Ato o orsodne([email protected]) correspondence for *Author Korea. of Republic 151-747, Seoul UK. 9HN, LE1 aaHardy Tara to Cep135 from disjunction it releases trigger C-Nap1 of phosphorylation Multisite ARTICLE RESEARCH ß eevd6Spebr21;Acpe 2Mrh2014 March 12 Accepted 2013; September 6 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This polarized a is 1 that observed single a cycle. usually has cell basis, centrosome the this are each throughout on G1, and, In two Centrosomes PCM possess the cells of 2005). normal markers using al., 2012; by (Bornens, experimentally et nucleated are a microtubules Doxsey into which arranged material from pericentriolar microtubules (PCM) by surrounded stabilized and of highly configuration cylinders pinwheel of short , triplets of in composed nine are centers They microtubule-organizing cells. primary animal the are Centrosomes INTRODUCTION C-Nap1, Nek2 Cep135 linker, Cep135, Centriolar disjunction, the and Centrosome WORDS: at KEY oligomerization disjunction centrosome both . precipitates of perturbs onset this of and Nek2 C- interaction in interaction, of by phosphorylation lost and multisite specifically that Nap1 is Cep135, propose therefore , We Cep135 mitosis. and centriolar C-Nap1 interaction core endogenous perturbs an the also to is C-Nap1 process. it with sufficient this of for that are required phosphorylation is suggesting that acidic Importantly, charge centrosome, negative more overall non-phosphorylatable the in increase domain from the in C-Nap1 release make Mutations and that oligomerization residues of association. loss to collectively, centrosome lead and, events C-Nap1 of phosphorylation domain these are C-terminal the multiple phosphorylation within phosphorylates Nek2 However,residues C-Nap1 that demonstrate C-Nap1. we triggered of Here, is of unknown. consequences and phosphorylation mechanistic disjunction Nek2-dependent the centrosome as the mitosis known of by onset process the a at linker occurs in filamentous disassembly proteinaceous the Linker and . a CEP250) as protein known by (also C-Nap1 tethered of composed interphase physically However, are instability. genetic centrosomes promote and perturb can segregation bipolar separation the centrosome establish in Delays to spindle. separate centrosomes entry, mitotic During ABSTRACT rnic Schultz Francisca eateto iceity nvriyo ecse,LnatrRa,Leicester Road, Lancaster Leicester, of University Biochemistry, of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,29–56doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2 eateto ilgclSine,SolNtoa University, National Seoul Sciences, Biological of Department 1 ienLee Miseon , 1 oneE Baxter E. Joanne , 2 eec .Hames S. Rebecca , 1 usoRhee Kunsoo , 1 uan .Prosser L. Suzanna , 2 n nrwM Fry M. Andrew and o en u noudrtnigtemcaim htla othe to is lead effort that mechanisms considerable the understanding and into cells centrosomes, put cancer being two now many than Importantly, more cells. through new possess offspring the into pass the mature of pro-centrioles then two matures daughters the and cells whereas appendages mother, a acquires As into centriole centrioles. daughter both the of daughter mitosis, end proximal and the pro- to a mother proximity as close known older in centriole, growing new the G2, a centriole, and with S identical; duplicated, into is two progress the centriole not cells each which the As are appendages, lacks. centriole sub-distal However, G1 daughter and younger end. distal in has distal present centriole mother and are that proximal centrioles a with structure age l,20) -a1i ag 21ka coiled-coil kDa) (281 large 2013; daughter and a al., mother of et is ends proximal He C-Nap1 the 2010; to localizes 2006). al., that protein al., et 1998a; Hadjihannas et al., 2007; et Yang Fry al., 2014; al., et et Fang Graser 2008; CEP250), al., et called and Bahmanyar centlein (also 2005; LRRC45, C-Nap1 Cep68, including rootletin, identified, been of have to untethering disjunction. The centrosomes centrosome as spindle. allow known mitotic is linker to the this establish is onset then and but mitotic separate duplicate, centrioles during as and G2 mother is disassembled and disengagement, tether S undergone physically through have to maintained they G1 after in centrioles is interphase. assembled daughter view in is centrosomes current two linker the the This connects Hence, linker 1992). physical al., a centrosomes that et the centrosomes Paintrand connecting 2002; However, seen (Nigg, and be 2004). cells, can cultured al., from material isolated electron-dense et when structures paired Thompson as remain 1999; Jean 1985; al., Schliwa, and the et (Euteneuer keep center to cell and cortex the microtubule cell in the throughout the centrosomes of against action push proximity the which to cytoskeletons, close due actin is this in part, In remain interphase. generally centrosomes Medema, lacking. and these are Tanenbaum details of mechanistic 2012; regulation the Schiebel, 2010), biochemical and the (Mardin been each events understanding has progress enabling into although to However, ‘disengages’, made centrioles separated centrosomes. now of two or are the which pair centrioles, apart form two the inherit splits to when cell pole daughter mitosis, exit each separation second cells within centrosomes, The as spindle. establish two occurs to mitotic the apart the event when move of centrioles, poles M, of opposing early pair the The a or containing progression. G2 now late mitotic each in during occurs events first separation distinct centrosomes two 2011). Stearns, amplified and Nigg or 2007; Glover, supernumerary and these (Bettencourt-Dias of generation ntrso t opsto,anme flne components linker of number a composition, its of terms In two the duplication, centriole after and before Both undergo cells G2 in centrosomes organization, their to Owing 1 on-ai Cheary Donna-Marie , 1, * 1 udaD Samant D. Mugdha , b ctnn(aee al., et (Bahe -catenin 2493 1 ,

Journal of Cell Science ntelne ean ob eemnd lhuhte might they although determined, be to al., remains et linker (Bahe and the linker centlein LRRC45, in intercentriolar dividing Cep68, for of the As major context 2005). of the the provide element that in filaments connecting However, entangling 2002). forms monociliated rootletin al., in cells, present et the rootlet (Yang of ciliary major cells striated recruitment a is the the rootletin of suggests, for name component platform its As a rootletin. as protein related acts it Here, centrioles. ARTICLE RESEARCH h ehnsi ai o o e2rgltscentrosome regulates Nek2 how 2494 detrimental for investigated have have basis we would mechanistic Here, separation stability. the genome centrosome on al., pathway consequences et Nek2 delay the Mardin in defects 2012; Hence, that al., 2012). al., et et Kaseda Silkworth 2013; 2007; Murray, errors and segregation of chromosome (Indjeian frequency to the lead enhances that it attachments as merotelic integrity, genome separation to centrosome detrimental delayed Nek2-dependent is Importantly, reduced assembly 2010). as al., spindle et apart, bipolar (Mardin linker and makes the requires rip this activity to Nek2, (Tanenbaum motor possibly of activity, absence motor sliding the robust in apart However, microtubule poles 2010). drive Medema, anti-parallel that force motors bipolar including through microtubule-based separation, prevent by centrosome not generation multiple to because do is contribute This depletion, processes progression. mitotic Nek2 or assembly as spindle such disjunction, 2011). al., (Mardin et the Smith Nek2 for 2011; PP1 of al., of upstream et affinity Plk1 the the putting by regulates complex, phosphorylated this Nek2–C-Nap1 is of and Mst2 Plk1, affinity