Journal of Cell Science n igt uete all 1 Teixido them Neus – rule nucleation to microtubule of ring how one and when where, The Commentary irtblsaehlo yidia oyesta r assembled are of composed that heterodimers from polymers cylindrical hollow are Microtubules Introduction words: Key Human nucleators. microtubule microtubules efficient new as of function nucleation complexes of The formation arrays. the these of over understood. which organization control in the Spatio-temporal way to arrays. central the ordered are highly properties into their of arrangement requires regulation their and on microtubules depends new microtubules of function The Summary 10.1242/jcs.106971 doi: 4445–4456 125, ß Science Cell of Journal ( correspondence for *Author 07.Ec yeo TChsasz,saeaddsrbto that distribution and shape size, a has MTOC Lu of with 2006; type associated Each Gundersen, 2007). of additionally and is (Bartolini (PCM) pair activity sites on other nucleation material Depending central type, 2012). pericentriolar cell Bornens, a the a 2007; the Bornens, centrosome, comprises and the by (Azimzadeh that is surrounded cells structure animal centrioles spherical in MTOC small main The (Lu 2007). (MTOCs) centers microtubule-organizing and initiated. when is microtubules where, new determines of polymerization which how regulation nucleation, the is microtubule strategy second of The 2010). McNally, and Roll- 2011; Mecak Akhmanova, and (Jiang disassembly and as severing well their their as bundling, controlling and by sliding transport, microtubules stabilization, elongation, existing of strategy first regulation The involves strategies. complementary two using This achieved geometry. defined is with arrays in organized of be to the microtubules for requirement segregation the share 2008). processes microtubule-dependent Heald, All and the Walczak 2011; Hoogenraad, Marshall, and and as Kapitein mediate (Ishikawa 2011; serve cilia motile and and flagella divisions, organelles, of blocks mitotic building and or meiotic during chromosomes molecules transport the for of tracks provide and Microtubules (Jiang 2011). dynamic highly Akhmanova, is end plus the whereas stable, relatively an with and microtubules with provides polarity, dimers intrinsic tubulin the of orientation unrelated additional c euainof regulation euaoyrlso the of roles regulatory irtbl ulainadorganization. and nucleation microtubule eladDvlpetlBooyProgramme, Biology Developmental and Cell tblncmlxcmoet GP)2 ,4 n ,wihaemmeso osre rti aiy eetwr a identified has work Recent family. protein conserved a of members are which 6, and 5 4, 3, 2, (GCPs) components complex -tubulin 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. irtbl ulaini yial ptal etitdto restricted spatially typically is nucleation Microtubule b tblntes-aldpu n.I iotemnsedis end minus the vivo In end. plus so-called the -tubulin c tbln nesnilpoenta sebe nomlisbntcmlxsadi on nalekroi raim.However, organisms. eukaryotic all in found is and complexes multi-subunit into assembles that protein essential an -tubulin, irtbl raiain irtbl nucleation, Microtubule organization, Microtubule c c tblnhsbe on ncmlxso aiu ie u eea ie feiec ugs htol ag,ring-shaped large, only that suggest evidence of lines several but sizes various of complexes in found been has -tubulin uCdpnetmcouuenceto sakymcaimo irtbl raiain pcfcly efcso the on focus we Specifically, organization. microtubule of mechanism key a as nucleation microtubule TuRC-dependent -Travesa ´ c tblncmlxsaergltdadhwti fet ulainad oetal,mcouuebhvo,i poorly is behavior, microtubule potentially, and, nucleation affects this how and regulated are complexes -tubulin c uCsbnt,a ela ag ubro oetransient more of number large a as well as subunits, TuRC [email protected] c a uCsbnt n neatr n rsn noeve fohrmcaim htregulate that mechanisms other of overview an present and interactors and subunits TuRC tblnfcn h ocle iu end minus so-called the facing -tubulin 1,2 onRoig Joan , a -and b tbln h longitudinal The -tubulin. 2 oeua eiiePorme nttt o eerhi imdcn IB,008Breoa Spain Barcelona, 08028 (IRB), Biomedicine in Research for Institute Programme, Medicine Molecular ) 2 n esLu Jens and dr n Stearns, and ¨ders dr n Stearns, and ¨ders c tblnrn ope,MO,Cnrsm,Spindle Centrosome, MTOC, complex, ring -tubulin ders ¨ c tblnrn opee ( complexes ring -tubulin 1, * iiso tblzsasalmcouuese omdfo multiple from formed seed microtubule but a small that a vitro, nucleator stabilizes a or requires in mimics process this spontaneously conditions physiological occurs under polymerization Microtubule h sebyo ihyodrdmcouueary n discuss and arrays microtubule ordered for highly the strategy of regulatory assembly fundamental the a as nucleation lattice microtubule microtubule 2005). the Chang, and onto Zimmerman 2006; proteins al., regulatory et (Cuschieri 1985) loading al., et by (Evans microtubule or the of structure the imposing on by constraints example, the for microtubules, modulate nucleated to the able of be properties also might (Lu sites structures Nucleation cellular 2007). other the Stearns, to sites and in nucleation is inactivation of differentiation centrosome transfer help cell gradual whereas with that coordinated poles, centrosomes frequently spindle the more-active point of larger organization time transition generate G2-M specific the and/or to at a assembled are additional assembly or sites example, nucleation For centrosomal site progression program. differentiation cellular cycle nucleation a during cell of to regulation activation the coupling microtubule of type particular a of array. organization the for suitable is h anmcouuencetr–the – nucleator microtubule main and The known organization. microtubule of affects framework overview regulatory an this how provide modulate that will mechanisms potential We to key regulation. the as this polymerization, microtubule nucleates that complex -tubulin– nti omnay ewl ihih h oto over control the highlight will we Commentary, this In by achieved is nucleation microtubule of control Temporal c tblnrn ope ( complex ring -tubulin b c tblnhtrdmr.Awl-nw microtubule well-known A heterodimers. -tubulin uCitrcos nti omnay edsusthe discuss we Commentary, this In interactors. 