, and they
are essential for the development and maintenance of neuronal
morphology. They function in the mitotic division of differen-
tiating neuronal precursors, in neurite outgrowth and migration,
and as substrates for the intracellular transport of organelles
and proteins. Mts may also play a role in neuronal plasticity
and possibly long-term potentiation involving memory and
learning. For reviews of several aspects of Mt function in
neurons see Mitchison and Kirschner (1988), Burgoyne (1991),

Received January 7, 1997. Revised April 7, 1997. Accepted April 7, 1997.

Abbreviations: Mts, microtubules; MAPs, microtubule-associated proteins; MTOC, microtubule organizing centre; C-terminus,
carboxy-terminus; CHO, Chinese hamster ovary.
N.B. Laferrire and D.L. Brown.1 Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5, Canada.
T.H. MacRae. Department of Biology, Dalhousie University, Halifax, NS B3H 4J1, Canada.
1
Author to whom all correspondence should be addressed. e-mail: dbrown@science.uottawa.ca

Biochem. Cell Biol. 75: 103117 (1997) 1997 NRC Canada

104
Cambray-Deakin (1990), Joshi and Baas (1993), Falconer et al.
(1994), Challacombe et al. (1996), and Heidemann (1996).
Early isoelectric focusing analyses of brain tubulin, the
major structural protein of Mts, showed multiple isoforms of
both - and -tubulin (Gozes and Littauer 1978; Marotta et al.
1978). The presence of several tubulin isoforms in a single
neuron raised the possibility that tubulins are sorted into
Mts that differ in composition and behaviour, the so-called
multitubulin hypothesis (Fulton and Simpson 1976; Gozes
and Sweadner 1981). Since these early studies it has become
clear that tubulin heterogeneity in neurons and in other cell
types is due to differential gene expression, posttranscriptional
processing of mRNA, and posttranslational modification of
- and -tubulin (Sullivan 1988; MacRae 1992; Luduea
1993; Raff 1994).
Microtubule organization in neurons
Long processes, the axons and dendrites, extend from neuronal
cell bodies to form contacts with target cells. Mts within these
processes must be tractable to permit neurite outgrowth and
respond to environmental cues and injury, and yet sufficiently
stable to maintain established neural circuitry. Temporal and
spatial changes in Mt dynamics are associated with the devel-
opment and maintenance of neuronal processes.
The structural subunit of all Mts is a 100-kDa protein
heterodimer termed tubulin, consisting of one 50-kDa - and
one 50-kDa -polypeptide. Tubulin molecules polymerize in
a head-to-tail fashion forming linear units known as proto-
filaments. Thirteen protofilaments form lateral associations to
create a Mt, an apparently hollow tube with a diameter of
24 nm. Protofilaments are assembled with - and -tubulin
subunits alternating, resulting in a polarized structure, with
-tubulin exposed at one end of the Mt and -tubulin at the
other. Assembly and disassembly of tubulin dimers take place
preferentially at one end of the Mt designated the plus (+) end,
with little assembly taking place at the other (minus, ) end,
which is generally embedded in the microtubule organizing
centre (MTOC).
Differences in Mt dynamics are operationally defined using
a variety of experimental manipulations, such as the relative
resistance of Mts to depolymerization induced by low tempera-
ture and by drugs such as colchicine (e.g., Black and Greene
1982). Mt dynamics have also been measured directly by
microinjection of labelled tubulin subunits into cells. Using
this approach the dynamic properties of Mts have been shown
to vary between cell types (Pepperkok et al. 1990) and even
between specific locations within a single cell (Schulze and
Kirschner 1987).
One model for Mt dynamics, the dynamic instability model,
predominates. Each - and -tubulin in the heterodimer con-
tains one bound guanosine triphosphate (GTP). At some point
after the incorporation of tubulin into the growing (+) end of
the Mt, the GTP associated with -tubulin is hydrolysed to
guanosine diphosphate (GDP) (David-Pfeuty et al. 1977). The
-tubulin GTP is not hydrolysed (Spiegelman et al. 1977;
Burns 1991; Burns and Farrell 1996). Both the mechanical and
structural properties of GTP-tubulin differ from those of GDP-
tubulin (Vale et al. 1994). Because GDP-tubulin dissociates
from a microtubule more readily than does GTP tubulin, ac-
cumulation of GDP-bound tubulin renders a Mt unstable and
Biochem. Cell Biol. Vol. 75, 1997
results in rapid depolymerization (Kirschner and Mitchison
1986). If more free GTP-tubulin is available and binds to the
growing end of the Mt before the GTP of earlier subunits is
hydrolysed, the Mt remains stable and assembly continues. In
this way, the concentration of free tubulin and the rate of GTP
hydrolysis are important factors in the regulation of Mt growth
and disassembly (Mitchison and Kirschner 1984).
Actively proliferating cells contain a radial array of
dynamic Mts that exhibit a half-life of approximately 5 min,
while the cell maintains a constant Mt mass (Schulze and
Kirschner 1986). Postmitotic neurons, on the other hand, es-
tablish less dynamic subpopulations of Mts with slower rates
of tubulin turnover (Kirschner and Mitchison 1986). In imma-
ture rat sympathetic neurons, for example, the average half-life
of axonal Mts is approximately 2.2 h (Li and Black 1996). Mts
within neuronal processes are not attached to the centrosome,
existing in overlapping segments along the shaft. During
initial neurite outgrowth the (+) or growing ends of all Mts
are oriented towards the periphery of the cell, but during de-
velopment a change in polarity occurs with the differentiation
of axons and dendrites (Baas et al. 1989). In mature neurons,
microinjection of biotin-labelled tubulin reveals the preferen-
tial addition of label to the fast-growing (+) ends of Mts
(Okabe and Hirokawa 1988), which in the axon are uniformly
oriented distal to the neuronal soma (Heidemann et al. 1981). In
dendrites, short Mts exhibit mixed polarity, with 50% of
(+) ends proximal to the cell body and 50% distal (Baas et al.
1988).Whether tubulin involved in neurite elongation is trans-
ported as Mt polymers and (or) as tubulin dimers has been
debated for many years (Joshi and Baas 1993; Baas and Yu
1996; Heidemann 1996). Two recent studies, concluding that
tubulin molecules are transported as heterodimers or oligom-
ers and not as Mts, continue the controversy. Miller and Joshi
(1996) microinjected fluorescent tubulin into the cell bodies
of neurons and observed the transport of a distinct wave of
tubulin subunits into the axon. In contrast, microinjected,
taxol-stabilized fluorescent Mts remained within the neuronal
cell body. Funakoshi et al. (1996) microinjected neurons with
tubulin labelled with caged fluorescein and photoactivated
tubulin molecules in the axons. The photoactivated tubulin
molecules were detected by electron microscopic immuno-
cytochemistry, using an antibody to fluorescein. While labelled
oligomeric and heterodimeric forms were abundant outside the
photoactivated area, labelled Mts were only detected within
the photoactivated area. Thus, it does not appear that intact Mts
are transported along the axon.
