Microtubules and microtubule-associated proteins
Eckhard Mandelkow and Eva-Maria Mandelkow
M a x Planck Unit for Structural Molecular Biology, Hamburg, Germany
Microtubule research is becoming increasingly diverse, reflecting the many
isoforms and modifications of tubulin and the many proteins with which
microtubules interact. Recent advances are particularly visible in four areas:
microtubule motor proteins (their structures, stepping modes, and forces);
microtubule nucleation (the roles of centrosomes and y-tubulin); tubulin
folding (mediated by cytoplasmic chaperones); and the expanding list of
microtubule-associated proteins, knowledge of their phosphorylation states,
and information on their effects on microtubule dynamics.
Current Opinion in Cell Biology 1995, 7:72-81
Introduction
Microtubules represent one of the fiber systems of the
eukaryotic cytoskeleton. They are essential for a wide
variety of cellular functions, notably cell motility, transport, cell shape and polarity, and mitosis. Microtubules
consist of a core cylinder built from heterodimers of etand ~-tubulin monomers. Three main classes of proteins
interact with tubulin. The 'inicrotubule-associated proteins', or MAPs, are sometimes called more specifically
'structural' MAPs because they bind to, stabilize and promote the assembly ofmicrotubules, and because they can
be copurified with tubulin through several cycles of
microtubule assembly and disassembly. Representatives
are MAPla and lb, MAP2a, 2b, and 2c, MAP4, tau
protein, 205 kDa MAP, and isoforms of these proteins
that are often generated by alternative splicing.
Another broad class comprises the motor proteins, so
called because they generate movement along microtubules using the chemical energy of ATP hydrolysis.
Representatives are kinesin (a motor moving towards the
distal, or plus, end of microtubules), dynein (a rearward
motor, also involved in the bending of cilia and flagella),
and their many relatives. The motors can bind tightly to
microtubules, for example in the absence ofnucleotides,
but they do not copurify through cycles of assembly and
disassembly.
A third and more heterogeneous class includes proteins that are not normally called MAPs but are often
found associated with microtubules and inay even copurify with them. Examples are glycolytic enzymes (e.g.
GAPDH and aldolase), kinases (e.g. protein kinase A,
GSK-3 and c-mos), proteins involved in biosynthesis
(elongation factor EF-lCt and even entire ribosomes),
proteins linking up to membrane receptors (dynamin and
G proteins), and ribonucleoproteins carrying mKNA.
These interactions are not always well defined, but they
point to the cytoskeleton as a (transient) anchor for many
cytoplasmic proteins.
The fields of microtubules, MAPs, and motors have expanded so nmch that they have become the subject of
separate reviews. For example, microtubule structure,
assembly and regulation have been reviewed recently
[1-4], as have MAPs [5,6] and motors [7-9]. General
surveys of microtubules and related proteins are given in
[10,11].
This review covers selected articles concerning microtubules, MAPs and motor proteins published in the last
eighteen months.
Structural aspects of microtubules revealed by
motors
The information on microtubule structure we have today
is derived mostly from electron microscopy combined
with image reconstruction, X-ray scattering, and video
microscopy; information on tubulin structure is indirect and derived mostly from biochemical experiments
(binding sites ofnucleotides or drugs, cross-linking studies, and so on). Novel insights on the microtubule lattice have recently come from studies of the interaction
between microtubules and kinesin. The head domain
of kinesin binds to [3-tubulin with a stoichiometry of
one kinesin head per tubulin heterodimer, generating an
axial periodicity of 8 nm (the height of the dimer) and
a 'B'-lattice in which adjacent protofilaments are staggered by about 0.9 nm [12°-14"]. The dimer polarity is
such that ~-tubulin points to the plus (fast-growing) end
of the microtubule and [3-tubulin points towards the minus (slow-growing) end (see Fig. 1). This implies that the
plus end has a crown ofet-tubulin subunits and the minus
end terminates with [3-tubulin [14°]. This polarity enables the ~-subunits to interact with T-tubulin, which is
a key component ofmicrotubule-organizing centers (reviewed in [3]). As the exchangeable GTP-binding site is
Abbreviations
GAPDH--glyceraldehyde-3-phosphate dehydrodgenase;GSK-3--glycogen synthasekinase-3; MAP~microtubule-associated protein.
