Teeatoscnrbtdeulyt hswork this to equally China. contributed 100101, authors Beijing Biology, *These Science, Developmental of and Academy Genetics Chinese of Institute Biology, Developmental 00 Australia. 3010, ihlsR Lim R. association Nicholas microtubules the regulating spindle in with A WDR62 Aurora of and JNK for roles Opposing ARTICLE RESEARCH eevd3Jn 04 cetd5Dcme 2014 December 5 Accepted 2014; June 3 Received ` in most the mutations Importantly, 2009). 1 Woods, (Mahmood pathogenic and Thornton proteins 2011; identified al., spindle-pole-associated et have and MCPH, the in centrosome- observed of studies birth determinants at size as molecular genetic brain and known defining head reduced condition In Thornton severely 2009]. hereditary (MCPH); Woods, the microcephaly and insufficiency primary in growth recessive observed in autosomal conversely, that observed or, as (as [such 2010) overgrowth tissue Knoblich, differentiation in cancer; result and might Bellaı proliferation mechanisms between and to determinants balance (Morin fate cell the of segregation control the cell regulate mammalian can during positioning division and assembly spindle Mitotic INTRODUCTION Mitosis Spindle, A, kinase Aurora JNK, WDR62, WORDS: KEY in of AURKA and AURKA-mediated association. JNK localization regulation spindle for its roles that complex determining spindle opposing highlight with localization, and studies the WDR62 Our of for complex mitosis. during required in WDR62 was were phosphorylation (AURKA) WDR62 kinase in A Aurora that resulted and identified cell we signaling contrast, of In irrespective JNK stage. cycle microtubules of to localization loss WDR62 regulated constitutive and negatively association, T1053 JNK- However, WDR62 microtubule pole. of spindle the phosphorylation to not mediated MAPK9), but as MAPK8), known as (also known JNK2 (also neurogenesis, JNK1 during recruits complex functional specifically WDR62 WDR62–JNK1 cell-cycle- a the with regulates for agreement requirement region association, In microtubule C-terminal compartmentalization. for dependent disordered required the is WD40- WDR62 whereas the of that demonstrate region we repeat Here, unknown. regulatory are the mechanisms although for distribution, requirement spatiotemporal crucial WDR62 a precise highlighting mislocalization, mitotic WDR62 during to in differentiation lead mutations neuroprogenitor Microcephaly-associated and development. division brain required cell protein pole normal spindle a for is (WDR62) 62 protein WD40-repeat ABSTRACT fMlore I 00 Australia. 3010, VIC Melbourne, of hn-egAng Ching-Seng uhrfrcrepnec ([email protected]) correspondence for Author eateto iceityadMlclrBooy nvriyo ebun,VIC Melbourne, of University Biology, Molecular and Biochemistry of Department 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,5750doi:10.1242/jcs.157537 527–540 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. 2 i2 oeua cec n itcnlg nttt,University Institute, Biotechnology and Science Molecular Bio21 ce 01.Dfcsi pnl regulatory spindle in Defects 2011). ¨che, 1,2, 2 ihlsA Williamson A. Nicholas , ,Yon .C Yeap C. Y. Yvonne *, 3 tt e aoaoyfrMolecular for Laboratory Key State 1,2, ,Trs .Zhao T. Teresa *, 2 hhn Xu Zhiheng , 3 ai .Bogoyevitch A. Marie , eet ihnisNtriu htaepeitdt form to predicted are that N-terminus its within repeats rm-hf uain dniid(ihlse l,2010). al., et (Nicholas identified mutations frame-shift with gene, associated ioi pnl sntflydfnd rvossuiso the on studies with Previous WDR62 expressed defined. ectopically the a missense fully of on suggests not activity WDR62 distribution which of intracellular is addition, 2014), JNK function progression spindle al., precise In require the et mitotic 2014). mitotic although Xu WDR62 role, al., 2012, signaling of al., normal et WDR62 et contributions Chen (Bogoyevitch 2010). mitosis for mitotic 2012, al., al., the during required et et Nicholas (Bogoyevitch spindle-pole-localized is 2012; al., expression but cytoplasmic dynamic et is predominantly (Bogoyevitch interphase WDR62 being of al., in cell-cycle-dependent, distribution et Wasserman intracellular and signaling 2011; on The stress-activated al., motif 2010). coordinate et defined to (Cohen-Katsenelson a through pathways region (JNK) C-terminal binds kinase Indeed, that the protein N-terminal 2010). scaffold c-Jun a al., as to et identified originally (Wasserman serine/threonine was kinases cyclin- WDR62 (CDKs) and contain proline-directed (MAPKs) kinases kinases to dependent protein by mitogen-activated predicted as targeted such is motifs WDR62 phosphorylation of C-terminal disordered largely region the contrast, In structures. propeller the towards WDR62 by contributions determination. fate key and neuroprogenitor highlight 2014). proliferation al., studies et (Xu reduced These of neurons immature in into support differentiation resulting premature In and regions, ventricular mutations. neurogenic subventricular the null in events be division neuroprogenitor to is predicted expansion this, are cell MPCH stem most to neural that requirement observations during the The by largely reinforced expression formation. albeit WDR62 brain multiple, for in during WDR62 functions for undefined, requirement cortical gross a of indicates spectrum broader (Bilgu a to malformations linked also are mutations euainaecuilfcosi erlse elsria and/or survival and/or cell stem architecture neural decisions. in spindle fate factors these mitotic crucial progression, are that disease regulation 2002; MCPH suggest (WD40- al., pole to observations spindle et contribute WDR62 of (Bond might functions MCPH2) and proteins non-mitotic as Although MCPH5) known 2010). also Wollnik, microcephaly as 62; spindle-like protein known repeat (abnormal also ASPM associated; as pole such spindle encode proteins genes MCPH-associated mutated commonly 1,2 D6,alre( large a WDR62, WDR62 a .Yip Y. Yan , WDR62 nutero in MCPH stescn otcmol uae MCPH- mutated commonly most second the is ee otismlil D0poeninteraction protein WD40 multiple contains gene, elto fWR2atr h ymtyof symmetry the alters WDR62 of depletion uain htatrdeouinrl conserved evolutionarily altered that mutations 1,2 , h .Wong C. Shu , vre l,21;Y ta. 00,which 2010), al., et Yu 2010; al., et ¨var 7 D)mlidmi rti rdc of product protein multi-domain kDa) 175 . 0nnes,mses n truncating and missense nonsense, 30 1,2 n oii .H Ng H. C. Dominic and 1,2 WDR62 a Xu Dan , uain linked mutations 3 , WDR62 1,2, ` 527 b -