turn, kinase the In mitotic centrosome. increases the region, for C-terminal occursNek2 non-catalytic which as phosphorylation, known the This (also 2010). in Mst2 al., kinase, et (Mardin upstream STK3) an subject by also phosphorylation is dimerization to Nek2 the regulated. that are date activity to autophosphorylation al., evidence or et no Fry is 2011; there to al., However, et 1999). response (Croasdale domain catalytic in of N-terminal downstream the present loop zipper leucine activation a through centrosome homodimerization its within and 2007). al., autophosphorylation et disassembly PP1 Mi 2002; 2001). linker al., physiological et Nigg, promoting (Eto phosphorylation a separation enhance and C-Nap1, could inhibitor-2, transition Meraldi during of G2/M C-Nap1, the 2000; of centrosomes at and inhibitor, al., binding of Nek2 et separation Furthermore, both (Helps untimely non- dephosphorylate interphase protein C-terminal the to can bound the with when preventing that, in PP1 interacts hypothesized motif is also It Nek2, a Nek2. their Nek2 of al., through domain centrosome et 1998b). catalytic (PP1) to Fry of al., 2003; 1 Fry, leads et loss phosphatase and Faragher Fry and overexpressed, 2005; Nek2 1998a; al., centrosome 2011). et when (Bahe the al., cohesion and, et from Moniz displacement proteins NIMA-related 2012; phosphorylate can human rootletin, al., these and the C-Nap1 et with of the interacts (Fry of physically member role family a the is kinase kinase, This transition? protein G2/M Nek2 the at 2007). al., disassembly et their Graser 2005; al., et cohesion (Bahe centrosome by of interphase depletion loss in their premature and to entry, leads the but interference mitotic centrosomes forming RNA upon interphase in poles on spindle assembly. present rootletin from are and lost they C-Nap1–rootletin C-Nap1 linker, the the for of role core of a stability with Consistent the to contribute oi hs rtiscnttt h ikrte htregulates what then linker the constitute proteins these if So xeietlapoce htitreewt centrosome with interfere that approaches Experimental undergoes Nek2 Nek2? of activity the controls what So b ctnn hi role their -catenin, csa ltomfrroltnfbrasml Bh ta. 2005). al., et (Bahe assembly fiber rootletin less for C-Nap1 and platform that a staining model C-Nap1 as the rootletin acts with in Furthermore, consistent reduction fibers, rootletin partial shown). prominent a not to and As split led (data on depletion 2012). similar was centrosomes al., Nek2 et in unsplit centrosome decrease induced (Spalluto the rootletin could however, the centriole and splitting; to C-Nap1 centrosome mother of localize the depletion the to expected, of reported at end recently Nek2 fraction distal small remaining the rootletin The represent with in of association C-Nap1. co- by dynamic localization reduction on their and centrosome dependent significant upon Nek2 that largely is a reduced conclude Nek2 we further to of of Hence, was led depletion. which intensity Depletion alone Nek2, C-Nap1 centrosomal 1D,E). centrosomal (Fig. or rootletin the microscopy rootletin and C-Nap1 immunofluorescence measured depleted we with components, and interaction stable upon more depends the Nek2 of recruitment centrosomal ijnto hog oso neato ihtecentriole the with interaction of and centrosome Cep135. protein triggers loss oligomerization end proximal C-Nap1 through perturbs in of model disjunction that a hyperphosphorylation support data domain which our Moreover, overall this suggest localization. in and centrosome change of a C-Nap1 multiple is of it phosphorylate charge modulation, (CTD) site-specific can domain than rather C-terminal Nek2 that, the that on show sites We disjunction. etooa e2 hc speoiaty( predominantly t is a with which of Nek2, ends proximal centrosomal the at capacity C- binding excess limited centrioles. released but the is microtubules structure, to along C- linker owing centrosome stable centrosomal the to a most trafficked inhibited into Nap1 that assembled was hypothesize is We C-Nap1 Nap1 S1A). of Fig. supplementary material 1B; recovery (Fig. depolymerization limited microtubule following The 1B,C). (Fig. htbecig(RP sa,w on httebl fC-Nap1 of after bulk recovery GFP-tagged the that at fluorescence found expressing a we were assay, Using (FRAP) rootletin cells proteins. photobleaching these and using of C-Nap1 versions restricted centrosomes dynamic We more ends. how interphase proximal the centriole examined to between, localizes then sat that C-Nap1 generally were of fibers staining and These 1A). This could from, (Fig. cells centrosomes all, distinct cells. two not the albeit each linking some, interphase in seen from that be and extend in centrosomes that two the structures of fibers filamentous the revealed intercentriolar confirm antibody to of rootletin against antibody we presence polyclonal disjunction, a centrosome raised regulating first mechanisms the study recruits To that structure stable a Nek2 is linker intercentriolar The RESULTS Fye l,19a.Uigti sa omr rcsl map precisely more fragment the to 2362–2442; binds (residues primarily C-Nap1 assay Nek2 of that residues this found 80 we C-terminal Using interaction, of 1998a). its region the al., assay of two-hybrid et yeast interaction the a in (Fry to through Nek2 with Nek2 1964–2442) identified recruits (residues CTD C-Nap1 was how C-Nap1 determine centrosome. to CTD sought C-Nap1 the next within We sites multiple phosphorylates Nek2 (t centrosomes, half-life ( the fraction a small with a associated exhibiting C-Nap1 stably with remained rootletin and h tblt ftelne opnnsi nsakcnrs to contrast stark in is components linker the of stability The ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal 1/2 of 1/2 , )of eod Hmse l,20) ots whether test To 2005). al., et (Hames seconds 5 , 0scns u orcvr frootletin of recovery no but seconds, 30 , 0)o unvrwith turnover of 10%) , 0)dynamic 70%)

Journal of Cell Science EERHARTICLE RESEARCH rsde 9424)mgtb hshrltdb e2by Nek2 by phosphorylated the be from might Nek2 dominant-negative 1964–2442) endogenous a (residues displace in act not S1B). Fig. to did material appear (supplementary it centrosome not as version did Myc-tagged manner, E a of fragment expression However, of 2A,B). Fig. in ‘E’ 10 bars: Scale respectively. c, and a components. panels linker in outlined intercentriolar regions stable the by of centrosome either and the (green) to rootletin against recruited antibodies is Nek2 1. Fig. treidpneteprmns 5cnrsmsprcniinprexperiment). per ( condition construct rootleti per each C-Nap1, centrosomes for GAPDH, or of 35 plotted (green), hours rootletin experiments, was 48 and independent profile for intensi (red) 1.5 RNAi recovery (three fluorescence C-Nap1 bar: by average The against depleted Scale the nm). or antibodies an shown. encompassing treated with 488 are and mock (ROI) stained laser, images ROI either and interest argon were the together, (100% of that rootletin for cells times region and HeLa seconds three A C-Nap1 from 300 bleached centrosomes microtubules. over was of depolymerize images ROI calculated to Immunofluorescence the then (D) nocodazole before was without collected pre-bleach were or to images with relative three treated and and selected (C) was rootletin centrosomes or (B) C-Nap1 tagged ete netgtdwihst()wti h nieCTD entire the within site(s) which investigated then We m .()Tecnrsm nest fNk nclsdpee si eaiet okdpee el.Dt hwtemean the show Data cells. mock-depleted to relative D in as depleted cells in Nek2 of intensity centrosome The (E) m. c tbln(,)o -a1(,)(e) n ocs 34 ovsaieDA(le.Pnl n r enlargements are d and b Panels (blue). DNA visualize to 33342 Hoechst and (red), (c,d) C-Nap1 or (a,b) -tubulin m .(,)FA nlsso 2Sclstasetytasetdfr2 or ihGFP- with hours 24 for transfected transiently cells U2OS of analysis FRAP (B,C) m. hsooia rti usrt.Ti niae tog letnot albeit strong, a mass or a on indicated phosphorylation serine This Nek2 us substrate. of by for protein allowed preference physiological sites this sequence result, 27 the a Analysis assess of As to 2D). identification (Fig. 2C). phosphorylation the (Fig. threonine to led phosphorylation spectrometry maximal performing ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal A muoloecneiae fHL el tie with stained cells HeLa of images Immunofluorescence (A) nvitro in c tbln(e)adNk gen,a niae.Merged indicated. as (green), Nek2 and (red) -tubulin iaeasy vr4hust achieve to hours 4 over assays kinase n 5 20). 2495 6 nor s.d. ty