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© Current Biology Ltd ISSN 0955-0674
Microtubules and microtubule-associated proteins Mandelkow and Mandelkow
on ~-tubulin and the microtubule-bound GTP is at the
plus end [15°], another consequence of this arrangement
of the microtubule is that the 'GTP-cap' sits on the plus
end in an inverted fashion so that the last ~-tubulin-GTP
is buried by a terminal layer of et-tubuhn (see Fig. 1).
region of ~-tubulin [39,40]. The stoichiometry of taxol
binding is one molecule per tubulin dimer; dimers stabilized by taxol are capable of assembling with bound
GDP, without the usual need for GTP hydrolysis [41].
Kinesin moves along the microtubule in 8 nm intervals
and produces forces in the pN range [16°-20"]. The direction of movement is parallel to the tracks of protofilaments [21], and the step size matches the separation
of the ~-tubulin 'stepping stones'. As kinesin works as
a dimer in vivo and shows alternating head catalysis (using each head of the dimer alternately to hydrolyze ATP)
[22°], one could integrate the structural and kinetic data
by assuming that a kinesin dimer would 'walk' or 'hop'
along one or two adjacent protofilaments. (Note that if
the center o f the kinesin molecule advances by 8 nm, this
may imply 16 nm steps for each head.) It also seems that
kinesin prefers rigid stepping stones over wobbly ones:
microtubules are fairly stiff structures, with persistence
lengths (a measure of their straightness) in the mm range
[23-25], but it is possible to make them even stiffer using
slowly hydrolyzable GTP analogues such as GMP-PCP,
in which case the movement of kinesin becomes 30%
faster [26].
GTP binding to tubulin
Microtubules contain a non-exchangeable GTP on
ct-tubulin and an exchangeable one on ~-tubulin (nucleotides on the latter site can be exchanged with nucleotides from the solution). Extending earlier work,
two recent studies showed that the exchangeable G T P
site is in the amino-terminal domain o f [~-tubulin; an
additional ATP-binding site (not involved in microtubule assembly) was found on et-tubulin [42,43]. That
microtubule stability is closely linked to GTP binding
and hydrolysis has now been demonstrated directly by
Davis et al. [44"], who made several mutant tubulins in
which altered GTPase activity is paralleled by altered
dynamic instability. Similarly, Reijo et al. [45 °] made
systematic mutations of clusters o f charged amino acids
for alanine in yeast [~-tubulin and deduced that three regions are critical for ~-tubulin function: near the carboxyl terminus, around residue 330, and around residue
150 (near the glycine cluster that is probably involved in
GTP binding).
The microtubule-motor interaction is surprisingly versatile: there are variants ofkinesin such as Drosophila ncd
or Saccharomyces cerevisiae Kar3 that can walk in the opposite direction (towards the minus end), and this property is inherent to the head domains [27°,28,29]. The
motor can twist its neck in order to attach to microtubules in the proper direction [30], and may even cause
rotation o f microtubules instead o f translation, as is suggested by the presence ofkinesin-like proteins on the C2
central microtubule in flagella in Chlamydomonas [31].
Finally, CENP-E, a kinesin-like protein that migrates
from kinetochores to the midzone of mitotic spindles,
cross-links the antiparallel microtubules and presumably
controls their rigidity or ghding during anaphase [32°].
Tubulin structure and interactions
Drugs that target tubulin
Tubulin has many applications as a target of drugs
(e.g. anti-cancer drugs) and therefore is a workhorse for
certain drug screening tests. The different modes of action and binding sites on tubulin of various drugs continue to be explored (see reviews [33,34]). Colchicine
and vinblastine are examples of drugs that destabilize
microtubules and thus disrupt mitosis; however, their effect can be felt even at drug concentrations too low to
cause net microtubule disassembly. This can be attributed
to a pronounced effect of the drugs on microtubule dynamic instability parameters [35-37], which are closely
linked to G T P binding and hydrolysis (see below). The
binding site for colchicine has been mapped near the
amino terminus of ~-tubulin [38]. By contrast, taxol
stabihzes microtubules, thereby also disrupting mitosis.
