γ-tubulin complexes and microtubule nucleation

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Microtubules are dynamic cytoskeletal polymers that assemblefrom α/β-tubulin and are vital for the establishment of cellpolarity, vesicle trafficking and formation of the mitotic/meioticspindle. γ-Tubulin, a protein related to α/β-tubulin, is requiredfor initiating the polymerization of microtubules in vivo.γ-Tubulin has been found in two main protein complexes: theγ-tubulin ring complex and its subunit, the γ-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 γ-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, USA†e-mail: [email protected]

Current Opinion in Structural Biology 2001, 11:174–181

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

AbbreviationsDgrip Drosophila gamma ring proteinγγTuRC γ-tubulin ring complex γγTuSC γ-tubulin small complex MTOC microtubule organizing center

IntroductionMicrotubules are cylindrical polymers of α/β-tubulindimers. The walls of the microtubule consist of 9–16 linearpolymers (protofilaments) of tubulin heterodimers thatassemble such that β-tubulin in one dimer contacts α-tubulin in the next (Figure 1a). Microtubules are thusinherently polar, with α-tubulin at one end of the polymer(the minus end) and β-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 followsα- or β-subunits laterally around the microtubule, theyform a ‘three-start’ helix. This means that the helix spansthree subunits of a protofilament before it completes oneturn. The three-start helix is not perfectly symmetrical,resulting in a ‘seam’ in the microtubule wall where eachhelix makes a complete turn. Thus, protofilaments interactwith each other laterally primarily through α–α and β–βcontacts, although at the seam α-tubulin meets β-tubulin(reviewed in [2•,3•]).

Microtubules polymerize spontaneously in vitro from highconcentrations of α/β-tubulin in the presence of GTP andMg2+. Polymerization occurs in a two-step process thatinvolves a rate-limiting ‘nucleation’ step followed by rapidelongation [4]. The nucleation step is thought to involvethe formation of a pair of short protofilaments, consisting of7 [4], 12 [5] or 18 [6] α/β-tubulin dimers. Once this nucle-us has formed, it rapidly grows laterally and longitudinally

as a sheet until about 1000 dimers have assembled; thesheet then closes into a cylinder. Sheets are also visible atthe growing ends of preformed microtubules, suggestingthat a two-dimensional polymer, rather than a helical poly-mer, is the mode of elongation [7,8]. It is presumed thatmicrotubules assemble in the same way in vivo. However,the early stages of nucleation have not actually beenobserved inside cells.

The concentration of α/β-tubulin inside cells is below thelevel required for spontaneous nucleation in vitro, so theprocess is assisted by microtubule organizing centers(MTOCs), such as the centrosome in animal cells and thespindle pole body in yeasts. The requirement for MTOCsallows the cell to control when and where microtubulesgrow. A large body of evidence — derived from geneticexperiments, antibody inhibition studies, in vitro comple-mentation assays, and fluorescence and electronmicroscopy — strongly implicates γ-tubulin as the key pro-tein responsible for microtubule nucleation in vivo. Thishighly conserved protein is approximately 30% identical toα- and β-tubulins, but does not assemble into the bulkmicrotubule polymer. Although its activity is confined tothe MTOC, most γ-tubulin is present in the cytosol(reviewed in [9•]).

Cytosolic γ-tubulin is found in two main complexes(reviewed in [10•,11•,12]): the large γ-tubulin ring complex(γTuRC) and the γ-tubulin small complex (γTuSC), which isanalogous to the Tub4 complex of Saccharomyces cerevisiae.The γTuRC was first isolated from Xenopus eggs [13] andsubsequently from Drosophila embryos, along with its sub-unit, the γTuSC [14•]. The γTuRC consists ofapproximately 10–14 γ-tubulin molecules and at least sixadditional proteins, resulting in a complex of roughly2 MDa. Similar protein complexes exist in mammalian cells[15–17], indicating that the γTuRC is highly conserved.The γTuSC consists of two copies of γ-tubulin and one copyeach of Dgrip84 and Dgrip91 (Dgrip: Drosophila gammaring protein), which are related to each other, as well as tothe yeast Spc97 and Spc98 proteins, and the XenopusXgrip109 and Xgrip110 proteins (reviewed in [10•,11•,12]).

