295Journal of Cell Science 110, 295-300 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS4362

COMMENTARY

Centrosome-microtubule nucleation

Gislene Pereira and Elmar Schiebel*

Max-Planck Institut für Biochemie, Genzentrum, Am Klopferspitz 18a, 82152 Martinsried, Germany

*Author for correspondence

In many cell types the formation of microtubules fromtubulin subunits is initiated at defined nucleation sites atthe centrosome. These sites contain the conserved γ-tubulinwhich is in association with additional not very well char-acterised proteins, identified as components of a γ-tubulinring complex from Xenopus egg extracts or from suppres-sor screens in the yeast Saccharomyces cerevisiae. In thisreview we discuss two recently proposed models of how theγ-tubulin complex assists in the assembly of tubulin to formmicrotubules. These models propose different roles for γ-tubulin and the other proteins in the complex in tubulin

assembly. While the structure and composition of a micro-tubule nucleation site is becoming clearer, it is stillunknown how the cell-cycle dependent regulation of micro-tubule nucleation sites is achieved and whether they disas-semble after microtubule formation in order to allowmicrotubule fluxes towards the centrosome which havebeen observed in mitotic cells.

Key words: Microtubule nucleation, MTOC, γ-Tubulin, Spc98p,Tub4p

SUMMARY

INTRODUCTION

Microtubule (MT) nucleation is defined as the de novoformation of MTs from tubulin, a heterodimer of α- and β-tubulin. In vivo MT nucleation is initiated at defined sites ofthe morphologically distinct centrosomes, basal bodies, spindlepole bodies (SPB), and nucleus-associated bodies. To collec-tively define the MT nucleation activity associated with thesequite heterogeneous organelles, Pickett-Heaps (1969) coinedthe generic term microtubule organizing centre (MTOC). Therecent discovery that γ-tubulin is a universal component ofMTOCs involved in MT nucleation, suggests a molecular basisfor this common MT nucleation activity of MTOCs (Horio etal., 1991; Liu et al., 1994; Lopez et al., 1995; Marschall et al.,1996; Oakley et al., 1990; Oakley and Oakley, 1989; Sobel andSnyder, 1995; Spang et al., 1996; Stearns et al., 1991; Zhenget al., 1991). γ-Tubulin is part of a large complex which isprobably the functional unit of MTOCs that nucleates MTs(Raff et al., 1993; Zheng et al., 1995). Knowledge about thecomposition of this complex is just beginning to emerge(Zheng et al., 1995) and a protein that may directly interactwith a γ-tubulin was recently characterized at a molecular level(Geissler et al., 1996). Despite this progress, the assembly ofthe γ-tubulin-containing complex, its activation, the number ofnucleation sites at the MTOC and the function of the non-tubulin components in the complex are only understood at arudimentary level.

It is important to note that MTs are nucleated and organizedin the absence of distinct MTOCs in plant cells and in meiosisof most animals (Lambert and Lloyd, 1994). Such a centrosome-independent MT organization process into spindle-like struc-

tures has also been observed in Xenopus egg extracts after theaddition of artificial chromosomes (Heald et al., 1996). However,the physiological relevance of this assembly remains to beexplored. Although the underlying molecular mechanism of MTnucleation with or without a defined MTOC may be similar,MTOC-independent MT nucleation is not covered in this review.

MT NUCLEATION BY THE MTOC

Tubulin is a heterodimer composed of α- and β-tubulin that selfassembles to form hollow cylinders known as MTs. In MTs, alltubulin subunits have the same orientation, resulting in a polarstructure with one end (the plus end) that assembles tubulin ata higher rate than the other, the minus end (Bergen and Borisy,1980). Tubulin self-assembles in vitro when a critical concen-tration is exceeded, indicating that MTOCs are not absolutelyrequired for MT nucleation. However, MTOCs provide MTnucleation sites at which MT assembly is initiated below thecritical concentration (Mitchison and Kirschner, 1984). MTnucleation is followed by the self-assembly of tubulin onto thistubulin seed, resulting in a defined orientation of all MTs withina cell such that their minus-ends are in contact with the MTOC(McIntosh and Euteneuer, 1984). The MT nucleation site alsoinfluences the structure of MTs: MTs formed at a MTOCcontain 13 protofilaments, while MTs assembled in vitro consistof 12-16 protofilaments (Amos, 1982).