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Fig. In function. their for required is nucleator 4446 eetdt togyspottes-aldtmlt nucleation template so-called the support of strongly addition data the Recent stabilizing promoting ends. by plus or the example at for subunits fragments a process, microtubule have microtubule assembly or nucleators the short true in as later function role proteins show these will research all Future 2004). whether Vernos, and and (Clarke Gruss TPX2 2008; as Zhang, during such factors, and, RanGTP-activated microtubule 2009) for Rogers, several mitosis, and proteins, (Rusan family proteins (TACC) plus-end-binding coil coiled acidic (Bouissou dynamics microtubule-plus-end 2009). of al., and et 2000) the modulation Zheng, capping and the Wiese by 2011; in 1999; Sawin, stabilization and al., microtubule (Anders ends et have in minus complexes Oegema implicated these 1998; nucleation, been 1998; to al., addition also al., In et 1995). et al., Murphy Moritz et 2001; Zheng 1995; al., al., et et Murphy (Moritz nucleators into microtubule assembles tubulin nipratqeto shwthe how is question important An transforming for described been also has activity Nucleation ora fCl cec 2 (19) 125 Science Cell of Journal c tbln hc oaie oalkonMOsadis and MTOCs known all to localizes which -tubulin, c tblni the in -tubulin c uC,wihaetemi cellular main the are which TuRCs, Drosophila c c uCrn tutr.Nceto fmcouueplmrzto novslniuia neatosof interactions longitudinal involves polymerization microtubule of Nucleation structure. ring TuRC uC(epaenceto model). nucleation (template TuRC c uCncetsmicrotubules. nucleates TuRC , Xenopus c tbln(blue). -tubulin n humans and c uCassembly. TuRC c uClk opee r sebe yrpaeeto C2ado C3wt GCP4, with GCP3 and/or GCP2 of replacement by assembled are complexes TuSC-like c - ( al okhssgetdta,aatfrom apart that, suggested has work Early oeua opsto of composition Molecular nucleation. microtubule of of al., regulation involvement the the et about known (Kollman is discuss what review briefly on focus only recent will excellent we an Here, 2011). in of covered aspects been structural other between have and models contacts Nucleation longitudinal tubulin. through for platform heterodimers assembly an of provides thereby arrangement and helical the the in that molecules proposes which model, oesbnt,termed subunits, core Fg A Gilte l,21) oercn tde aeidentified have studies More-recent 2011). al., domains’ et ‘Grip (Guillet C-terminal 1A) will and (Fig. N- we as structure, regions crystal conserved the GCP4 to the refer from obtained the insight on of and, motifs basis these beyond extends similarity sequence the but described initially were 2001). 2–6 as al., GCPs protein et in Murphy conserved sequences 2000; conserved a al., Highly et to (Gunawardane belong 1A) humans), (Fig. family in TUBGCPs as known A B naineto ua C2 C3 C4 C5adGP sn the using GCP6 and GCP5 GCP4, GCP3, GCP2, human of alignment An ) c pcltv oe of model Speculative ) tblnrn rti Gi)mtf Gnwraee l,2000), al., et (Gunawardane motifs (Grip) protein ring -tubulin c uCmthstesmer famicrotubule a of symmetry the matches TuRC c uCassembly. TuRC c tblncmlxpoen GP,also (GCPs, proteins complex -tubulin c TuRCs c uC r opsdo GCP2 of composed are TuSCs c uCsrcueadwill and structure TuRC c c tbln l complexes All -tubulin. tbln all -tubulin, a a c -tubulin– -tubulin– uCsbnt in subunits TuRC c a define can e b -and b c -tubulin c -tubulin -tubulin c TuRCs TuRC eof a - Journal of Cell Science eeane l,21;Hrbc ta. 04 mm ta. 08 ay ta. 09 le ta. 00 pemn ta. 09 ibl ta. 01 Rik 2011; al., et Rigbolt 2009; al., 2008). et al., Oppermann et 2010; Dephou al., Wang 2008; et 2009; al., Olsen et al., 2009; Daub et al., 2004; Hoof et al., Mayya Van et 2008; 2005; (Beausoleil al., asterisk al., et an t et Imami by in 2004; Rush referenced marked al., studies 2007; are et in vivo Hornbeck identified in 2011; been identified al., have been et that have Hegemann sites that phosphorylation Sites of indicated. positions are The colored. analyses proteomic are motifs sequence and domains mass, molecular htaesmlrt h C aiymmes ofatoaewith co-fractionate members, family GCP the c to similar ‘ are with as co-purify that that define proteins We all 2010). Lu components’ al., 2010; al., et et Hutchins Teixido-Travesa 2006; 2006; al., Gunawardane et 2010; Haren al., 2003; et al., (Choi 1) et Table 2; (Fig. members family additional i.2. Fig. Ve 2008; al., et (Izumi roles structural important c of Depletion Grip-GCPs soitdpoen ilb osdrditrcos hc might which conditions. interactors, considered to be bind will other proteins All Grip- ‘Grip-GCPs’. associated the to as and 2–6 ‘GCPs’ as GCPs general domain-containing in subunits core to refer will We uC nscoegainsadclclz with colocalize and gradients sucrose in TuRCs uCi urs rdet,sgetn htte l have all they that suggesting gradients, sucrose in TuRC c uCcr opnnsdmisadpopoyainsites. phosphorylation and domains components core TuRC c uC estgtyo idol ne eti cellular certain under only bind or tightly less TuRCs c uCsbnt htaentrltdt hs ieGCP five these to related not are that subunits TuRC c tblno n fteGi-Csdestabilizes Grip-GCPs the of any or -tubulin c uC tamounts at TuRCs c tblni cells. in -tubulin ´ olte al., et rollet c uCcore TuRC dr tal., et ¨ders c TuRC- Human c tblnadGP r hw nshmtcdarm htaesae rmtheir from scaled are that diagrams schematic in shown are GCPs and -tubulin 06 ote l,20;XogadOke,20;Zage al., et Zhang so-called the 2009; subcomplexes, Oakley, Y-shaped and Xiong 2006; 2000). al., et Vogt 2006; fsrcua oe.Ide,too hm C-D(lokonas known (also GCP-WD them, instead of regulatory two have Indeed, might roles. and structural 1) of Table 2; (Fig. Grip-GCPs to Human GCPs Other Cs(i.1) n htGP,GP n C6mgtb part be Grip- might all GCP6 to and GCP5 common GCP4, the is that of 1). that and Box 1A), core five (Fig. the 1B; structural GCPs in (Fig. a regions GCP3 conserved form the and Grip-GCPs that GCP2 suggested has of work each Recent molecule one and ( ntaoso emntr Fg B Gilte l,21;Kollman assembly 2011; ring al., et as 2011). (Guillet al., example, 1B) et (Fig. for terminators function, or initiators to positions specific c uC) hc r opsdo w oeue of molecules two of composed are which TuSCs), c c uCrn tutr ysbtttn o C2o C3at GCP3 or GCP2 for substituting by structure ring TuRC c uC r omdb h eia ragmn fsmaller of arrangement helical the by formed are TuRCs uC oti eea oesbnt