hc csb ouaigtehmotsso L4activity. PLK4 of homeostasis the modulating by acts which suggest findings binding our Together, PIPKI this PLK4. that that of PIPKI and activity PLK4 kinase that of the domain found reduced overexpression, polo-box cryptic we the Importantly, to PLK4 bound duplication. PIPKI endogenous or restrain of PIPKI treatment result that suggesting a hydroxyurea as homolog depletion, occurred PIPKI 6 exogenous that of abnormal amplification expression assembly The spindle (SAS6). and PLK4 of depletion upon the phase, was M localization or S This PIPKI in phase. cells KDa M detaining 152 Without in of (CEP152). protein not centrosomal with but association in an phases, upon centriole dependent G2 the and of S end proximal G1, the around materials pericentriolar Ato o orsodne([email protected]) correspondence for USA. *Author 55902, MN Rochester, SW, Street First 200 yetninadNprlg,My lnc 0 is tetS,Rochester, SW, Street First USA. 200 55902, Clinic, MN Mayo Nephrology, and Hypertension ige Xu Qingwen duplication PIPKI ARTICLE RESEARCH ß eevd2 uut21;Acpe 9Dcme 2013 December 19 Accepted 2013; August 23 Received Pihan 2006; Nigg, 2002; al., 1 et Lingle many 2000; to tumors al., aneuploid anomalies et different of centrosomal (Ghadimi variety a linking including 2009), diseases, human al., et cells daughter (Ganem aneuploid and to instability rise genomic mis- give mis-segregation, in frequently results these The and mitosis. spindles, of that cell assembled after number so abnormal In an phase one 2009). of and inherit S possession Raff, can in cell once and ciliogenesis in daughter duplicates Nigg each centrosome major role 2010; the primary cells, al., crucial the cycling et a assembly, (Debec the is plays morphogenesis spindle and and (PCM), center mitotic of material organization pair microtubule pericentriolar one comprising surrounding centrosome, The INTRODUCTION gamma type-1 5-kinase CEP192, 4-phosphate CEP152, phosphatidylinositol PLK4, duplication, Centriole WORDS: KEY PIPKI fidelity. PIPKI as to referred hereafter that report (PIP5K1C, we gamma that type-1 Here, 5-kinase cycle. ensure 4-phosphate phosphatidylinositol cell to per essential once occurs is duplication centrioles centriole of pre-existing regulation at spatiotemporal The proteins. these a structural and (PLK4) of kinase group polo-like small the on depends biogenesis Centriole ABSTRACT eateto iceityadMlclrBooy n iiinof Division and Biology, Molecular and Biochemistry of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,19–35doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. c e ocnroeapiiaini anrta a dependent was that manner a in amplification centriole to led c c sanvlngtv euao fcnroeduplication, centriole of regulator negative novel a is c 2 iiino yetninadNprlg,My Clinic, Mayo Nephrology, and Hypertension of Division 1 oaie narn-iepteni h intermediate the in pattern ring-like a in localized agt otecnrsm n etan centriole restrains and centrosome the to targets ui Zhang Yuxia , c slkl ofnto ttePK ee to level PLK4 the at function to likely is c ly nipratrl ncentriole in role important an plays ) 2 uhoXiong Xunhao , c eue centriole reduced 1 a Huang Yan , c c 1 efr .Salisbury L. Jeffery , agtdt itntsbellrsts yrgltn h dynamics the regulating By PI(4,5)P regional sites. of subcellular distinct to targeted ced ta. 01 lyenSh ta. 07 ig 07 Nigg 2007; 2005; Nigg, 2007; al., al., et et Kleylein-Sohn 2011; (Bettencourt-Dias al., et amplification Eckerdt activity centriole PLK4 excessive to that leads and formation 2011). centriole Kleylein- Stearns, of and regulator 2011; Nigg al., 2007; duplication Nigg, et to 2007; centrosome