γ-Tubulin complexes and microtubule nucleation

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Microtubules are dynamic cytoskeletal polymers that assemble

from /-tubulin and are vital for the establishment of cellpolarity, vesicle trafficking and formation of the mitotic/meioticspindle. -Tubulin, a protein related to /-tubulin, is requiredfor initiating the polymerization of microtubules in vivo.-Tubulin has been found in two main protein complexes: the-tubulin ring complex and its subunit, the -tubulin smallcomplex. The latter is analogous to the yeast Tub4 complex. Inthe past year, important advances have been made inunderstanding the structure and function of the -tubulin ringcomplex and how it interacts with microtubules.

Addresses*Department of Biochemistry and Biophysics, Howard HughesMedical Institute, University of California San Francisco, 513Parnassus Avenue, San Francisco, CA 94143-0448, USAe-mail: [email protected]

Current Opinion in Structural Biology 2001, 11:174181

0959-440X/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved.

AbbreviationsDgrip Drosophilagamma ring proteinTuRC -tubulin ring complexTuSC -tubulin small complexMTOC microtubule organizing center

IntroductionMicrotubules are cylindrical polymers of /-tubulindimers. The walls of the microtubule consist of 916 linear

polymers (protofilaments) of tubulin heterodimers that

assemble such that -tubulin in one dimer contacts -tubulin in the next (Figure 1a). Microtubules are thus

inherently polar, with -tubulin at one end of the polymer(the minus end) and -tubulin at the other (the plus end).

In vivo, microtubules consist primarily of 13 protofilaments

[1], which are offset from one another so that if one follows

- or -subunits laterally around the microtubule, theyform a three-start helix. This means that the helix spans

three subunits of a protofilament before it completes one

turn. The three-start helix is not perfectly symmetrical,resulting in a seam in the microtubule wall where each

helix makes a complete turn. Thus, protofilaments interact

with each other laterally primarily through and contacts, although at the seam -tubulin meets -tubulin(reviewed in [2,3]).

Microtubules polymerize spontaneously in vitro from high

concentrations of/-tubulin in the presence of GTP andMg2+. Polymerization occurs in a two-step process that

involves a rate-limiting nucleation step followed by rapid

elongation [4]. The nucleation step is thought to involve

the formation of a pair of short protofilaments, consisting of

7 [4], 12 [5] or 18 [6] /-tubulin dimers. Once this nucle-us has formed, it rapidly grows laterally and longitudinally

as a sheet until about 1000 dimers have assembled; the

sheet then closes into a cylinder. Sheets are also visible atthe growing ends of preformed microtubules, suggesting

that a two-dimensional polymer, rather than a helical poly-

mer, is the mode of elongation [7,8]. It is presumed that

microtubules assemble in the same way in vivo. However,

the early stages of nucleation have not actually been

observed inside cells.

The concentration of/-tubulin inside cells is below thelevel required for spontaneous nucleation in vitro, so the

process is assisted by microtubule organizing centers

(MTOCs), such as the centrosome in animal cells and the

spindle pole body in yeasts. The requirement for MTOCs

allows the cell to control when and where microtubules

grow. A large body of evidence derived from genetic

experiments, antibody inhibition studies, in vitro comple-

mentation assays, and fluorescence and electron

microscopy strongly implicates -tubulin as the key pro-

tein responsible for microtubule nucleation in vivo. This

highly conserved protein is approximately 30% identical to

- and -tubulins, but does not assemble into the bulkmicrotubule polymer. Although its activity is confined to

the MTOC, most -tubulin is present in the cytosol(reviewed in [9]).

Cytosolic -tubulin is found in two main complexes(reviewed in [10,11,12]): the large -tubulin ring complex(TuRC) and the -tubulin small complex (TuSC), which isanalogous to the Tub4 complex of Saccharomyces cerevisiae.

The TuRC was first isolated from Xenopus eggs [13] andsubsequently fromDrosophila embryos, along with its sub-

unit, the TuSC [14]. The TuRC consists ofapproximately 1014 -tubulin molecules and at least six

additional proteins, resulting in a complex of roughly

2 MDa. Similar protein complexes exist in mammalian cells

[1517], indicating that the TuRC is highly conserved.The TuSC consists of two copies of-tubulin and one copyeach of Dgrip84 and Dgrip91 (Dgrip: Drosophila gamma

ring protein), which are related to each other, as well as tothe yeast Spc97 and Spc98 proteins, and the Xenopus

Xgrip109 and Xgrip110 proteins (reviewed in [10,11,12]).