It was shown using photoactivatable taxol derivatives
that taxol binds predominantly to the amino-terminal
Modifications of tubulin
An enigmatic feature of tubulin is its heterogeneity. Not
only are et- and [~-tubulin each encoded by up to six
different genes, but the protein can also be modified
in several ways: phosphorylation, acetylation, detyrosination, and glutamylation. The complexity of neuronal
tubulins is particularly great and appears to increase with
differentiation. In other cells, such as avian erythrocytes,
the degree o f heterogeneity is conspicuously low [46].
One function of neuronal tubulin modification may be
the selective stabihzation of certain microtubules [47].
One of the modifying enzymes, the tubulin tyrosine
ligase, has now been sequenced and characterized in
detail [48]. This enzyme restores a carboxy-terminal
tyrosine to et-tubulin after it has been cleaved by a
tubulin carboxypeptidase. However, et-tubulin can also
lose two carboxy-terminal residues (glutamic acid and
tyrosine). This A2-tubulin can no longer be retyrosinated by the ligase and is enriched in very stable neuronal
microtubules [49]. Another sign of neuronal differentiation is the increasing length ofpoly-glutamic acid chains
attached to glutamic acid residues near the carboxyl terminus of 0t- and ~-tubulin; this makes the already very
acidic carboxyl termini even more negatively charged
[50]. The functions of these post-translational modifications have been ditlqcult to ascertain, but Gurland
and Gundersen [51] found that long-lived stable microtubules (known as 'Glu-microtubules' because they
have lost the carboxy-terminal tyrosine from ct-tubulin)
selectively break down when phosphatases are inhibited
by okadaic acid. One interpretation of this observation
is that stabilization factors (caps) that are normally associated with stable microtubules can be lost as a result of
unchecked phosphorylation.
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Cytoskeleton
Regulation of tubulin synthesis
The level of tubulin synthesis is regulated via the soluble
tubulin pool, whose size is in turn coupled to the degree
of microtubule assembly. In the case of ~-tubulin this
autoregulation involves the four amino-terminal residues
(MR_El, in the single letter code for amino acids) [52].
In an extension of their earlier work, Cleveland and coworkers [52] showed that 0t-tubulin follows a different
pathway (even though both subunits are downregulated
by degrading their mRNAs).
Prokaryote homologs of tubulin?
Tubulin is thought to be restricted to eukaryotic cells,
but there is a curious homology of the conserved
glycine-rich cluster (GGGTGSG) to the bacterial FtsZ
protein from Escherichia coll. Like tubulin, this protein
is also involved in cell division and binds GTP, and
Bramhill and Thompson [53] have now shown that
it even polymerizes in a GTP-dependent fashion, suggesting that the similarity is more than accidental, even
though the overall homology is very low.
Centrosomes and chaperones
(D 1995 Current Opinion in Cell Biology
cz-tubulin
O [3-tubulin-GTP
0 J3-tubulin-GDP
Fig. 1. Several features of the microtubule structure deduced from
labeling microtubules with the kinesin motor domain (for details,
see [14"]). The plus (fast-growing, distal) end is up, the minus
end (slow-growing, proximal) is down. The minus end is normally
buried in the microtubule-organizing structure containing y-tubulin (not shown here). An ct[B-tubulin heterodimer is shown near the
top (height 2 x 4 = 8 nm). The plus end of a microtubule terminates
with a crown of (~-tubulin, the minus end with a crown of ifl-tubulin. [3-Tubulin carrying GTP occurs in solution and at the plus end
[15"], but not in the interior of the microtubule. Note that the [3tubulin-GTP cap is covered by the terminal layer of ct-tubulin. The
protofilaments are shifted axially by 0.9 nm, corresponding to the
'B4attice' which results in a discontinuity in the microtubule wall
(moved to the back and thus not shown in this diagram). In the fully
saturated microtubule lattice every tubulin heterodimer binds one
kinesin head, thus emphasizing the dimer lattice of microtubules.