Eckerdt al., controlled and et 2005; carefully Sohn centriole al., be et of to (Bettencourt-Dias fidelity have the centrosome these maintain of the assembly activity and at with availability centriole, The is proteins cycle. pre-existing and cell per proteins per once conserved one occuring is highly to procentriole of limited The group 2009). strictly small Raff, a and by Nigg assembled syndromes 2011; microcephaly al., and et 2004) al., (Megraw et Yamamoto 1998; al., et PPKC eefe eerdt sPIPKI as to referred hereafter (PIP5K1C, of understood. importance fully from the is during to mechanism far of underlying the PLK4 spite accurately, activities activity of In PLK4 al., controlling machinery. importance phosphatase assembly et the centriole highlight and Song centriole the and 2011; kinase duplication nascent by al., centriole between important et intrinsic formation the an of Kitagawa balance existence the centriole support 2011; at results These al., 2011). promote accumulation centriole et to the (Brownlee SAS6 2 of reported phosphatase protein facilitating components was contrast, 2011), By other al., number. (PP2A) et of centriole (Puklowski Rogers restrict SAS6) that 2010; to as al., or (such et machinery Holland 2009) duplication 2010; Cunha-Ferreira al., al., et 2012; et Guderian Rogers, 2009; al., and both et (Brownlee its for control FBXW5) out availability can as PLK4 way, own (such carry this substrate In destruction. a can and or ubiquitinylation itself PLK4 substrates, either other priming 2010). of thereby phosphorylation al., the and al., et autophosphorylation et (Cizmecioglu centrosome Dzhindzhev the by to 2010; bind centrosome to protein CPAP) centromere or the for (CENPJ platform J a to provides with also together which recruited assembly protein, centriole this of is onset the PLK4 controls and CEP152 2011). Stearns, and aehe l,20;Ln ta. 07;Ln ta. 02 Sun 2002; al., PIPKI et al., Moreover, Wang 2012). et 2009; al., al., Ling et et El Thieman 2007a; 2012; Xiong 2002; al., al., 2004; al., et et Thapa et 2007; Ling Paolo al., al., 2007; et (Di et al., migration Thieman (Wang et 2012; and signaling al., Sayegh adhesion calcium et 2012), cell al., Thapa 2006; or et 2013; 2004) al., Xiong al., 2009; et et al., Sun (Bairstow et 2007; trafficking al., et vesicular Ling as such processes Guiie l,20;Guiie l,20;Ihhr ta. 1998; al., et PIPKI Ishihara 2011). al., 2006; et al., Xia 2009; et Anderson, Giudici and PIPKI Schill 2004; al., of et isoforms (Giudici spliced alternatively hshtdlnstl(,)bshsht [PI(4,5)P generate (4,5)-bisphosphate to phosphatidylinositol 4-phosphate phosphatidylinositol phosphorylates that thsbe eosrtdta h oolk iaePK sakey a is PLK4 kinase polo-like the that demonstrated been has It hshtdlnstl4popae5kns ye1gamma type-1 5-kinase 4-phosphate Phosphatidylinositol 2 ol,te r mlctdi itntcellular distinct in implicated are they pools, 1 igu Hu Jinghua , c 1,2 c c aebe reported been have salpdkinase lipid a is ) n u Ling Kun and a euaesome regulate can 2 .T ae six date, To ]. c sfrsare isoforms 1293 1, *

Journal of Cell Science ytecnrsmlmre eti nHL el.B contrast, By cells. HeLa in 2 highlighted PIPKI is marker which against centrosomal centrosome, the antibody the stained by monoclonal panel) previous left a 1A, the and (Fig. from S1B) (supplementary artifact Fig. antibodies an material polyclonal purified not that the Both ensure was antibody. to pattern antibodies localization the monoclonal this investigate and PIPKI To polyclonal centrosome. of rabbit the new localization as 2007; well centrosomal al., as et unexpected 2002), (Ling al., endosomes et adhesions, Ling recycling focal