htaentrelated not are that subunits core several contain TuRCs c TR euain4447 regulation -TuRC c tblnsalcomplexes small -tubulin etx n from and text he ee l,2008; al., et re c v tal., et ova -tubulin Journal of Cell Science MZT1 MZT2A NEDD1 TUBGCP6 ED)adGP as nw sMZR2,hv been have MOZART2), for as non-essential known be to (also shown GCP8 and NEDD1) TUBGCP5 symbol gene Official 4448 TUBGCP4 TUBGCP3 TUBGCP2 TUBG1 C-Dmdae t lgmrzto n idn othe to binding and oligomerization 2003; its al., Lu mediates et GCP-WD 2006; (Gunawardane al., fungi in et not found Haren but is plants It and progression. animals and in assembly spindle meiotic and mitotic Lu 2006; al., 2010). al., et et Haren Travesa 2003; al., et hog h ietitrcinwith interaction direct the through C8seiial otiue to contributes specifically GCP8 2010). al., alga in green et unicellular and (Choi Teixido-Travesa the in 2010; motifs found al., also sequence are et Homologs or Hutchins domains 2010; known al., et any contain not does 2009). al., et Zeng the 2010; al., al., target et Lu et 2008; Manning (Haren Wiese, to and MTOCs a Liu non-centrosomal of required 2006; blades other the and are form centrosomes to structure, predicted are propeller which repeats, WD40 00 exd-rvs ta. 00.I ua el,MOZART1 cells, human In 2010). al., et Teixido-Travesa no 2010; 2010). has al., but et (Teixido-Travesa centrosomes, mitosis during interphase role obvious at nucleation microtubule ,cmoeti rsn;– opnn sntpeet ,ukon r oeua asi kDa. in mass molecular Mr, unknown; ?, present; not is component –, present; is component +, MZT2B TUBG2 C-Di a is GCP-WD C8i ml rti hti osre ndueotmsbut deuterostomes in conserved is that protein small a is GCP8 Another Hymenoptera and and ora fCl cec 2 (19) 125 Science Cell of Journal c uCcr uui sMZR1(ucise al., et (Hutchins MOZART1 is subunit core TuRC oecaueMr nomenclature C985+++++ + + + + + 8.5 + GCP9 + and GCP8A 71.9 GCP-WD C6205+++++ + + + + 200.5 GCP6 C5183+++++ + + + + 118.3 GCP5 C3136++++++YsCmoetof Component of Component Yes Yes + + + + + + + + + + + + + + + + 76.1 + 103.6 GCP4 102.5 GCP3 GCP2 c tbln1 -tubulin GCP8B uui 2 tubulin and c GCP uCtreigfco hti nipnal for indispensable is that factor TuRC-targeting c u,crosy o nohrpat rinsects. or plants other in not curiously, but, - dr ta. 06.TeCtria afof half C-terminal The 2006). al., et ¨ders dr ta. 06 ae l,2010; al., et Ma 2006; al., et ¨ders 62+ 16.2 11++++++YsCmoetof Component Yes + + + + + + 51.1 c uCasml (Gunawardane assembly TuRC al .Cr uuiso human of subunits Core 1. Table c dr ta. 06 Teixido- 2006; al., et ¨ders uCrcutett and to recruitment TuRC tropicalis c tbln h N-terminal The -tubulin. X. melanogaster Micromonas D. 22222 c uCto TuRC c TuRC b thaliana - A. tutr.Rgltr ucin aebe ugse o oeof some for suggested been have functions Regulatory structure. and Grip-GCPs regulatory versus Structural as qualify LGALS3BP and NDK7 c whether unknown However, lectin, al., 2010). currently et al., is (for et (Choi it Teixido-Travesa 1) 2010; LGALS3BP al., (Table et interactions and Hutchins cell–matrix 2010; 2010), and a cell–cell have al., in might (Lai role which et protein), motility binding and Vogel 3 soluble, transport galactoside-binding, 2011; ciliary al., in functions et which known NME7), (also NDK7 as member family kinase nucleoside-diphosphate human Purified Other srqie o erimn of recruitment for required is osre nfsinyatbtnti udn es,wihmight which yeast, involving budding function in in not a but indicate role yeast a fission of in has conserved none MOZART1 However, whether 2012). analyzed c al., have proper et mitosis studies Nakamura for during these 2012; required segregation al., is et chromosome (Janski interphase, active and to assembly in localizes spindle sites and Similarly, GCP3 nucleation 2010). to al., cortical binds et which (Hutchins MOZART1, assembly plant spindle bipolar for c uCcr uuisadwa hi oei the in role their what and subunits core TuRC uCasml n/rsaiiy neetnl,MZR1is MOZART1 Interestingly, stability. and/or assembly TuRC uC n hi properties their and TuRCs nidulans A. 22 22 c uCascae proteins TuRC-associated pombe S. c tblnaecniee seta o the for essential considered are -tubulin c uC oti w diinlpoen,the proteins, additional two contain TuRCs cerevisiae 2 2 2 2 S. c c uCt ioi etooe and centrosomes mitotic to TuRC uClk complexes. TuRC-like c uCsubunits TuRC c n/rsaiiyComments stability and/or uCassembly TuRC eurdfor Required e io oein role Minor Yes e io oein role Minor Yes e io oein role Minor Yes oCentrosome No oRl in Role No eurdfor Required ? c uCis. TuRC assembly spindle bipolar targeting centrosome specific interphase- factor targeting spindle and pombe and nidulans A. pombe and nidulans A. pombe and nidulans A. c c c TuSC TuSC TuSC c S. S. S. TuRC Journal of Cell Science GNPo KP)(aaah ta. 02 n CDK5RAP2 and 2002) al., et as (Takahashi known AKAP9) (also or AKAP450 2004), CG-NAP including al., proteins, et centrosomal (Zimmerman several pericentrin that proposed been has It centrosomes to Targeting the of Regulation following the in discuss non-structural of will analysis the we al., from altered As et (Gregoretti 2004). into insight system al., However, section, closed et a Sawin 2005). changes 2006; in of nucleation effect Chang, indirect microtubule an in be also and might al., dynamics et microtubule Tange 2000; Zimmerman al., et Paluh 2001; 2004; al., et Jung 2002; al., 2009; et al., Fujita et (Bouissou dynamics and stability depletion microtubule RNAi-mediated alter can or mutation their because proteins these ftecmlxt pcfcMOs(i.2 al 2). Table 2; (Fig. MTOCs specific to complex the of c Several proteins uCrglto,feunl ymdaigsbellrtargeting subcellular mediating by frequently regulation, TuRC acaoye cerevisiae Saccharomyces cioacaoye pombe Schizosaccharomyces nidulans Aspergillus ekta ta. 2004). al., et Venkatram 2002). hsooia ufrcniin eeldmsl small, mostly ( revealed complexes sized conditions more under buffer fractionation physiological gradient sucrose However, complexes. as to referred commonly now ( GCP2 complexes are yeast, small budding tubulin complexes in has Such present GCP3. members yeast family and GCP budding two only of in the molecules work two of Early composed sizes. various identified of complexes c of size and in composition differences Organism-specific 1. Box 01 upye l,19;Ogm ta. 99 hn ta. 