Journal of Cell Science ioi ucin htlkl otiuet iaycl fate cell binary to its for contribute required likely is that localization WDR62 functions of Thus, mitotic 2014). divisions control al., cell et precise (Xu neuroprogenitor the depletion WDR62 altered embryonic MCPH-associated by rescue 2013). induced to al., an failed from et isolated mutants pole (Farag cells spindle primary patient spindle in in defects MCPH reported reduced Similar also 2010). were substantially al., localization or et (Nicholas lost accumulation revealed residues ARTICLE RESEARCH o-fiiyascaino D6 ihmcouue in microtubules with 528 WDR62 cells of live indicating shown), association in (not fixation observed chemical low-affinity only following association lost was was and WDR62 microtubules interphase Moreover, or with 1D,E). tubulin–GFP (Fig. by 1C). marked tubulin–mCherry (Fig. microtubules filaments with cytoplasmic colocalized These as a WDR62–GFP revealed of However, microtubules 1C). proportion interphase (Fig. of nucleus stabilization the taxol-induced from excluded and cytoplasmic transition. metaphase-to-anaphase spindle and entry with precisely mitotic correlating specific, association with stage anaphase is WDR62 by mitosis during Thus, cytoplasm microtubules the 1B). to (Fig. returned II/telophase WDR62 at 1A; transition I. (Fig. and anaphase localization segregation into chromosome entry metaphase, following WDR62 and decreased prometaphase mitotic and S1A). throughout of persisted Fig. poles and separation spindle material breakdown following after markedly supplementary envelope evident increased We this first nuclear and mechanisms. centrosome-nucleated centrosomes was with duplicated signaling how cell microtubules association mitotic with reveal WDR62 (astral) coordinated that to and observed are stages progression or movements mitotic cycle GFP intracellular addressed expressing during of WDR62 have cells resolution distribution temporal single We improved WDR62 of known. with imaging WDR62 not mCherry-tagged live rapidly are how through between and occur this when shifts precisely transitions but 2010) these localization al., al., et et (Bogoyevitch Nicholas WDR62 2012; compartments cytoskeletal division, and cytoplasmic cell During metaphase-anaphase and transition entry mitotic is with microtubules coordinated with association WDR62 Increased RESULTS decisions rca n poigrlspae yJK n AURKA- and JNK- by mechanisms played signaling roles define phosphorylation. opposing mediated to the and highlighting entry. first distribution, crucial mitotic spatiotemporal the WDR62 during are regulating localization studies that spindle These (AURKA) WDR62 kinase demonstrated A Aurora increased mitotic by association we by, association phosphorylation regulated WDR62 and JNK with, regulation, contrast, negatively In although was phosphorylation. mitotic JNK-mediated association Furthermore, for microtubule 2014). WDR62 mitotic required 2012; al., al., regulation, is for et et spindle (Bogoyevitch requirement development Xu in neural essential and interaction spindle progression the to WDR62 WDR62–JNK MAPK8) with that cytoskeleton. the as time consistent known first microtubule (also microtubules, the JNK1 the recruits for specifically demonstrated with we Importantly, association cell-cycle- WDR62 on impact dependent events signaling by regulation their undefined. remain WDR62 of distribution spatiotemporal nnndvdn el,WR2GPwspredominantly was WDR62–GFP cells, non-dividing In nti td,w eeldhwtedmiso D6 and WDR62 of domains the how revealed we study, this In nvivo in oee,temcaim htrglt the regulate that mechanisms the However, . WDR62 eeldb ao ramn,wihddntehnemicrotubule enhance cytoplasmic as not binding, did remained which microtubule treatment, and in taxol by defects pathogenic pole revealed these showed failed addition, spindle mutations mutants In 1F). WDR62 MCPH the (Fig. division WDR62 cell to throughout that resulting localize observed defects to also proliferation we depletion neural study, rescue WDR62 small to from the fail that mutants likely fixation. also chemical following observed is preserved would interphase be It in not previously protein 2012). microtubule-associated of not samples al., proportion to fixed et in had bind tubulin microtubules (Bogoyevitch with colocalization We to interphase protein to to endogenous WDR62 undetermined. extent binds the of WDR62 remains However, capacity endogenous interphase. which the during even indicate microtubules, microtubules. in studies with tubulin S1C), WDR62 fraction of Fig. Our association alter tubulin direct material a polymerized with (supplementary not consistent the analysis did sedimentation with our proportion pelleted WDR62 a WDR62 S1B), of of Fig. material inclusion (supplementary polymerization the Although euae n slkl motn o D6 ucin in functions WDR62 for tightly important of is the likely the development. that WDR62 is association neural demonstrate notion of and the microtubule localization thus regulated reinforcing intracellular the entry, findings stage-specific mitotic of as supplementary during Our WDR62 1F; enhancement mutants (Fig. S1D). WDR62 specific Fig. WDR62 wild-type the material MCPH-associated for observed of colocalization ihmcouue,w eosiue eobnn D6 with WDR62 WDR62 of recombinant tubulin association reconstituted purified direct we the microtubules, assess with To cells. non-dividing -emnlW4 eetrgo fWR2wsnecessary C- the WDR62. cell-cycle- was to of attributed whereas terminus be WDR62 could association, regulation of microtubule spatiotemporal the dependent for Thus, region 2B,C). sufficient (Fig. repeat and stage GFP-tagged WD40 cycle contrast, the cell N-terminal in In of localized was regardless 2). region, (Fig. cytoplasm repeat WD40 region the an lacking required repeat WDR62-C, microtubules Fig. WD40 with material association intact supplementary WDR62 in Thus, cells images S1E). non-dividing complete and representative a mitotic 2A; in and (Fig. association WDR62–GFP localization microtubule the of cytoplasmic loss from in deletion resulted domain construct, of WD40 truncation Further or 2C). WDR62-N, (Fig. localized 2B). interphase WDR62-N (Fig. during of microtubules cells 2012). proportion to live the decorated increased in al., treatment interphase Taxol prominently during previous et filaments WDR62-N our microtubule with GFP-tagged (Bogoyevitch accord Interestingly, in analysis localization not immunofluorescence was spindle Ser/ 2010), for al., putative C-terminal et and required (Wasserman the JNK-binding sites that includes phosphorylation which Thr-Pro indicating N- WDR62, C-terminal 2A,B), of (Fig. the half 842– decorated spindle the WDR62-N mitotic residues or GFP-tagged that the (WDR62-C, lacking showed 1–841) we microtubule region Firstly, residues 1523). rich 2A) Fig. truncation in WD40-repeat (WDR62-N, terminal WDR62 in region involved GFP-labeled disordered (illustrated domains generated mutants WDR62 we association, identify To association microtubules WDR62 with of analysis mapping Domain rvossuishv hw htMP-soitdWDR62 MCPH-associated that shown have studies Previous ora fCl cec 21)18 2–4 doi:10.1242/jcs.157537 527–540 128, (2015) Science Cell of Journal nvitro in splmnaymtra i.S1B,C). Fig. material (supplementary nvivo in X ta. 04.I this In 2014). al., et (Xu