and including junctions sites adherens subcellular multiple to localizes (PIPKI hwsann rudtecnrsm Fg A.Tecentrosomal The anti-PIPKI 1A). of (Fig. binding centrosome recognizing the around staining specifically show antibodies polyclonal PIPKI rabbit purified eso htls fPIPKI of loss that show We CPAP, and PIPKI of PLK4 residence to just the Similar manner, proteins. cell-cycle-dependent centrosomal other a many in like centrioles parental directly of proximal ends the by PIPKI with associated kinase that 2007), lipid This duplication. report centriole al., we et Here, (Ling them. E-cadherin with interacting as such proteins, ARTICLE RESEARCH 1294 PIPKI a with treatment PIPKI the that of antibodies all polyclonal recognize rabbit characterized previously Using PIPKI RESULTS degradation. and centriole ubiquitinylation and protein phosphorylation-dependent activity with PLK4 combination in of acting regulation existence biogenesis, of the level suggest additional results an These of activity. its that inhibits indicating and PIPKI PLK4 phase, that G2/M revealed investigation or further S in cells PIPKI the blocking without n - Sclsadi uieIC3adNIH3T3 and IMCD3 shown), murine not data in and PIPKI of role and S1E functional potentially pervasive, Fig. a cells suggesting material also (supplementary OS we cells cells, U-2 HeLa in and presence its PIPKI results to observed addition These In cytoskeleton. centrosome. microtubule PIPKI that the demonstrated of independent is PIPKI between association the that indicating S1D), Fig. material (supplementary integrity microtubule disrupted etooe,w ue nH a oteNtriu falsix all of N-terminus the to tag PIPKI HA an known fused we centrosomes, centrosome. no had with polymerization, PIPKI microtubule on treatment of effect inhibitor Moreover, an nocodazole, microscopy. immunofluorescence PIPKI that CPAP HsSAS-6), to and referred (hereafter SAS6 human gradient kidney components PIPKI 2004), sucrose al., mouse method et this using (Kaplan identified Similar using were or blot. that proteins western centrosomal other by S1C) to 1C) fractions (Fig. the analyzed Fig. and cells the centrifugation material supporting HeLa (supplementary S1B), from centrosomes Fig. PIPKI PIPKI this excess material of of authenticity presence (supplementary the protein in or 1B), (Fig. odtriewihPIPKI which determine To c c c tbln–arsl htfrhrspotdtehypothesis the supported further that result a – -tubulin a oaie ttecentrosome the at localizes c i)i agtdt h etooeadngtvl regulates negatively and centrosome the to targeted is _i3) sangtv euao fcnroedpiain Indeed, duplication. centriole of regulator negative a is rPIPKI or c c soitswt etooe ssgetdby suggested as centrosomes with associates c c pievrat.A hw nFg Dand 1D Fig. in shown As variants. splice inla h etooe lhuhi efficiently it although centrosome, the at signal b ttecnrsm nhmnMDA-MB-231 human in centrosome the at splmnaymtra i.SA i not did S1A) Fig. material (supplementary c c c ttecnrsm eedduo CEP152. upon depended centrosome the at nioywscmltl blse after abolished completely was antibody iN httree l PIPKI all targeted that siRNA c c c c a oafd opnn fthe of component fide bona a was oaiain nadto,w isolated we addition, In localization. sfrs eosre htPIPKI that observed we isoforms, c ofatoae ihtecentrosome the with co-fractionated eut ncnroeamplification centriole in results c sfr a agtdto targeted was isoform c ietyitrcswith interacts directly c n h centrosome the and c c u o PIPKI not but egenerated we , c c sfr 3 isoform isoforms c tthe at a c c ihtecnrsm,w olbldHL el ihantibodies with cells HeLa co-labeled PIPKI we of centrosome, association the the