Native kinesin is a dimer of two heavy chains, linked via a flexible
neck and a coiled-coil stalk (simplified here as a V-shape), and two
light chains. The diagram shows three hypothetical modes of attaching the kinesin dimer to the microtubule: (a) on two adjacent
protofilaments side by side; (b) on one protofilament (kinesin heads
displaced axially by one tubulin dimer, or 8 nm); (c) on two adjacent protofilaments (heads staggered by one tubulin dimer). Each
of these modes would be compatible with the observed structure
of the microtubule decorated with kinesin.
Components of the centrosome
For microtubules to operate properly they must be nucleated at the right time and in the right place. This
normally happens at the centrosome, which consist of
a pair of centrioles surrounded by periocentriolar material; the latter is where microtubule nucleation actuary occurs. The stages and components of centrosome
assembly are beginning to be understood. One of the
components is 7-tubulin, which exists in the cytoplasm
as part of a multiprotein complex ('gammasome') with
several other proteins. 7-Tubulin is recruited to the centrosome for nucleation of microtubules at their minus
ends; nucleation is blocked when 7-tubulin is depleted or
inactivated. Other components of the centrosome precursor include ct- and ~-tubulin, actin, and the molecular chaperone hsp70 [54"-57"]. Additional proteins are
required to assemble the pericentriolar material, such
as pericentrin, an elongated 220kDa protein with a
coiled-coil stalk and two globular ends, identified using antisera from patients with scleroderma [58"]. This
general domain structure is shared with other proteins
found in microtubule-organizing structures, such as the
SP-H antigen/NuMa [59], and variants of tektin, an
(~-helical protein first found in flagellar axonemes and
recently in centrosomes [60]. The microtubule-nucleating function of 7-tubulin in centrosomes appears to
be highly conserved as human 7-tubulin functions even
when introduced into yeast [61].
Chaperones
Before microtubules can be nucleated, the tubulin must
first be folded; this is achieved by another oligomeric
complex, the cytoplasmic chaperone complex TCP-1,
Microtubules and microtubule-associated proteins Mandelkow and Mandelkow
also known as TriC. This 800 kDa structure contains several homologs of the heat shock protein Hsp60 (termed
CCT0t, -~, and so on) arranged in the two-layered
ring structure also found in bacterial and mitochondrial chaperones; this complex assists in the folding o f
relatively few proteins, among them the three tubulins
and actin [62"-64"]. TCP-1 subunits are the cytosolic
equivalents of the bacterial chaperone GroEL and mitochondrial Hsp60. The folding takes place in several steps
such that the initial association occurs with ADP bound
to the complex, followed by a later release involving
ATP hydrolysis [65"]. Yeast have equivalent chaperones
(BIN2, BIN3 and so on); even though they are related,
they appear to serve somewhat different functions and
do not replace one another [66"].
Location of assembly and transport
A related and more specialized problem is the nucleation
of microtubules in nerve cells, whose long processes are
filled with microtubules that serve as tracks for intracellular transport. Are the microtubules assembled in the
axon or in the cell body? The latter appears to be the
case: nucleation sites containing ~,-tubulin are found only
in the cell body, and microtubules are then transported
into the axons or dendrites (see below). Superimposed
on this transport process is a length redistribution which
allows microtubules to change their length, presumably
by dynamic instability, in the axon. The plus ends of the
transported microtubules are oriented distally, and this
end is where incorporation o f new subunits can occur
[67].
Microtubule-associated proteins
The majority of studies have traditionally been done
with brain MAPs, partly because brain tissue was the
most abundant source of microtubules, and partly because the MAPs provide selective markers for brain
development and cell type. The best known MAPs are
the heat-stable proteins MAP2 and tau; they are localized (mainly) to the dendritic and axonal compartment,
respectively, and they share homologous repeats in the
carboxy-terminal region that contribute to microtubule
binding (reviewed by Schoenfeld and Obar [51).