Journal of Cell Science htNk o nypopoyae aystswti h T of CTD the within identified sites many we phosphorylates only that not Nek2 sites that match others EERHARTICLE RESEARCH 2496 the on Nek2 by curated phosphorylated were ten and that these, sites Of match studies (www.phosphosite.org). database phosphoproteome reported PhosphoSitePlus been global also have C-Nap1 from of region this in phosphorylation or Nek2 by vitro phosphorylated serine LxxS/T being sample of the immunoprecipitated the sites the had between 14 overlap which identified considerable sites of was There of 2D). identification (Fig. four consensus the phosphorylation, to transfected threonine led To mass from to This position. CTD immunoprecipitation cells. C-Nap1 this the following of at version spectrometry Myc-tagged residue a phosphorylated are subjected hydrophobic sites we a these of of a also out some 16 whether was having with determine +1, There sites at 2E). residue (Fig. hydrophobic 27 a consensus for preference LxxS/T strong an into at leucine falling a for requirement absolute, Fg F.Frhroe 5stso eieo threonine or serine of sites 15 Furthermore, 2F). (Fig. nvitro in and nvivo in ihegtstsietfe from identified sites eight with , 2 ,wt 4o h 7sites 27 the of 14 with 3, nvivo in nvitro in econclude We . n three and nvivo in in W , -a1 u hti slkl epnil o h ukof bulk the in for domain this responsible phosphorylate also likely can cells. kinases other is domain that possible this it on that phosphorylation but C-Nap1, uatwsgnrtdi hc 0o h 7srnsand When 3A). serines (Fig. diffusely 27 was residues CTD-S10D, construct, acidic the this to cells, of HeLa mutated in expressed 10 were domain which phosphomimetic this in how Nek2 a by rootletin examine phosphorylated generated To threonines C-Nap1, 2006). was al., triggering regulate et mutant patches Yang might al., 2000; while et al., phosphorylation oligomeric (Mayor et splitting Mayor large self-assembly, centrosome 2002; premature when forms and Also, of displacement 2000). mainly CTD al., is indicative et the which (Mayor overexpressed, centrosomes CTD, of C-Nap1 with structure isolated associate the secondary that indicated the have studies Previous the for affinity its centrosome regulates C-Nap1-CTD of Phosphorylation ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal a1CDpoenb eobnn Nek2A. indicates recombinant Graph by protein C- CTD GST-tagged Nap1 purified a of phosphorylation of Timecourse A. (C) panel experiments). in mean shown the those show are Data shown. E are to alone A DBD Fragments the AD with with Nek2 CTD and the alone, SNF4, and SNF1 of known interaction the (PJ69-4A), from yeast GAL4 Activity untransformed a fusion. is (DBD) protein domain second DNA-binding domain the activation and GAL4 fusion a (AD) is pair first each The in 1). protein as of assigned interaction (arbitrarily homodimeric Nek2A the is to indicated relative constructs shown of pairs activity the interaction between two-hybrid (2362– Yeast E (B) and 2442). (2262–2368) D (2162–2268), (2062–2168), C are B region (1964–2070), this A of indicated: sub-fragments five 1964– and residues 2442, (CTD), domain C-terminal (blue). The coiled-coils predicted with protein C-Nap1 Nek2. C- by the CTD of Nap1 phosphorylation Multisite 2. Fig. h -a1CDhglgtn eiusta are that residues phosphorylated highlighting CTD of sequence C-Nap1 acid the Amino SDS- (D) an gel. from polyacrylamide excised bands scintillation of by counting determined as (a.u.), units on nteCDpopoyae yNek2 by phosphorylated vitro CTD the sites on phosphorylation found of proportion relative the C- the within CTD Nek2 Nap1 by phosphorylated sites based on of site, phosphorylation analysis consensus Weblogo Nek2 (E) the (purple). both and (red) n hs identified those and nvitro in nvivo in nvitro in nvivo in 32 F endarmshowing diagram Venn (F) . noprto narbitrary in incorporation P 6 oee,i remains it However, . A ceai iga of diagram Schematic (A) ..(he independent (three s.d. (blue), n itiue across distributed and nvivo in nvitro in a clr si D). in as (colors nvitro in hlcl can -helical, yNek2 by in

Journal of Cell Science niGPatbde.Mlclrms ka sidctdo h et I h ecnaeo rnfce el nwihtercmiatCDpoenwsd was protein CTD recombinant the which as an in hours, with cells transfected blotted 24 of western for percentage were cells The immunoprecipitates ( (I) U2OS and left. shown in inputs the is CTD-S10D on and centrosome indicated or Myc, is the Myc–C-Nap1-CTD against (kDa) antibodies with mass using coexpressed Molecular antibodies. (IP) were anti-GFP immunoprecipitated (H) were GFP–Nek2A lysates or Cell (G) indicated. GFP–C-Nap1-CTD (G,H) fusion. AD cerevisiae ( indicated EERHARTICLE RESEARCH itiue ntectpamadfie olclz othe to It localize levels high S2A). at to expressed when Fig. patches failed into material assemble to and supplementary failed also cytoplasm 3B,C; (Fig. the centrosome in distributed ( shown ( is shown Myc, against is 5 against antibodies centrosome bars: with antibodies the Scale stained with construct shown. at stained each are detected centrosomes and mut was of S5D.2 indicated protein views (Student’s the constructs Magnified recombinant in indicated C-Nap1-CTD (blue). mutated the DNA Myc-tagged sites which visualize the blue, to in mutant; with 33342 cells S5D.1 hours Hoechst the with 24 in and for mutated (green), transfected sites Myc Red, were mutant. localization. cells C-Nap1-CTD-S10D centrosome U2OS Myc-tagged and (B) the oligomerization in C-Nap1-CTD mutated sites prevent phosphorylation mutations Phosphomimetic 3. Fig. n niaigitrcinatvt mu,Mle nt)btentepoen hw hr h pe rti a eADDfso n h oe VP16- a lower the and fusion LexA-DBD a was protein upper the where shown proteins the between units) Miller (m.u., activity interaction indicating 5 0) T nrnfce el.Clstasetdwt y–e2 eeue sapstv oto.()Yattohbi sa nL40 in assay two-hybrid Yeast (F) control. positive a as used were Myc–Nek2A with transfected Cells cells. untransfected UT, 100). t ts) D h ecnaeo rnfce el oiiefrlrepthso eobnn rti,tgte ihrpeettv mgsof images representative with together protein, recombinant of patches large for positive cells transfected of percentage The (D) -test). n 5 0.Dt nCFadIso h mean the show I and C–F in Data 50). n 5 0.()Tepretg ftasetdclsi hc h w etooe eeslt( split were centrosomes two the which in cells transfected of percentage The (E) 30). 6 ..(tlattreidpnetexperiments). independent three least (at s.d. eandroltna h etooe rsmbyrcutdby recruited presumably centrosome, the still splitting protein at centrosome CTD-S10D rootletin of the expressing level retained cells low Indeed, relatively 3D,E). a (Fig. only induced and n 5 50). P ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal vle o ahcntutcmae ihtewl ye(T are (WT) type wild the with compared construct each for -values A ceai iga hwn h ten the showing diagram Schematic (A) m .()Tepretg ftransfected of percentage The (C) m. c tbln(e)and (red) -tubulin S. iMcand ti-Myc . tce at etected 2 m 2497 )is m) ant.