of details with structural fine the examine To manner dependent PIPKI acids. the amino in 445 C-terminal embedded first motif centrosome-targeting unique the expose the to acts that that possible PIPKI is of insertion 445 it N-terminal targeting, the within centrosome located PIPKI Because be acids. to amino likely centrosome- is the motif that suggest targeting results These panel). middle 1D, (Fig. etooewsosre nclsa 1 n 2phase PIPKI G2 and PIPKI when until the centrosomes S telophase, from prophase, absent G1, remained late At it at and S1G). diminished cells signal Fig. in material PIPKI observed (supplementary arrangements). was varying centrosome of chromosome condensed in pairs of and stages membrane DNA separated various nuclear and (broken (two membrane) and phase nuclear M foci phase intact and 2 intact G2 foci and centrin membrane), foci clustered 2 were (four nuclear centrin cells phase intact (two DAPI, S phase and membrane), G1 2 immunofluorescence nuclear in centrin being of to as staining categorized the cells on PIPKI Based centrosome HeLa DAPI. visualize other to many subjected for microscopy case we the is proteins, as dependent, cell-cycle diameter a . with of centrioles to that of end likely than proximal appeared smaller these the and around In ring matrix the a pericentriolar 2C). form the of (Fig. in fiber 2000) appendage wall al., dense centriole distal et outer the PIPKI (Piel and images, marks centriole C-Nap1 Further which mother between mature (ODF2), as 2A). 2 2000) (Fig. well al., protein et as (Mayor PIPKI proteins centrioles 2B), between (Fig. that protein two linker a showed C-Nap1, PIPKI these 1996) that indicated centriole examination al., the below of al., end et subdistal distal positioned et the (Paoletti (Nakagawa (a marks illumination (which centriole) cylinder) 2 ninein mother centrin structured and the for were 2001) on dimensional centrosome marker Co-staining appendage the (3D-SIM). three around microscopy using proteins these analyzed of localizations PIPKI against nsnhoie eacls(i.2) hsfloe h same CEP152-interacting the a 2D), followed (Fig. This 2009) al., 2D). et (Tang (Fig. CPAP as cells trend exit HeLa mitotic synchronized at recovered in then and PIPKI mitosis through of progressed cells level cell protein daughter the centrosome each its targeting, with by Consistent inherited S1G). Fig. be material (supplementary soon would that centrosomes upeetr aeilFg 1,ol PIPKI only S1F, Fig. material supplementary neto aioais6166 tteCtriu,btenthe between PIPKI C-terminus, of end the the adhesions at representing sequence 641-666) PIPKI focal acids (amino 2002). to insertion al., localized et or vesicle- targeting, (PIPKI showed cytoplasmic isoforms like Other signal. centrosome-associated h PIPKI the rdmnnl yolsi Fg D ih ae) h N- The panel). right PIPKI of 1D, fragment (Fig. PIPKI terminal cytoplasmic of predominantly region terminal omnt l PIPKI all to common oadeswehrtecnrsmllclzto fPIPKI of localization centrosomal the whether address To c stree otepoia n fcnroe nacl cycle- cell a in centrioles of end proximal the to targeted is c ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal i) srpre rvosy(iPooe l,20;Ling 2002; al., et Paolo (Di previously reported as _i2), c i -emnletnin(i.1) oee,teC- the However, 1E). (Fig. extension C-terminal _i2 c c emdt edsrbtdaogtesd fthe of side the along distributed be to seemed c n aiu etooecmoet.The components. centrosome various and i ih rvk ofrainladjustment conformational a provoke might _i3 c c i otisauiu 26-amino-acid unique a contains _i3 ains oaie otecentrosome the to localized variants, c c c i steol sfr htshowed that isoform only the is _i3 i aioais145,wihis which 1-445), acids (amino _i3 i aioais45Ed is 445-End) acids (amino _i3 c c c a erie akt both to back recruited was oehrwt eti and 2 centrin with together oaie ewe ienand ninein between localized c erae infcnl as significantly decreased c i n h einn of beginning the and _i1 c tiigaon the around staining c i ipae a displayed _i3 c was c is c c