Journal of Cell Science EERHARTICLE RESEARCH hshrltsrsde ihnteCtria afof half the cells, C-terminal In 2012). the al., et within (Bogoyevitch (WDR62-C) WDR62 residues previous characterized phosphorylates Our been 2010). vitro al., not et in Wasserman have 2012; al., potential function et (Bogoyevitch and to by the contributions localization their phosphorylated and targeted mitotic is investigated sites specific WDR62 of the although signaling. we association JNK, JNK the microtubules, by regulating contributions with mechanisms the WDR62 considering In regulates association negatively microtubule WDR62 of phosphorylation JNK hshrlto tde aeidctdta JNK that indicated have studies phosphorylation apn yms pcrmtyo eobnn WDR62-C stress recombinant of or spectrometry mitotic mass by phosphorylated Phosphopeptide by altered S2A). WD40 Fig. mapping not material N-terminal the (supplementary was the WDR62 stimulation of containing (WDR62-N) endogenous mobility WDR62-C mutant Moreover, the repeats for WDR62 contrast, truncated In observed of S2A). a S2A). shift Fig. material shift the (supplementary Fig. with stimulation mobility stress correlated hyperosmotic material was or phosphorylation-dependent arrest WDR62-C mitotic (supplementary of by shift induced mobility phosphorylation-dependent ora fCl cec 21)18 2–4 doi:10.1242/jcs.157537 527–540 128, (2015) Science Cell of Journal nvitro in yatv N1ietfe he putative three identified JNK1 active by soitdWR2mses (V65M, missense WDR62 associated MCPH- GFP-tagged by microtubule localization Defective (F) cells. AD293 non-mitotic taxol-treated in bundles (arrows) microtubule with colocalized WDR62 is Filamentous (D,E) the panel. in lower magnification is higher panel at middle shown the in indicated of interest area highlighted The interphase. during (arrows) filaments to cytoplasmic localized partially was GFP (10 treatment Taxol (C) WDR62. centrosome- associated of levels reduced highlight Arrows coexpression. by H2B–mCherry determined were transition anaphase and separation Chromosome transition. metaphase-anaphase undergoing in cells WDR62 GFP-tagged of pole localization spindle Decreased (B) association. spindle indicate Arrows fluorescence imaging. live-cell by revealed entry was mitotic during with microtubules association astral their and cells AD293 in coexpressed were tubulin–GFP mitosis. and interphase during with microtubules associates WDR62 1. Fig. as 20 bars: taxol-treated (10 or mitotic in mutants L1414LfsX41) (V1314RfsX18, frame- shift truncated and D511N) R438H, m m ,3 i)rvae htWDR62– that revealed min) 30 M, ,3 i)itrhs el.Scale cells. interphase min) 30 M, m A D6–Cer and WDR62–mCherry (A) m. 529

Journal of Cell Science EERHARTICLE RESEARCH 530 following mitotic phosphorylated T1053 modestly nocodazole-induced also WDR62 to was but endogenous response synchronization that in phosphorylated demonstrated was and specific material S2D) (supplementary with Fig. T1053A this substantially specificity WDR62 its expressed validated ectopically was on antibody, WDR62 mutant phospho-T1053 the investigation our T1053A in of reduced our WDR62-C phosphorylation JNK1-mediated recombinant focused site. highly S2B,C). phosphorylation we the Fig. material including (supplementary S1353), Thus, residue T1273, T1053 (T1053, conserved sites target JNK nvitro in iaeasy(i.3) egnrtda generated We 3A). (Fig. assay kinase yJKihbto Fg C.Tu,WR2T03was T1053 JNK WDR62 by targeted Thus, site phosphorylation attenuated 3C). novel (Fig. T1053 vitro was a inhibition as WDR62 cells identified arrested JNK Furthermore, mitotically by 3B). in (Fig. phosphorylation stimulation stress 17Ao 15Amtnsddntso lee cytoplasmic altered show with not did T1053A mutants WDR62 S1353A WDR62 whereas or cells, alanine GFP-tagged non-dividing T1273A a in found generated of microtubules We interphase next WDR62–GFP. association we full-length striking of WDR62, mutants of substitution localization mitotic odtrietecnrbto fnvlJKtre ie othe to sites target JNK novel of contribution the determine To n ncells. in and ora fCl cec 21)18 2–4 doi:10.1242/jcs.157537 527–540 128, (2015) Science Cell of Journal D9 el.Saebr:20 bars: Scale cells. AD293 (10 taxol-treated in respectively, compartments, and cytoplasmic microtubule to localized are WDR62-C–GFP and WDR62-N–GFP interphase. and (C) mitosis during cells in AD293 determined was association microtubule and tubulin–mCherry, with coexpressed were mutants acids truncation (amino 842–1523) WDR62-C or 1–841) (amino acids WDR62-N GFP-tagged Fig. (B) material S1E. supplementary in shown are 2 association; MT strong +++, MT association; +, binding. of (MT) extent microtubule the and MCPH tested the mutations deletion, or Schematic truncation cells. depicts mitotic and interphase determined in was deletions and truncations progressive with GFP- WDR62-N of tagged localization microtubule The (A) localization. microtubule and for required sufficient is WDR62 N-terminal of rich region WD40-repeat The 2. Fig. oascain ersnaieimages Representative association. no , m ,3 i)non-mitotic min) 30 M, m m. in