1995). al., et Zheng 1999; al., et Oegema 1998; al., et Murphy 2001; ( termed assemblies, ( larger complexes much forms also tubulin oi teghbfe odtossoe ag ( large showed conditions buffer strength ionic ( complexes larger ta. 01 upye l,19;Ogm ta. 99 hn tal., et melanogaster Zheng 1999; al., Drosophila et Oegema 1998; al., 1995). et Murphy 2001; al., et mostly organisms. sapiens different in Homo fraction cellular soluble the nucleation. organisms microtubule support some can in alone that subunits suggests which viability, for essential c the Whereas than stable organisms less the these or than abundant in size less in However, larger are yeast. that budding complexes tubulin than other found also fungi are proteins in These humans). in GCP6 and GCP5 GCP4, c . tblnadmmeso h C aiycnasml into assemble can family GCP the of members and -tubulin uC the TuSC, (termed members family GCP additional three contain TuRCs eo,w uln h ye n ie of sizes and types the outline we Below, 1)(oize l,19;Mrt ta. 98 upye al., et Murphy 1998; al., et Moritz 1995; al., et (Moritz 31S) c c uC( TuRC uCascae rtishv enipiae in implicated been have proteins TuRC-associated c uCi osdrdt eamr ciencetrthan nucleator active more a be to considered is TuRC c c tblncmlxsa eeoermr htare that heterotetramers as complexes -tubulin c uCseii C4 C5adGP nfniaenot are fungi in GCP6 and GCP5 GCP4, TuRC-specific uC) nadto to addition In TuRCs). , , 2)(oize l,19;Mrt ta. 98 Murphy 1998; al., et Moritz 1995; al., et (Moritz 32S) eou laevis Xenopus , , c –S Adr ta. 06 uiae l,2002; al., et Fujita 2006; al., et (Anders 8–9S) uCrglto a rmrl enobtained been primarily has regulation TuRC 1)(in n aly 2009). Oakley, and (Xiong 21S) : c otysalcmlxs( complexes small mostly c uCtruhassociated through TuRC uC) In TuSCs). : : c only c c tblnadoemlcl ahof each molecule one and -tubulin uC( TuSC : tblncomplexes -tubulin : e itainaayi ne low under analysis filtration gel c uCsbnt n interactors. and subunits TuRC oesalrcmlxs but complexes, smaller some c Drosophila c c uC( TuSC uC nhge eukaryotes. higher in TuRCs tbln C2adGCP3, and GCP2 -tubulin, , 01S and 10–13S) c tblncmlxsin complexes -tubulin , c 2)(ihe al., et (Vinh 12S) uCapa obe to appear TuSC n vertebrates, and , –4) some 7–14S), c . tblnring -tubulin 00kDa) 2000 c c c TuSC- TuRC TuSC c c c - - - rtisaeipratfrcnrsm tutr n therefore and structure affect indirectly centrosome also for might recruit important these 2008) PCM, are the al., of proteins et components integral (Fong as However, Cep215) centrosomes. as known (also hn,20;Mrsae l,20) hra the Whereas 2009). and al., et (Clarke Maresca chromosomes 2008; Zhang, mitotic promote around independently assembly microtubule (CPC) complex passenger chromosomal 1996). al., et (Moudjou 3A) (Fig. sites that suggests which c the of targeting centrosome Whereas sites non-centrosomal to Targeting atrta isms rxmlt the to proximal most lies that 2011). factor Rhee, and Lee 2009; al., et Haren 2007; ahrta eea hoai hthv h oiatrl in al., role et dominant (O’Connell the assembly Although have spindle 2009). and that formation chromatin kinetochores microtubule general the is than it Interestingly, rather established. been has CPC the al., Lu et (Groen 2009; sites non-centrosomal these at nucleation for required organization, microtubule centrosomal ta. 06 Lu the 2006; of al., independently complex, et centrosomes the to of subunits localizes of other it unlike localization but, mitosis, centrosomal and interphase the for indispensable hs uhr i o eosrt h bec fmcouue in microtubules of However, absence the 2010). demonstrate al., not the did et authors (Mishra those that and recruits microtubules 107 suggested of complex protein independently has complex respectively) study pore 160, nuclear recent (for a NUP107–NUP160 microtubules, bound osntaoihcnrsmlrcutetof in recruitment GCP6 and contrast,GCP5 centrosomal (Ve in By nucleation abolish GCP6 microtubule 2008). and not GCP5 al., GCP4, does GCP-WD, et of Izumi depletion observations; intact unpublished an requires humans on depends 2010). also al., itself et GCP8 (Teixido-Travesa of GCP-WD localization centrosomal the but to contributes uui erimn n irtbl ulaina pnl pole spindle at nucleation microtubule and recruitment tubulin pombe C5adGP n h blt oassemble GCP4, to ability of the presence ring-like and The GCP6 2010). of and al., GCP5 assembly et (Kollman promotes vitro body, in linksoligomers pole which Spc110, observation spindle yeast the budding the by of supported fragment is a view that This bodies. centrosomes pole with spindle interaction or their following scenarios only that but aforementioned assemblies, possible is the It cells? 2002; in in al., nucleation the microtubule et does support How 1999), Fujita 2009). 2006; Oakley, and al., Xiong c et 2004; al., (Anders et species Venkatram that some demonstrates in which sites GCP6, and GCP5 the GCP4, of orthologs Moreover, bodies. eipratfrnceto rmcrantpso MTOCs. of types certain from nucleation for important be uC hc savr orncetri ir Ogm tal., et (Oegema vitro in nucleator poor very a is which TuSC, tblni rsn ntenncnrsmlctslcfraction, cytosolic non-centrosomal the in present is -tubulin uigmtssbt hoai-eeae aGPadthe and RanGTP chromatin-generated both mitosis During nhmncls the cells, human In nadto oGPW,cnrsmltreigof targeting centrosomal GCP-WD, to addition In c uCpoen ln aeteaiiyt sebenucleation assemble to ability the have alone proteins TuSC r o seta o iblt n r ipnal for dispensable are and viability for essential not are dr ta. 06,n ietrgltr ikt aGPor RanGTP to link regulatory direct no 2006), al., et ¨ders c dr ta. 06.The 2006). al., et ¨ders tblnrcutett nepaecentrosomes, interphase to recruitment -tubulin c tblni nw olclz okinetochore- to localize to known is -tubulin seglu nidulans Aspergillus c acaoye cerevisiae Saccharomyces tblnmgtas ucina te cellular other at function also might -tubulin c uCsbntGPW steattachment the is GCP-WD subunit TuRC ´ olte l,20) iial,GCP4, Similarly, 2006). al., et rollet c c uC(.T-. .R n .L., J. and R. J. T.