Electron microscopic images suggest that theXenopus and

Drosophila TuRCs have a flexible, open-ring structureapproximately 25 nm in diameter [13,14]. Individual sub-

units visible within the ring walls have been proposed to

be TuSCs [14]. Centrosomes ofDrosophila [18] and thesurf clam Spisula [19] contain similar ring structures, and

these rings contact the minus ends of microtubules and

contain -tubulin [20].

How does the TuRC nucleate microtubules? Its structuresuggested to Zheng and co-workers [13] that it may act as

-Tubulin complexes and microtubule nucleationMichelle Moritz* and David A Agard


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-Tubulin complexes and microtubule nucleation Moritz and Agard 175

a template out of which the microtubule grows

(Figure 1b). As microtubules inside cells usually contain

13 protofilaments [1], the model proposed that the TuRCcontains 13 laterally interacting -tubulins, each of whichcontacts one - (or -) tubulin longitudinally at the minusend of a protofilament.

A second, protofilament, model was proposed based onearlier observations of rings that form from pure /-tubulinor from its bacterial homolog, FtsZ [21]. In this model, the

-tubulins in the TuRC interact longitudinally with oneanother, in the same way that - and -tubulin or FtsZinteract in a protofilament or ring (Figure 1c). The TuRCunwinds to form the first protofilament of the microtubule

and the -tubulins interact laterally with - and -tubulin,stabilizing a pair of protofilaments that could then seed

further growth of the microtubule.

In the past year, four papers presented exciting new

evidence regarding -tubulin-mediated microtubule nucle-ation. Three groups used fluorescence or electron

microscopy to examine Xenopus or Drosophila TuRCs on

their own or in complex with microtubules [2224].

The consensus of these three studies is that a template

mechanism, albeit modified from the original model, is

more consistent with the data. In the fourth paper, a bio-

chemical study indicates that a single -tubulin is sufficientto nucleate microtubule assembly [25]. A compelling

argument has been made for how these new findings fit

into the protofilament model [26]. The focus of thisreview is to discuss these four papers and their implica-

tions for the mechanism of microtubule nucleation.

Structure of isolated TuRCsIn the initial characterizations of TuRCs isolatedfrom Xenopus [13] and Drosophila, the complexes were

examined by negative-stain electron microscopy or cryo-

electron microscopy and were found to be structurally very

similar. However, these techniques yielded only relatively

low-resolution two-dimensional information. In a recent

study [24], electron microscopic tomography and plat-

inum shadowing were used to gain insight into the

three-dimensional structure ofDrosophila TuRCs. Thetomography revealed that the subunits comprising the ring

Figure 1

a

/-tubulin a

TuRCTuRC

(a) (b) (c)

() end

(+) end

Current Opinion in Structural Biology

Models of microtubule nucleation. (a) A microtubule nucleatedspontaneously from pure /-tubulin. The microtubule is polar:-tubulin is minus-end proximal and -tubulin is plus-end proximal.

Note the three-start helix and the seam. (b) The template modelpredicts that the -tubulins of the TuRC interact with each otherlaterally and contact -tubulins longitudinally at the minus end of themicrotubule. This results in the stabilization of a small number of tubulin

subunits, so that elongation is favored. The TuRC determines thenumber of protofilaments in the microtubule. (c) The protofilamentmodel proposes that the -tubulins in the TuRC interact with each

other longitudinally and with /-tubulins primarily laterally. The TuRCunwinds to form the first protofilament of the microtubule, promotingformation of a small sheet that then grows into a microtubule.


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176 Macromolecular assemblages

walls are arranged in pairs with a distinct U or V shape,with the pairs separating on one ring face and converging

on the other (Figure 2). A globular structure sits asymmet-

rically atop the face of the ring where the subunit pairs

meet and does not extend very far into the ring lumen

(Figure 2ad). Platinum shadowing revealed a similar

structure that, in addition, nicely displayed the helical

nature of the complex (Figure 2e,f). It was not possible in

this study to determine unequivocally how many subunits

are in the ring walls, although preliminary data indicate

that there are approximately 12.