Electron microscopic images suggest that the Xenopus andDrosophila γTuRCs have a flexible, open-ring structureapproximately 25 nm in diameter [13,14•]. Individual sub-units visible within the ring walls have been proposed tobe γTuSCs [14•]. Centrosomes of Drosophila [18] and thesurf clam Spisula [19] contain similar ring structures, andthese rings contact the minus ends of microtubules andcontain γ-tubulin [20].

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

γγ-Tubulin complexes and microtubule nucleationMichelle Moritz*† and David A Agard‡

γγ-Tubulin complexes and microtubule nucleation Moritz and Agard 175

a template out of which the microtubule grows(Figure 1b). As microtubules inside cells usually contain13 protofilaments [1], the model proposed that the γTuRCcontains 13 laterally interacting γ-tubulins, each of whichcontacts one α- (or β-) tubulin longitudinally at the minusend of a protofilament.

A second, ‘protofilament’, model was proposed based onearlier observations of rings that form from pure α/β-tubulinor from its bacterial homolog, FtsZ [21]. In this model, theγ-tubulins in the γTuRC interact longitudinally with oneanother, in the same way that α- and β-tubulin or FtsZinteract in a protofilament or ring (Figure 1c). The γTuRCunwinds to form the first protofilament of the microtubuleand the γ-tubulins interact laterally with α- and β-tubulin,stabilizing a pair of protofilaments that could then seedfurther growth of the microtubule.

In the past year, four papers presented exciting new evidence regarding γ-tubulin-mediated microtubule nucle-ation. Three groups used fluorescence or electronmicroscopy to examine Xenopus or Drosophila γTuRCs on

their own or in complex with microtubules [22••–24••].The consensus of these three studies is that a templatemechanism, albeit modified from the original model, ismore consistent with the data. In the fourth paper, a bio-chemical study indicates that a single γ-tubulin is sufficientto nucleate microtubule assembly [25••]. A compellingargument has been made for how these new findings fitinto 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 γγTuRCsIn the initial characterizations of γTuRCs isolated from Xenopus [13] and Drosophila, the complexes wereexamined by negative-stain electron microscopy or cryo-electron microscopy and were found to be structurally verysimilar. However, these techniques yielded only relativelylow-resolution two-dimensional information. In a recentstudy [24••], electron microscopic tomography and plat-inum shadowing were used to gain insight into thethree-dimensional structure of Drosophila γTuRCs. Thetomography revealed that the subunits comprising the ring

Figure 1

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Current Opinion in Structural Biology

Models of microtubule nucleation. (a) A microtubule nucleatedspontaneously from pure α/β-tubulin. The microtubule is polar:α-tubulin is minus-end proximal and β-tubulin is plus-end proximal.Note the three-start helix and the ‘seam’. (b) The ‘template’ modelpredicts that the γ-tubulins of the γTuRC interact with each otherlaterally and contact α-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 γTuRC determines thenumber of protofilaments in the microtubule. (c) The ‘protofilament’model proposes that the γ-tubulins in the γTuRC interact with eachother longitudinally and with α/β-tubulins primarily laterally. The γTuRCunwinds to form the first protofilament of the microtubule, promotingformation of a small sheet that then grows into a microtubule.

176 Macromolecular assemblages

walls are arranged in pairs with a distinct ‘U’ or ‘V’ shape,with the pairs separating on one ring face and convergingon the other (Figure 2). A globular structure sits asymmet-rically atop the face of the ring where the subunit pairsmeet and does not extend very far into the ring lumen(Figure 2a–d). Platinum shadowing revealed a similarstructure that, in addition, nicely displayed the helicalnature of the complex (Figure 2e,f). It was not possible inthis study to determine unequivocally how many subunitsare in the ring walls, although preliminary data indicatethat there are approximately 12.