-T., (N. TuRC uCrcutet(rsre al., et (Graser recruitment TuRC c TR euain4449 regulation -TuRC c uC tl omring-like form still TuSCs c uCi lal rca for crucial clearly is TuRC , c and 0 ftettlcellular total the of 80% uCt kinetochores to TuRC c c uCsbntGCP8 subunit TuRC uC C-Dis GCP-WD TuRC. Schizosaccharomyces c uC ih,thus, might, TuRCs c uCi clearly is TuRC aual lacks naturally c c uC(Haren TuRC tblnand -tubulin c c tblnin -tubulin Drosophila c tblnin -tubulin c uC to TuSCs uCto TuRC c TuSC c - Journal of Cell Science GL3PCandidate LGALS3BP h oaiainof localization the been yet not has 2005), vertebrates. al., in et in Murata described and 2005; plants al., et in (Janson described yeast as fission microtubules, 2010). existing to Kimura, bound by and laterally nucleation (Goshima pathway microtubule this However, important into provided insight has recruits GCP-WD, molecular that adaptor the complex through microtubules protein multi-subunit (Lu a 3A) Lu (Fig. 2006; microtubules al., et additional assembly nucleate spindle to during microtubules proposed: been c has model the amplification in density microtubule reduces (Lu spindle and assembly spindle proper with l4Bnsadpopoyae C6t euaecnroedpiain(at ta. 2012) al., et (Bahtz Candidate ubiquitylates activity ligase E3 duplication centriole regulate to GCP6 phosphorylates and Binds phosphorylates and with Associates NME7 BRCA1 family Src and Syc Plk4 SADB assembles and GCP6 to with directly interaction Binds Enhanced the of stability for Required Nup107–Nup160 the tethering for scaffold complex Augmin a provides GCP3; and GCP2 to directly Binds of folding Promotes Keratin HCA66 chaperonin TRiC AKAP9 hsrglto eanucer u h agtn lorequires also targeting the c but unclear, Drosophila remain of regulation details molecular this The dynamics. plus-end microtubule regulate disrupts specifically that mutant GCP-WD a of targeting of expression and issues. these resolve to needed the thus case is not investigation this and Further ch- 2010). CKAP5) to in as related but known protein (also yeast, a TOG References budding Stu2, is in nucleator only kinetochore-associated mechanism described a such far, been So mechanism. has specific have a would by orientation corrected reversed be this to and kinetochore, the kinetochore- to distal by microtubules of bound Nucleation experiments. their to contribute might mitosis; in subunit GCP-WD phosphorylates and Binds Role Plk1 the the tethering tethering for for scaffold scaffold a a provides Provides GCP3; and GCP2 to directly Binds CDK5RAP2 Pericentrin Interactor 4450 uC ih soit aeal ihpeiul formed previously with laterally associate might TuRCs uCisedof instead TuRC yoiekinases tyrosine complex In uigmtssthe mitosis During Drosophila c uCwudrsl nmcouue ihpu nsta lie that ends plus with microtubules in result would TuRC dr ta. 06.O h ai fti idn,teso-called the finding, this of basis the On 2006). al., et ¨ders ora fCl cec 2 (19) 125 Science Cell of Journal c C4 hc ugssta hspoesinvolves process this that suggests which GCP4, dr n tan,20) h dniiaino augmin, of identification The 2007). Stearns, and ¨ders uCt pnls u o ocnrsms interferes centrosomes, to not but spindles, to TuRC 2clsteGPW rhlgi eurdfor required is ortholog GCP-WD the cells S2 c uC(oisue l,2009). al., et (Bouissou TuSC c c uCi lotree osidemicrotubules, spindle to targeted also is TuRC uC ln nepaemcouue to microtubules interphase along TuRCs soit with Associate Tethers n elmgain h oein role the migration, cell and c nucleation microtubule by disrupted is GCP6 with phosphorylation interaction GCP6 cells; CDK1-dependent epithelial of domain apical microtubules generation microtubule intra-spindle promote to GCP-WD through microtubules Golgi and centrosomes mitotic c centrosomes mitotic uCi unknown is TuRC uCrcutett h centrosome the to recruitment TuRC c uC ouatce ieohrst upr ulaino kinetochore of nucleation support to kinetochores unattached to TuRCs c c uCsbnt oeta oe ncl–eladcl–arxinteraction, cell–matrix and cell–cell in roles potential subunit; TuRC uCsbnt D iaewt ucini oiecla h oein role the cilia, motile in function with kinase NDP subunit; TuRC al .Selected 2. Table c c uC n phosphorylate and TuRCs tblncmlxsta are that complexes -tubulin c tbln(iauae al., et (Kitamura -tubulin c tblnadGPW Mlie l,19;TiioTaeae al., et Teixido-Travesa 1993; al., et (Melki GCP-WD and -tubulin c uCdrn ioi;recruits mitosis; during TuRC c c uCt spindle to TuRC uCsubunits TuSC c tblnt nii etooa ulainatvt Snaa ta. 05 trt ta. 2004) al., et Starita 2005; al., et (Sankaran activity nucleation centrosomal inhibit to -tubulin c uCi unknown is TuRC c c c tblnt euaecnroedpiain(laaoKitnsne l,2009) al., et (Alvarado-Kristensson duplication centriole regulate to -tubulin uCitrcosadterpooe roles proposed their and interactors TuRC uC activates TuRC; c tblncnann ulainstsi the in sites nucleation -tubulin-containing c ¨ders uCascae rtist promote to proteins TuRC-associated c tbln C2adGP Fn ta. 2009b) al., et (Fant GCP3 and GCP2 -tubulin, c fiin ulainrqie ciainof activation requires of nucleation targeting efficient specific of to number addition substantial a where cytoplasm, the c in sites random and at MTOCs not at predominantly occur to nucleation microtubule For of Modulators 06.I amla pteilclsGP mediates GCP6 cells epithelial mammalian In 2006). has AKAP450 and Golgi, the recruit the to to shown localize been as also CDK5RAP2, described centrosome, and been overall the AKAP450 both the Interestingly, have to 2009). Su which and al., contribute in (Kodani et and complex help Miller Golgi stacks complex the Golgi Golgi of organization the of at positioning nucleated the are that Microtubules c1 epciey Fn ta. 08 ai ta. 04.Full- 2004). by motif, al., conserved nucleation et the microtubule comprising Sawin stimulates fragment 2008; a or al., CDK5RAP2, et length (Fong respectively) the related Pcp1, to in binding conserved in mediates is proteins that and CDK5RAP2, motif complex protein sequence tubulin scaffold a centrosomal contains an the Such which 3B). be (Fig. might MTOCs at activator present only are molecules activating oaiaint h pclsbebaergo hog its through region 2007). submembrane al., et (Oriolo apical keratin with the interaction to localization target during GCP5 MTOCs and centrosomal GCP4 of Orthologs targeting. 