Journal of Cell Science T-REadsandwt niMcatbde ihihigtedfuectpamcsani hw npnlE T idtp;U,utetd aashow Data untreated. UT, type; wild Myc–C-Nap WT, with E. transfected panel cell in a shown of is image stain immunofluorescence cytoplasmic representative diffuse A the 3C–E. highlighting Fig. antibodies in anti-Myc mean described with as stained determined and were CTD-KR5E and shown, are constructs fNk ihnteCtria 0rsde fCNp snot is C-Nap1 of residues 80 this C-terminal that the confirmed within site interaction only Nek2 the that of not indicated but self-association. functionality, mutant This retains S10D co-immunoprecipitation protein for 3F–H). the or (Fig. two-hybrid of capacity yeast association experiments either of reduced in loss Nek2 no with a was there had However, mutant Yeast the that S10D S2B). confirmed assays Fig. co-immunoprecipitation and material two-hybrid (supplementary C-Nap1 endogenous ARTICLE RESEARCH 2498 5 a bar: Scale antibodies shown. using are stained centrosomes and of views constructs Magnified (C; indicated (blue). the localization 33342 with centrosome Hoechst stain transfected exhibiting DNA were cells the localization. cells transfected with centrosome U2OS along of (green), and (B) Myc mutant. oligomerization and KR5E C-Nap1-CTD (red) the prevent tubulin in to made sufficient substitutions is basic-to-acidic acidity Increased 4. Fig. of three All S2394. and it S2234 although at (Ahmed S2392, C-Nap1 phosphorylates centrosome in phosphorylates primarily the reduced SIK2 also to 2010). localization is al., its et centrosome regulate to the reported phosphorylated. for sites of of the domain number that the number conclude to this the proportion 3I; we (Fig. of Thus, as ten S2C). affinity to association Fig. five material centrosome from supplementary increased of Fig. mutations loss phosphomimetic material in response of supplementary dose-dependent clear terms a of 3C–E; observed (Fig. that we with Moreover, CTD S2A). compared reduced wild-type modestly a at the only with centrosome was expressed other and that formation each were patch splitting to association, constructs results centrosome similar of these level gave that and levels found comparable mutated S10D We sites the five in 3A). – other mutated the (Fig. had sites mutants which ten CTD-S5D.2, phosphomimetic the and of construct, further five had two which CTD-S5D.1, generated we crucial, these from results that charge mutations. negative increased the by perturbed nte iae I2 a hshrlt -a1adhsbeen has and C-Nap1 phosphorylate can SIK2, kinase, Another be might sites specific more or one whether determine To 6 ..(tlattreidpnetexperiments). independent three least (at s.d. n 5 0,cnrsm pitn (D; splitting centrosome 50), hehl fCNp hshrlto eesr otigrlinker trigger C- to achieve necessary regulate disassembly. phosphorylation to C-Nap1 to Nek2 of Hence, with insufficient threshold cooperate a S3). is might SIK2 the alone Fig. Nevertheless, site Nap1. S2A; to this centrosome Fig. strongly at inducing phosphorylation material localizing and (supplementary protein, patches splitting into wild-type assembling the centrosome, point similar phosphomimetic to a in single behaved manner mutant a the this generated However, of CTD-S2392D. importance we mutant, the alone, test site To cells. S2392 in S2392 phosphorylates that sites phosphorylation Nek2 vitro as in identified our were S2394) in and identified (S2234 were sites these a neuvln hnei hre oaiigdfueyi the in diffusely localizing that charge, mutant in CTD-KR5E, CTD-S10D change the construct, to equivalent manner an this to similar has very CTD, a Strikingly, the in behaved across particular 4A). (Fig. with distributed five associated were mutated glutamate not and we were sites hypothesis, which phosphorylation this displacement arginines, its test or causes To lysines CTD centrosome. increase C-Nap1 an the the that from on reasoned charge we above, negative presented in data the the for on CTD Based C-Nap1 the of affinity centrosome the regulates charge Bulk igekyst fpopoyainta euae C-Nap1 regulates that phosphorylation splitting of a be centrosome to site centrosome unlikely induce is there key not it the that argue did did data single with also these neither Hence, It 4D,E). associate and (Fig. S2A). Fig. patches to material form supplementary failing 4B,C; (Fig. and cytoplasm n ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal hsi ossetwt I2bigtepiaykinase primary the being SIK2 with consistent is This . 5 0)adpthfrain(E; formation patch and 100) A ceai iga hwn h five the showing diagram Schematic (A) nvivo in n 5 0 o h indicated the for 30) nlssbtol two only but analysis m .(–)Percentages (C–E) m. gainst the 1- c -

Journal of Cell Science uati hc h aetnpopoyainststa were that sites phosphorylation ten same the CTD-S10A which a generated in we mitosis, mutant in regulates centrosomes Nek2 by for C-Nap1 affinity of its poles phosphorylation spindle the whether from test it To displaces C-Nap1 of partner. centrosomal Phosphorylation a threshold with a associating reaches from it it until prevents charge that negative overall phosphorylation the cumulative that increases propose we Rather, localization. ARTICLE RESEARCH ees Fg F.Tkntgte,teedt hwta centrosomal that show data these together, Western Taken 6F). post- 6E). time-points (Fig. 9–12-hour (Fig. release the that G1 at confirmed mitosis (pH3) through or weak phospho-histone-3 progressed phase cells and with B cells S cyclin with either for compared blotting of as indicative G2, B, late cyclin in staining, cells B cyclin of strong indicative with fourfold cells revealed in At B pC-Nap1 (12– centrosomal 6C,D). cyclin higher time-points (Fig. with G1 co-staining later time-point, into mitosis 9-hour through at the pass cells decreased when centrosome hours) pC-Nap1 15 the However, hours). at (0–9 staining mitosis towards arrest phase progressed S an and from released were increase cells as steady intensity centrosome a in synchronized revealed antibodies in phospho-specific we Both phosphorylation markers, cells. surrogate C-Nap1 as centrosomal antibodies LLEK 6A,B). analyzed (Fig. this and Nek2 AQDL in or and the C-Nap1 centrosomes Using of microscopy, depletion by detects the immunofluorescence abolished is with antibody staining by As LLEK cells S2064. interphase the against antibody, directed AQDL ‘LLEK’, anti-pC- third antibody, a describe beNap1 we Nek2 Here, a 2012). could of al., addition et that by (Innocenti inhibitor or depletion, cells Nek2 microscopy, for and interphase C-Nap1 by specific in immunofluorescence prevented staining was centrosome in S2179) revealing C-Nap1 against (directed phosphorylated anti- in antibody ‘AQDL’ specific the not pC-Nap1 contrast, was antibody By this microscopy. However, it immunofluorescence 2010). and al., cells blotting, et mitotic in (Mardin site western this is at in C-Nap1 S2421) of phosphorylation the C-Nap1 and confirmed phosphorylated S2417 both for containing specific peptide a against antibody (directed (p)C-Nap1 anti-phosphorylated ‘RRLD’ these The of two antibodies. reported previously have We Nek2. sites cell by against phosphorylated antibodies the phospho-specific