Along the neurite shaft, with increasing distance from the
soma, there is a progressive stabilization of Mts (Lim et al.
1989); however, even within the shaft of the axon the distal
(+) ends of Mt segments are labile whereas the proximal
regions become progressively more stable (Baas and Black
1990; Li and Black 1996) and serve as nucleating sites for Mts
within the axon during growth (Baas and Ahmad 1992). On
the other hand, Mts in the growth cone are highly dynamic
(Tanaka and Kirschner 1991; Edson et al. 1993; Li and Black
1996) and function in growth cone motility and axonal growth
(Bamburg et al. 1986; Tanaka and Kirschner 1995; Tanaka
et al. 1995; Rochlin et al. 1996). These Mts are highly sensitive
to Mt-depolymerizing drugs (Bamburg et al. 1986) and low
doses of nocodazole reduce the dynamic instability of growth
cone Mts and interfere with axon elongation (Rochlin et al.
1997 NRC Canada
Review / Synthse
1996). Stabilization of growth cone Mts with taxol also dis-
rupts axon extension (Jordan et al. 1993; Laferrire and
Brown 1995).
The molecular mechanisms regulating the subcellular
differential stability of Mts in neurons are unknown. Tubulin
isotype composition, posttranslational modifications of tubu-
lin, and microtubule-associated proteins (MAPs) have, how-
ever, been implicated in modulating Mt dynamics in neurons.
Genes for tubulin isotypes
The - and -polypeptides of tubulin constitute a multigene
family of biochemically distinguishable isotypes. With respect
to tubulin, the term isotype refers to a polypeptide sequence
that varies by at least one amino acid substitution from other
tubulins within the same organism, and that shows sequence
conservation across species lines (Little and Seehaus 1988).
An additional tubulin isotype, -tubulin, is associated primar-
ily with the pericentriolar material of the centrosome and other
MTOCs (Oakley 1994; Joshi 1994). Immunoelectron micro-
scopic tomography has now localized -tubulin to ring struc-
tures within the centrosome and in vitro studies have shown
that -tubulin ring complexes nucleate MT assembly and cap
the () ends of Mts (Moritz et al. 1995; Zheng et al. 1995).
Much of the heterogeneity among tubulin isotypes is lo-
calized within the 50 amino acids of the carboxy-terminus
(C-terminus). In particular, the last 15 residues of the extreme
C-terminus of -tubulins (Sullivan and Cleveland 1986) and,
to a lesser degree, -tubulins (Villasante et al. 1986), contain
the greatest variation, and this region is termed isotype defin-
ing. Six -tubulin genes occur in mouse (Table 1), with the
products of m3 and m7 expressed in testis only. In mouse
brain, the m1 and m2 genes are expressed at high levels,
while only trace amounts of the products of m4 and m6
genes can be detected (Villasante et al. 1986). In rat, T1
(equivalent to mouse m1) is the major -tubulin mRNA
present in embryonic brain; however, by postnatal day 25,
T1 mRNA levels are greatly reduced. The other major rat
-tubulin isotype, T26, is constitutively expressed in brain
(Miller et al. 1987).
Six mouse -tubulin genes have been identified (Table 2).
The C-terminal amino acid sequence differences are used to
classify the -tubulin gene products, yielding six major isotype
classes (Lopata and Cleveland 1987). A mouse gene corre-
sponding to the class V -tubulin gene found in chicken has
not been identified. Each isotype exhibits a characteristic
pattern of cellular expression (Table 2) and except for class
VI, which is found only in hematopoietic cells, and class IVb,
which is testis specific, all are expressed in neural tissues.
Class II -tubulin is the major brain -tubulin, and class III is
neuron specific in mammalian and avian brain (Frankfurter
et al. 1986).
Regulation of tubulin synthesis
In cells such as fibroblasts, tubulin gene expression is thought
to be constitutive (Cleveland and Havercroft 1983). However,
the cell-specific pattern of tubulin isotype synthesis (Lopata
and Cleveland 1987) suggests that tubulin gene expression is
regulated during differentiation. Only the rat neuronal T1
-tubulin gene promoter has been examined in mammals and
it is sufficient to confer neuron specificity to a T1:nlacZ
105
Table 1. Mouse -tubulin genes and tissue of expression.
Gene Tissue
m1 Major neuronal
m2 Constitutive, all tissues
m3 Testis specific
m4 Muscle
m6 Constitutive, low levels, all tissues
m7 Testis specific
Table 2. Mouse -tubulin isotypes, genes, and tissue of expression.
Isotype class Gene Tissue
I m5 Constitutive
II m2 Major brain
III m6 Neuron specific in brain;
low levels in testis
IVa m4 Brain specific
IVb m3 Mainly testis
VI m1 Hematopoietic cells only
transgene during development and regeneration (Miller et al.
1994). Similarly, we have used the T1 -tubulin promoter
to direct neuron-specific expression of a transgene in P19
embryonal carcinoma cells induced to differentiate along
the neuronal pathway by retinoic acid (Rogers et al. 1994).
Analysis of the specific promoter elements implicated in
this neuron-specific activation is underway (Miller et al. 1996).
Posttranscriptional mechanisms are thought to regulate the
amount of tubulin mRNA in developing rat brain and selective
stabilization of mRNA for specific isotypes of tubulin has
been observed. For example, the steady-state mRNA levels
for rat -II tubulin (the major neuronal tubulin isotype) at
postnatal day 5 were 168 times higher than the adult levels,
while the transcription rate was only 3.6 times greater
(Moskowitz and Oblinger 1995). Thus, processing of tubulin
mRNA during neurogenesis appears to regulate tubulin
synthesis (Bhattacharya et al. 1991).
During translation, appropriate quantities of soluble -
tubulin are maintained via an autoregulatory mechanism that
employs the strictly conserved, first four amino acid residues
MREI (Met-Arg-Glu-Ile) at the -tubulin amino terminus
(Yen et al. 1988). Excess soluble -tubulin, along with an un-
identified effector molecule (Theodorakis and Cleveland 1992),
are hypothesized to bind the first four amino acids of the nas-
cent polypeptide, destabilizing the tubulin mRNA and termi-
nating translation (reviewed by Cleveland and Theodorakis
1994). The production of -tubulin is thought to undergo
autoregulation independent of -tubulin (Bachurski et al. 1994),
although the mechanism is unknown. However, in this context,
when a -tubulin isotype normally expressed in Chinese hamster
ovary (CHO) cells was epitope tagged with a hemagglutinin
antigen and expressed at high levels in CHO cells, the ex-
pression of the endogenous (untagged) -tubulin isotype de-
creased, and -tubulin synthesis increased to match the
increase in total -tubulin (Gonzalez-Garay and Cabral 1995).
This result suggests that there is coregulation of - and
-tubulin within these cells. Additionally, in developing and
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regenerating rat neurons T1 tubulin (Miller et al. 1987, 1989)
and class III -tubulin (Moody et al. 1989; Lee et al. 1990;
Memberg and Hall 1995; Moskowitz and Oblinger 1995)
appear to be coordinately upregulated.