Although the definitive assignment ofTuRC proteins tospecific substructures awaits immuno-labeling experi-

ments, the images obtained begin to provide structural

evidence for the models proposed previously on the basisof biochemistry [12,14,16,27]. It seems very likely that six

or seven TuSCs make up the wall of the ring and that the-tubulins are positioned on the face of the ring away fromthe asymmetric cap. The cap is probably made up of the

proteins Dgrips 163, 128 and 75s, which biochemistry has

shown to be of lower stoichiometry in the TuRC [14](Figure 2g). The position of the cap suggests that it may be

involved in attachment of the TuRC to the centrosome,regulation ofTuRC activity and/or stabilization of the ring.

Structure of TuRCs in complex withmicrotubulesA motivating assumption in the three recent structural

studies was that the template and protofilament models

Figure 2

Structure of isolated DrosophilaTuRCs.(ad) Selected views of a reconstructedTuRC obtained by electron microscopictomography. In each image, several sections

from the reconstruction were stacked into asingle volume. (a) View of the TuRC facecontaining the asymmetric cap. (b) Middlesection of the TuRC. Note the ring wallsubunits and that the cap does not extendvery far into the ring lumen; a cap remnant canbe seen spanning the ring lumen. (c) Sideview of the TuRC. Note the invertedV-shaped ring wall subunits and the capstructure. The blue-dashed line outlines onepaired subunit, which is proposed to be oneTuSC. (d) Alternative side view of the TuRC.Bar = 10 nm. (e) Platinum replicas of TuRCs.The helical structure and ring wall subunitsare apparent in the upper three and lower leftpanels. The lower right and middle panels

show the ring face topped by the asymmetriccap. Bar = 10 nm. (f) Platinum replicas ofpure bovine-brain /-tubulin. Note that thestructures of the tubulin polymers are distinctfrom those of TuRCs (compare withFigure 2e). (g) Model of the helical TuRCstructure, showing the ring opening (left) andthe opposite side (middle). The modelincorporates features of the reconstructionsand of the platinum replicas. Ring walls areproposed to consist of repeating TuSCsubunits (outlined in blue), each comprisingtwo -tubulins (pink) and one copy each ofDgrips 84 and 91 (green). Dgrips 163, 128and 75s (gray) are proposed to make up the

cap. The right panel shows a tilted view of theimage shown in (c), showing the TuRC as itmight be expected to appear in the absenceof helix flattening caused by binding to thegrid. Reproduced from [24] with permission.

Dgrips 163,128, 75s

Dgrips 84,91

-tubulin

(a) (b)

(c) (d)

(e)

(g)

(f)


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should be distinguishable ifTuRC proteins, and -tubulinin particular, could be localized with respect to micro-

tubule ends. The template model predicts that the TuRC

forms a cap at one end of the microtubule and that the

-tubulins are confined to a narrow (12 nm) zone at thatend. In the protofilament model, the TuRC either mightbe fully incorporated into the wall of the microtubule, and

thus extend approximately 50 nm up the polymer wall, or

it may be partially incorporated, with the remainder curl-

ing away from the end. Several different approaches were

taken to distinguish these possibilities.

Keating and Borisy [22], and Wiese and Zheng [23]

studied the position of some Xenopus TuRC componentswith respect to microtubule ends in a similar manner. In

the former study, gold labeling of-tubulin or Xgrip109 andnegative-stain or platinum-replica electron microscopy

were used to localize these proteins at microtubule ends. In

the latter study, the entireXenopus TuRC was biotinylated

and detected by streptavidingold conjugates using nega-

tive-stain electron microscopy. In both studies, distances

were measured between the ends of the microtubules and

large numbers of gold particles. In both cases, the gold was

mainly confined to one end of the microtubule, in a zone

more consistent with the template model. It is unlikelythat these studies missed -tubulins in the wall of themicrotubule because, in the Keating study [22], the anti-

bodies were directly labeled with gold and were raised

against a C-terminal -tubulin peptide that is known to beaccessible in the native TuRC and, in the Wiese study[23], the entire TuRC was biotinylated and detected bystreptavidingold conjugates (see also Update).