γ-TUBULIN IS A UNIVERSAL COMPONENT OFMTOCs THAT FUNCTIONS IN MT NUCLEATION

How the structurally diverse MTOCs fulfil their common

296 G. Pereira and E. Schiebel

microtubule organizing function is becoming clearer. A geneticapproach identified a new class of tubulin, named γ-tubulin, asa suppressor of a temperature sensitive β-tubulin mutation inthe fungus Aspergillus nidulans (Oakley and Oakley, 1989;Oakley et al., 1990; Weil et al., 1986). Since then γ-tubulins have been identified in many different organisms,including human, Schizosaccharomyces pombe, Drosophilamelanogaster, Xenopus laevis and plant cells (Horio et al.,1991; Liu et al., 1994; Lopez et al., 1995; Stearns et al., 1991;Sunkel et al., 1995; Zheng et al., 1991). These γ-tubulins revealsequence identities of about 65 to 70% among themselves,while their identities to the α- and β-tubulin families are in therange of 30% (Burns, 1995). γ-Tubulins also appear to beconserved at the functional level, since the human γ-tubulinfunctions in S. pombe (Horio and Oakley, 1994). A moredivergent γ-tubulin is that of Saccharomyces cerevisiae, namedTub4p, indicated by the moderate identity of about 40%compared to other γ-tubulins (Burns, 1995) and by the non-functionality of Xenopus γ-tubulin in S. cerevisiae (Spanget al., 1996).

γ-Tubulin’s function in MT nucleation can be shown by theMT defects seen in A. nidulans, S. pombe and S. cerevisiaeafter its depletion in these organisms (Horio et al., 1991;Oakley et al., 1990; Sobel and Snyder, 1995). The phenotypeof a conditional lethal tub4(ts) mutant strain of S. cerevisiae isalso consistent with a function of Tub4p in MT nucleation.tub4(ts) cells incubated at the non-permissive temperatureduplicated the SPB, but the newly formed SPB failed tonucleate MTs (Marschall et al., 1996). Further evidence hasbeen provided by the microinjection of anti-γ-tubulin anti-bodies into mammalian cells. This prevented MT nucleation bythe centrosome throughout the cell cycle (Joshi et al., 1992).In addition, a purified γ-tubulin-containing ring complexnucleated MTs in vitro (Zheng et al., 1995). While these resultsstrongly support a direct role of γ-tubulin in MT nucleation, γ-tubulin-independent MT nucleation cannot be excluded. Fur-thermore, γ-tubulin may have additional functions. Forexample, depletion of γ-tubulin in Drosophila neuroblastsresults not only in a reduced number of MTs but also in highlyabnormal centrosomes, suggesting that it may have a structuralrole in MTOCs (Sunkel et al., 1995).

LOCALIZATION OF γ-TUBULIN WITHSUBSTRUCTURES OF MTOCs

The MTOC of mammalian cells is the centrosome, whichcomprises a pair of centrioles surrounded by the pericentriolarmaterial. The centrioles consist of nine triplet microtubuleswhich are arranged in cylindrical form. The pericentrolarmaterial is the protein mass that is responsible for MT nu-cleation (Gould and Borisy, 1977). In agreement with itsmicrotubule nucleation activity, Stearns et al. (1991) localizedγ-tubulin to the pericentriolar region of mammalian centro-somes. In these studies no γ-tubulin staining of the centrioleMTs was observed. However, the authors pointed out that theanti-γ-tubulin antibody may not have accessed the inside of thecentrosome. Further ultrastructural immunolocalization studiesusing isolated human centrosomes localized γ-tubulin to thepercentriolar material and in addition on the centrioles andwithin the lumen of the centrioles (Moudjou et al., 1996). The

centriolar γ-tubulin may function in the nucleation and stabil-isation of the centriolar MTs (Moudjou et al., 1996) whichwould fit well with the structural role of γ-tubulin reported forDrosophila neuroblast centrosomes (Sunkel et al., 1995).Three-dimensional structural characterization of centrosomesfrom early Drosophila embryos revealed numerous ring-likestructures with a similar diameter to microtubules at all levelsin the pericentriolar material (Moritz et al., 1995a). Since theserings contain γ-tubulin (Moritz et al., 1995b), they most likelyrepresent ΜΤ nucleation sites associated with centrosomes.