Journal of Cell Science EERHARTICLE RESEARCH esse hogotmtss nldn nerypohs n late and prophase early in including S1353, mitosis, throughout or persisted T1273 not but microtubules. with T1053, association S3A). WDR62 of Fig. increased substitution material (supplementary alanine WDR62 Thus, wild-type comparable in of more microtubules that was association to to of 3D; localized extent (Fig. the also cells, mutants taxol-treated microtubules WDR62 S1353A although and contrast, and with treatment, In T1273A S3A). colocalized taxol Fig. material by protein supplementary distribution exacerbated mutant filamentous was this T1053A enhanced WDR62 The of 3D). (Fig. localization utemr,temcouueascaino D6 T1053A WDR62 of association microtubule the Furthermore, ie D6 15,atgnzdteascaino D6 with WDR62 of association microtubules. the target antagonized WDR62 T1053, novel WDR62 the site, of phosphorylation JNK material Thus, S3A). supplementary Fig. 3F; (Fig. wild-type interphase of during association microtubule WDR62–GFP the activity increase JNK supplementary to of sufficient 3F; inhibition was chemical (Fig. Similarly, cells S3A). taxol-treated Fig. material in defined and in cytoplasm as association microtubule the (L1299/L1301A) clear showed conditions mutant live-cell under JNK-binding of distribution WDR62 intracellular the the Moreover, 3E). (Fig. cytoplasmic predominantly was localization WDR62 when anaphase/telophase ora fCl cec 21)18 2–4 doi:10.1242/jcs.157537 527–540 128, (2015) Science Cell of Journal elepesn h F-agdWDR62 GFP-tagged AD293 the single expressing a cell of division and progression taxol- (10 following treatment evident or more T1273A was WDR62 S1353A) of that to microtubules (compared with mutant the T1053A of WDR62 association increased AD293 The non-dividing cells. in evaluated was H2B, mCherry-tagged with coexpressed (WT) WDR62, wild-type or S1353A) T1273A, (T1053A, mutants The WDR62 (D) substituted h). alanine- 16 GFP-tagged nM, of 350 localization (NZ, cells mitotic in phosphorylation T1053 WDR62 decreased JNK 20 (C) (SP600125, indicated. inhibition as blotted the were and lysates left (AS), or asynchrony min) in 30 untreated M, 0.5 hyperosmotic (Sorbitol, with stress stimulated h), 16 (NZ, nM, mitosis 350 in synchronized were cells mean the ( as expressed are (pmol) and incorporation phosphate JNK1. show by Data WDR62-C or T1053A WDR62-C expressed recombinantly of phosphorylation interphase. during localization cytoplasmic maintains of WDR62 phosphorylation JNK-mediated 3. Fig. cl as 20 bars: 20 Scale inh.), (JNK [JNK VIII inhibitor inhibitor JNK with treated wild- WDR62 or type (L1299/1301A) WDR62 of mutant JNK-binding-domain GFP-tagged of the association microtubule Cytoplasmic (F) H2B–mCherry. and mutant T1053A n 5 ) * 3); P , .5(Student’s 0.05 m m ,3 i) E Mitotic (E) min). 30 M, m. m ,6 min) 60 M, t ts) B AD293 (B) -test). m ,6 min]. 60 M, 6 (A) s.e.m. nvitro In 531

Journal of Cell Science aoiyo rti a ihymbl n edl diffusible readily and mobile highly was protein the of that of recovery indicating majority fluorescence interphase, complete during cells. near WDR62–GFP non-dividing cytoplasmic and and rapid mitotic observed in We (FRAP) photobleaching with measurements association after recovery WDR62 fluorescence of conducted mobility we and microtubules, dynamics the probe To regulated phosphorylation is T1053 microtubules by with association WDR62 Dynamic ARTICLE RESEARCH 532 recovery maximum t to the comparison, half-time In the WDR62-WT calculated (t spindle-localized microtubule We spindle- of further 4B). that WDR62 of was (Fig. to metaphase comparison mobility of in during the reduced T1053A kinetics for Interestingly, WDR62 the role pole-localized 4B). (Fig. a regulating with association in consistent spindle-pole-localized (WDR62-WT), T1053 of of WDR62 curve recoveries interphase recovery wild-type in fluorescence the measurements our with the that superimposed found compared and next T1053A WDR62 We binding microtubule 4A). greater in (Fig. a participating to during WDR62–GFP attributed WDR62–GFP of we of proportion which slower, recovery substantially the was mitosis comparison, In 4A). (Fig. irtbl-on ouain.Orrslsidct that indicate results Our Thus, and and 4A,B). populations. cytoplasmic (Fig. between interphase mutant exchange rapid microtubule-bound T1053A with in WDR62 mobile ( is the WDR62–GFP WDR62 for fraction or for mobile mitosis different The significantly 4C). regions (Fig. in recovery fluorescence measured postbleach 11.2 and prebleach at depict extended, substantially K fmtpaesidelclzdWR2W s4.7 as WDR62-WT metaphase-spindle-localized of ) K fsidelclzdWR2T03 was T1053A WDR62 spindle-localized of 6 . .Rpeettv images Representative s. 0.4 , 5)wsnot was 95%) 6 . s. 0.2 D6 a rdmnnl yolsi,adcepeso with However, coexpression 5A). and (Fig. cytoplasmic, predominantly compartments was cytoplasmic WDR62 present and were nuclear JNK2–GFP or in JNK1–GFP interphase, GFP-tagged ectopically of During of distribution JNK. intracellular effect the the on WDR62 investigated the expressed to we JNK Firstly, recruiting Therefore, spindle. in WDR62 2012). mitotic of al., involvement the WDR62 et Centrosome evaluated of (Bogoyevitch we functions mitotic interaction that MacCorkle-Chosnek JNK shown partners. have required 2011; we direct and al., 2001) been al., et binding have et including (Huang JNK for previously functions mechanisms, protein described regulatory spindle complex and with localization by diverse is interaction members regulated family JNK and of pole distribution spindle intracellular the The to JNK1 recruits WDR62 loecne(nc eeldta oxrsino WDR62 of a coexpression to that nucleus JNK1–GFP revealed cytoplasmic the to (Fn/c) nuclear from of fluorescence ratio kinase the Quantitative the of 5A,B). measurements to (Fig. of sufficient distribution was cytoplasmic localization MAPK9), predominantly as the known (also alter JNK2 not but JNK1, D6 soito ihmcouue nadto oJNK to addition in of kinetics microtubules our at the with determine signaling. Furthermore, retention that association factors WDR62 phosphorylation. WDR62 mitotic of T1053 highlight results regulation by which negative microtubules, microtubules with the associated mobile stably less reinforces is more mutant therefore T1053A WDR62 and the division. that conclude cell also during We contribute localization might spindle-microtubule mitosis WDR62 to during retention microtubule increased ora fCl cec 21)18 2–4 doi:10.1242/jcs.157537 527–540 128, (2015) Science Cell of Journal ein cl as 20 bars: Scale region. bleached the indicate Circles cells. mitotic and dividing non- in T1053A WDR62 or WDR62 GFP-tagged of images post-bleach and pre-bleach Representative (C) curves. fitted indicated lines mean the as recovery fluorescence percentage the show Values comparison. for included also is metaphase during WDR62 (WT) wild-type of recovery post- (s) Fluorescence time bleach. of function a plotted as is cells metaphase or interphase in T1053A WDR62 mutant dephosphomimetic GFP-tagged the of intensity Fluorescence (B) post- bleach. (s) time of a function as plotted is metaphase and interphase in WDR62 GFP-tagged of intensity Fluorescence (A) analysis. FRAP to subjected cells were single and cells, AD293 in expressed were mutant T1053A or WDR62-WT microtubules. with association dynamic regulates T1053 WDR62 4. Fig. 6 ..( s.d. n 5 GFP-tagged 5.Dashed 15). m m.