In addition, in all three structural studies, most TuRC-nucleated microtubules exhibited a cap-like structure at

one end and, in some cases, the TuRC encircled the endof the microtubule (Figure 3), as expected if the TuRCacts as a template. The cap structure was observed on

microtubules nucleated from TuRCs inXenopus extracts,

from isolatedXenopus orDrosophila TuRCs and pure tubu-lin, and from isolated Drosophila centrosomes. Structures

such as rings or curled protofilaments projecting from

microtubule ends, as would be expected if the TuRC actsas a protofilament, were not observed [2224]. It is

unlikely that the observed cap-like or ring-like arrange-

ment of the TuRC at microtubule ends is an artifact ofelectron microscopy because of the different approaches

taken and the different organisms used in these studies.

A new capping activity for the TuRC

Given the appearance of the TuRC on microtubules inthe electron microscopic images, the complex might be

expected to cap the minus end of the microtubule, func-

tionally inhibiting further growth at that end. This

possibility was investigated by Wiese and Zheng [23]

using fluorescently labeled Xenopus TuRCs and micro-tubules that were marked by nucleating in the presence of

a high ratio of rhodamine-labeled to unlabeled tubulin.

The microtubules were then elongated with a dim mix of

tubulin, that is, one with a lower ratio of labeled to unla-

beled tubulin. Thus, if both ends of the microtubule grow,

it would contain a central bright region flanked by two dim

ends. As the minus end grows more slowly than the plus

end, one dim end would usually be shorter than the other.It was found that the TuRC prevents minus-end growthon the microtubules it nucleates, as well as on the pre-

formed microtubules to which it binds. The complex can

also prevent minus-end depolymerization. Thus, this

study revealed that the TuRC not only nucleates micro-tubules, but also has a separate capping activity that may

be very important for modulating minus-end dynamics.

The template model revisitedThe simplest model to explain the structural and functional

data in these three studies would have most or all of the

-tubulins in the TuRC in direct, longitudinal contact withthe tubulin at the minus ends of microtubules (Figure 4), as

the original model proposed [13]. The original model must,

Figure 3

One end of Xenopusor DrosophilaTuRC-nucleated microtubules displays a capor ring structure. (a) Electron microscopicimage of a negative-stained microtubule

nucleated in a Xenopusegg extract(reproduced from [22] with permission).(b) Negative-stained microtubule nucleatedfrom an isolated, biotinylated XenopusTuRC.One streptavidingold conjugate labels themicrotubule end (reproduced from [23] withpermission). Bar = 20 nm. (c) Reconstructionfrom electron microscopic tomography of amicrotubule nucleated by an isolatedDrosophilaTuRC (reproduced from [24]with permission). Compare the cap-likestructures visible in (ac). Bar = 25 nm.(d) Reconstruction from electronmicroscopic tomography of a microtubule

nucleated by an isolated DrosophilaTuRC (reproduced from [24] withpermission). In this example, the ring

structure is more prominent than the cap andappears to encircle the end of themicrotubule. Bar = 25 nm.

(a)

(b)

(c)

(d)


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however, be modified to incorporate biochemical data sug-

gesting that the TuRC is assembled from preformedTuSCs, which contain two copies of-tubulin and one copyeach of the homologs of theS. cerevisiaeSpc97 and Spc98 pro-

teins [14]. This implies that the TuRC must contain aneven number of-tubulins, not the 13 that were originallyproposed. It is therefore possible to hypothesize at least three

likely arrangements of the -tubulins within the complex.

If the TuRC contains 14 -tubulins, two of them may

overlap, maintaining 13-fold symmetry (Figure 4a)

[22,23]. In this arrangement, all of the -tubulins inter-act with -tubulins. In order to explain the original geneticdata suggesting that -tubulin interacts with -tubulin,

Keating and Borisy [22] presented an alternative arrange-

ment in which the TuRC helix is split on one side andoverlaps on the other, so that the -tubulins interact withboth - and -tubulins (Figure 4b). It is worth noting, how-ever, that the original genetic interactions were not

allele-specific and, in fact, involve three separate regions of

-tubulin, calling into question the need to invoke a directinteraction between -tubulin and -tubulin [9].