Although the definitive assignment of γTuRC 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 sixor seven γTuSCs make up the wall of the ring and that theγ-tubulins are positioned on the face of the ring away fromthe asymmetric cap. The cap is probably made up of theproteins Dgrips 163, 128 and 75s, which biochemistry hasshown to be of lower stoichiometry in the γTuRC [14•](Figure 2g). The position of the cap suggests that it may beinvolved in attachment of the γTuRC to the centrosome,regulation of γTuRC activity and/or stabilization of the ring.

Structure of γγTuRCs in complex withmicrotubulesA motivating assumption in the three recent structuralstudies was that the template and protofilament models

Figure 2

Structure of isolated Drosophila γTuRCs.(a–d) Selected views of a reconstructedγTuRC obtained by electron microscopictomography. In each image, several sectionsfrom the reconstruction were stacked into asingle volume. (a) View of the γTuRC facecontaining the asymmetric ‘cap’. (b) Middlesection of the γTuRC. 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 γTuRC. Note the invertedV-shaped ring wall subunits and the capstructure. The blue-dashed line outlines onepaired subunit, which is proposed to be oneγTuSC. (d) Alternative side view of the γTuRC.Bar = 10 nm. (e) Platinum replicas of γTuRCs.The helical structure and ring wall subunitsare apparent in the upper three and lower leftpanels. The lower right and middle panelsshow the ring face topped by the asymmetriccap. Bar = 10 nm. (f) Platinum replicas ofpure bovine-brain α/β-tubulin. Note that thestructures of the tubulin polymers are distinctfrom those of γTuRCs (compare withFigure 2e). (g) Model of the helical γTuRCstructure, 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 γTuSCsubunits (outlined in blue), each comprisingtwo γ-tubulins (pink) and one copy each ofDgrips 84 and 91 (green). Dgrips 163, 128and 75s (gray) are proposed to make up thecap. The right panel shows a tilted view of theimage shown in (c), showing the γTuRC 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

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should be distinguishable if γTuRC proteins, and γ-tubulinin particular, could be localized with respect to micro-tubule ends. The template model predicts that the γTuRCforms a cap at one end of the microtubule and that theγ-tubulins are confined to a narrow (12 nm) zone at thatend. In the protofilament model, the γTuRC either mightbe fully incorporated into the wall of the microtubule, andthus extend approximately 50 nm up the polymer wall, orit may be partially incorporated, with the remainder curl-ing away from the end. Several different approaches weretaken to distinguish these possibilities.

Keating and Borisy [22••], and Wiese and Zheng [23••]studied the position of some Xenopus γTuRC componentswith respect to microtubule ends in a similar manner. Inthe former study, gold labeling of γ-tubulin or Xgrip109 andnegative-stain or platinum-replica electron microscopywere used to localize these proteins at microtubule ends. Inthe latter study, the entire Xenopus γTuRC was biotinylatedand detected by streptavidin–gold conjugates using nega-tive-stain electron microscopy. In both studies, distanceswere measured between the ends of the microtubules andlarge numbers of gold particles. In both cases, the gold wasmainly confined to one end of the microtubule, in a zonemore consistent with the template model. It is unlikelythat these studies missed γ-tubulins in the wall of themicrotubule because, in the Keating study [22••], the anti-bodies were directly labeled with gold and were raisedagainst a C-terminal γ-tubulin peptide that is known to beaccessible in the native γTuRC and, in the Wiese study[23••], the entire γTuRC was biotinylated and detected bystreptavidin–gold conjugates (see also Update).