Journal of Cell Science EERHARTICLE RESEARCH nrae hni a oxrse ihWR2adddnot did and WDR62 the with JNK1 coexpressed Moreover, fluorescent-protein-tagged was 5C). it of (Fig. when localization increased JNK2–GFP spindle pole the expressing spindle observed to not cells was localized this was Interestingly, with 5C). JNK1–GFP (Fig. of mitosis during subset pole small a specifically. that the JNK1 isoform recruit determine also pole. JNK1 might to spindle WDR62 the the WDR62 that to postulated for of Our we not Therefore, 5B). capacity was localization (Fig. JNK2–GFP a coexpression subcellular of indicate WDR62 Fn/c by the findings contrast, altered In significantly 5B). of retention (Fig. cytoplasmic the JNK1 and Fn/c both decreased substantially ieiaigo iiigclsepesn N1GPindicated JNK1–GFP expressing cells dividing of imaging Live –4)mtn,lcigteJKbnigmtf localized motif, coexpressed recruit JNK-binding to failed the but microtubules lacking acids to (amino constitutively WDR62-N mutant, the contrast, In 1–841) 5F,G). interphase (Fig. during we filaments mitosis microtubule and JNK1, resulted to JNK1 of colocalization and their distribution T1053A in WDR62 intracellular of capacity the coexpression the that direct showed on to findings our WDR62 JNK2 when extending of WDR62 In pole, with 5E). spindle colocalize (Fig. the not coexpressed did at and K55A colocalized cytoplasmic were JNK1 remained as WDR62 inactive activity, whereas and addition, the JNK In JNK1 S3B,C). on with Fig. material dependent obtained (supplementary not mutant were was H2B results This as identical 5D). such (Fig. controls negative GFP of or coexpression the with increase ora fCl cec 21)18 2–4 doi:10.1242/jcs.157537 527–540 128, (2015) Science Cell of Journal loecn-rti-agdJNK1 Fluorescent-protein-tagged cells. mitotic in determined was WDR62 with coexpressed of JNK1 localization fluorescent-protein-tagged The (D) poles. spindle to localized JNK1–GFP Arrows indicate cells. mitotic H2B– in with mCherry, JNK2, or JNK1 GFP- tagged of localization Intracellular (C) (Student’s non-significant * analyses); independent as 20 Scale bars: 1–841). acids (amino WDR62-N GFP-tagged when with microtubules coexpressed (I) spindle or interphase (H) with associate not does JNK1–mCherry (G,H) poles. spindle mitotic (G) and microtubules interphase on T1053A–GFP WDR62 and JNK1– mCherry of Colocalization (F) mitotic cells. in determined was WDR62 with coexpressed JNK2 fluorescent- protein-tagged of localization The controls. (E) negative as served H2B– mCherry or GFP with coexpressed mean the show Data distribution. cytoplasmic values whereas localization, cytoplasmic stencert yolsi ai (Fn/ Values ratio c). cytoplasmic to nuclear the as expressed and of quantified absence was or WDR62, presence the in or JNK2, JNK1 GFP-tagged of fluorescence cytoplasmic and Nuclear (B) interphase. in evaluated isoforms was JNK of or localization cytoplasmic nuclear The control. negative as a (H2B), 2B histone or WDR62 tagged mCherry- with coexpressed were JNK2 the to pole. JNK1 spindle recruits WDR62 5. Fig. 6 , ..(5clseautdi three in evaluated cells (25 s.d. m niaepredominantly indicate 1 m. , niaencerand nuclear indicate 1 A F-agdJK or JNK1 GFP-tagged (A) P t , -test). .5 ns, 0.05; 533

Journal of Cell Science oei ieyrqie o ioi rsiderglto n is 2014). al., and et regulation WDR62–JNK1 (Xu the spindle neuroprogenitors for or in role spindle complex described mitosis recently to for the with required specifically consistent spindle likely the isoform Taken is directing at JNK1 pole localization in 5H,I). JNK1 WDR62 the (Fig. WDR62-directed for of microtubules. role compartment localization a cytoskeletal reinforce the findings the our together, to JNK1 ARTICLE RESEARCH 534 of addition, analysis In been 2011). mitotic phosphoproteomic previously al., of quantitative had et profiling (Kettenbach WDR62 our phosphoproteomic substrates of global AURKA S52) in association, N-terminus and T50 identified microtubule the S49, S33, regulating (S32, within residues in Ser/Thr phosphorylated involved potentially AURKA by sustained and enhanced localization activity. is localization mitosis microtubule spindle during mitotically mitosis the WDR62 that WDR62 of indicate in results during decreased These AURKA 6H). rapidly (Fig. administered of cells inhibition MLN8237 arrested was Furthermore, WDR62 by 6G). endogenous inhibitor (Fig. disrupted of AURKA-selective accumulation Fig. also pole material an supplementary Spindle was (MLN8237, with S4C). 2011) microtubules al., treatment et with (Manfredi by association WDR62–GFP AURKA- reversed Moreover, alter WDR62–GFP AURKA 6F). not (Fig. enhanced inactive cells did non-dividing controls, overexpression in distribution JNK1 negative AURKA–mCherry or As during (K162R) of 6F). WDR62 WDR62–GFP (Fig. of expression regulated association interphase activity microtubule ectopic the AURKA augmented The that found localization. In of S4A,B). we Fig. depletion material contrast, mitotic (supplementary or AURKA, AURKA expression of AURKA regulating localization alter substantially in not did the spindle WDR62 WDR62 investigating In of 6E). on WDR62 (Fig. requirement colocalized spindle-associated AURKA closely spindle, contrast, were JNK1 mitotic In with and 6E). the (Fig. colocalized and Although microtubules centrosomes specifically 6D,E). (Fig. to WDR62 pole localized spindle were is AURKA the and AURKA at WDR62 target that colocalized phosphorylation found We partially direct 6C). a (Fig. AURKA was of We WDR62 that 6B). and (GST–WDR62- (Fig. demonstrated WDR62 for cells WDR62 full-length and arrested FL) recombinant endogenous mitotically purified 6A) Myc– between (Fig. in next proteins interaction and studies AURKA the HA–WDR62 co-immunoprecipitation the verified demonstrated in coexpressed We analyses AURKA mitosis-specific between S3D,E). and repeated significant in Fig. interaction cells spectrometry highly material mass synchronized (supplementary a using Myc- partner mitotically as of binding pulldown AURKA the from affinity into identified performed WDR62 insights WDR62 we of tagged not entry, gain accumulation WDR62 mitotic was spindle To rapid upon JNK-mediated this localization. triggers that mitosis, spindle although mechanism during for that increased required was show phosphorylation findings localization spindle Our WDR62 promotes activity AURKA vrxrsinddntehnemcouueascainof association microtubule enhance these of AURKA not that contribution showed did we the WDR62, of overexpression determining localization the In to residues S4D). (supplementary Fig. significantly AURKA active material as of S33 presence the and in phosphorylated S32 identified WDR62 phosphorylated ncnieigteARAtre eiuso WDR62 on residues target AURKA the considering In nvitro in iaeasy ihatv UK and AURKA active with assays kinase in-vitro - usto N1GPwsosre tsideplsadthis and