Posttranslational regulation of tubulin appears to exist, re-
sulting in selective degradation of tubulin isotypes. When the
chicken class IV -tubulin gene is overexpressed in CHO cells,
transcription and translation of the chicken class IV mRNA
increase almost fourfold relative to endogenous tubulin iso-
types, yet the class IV isotype does not accumulate to greater
than 10% of total tubulin. In addition, a selective decrease in
endogenous class IV -tubulin transcription is noted (Sisodia
et al. 1990).
Functional significance of tubulin isotype
heterogeneity
The multitubulin hypothesis proposed by Fulton and Simpson
(1976) suggests that specific tubulin isotypes within a cell
form Mts with a particular function. However, there is evi-
dence from microinjection and transfection studies (Luduea
1993; Raff 1994) that isotype usage is indiscriminate and all
intracellular tubulin isoforms are uniformly incorporated into
all Mts. For example, purified bovine brain tubulin coassem-
bles with endogenous tubulin into typical cytoplasmic Mts and
spindles when microinjected into 3T3 fibroblast cells (Keith
et al. 1981), sea urchin embryos (Salmon et al. 1984), and
Drosophila embryos (Kellog et al. 1988). In addition, when a
chimeric chickenyeast -tubulin, constructed by replacing the
chicken C-terminal sequence with an extremely divergent yeast
C-terminal sequence, is expressed in 3T3 fibroblast cells it is
incorporated into all Mt arrays, with no apparent loss of Mt func-
tion (Bond et al. 1986). Transfection of the erythrocyte-specific
VI tubulin into HeLa cells yields similar results (Lewis
et al. 1987). These studies indicate that at least in cultured
cells, all isotypes are incorporated into all Mts without loss of
function and that tubulin isotypes are functionally redundant.
However, the conservation of protein sequence, of pattern
of expression, and of posttranslational modifications noted
within vertebrate -tubulins argues that selection has played a
role in the maintenance of isotype specificity (Sullivan and
Cleveland 1986; Sullivan 1988), supporting the idea that cyto-
plasmic distribution and properties of Mts may indeed be in-
fluenced by isotypic differences. There are several examples
in multicellular organisms where specific isotypes clearly play
a role in the formation of functionally distinctive Mt arrays.
For example, while most neuronal Mts in Caenorhabditis
elegans contain 11 protofilaments, six touch-receptor neurons
have Mts with 15 protofilaments. When the tissue-specific
-tubulin gene mec-7 is mutated, the number of Mt protofila-
ments in these specialized neurons drops to 11, and the animals
are touch insensitive (Savage et al. 1989).
Of the four -tubulin isotypes synthesized in Drosophila
spp., 2 is testis specific. When the minor 3 isotype is ex-
pressed in testis in place of the major 2 isoform, some of the
resultant Mts lose function. When 3 is expressed either alone
or at levels exceeding 20% of the total tubulin pool, axoneme
assembly is disrupted, resulting in male sterility (Hoyle and
Raff 1990), showing that the two -tubulin isotypes are not
functionally equivalent. It is noteworthy that phenotypic dif-
ferences are not seen until 3 exceeds 20% of the total tubulin.
Biochem. Cell Biol. Vol. 75, 1997
This suggests that the ectopic tubulin must be present at suffi-
ciently high threshold levels within transfected or microin-
jected cells for function to be assessed. A threshold effect may
explain the lack of phenotype variation seen in other tubulin
transfection and microinjection studies (Raff 1994).
The -tubulins TUB67C and TUB84B that are coex-
pressed in Drosophila embryos also possess distinctive func-
tional properties. When the quantity of TUB67C is reduced,
the function of both meiotic and mitotic spindles is disrupted.
Elevated amounts of TUB67C did not affect meiosis, but this
isotype did disrupt mitosis and cleavage in the early embryo.
However, when the level of TUB84B is increased simulta-
neously, mitosis and cleavage return to normal. Thus, both the
67C and 84B -tubulins are required for normal spindle func-
tion in the oocyte and early embryo and they are not function-
ally equivalent (Matthews et al. 1993).
In vitro assembly studies have shown that among tubulin
isotypes, intrinsic biochemical differences and the composi-
tion of the isotype pool alter the assembly kinetics of Mts. For
example, Mts made from purified III are more dynamic
than Mts made from unfractionated tubulin preparations con-
taining all brain isotypes, or purified samples of II or IV
tubulin dimers. Assembly of a mixture of 20% III and 80%
II tubulin, however, produces Mts that are less dynamic
than Mts made from pure II tubulin (Panda et al. 1994).
Class II -tubulin represents 58%, and class III -tubulin 25%,
of the -tubulin in bovine brain preparations (Banerjee et al.
1988). While these values may not reflect actual isotype ratios
for individual cells, immunofluorescence microscopy using
isotype-specific antibodies to -tubulins does demonstrate the
coexistence of multiple -tubulin isotypes within the cell body
and along the length of neurites (Joshi and Cleveland 1989;
Falconer et al. 1992), suggesting that the -tubulin isotype
ratio may contribute to the temporal increase in Mt stability
noted in differentiating neurons. In all of these studies, the
-tubulin isotype composition is not known and it may also
influence Mt dynamics.
The divergent erythrocyte class VI -tubulin forms Mts
that are more stable and less affected by changes in ionic
strength and pH than Mts assembled from a mixture of brain
-tubulin isotypes (classes I, II, III, and IV). By
combining varying amounts of erythrocyte and brain -
tubulin isotypes, it is possible to predictably alter the assembly
properties of the resultant mix. In addition, rather than observ-
ing a random distribution of tubulin isotypes in individual
Mts, an isotype gradient with a changing ratio of tubulin iso-
types is evident along the length of the resultant Mt copolymers
(Rothwell et al. 1986; Baker et al. 1990). Sorting of class III
-tubulin into short segments along the Mts has been noted
in cultured PC12 cells (Joshi and Cleveland 1989). In addition,
-III tubulin is excluded from colchicine-stable Mt arrays
during early stages of differentiation of hippocampal py-
ramidal neurons and cerebellar macroneurons (Ferreira and
Caceres 1992). Similarly, there is selective sorting of class II
-tubulin into colchicine-stable Mt arrays whereas class III
-tubulin is excluded immediately prior to neurite outgrowth
in retinoic acid-induced P19 neurons (Falconer et al.1992).
Interestingly, 3 days after addition of retinoic acid, when
neurite outgrowth is evident, III tubulin is first detected
in colchicine-resistant Mts in developing neurons and by day
12, III tubulin is preferentially included in colchicine-stable
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Review / Synthse
Table 3. Summary of tubulin posttranslational modifications.