In addition, in all three structural studies, most γTuRC-nucleated microtubules exhibited a cap-like structure atone end and, in some cases, the γTuRC encircled the endof the microtubule (Figure 3), as expected if the γTuRCacts as a template. The cap structure was observed onmicrotubules nucleated from γTuRCs in Xenopus extracts,

from isolated Xenopus or Drosophila γTuRCs and pure tubu-lin, and from isolated Drosophila centrosomes. Structuressuch as rings or curled protofilaments projecting frommicrotubule ends, as would be expected if the γTuRC actsas a protofilament, were not observed [22••–24••]. It isunlikely that the observed cap-like or ring-like arrange-ment of the γTuRC at microtubule ends is an artifact ofelectron microscopy because of the different approachestaken and the different organisms used in these studies.

A new capping activity for the γγTuRCGiven the appearance of the γTuRC on microtubules inthe electron microscopic images, the complex might beexpected to cap the minus end of the microtubule, func-tionally inhibiting further growth at that end. Thispossibility was investigated by Wiese and Zheng [23••]using fluorescently labeled Xenopus γTuRCs and micro-tubules that were marked by nucleating in the presence ofa high ratio of rhodamine-labeled to unlabeled tubulin.The microtubules were then elongated with a ‘dim’ mix oftubulin, 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 dimends. As the minus end grows more slowly than the plusend, one dim end would usually be shorter than the other.It was found that the γTuRC prevents minus-end growthon the microtubules it nucleates, as well as on the pre-formed microtubules to which it binds. The complex canalso prevent minus-end depolymerization. Thus, thisstudy revealed that the γTuRC not only nucleates micro-tubules, but also has a separate capping activity that maybe very important for modulating minus-end dynamics.

The template model revisitedThe simplest model to explain the structural and functionaldata in these three studies would have most or all of theγ-tubulins in the γTuRC in direct, longitudinal contact withthe tubulin at the minus ends of microtubules (Figure 4), asthe original model proposed [13]. The original model must,

Figure 3

One end of Xenopus or DrosophilaγTuRC-nucleated microtubules displays a capor ring structure. (a) Electron microscopicimage of a negative-stained microtubulenucleated in a Xenopus egg extract(reproduced from [22•• ] with permission).(b) Negative-stained microtubule nucleatedfrom an isolated, biotinylated Xenopus γTuRC.One streptavidin–gold conjugate labels themicrotubule end (reproduced from [23•• ] withpermission). Bar = 20 nm. (c) Reconstructionfrom electron microscopic tomography of amicrotubule nucleated by an isolatedDrosophila γTuRC (reproduced from [24•• ]with permission). Compare the cap-likestructures visible in (a–c). Bar = 25 nm. (d) Reconstruction from electron microscopic tomography of a microtubule

nucleated by an isolated Drosophila γTuRC (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)


however, be modified to incorporate biochemical data sug-gesting that the γTuRC is assembled from preformedγTuSCs, which contain two copies of γ-tubulin and one copyeach of the homologs of the S. cerevisiae Spc97 and Spc98 pro-teins [14•]. This implies that the γTuRC must contain aneven number of γ-tubulins, not the 13 that were originallyproposed. It is therefore possible to hypothesize at least threelikely arrangements of the γ-tubulins within the complex.

If the γTuRC contains 14 γ-tubulins, two of them mayoverlap, maintaining 13-fold symmetry (Figure 4a)[22••,23••]. In this arrangement, all of the γ-tubulins inter-act with α-tubulins. In order to explain the original geneticdata suggesting that γ-tubulin interacts with β-tubulin,

Keating and Borisy [22••] presented an alternative arrange-ment in which the γTuRC helix is split on one side andoverlaps on the other, so that the γ-tubulins interact withboth α- and β-tubulins (Figure 4b). It is worth noting, how-ever, that the original genetic interactions were notallele-specific and, in fact, involve three separate regions ofβ-tubulin, calling into question the need to invoke a directinteraction between γ-tubulin and β-tubulin [9•].