poles spindle at a observed this, was with agreement JNK1–GFP mitotic-stage-specific In spindle- pole. of spindle directing coordinate the subset to by might JNK of events WDR62 recruitment defined. that signaling completely postulated localized although been 2004), not we Tan, have Thus, and al., mechanisms MacCorkle et of underlying 2011; (Gutierrez regulation the al., progression the et Huang in metaphase 2010; al., implicated and et been assembly (Bogoyevitch has spindle binding signaling JNK JNK mitotic require the 2012). that WDR62 previously of shown have functions We mitosis. in WDR62 of of 7D,E). control (Fig. spatiotemporal intracellular events mediating signaling for in mitotic AURKA association the and have microtubule JNK we regulating WDR62 of Specifically, roles protein. opposing mechanisms MCPH study demonstrated a Our 2014). WDR62, the al., of et distribution 2012; Xu revealed 2010; al., al., spindle et et has Higgins neural (Bogoyevitch for 2006; for al., maintenance required et necessary or Fish is is expansion that WDR62 cell pole regulation stem and spindle mitotic ASPM and MCPH-associated organization as of such accumulation proteins mitotic The DISCUSSION 5-AA Fg C.Tesidepl soito fWDR62 of association pole spindle D The 7C). (Fig. S52-AAAAA WDR62 of accumulation of localization phosphorylation-mediated microtubules. to in WDR62 involved be residues in multiple might redundancy that alternatively, indicates or, This residues AURKA-targeted S4E). 7B; Fig. ectopically (Fig. material phosphorylation of supplementary AURKA taxol in of absence defective association mutants the Similarly, microtubule WDR62 reduced in 7A). revealed cells, (Fig. AURKA, expressed non-dividing AURKA of with treatment microtubules interphase coexpressed with S49/T50/S52-AAA associate when to WDR62 capacity or some ( association retained S32/S33-AA WDR62 our WDR62 truncated microtubule recapitulating contrast, N-terminally 7A), (Fig. interphase with coexpression observations AURKA attenuated by induced WDR62 (S32/S33/S49/T50/S52-AAAAA) in of five sites AURKA-mediated the specific of in WDR62 phosphorylation replacement Alanine mutants putative the WDR62. of of alanine-substituted for region identify N-terminal generated the region we To requirement involved, N-terminal association. residues microtubule the a promoting of with phosphorylation ( WDR62 consistent truncated N-terminally Fg DE.Orfnig niaea AURKA-directed an WDR62– of 7E). indicate regulation (Fig. mitosis spatiotemporal during findings JNK1 for AURKA-mediated crucial association Our mechanism microtubule JNK that promotes contrast, 7D,E). regulates (Fig. In region negatively 7D,E). indicate (Fig. N-terminal phosphorylation Taken association WDR62 localization. microtubule results the WDR62 pole of spindle our phosphorylation lost microtubule likely in to together, bind region to resulting WDR62 N-terminal of filaments, capacity the intrinsic an in microtubule on impact mutations promote MCPH can association, This phosphorylation whereas 7C). N-terminal Thus, (Fig. of 1F). WDR62 observed (Fig. spindle localization proportion mutants spindle-pole WDR62 the MCPH-associated a of with loss on although complete observed the WDR62-WT, contrasts was of WDR62 that mutant to compared –2adWR2S2S3S9T0S2AAAwsreduced was S32/S33/S49/T50/S52-AAAAA WDR62 and 1–52 u tde aeadesdteottnigqeto fterole the of question outstanding the addressed have studies Our uigmtss eosre etre pnl pole spindle perturbed observed we mitosis, During ora fCl cec 21)18 2–4 doi:10.1242/jcs.157537 527–540 128, (2015) Science Cell of Journal D –2adWR2S32/S33/S49/T50/ WDR62 and 1–52 D –2 Fg A.Ti is This 7A). (Fig. 1–52) D –2.In 1–52).

Journal of Cell Science n h irtbl soito fWR2wsdtrie sn iecl irsoy h seik()idctsacl ihehne irtbl ass WDR62, microtubule GFP-tagged enhanced with with cell coexpressed a were indicates JNK1 (*) 20 asterisk or of bars: The (K162R) Scale localization microscopy. mutant live-cell the coexpression. using AURKA determine AURKA determined assessed kinase-dead to with was localization AURKA, WDR62 their immunostained WDR62 and mCherry-tagged of were cells, association (F) cells AD293 microtubule imaging. in the AD293 coexpressed live-cell and Mitotic were by JNK1–mCherry (D) cells or JNK1. AURKA–mCherry mitotic or and in WDR62–GFP AURKA (E) by AURKA. and FL) WDR62 (GST–WDR62 endogenous WDR62 full-length of phosphorylation EERHARTICLE RESEARCH h irtbl yokltn(dlaue l,20;Verhey 2004; al., et with indirectly, (Gdalyahu or cytoskeleton directly either microtubule associated, the are that DCX) and regulation. cell for neuroprogenitor in role in a complex with functions consistent WDR62–JNK1 WDR62 are isoform-selective the and processes behind JNK disease and basis of biological the (Xu support molecular studies appreciation embryo our unclear, the mouse increased is the isoforms required Although JNK in of specifically 2014). discrimination WDR62 was al., of that functions isoform et reported neural JNK1 recently the the for was it with Additionally, interaction spindle. in at mitotic WDR62 signaling spindle. JNK WDR62 for the isoform-specific function mitotic that of scaffolding the coordination found a spatiotemporal to suggest we findings specifically 2010), our isoform al., Thus, JNK1 et the of Wasserman (Cohen-Katsenelson recruited 2011; isoforms colocalization al., JNK2 and and et JNK1 biochemical expression with described association previously ectopic despite wa cells Interestingly, by h) WDR62. 16 increased nM, 10 (taxol, was M-phase-arrested (0.5 in MLN8237 WDR62 following endogenous intervals of time localization indicated spindle-pole the The at (H) evaluated staining. immunofluorescence by determined mitoticall (C) in control. WDR62 negative with a co-immunoprecipitation as AURKA served Endogenous antibodies (B) IgG (I blot. co-immunoprecipitation with by western Immunoprecipitation evaluated WB, h). was control. 16 cells negative h) nM, a 16 350 nM, as (NZ, 350 served cells (NZ, vector arrested arrested Myc microtubules. mitotically spindle a or on (AS) with accumulation asynchronous Coexpression its in regulates interaction and their WDR62 and with coexpressed, interacts AURKA 6. Fig. D6 on ito N-idn ates(o xml,JIP example, (for partners JNK-binding of list a joins WDR62 m .()FloigMN27(0.5 MLN8237 Following (G) m. m )treatment. M) oprmnaiaino D6,adWR2T1053 WDR62 to binding and microtubule regulated spindle WDR62, negatively on and of phosphorylation cytoplasm WDR62 intracellular steady-state the the compartmentalization regulated while reciprocally its in activity Thus, JNK localization microtubules, to microtubules. WDR62 JNK1 leading increased interphase directed microtubules, observed of we on decoration microtubule-associated In activity, times binding. JNK for microtubule retention of low-affinity absence rapid kinetics its the of the recovery indicative indicated WDR62, and studies stage. cycle photobleaching (T1053A), turnover cell our of residues decorated irrespective Furthermore, target WDR62 constitutively signaling, inhibition, microtubules of JNK kinase the was of mutation or absence (WDR62-N) interphase through truncation the during In achieved signaling. as WDR62 JNK by of regulated steady-state distribution was the addition, WDR62 region, In cytoplasmic repeat of regulated. tightly WD association and its dynamic the highly through that mediated demonstrated microtubules, We with 2001). al., et m )tetet pnl oeascaino noeosWR2was WDR62 endogenous of association pole spindle treatment, M) ora fCl cec 21)18 2–4 doi:10.1242/jcs.157537 527–540 128, (2015) Science Cell of Journal A y–UK n Atge D6 eetransiently were WDR62 HA-tagged and Myc–AURKA (A) nvitro In iaeasy odtriethe determine to assays kinase )analysis. P) cainof ociation s 535 y