2010). al., et Wang 2009; al., et (Rivero uCnceto ciiy(are l,21;Co ta. 00 Fong 2010; al., et Choi 2010; al., et (Barr activity nucleation TuRC uC r lopeet el eur euaoymcaimin mechanism regulatory a require cells present, also are TuRCs nte o-etooa TCi h og complex. Golgi the is MTOC non-centrosomal Another nsm ae rpGP aeas enipiae in implicated been also have Grip-GCPs cases some In c uCt spindle to TuRC Drosophila c c uCto TuRC uCto TuRC c uCnceto activity nucleation TuRC n iso es nml n,adMo and Mto1 and Cnn, (namely yeast fission and c Gasdnae l,20;Hthn tal., et Hutchins 2004; al., et (Grassadonia Co ta. 00 ucise l,2010; al., et Hutchins 2010; al., et (Choi (Dra 2007) al., et (Oriolo Hrne l,20;Jhuae l,2011; al., et Johmura 2009; al., et (Haren 2010) al., et (Mishra 2009; al., et Lawo 2008; al., et (Goshima al., et Zimmerman 2002; al., et (Takahashi uCt h i-og compartment cis-Golgi the to TuRC LeadRe,21;Tkhsie al., et Takahashi 2011; Rhee, and (Lee Drosophila 00 exd-rvs ta. 2010) al., et Teixido-Travesa 2010; 00 oe ta. 2010) al., et Vogel al., 2010; et Teixido-Travesa 2010; Ikeda, 04 aue ta. 08 Sulimenko 2008; al., et Macurek 2004; 2009) al., et Zhang 2008) al., et Zhu 2009; al., et Uehara 2008) al., et Yam 2010; 2004) 2008) al., et ta. 2006) al., et 02 imra ta. 2004) al., et Zimmerman 2002; ´ berova c uC.Oepsiiiyi that is possibility One TuRCs. ´ c c uC ntectpamof cytoplasm the in TuRCs ta. 99 uhrkye al., et Kukharskyy 1999; al., et uCtteigfcosat factors TuRC-tethering oeei Vg tal., et (Vogt oogenesis c uC n htthe that and TuRCs c tblntethering -tubulin c uC onon- to TuRCs teln 2009; ¨tterlin, c c TuRC TuRC c - Journal of Cell Science sebyit opee and/or complexes into assembly of function aiblt.( generate variability. thus and interchangeable be ciiyof activity diin h agtn atrcudas ucina natvtro the of activator an as function also could factor targeting the addition, ouaino h oesbntcomposition subunit Human core the of Modulation 2011). al., et (Kollman lattice microtubule the of position flexible the the a adjust in would by molecules which mediated GCP3, in possibly region switch, hinge conformational the cytoplasmic a of Activation involve the 2010). al., of et Teixido-Travesa most 2010; that cytoplasmic with idea co-purifies CDK5RAP2 the with c consistent is result isolated from and cells of Mechanisms 3. Fig. TiioTaeae l,21) u C6i betfo fraction a from absent is is GCP6 of but 2010), stoichiometry al., et exact (Teixido-Travesa Grip-GCPs of in their stoichiometry similar The but is 2001). al., Grip-GCPs, et (Murphy other unknown of copies rget(hie l,21) Some 2010). al., et (Choi fragment uC r o soitdwt natvtr ned eyltl rno or little very Indeed, activator. an with associated not are TuRCs c uC htaeascae iharcmiatCDK5RAP2 recombinant a with associated are that TuRCs c c uC oti igeGP oeueadmultiple and molecule GCP5 single a contain TuRCs c uC olwn h neato ihMOs ( MTOCs. with interaction the following TuRCs D uC.( TuRCs. ycnrligboeei n tblt fspecific of stability and biogenesis controlling By ) c uC rmaycrnu n ioi eacells HeLa mitotic and asynchronous from TuRCs c uCt oeacrtl ac h emtyof geometry the match accurately more to TuRC E ottasainlmdfctoscudrglt h ulainatvt of activity nucleation the regulate could modifications Post-translational ) c uCregulation. 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TuRC c uC ihdsic rpris noprto of Incorporation properties. distinct with TuRCs ( A c tblncmlxsalso complexes -tubulin ) c c uC Hthn tal., et (Hutchins TuRCs uCascae agtn atr gen iettecmlxt ifrn uclua oain sidctd In indicated. as locations subcellular different to complex the direct (green) factors targeting TuRC-associated C c rpGP ih cuyfxdpstosi the in positions fixed occupy might Grip-GCPs ) uCmight TuRC c c -tubulin uCsbnt,poenfligaddgaainmcieiscudmdlt sebyand assembly modulate could machineries degradation and folding protein subunits, TuRC c uCnceto ciiy ( activity. nucleation TuRC h ignssadfnto of function and biogenesis The degradation and machineries folding protein through Regulation odn n erdto ahnre Lni ta. 2010). al., et (Lundin machineries degradation and folding ae oehrteefnig ugs htdistinct that suggest findings 2012). these al., et Nakamura together 2010; al., et Taken (Choi GCP-WD lack to seem ihvral tihoere fGi-Cs(i.3C). (Fig. Grip-GCPs of stoichiometries of of variable Incorporation be assembly could the allow 2011) with positions, would al., some This in occupy et least interchangeable. at Grip-GCPs are (Kollman Grip-GCPs, that ring all assuming by the that extended in model positions The specific exist. subpopulations is,20;Wlo ta. 97 uaKb ta. 2005). al., et Yuba-Kubo 2012; Raynaud- 1997; al., al., 2012; et Vinopal et al., 1997; Wilson et al., 2008; et (Nakamura Wiese, Tavosanis generate type 2001; al., cell to et or Messina variability stage cycle additional cell create of would subpopulations GCPs, other c tblnadGi-C sfrsadohrGP rae additional creates GCPs other and isoforms Grip-GCP and -tubulin c B uC sona neape,tesaiiyo uuis their subunits, of stability the example), an as (shown TuRCs ciiymdltr prl)mgtsiuaetenucleation the stimulate might (purple) modulators Activity ) c c tblnadGi-C sfrs swl as well as isoforms, Grip-GCP and -tubulin uC ihrlsta r pcfct certain a to specific are that roles with TuRCs c uCsrcue lentvl,Gi-Csmight Grip-GCPs Alternatively, structure. TuRC a c and - TR euain4451 regulation -TuRC b tbln r euae by regulated are -tubulins c c TuRCs TuRC Journal of Cell Science are l,20;ORgne l,20) nfis centrosome flies, In 2007). al., et O’Regan as well 2004; (as maturation al., et Barr promote PLK1, kinases, of to NIMA-family organizers. transition, accumulation and centrosomal spindle activity mitotic A other Aurora several mitotic G2-M and including nucleation process, kinases, this as of regulator and major the role a is which their size at for their ‘prepare’ maturation increase centrosomes centrosome During maturation centrosome and Phosphorylation by binding GTP GTP of role The the folding, of incorporation to the into addition in subunits subunits, involved In be key also modulate 3D). could complex of (Fig. chaperonin could function availability machineries and/or the degradation assembly controlling and By Yam folding 2010; 2008). al., et al., Teixido-Travesa also 1993; et al., with complex, et co-purifies (Melki (TCP1-ring cells which HeLa TRiC CCT), chaperonin as known the of substrates both Similarly, 4452 nmttcknsssc scci-eedn iae1(CDK1), A. 1 Aurora kinase and (PLK1) cyclin-dependent 1 as depends kinase such Polo-like or kinases mitosis mitotic in on specifically occurs phosphorylation cases the of Most modification