Journal of Cell Science EERHARTICLE RESEARCH eemnduigac-muorcptto sa.Ide,HA- Indeed, PIPKI assay. co-immunoprecipitation tagged a using PIPKI PIPKI CEP152 determined between of whether association targeting physical examined the The we for centrioles, responsible of al., was et end PIPKI Dzhindzhev Because proximal 2010). 2010; the of al., al., localization et et centrosomal Hatch (Cizmecioglu 2010; the CPAP for end and required proximal PLK4 is the and around localizing centrioles protein of scaffold a is CEP152 CEP152 with PIPKI is (Strnad which phase 2D), G1 centrosome. (Fig. in the absent at HsSAS-6 PIPKI is that suggesting of and 2007), al., phase et that M late from during degraded different this however, is duplication; centriole trend in early functions that protein nce onb E12seii iN Fg D,suggesting 3D), CEP152 (Fig. The all siRNA be other. CEP152-specific could by each which down 3B), to knocked (Fig. bind two bands these multiple to recognized that potential antibody suggesting the 3A), have (Fig. proteins cells HEK293T in CEP152 c agtn otecnrsm sdpneto nassociation an on dependent is centrosome the to targeting c i a ulddw ihoeepesdGFP– overexpressed with down pulled was _i3 c ih aeaseii function specific a have might c c n E12wsfirst was CEP152 and c lolclzsaround localizes also otecentrosome. the to eiiiecnlso eas ftehaydgaaino our of degradation heavy polypeptides. the CEP152 of obtain recombinant because to needed conclusion is definitive investigation a further but PIPKI CEP152, of that terminus suggest data PIPKI These with interact not did i.SA.Hwvr h -emnl27rsde fCEP152 of residues 217 N-terminal the (CEP152 However, S2A). Fig. (CEP152 htedgnu E12eit nvratfrs Endogenous forms. variant in exists PIPKI CEP152 endogenous that lhuhi sntkonwehrti PIPKI this 3B). whether (Fig. gels known SDS-PAGE not on is faster it migrated Although that protein oaGT i rmloebnigpoen(B)tg we tag, (MBP) and (CEP152 protein fragments residues maltose-binding CEP152 His–PIPKI with interaction MBP-tagged or their tested purified His fused and was GST, it constructed whether a matter no the insoluble to or Because in degraded proteins. highly expressed was two CEP152 an spliced of full-length these existence alternatively recombinant between the or support connection results truncated physical our CEP152, unmodified, of an form is CEP152 c ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal oimnpeiiae ihteedgnu CEP152 endogenous the with co-immunoprecipitated 1-217 749-1654 ,t hc L4drcl id Hthe l,2010), al., et (Hatch binds directly PLK4 which to ), ulddw PIPKI down pulled ) 1-748 n h -emnl96residues 906 C-terminal the and ) c tie ihteidctdantibodies. Anti- indicated the with stained (siPIPKI PIPKI specific or (siNC) control nonspecific with anti- PIPKI by recognized signal centrosome PIPKI siRNA-mediated of (B) depletion (red). 2 centrin with (HA–PIPKI PIPKI (HA–PIPKI full-length HA-tagged the with were transfected cells HeLa (D) antibodies. indicated the using and immunoblotted cells HeLa from isolated were fractions Centrosome sucrose gradients. 