Journal of Cell Science EERHARTICLE RESEARCH 536 JNK aberrant by regulation signaling. negative microtubule involve of might remains mutants but loss MCPH undefined truncated the 2011). C-terminally underlying the al., by basis association et mechanistic dispensable (Cohen-Katsenelson the is Thus, this association that microtubule dimerization, suggest protein also for for would findings required our is although WD40 WDR62 microtubules. of requisite spindle C-terminus to the localization The contain their L1414fsX41, in but defective WDR62 and are regions repeats, C-terminal V1314RfsX18 pole of lack mutants which spindle MCPH-associated truncation region the and why further of WDR62 microtubule question the WD40-repeat for raises We This association. sufficient the 7D,E). was (Fig. (WDR62-N) that localization demonstrated cytoplasmic promote eedn n UK inln.ARAi etooe and centrosome- a is AURKA and signaling. with, AURKA coordinated on, was enhanced revealed WDR62 dependent of we promote localization Indeed, mitotic the mechanisms entry. that mitotic distinct upon that association microtubule suggests this T1053A, WDR62 and spindle-associated on of impacted recovery further fluorescence our mitosis the Moreover, that localization. showed WDR62 analysis regulating photobleaching likely in are involved events be that signaling to this anaphase-promoting fact and the and entry microtubule-associated highlighting mitotic breakdown, segregation, chromosome envelope to until persists nuclear cytoplasmic following immediately predominantly shifts living rapidly WDR62 In of from entry. distribution intracellular mitotic the during cells, single triggered is microtubules spindle u urn td niae htWR2ascainwith association WDR62 that indicates study current Our ora fCl cec 21)18 2–4 doi:10.1242/jcs.157537 527–540 128, (2015) Science Cell of Journal Cer n ao rae (10 treated taxol and H2B– mCherry with coexpressed were mutants WDR62 alanine-substituted or truncated N-terminally GFP-tagged (B) evaluated. was localization and with mCherry–AURKA coexpressed were mutants S52-AAAAA S32/S33/S49/T50/ WDR62 or S49/ T50/S52-AAA WDR62 S32/S33-AA, WDR62 (WT) WDR62, wild-type full-length WDR62, ( 52) truncated N-terminally GFP-tagged (A) association. microtubule N-terminal regulates WDR62 region the within residues of phosphorylation AURKA-mediated 7. Fig. cn ietn pnl oe S) Scale 20 (SP). bars: poles spindle line bisecting along scans quantified GFP were Relative intensities cells. signal mitotic in proteins determined WDR62 was of association pole the spindle and H2B–mCherry with coexpressed were mutants WDR62 or alanine-substituted truncated N-terminally GFP-tagged evaluated. (C) was localization protein and UK.Cnrsm-soitdAURKA Centrosome-associated and AURKA. JNK by WDR62 regulation of spatiotemporal model Proposed (E) with microtubules. association protein on respective effects their JNK and and sites AURKA phosphorylation of position the depicting irtblsfrctpamclocalization. cytoplasmic for from microtubules dissociation promotes WDR62 of phosphorylation JNK-mediated pole. the spindle at of complexes accumulation signaling the WDR62–JNK for required is entry.This mitotic following microtubules astral association with trigger to WDR62 phosphorylates m .()Shmtco WDR62 of Schematic (D) m. m ,3 min), 30 M, D 1–

Journal of Cell Science xrsino UK surgltdi 2adisatvt is activity its and G2 in upregulated The 2013). is al., spindle AURKA et Reboutier central of 2013; expression al., in and et (Lioutas Nikonova interphase roles during 2013; and Vernos, important stages mitotic spindle revealed late in bipolar assembly also Recent 2013). and have al., et entry Nikonova studies 2007; Gergely, mitotic and centrosome (Barr separation, of assembly regulation functions and including well-defined phases, maturation mitotic with early kinase during mitotic spindle-associated ARTICLE RESEARCH D6-ietdsgaigeet ntemttcsidewill spindle of mitotic characterization the further on The spindle-associated events directing 7D,E). signaling in (Fig. WDR62-directed WDR62 signaling for role JNK1 a of association and spindle-microtubule WDR62 the regulating MCPH- in AURKA and and clarify AURKA will between studies links Further WDR62. mechanistic associated 2014). precise al., the et neural (Chen to downstream AURKA–WDR62 of development diverse contribution the Nevertheless, supports the study activity. our regulation AURKA into mitotic spindle by insights of regulated in mechanisms additional aspect involved provides another network and represent signaling restricted AURKA could of were the recruitment complex spindle and WDR62–JNK the Therefore, severe the 2012). al., less a progression et as and were (Bogoyevitch organization defects spindle loss metaphase mitotic to WDR62 predominantly comparison, spindles In of defects, 2007). monopolar result spindle Gergely, and and and centrosome chromosomes (Barr mitotic formation, misaligned profound microtubule expansion, to astral has attenuated lead Gergely, signaling including to and AURKA Abrogated found (Barr 2013). centrosome been al., formation et regulates for spindle Nikonova signaling components 2007; bipolar AURKA spindle and pathway. and maturation signaling centrosome mitotic WDR62 multiple AURKA of prevented a downstream activity or in WDR62 AURKA placing expression Rather, accumulation, of WDR62. AURKA spindle inhibition of altered specific depletion specific embryonic observe the experimental with not In mutant localization alleles. and did our spindle hypomorphic in we with in expression progenitors observed contrast, neural differences AURKA and were fibroblasts by accumulation reduced likely for are spindle Previously, The they accounted but localization. approaches. unclear be are in mitotic discrepancies WDR62 to or for these al. requirement for et expression a reasons Chen excluded AURKA unlike we However, 2014), regulating 2010). al., al., et such et (Chen proteins (Joukov centrosomal is CEP192 with AURKA as interaction of an