nyof only not recruitment centrosomal promotes PLK1 Importantly, difficult. directly al., that substrate et PLK1 controls (Dobbelaere a of mitosis is identification contrast, which in By Plk1, phosphorylation 2008). and Cnn CDK5RAP2, for of homolog required fly a Cnn, proteins: 1997). Mitchison, by hydrolysis and ‘dynamic and/or (Desai modulate binding so-called GTP microtubules the Similarly, of and polymerization instability’ promote tubulin and assembly with interferes and GCP3, of function and RNA- GCP2 destabilizes nucleolar HCA66 small tubulin, of Depletion U3 (UTP6)]. as 6 known protein [also associated HCA66 protein the for anr(etnahe l,21;Snaai ta. 2011). includes controls which al., mechanism, probably one et than PLK1 Santamaria more vertebrates, through 2011; recruitment al., in PLK1-dependent of a all et Therefore, in and vivo 2011), (Kettenbach in al., al., manner phosphorylated et et are Santamaria proteins 2011; (Haren these Rhee, CDK5RAP2 and Lee and 2009; pericentrin Cep192, including post-translational the involve section. of following Regulation the also in of discuss will functions activity we which and modifications, these composition However, this assembly, solve to targeting, required is work further Thus, 2008). issue. al., et Rice the to monomeric response neither the However, mutations 2001). nor the al., al., identified et et (Hendrickson Jung dynamics has of microtubule 2001; affect or fungi lethal analysis are in that Indeed, domain nucleotide-binding microtubule. nucleated nsummary, In c c c ora fCl cec 2 (19) 125 Science Cell of Journal uC u loo eea tutrlPMproteins, PCM structural several of also but TuRCs uCrcuteti etbae a rvnt be to proven has vertebrates in recruitment TuRC c uCudroamjrcnomtoa hnein change conformational major a undergo TuSC c uCnceto ciiyo h rpriso the of properties the or activity nucleation TuRC c uCand TuSC uCsbnt r hshrltd(i.2.I many In 2). (Fig. phosphorylated are subunits TuRC c uCor TuSC c c c tblnadGPW aebe dniidas identified been have GCP-WD and -tubulin tblnncetd tt Klmne l,2008; al., et (Kollman state nucleotide -tubulin uCascae rtisaeal ocontrol to able are proteins TuRC-associated a and - c tblnrcutet eed nol two only on depends recruitment) -tubulin c c c uC(ate l,2009a). al., et (Fant TuRC uC iia oehsbe proposed been has role similar A TuRC. uCb posttranslational by TuRC b tbln n T yrlssby hydrolysis GTP and -tubulin, c tbln(aradGrey 2007; Gergely, and (Barr -tubulin c uC sltdfrom isolated TuRCs c tblncould -tubulin c c c -tubulin -tubulin TuRCs. c c TuRC TuRC b c - - hshrlto of Phosphorylation of Phosphorylation D1adPK otiuet t hshrlto nmitosis in phosphorylation both its and to vivo contribute in PLK1 sites and multiple on CDK1 phosphorylated is GCP-WD GCP-WD of Phosphorylation ta. 01.Yatexpressing Yeast (Keck 2011). Cdk1 by al., bodies pole et spindle at in phosphorylated is Ser360 It humans, that 2001). al., shown et been (Vogel has assembly organization microtubule astral microtubule the promoting regulates by phase near G1 tyrosine the during conserved terminus a of Phosphorylation h euaino etooesz hog hshrlto of 2011). Rhee, phosphorylation and Lee through 2009; al., et size (Haren proteins centrosome PCM structural of regulation the n rsn ntectpam(i ta. 01,mgtprovide might 2011), al., of et regulation 2011) the (Lin al., into et cytoplasm insight Lin the further 2011; al., in et present (Keck yeast and bodies pole in spindle to identified bound were phosphorylation additional which of of Characterization sites, destabilization 2011). by bipolar al., part, et in short (Lin caused, these with be However, to mitosis seem microtubules. defects in disorganized At containing arrest 2011). spindles al., cells et temperatures (Keck at higher dynamics viable microtubule in are changes spindle involving site defects anaphase spindle this display of but temperatures phosphorylation low mimics that mutation etil ignss a o enrvae.I pteilcells, epithelial The of interaction In the 2012). revealed. disrupt to been al., GCP6 phosphorylates not to CDK1 et linked has are biogenesis, (Bahtz events centriole phosphorylation centriole these biogenesis which addition, by known centriole mechanism, a In Plk4, of by 2009). GCP6 regulator of phosphorylation al., requires duplication by et possibly duplication, (Alvarado-Kristensson centrosome control regulating to residue phosphorylates Ser131 BRSK1) as known n specific of and stability the affect Sulimenko al., 2008; al., et family et (Colello 2006). Src Macurek al., identified 2004; et and al., been et Syc not Kukharskyy the have 2010; substrates of but of members regulation kinases, been involves The 2011). have also Kettenbach al., contain et complexes these 2011; Santamaria 2011; al., also of al., et et none GCP4 (Hegemann but characterized and functionally sites, GCP3 phosphorylation PCM GCP2, other multiple involves or Human GCP5 whether of however, of level components. unclear, the at is occurs It amount regulation 2008). this al., the et (Izumi centrosomes regulates (GSK3 beta GCP5 3 kinase human negatively synthase vitro, glycogen In for 2007). microtubule substrate al., a the et is of (Oriolo entry remodeling mitotic the on for array domain, important apical be the from might complexes which the remove to and keratin with ofrainlcag htmgtb eurdfor required be might that cases change few conformational of a arrangement in c the and characterized coordinate poorly still controls is of Phosphorylation se. Grip-GCPs per activity nucleation microtubule control ciain(ola ta. 2011). al., et (Kollman activation uC hshrlto fGi-Cscudas euaea regulate also could Grip-GCPs of phosphorylation TuRC, nhmncls h eietroiepoenkns AB(also SADB kinase protein serine/threonine the cells, human In nsmay the summary, In c c uClclzto.Hwvr eas Grip-GCPs because However, localization. TuRC uCdpnetnceto fcnroa microtubules centriolar of nucleation TuRC-dependent c uCdpnetpoess u ontse to seem not do but processes, TuRC-dependent c c tblnpopoyainstssuids far so studied sites phosphorylation -tubulin tblnwsfrtsuidi udn yeast. budding in studied first was -tubulin c c tblnadGrip-GCPs and -tubulin tbln h rpriso microtubules, of properties the -tubulin, c tbln hc scnevdin conserved is which -tubulin, c c tblnwt Ser360Asp a with -tubulin tblnmlclsi the in molecules -tubulin c c