If the complex contains 12 -tubulins, the TuRC accessory

proteins may hold the TuSCs in a three-start helix with13-fold symmetry, with one part of the symmetry defined

by a gap in the ring (Figure 4c) [24]. It is also possible

that TuRC-nucleated microtubules begin with 12 or 14

Figure 4

(c)(a)

(d)

(b)

(e)TuSC

Dgrips 163,128, 75s

TuRC

-tubulin

-tubulin

-tubulin

Dgrips 91, 84X

X

Current Opinion in Structural Biology

New models of microtubule nucleation by the TuRC or monomeric-tubulin. (ac) Possible template mechanisms for nucleation. (a) Amodel to accommodate a TuRC containing 14 -tubulins (based onmodels presented in [22,23]). An overlap of one half of a TuSC oneach end of the helix would maintain 13-fold symmetry. The -tubulinswould all contact -tubulin longitudinally. (b) A split-helix model [22]that would accommodate 14 -tubulins in the TuRC and allow a directinteraction between -tubulin and -tubulin. The seam side of themicrotubule contains an overlap of two -tubulins (left), with the D/Xgripsubunit perhaps folding inward to allow the interaction. The TuRC helixon the nonseam side of the microtubule (right) would also be split toallow an interaction between -tubulin and -tubulin. (c) A model toaccommodate a 12 -tubulin-containing TuRC (reproduced with

permission from [24]). The D/Xgrip subunits are proposed to hold the-tubulins in a helix with 13-fold symmetry, with the thirteenth point ofsymmetry created by a gap in the ring. The thirteenth protofilamentmight form through stabilizing lateral interactions with the twelfth andfirst protofilaments. (d) A model of how a single -tubulin might nucleateand cap a microtubule (based on [25]). The single, strong interactionbetween -tubulin and -tubulin is proposed to stabilize the initial fewtubulin subunits in the polymer, facilitating subsequent elongation. Apossible nucleus is outlined in green. The Xs indicate that furtheraddition of subunits cannot occur in these two directions, explaining theminus-end capping activity of the -tubulin. (e) An example of howbinding of the D/Xgrip proteins of the TuSC (or Spc97/Spc98 of theyeast Tub4 complex) might shield -tubulin-binding sites on -tubulin.


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protofilaments and shift to 13 protofilaments further along

the polymer [28]. In all models, the non-TuSC compo-nents of the TuRC are proposed to make up the cap,which may regulate activity, impart stability to the helix

and/or attach the TuRC to the centrosome.

Microtubule nucleation and capping bymonomeric -tubulinAlthough the recent structural papers support the idea that

the TuRC acts as a template in which all -tubulin mol-ecules in the complex play a role in nucleation, a recent

biochemical study by Leguy et al. [25] raises intriguing

new possibilities regarding the mechanism of nucleation.

In this study, -tubulin was produced in a reticulocytelysate and the monomeric portion was partially purified on

a sizing column. The effect of this -tubulin on micro-tubule nucleation was followed by turbidity monitoring

for a decrease in the lag time of assembly and an increase

in microtubule number. Remarkably, -tubulin concentra-tions of 0.60.8 nM were found to induce microtubule

nucleation at low (1217 M) tubulin concentrations,decreasing the size of the nucleus from seven to three

tubulin heterodimers. The binding stoichiometry was one

-tubulin per microtubule and this was sufficient to blockfurther growth from the minus end. The interaction

between -tubulin and the microtubule was high affinity(1010 M1). In a blot-overlay experiment, -tubulin boundmore strongly to -tubulin than to -tubulin.

These data suggest a model in which a single -tubulin

forms a tight, lateral bond with a -tubulin in an oligomer ofthree tubulin dimers. On the basis of the capping data, fur-

ther growth is possible only in the plus-end direction

(Figure 4d). The authors propose that their observations can

explain microtubule nucleation in the context of either the

template or the protofilament models. Within the template

model, one of the -tubulins in the TuRC might act in thesame manner as monomeric -tubulin, binding laterally to-tubulin, and the additional -tubulins would interact withthe -tubulins at the microtubule minus end. These lessstrong associations might be stabilized by the Spc97/Spc98

homologs. This model is similar to the split-helix model

described above (Figure 4b). Within the protofilament

model, the accessory proteins might strengthen the interac-tion of the key -tubulin with the protofilament.

ConclusionsIn the past year, several exciting advances in understanding

microtubule nucleation by -tubulin complexes have beenmade at both structural and biochemical levels. It is now fair-

ly certain that the TuRC caps the minus end of themicrotubules it nucleates both structurally and functionally.