Tau
Tau is thought to stabilize microtubules in axons and
thus provide the basis for axonal transport. Consistent
with this idea, it was found that when tau is transfected
into culture cells microtubules become more stable and
the degree of assembly increases [68,69]. This effect can
be observed even more dramatically in insect Sf0 ceils
transfected with tau using the baculovirus system: these
normally round cells develop 'neurite-like' processes. In
this system, tau also protects microtubules against druginduced disassembly [70",71"]. The simple picture does
not always hold, however, as transfection oftau into Chinese hamster ovary cells did not result in an increase in
microtubule stability or mass [72"]. Even more surprising
is the fact that tau-null transgenic mice are viable; in this
case, the stabilization ofmicrotubules may be achieved by
other MAPs [73"].
Functions of tau
Tau has several distinct effects on microtubules in vitro:
it binds to them, promotes their nucleation and elongation, protects against disassembly, and induces them
to form parallel arrays (bundles). These functions can
be traced back to different domains of tau. For example, efficient assembly of microtubules requires not only
the internal repeat domain, but also regions on either
side, acting as 'jaws'; the flanking regions in turn tend
to promote the formation of bundles [74-76]. Bundles
of microtubules may be important not only in neurite outgrowth, but also in other functions such as in
the erythrocyte marginal band, where tau is one of the
MAPs responsible for cohesion [77]. Although the contribution o f the repeat domain to microtubule binding is
generally weak, there are certain 'hotspots' (e.g. the region between repeats 1 and 2) which could regulate the
affinity, especially during the developmentally regulated
alternative splicing o f tau [78].
Distribution
Another characteristic feature o f tau is its heterogeneous
distribution, both within cells and between different cell
populations. Whereas six tau isoforms are found in central nervous tissue (in humans), 'big' tau (distinguished
mainly by the presence o f an additional exon, 4a) occurs in peripheral nerves. Within neurons, tau is mainly
axonal (in contrast to the somatodendritic MAP2), but
there is also a nuclear variant of tau. This intracellular
sorting may take place at the level of the transcripts,
which come in sizes between 2.5 and 8 kilobases and may
contain targeting information. Microtubules may play a
role in distributing tau, because m R N A is transported
along them [79-82].
AIzheimer's disease
Apart from its cell biological role, tau has generated
considerable interest because of its relationship with
Alzheimer's disease. Tau is the main component of the
paired helical filaments that make up the neurofibrillary tangles in the brains of patients with Alzheimer's
disease. In these tangles, tau is not only abnormally
aggregated, but also modified in several other ways fieviewed in [83]). The most conspicuous modification is
excess phosphorylation, frequently at Set-Pro or ThrPro motifs (indicating the influence of proline-directed
kinases). There are other sites (such as Ser262) that
strongly influence the affinity of tau for microtubules
[84"] and are phosphorylated in the tau protein found in
Alzheimer's disease [85]; phosphorylation might explain
why this tau does not bind to microtubules and ceases
to stabilize them [86]. This effect contrasts with that resulting from 'fetal' phosphorylation of tau, which shares
some of the phosphorylation sites of Alzheimer's disease,
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Cytoskeleton
but which still binds well to microtubules [87]. Several
phosphorylation sites in fetal and adult rat tau have now
been determined directly by mass spectrometry and sequencing [88]. From a practical point of view, most studies of phosphorylation rely on phosphorylation-dependent antibodies, which can also be used with cell models
(epitopes are summarized in [83]). All of the sites found
so far can be dephosphorylated by phosphatases PP-2A
and PP-2B (calcineurin), both of which are abundant in
brain [89]. Other modifications include ubiquitination at
several lysines [90] and glycation [91].
Structure
How the normal and pathological properties o f tau are
related to its structure remains enigmatic. In solution, tau
lacks secondary structure or even a defined shape and is
best described as a random coil; the lack of secondary
structure is observed even in paired helical filaments,
suggesting that these may not assemble by way of interacting ~-strands, in contrast to other fibers found in
pathological states [92]. Nevertheless, tau tends to associate into antiparallel dimers, and in this form it is prone
to assembly into paired helical filaments [93].