Modification Amino acid residue affected
Detyrosination Carboxy-terminal tyrosine
Nontyrosination Carboxy-terminal tyrosine
and glutamic acid
Acetylation Lysine 40
Phosphorylation Serine 441
Serine 444
Serine 444
Tyrosine
Polyglutamylation Glutamic acid 445
Glutamic acid 438
Glutamic acid 434
Glutamic acid 441
Glutamic acid 435
Polyglycylation Glutamic acid 445
Glutamic acid 437
Note: Tubulin heterogeneity is increased by multiple posttranslational changes to each isotype and (or) by attachment of side chains of different lengths and linkages.
*A more complete citation of references is given in the text.
Mts (Laferrire and Brown 1996). III tubulin protein and
mRNA increase concurrently until approximately day 7, when
III mRNA begins to decrease while the protein level remains
high, suggesting that III tubulin is stabilized at later stages of
development. A similar observation has been made for the
cerebral cortex where III mRNA decreases more dramati-
cally than its protein product during development (Jiang and
Oblinger 1992).
The biological significance of tubulin-isotype sorting is un-
certain. In addition to affecting Mt dynamics, it may permit
cell proteins, such as structural and motor MAPs, to interact
with a selected population of Mts (see below). In this way,
specific Mts are targeted for further stabilization, either through
enhanced MAP binding, or to serve as markers or tracks for
organelle localization or intracellular transport. Such a mecha-
nism may explain why only 25% of the Mts in lobster axons
are utilized for vesicle transport (Miller et al. 1986).
Posttranslational modifications of tubulin
Tubulin is modified posttranslationally by enzymatically me-
diated mechanisms that are usually reversible (Barra et al. 1988;
MacRae and Langdon 1989; Bulinski and Gundersen 1991;
Paturle-Lafanechre et al. 1991; Redeker et al. 1991, 1994;
MacRae 1992; Luduea 1993). Posttranslational changes oc-
cur preferentially on either soluble (/-dimer) or assembled
tubulin. Additionally, all but one of the posttranslational modi-
fications described in this report affect amino acids at the
glutamate-rich C-termini of - and -tubulins, the most vari-
able domains within the protein, and they tend to add to the
negative change of this region (Table 3) (Burns and Surridge
1990; Burns 1991; Paturle-Lafanechre et al. 1991; Rudiger
and Weber 1993; Audebert et al. 1993). The C-terminus of
tubulin is thought to project outward from the Mt lattice and
be the major location for binding of MAPs and Ca2+ (Little and
Seehaus 1988; Burns 1991; Luduea et al. 1992; Chapin and
107
Tubulin
isotype affected References*
Bulinski and Gunderson 1991; Webster et al.
1992; Ersfeld et al. 1993
Paturle-Lafanechre et al. 1991, 1994
MacRae and Langdon 1989; Kozminski et al. 1993
Rudiger and Weber 1993
Diaz-Nido et al. 1990
Alexander et al. 1991
and Matten et al. 1990
Edd et al. 1992
Alexander et al. 1991
Mary et al. 1996
Mary et al.1996
Redeker et al. 1992
Redeker et al. 1994
Redeker et al. 1994
Bulinski 1992; Schoenfeld and Obar 1994). Removal (Serrano
et al. 1984) and neutralization of the C-terminus by reaction
with carbodiimide promote Mt polymerization and lower MAP
affinity for Mts, suggesting that the negative charge in this
region regulates tubulin assembly (Mejillano and Himes 1991;
Luduea et al. 1992). The C-terminus may also contribute to
isotype-specific functions of the different -tubulins (Joshi and
Cleveland 1990; Padilla et al. 1993). For example, 2 tubulin
is necessary for assembly of axonemes in Drosophila spp. and
when a truncated 2 protein missing the last 15 amino acids
is synthesized, it assembles into Mts, but these Mts fail to form
organized axonemal structures. The truncated 2 tubulin is
less stable than the endogenous 2 tubulin (Fackenthal et al.
1993), suggesting a role for the C-terminus in the folding of
the tubulin molecule after translation, perhaps through a direct
reaction with the tubulin GTP-binding site (Fontalba et al. 1995).
Isotype-specific differences in the rate of tubulin folding and
dimer formation have been identified (Zabala and Cowan 1992).
Another consequence of the proposed interaction between the
C-terminus and the GTP binding site is an alteration of GTP
binding or hydrolysis, directly influencing dynamic instability
(Burns and Surridge 1990; Padilla et al. 1993). Biochemical dif-
ferences within the isotype-defining region alone, or in com-
bination with posttranslational modifications, may therefore
affect molecular conformations and tubulin dynamics (Burns
and Surridge 1990).
The effects of posttranslational changes on the C-terminus
of tubulin within Mts are uncertain, but they clearly have the
potential to shape the surface of these polymers and influence
their spatial distribution, perhaps with functional implications.
Not all cells, however, carry out all tubulin posttranslational
modifications, and it is not clear if any one modification is
essential for survival. Such observations suggest that post-
translational changes may be functionally redundant, similar
to the situation with some MAPs (Goldstein 1993), and that
their role in the cell is subtle (Koziminski et al. 1993).
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Detyrosination and acetylation
Two posttranslational modifications of tubulin examined in
detail are detyrosinationtyrosination and acetylation
deacetylation (Barra et al. 1988; MacRae and Langdon 1989;
Bulinski and Gundersen 1991; MacRae 1992, 1997; Kozminski
et al. 1993; Lai et al. 1994; Gaertig et al. 1995). In the former,
a C-terminal tyrosine found on most but not all -tubulin gene
products (Lai et al. 1994; Delgado-Viscogliosi et al. 1996) is
removed by a tubulin carboxypeptidase (Webster et al. 1992;
Xiang and MacRae 1995; Webster and Oxford 1996) and
reattached by tubulin tyrosine-ligase (Ersfeld et al. 1993).
Detyrosination occurs mainly on Mts, whereas tyrosination
is restricted to soluble tubulin. Detyrosinated tubulin is en-
riched in stable Mts and it is thought to play a role in the
polarity of cells such as melanophores (Nilsson et al. 1996)
and neurons where MAPs influence assembly disassembly of
Mts (Bulinski and Gundersen 1991; Bosc et al. 1996). The
intracellular arrangement of membrane-bound organelles and
other cytoskeletal elements such as vimentin intermediate
filaments is also influenced by Mt stability and thus by the
posttranslational modification of tubulin (Mizuno and Singer
1994; Gurland and Gundersen 1995). As shown by analysis of
macrophage Mts (Robinson and Vandr 1995), the correlation
between detyrosination and stability is not absolute nor is the
stabilization of Mts a prerequisite for their use in intracellular
transport, at least in fish melanophores (Rodionov et al. 1994).