The SPB of S. cerevisiae does not contain centrioles; insteadit consists of three plaque structures of which functions havebeen deduced by electron microscopy (Byers, 1981; Byers andGoetsch, 1975). The outer and inner plaques organize the cyto-plasmic and nuclear microtubules, while the central plaqueanchors the SPB in the nuclear membrane. SPB componentsinvolved in MT nucleation are therefore expected to be asso-ciated with the outer and inner plaques. In agreement with thisconsideration, yeast γ-tubulin, Tub4p has been localized to theouter and inner plaques (Spang et al., 1996).

γ-TUBULIN IS PART OF A LARGER COMPLEX

Soon after the discovery of γ-tubulin, it became apparent thatγ-tubulin is part of a larger complex which may be the func-tional unit in MT nucleation. γ-Tubulin was found in a multi-protein complex in Drosophila embryos, containing at leasttwo centrosomal MT-associated proteins now called CP190and CP60 (Raff et al., 1993). This γ-tubulin complex wascapable of MT binding. It is important to note that unlikeCP190 and CP60, only 10-50% of the γ-tubulin bound to MTs,suggesting that γ-tubulin is also present in other forms inDrosophila embryos. The understanding of the function ofCP60 and CP190 in association with γ-tubulin is complicatedby the cell-cycle dependent centrosomal localization of CP60and CP190. The zinc-finger protein CP190 was associated withcentrosomes during mitosis and relocated to the chromatinduring interphase (Whitfield et al., 1995). CP60 was detectedat the centrosome maximally during anaphase and telophase,and then at lower levels during late telophase or early inter-phase (Kellogg et al., 1995).

γ-Tubulin is present as a large 25 S complex in the cytoplasmof frog eggs and vertebrate somatic cells. This complex andATP are required for the formation of centrosomes aroundsperm centrioles in frog egg extracts (Stearns and Kirschner,1994). The 25 S γ-tubulin complex has been purified frommitotic frog eggs. It contains at least seven proteins includingα-, β- and γ-tubulin. The additional proteins have apparentmolecular masses of 195, 133, 109 and 75 kDa. The γ-tubulincomplex is seen in the electron microscope as an open ringstructure with a diameter of 25-28 nm (Zheng et al., 1995).That γ-tubulin is also organized into ring-like structures in situis supported by immunoelectron microscopic tomographystudies using isolated Drosophila centrosomes (Moritz et al.,1995a,b). These γ-tubulin-containing clusters were identifiednear the minus end of MTs after MT nucleation by the purifiedcentrosomes.

The more divergent γ-tubulin Tub4p of S. cerevisiae is partof an oligomeric complex containing the SPB componentSpc98p (Geissler et al., 1996) and at least one additional SPB

297Centrosome-microtubule nucleation

protein (Knop et al., unpublished). SPC98 has been cloned asa dosage-dependent suppressor of temperature sensitive tub4-1cells. It encodes the 90 kDa SPB component, previously iden-tified by a monoclonal antibody approach (Rout and Kilmartin,1990). The following experiments showed that both proteinsare found in the same complex and that they function together:temperature sensitive tub4 and spc98 alleles showed a synthetictoxic defect, Spc98p interacted with Tub4p in the two-hybridsystem (Fields and Song, 1989) and Tub4p co-immunoprecip-itated with Spc98p. By analyzing temperature sensitivemutants of SPC98 it could be determined that Spc98p alsofunctions in MT organization. Consistent with this microtubuleorganizing function, Spc98p was associated with the outer andinner plaques, the SPB substructures that organize the cyto-plasmic and nuclear microtubules (Rout and Kilmartin, 1990).The existence of a γ-tubulin ring complex in Drosophila cen-trosomes (Moritz et al., 1995b) raises the question of whethera similar structure exists in the yeast SPB. Conventionalelectron microscopy of MT ends which are in contact with theSPB revealed that these ends are sealed by a cap-like terminalcomponent connecting the walls of the microtubule cylinder(Byers et al., 1978). Noteworthily, this sealed cap is somewhatdifferent in structure compared to the isolated γ-tubulin ringcomplex of Xenopus. It is tempting to speculate that theTub4p/Spc98p complex is part of the cap-like terminalcomponent which may function as a MT nucleation unit at theSPB.