three through generated phosphorylation we cycle, C-Nap1 centrosomal mitosis assess towards To increases C-Nap1 of not Phosphorylation its although to from contribute it and, might this displaces progression separation, timing. Nek2 centrosome hypothesis mitotic for blocking by the essential support during C-Nap1 without data of centrosomes these of material poles phosphorylation together, accumulation supplementary that Taken spindle enhanced 5G,H; S4A). (Fig. greatly Fig. at to separation led centrosome (Innocenti C-Nap1 also (R)-21 separation 2012), endogenous small aminopyridine a centrosome al., Nek2, with cells prevent et of of inhibitor treatment not this, molecule with did Consistent 5E,F). it to wild-type (Fig. the poles although of spindle those than protein, on higher strongly threefold the occupied approximately cells, accumulated mitotic levels that higher protein transfected In patches CTD 5D). at large mutant (Fig. cytoplasm into expressed the assembled of when much protein split However, the of level levels, 5C). similar (Fig. a induced and centrosomes the phospho- 5A,B) as this (Fig. centrosome the cells, protein to wild-type interphase recruitment similar in exhibited alanine. levels mutant to null low converted at were expressed residues When acidic to mutated previously ooecm h hehl fpopoyainncsayfor necessary mitosis. phosphorylation of onset of cells the threshold at as required disjunction is the centrosome PP1 increases of overcome inactivation phosphorylation that to Hence, but C-Nap1 mitosis, 2011). towards that al., progress et the propose (Mardin to contributing Nek2 we Plk1 of with recruitment in consistent 2012). centrosomal reduced Plk1, al., was activity of pC-Nap1 et inhibition of (Prosser having upon intensity laboratory centrosomal despite our no the in had Interestingly, pC-Nap1, performed A both assays Aurora centrosomal other or with Cdk2 on staining of to effect Inhibitors pC-Nap1 fourfold 6G,H). a centrosomal (Fig. to led antibodies in This A. increase calyculin G2-arrested inhibitor fivefold treated PP1 we the regulation, with additional cells to subject G1. be and mitosis might late in decreases in it increase before an phases, with G2 dependent and cycle S cell is phosphorylation C-Nap1 neat ihteCtria ein(eius6814)of 648–1141) (residues 2008). region al., C-terminal et C-Nap1 the (Kim of CTD with C-Nap1 the that interacts with indicated associate had studies Structure-function to has reported that marker its centriole been examined proximal-end a we Cep135, centrosome, with the interaction from phosphorylation displacement C-Nap1 which its by regulates Cep135 mechanism for the affinity understand its To regulates C-Nap1 of Phosphorylation oe nwihcnrsm ijnto stigrdb the by support triggered data is Our This component. disjunction linker 2012). major centrosome a Schiebel, C-Nap1, which of and (Bruinsma hyperphosphorylation in Mardin interphase model 2012; during a linker process together al., proteinaceous the the centrosomes et into breaks holds insight that mechanistic that disjunction provide centrosome we of study, this In DISCUSSION inhibitor nocodazole Eg5 with phase the M in arrested or cells in not S-phase- but in cells, associated arrested Cep135 and C-Nap1 endogenous that from found we experiments, Importantly, lost co-immunoprecipitation S4C). reciprocal performing Fig. was was by material Cep135 supplementary whereas C-Nap1 7E; M, direct into (Fig. that G2 retained from Moreover, proteins passed confirmed they two as 7C,D). these cells centrosomes (Fig. of levels synchronized intensities the unchanged centrosomal in centrosomes, the were of split although comparison that the Cep135 found from we of displaced treatments, was these to C-Nap1 al., response et epidermal Mardin In 2003; of Fry, 2013). and in addition (Faragher splitting manner centrosome or Nek2-dependent induce a which overexpression of both (EGF), Nek2 factor split growth prematurely to were centrosomes response which in C-Nap1 in of cells localization in the S5D.2 Cep135 examined and then and We supplementary S5D.1 S4B). 7A,B; Fig. (Fig. the material interaction whereas reduced and exhibited interaction, S10D mutants the lost both Cep135, mutants to KR5E bound although CTD that C-Nap1 found wild-type we Strikingly, the CTD mutants. C-Nap1 therefore CTD wild-type different the we the with and construct, co-precipitate to C-terminal ability its Cep135 tested this Using Cep135. a1itrcinwt e15ta eut rmNek2-induced from results C- we of that Thus, C-Nap1. loss Cep135 cells. of by hyperphosphorylation interphase with triggered in interaction is centrosome Nap1 disjunction centrosome the that at conclude that only Cep135 of found fraction modified is a to that binds possibility preferentially the C-Nap1 raises This shift. with gel-mobility Cep135 partial a of performed exhibited population co-precipitated was the antibodies, immunoprecipitation anti-C-Nap1 when Interestingly, odtriewehrteetn fCNp phosphorylation C-Nap1 of extent the whether determine To ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal S -trityl- L csen SL)(i.7F–H). (Fig. (STLC) -cysteine 2499

Journal of Cell Science EERHARTICLE RESEARCH 2500 10 with medium sta fresh Myc into on release based before against indicated, hours are antibodies cells 16 with metaphase for stained in RO-3306 were proteins tra with Cells transfected were G2 hours. of cells in units) 3 U2OS synchronized arbitrary for (E) (a.u., were protein. intensities cells CTD-S10A pole U2OS the spindle (G) by Mean against formation (F) antibodies shown. patch using are extensive stained cells the and revealing constructs (grayscale) indicated the antibodies with anti-Myc with stained and indicated poles. spindle on against retained antibodies is using C-Nap1-CTD Non-phosphorylatable 5. Fig. si .Aldt hwtemean 5 the bars: show Scale data G. All and G. E in A, as in shown are centrosomes ( ( split shown were is centrosomes centrosome two the the at detected which was in protein indicated recombinant the which in cells c tbln(e)adMc(re) ln ihteDAsanHeht332(le.W,wl ye B h ecnaeo transfected of percentage The (B) type. wild WT, (blue). 33342 Hoechst stain DNA the with along (green), Myc and (red) -tubulin 6 ..(tlattreidpnetexperiments). independent three least (at s.d. . 2 m c m .()Sidepl nest fedgnu -a1wsdtrie n2 eahs el treated cells metaphase 20 in determined was C-Nap1 endogenous of intensity pole Spindle (H) m. tbln(e)adCNp gen,adteDAsanHeht332(le.Mgiidvesof views Magnified (blue). 33342 Hoechst stain DNA the and (green), C-Nap1 and (red) -tubulin )i hw ( shown is m) c tbln(e)adMc(re) ln ihteDAsanHeht332(le.Metaphase (blue). 33342 Hoechst stain DNA the with along (green), Myc and (red) -tubulin n 5 0) T nrnfce el.()UO el eetasetdwt h constructs the with transfected were cells U2OS (D) cells. untransfected UT, 100). A 2Sclswr rnfce ihtecntut niae n stained and indicated constructs the with transfected were cells U2OS (A) n 5 0.()Tepretg fitrhs el rnfce ihteconstructs the with transfected cells interphase of percentage The (C) 50). ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal m e2ihbtr 2,(rDS scontrol) as DMSO (or R21, inhibitor, Nek2 M nn ( ining nsfected n 5 20).