40–70% in centrosomes with PIPKI (C) centrosomes. visualize PIPKI to with antibodies stained indicated and the fixed were cells HeLa PIPKI or centrosome. rmeo 6 fteppkcgn.Scale 5 . bars: pip5k1c the of 16c exon from translated polypeptide shading the hatched represents the 16b, exon from translated polypeptide the represents shading white The centrosomes. PIPKI C-terminus. CT, domain; kinase KD, euneadcnrsm agtn of targeting centrosome and sequence the comparing (E) diagram antibodies. Schematic (red) 2 (green) anti-centrin anti-HA and with stained then were PIPKI 1. Fig. net.DAwssandwt DAPI. with stained was DNA as inserts. shown were images centrosome PIPKI known splmnaymtra i.S2A). Fig. material (supplementary c c ih ietybn oteC- the to bind directly might tblnwsue olabel to used was -tubulin c c c c PIPKI , i steol sfr oaie to localized isoform only the is _i3 nioy eaclswr treated were cells HeLa antibody. -4)adCtria fragment C-terminal and 1-445) m b ABD.Enlarged (A,B,D). M stree otecentrosome. the to targeted is , c c c c ohteNtria 748 N-terminal the Both . ,Ntria rget(HA– fragment N-terminal ), 4-n)o PIPKI of 445-End) splmnaymaterial (supplementary c c a A PIPKI (A) sfrs T N-terminus; NT, isoforms. soitswt the with associates rPIPKI or c iN n then and siRNA ) c blse the abolishes shrci coli Escherichia c c b u o PIPKI not but , co-sedi-ments gen along (green) c -associated c i.Cells _i3. nvivo in 1295 c a -

Journal of Cell Science niaeta PIPKI that indicate ls otepoia n fcnroe.Saebr:0.5 bars: Scale centrioles. of end proximal the to close the show centrosomes ODF2/C-Nap-1 representative PIPKI and of of (B) images positions ninein/C-Nap-1 SIM (A), 3D-SIM. 2 using centrin (C) PIPKI and visualize ninein to with microscopy along cycle. immunofluorescence cell indirect the to by subjected regulated is level protein fCP5 Fg D e oals fPIPKI of loss a to association led depletion functional 3D) siRNA-mediated (Fig. a Indeed, CEP152 proteins. of suggested two these and between interaction physical EERHARTICLE RESEARCH 1296 Asy, synchronization. successful cells. iodide-stained confirming asynchronous propidium time-point, of each analyses at cytometry antibodies. cells flow appropriate shows using time- panel immunoblotting indicated lower the by The at analyzed collected were were and block points thymidine double a from released PIPKI 2. Fig. onne l,21) oeipraty h colocalization the 2012; importantly, al., PIPKI et More Mennella and 2012). 2012; CEP152 al., al., between et et (Fu Lawo CEP152 to Sonnen 2012; similar manner Glover, a in and centrioles of end proximal al., the ring-like et PIPKI a Sonnen that suggesting supported 2012; PIPKI images al., between 3D-SIM et Glover, colocalization Mennella and our (Fu 2012; Consistently, centriole al., 2012). the et CEP152, around Lawo including by structure 2012; proteins, recently ring-like reported a centrosomal been form that has groups It centrosome. multiple the at colocalized PIPKI between oudrtn h ipyia infcneo h association the of significance biophysical the understand To c oaie rudtepoia n fcnroe n its and centrioles of end proximal the around localizes c c c n h niae etooemres hs images These markers. centrosome indicated the and oaie eo h itladsbitlapnae and appendages subdistal and distal the below localizes n E12 efrttse hte they whether tested first we CEP152, and c oaie oteitreit C around PCM intermediate the to localizes c c enocdteeiec o a for evidence the reinforced n LGCP5 Fg 3C), (Fig. FLAG–CEP152 and AC eaclswere cells HeLa (A-C) c m .