Acetylation entails the addition of an acetyl group to lysine
40 of -tubulin by an acetyltransferase, which utilizes either
soluble or polymerized tubulin as substrate, but prefers the
latter (Maruta et al. 1986; MacRae and Langdon 1989;
Kozminksi et al. 1993; Lloyd et al. 1994). The acetyl group is
removed by a second enzyme, tubulin deacetylase. Acetylated
tubulin is usually in stable Mts, but there are several excep-
tions to this rule (MacRae and Langdon 1989; Delgado-
Viscogliosi et al. 1996) and there are examples of -tubulins
where a lysine at position 40 is not acetylated, perhaps owing
to rapid assemblydisassembly of Mts (Wolf et al.1988; Read
et al. 1993). Interestingly, extensive synthesis of nonacetylat-
able tubulin in Chlamydomonas reinhardtii does not cause
gross phenotypic effects in spite of its coassembly with endo-
genous tubulin (Kozminski et al. 1993). Because the gene for
acetylatable tubulin was expressed in these cells, threshold
effects must be considered (MacRae and Langdon, 1989;
Kozminski et al. 1993; Raff 1994). That is, sufficient endo-
genous acetylatable tubulin may be incorporated into Mts, even
in the presence of excess nonacetylatable tubulin, to allow
normal function. In similar experiments, Tetrahymena
thermophila -tubulin, with lysine at position 40, was com-
pletely replaced by an -tubulin with arginine at this position
(Gaertig et al. 1995). Again, mutants and wild-type cells could
not be distinguished from one another. The critical experiment
required to reveal the effect of acetylation, namely elimination
of the tubulin acetyltransferase while leaving lysine 40 intact,
has yet to be done. It has been speculated, however, that
acetylation may block the potentially reactive -amino group
of lysine (Kozminski et al. 1993), or as suggested by the study
of Xenopus laevis oocytes, stabilized acetylated Mts may be
MTOCs (Gard et al. 1995), acting in a manner similar to that
proposed for Mts in neurons (Baas and Ahmad 1992).
Posttranslationally modified tubulins exhibit an interesting
distribution within highly polarized neuronal cells. Neurites
Biochem. Cell Biol. Vol. 75, 1997
are usually characterized by acetylated and (or) detyrosinated
tubulins, isoforms missing from the cell body and growth cone
(Lim et al. 1989), whereas tyrosinated tubulin is distributed
throughout the growth cone of cultured dorsal root ganglion
neurons. Acetylated and detyrosinated tubulin occur promi-
nently in the growth cone only if Mts are taxol stabilized
(Robson and Burgoyne 1989). Moreover, axonal Mts consist
of two abruptly separated domains, enriched in either acety-
lated or tyrosinated tubulins (Brown et al. 1993). The plus
ends of these Mts, from the transition point onward to the
growth cone, are characterized by a proportion of tyrosinated
tubulin that increases distally from the cell body. This distri-
bution of tubulin isoforms shows the history of Mt growth with
more recently assembled and more dynamic domains enriched
in tyrosinated tubulin. The stable domains, poor in tyrosinated
tubulin but with increased detyrosinated and acetylated iso-
forms, have a longer turnover time and nucleate assembly
locally within the axon (Li and Black 1996).
Nontyrosinatable tubulin
In addition to tyrosinated and detyrosinated tubulin, brain is
characterized by large amounts of a related isoform termed
nontyrosinatable or delta 2-tubulin (Paturle-Lafanechre et al.
1991, 1994; del C. Alonso et al. 1993). This -tubulin isoform
arises by the loss of C-terminal tyrosine and glutamic acid
residues, removing the protein from the detyrosination
tyrosination cycle. Delta 2-tubulin constitutes 3550% of brain
tubulin, it occurs only in neuronal cells in this tissue, and it
appears early in growth. The production of delta 2-tubulin is
developmentally regulated, and it is found in neuronal growth
cones (Paturle-Lafanechre et al. 1994), dynamic structures
devoid of detyrosinated, but not tyrosinated, tubulin. This com-
bination of isoforms suggests that growth cone Mts go from
the tyrosinated to the nontyrosinatable state, without being
stabilized and detyrosinated. In nonneuronal cells, delta 2-
tubulin is either absent or found in limited amounts in stable
structures such as primary cilia, flagella, and centrioles (del
C. Alonso et al. 1993; Paturle-Lafanechre et al. 1994; Mary
et al. 1996). Moreover, stabilization of Mts by exposing cells
to high taxol concentrations (Edd et al. 1992) causes them to
bundle and react with antibody to nontyrosinatable tubulin, the
latter not observed upon treatment with lower drug concentra-
tions (Paturle-Lafanechre et al. 1994). These results indicate
that the terminal condition of -tubulin in mature Mts is re-
moval of the two C-terminal amino acid residues, and that this
occurs slowly in either the presence or absence of drugs. One
possibility is that tyrosinated -tubulin is sequentially modified
to give detyrosinated, then nontyrosinatable tubulin, or a dipep-
tidyl carboxypeptidase cleaves both amino acid residues from
-tubulin simultaneously (Paturle-Lafanechre et al. 1991, 1994).
Polyglutamylation
Polyglutamylation, the covalent attachment of one to six glu-
tamyl moieties to C-terminal glutamic acid residues of both
- and -subunits, is a major source of tubulin heterogeneity
(Redeker et al. 1991; Edd et al. 1990; Wolff et al. 1994). For
class III -tubulin, an isotype found in neurons and testis,
but which increases in isoform complexity only during de-
velopment of the former, glutamylation is at Glu-438
(Alexander et al. 1991). In mouse brain and cultured neurons,
the m1m2 gene products are glutamylated at Glu-445
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Review / Synthse
(Edd et al. 1990, 1992), while class IVa and I -tubulin
isotypes are glutamylated at Glu-434 and Glu-441, respec-
tively (Mary et al. 1996). Young cultured neurons from mouse
possess extensively glutamylated -tubulin, whereas -
tubulin glutamylation increases in extent and complexity,
leading to greater isoform complexity as the neuronal differ-
entiation proceeds (Audebert et al. 1994). Major isotypes of
pig brain - and -tubulin are glutamylated at Glu-445 and
Glu-435, respectively, with the latter a class II -tubulin
(Redeker et al. 1992). Additionally, m3/7 and m3, the ma-
jor testis-specific isotubulins, are glutamylated (Fouquet et al.
1996). These modifications are associated with stable Mts and
they may exhibit a differential distribution along the axoneme
(Fouquet et al. 1994, 1996; Kann et al. 1995). Other non-
neuronal mouse tissues possess small amounts of glutamylated
-tubulin (Wolff et al. 1992). These results were obtained through
use of the antibody, GT335, which recognizes the glutamyla-
tion motif in combination with elements of tubulin polypeptide
structure. Lastly, Mts of Paramecium (Br et al. 1994), Tricho-
monas vaginalis (Delgado-Viscogliosi et al. 1996), sea urchin
sperm (Mary et al. 1996), and melanophores of Atlantic cod
(Nilsson et al. 1996) display this modification, but tubulin pu-
rified from turkey erythrocyte marginal bands is not glutamy-
lated in either the - or -subunit (Rudiger and Weber 1993).
Polyglutamylated -tubulin participates in tyrosination
detyrosination reactions and is withdrawn from this cycle by
removal of its two C-terminal amino acids, yielding the delta
2 isoform (Paturle-Lafanechre et al. 1991; Edd et al. 1992).