MAP2, MAPla, MAPlb and MAP4
MAP2
MAP2 is a relative o f tau in that it has a homologous repeat domain near the carboxyl terminus but has a much
larger extension towards the amino terminus. The middle part can be spliced out to give MAP2c, a 'juvenile'
isoform [94]. Whereas tau can have either three or four
repeats, MAP2 used to be found with three repeats only;
however, this gap has now been closed by the discovery
o f a four-repeat MAP2c. Unlike the neuronal MAP2,
this isoform occurs in gila cells [94]. The question of
how MAPs bind to microtubules, and how they interfere
with motor proteins, continues to-be a matter of study.
Wallis et al. [95] showed that MAP2 binds in a positively cooperative manner, leading to the clustering of
MAP2 on the microtubule surface (which makes it distinct from tau [76]). Echoing an earlier debate, Hagiwara
et al. [96] reported that MAP2 and motor proteins compete for the same binding sites (in the carboxy-terminal
domain o f tubulin), whereas Marya et al. [97] found distinct sites and no competition. A compromise solution
was offered by Lopez and Sheetz [98], who found distinct sites but steric hindrance, as might be expected with
molecules as large as MAP2. Whatever the explanation
o f the results, in practice there seems to be no doubt that
motors can work their way along microtubules quite efficiently, even when MAPs are present.
M A P l a and M A P l b
MAPla and M A P l b are two other high molecular
weight neuronal MAPs; they do not belong to the
same class as MAP2 and tau because they have different microtubule interaction motifs. MAPla and M A P l b
share partial sequence homology, and both are syn-
thesized as a polyprotein coupled to a light chain
(LC; M A P l b - L C 1 , 34 kDa, and MAPla-LC2, 28 kDa,
respectively [99,100]). As M A P l b binds to microtubules
through a series of basic repeats o f the type KKE,
the same mode of binding had also been assumed for
MAPla. This is not the case, however, as Cravchik et al.
[101] have discovered that MAPla has a novel binding
motif which is unique, as it is acidic rather than basic.
They proposed that MAPla has a bipolar helical structure, with positive and negative charges separated on the
two sides. The third light chain, LC3 (18kDa), is encoded separately and has now been cloned [102]. It is
a basic protein, abundant in neurons, that binds to both
M A P l a and M A P l b and could modulate their interaction with microtubules.
MAP4
In contrast to the neuronal proteins discussed above,
MAP4 has a ubiquitous distribution. It resembles MAP2
and tau in having a homologous repeat domain that
interacts with microtubules. Contrary to the view that
MAPs are freely diffusible in cells, Chapin and Bulinski
[103] have observed a surprising extent o f microheterogeneity of MAP4-microtubule interactions in cell cultures, suggesting the selective stabilization of subsets of
microtubules within cells. A related MAP4-1ike protein
has been purified from Xenopus eggs [104]. This protein becomes phosphorylated during mitosis and thus is
a candidate for MAP4-dependent regulation of microtubule dynamics.
Plant MAPs
Compared to the proteins mentioned so far, the study of
plant microtubules has been lagging behind, partly because o f the difficulty in preparing sufficient quantities
o f protein. Recent work shows that plant microtubules
are quite comparable with those of animals; for example,
plant tubulin can be assembled reversibly using taxol, hybrid systems o f maize tubulin assembled with neuronal
MAP2 show tight binding (dissociation constant in the
micromolar range), and some plant MAPs appear to be
related to tau [105-107].
Other proteins associated with microtubules
As mentioned in the Introduction, not all associated proteins can be classified as structural or motor MAPs;
conversely, as microtubules are 'sticky', not all associations may be meaningful. As phosphorylation regulates
many intracellular interactions, it is intriguing that microtubules associate with a number ofkinases (such as the
mitogen-activated protein kinase cdk5, and GSK-3; see
[83]) that can cause the 'pathological' phosphorylation of
tau protein. The kinases often bind to microtubules via
MAPs. An interesting case is that o f protein kinase A associated with MAP2: phosphorylation of the regulatory
subunit of protein kinase A by the cell cycle kinase cdc2
releases the catalytic subunit and presumably allows it to
phosphorylate other targets [108]. Dynamin is a GTPase
involved in endocytosis; it has two microtubule-bind-
Microtubulesand microtubule-associatedproteinsMandelkow and Mandelkow
ing sites near the amino terminus and a proline-rich
domain (resembling Src homology region 3 domains)
near the carboxyl terminus whose affinity is regulated
by phosphorylation [109].