Journal of Cell Science EERHARTICLE RESEARCH niio,R-36 o 6hust retclsi 2 n hnihbtr i gis P clclnA,Ck N62) L1(I23)o uoaA(AuA A mean Aurora the or show 2536) Data (BI untreated. PLK1 UT, (NU6120), shown. Cdk1 Cdk2 are the A), G) a with (calyculin against panel C,D treated PP1 antibodies AQDL, in were against with F; as cells (i) stained panel treated U2OS inhibitors were (LLEK, cells (G,H) experiments). then antibod Cells pC-Nap1 from indicated. and anti-pC-Nap1 minutes. of prepared is G2, 30 the intensities were (kDa) in with for centrosome Lysates mass cells co-stained added (F) Molecular C,D arrest were were staining. GAPDH. in to 8054) Cells B as and hours MLN arrest. cyclin plots (pHH3) intens 16 phase whisker weak H3 centrosome and for S or of phospho-histone Box RO-3306, an strong (E) spread B, inhibitor, from having units. the cyclin hours arbitrary as represent for a.u., 9 scored plots blotted values. for and whisker maximum western released antibodies, Cent to and cells (D). minimum anti-cyclin-B box AQDL the in and The or show intensity indicated (C) indicated. whiskers and centrosome LLEK the as fixed antibodies pC-Nap1 and scored were anti-pC-Nap1 25–75% showing were the hours from except with cells data 48 stained represents 100 for and box in fixed down the times, antibodies 5 knocked where specified bars: anti-pC-Nap1 was at Scale the Nek2 collected insets. of were which as Cells intensities shown in medium. are line fresh centrosomes cell into of released against U2OS views and antibodies Magnified Nek2-shRNA-expressing with A. doxycycline-inducible in microscopy a as immunofluorescence and analyzed by cells analyzed cells. U2OS and G2 Parental fixed in (B) being increases before C-Nap1 hours of 48 phosphorylation Centrosomal 6. Fig. A 2Sclswr rnfce ihsRAoiouloie,a niae,for indicated, as oligonucleotides, siRNA with transfected were cells U2OS (A) c tbln odtc h etooe n nip-a1atbde.The antibodies. anti-pC-Nap1 and centrosome, the detect to -tubulin, m .(,)UO el eetetdwt piioi o 6hus washed hours, 16 for aphidicolin with treated were cells U2OS (C,D) m. ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal c tblnadteat-CNp nioyLLEK. antibody anti-pC-Nap1 the and -tubulin 6 ..(tlattreindependent three least (at s.d. rosome ities, 2501 nd ; ies ,

Journal of Cell Science EERHARTICLE RESEARCH 2502 with stained and fixed being before hours 4 for EGF (B with same. stimulated the or are IPs OE) two (Nek2 the Nek2 for overexpress type. exposures wild but to WT, inputs, antibodies. hours ag the anti-Myc antibodies 24 with for with for performed shown against blotted only transfected are antibodies western were exposures treated, and immunoprecipitations shorter mock and HA, blot, indicated or were anti-Myc constructs Myc cells the Myc–C-Nap1-CTD against the in antibodies using that using except Note (IP) A, indicated. for immunoprecipitated as were mitosis. HA, lysates in or Cell Myc Cep135 cells. C-Nap1, with U2OS interaction into CTD-S10D its or disturbs CTD phosphorylation C-Nap1 7. Fig. rmFadGsoigteetn fc-rcptto nmttclyarse el eaiet htse nSpaearse el.Nc nocodazole; Noc, cells. of S-phase-arrested Quantification (H) in G. seen and that F to B, relative A, panels cells of mitotically-arrested we left in the were co-precipitation on immunoprecipitates ar shown of and is and extent (input) (kDa) measured the blotted. Lysates mass were showing western Molecular antibodies. intensities G indicated. (G) as and Centrosome anti-C-Nap1 C-Nap1, F B1. fixe or and from cyclin were Cep135 (F) cells for against anti-Cep135 case, staining antibodies ( and each with by cells In (PI) blotted confirmed status. metaphase serum was chromosome of pre-immune by status that using population cycle to immunoprecipitated unsynchronized D cell relative an units. and means in arbitrary antibodies, identified a.u., as cells. C-Nap1 were presented mock-treated cells and the telophase Cep135 to and with relative Metaphase stained determined (G2). were hours EGF 9 with or treated (S) or Nek2 overexpressing cells mean the the in C-Nap1 and Cep135 of 6 ..(he needn xeiet) E eaclswr ycrnzdwt obetyiiebokadrlae o or G/) hours 4 (G1/S), hours 0 for released and block double-thymidine a with synchronized were cells HeLa (E) experiments). independent (three s.d. c tbln(e)adCp3 rCNp gen,aogwt h N ti ocs 34 bu) cl a:5 bar: Scale (blue). 33342 Hoechst stain DNA the with along (green), C-Nap1 or Cep135 and (red) -tubulin n 5 0.(,)HK23 el htwr retdi h elccea niae eelsdand lysed were indicated as cycle cell the in arrested were that cells 293T HEK (F,G) 50). A ACp3-T 6814)wsc-rnfce ihMyc–C-Nap1- with co-transfected was (648–1141) HA–Cep135-CTD (A) ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal m .()Cnrsm intensities Centrosome (D) m. WB, C U2OS (C) t show ata stern and d ainst data )As e

Journal of Cell Science rknadcnrsmsaefe osprt n sals the establish and 8). (Fig. separate spindle to mitotic the free is of and linker poles are centriolar opposing oligomerization centrosomes the of of and integrity broken loss this the rootletin, of upon with loss association potentially Upon and Cep135. the protein, interaction, of proximal-end the activities with centriole interaction of crucially, core and, balance oligomerization both the of loss in the shift triggers hyperphosphorylation C-Nap1 a switch. bi-stable to Nek2–PP1 response in occurs ARTICLE RESEARCH idn oCp3,adta erdto fSs6a h n fmtsswudeal -a1rcutetadr-salsmn ftelne sclspass cells as linker the of re-establishment and co recruitment might C-Nap1 C-Nap1 enable l and would rootletin, Sas-6 mitosis with that of possibly hypothesize end centrioles. and the we C Blue, itself at side, to G1. C-Nap1 Sas-6 right-hand leads into with of the kinases, back Cep135, degradation On other mitosis that disjunction. with by and centrosome C-Nap1 phosphorylation Cep135, driving of to and potentially interaction binding linker and the intercentriolar PP1 disturbs the of This of inactivation transition. disassembly with G2/M together the Nek2, at hyperphosphorylation. of C-Nap1 hyperphosphorylation recruitment by Mst2-driven disjunction that centrosome understanding of Induction 8. Fig. One is rootletin component charge. linker presumably related well closely negative oligomerization, the as with C-Nap1, association prevent in as of Self-assembly to increase repulsion. electrostatic is local through this a of through consequence phosphorylation, site-specific rather through C-Nap1 but regulate to not Nek2 due that does suggest change strongly data charge These C-Nap1 interactions. equivalent and on localization consequences an similar surface. had had that domain substitutions as basic-to-acidic construct the mutants, a phospho-site across of as sets distributed well different that are cannot found sites we we However, that these means how information structural tell phosphoproteome of (Helps global lack in domain be The or to this us studies. found by were of either sites cells same mole the in per phosphorylated of many phosphate Importantly, 2000). of al., et moles 13 of incorporation least the catalyzes at confirms Nek2 phosphorylation that suggest multisite that studies of earlier notion This CTD. C- al., C-Nap1 for et Nek2 is of Mardin non- affinity 2005; the Nek2 al., C-terminal regulate et Nap1. directly of its might (Hames Mst2 Mst2 recruitment of 2010), by rootletin. domain phosphorylation centrosome and catalytic through the C-Nap1 stimulated of as C- linker the phosphorylation regulate turn, Furthermore, to with data; it in through interacts enabling (our C-Nap1, specifically are, integrity of Nek2 acids Nek2 amino components kinase 80 2012). terminal protein linker al., the et These Spalluto recruiting 2006). 2005; for al., et al., responsible (Bahe filaments centrosomes forms et two rootletin centrioles the 2000), Yang of between al., ends extend et can proximal Mayor that that 1998a; the evidence al., at the et further focused of (Fry provide is components C-Nap1 also stable although They as act linker. rootletin intercentriolar and C-Nap1 proteins e2cnpopoyaemn ie ( sites many phosphorylate can Nek2 coiled-coil large the that demonstrate firstly results Our . 5 ihnthe within 25) , 50-kDa fteetrersde tti oiin lo h etd screen peptide the none Also, had sites position. 