Acetylation of Lys-40 (Edd et al. 1991) and phosphorylation
of Ser-444 (Alexander et al. 1991) also occurs on polyglu-
tamylated - and -tubulins, respectively, with the latter pos-
sibly priming the glutamylation reaction (Boucher et al. 1994).
Drug-induced modulation of Mt assembly in differentiating
mouse neurons indicates that Mts are the preferred substrate
for glutamylation, while deglutamylation happens on either
soluble or polymerized tubulin (Audebert et al. 1993; Br
et al. 1996). Both stable and labile Mts contain glutamylated
tubulin (Audebert et al.1993; Bressac et al. 1995) in contrast
to detyrosinated and acetylated tubulin, which are usually
found in stable Mts. The rate of -tubulin deglutamylation
varies, with the distal (4th6th) residues removed more quickly
than the proximal three. Moreover, side chains of four or more
glutamyl residues are restricted to Mts, while shorter chains of
one to three residues are spread evenly between polymerized
and nonpolymerized -tubulin. Cells apparently maintain ap-
preciable levels of glutamylated tubulin, independent of its
assembly and disassembly, perhaps employing different en-
zymes to glutamylate - versus -tubulin (Audebert et al. 1993).
Polyglycylation
Polyglycylation affects the C-terminus of axonemal - and
-tubulins from several species and a subpopulation of cortical
Mts in Paramecium (Redeker et al. 1994; Bressac et al. 1995;
Levilliers et al. 1995; Rudiger et al. 1995; Br et al. 1996;
Mary et al. 1996). Three to 34 glycyl units are added cova-
lently to the carboxyl group of glutamyl residues at positions
445 and 437 of Paramecium - and -tubulins, respectively,
but other sites may be involved. For bull sperm axonemal Mts,
however, only -tubulin is glycylated whereas -tubulin lacks
this modification. Moreover, 60% of the -tubulin is glycy-
lated in bull sperm as opposed to 100% modification in
109
Paramecium. The acidity of the tubulin C-terminal region is
decreased by glycylation, perhaps favouring assembly. Also,
potential exists for variation in the extent of glycylation to
affect MAP binding to Mts in the axoneme. Thus, glycylation,
thought to be a postpolymerization process, may either stabi-
lize axonemal and cortical Mts or link these Mts to the cyto-
plasmic membrane. To our knowledge, this modification has
not been examined in neurons.
Phosphorylation
Phosphorylation was the first posttranslational modification
reported for tubulin (Goodman et al. 1970) and since this
observation, many different kinases have been shown to use
tubulin as substrate (MacRae 1997). These include cyclic AMP
(cAMP) and kinases stimulated by Ca2+calmodulin, casein
kinases I and II, and the tyrosine kinases. These enzymes, as
a group, have the potential to phosphorylate serine, threonine,
and tyrosine residues. Many of the kinases have been isolated
from neural tissue and they utilize tubulin from this source,
both in vivo and in vitro. However, the physiological rele-
vance of tubulin phosphorylation in some of these cases is
difficult to assess. Precautions were not always taken to inhibit
endogenous kinase and phosphatase activities in cell-free homo-
genates employed in kinase assays and (or) as sources of tubu-
lin. This is especially critical in experiments where tubulin was
purified by assemblydisassembly at elevated temperatures,
providing the opportunity for adventitious enzymatic modifi-
cation of the protein (Edd et al. 1989; MacRae 1997).
In spite of the difficulties just mentioned, there has been a
concerted effort to understand tubulin phosphorylation and its
physiological consequences, with much of this work focused
on neuronal systems. Gard and Kirschner (1985) demonstrated,
in a pioneering set of experiments designed to limit artifactual
incorporation of phosphate into tubulin, that there is a fourfold
enhancement of -tubulin phosphorylation upon differentia-
tion of N115 neuroblastoma cells. Only one of three -tubulins
resolved on two-dimensional gels is phosphorylated and this
isoform is termed 2. It was concluded that phosphorylation
is regulated by the extent of tubulin assembly and not by
modification of kinase activity (Gard and Kirschner 1985).
Subsequently, Edd et al. (1987, 1989) demonstrated that a
-tubulin isoform, 1, a primary gene product synthesized
early in development of mouse neuronal cells, is phosphorylated
during neurite extension yielding the isoform 2, equivalent
to 2 of Gard and Kirschner (1985). The phosphorylated -
tubulin was then identified as class III (Luduea et al. 1988),
a result confirmed by chemical analysis of tubulin phosphory-
lation rather than by incubation of cells with radioactive phos-
phate (Eipper 1974).
The identity of the phosphorylated residue(s) in neural
tubulin has been examined in detail. Brain tubulin labelled
in vivo with 32P contains a single phosphorylated serine in an
acidic region of the protein (Eipper 1974). Labelling of neuro-
nal cells in culture, as just described, indicates that a single
serine is labelled and this was proposed to be either residue
444 or 446, with the former more likely than the latter
(Luduea et al. 1988). To pursue this question Diaz-Nido et al.
(1990) injected rats intracraneally with 32P and sequenced a
labelled peptide obtained by digestion of purified tubulin with
V8-protease. The peptide was identical to the C-terminus of
class III -tubulin and it was phosphorylated at serine 444,
1997 NRC Canada
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a result also obtained by analysis of phosphorylated tubulin
from differentiated N115 neuroblastoma cells. These data were
verified by Alexander et al. (1991) who isolated the C-termi-
nal portion of class III -tubulin by affinity chromatography
using the isoform-specific antibody TuJ1 and analyzed its
composition by peptide sequencing and mass spectrometry. A
synthetic peptide representing this region of class III -tubulin
is phosphorylated by casein kinase II at a serine that corre-
sponds to residue 444 (Diaz-Nido et al. 1990), suggesting that
this kinase is responsible for in vivo phosphorylation, a proposal
made earlier by the same group (Serrano et al. 1987; Avila
et al. 1988). More recently, chemical analysis of class III -
tubulin indicates that other residues, such as tyrosine 437 and
serine 239, are phosphorylated as well (Khan and Luduea 1996).
In contrast to Ca2+calmodulin-dependent kinases, which
phosphorylate both - and -tubulin at several threonine and
serine residues producing tubulin with reduced ability to
polymerize (Yamamoto et al. 1985; Wandosell et al. 1986),
modification by casein kinase II yields assembly-competent
tubulin (Serrano et al. 1987). Indeed, polymerized tubulin is a
better substrate for casein kinase II than is soluble tubulin
and the modified -isoform is found preferentially in Mts
(Diaz-Nido et al. 1990; Serrano et al. 1987; Avila et al. 1988).
Denoulet et al. (1989) also demonstrated that 2, the phos-
phorylated isoform of -tubulin, is always in the assembled
fraction of the cytoskeleton in rat sciatic nerve motor axons.
Moreover, this isoform is more prominent in the slow compo-
nent of axonal transport labelled b (SCb) than in the component
labelled a (SCa), suggesting that phosphorylation regulates
tubulin assembly and the interaction of Mts with other cell
components.