NUMBER AND ACTIVATION OF MT NUCLEATIONSITES

That MTOCs contain a defined number of nucleation siteswas first reported by Brinkley et al. (1981), who showed thatthe number of MTs organized by the centrosome of perme-abilized mammalian cells reached saturation after theaddition of excess tubulin. However, most cells have the capa-bility to regulate the number of active MT nucleation sites atthe MTOC as well as to assemble new sites after the cell-cycle dependent duplication of the MTOC. For the assemblyof new sites the cytosolic γ-tubulin complexes may functionas a storage form which binds to the newly formed centro-some to become an active MT nucleation site. A number ofobservations support this hypothesis. Firstly, the γ-tubulinring structures within purified Drosophila centrosomes aresimilar in appearance to the γ-tubulin complex from Xenopuseggs (Moritz et al., 1995b; Zheng et al., 1995). Secondly,Moudjou et al. (1996) estimated a minimum figure of 80% oftotal human γ-tubulin that exists as a cytosolic form, most ofit present in large complexes. Finally, the Tub4p/Spc98pcomplex is already formed in the cytoplasm of yeast cells,followed by its binding to the inner and outer plaques of theSPB (G. Pereira, unpublished). Since, in many cell types,microtubule nucleation is restricted to the MTOC the γ-tubulin complex must become activated for microtubulenucleation after binding to the MTOC. Activation mayinclude binding of the complex to a docking protein at thecentrosome or post-translational modification of one of theproteins in the complex after the binding step.

The number of MTs at mammalian centrosomes increasedfivefold at the onset of mitosis (Kuriyama and Borisy, 1981).

This may be achieved either by the assembly of new nucleationsites (see above) or activation of existing sites. Evidence for acell-cycle dependent activation of MT nucleation sites comesfrom S. pombe. The S. pombe SPB nucleates MTs only atmitosis (Hagan and Hyams, 1988). Remarkably, cell-cycledependent MT nucleation seems not to be regulated by bindingand release of γ-tubulin to the SPB, taking into considerationthe fact that S. pombe γ-tubulin is also associated with the inter-phase SPB in the absence of MTs (Horio et al., 1991). Mostinterestingly, the nucleation activity of S. pombe SPBs wasactivated in vitro by cytostatic factor-arrested Xenopus eggextracts containing high Cdc2-kinase activity (Masuda et al.,1992). The nucleation-competent SPBs were then recognisedby the MPM-2 antibody, which is directed against mitosisspecific phosphoepitopes (Davis et al., 1983). However,purified Cdc2 kinase/cyclin B complex in itself was unable toactivate MT nucleation. Therefore, a protein regulated by Cdc2kinase was most likely responsible for the activation processby the phosphorylation of a protein in the γ-tubulin complex,thereby activating existing nucleation sites.

In contrast to S. pombe, the S. cerevisiae SPB is associatedwith a relatively constant number of MTs throughout the cellcycle. In mitosis, each of the 16 centromeres of a haploid cellis in contact with one MT and eight MTs connect both poles(Winey et al., 1995). An increase in the chromosome numberduring karyogamy of haploid yeast cells or endomitosis incdc31 mutants (Schild et al., 1981) results in an increasingnumber of MTs per nucleus and hence faithful segregation ofthe chromosomes in mitosis. This is accompanied by adoubling in the size of the SPB (Byers, 1981), keeping thenumber of MTs per area of inner plaque approximatelyconstant. Since MT ends appear densely packed along thesurface of the disc-like inner plaque (Byers and Goetsch,1975), the number of nuclear MTs in S. cerevisiae may simplyreflect the ratio of the area of the inner plaque to that of a nucle-ation site. Alternatively, the number of docking sites for theTub4p complex may correlate with the area of the inner plaque.

MT NUCLEATION BY THE γ-TUBULIN COMPLEX

The finding that γ-tubulin is associated with MTOCs in diverseorganisms and is involved in MT nucleation led Oakley topropose that a ring of γ-tubulin molecules at the MTOCfunctions as a template in MT organization (Oakley, 1992;Oakley et al., 1990). Based on the structure of the isolated γ-tubulin complex, this model was extended by Mitchison andcolleagues (Zheng et al., 1995). They suggested that the non-tubulin components of the complex form a scaffold on whichthe 13 molecules of γ-tubulin are arranged (Fig. 1A). Themodel by Oakley and Mitchison implies that γ-tubulin directlyinteracts with the tubulin subunit which is at the minus end ofMTs. Whether this is α- or β-tubulin has been a controversialquestion. Experiments by Mitchison suggested that β-tubulinis at the plus end of MTs (Mitchison, 1993). This was deducedfrom the binding of beads coated with GTP to the plus end ofMTs. Since β- but not α-tubulin has an exchangeable GTP(Kirchner and Mandelkow, 1985), it was concluded that thepolarity of MTs is such that β-tubulin is at the plus and α-tubulin at the minus end. In contrast, Song and Mandelkow(1995) identified the β-tubulin subunit at the minus end basedon its preferred interaction with the head domain of the motor