27 this the at of 13 residues fact, three In position. these this of at M or strong position F the having at was leucine analysis al., our for et from (Alexander preference clear studies was what our However, from 2011). identified Nek2 consensus the with for LxxS/ of sequence motif T consensus a preferred of peptide definition than T the the rather to led substrate This protein analyze libraries. a on to based phosphorylation us allowed further (multi)-site would to events disjunction. centrosome subject we these and disassembly of be Hence, linker Either promote equally 2014). Nek2. might by al., these phosphorylation while proteins et centlein, linker and rootletin (Fang other with perturb C-Nap1 Cep68 might of interaction phosphorylation to the Nek2-mediated bridge and that CTD a hypothesize C-Nap1 the as with group al., acts interacts et Yang another centlein 2005; N-terminal al., Meanwhile, et whereas (Bahe an 2006). C-Nap1 of with rootletin, CTD, the rootletin not but with of domain, interaction – preferential – CTD C-Nap1 reported of group the domains One multiple interact. including between rootletin interaction It and linker. strong C-Nap1 the found of how integrity though to unclear factor contributing is major a be to likely nraei hspopoyaincudrfeticesdcentrosome increased reflect steady could The phosphorylation centrosome. this the in to increase recruited and when expressed onwards, first in phase is S Nek2 increase from substantial present is level further phosphorylation low C-Nap1 a a of that to propose led We phosphorylation. of inhibitor C-Nap1 incubation centrosomal PP1 phosphorylation Thirdly, a G2. of to with S cells from extent progressed cells the as the increased Secondly, about of occurred. centrosome points phosphorylation before disjunction interphase interesting in Firstly, detected was cohesion. of C-Nap1 centrosomal centrosome number of in a centrosomes regulation revealed at phosphorylation This C-Nap1 cells. of extent phospho-site the address influence can proteins. position. factors intact this in at additional consensus selection proline on a but information had useful sequences, identified provide we screens and peptide sites +1, Hence, position the at of proline two against yet discrimination strong a found W W h ethn ieo hsshmtcmdlsmaie u current our summarizes model schematic this of side left-hand The dniiaino ag ubro hshrlto sites phosphorylation of number large a of Identification h eeaino hsh-pcfcatbde loe sto us allowed antibodies phospho-specific of generation The RHX steotmlmtffrNk,wihft eywell very fits which Nek2, for [F/L/M]xxS/ motif identified optimal screen the as peptide-library [R/H/X] oriented An . ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal 2 ,wt oeo h 7sites 27 the of none with 3, aigto eading -Nap1 pt for mpete 2503 from

Journal of Cell Science hstm hog d1mdae erimn fihbtr2 and inhibitor-2, of recruitment Cdk1-mediated through at time inhibited this both is PP1 does Indeed, to disjunction. mitosis required centrosome threshold at precipitate the PP1 overcome C-Nap1 of of centrosome of removal phosphorylation and/or the trigger inhibition stoichiometry upon to phosphatase counteracting Only the PP1. overall required of result a as that Mst2. likely the most disjunction, by below phosphorylation interphase, is to phosphorylation during response in However, Nek2 of recruitment ARTICLE RESEARCH 2504 anti-parallel through through bipolar centrosomes amplified forcing with apart cells of in capable even centrosomes assembly are spindle motors drive known of These (also sliding. action to microtubule hKlp2 the and or KIF15), on Eg5 prophase as depend as in such both motors, centrosomes they microtubule-based the of differ, the might separation to Although prometaphase for 2010). access required Medema, have events and microtubules (Tanenbaum once prometaphase, or breakdown, envelope during nuclear to prior can prophase, Centrosomes in 2012). early al., separate et al., Silkworth 2013; et al., Kaseda et Mardin 2007; 2012; Murray, and (Indjeian separation centriole centrosome next the with of of start the recruitment concomitant at the linker cycle. the cell allows mitosis of 2007), re-establishment Sas-6, and late al., C-Nap1 of et in degradation (Strnad are disengagement that We occurs centriole hypothesis manner. the exclusive which the in nascent mutually proteins exploring times a two of different in currently these potentially, at ends and, Hence, Cep135 is cycle centrioles. with but proximal interact parental duplication might the on centriole of present to process not the al., et localizes during (Lin procentrioles protein Sas-6 Sas-6 C-terminal the with same Nek2-induced 2013). interacts also the from key Cep135 Intriguingly, of results region interaction a C-Nap1. that of of that Cep135 not loss phosphorylation and the propose but is C-Nap1 therefore cells disjunction between centrosome asynchronous We in in cells. mechanism endogenous interact arrested that show the Cep135 mitotically we of Moreover, and nature 5.72). C-Nap1 acidic of Cep135. to (pI the due CTD is of with this Cep135 that consistent domain suggests repulsion, mutant C-terminal KR5E electrostatic the with the data specifically our with CTD Again, hypothesis, C-Nap1 this interaction the extend and of disrupts C-Nap1 support phosphorylation of data that recruitment Our the revealing 2008). for al., the platform et at a present (Kim as protein It acts centriolar ends, centrosome. core a proximal the Cep135, from that displacement reported was its to led negative phosphorylation of threshold the disassembly. or linker achieve the for help localization required site with to charge can centrosome this Nek2 consistent sites different with on of is phosphorylating cooperate kinases effect mutation model additional that no that our notion found or However, We little oligomerization. 2010). had kinase SIK2 al., alone the by et phosphorylated be (Ahmed to reported was Nek2 that by S2392 phosphorylated not were that mitotic analysis at peak not 1995). does al., and et G2 (Fry and entry Nek2 S of somewhat through activity constant the catalytic intrinsic remains explain the data that Plk1-mediated These observation was 2011). surprising inhibition. phosphorylation al., C-Nap1 Plk1 by et that upon Mardin found reduced complex 2002; we al., this, with et Nek2–Mst2 Consistent (Eto Mst2 the of phosphorylation from released swl ssiuaigls foioeiain C-Nap1-CTD oligomerization, of loss stimulating as well As the from identified were sites other of number A cuaesgeaino hoooe eurstimely requires chromosomes of segregation Accurate nvitro in including , nvivo in (Invitrogen). CGCAAUU-3 CCAGGAGAAGAUCAGCAAUU-3 infcn otiuint uo rgeso HyadadFry, 2013). and al., et (Hayward Zhou progression 2006; tumor a make to could cancers contribution human in significant observed frequently overexpression is affect the that Nek2 Thus, could of division. that cell and separation migration Nek2 centrosome polarity, of cell premature expression to elevated lead Equally, 2010). might al., al., inhibitors), et et for Mardin (Mardin kinesin essential stability 2013; become chromosome low-dose activity and assembly timely Nek2 of spindle and bipolar its presence linker is the when the activity of apart disassembly motor in linker when (e.g. However, these the reduced occurred. by tear not provided has to forces disassembly Nek2, sufficient separating the are of that motors absence speculate the can progression. mitotic One block in not 2009). does can depletion separation Nek2 motors al., why explaining centrosome and et microtubules full (Ganem of promote action clustering concerted centrosome the Moreover, of mechanism a rti MP ysblnn notepAcXvco,epesdin Cep135 maltose-binding expressed chromatography. affinity vector, N-terminal resin pMALc2X amylose an the by purified into to and CTD subcloning bacteria rootletin by fused The (MBP) was respectively. protein tag, or 1651–2018) GST tag Myc N-terminal (residues N-terminal an an with with or cells 20 bacteria mammalian pLeics in into expression subcloned for was pGEX 1964–2442) (residues CTD C-Nap1 The mutagenesis and construction Plasmid METHODS AND MATERIALS yae eepecerdfr3 iue t4 at minutes 30 for immunoprecipitation, pre-cleared For blotting. were western lysates were and lysates SDS-PAGE and assay, by (BCA) analyzed acid 0.5% bicinchoninic X-100, the Triton using 1% by SDS, determined were 0.1% concentrations EDTA, Protein inhibitors. mM protease Tris-HCl NaCl, with 1 mM deoxycholate) sodium mM NaCl, (50 mM 150 buffer 150 RIPA 8.0, 7.5, or pH X-100) pH Triton 2012), Tris-HCl 1% al., azide, mM et sodium (50 (Zalli 0.02% buffer buffer NEB in lysis indicated single-detergent as prepared