How might phosphorylation of class III -tubulin influence
its assembly? Interestingly, treatment of class III -tubulin
with human erythrocyte phosphatase 2A causes a 32% reduc-
tion of its assembly in the presence of MAP2, but has no effect
if polymerization is driven by 4 M glycerol 6 mM MgCl2,
indicating a role for phosphorylation in tubulinMAP interac-
tions (Khan and Luduea 1996). In this context, the C-terminal
region of class III -tubulin is a MAP-binding site, and this
area is less negatively charged than the corresponding regions
of other -tubulin isoforms (Luduea et al. 1988; Khan and
Luduea 1996). Because tubulinMAP interactions are partly
electrostatic, the addition of a negatively charged phosphate
may enhance interaction with positively charged MAPs, thus
stimulating assembly. Another proposal is that phosphoryla-
tion alters glutamate side chains known to attach to Glu-435
and in this way modulates tubulinMAP interactions (Khan
and Luduea 1996). Whatever the case, phosphorylation of
neuronal class III -tubulin, possibly by casein kinase II and
in concert with other posttranslational changes including glu-
tamylation, seems to have an important effect on tubulin
assembly and Mt function.
In addition to serine and threonine, tyrosine residues of
tubulin are phosphorylated by several receptor and nonreceptor
tyrosine kinases, some of which bind with tubulin in stable
complexes (Alexandrova et al. 1995; Peters et al. 1996; MacRae
1997). The consequences of tyrosine phosphorylation are var-
ied. In vitro phosphorylation by the insulin receptor kinase
inhibits tubulin assembly, perhaps through modification of the
C-terminal tyrosine (Wandosell et al. 1987). Phosphorylation
of tyrosine residues may also influence differentiation of cells.
Biochem. Cell Biol. Vol. 75, 1997
For example, Fyn and Lyn, members of the src protooncogene
family, are induced during monocytic differentiation of cul-
tured HL-60 cells exposed to tetradecanoyl phorbol acetate
and tubulin is phosphorylated (Katigiri et al. 1993). If phos-
phorylation is prevented the cells fail to differentiate. Simi-
larly, stimulation of Jurkat T cells by way of the T cell receptor
enhances tyrosine phosphorylation on a small portion of their
tubulin (Ley et al. 1994). Although the responsible enzyme(s)
has (have) yet to be identified, Jurkat T cells possess several
tyrosine kinases and two of them, ZAP-70 and Vav, interact
with tubulin (Huby et al. 1995).
Phosphorylation of tubulin at tyrosine residues occurs in the
nerve growth cone. Matten et al. (1990) have shown through
use of phosphotyrosine-specific antibodies that this tubulin is
phosphorylated by pp60c-src, the prototype member of the src
protooncogene family of tyrosine kinases found at the plasma
membrane inner surface. Phosphorylation is very modest, with
0.068 and 0.045 mol of phosphotyrosine incorporation per mol
of - and -tubulin, respectively, in a reaction inhibited by
antibodies to pp60c-src. The phosphorylation, although limited,
could have physiological significance if it affects a subpopu-
lation of tubulin within cells, if the modified tubulin is added
to the elongating ends of Mts, or if phosphorylation causes
incorporation of tubulin into membranes (Matten et al. 1990).
In this functional context, phosphorylation of tubulin in the
growth cone membrane at tyrosine residues is reduced by in-
teraction of neural cell adhesion molecules with antibodies
specific to these proteins or with their ligands (Atashi et al.
1992). Thus, changes in pp60c-src kinase activity by the inter-
action of surface receptors with ligands may allow signals to
pass into the cell, modulating neurite extension and growth
cone migration through an effect on cytoskeleton dynamics.
Coordination between MAPs and tubulin
MAPs comprise a diverse family of proteins that copurify with
tubulin in a stoichiometric fashion during in vitro assembly
and associate with Mts in vivo. In neurons, these proteins in-
clude high molecular mass MAPs (270350 kDa) such as
MAP1A, MAP1B, and MAP2, and MAPs with a molecular
mass between 55 and 82 kDa, including tau proteins, Map 2c
and 2d, chartins, and STOP (stable tubule only protein). MAPs
promote tubulin assembly and stabilize Mts (Hirokawa 1994;
Matus 1994; Schoenfeld and Obar 1994; Maccioni and Cam-
biazo 1995; Mandelkow and Mandelkow 1995). They form
cross-bridges that appear to link neuronal Mts (Lee and Brandt
1992) and suppress their dynamic properties (Pryer et al. 1993;
Dhamodharan and Wadsworth 1995). They may also link Mts
to actin filaments (Cross et al. 1993) and intermediate fila-
ments (Capote and Maccioni 1994). Their high quantity and
developmental regulation suggest that MAPs play an impor-
tant role in regulating the differential stability exhibited by
neuronal Mts (Cambray-Deakin 1990; Matus 1991).
Two structurally related MAPs (MAP1A and MAP1B) ex-
hibit complementary patterns of synthesis during neuronal de-
velopment. MAP1B peaks early in development and declines
after maturation, while MAP1A appears much later and per-
sists in mature neurons (Schoenfeld et al. 1989). Synthesis of
MAP2 and MAP2c is similarly regulated during neurogenesis,
with MAP2c synthesized at high levels only during neuronal
development and MAP2 abundant only in mature neurons
(Matus 1988).
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Review / Synthse
Several strategies have been used to examine MAP func-
tion. For example, expression of tau and MAP2 by transfec-
tion, or microinjection into cells that do not normally express
these MAPs, results in increased Mt stability and bundling,
and outgrowth of axon-like processes. Tau and MAP2 anti-
sense experiments, showing inhibition of process outgrowth
from neurons, also indicate the importance of these proteins in
neuronal morphogenesis (for a recent detailed discussion of
these studies see Heidemann 1996). However, results from
some experiments with transgenic mice are more ambiguous.
As a case in point, the nervous system of mice lacking the tau
gene appears immunohistologically normal, except for a de-
crease in Mt stability and change in Mt organization in some
small-calibre axons. Interestingly, these mice exhibit increased
amounts of MAP1A, perhaps compensating for the loss of tau
in the large-calibre axons (Harada et al. 1994). In another trans-
genic study, the presence of high quantities of embryonic
MAP2c in adult brain had no obvious effect on the arrange-
ment or morphology of neurons, suggesting that MAP2c
does not function in regulation of neuronal morphogenesis
(Marsden et al. 1996). In contrast, in MAP1B mutant mice
generated by gene targeting methods the mutation was lethal
in the homozygous state and resulted in a variety of neuronal
abnormalities in some of the heterozygous animals, empha-
sizing the importance of this MAP in normal development
(Edelmann et al. 1996).