If the complex contains 12 γ-tubulins, the γTuRC accessoryproteins may hold the γTuSCs in a three-start helix with13-fold symmetry, with one part of the symmetry definedby a gap in the ring (Figure 4c) [24••]. It is also possiblethat γTuRC-nucleated microtubules begin with 12 or 14

Figure 4

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Current Opinion in Structural Biology

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

permission from [24•• ]). The D/Xgrip subunits are proposed to hold theγ-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 γ-tubulin might nucleateand cap a microtubule (based on [25•• ]). The single, strong interactionbetween β-tubulin and γ-tubulin is proposed to stabilize the initial fewtubulin subunits in the polymer, facilitating subsequent elongation. Apossible nucleus is outlined in green. The ‘X’s indicate that furtheraddition of subunits cannot occur in these two directions, explaining theminus-end capping activity of the γ-tubulin. (e) An example of howbinding of the D/Xgrip proteins of the γTuSC (or Spc97/Spc98 of theyeast Tub4 complex) might shield β-tubulin-binding sites on γ-tubulin.


protofilaments and shift to 13 protofilaments further alongthe polymer [28]. In all models, the non-γTuSC compo-nents of the γTuRC are proposed to make up the cap,which may regulate activity, impart stability to the helixand/or attach the γTuRC to the centrosome.

Microtubule nucleation and capping bymonomeric γγ-tubulinAlthough the recent structural papers support the idea thatthe γTuRC acts as a template in which all γ-tubulin mol-ecules in the complex play a role in nucleation, a recentbiochemical study by Leguy et al. [25••] raises intriguingnew possibilities regarding the mechanism of nucleation.In this study, γ-tubulin was produced in a reticulocytelysate and the monomeric portion was partially purified ona sizing column. The effect of this γ-tubulin on micro-tubule nucleation was followed by turbidity — monitoringfor a decrease in the lag time of assembly and an increasein microtubule number. Remarkably, γ-tubulin concentra-tions of 0.6–0.8 nM were found to induce microtubulenucleation at low (12–17 µM) tubulin concentrations,decreasing the size of the nucleus from seven to threetubulin heterodimers. The binding stoichiometry was oneγ-tubulin per microtubule and this was sufficient to blockfurther growth from the minus end. The interactionbetween γ-tubulin and the microtubule was high affinity(1010 M–1). In a blot-overlay experiment, γ-tubulin boundmore strongly to β-tubulin than to α-tubulin.

These data suggest a model in which a single γ-tubulinforms a tight, lateral bond with a β-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 canexplain microtubule nucleation in the context of either thetemplate or the protofilament models. Within the templatemodel, one of the γ-tubulins in the γTuRC might act in thesame manner as monomeric γ-tubulin, binding laterally toβ-tubulin, and the additional γ-tubulins would interact withthe α-tubulins at the microtubule minus end. These lessstrong associations might be stabilized by the Spc97/Spc98homologs. This model is similar to the split-helix modeldescribed above (Figure 4b). Within the protofilamentmodel, the accessory proteins might strengthen the interac-tion of the key γ-tubulin with the protofilament.

ConclusionsIn the past year, several exciting advances in understandingmicrotubule nucleation by γ-tubulin complexes have beenmade at both structural and biochemical levels. It is now fair-ly certain that the γTuRC 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 γ-tubulins in the complex con-tact the minus end of the microtubule, it is tempting toassume that they are all directly involved in promotingnucleation through a template mechanism. However, the finding that a single γ-tubulin molecule is sufficient to

promote nucleation raises the possibility that only oneγ-tubulin in the complex is directly involved in nucleation;the others would have a ‘supporting’ role. For example, themechanism could involve a combination of nucleus assemblythrough a single γ–β lateral interaction and template forma-tion, and/or further complex stabilization through theremainder of the γ-tubulins. This could occur in a mannersimilar to that proposed in the split-helix model (Figure 4b).However, it is important to keep in mind that most, if not all,γ-tubulin inside cells is bound up in the γTuRC or the γTuSC.