What this means for the mechanism of nucleation, however,

is unclear. If most or all of the -tubulins in the complex con-

tact the minus end of the microtubule, it is tempting to

assume that they are all directly involved in promoting

nucleation through a template mechanism. However,

the finding that a single -tubulin molecule is sufficient to

promote nucleation raises the possibility that only one

-tubulin in the complex is directly involved in nucleation;the others would have a supporting role. For example, the

mechanism could involve a combination of nucleus assembly

through a single lateral interaction and template forma-

tion, and/or further complex stabilization through theremainder of the -tubulins. This could occur in a manner

similar to that proposed in the split-helix model (Figure 4b).

However, it is important to keep in mind that most, if not all,

-tubulin inside cells is bound up in the TuRC or the TuSC.

If these complexes nucleate microtubules similarly to

monomeric -tubulin, one would expect the TuSC to be apotent nucleator. In fact, the TuSC is a very poor nucleator[14]. This suggests that the binding of Dgrips 84 and 91

(homologs of Spc97, Spc98 and Xgrips 109, 110) shields

-tubulin from interacting with -tubulin (Figure 4e). Asthe TuRC is a much better nucleator than the TuSC [14],assembly of the TuSC into the TuRC must either exposecritical interaction sites required for nucleation, strengthen

activity by simply providing a greater number of interaction

sites or form a special structure that promotes nucleus for-

mation and microtubule growth through a unique

mechanism. It is possible that monomeric -tubulin is amore potent nucleator than the TuSC and TuRC, and thatcells have evolved a way of modulating this activity by

sequestering -tubulin in these complexes. Thus, it couldbe that the nucleating activity of monomeric -tubulin doesnot reflect the mechanism used by the TuSC and TuRC.

The recent work on -tubulin, TuRC and TuSC has pro-vided new insights into the structure and activity of these

microtubule nucleators, but it has also raised new ques-

tions. It is now very important to perform parallel

comparisons of the nucleating (and capping) activities of

pure -tubulin, TuRC, TuSC and perhaps even the yeastTub4 complex, as it forms a similar closed structure at

microtubule minus ends [29,30] and therefore might act in

the same way. Other important experiments include deter-

mining the number of subunits in the TuRC and thenumber of protofilaments in microtubules nucleated by

them; localizing specific proteins within the complex and

determining their function; and cross-linking or mutagen-

esis experiments to explore further the proposed contactbetween -tubulin and -tubulin (this last possibility isdescribed more fully by Erickson [26]). Preliminary

experiments are also beginning to indicate the regions of

interaction between -, - and -tubulin [31]. As many ofthe tools needed to carry out these experiments are avail-

able, the prospects for obtaining a detailed, molecular

understanding of microtubule nucleation by -tubulincomplexes in the near future are good.

UpdateIn a recent study, an additional component of theXenopus

TuRC, Xgrip210, was localized with respect to the micro-tubule minus end by labeling with Xgrip210 antibodies

that were directly conjugated to gold particles [32].


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Xgrip210 is a TuRC component that is present at a lowerstoichiometry in the complex than -tubulin and theSpc97/Spc98 homologs (Xgrip109, Xgrip110). The distances

of the Xgrip210 gold particles from the microtubule ends

were measured in the same manner as in the Keating and

Borisy study [22], and were found to occupy a micro-tubule-distal region of the TuRC-capped end [32]. Inaddition, Xgrip210 was found to be required for TuRCassembly and for the recruitment of-tubulin and Xgrip109to the centrosome. Similarly, Xgrip109 is required for the

localization of Xgrip210 to the centrosome. These data sug-

gest that Xgrip210 is a component of the TuRC capstructure and is involved in attaching the TuRC to the cen-trosome. This study also showed that the TuSC can notattach to the centrosome on its own, supporting the idea that

the attachment is made through the TuRC cap structure.

AcknowledgementsThanks to T Keating and C Wiese for permission to use their images in thisreview. We are grateful to L Rice, P Dias, H Aldaz and M Trammell forinsightful discussions and comments on the manuscript. Our work issupported by the National Institutes of Health (NIH Grant GM31627) andthe Howard Hughes Medical Institute.

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γ-Tubulin complexes and microtubule nucleation