Differences in MAPtubulin binding affinities have been
observed. The affinity of MAP2 for chicken brain Mts is two
times higher than for erythrocyte Mts, suggesting that MAPs
interact preferentially with specific tubulin isotypes (Murphy
1991). Br et al. (1994) proposed that glutamylation enhances
MAP binding to Mts, causing their stabilization, eventual
acetylation, and functional designation. In this context, the
interaction of tau and MAP2 with Mts, as measured by an
in vitro ligand binding assay, is regulated by glutamylation
chain length (Boucher et al. 1994). MAP binding is enhanced
as chain size increases, reaching a maximal value at three
glutamyl units, then declining upon further addition of glu-
tamic acid. Moreover, the proximal three glutamyl units are
released the slowest (Audebert et al. 1993), ensuring an intra-
cellular pool of tubulin with high affinity for tau. Perhaps the
assembly of this tubulin into growing Mts accounts for the
enrichment of tau on dynamic Mts in growing axons (Black
et al. 1996). Recently, Larcher et al. (1996) have shown, using
a blot overlay assay, that masking of the polyglutamyl chains
on tubulin with a monoclonal antibody specific to the glutamy-
lated epitope inhibits kinesin binding, indicating that tubulin
modification regulates the binding of motor MAPs. The MAPs
probably do not interact directly with the glutamyl residues;
rather, the conformation of the C-terminus is modified, poten-
tiating MAP binding or release. Whatever the situation, the
results indicate clearly that MAP binding is influenced by the
posttranslational glutamylation of tubulin, thus regulating Mt
organization and function. Glutamylation, and other posttrans-
lational modifications, may signal the binding of subcellular
organelles and vesicles to Mts.
A temporal correspondence of tubulin isotype and MAP
expression is observed in developing hamster brain (Oblinger
and Kost 1994) and in P19 neurons induced to differentiate
in culture (Falconer et al. 1994). This raises the possibility of
coordinate gene regulation to match different tubulins and
111
Fig. 1. Interactions of tubulin and MAPs to yield functional Mt arrays.
MAPs for the assembly of functionally distinct Mts at stages
of neurogenesis. In addition, as discussed above, the occur-
rence or extent of several of the posttranslational modifica-
tions of tubulin is developmentally regulated. For example,
the increase in the amounts of moderately glutamylated -III
tubulin isoforms detected in the colchicine-stable Mts of
differentiating P19 neurons (Laferrire and Brown 1996) is
coincident with the expression of MAP2 and adult tau
(Falconer et al. 1994). This suggests that the enzyme(s) re-
sponsible for tubulin posttranslational modifications is (are)
coregulated with MAPs, permitting enhanced tubulinMAP
interaction.
Posttranslational modification of MAPs may provide an
additional mechanism to regulate their association with
tubulin (Matus 1988; Schoenfeld and Obar 1994). In primary
cultures of rat cortical neurons, phosphorylated MAP1B colo-
calizes with Mts containing detyrosinated -tubulin, while
nonphosphorylated MAP1B associates with Mts containing
tyrosinated -tubulin (Mansfield et al. 1991). Phosphorylation
thus appears to modulate the affinity of MAP1B for Mts (and
for microfilaments, Pedrotti and Islam 1996) and ultimately
the stabilizing characteristics of MAP1B because detyrosinated
Mts are more resistant to depolymerization than tyrosinated
Mts (Baas and Black 1990). In related studies, differences in
the subcellular distribution of MAPs were characterized
(Matus 1988; Hirokawa 1991, 1994; Schoenfeld and Obar 1994).
MAPs 2a and 2b are found along with juvenile MAP2c within
the somatodendritic compartment, while only MAP2c is pre-
sent in developing axons (Tucker and Matus 1987). Tau pro-
teins are thought to be sequestered within axons (Matus 1988).
Similarly, differences in the localization of posttranslationally
modified MAPs have been noted. MAP1B is present in both
axons and dendrites, but is phosphorylated within developing
axons only (Diaz-Nido et al. 1991) and tau is normally phos-
phorylated in dendrites and nonphosphorylated in axons
(Papasozomenos and Binder 1987). These variations among
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MAPs, in combination with the presence of distinctive tubulin
isotypes and isoforms, may be responsible for the differences
in inter-Mt distances in axons and dendrites.
Concluding remarks
Tubulin isotypes, their modifying enzymes, and MAPs vary
owing to regulated differential gene expression. Tubulin
diversity within cells where the cytoskeleton has a small role
is restricted because there is no need for the expression of
multiple, slightly modified genes to produce adequate amounts
of tubulin. Further, in this case, Mts need only react with a
limited variety of MAPs, probably structural in nature, and
extensive posttranslational modification of assembled tubulin
is unnecessary. On the other hand, cells with functionally
complex cytoskeletons, such as neurons, continually require
large amounts of tubulin for assembly into arrays of Mts spe-
cialized for morphogenesis and intracellular transport. In these
cells, several tubulin genes are expressed, yielding a mixture
of isotypes that varies in composition during development.
Differences in isotype composition can directly affect Mt dy-
namics and function. Posttranslational modifications, either
before or after assembly, also increase tubulin heterogeneity
in a developmentally regulated progression.
What is accomplished by the posttranslational modifica-
tions of tubulin and Mts? The only established function of the
tubulin / heterodimer is to form Mts, and posttranslational
changes to soluble tubulin could influence its synthesis, deg-
radation, and assembly properties (Fig. 1). For example, post-
translationally modified isoforms may no longer function in
feedback mechanisms, leading to higher or lower rates of
tubulin synthesis. Modified accessibility to proteolytic en-
zymes caused by covalent attachment of chemical groups
might change turnover rates, either increasing or decreasing the
amount of tubulin within a cell. Enhanced assembly and sta-
bilization of Mts owing to posttranslational changes may also
remove tubulin from a feedback loop and lead to enhanced
synthesis. Moreover, assembled tubulin is potentially less
susceptible to proteolytic enzymes than when it is soluble,
increasing the longevity of the protein and ensuring the avail-
ability of Mts in the cell.
The phenotypic consequences of Mt function are varied,
but these functions may have similar molecular foundations
(Fig. 1). Posttranslational modifications to tubulin and (or)
MAPs could promote MAP binding, or MAPs, once bound,
stabilize Mts and enhance posttranslational changes. This may
be sufficient to organize Mts into discrete bundled arrays with
a role in cellular morphogenesis, including the formation of
neurites. Or, it is possible that a sequence of proteinprotein
interactions occurs, wherein Mts stabilized by MAPs are post-
translationally modified, leading to the attachment of one or
more extra layers of MAPs. The additional MAPs then stabi-
lize discrete cellular distributions of Mts, solidifying their
structural role, or motor MAPs could facilitate Mt-dependent
movement of cell components. The morphogenetic and trans-
port functions are often interrelated, with Mts in specific arrays
facilitating directed transport by the motor MAPs, kinesin, and
cytoplasmic dynein.
Ultimately, the developmental regulation of Mt assembly in
neurons may require the coordinated expression and posttransla-
tional modifications of tubulin and MAPs to provide biochemi-
Biochem. Cell Biol. Vol. 75, 1997
cal forms of each that favour specific interactions, each combi-
nation conferring specific dynamic and functional properties.
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