()HL cells HeLa (D) m. inlfo the from signal c ifrne EG nagdcnrsm mgsaesona net.Scale inserts. as shown 0.5 significant are bars: no images N.S., centrosome s.d.; Enlarged indicate (E,G) bars difference. Error experiment. each in examined rma es he needn xeiet eeaaye,with analyzed, were not experiments independent but CEP152, three of least loss at the from that PIPKI indicates eliminates G significantly and CEP192, E in described (H) PIPKI group centrosomal cells. each robust these with cells in of decreased number the strongly of was Quantification and CEP152 centrosome of the signal from centrosomal abolished the completely was CEP192 CEP192-depleted although in affected cells, significantly not was PIPKI centrosomes (G) at CEP192. localization CEP192-specific endogenous (F) depleted shown. efficiently are (siCEP192) cells siRNA CEP152-depleted Representative or antibodies. control indicated of the images with stained then and siCEP152 endogenous eliminates CEP152 of PIPKI Loss siNC, (E) cells. siRNA. HeLa control from non-specific siRNA CEP152 CEP152-specific endogenous (D) depleted 3D-SIM. efficiently by (siCEP152) analyzed then and antibodies anti-PIPKI FLAG with stained were FLAG–CEP152 expressing cells HeLa the with immunoblotting PIPKI by (C) analyzed antibodies. were indicated B and A from Co- precipitates (B) The cells. lysate. cell PIPKI Lys., endogenous antibodies. of (GFP) precipitation anti-GFP rabbit or rabbit (rIgG) normal IgG using immunoprecipitation to HA–PIPKI subjected encoding were GFP–CEP152 vector and with co-transfected cells HEK293T (A) another. centrosome. the fCP5 loeiiae h etooetreigo exogenous of targeting centrosome the eliminated also for CEP152 necessary of is PIPKI of CEP152 recruitment that the indicating 3E,H), (Fig. centrosome i.3 E12ascae ihPIPKI with associates CEP152 3. Fig. c oaiaina etooe.HL el eetetdwt iCor siNC with treated were cells HeLa centrosomes. at localization ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal m C 5 (C) M AB E12adPIPKI and CEP152 (A,B) m ,(,) N a tie ihDAPI. with stained was DNA (E,G). M, c c otecnrsm.Mroe,depletion Moreover, centrosome. the to n E12clclz ttecentrosome. the at colocalize CEP152 and c n noeosCP5 rmHeLa from CEP152 endogenous and c c agtn otecnrsm.Results centrosome. the to targeting n euae PIPKI regulates and c oetal soit ihone with associate potentially c agtn to targeting c . c tiigin staining 0 cells 200 n anti- and c c

Journal of Cell Science utemr,teepeso fbt idtp n kinase-dead and wild-type both of PIPKI significantly HU-induced expression the was 4G, Furthermore, Fig. cells in HA–PIPKI OS PIPKI of shown overexpression U-2 the As by in suppressed 2009). cycle amplification cell al., the centrosome of et phases other (Hemerly on influence potential the excluded eetetdwt Ufr2 eoebigtasetdwt or PIPKI with overexpressed transfected of impact being the before limited cells HA–PIPKIdesign h kinase-dead OS 24 or U2 for wild-type 4F, without HU Fig. with in treated shown were As treatment. (HU) hydroxyurea in observed also was foci 2 PIPKI distinct centrin two with of treated cells number HeLa the in increase h etooe(i.3,) hs eut ugs htavery a the for that sufficient PIPKI be of suggest could recruitment results CEP152 centrosomal centrosomal these of amount 3E,H), small (Fig. centrosome the 3,) hsfrhrcnimdta h hntp a asdby caused was phenotype the that confirmed further This S3F,G). PIPKI of depleted cells siPIPKI and PIPKI amplification RNAi-resistant centriole PIPKI of Importantly, of expression depletion 4B,C). (Fig. by in cells cell) caused per four the than (more of PIPKI foci of 2 centrin loss of the augmentation Strikingly, PIPKI 4A). PIPKI down (Fig. lentivirus-based knocked (sh)RNA using we cells purpose, this HeLa For centrosome. the fPIPKI PIPKI of that suggest results duplication. these centriole inhibits Together, likely S3J). Fig. (supplementary IMCD3 material line cell epithelial duct and collecting