were blotting lysates western Cell and immunoprecipitation lysis, Cell C-Nap1 AM4631), (Ambion, GAPDH (5 against oligonucleotides siRNA RNAi serum, calf fetal 10% 100 modified 37 and with Dulbecco’s penicillin in supplemented IU/ml grown 100 (DMEM) were cells medium 293T Eagle’s HEK and U2OS HeLa, transfections and culture Cell Genetailor Site- the 2008). using al., et performed (Kim was previously mutagenesis described directed been have constructs atra xrsinadprfcto eepromdacrigto according and performed sequencing, were DNA purification by procedures. and verified standard expression were manufacturer’s the constructs bacterial to All according (Invitrogen) instructions. Kit Mutagenesis Directed ;MrliadNg,20) irtblswr eoyeie with 2005). depolymerized al., were et (Hames Microtubules previously 2001). described as Nigg, nocodazole and Meraldi A; (50 (1 Cdk2 A (10 aurora 2012), Nek2 Chem), for al., Med indicated et Axon as 2536; Prosser used BI incubated were nM were inhibitors (100 Specific cells Plk1 respectively. arrest, hours, G2 16 and for G1/S For 1.6 were cells with hours. and 24 instructions, after manufacturer’s the analyzed to according (Roche) HD nuae ihatbde gis y 110;Cl Signaling Cell (1:1000; Myc against were pre- antibodies supernatants and been removed with then had were that Beads incubated buffer. Healthcare) lysis in (GE washed Protein-A–Sepharose or (Sigma) 9 ˚ -GGAAGAGCGUCUAACUGAU-3 ne %CO 5% under C ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal m m /lahdcln(im)ad10 and (Sigma) aphidicolin g/ml R-1 noet ta. 02 n P 1 Mcalyculin nM (10 PP1 and 2012) al., et Innocenti (R)-21; M 9 hrao)wr rnfce sn Oligofectamine using transfected were Dharmacon) ; 2 rnin rnfcin eepromdwt Fugene with performed were transfections Transient . m /lsrpoyi.Clswr rw at grown were Cells streptomycin. g/ml m U10 adatee l,2004), al., et Hardcastle NU6120; M 9 ;5 9 ign n otei (5 rootletin and Qiagen) ; 9 -CAGGGAGAUUGUCACC- m ˚ O30 (Calbiochem) RO-3306 M ihProtein-G–agarose with C m L 8054; MLN M TM 9 -CAG- Site-

Journal of Cell Science xie rmdidgl.Ecsdbnswr mesdi lof ml 3 in and immersed Elmer) (Wallace-Perkin were scintillant bands liquid Excised 2 Substrate gels. HiSafe autoradiography. 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S., Bahmanyar, Leiss, J., C. Wilkinson, D., Y. R., Stierhof, J. S., Bahe, Hutchins, B., Hegemann, A., B. Joughin, D., Lim, J., Alexander, rm meitl eoepoolahn n a xrse sa as Adobe expressed with processed was were Images 4.0. and 2004). Photoshop the al., of photobleaching et value (Thompson intensity before percentage fluorescence the intensity immediately by divided fluorescence frame frame the given as a calculated of value then was recovery fluorescence neato ciiy(ilruis a esrdusing measured was plasmids units) assays. (Miller and (VP16-AD) acids, activity amino appropriate interaction lacking medium pLEICS-82 dropout synthetic on growth and (GAL4-AD) into transformed pACTII (LexA-DBD) and constructs pLEICS-81 (GAL4-DBD) into using pGBDU transformed performed plasmids either were in assays generated interaction two-hybrid Yeast analysis two-hybrid Yeast he,A . u . enns .B,Eeamgaa,D,Cplo L., Capalbo, D., Etemadmoghadam, B., N. Jennings, Z., Lu, A., A. Ahmed, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.142331/-/DC1 online available material Supplementary material Supplementary Deposited release. Research. immediate Cancer for International PMC for Cancer in Association to Council; the 082828 Research and number Sciences UK; [grant Research Biological Trust and Wellcome Biotechnology The the by A.M.F.]; supported was work This manuscript. Funding the wrote and work experimental the hypothesis, of the bulk devised A.M.F. the revision. supervised manuscript to contributed and M.L. of experimental work supervised the K.R. revisions. performed manuscript J.E.B. to contributed and and F.S. experiments M.D.S., D.-M.C., S.L.P., R.S.H., M.L., T.H., contributions Author interests. competing no declare authors The interests Competing mapping. imaging, phospho-site with and support sequencing for DNA (CBS) construction, acknowledge Services plasmid We Biotechnology prediction. Core Winter structure Leicester secondary Anja the for and UK) cDNA Leicester, Erich rootletin of production, for (University antibody Switzerland) Balca with Basel, and help (Biozentrum, Schiebel for Nigg Elmar Germany) UK), Heidelberg, Leicester, (DKFZ, of Mardin (University Tsai Alex thank We Acknowledgements hr niae sn Student’s using indicated where o h O tagvntm seult In to equal is time given a this at and intensity ROI photobleaching, fluorescence the after normalized that the frame such a frames, first for all the from and subtracted as an was fluorescence (U2) set value background determined of was The P1/U2. (Bk) was intensity using intensity was cell calculated fluorescence was (P1) the (In) The ROI intensity in software. photobleached ROI the Leica unbleached of using time- intensity each For determined fluorescence period. 30–90-second the a laser over point, argon bleaching (488-nm after second power) every 4% taken at was image An interval. 1-second 451-463. ucin etooisbig e understanding. new brings centrosomics separation. function: centrosome in I. A. involved Barth, and substrate J. Nek2 W. Dev. Nelson, a J., P. is Casey, beta-Catenin D., E. centrosome Salmon, M., in Winey, functions and filaments centriole-associated of cohesion. signaling. forms selection mitotic Rootletin context-dependent negative for and al. basis et positive the A. Signal. Sci. E. is with Nigg, motifs combined O., phosphorylation Hudecz, exclusivity F., Sessa, Spatial F., Ivins, T., Ehrenberger, aao .O,BroaMri,N,L,X . ia-ei,P,Lopez- cancer. P., ovarian provides in that Vivas-Mejia, therapy formation for spindle F., target mitotic bipolar X. potential for Group a required Study Le, kinase Cancer centrosome Ovarian N., Australian a al.; Barbosa-Morais, et G. O., Berestein, R. Jacamo, 22 ora fCl cec 21)17 4320 doi:10.1242/jcs.142331 2493–2506 127, (2014) Science Cell of Journal 91-105. , .Cl Biol. Cell J. 4 ra42. , P vle eecluae for calculated were -values .cerevisiae S. 171 27-33. , acaoye cerevisiae Saccharomyces 4.Itrcigcoe eeslce by selected were clones Interacting L40. , t -test. 20) etooeboeei and biogenesis Centrosome (2007). acrCell Cancer t 1/2 a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 2 rpretg recovery percentage or k h mutof amount The Bk. J94,o in or PJ69-4A, , 18 b 109-121. , -galactosidase 21) I2is SIK2 (2010). (2005). (2008). (2011). 2505 8 ,

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Faragher, ua e2poenkns,amme fteNM aiyo elcycle cell of family NIMA the of member a kinase, protein candidate regulators. Nek2 and Nek2. human protein kinase protein coiled-coil cycle-regulated centrosomal cell 1563-1574. the novel of a substrate C-Nap1, (1998a). homolog human nidulans. potential Aspergillus a of 12905. NIMA kinase, regulator protein mitotic Nek2 the the of of regulation cycle spindles. cell and mitotic bipolar of formation for required Cell is and disjunction and C-Nap1 between interaction an Cep68. bridging by cohesion centrosome maintains centrosomes. of motility and positioning aea nrni ist ireto h spindle. the on biorient to is bias centrosomes, intrinsic an to have localized kinase 1. phosphatase protein protein cell-cycle-regulated to complexed a (Nek2), 2 cohesion. centrosome for required 1100-1107. component linker centrosome cancer. al. et 2. J. and R. cyclin- 1 Griffin, of T., kinases inhibitors B. dependent potent Golding, derivatives: E., O6-cyclohexylmethylguanine A. substituted Gibson, A., J. Endicott, cohesion. centrosome regulates signalling Wnt euae rti hshts- opee ihNm-eae iaet induce to kinase separation. NimA-related centrosome with complexed phosphatase-1 protein regulates 426. ltompoeno -A1friscnroa localization. centriolar its for C-NAP1 3692-3700. of protein platform mitosis. of the fidelity the and by speed the both increases together assembly maintained units cytoskeleton. microtubule independent structure-activity the of : two dynamics kinase centrosome: mitotic interphase the of activity. cellular inhibitors and biology, al. structural hybrid relationship, et T. Hardy, selective L., Pickard, and M., Westlake, K., Boxall, regulation. space. and time in off and on kinase-1 like ecn ipro e2kns xiisayia ofrainlexchange conformational atypical exhibits kinase Nek2 of M. dynamics. Pfuhl, zipper and M. 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