tblna h conserved the at -tubulin tblncomplexes. -tubulin c uCsbnt htare that subunits TuSC c tblna mitotic at -tubulin c tblnC- -tubulin b c c c ,which ), -tubulin -tubulin c TuRCs TuRC Journal of Cell Science hti iia oagi elto,aoihslclzto of localization Lu abolishes 2011; al., depletion, et (Johmura generation augmin to c similar manner a alanine is in to and, A) augmin that isoform with GCP-WD longer of the interaction at in the site disrupts Ser418 consensus Lu B; CDK1 2011; isoform the (in of al., Ser411 Mutation et 2011). al., Johmura et 2009; Santamaria al., et (Haren ifrn eltpsadognss oee,w tl nwvery of know regulation still the we However, about organisms. little and types understanding cell in of molecular different made remarkable been targeting this has the Progress how regulated. unravel the is of to machine discovery beginning the just after years only 15 than more At Conclusions 2010). Wagner al., 2011; et al., Xu et 2011; (Kim al., characterized et functionally been not other has of Ubiquitylation 2009). (Parvin, 1 type cancer c breast ubiquitylates the (BRCA1) of protein activity ligase susceptibility (Choudhary ubiquitin the determined The but be 2009). Lys827 al., to at et remains acetylation modification by this modified of is function GCP2 that known is for described it been not have modifications glutamylation, such and glycylation acetylation, including modifications, Whereas modifications post-translational Other weakens to alanine to GCP-WD vitro, of in binding PLK1-dependent identified four of sites, group a (Johmuraphosphorylation or Hice1) Thr550 Mutating as 2011). known al., (also et HAUS8 complex, control subunit augmin Interestingly, a indirectly the of 2009). of to phosphorylation through seems al., GCP-WD GCP-WD of et binding on spindle Zhang Ser460 to 2011; bound al., PLK1 al., et by et (Haren phosphorylated Johmura box polo are 2009; PLK1 the GCP-WD with interaction on mediate to Thr550 CDK1 and Ser460 2009). ucinlcaatrzto fpopoyainstsin sites phosphorylation of characterization of functional activation the studying from of function a the as with protein consistent of is this which localization sites, the nucleation specific affect mutants phosphorylation spindle. bipolar functional to centrosomes. key mitotic is at which Ser377, Plk1 recruiting at by GCP-WD activated phosphorylates Nek9 is (Sdelci Active which Nek9 kinase 2012), NIMA-family al., the which has et be GCP-WD substrates these to of of centrosomes, shown One been 2009). upstream PLK1-dependent al., recently et substrates mitotic Zhang 2009; PLK1 the al., et are at (Haren there for GCP-WD that required suggests of is accumulation sites phosphorylation previous targeting which for 2009), essential a not al., is of in Thr550 et to (Haren binding observed PLK1 mutation that suggests not Thr550Ala the was of analysis phenomenon this However, of localization oeo uloie nterglto fthe of regulation the in nucleotides and of structural role systematic a of Similarly, analysis functional required. be will subunits tblnlclzto n ulainatvt tcentrosomes at activity nucleation and localization -tubulin uCt pnl irtblsaditasidemicrotubule intra-spindle and microtubules spindle to TuRC ae oehr h vial aasgetta GCP-WD that suggest data available the together, Taken c uC omttccnrsms motnl,nn fthese of none Importantly, centrosomes. mitotic to TuRCs a c -and uCt ioi etooe n sebiga assembling and centrosomes mitotic to TuRC c c uCt etooe Zage l,2009). al., et (Zhang centrosomes to TuRC uCtreigfactor. TuRC-targeting b c tblncmlxst aiu TC in MTOCs various to complexes -tubulin tblnaehaiyatrdb ag of range a by altered heavily are -tubulin c tblnmtnsi eesr ocaiythe clarify to necessary is mutants -tubulin c tblnadmdrtl reduces moderately and -tubulin c uCnceto ciiy Apart activity. nucleation TuRC c uCb neatn rtis a proteins, interacting by TuRC dr ta. 06 eaae al., et Uehara 2006; al., et ¨ders c uCsbnt cusbut occurs subunits TuRC c c TuRC. tblnadreduces and -tubulin c dr ta. 2006; al., et ¨ders tbln However, -tubulin. c uC eare we TuRC, c uC to TuRCs c TuRC hi .K,Lu . z,S . a,C n i .Z. R. Qi, and C. Dai, K., S. Sze, P., Liu, K., Y. Choi, Ve A., Bouissou, Ville E., J. Elias, D., Schwartz, M., Jedrychowski, A., S. Beausoleil, F. Gergely, Lehmann, and C., R. A. Antony, Barr, U., Haselmann-Weiss, M., Arnold, J., Seidler, R., Bahtz, MCN,Spain). (MICINN, onn,M. Bornens, G. G. Gundersen, and F. Bartolini, Sillje F. A., F. Gergely, Barr, and V. J. Kilmartin, R., A. Barr, M. Bornens, and J. Azimzadeh, nes . Lourenc A., Anders, E. K. Sawin, and A. Anders, Rodrı M., Alvarado-Kristensson, Ramo the References Re- from FP7-PEOPLE-2007-4-3-IRG, International and number Curie 224835]; J.L. Marie [grant no. Barcelona). a project IRB Grant from Group, integration Research support Carme intramural of Signaling support acknowledges Barcelona continuous (Cell IRB the acknowledges and Caelles J.R. J.L.). J.L.)]; (to funds (to de BFU2009-08522 (Ministerio I+D+I J.R.), of Innovacio Nacional Plan e by Ciencia supported are J.L. and J.R. comments. Funding and reading critical for Kiffin Roberta thank We Acknowledgements hrceiaino potential of characterization ifri opsto rps-rnltoa modification. post-translational or composition in differ mgn ehd,i ih epsil ocmaetesubunit single-molecule the compare using to By possible individual be of tags. might composition it affinity subunits methods, different or imaging various of antibodies down pull using through and/or types stages cell cycle different cell from obtained be could subpopulations atr fetmcouuenceto,w edabetter a structural need Careful we process. of analysis this functional nucleation, and of microtubule understanding mechanistic affect other and factors nucleotides, modifications, post-translational proteins, oe ol n sasta ol allow would that assays of development and the be tools would achieving milestone to novel important crucial An be goal. will this proteins, recombinant purified from ognrt irtbl raso ra tutrladfunctional and structural great of diversity. arrays microtubule the generate how to and when where, of picture elongation and stabilization microtubules. from of distinguished be to nucleation irtbl ulainb h am-uui igcomplex. ring gamma-tubulin 1095. the by nucleation microtubule I. duplication. 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