298 G. Pereira and E. Schiebel

A

B

Nucleation

Nucleation

Poly-merization

Poly-merization

MTOC

MTOC

MTOC

MTOC MTOC

MTOC

Fig. 1. Models for the nucleation of MTs by the γ-tubulin ring complex. (A). According to Oakley(Oakley, 1992; Oakley et al., 1990) and Mitchisonand colleagues (Zheng et al., 1995). The proteinsin the γ-tubulin complex provide a scaffold (greenspiral) on which 13 γ-tubulin subunits (red circles)are aligned. α-Tubulin (yellow circles) interactsdirectly with γ-tubulin, thereby determining thepolarity of MTs. β-Tubulin is shown as bluecircles. At least one of the scaffold proteinsinteracts with other components of the MTOC.(B). In the model proposed by Erickson andStoffler (1996) γ-tubulin forms a protofilamentterminating in a helical structure. The polarity ofthe γ-tubulin helix (red circles) may be determinedby accessory proteins (green, brown, pink and bluerectangles) which may also provide contact pointswith other proteins of the MTOC. γ-Tubulininteracts with α- (yellow circles) and β-tubulin(blue circles). The γ-tubulin protofilamentfunctions as stable seeds upon which additionaltubulin subunits assemble. The extended MT sheetis closed to a cylinder as soon as it contains 13protofilaments.

protein kinesin. Re-evaluation of their data (Hirose et al., 1995)confirmed the previous conclusion by Mitchison. Finally, aphage display antibody specific to α-tubulin labelled the minusend of MTs (Fan et al., 1996), providing further evidence forthe localization of α-tubulin at the minus end. For the Oakleyand Mitchison model, it means that γ-tubulin directly interactswith α-tubulin. This was surprising, since the identification ofthe Aspergillus γ-tubulin gene as a suppressor of a β-tubulinmutant (Weil et al., 1986) has been used as an argument for thebinding of γ- to β-tubulin. However, suppression of a mutationin β-tubulin by γ-tubulin via the interaction with α-tubulinseems easily possible, considering that the functional unit oftubulin is the heterodimer. It is worth mentioning that no con-vincing evidence for a direct interaction between γ-tubulin andα- or β-tubulin has hitherto been provided. Even the bindingof radiolabelled γ-tubulin to the minus-end of MTs (Li andJoshi, 1995) does not prove a direct interaction, since the γ-tubulin has not been purified.

An alternative model for γ-tubulin’s function has beenproposed by Erickson and Stoffler (1996). They studied thestructure of rings formed by FtsZ, a prokaryotic homologueof tubulin (Erickson, 1995), and pointed out that FtsZ ringsand the γ-tubulin complex are very similar in diameter (Zhenget al., 1995). Interestingly, α- and β-tubulin also form ringstructures which are continuous with the protofilaments ofMT walls. These observations led Erickson and Stoffler topropose that γ-tubulin rings are curved protofilaments. Theyfurther suggest that the γ-tubulin protofilament provides theseed for nucleating the MT wall, meaning that γ-tubulininteracts with both α- and β-tubulin at this point (Fig. 1B).This model coincides with studies of early tubulin assembly,showing that the crucial event in MT nucleation is theformation of a stable seven-subunit tubulin nucleus. The latter

probably contains two tubulin protofilaments, one of four andanother of three dimers (Voter and Erickson, 1984). Since thefirst protofilament disassembles easily before the secondforms, a stable γ-tubulin protofilament would greatly enhanceformation of a microtubule sheet. The extended MT sheet isthen closed to a cylinder as soon as it contains 13 protofila-ments.