WDR62 accumulation through and mitotic mediated The AURKA interactions. of an signaling understanding AURKA provides and confirms mechanistic WDR62 study of expanded Our phosphorylation 2014). and with al., interaction et mitosis cell (Chen stem revealed neural murine of were in preparation involved between WDR62 interactions the and target genetic AURKA during and phosphorylation biochemical Moreover, the a manuscript, 2011). this as al., et WDR62 (Kettenbach identified previously mitosis. during has activation AURKA and of Littlepage correlates kinetics transition the 2002; spindle anaphase with to closely with al., entry association mitotic et from WDR62 microtubules (Castro Therefore, complex is 2002). AURKA Ruderman, APC/C 2002). anaphase the al., during mitotic and et initiated by degradation Littlepage facilitate proteasomal activators 2003; by to al., inactivated protein et primarily (Bayliss with autophosphorylation entry kinase interactions by through increased uniaiepoportoi ceno UK substrates AURKA of screen phosphoproteomic quantitative A nsmay u td a dniidopsn oe o JNK for roles opposing identified has study our summary, In otiuet erpoeio eldvso n ri size brain and division cell neuroprogenitor determination. to networks phosphorylation mitotic contribute how into insights novel provide epae lnn usiuin fSrTrpopoyainstswere sites phosphorylation using and Ser/Thr mutagenesis PCR site-directed of by as made by substitutions (WDR62-FL) generated Alanine WDR62 were template. full-length 2A) codon-optimized (Fig. siRNA-resistant mutants truncation WDR62 Plasmids rabbits ( of phosphopeptides inoculation through SSLPQ(pT)PEQEK Abmart WDR62) with by (pT1053 generated custom phospho-T1053 from was were WDR62 antibody antibodies Biotechnology. HA and Cruz Myc Santa Technology. Signaling Anti-cdc25C Cell (A301-560A). from Laboratories was Bethyl from was reagents Anti-WDR62 and inhibitors enzymes, Antibodies, METHODS AND MATERIALS %(/)Tio -0,02 wv a n 100 and NaF (w/v) Tris-HCl 0.2% mM [50 deoxycholate, X-100, sodium buffer (w/v) Triton 1% RIPA EDTA, (v/v) mM in 0.1 NaCl, prepared 1% mM 150 were 7.3, lysates pH cell immunoprecipitation Total and immunoblots lysates, Cell CO 5% Lipofectamine with humidified cells performed and a was penicillin-streptomycin, transfection in U/ml mediated with 100 cultured supplemented and DMEM serum were in calf maintained fetal were 10% cells AD293 and and transfection HeLa autoradiography transient and by culture Cell analyzed and (Pierce) counting. reagent Gelcode Cerenkov with stained Stain SDS-PAGE, by Blue resolved then were Samples buffer. (10 GST–WDR62 recombinant Purified Addgene the vitro In from obtained Origene. were from as repository. obtained tubulin–GFP and plasmid was vectors digestion and cDNA restriction AURKA pXJ40-Myc H2B–mCherry by analysis. validated sequencing or were full cloning constructs by pmCherry-N1/C1 All fluorescent tags appropriate. epitope with pEGFP-N3/C3, (Myc) labeled affinity were small into mutants or al., WDR62 mCherry) et 2014). 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Journal of Cell Science oe-asnlo,K,Wsemn . htb . htas,A .and J. A. Whitmarsh, S., Khateb, T., Wasserman, K., Cohen-Katsenelson, A. I. Blair, and J. Hevko, E., Ciccimaro, hn .F,Zag . id,J,Hne,K . a,F n iwne,L. Niswander, and F. Lai, C., K. Hansen, J., Wilde, Y., Zhang, J.-F., Chen, ato . ro-onmis . inrn . Labbe S., Vigneron, Y., Arlot-Bonnemains, A., Askham, Castro, S., Scott, J., D. Hampshire, H., G. Mochida, T., E., T. Roberts, Zhao, J., Y., Y. Bond, Yip, R., K. Ngoei, Z., Qu, Y., Y. Yeap, A., M. Bogoyevitch, Bilgu eoyeie ih2m CaCl mM 2 with depolymerized uiidprietbln(300 tubulin porcine Purified vitro 2011). In al., et (Taus requiring confidence PhosphoRS, 90% least with and applied, at validated was was 1% of identification threshold rate phosphopeptide discovery false A database. Uniprot ARTICLE RESEARCH als,R,Sro,T,Vro,I n ot,E. Conti, and I. Vernos, T., Sardon, R., Bayliss, F. Gergely, and R. A. Barr, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.157537/-/DC1 online available material Supplementary material Supplementary and APP1046032]. Health number National [grant a Grant by Project supported Council was Research [grant work Medical Fellowship this Future and Council Research FT120100193]; Australian number an of recipient a is D.C.H.N. Funding manuscript. of the conceived wrote D.C.H.N. and preparation. experiments manuscript contributed and Z.X. design and experimental M.A.B. towards the N.A.W., to analyses. contributed phosphoproteomic C.-S.A. generated quantitative mutants. S.C.W. WDR62 and MCPH-associated D.X. characterized analysis. FRAP and identified T.T.T. to work. Y.Y.Y. contributed experimental sites. of target JNK majority the Y.Y.C.Y. performed and N.R.L. contributions Author interests. financial or competing no declare authors The interests Competing in assistance for facility P. Platform and Microscopy imaging. technical WDR62, Optical cellular for and Biological Cheng JNK1 the H.-C. recombinant and and of McMillan Lo expression D. Australia) baculoviral construct, VIC, in K162R Clayton, assistance University, AURKA (Monash the Plotnikova sharing Olga for to grateful are We Acknowledgements ubdt nlss ape eefatoae osprt oul and of soluble completion separate (100,000 ultracentrifugation to the 37 by fractionated At pelleted were tubulin plate Labtech). polymerized samples temperature-controlled BMG a analysis, on Optima, turbidity min (POLARstar 100 for reader minute every measured uui eersle ySSPG,dtce n uniidby quantified and detected SDS-PAGE, tubulin. and by WDR62 for resolved blotting were tubulin ntae ihicbto t37 was polymerization at Tubulin incubation plates. 96-well with clear initiated on glycerol) v/v 10% 8 MPPSp .,05m GA MMgCl mM 2 EGTA, mM 0.5 6.8, pH PIPES mM (80 (20 WDR62 ice.J. Biochem. A. chromatography/multiple Aronheim, liquid nanospray using spectrometry. 3681-3692. kinase mass monitoring adhesion reaction focal on 21) ircpaydsaegn d6 euae ioi rgeso of progression mitotic size. brain regulates and Wdr62 cells stem gene neural embryonic disease Microcephaly (2014). .M,Srnel . aaea,M,Co,Y . aka,A .e al. et F. A. Markham, J., Y. Crow, and M., Mahadevan, K., maintenance Springell, M., spindle J. for C. required D. progression. protein Ng, mitotic pole timely and spindle I. phosphorylated J. Heng, MORep. EMBO T. size. Lorca, cortical cerebral of determinant major 320. a is ASPM xm eunigietfe eesv D6 uain nsvr brain severe in mutations WDR62 recessive identifies malformations. sequencing exome Yalnizog uoaAatvto yTX ttemttcspindle. mitotic the at TPX2 by activation Aurora-A poles. ˚ ) oul rcin eermvd elt eewse and washed were pellets removed, were fractions Soluble C). a,K,Oztu K., var, ¨ .Cl Sci. Cell J. uui ubdt n eietto assay sedimentation and turbidity tubulin u . 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