Several proteins involved in signal transduction bind
tightly to tubulin or microtubules. Recent examples include neurofibromin (a neuronal GTPase-activating protein) [110], the ~-subunits of several G proteins (which
can take up GTP from tubulin) [111], and gephyrin (a
protein linked to the glycine receptor) [112]. Associations with microtubules are not restricted to proteins;
the binding of mRNAs was mentioned above [80], and
recently Surridge and Burns [113] reported that the middie part of MAP2 has a high affinity for phospholipids,
a property not found in tau or MAP2c.
O f special interest in this context are proteins that are
capable ofmicrotubule severing, which could contribute
to their rapid reorganization, for example during mitosis. Examples include katanin, a heterodimeric protein
(60 and 81kDa) that severs microtubules in an ATPdependent fashion [114"], and elongation factor EF-I~,
a GTP-binding protein involved in protein synthesis
[115°]. On the other hand, a protein like EF-10t has
also been found to be able to cause the formation of
bundles of microtubules [116 °] and has been found in
the pro-centrosomal protein complex (see above; [54°]).
It will be interesting to see how these seemingly contradictory activities are related to one another. Finally,
flagellar microtubules can be excised near their base,
and it turns out that this is achieved by contractile fibers
consisting of centrin, a relative of the calcium-binding
protein calmodulin ([117"]; see also Salisbury, this issue,
pp 39-45).
Microtubule dynamics
One of the most fascinating aspects of microtubules is
their rapid turnover and potential for reorganization,
unexpected for a cyto-'skeletal' polymer. The half-lives
of most microtubules are in the range of minutes, and
the predominant mechanism of redistribution appears to
be dynamic instability (reviewed by Cassimeris, [118]),
perhaps in combination with the severing factors just
mentioned [114",115°]. Dynamic instability can also
be observed in plant cells [119]. On the other hand,
many cellular microtubules are intrinsically stable [120],
suggesting that the observed dynamics are tightly regulated by factors controlling catastrophes and rescues,
including kinases such as the cell cycle dependent kinase cdc2. Independent of these mechanisms is a steady
flux (or treadmill) of subunits, such that labeled subunits migrate slowly from the plus to the minus end
of the microtubule [121]. In spite of this activity, and
contrary to intuition, the assembled microtubule mass
does not change nmch during mitosis, indicating that
reorganization does not require net disassembly [122].
A reconstruction of an entire spindle microtubule assembly shows that the nearest-neighbor interactions are
mainly between antiparallel microtubules, supporting the
potential for bidirectional gliding during anaphase [123].
Plant ceils have the capacity for reorienting their entire
microtubule network in response to extracellular factors
and gravity [124]. Some of these features can be reproduced in vitro, where microtubules can be induced to
display elaborate waves of assembly and orientation in
time and space [125"].
Conclusions
In contrast to the actin-based cytoskeleton, where the
structures of the main players (actin, myosin and their
complex) have been solved by X-ray crystallography
[126,127], the microtubule system does not yet offer
such insights. A direct structural analysis may be possible by electron crystallography, because zinc induces
the formation of tubulin sheets that diffract to nearatomic resolution [128°]. For the time being, we have
to learn by analogy. For example, the structure of the
actin-binding domains of gelsolin [129] illustrates that
repetitive sequences need not reflect repetitive binding; the interactions may in fact be based on the variable
parts, whereas the repeats could simply provide a framework for protein folding (this argument might apply to
models of rnicrotubule-MAP interactions). Tubulin is a
GTP-binding protein that shares weak homology with
signal-transducing G proteins. The structure of the ctsubunit of transducin has become available [130]; part
of it folds in a manner similar to the small G protein
1Las/p21 (reviewed in [131]). Finally, if one is interested
in an example of an assembly of 0r- and [~-subunits, with
nucleotides bound in a non-exchangeable and exchangeable fashion, then the recent solution of the F1-ATPase
structure may be worth studying [132]. Even though
these proteins have different functions from tubulin, they
provide intriguing food for thought.