Besides structural differences and the interaction of γ-tubulinwith α- or β-tubulin, both models explain essential aspects ofMT nucleation quite differently. In the Oakley and Mitchisonmodel (Fig. 1A), γ-tubulin fulfils an adaptor role for tubulinbinding. The important components for defining the lattice arethe non-tubulin proteins in the complex. MT polarity is deter-mined by the preferred interaction of γ- with α-tubulin. Finally,the number of protofilaments per MT is determined by the γ-tubulin ring complex containing this number of γ-tubulinsubunits (Fig. 1A). In Erickson and Stoffler’s model (Fig. 1B)γ-tubulin forms a stable protofilament which favours theformation of a tubulin sheet. Microtubule polarity is a directconsequence of the polarity of the γ-tubulin protofilamentwhich may result from the binding of accessory proteins. It ismore difficult to imagine how the number of protofilaments isdetermined by this model.

How can we discriminate which model reflects the in vivosituation? In the model by Erickson and Stoffler, the axis ofthe γ-tubulin ring structure is perpendicular to MTs and notparallel as suggested by Oakley and Mitchison. Unfortunately,the limitation of the resolution of the γ-tubulin complex in thecentrosome (Moritz et al., 1995b) or associated with MT ends(Zheng et al., 1995) does not yet allow a differentation betweenboth models. In addition, crosslinking experiments will showwhether γ-tubulin only interacts with α-tubulin or both tubulinsubunits.

299Centrosome-microtubule nucleation

γ-TUBULIN AND MT DYNAMICS

MTs are dynamic polymers in mitosis: tubulin polymerizes atthe plus and depolymerizes at the minus end (Mitchison, 1989;Sawin and Mitchison, 1994). Since the γ-tubulin complex sta-bilizes the minus end of MTs, this dynamic behaviour raisesthe question of how MT depolymerization occurs. Possibly, theγ-tubulin complex may rearrange after MT nucleation,allowing the depolymerization of tubulin. For instance, the S.cerevisiae γ-tubulin mutant tub4-1 does not have a defect inMT nucleation but rather in mitotic spindle formation (Spanget al., 1996), which may reflect a deficiency of the tub4-1mutant to restructure the γ-tubulin complex. Alternatively, theTub4p complex may also be required for the attachment ofMTs to the yeast SPB. Mitotic specific pulling forces(McIntosh and Koonce, 1989) could therefore result in detach-ment of MTs assuming that their attachment to the SPB isweakened in the tub4-1 mutant. As discussed by Erickson andStoffler (1996), a further argument for a function of γ-tubulinin MT dynamics arises from the phenotype of the β-tubulinmutation benA33 in Aspergillus. MTs of this mutant are hyper-stable and this defect is either suppressed by MT depolymer-ization drugs (Oakley and Morris, 1981) or by a mutation inthe γ-tubulin gene (Weil et al., 1986).

In the extreme case, MTs may be released from the nucle-ation complex, followed by their recapture at other sites at theMTOC. A total release of nucleated MTs from the centrosomehas been observed in neuronal cells (Ahmad and Baas, 1995)and grasshopper spermatocytes (Ault and Nicklas, 1989),showing that the detachment of MTs from the centrosome ispossible. The nature of the putative capture site is quite specu-lative. Motor proteins associated with MTOCs (Blangy et al.,1995; Meluh and Rose, 1990; Page et al., 1994), or centroso-mal proteins which have been implicated in MT nucleation(Moudjou et al., 1991) may play a role here.

CONCLUSION

MT nucleation is one of the principle functions of centrosomes.For decades, the molecular mechanism of this process wasobscure. However, a recently discovered γ-tubulin ringcomplex, which may function as the MT nucleation unit at thecentrosome, provides the opportunity for understanding thiscentral aspect in cell biology at the molecular level. Moreover,it illustrates the fundamental importance of the centrosome,which has received too little attention until now. The centro-some may have additional, essential functions, for example, incell cycle regulation; Cdc16p and Cdc27p, components of theanaphase promoting complex, have been localized to the cen-trosome (King et al., 1995; Tugendreich et al., 1995). Consis-tent with this role in cell cycle regulation is also the associa-tion of the cell cycle regulators Cdc2 (Bailly et al., 1989),cyclin B1 (Bailly et al., 1992) and cyclin B2 (Gallant and Nigg,1992) with centrosomes.

We thank Drs Michael Bornens and Bill Wickner for reading themanuscript and helpful discussions. Drs Erickson and Stoffler areacknowledged for communicating results prior to publication. Ourwork is supported by grants from the DFG and the BMBF. G.P. issupported by a fellowship from the DAAD.

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