If these complexes nucleate microtubules similarly tomonomeric γ-tubulin, one would expect the γTuSC to be apotent nucleator. In fact, the γTuSC is a very poor nucleator[14•]. This suggests that the binding of Dgrips 84 and 91(homologs of Spc97, Spc98 and Xgrips 109, 110) shieldsγ-tubulin from interacting with β-tubulin (Figure 4e). Asthe γTuRC is a much better nucleator than the γTuSC [14•],assembly of the γTuSC into the γTuRC must either exposecritical interaction sites required for nucleation, strengthenactivity by simply providing a greater number of interactionsites or form a special structure that promotes nucleus for-mation and microtubule growth through a uniquemechanism. It is possible that monomeric γ-tubulin is amore potent nucleator than the γTuSC and γTuRC, and thatcells have evolved a way of modulating this activity bysequestering γ-tubulin in these complexes. Thus, it couldbe that the nucleating activity of monomeric γ-tubulin doesnot reflect the mechanism used by the γTuSC and γTuRC.

The recent work on γ-tubulin, γTuRC and γTuSC has pro-vided new insights into the structure and activity of thesemicrotubule nucleators, but it has also raised new ques-tions. It is now very important to perform parallelcomparisons of the nucleating (and capping) activities ofpure γ-tubulin, γTuRC, γTuSC and perhaps even the yeastTub4 complex, as it forms a similar closed structure atmicrotubule minus ends [29,30] and therefore might act inthe same way. Other important experiments include deter-mining the number of subunits in the γTuRC and thenumber of protofilaments in microtubules nucleated bythem; localizing specific proteins within the complex anddetermining their function; and cross-linking or mutagen-esis experiments to explore further the proposed contactbetween γ-tubulin and β-tubulin (this last possibility isdescribed more fully by Erickson [26••]). Preliminaryexperiments are also beginning to indicate the regions ofinteraction between γ-, α- and β-tubulin [31•]. As many ofthe tools needed to carry out these experiments are avail-able, the prospects for obtaining a detailed, molecularunderstanding of microtubule nucleation by γ-tubulincomplexes in the near future are good.

UpdateIn a recent study, an additional component of the XenopusγTuRC, Xgrip210, was localized with respect to the micro-tubule minus end by labeling with Xgrip210 antibodiesthat were directly conjugated to gold particles [32].

Xgrip210 is a γTuRC component that is present at a lowerstoichiometry in the complex than γ-tubulin and theSpc97/Spc98 homologs (Xgrip109, Xgrip110). The distancesof the Xgrip210 gold particles from the microtubule endswere measured in the same manner as in the Keating andBorisy study [22••], and were found to occupy a micro-tubule-distal region of the γTuRC-capped end [32]. Inaddition, Xgrip210 was found to be required for γTuRCassembly and for the recruitment of γ-tubulin and Xgrip109to the centrosome. Similarly, Xgrip109 is required for thelocalization of Xgrip210 to the centrosome. These data sug-gest that Xgrip210 is a component of the γTuRC capstructure and is involved in attaching the γTuRC to the cen-trosome. This study also showed that the γTuSC can notattach to the centrosome on its own, supporting the idea thatthe attachment is made through the γTuRC 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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• of special interest••of outstanding interest

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22. Keating TJ, Borisy GG: Immunostructural evidence for the •• template mechanism of microtubule nucleation. Nat Cell Biol

2000, 2:352-357.The authors present the first evidence (with [23•• ]) that the Xenopus γTuRCstructurally caps the microtubule end and that γ-tubulin is confined to a nar-row zone at that end, as predicted by the template model.

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2:358-364. This paper (with [22•• ]) provides evidence that the γTuRC functionally capsthe minus ends of microtubules, preventing both further growth and depoly-merization at that end.

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γ-tubulin complexes and microtubule nucleation

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