Acknowledgements
We thank Alexander Marx for help in compiling the references,
and Stefan Sack for the preparation of Fig. 1.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
*
of special interest
**
of outstanding interest
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Cytoskeleton
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Lee G: Non-motor microtubule-associated proteins. Curt Opin
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Goldstein LSB: With apologies to Scheherazade: tails of 1001
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Gittes F, Mickey B, Nettleton J, Howard J: Flexural rigidity
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Vale RD, Coppin CM, Malik F, Kull FJ, Milligan RA: Tubulin
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Kreis 1-, Vale R (Eds): Guidebook to the cytoskeletal and motor
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Hyams JS, Lloyd CW (Eds): Microtubules. New York: Wiley-Liss;
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12.
•
Song Y-H, Mandelkow E: Recombinant kinesin motor domain
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Decoration of microtubules with recombinant kinesin head domains
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is one kinesin head per tubulin dimer, and chemical cross-linking connects kinesin to [[]-tubulin.
13.
•
Harrison B, Marchese-Ragona S, Gilbert S, Cheng N, Steven
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S o n g Y-H, Mandelkow E: Anatomy of flagellar microtubules:
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Analysis of flagellar outer doublet and central pair microtubules by decoration with kinesin heads, showing the lattices (B-type in all cases) and
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14.
•
15.
Mitchison T): Localization of an exchangeable GTP-binding site
•
at the plus end of microtubules. Science 1993, 261:1044-1047.
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Kuo SC, Sheetz MP: Force of single kinesin molecules meas•
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the tubulin dimer repeat, and the forces are in the picoNewton range per
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Demonstration by optical trapping that kinesin moves along microtubules
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17.
•
18.
Svoboda K, Block SM: Force and velocity measured for single
•
kinesin molecules. Cell 1994, 77:773-784.
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19.
•
Hall K, Cole DG, Yeh Y, Scholey JM, Baskin RJ: Forcevelocity relationships in kinesin-driven motility. Nature 1993,
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20.
*
21.
Ray S, Meyhofer E, Milligan RA, Howard J: Kinesin follows the microtubules protofilament axis. J Cell Biol 1993,
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the head domain of a motor, independently of its arrangement in the
protein.
27.
•
28.
Endow S, Kang S, Satterwhite L, Rose M, Skeen V, Salmon ED:
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Middleton K, Carbon J: Kar3-encoded kinesin is a minus-enddirected motor that functions with centromere-binding proteins (cbf3) on an in-vitro yeast kinetochore. Proc Natl Acad
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Hunt AJ, Howard J: Kinesin swivels to permit microtubule
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An example of how a kinesin-like head domain can cooperate with
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32.
•
33.
Sackett DL: Podophyllotoxin, steganacin and combretastatin:
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44.
•
Reijo RA, Cooper EM, Beagle GJ, Huffaker TC: Systematic mutational analysis of the yeast [3-tubulin gene. Mol Biol Cell
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45.
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47.
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55.
•
56.
•
Felix MA, Antony C, Wright M, Maro B: Centrosome assembly
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Ahmad FJ, Joshi HC, Centonze VE, Baas PW: Inhibition of microtubule nucleation at the neuronal centrosome compromises
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Doxsey SJ, Stein P, Evans L, Calarco PD, Kirschner M: Pericentrin, a highly conserved centrosome protein involved in
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58.
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59.
Maekawa T, Kuriyama R: Primary structure and microtubuleinteracting domain of the SP-H antigen: a mitotic MAP located
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Sternlicht H, Farr GW, Sternlicht ML, Driscoll JK, Willison K,
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A pulse-labelling analysis of actin and tubulin biogenesis in Chinese
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Melki R, Cowan NJ: Facilitated folding of actins and tubulins
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Chen XY, Sullivan DS, Huffaker TC: Two yeast genes with similarlty to TCP-1 are required for microtubule and actin function
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