S3H,I) renal Fig. the material (supplementary NIH3T3 as such types cell two than (more number centrosome depletion Furthermore, PIPKI phase. M or of G2 S, sustained PIPKI a from in resulted foci treated significant 2 cells centrin between no excessive observed S3C-E, was siPIPKI As markers Fig. with phase. these G2/M material in in supplementary difference cells (supplementary identify in to H3 (PCNA) used shown was phospho-histone antigen S3E) S Fig. the S3C) and nuclear material determine to population, Fig. cell used were phase proliferating S3D) material Fig. material for (supplementary (supplementary staining BrdU and we phase, of G2/M PIPKI or of incorporation phase distribution S cycle in cell arrest the examined cycle cell from resulted foci PIPKI opeels fCP5 lce h agtn fPIPKI that of targeting observation the previous blocked CEP152 our of with loss complete Combined found however, 3G,H). eliminated 2013); (Fig. we al., largely et PIPKI was (Sonnen cells, proteins previously the CEP152 reported CEP192-depleted centrosomal of as 3G), signal In of (Fig. centrosomal 2013). recruitment the al., the that for et important (Sonnen the be for to platform structural PIPKI necessary of a association stable provides CEP152 that PIPKI ARTICLE RESEARCH h pcfccl-yl-eedn etooa oaiainof localization centrosomal cell-cycle-dependent PIPKI specific The PIPKI ots hspsiiiy eeaie hte h expression the whether examined we possibility, this test To nadto oCP5 Fg F,CP9 a eetyshown recently was CEP192 3F), (Fig. CEP152 to addition In c c c c c c eaieyrgltscnroeduplication centriole regulates negatively _i3 ol ececnroeoe-ulcto asdb PIPKI by caused over-duplication centriole rescue could elto.T eemn hte h nrae eti 2 centrin increased the whether determine To depletion. e st netgt h oeta ucino PIPKI of function potential the investigate to us led sangtv euao fcnrsm duplication. centrosome of regulator negative a is c c 1-445 c u o PIPKI not but , c ol ees etooeapiiaincue by caused amplification centrosome reverse could c inlaon h etooewsmsl retained mostly was centrosome the around signal O)(upeetr aeilFg 3,)btntin not but S3A,B) Fig. material (supplementary -O2) n el rae ihcnrlsRA hrfr,the Therefore, siRNA. control with treated cells and splmnaymtra i.SB,frhrsuggesting further S2B), Fig. material (supplementary a rPIPKI or a c rPIPKI or ihtecentrosome. the with c c c b a lotflyrsudby rescued fully almost was dpee el eentlikely not were cells -depleted c . splmnaymtra Fig. material (supplementary tblnlc e el nother in cell) per loci -tubulin c b asda nraein increase an caused , Fg DE.Asimilar A 4D,E). (Fig. c c seii hr hairpin short -specific iNs(siPIPKI siRNAs c c hsexperimental This . dpee el.The cells. -depleted c ugsigthat suggesting , c c oSpaeand phase S to eutdi an in resulted , c 20% c c c -O1 to in at c c fP(,)2mgtntpa nipratrl ncentriole in role production important PIPKI an the play that not suggesting might biogenesis. treatment, PI(4,5)P2 HU of or depletion ee fPK sudtcal yimnbotn,w examined we immunoblotting, by endogenous undetectable the is functional Because PLK4 the another. of investigate one level To with 2010). associate physically al., et PIPKI a Hatch between correlation in 2010; centrioles al., of end et Dzhindzhev 2010; proximal al., the et (Cizmecioglu around manner CEP152-dependent PCM the PIPKI in with localizes interacts directly PIPKI PLK4 of Like domain polo-box cryptic The oee,tecnrsm ubr(ersne by (represented PIPKI number in four centrosome other than of the each (more However, with foci ends associated 2 that centrin cell) proximal and per PIPKI CP110 the cells or excessive HeLa control observed at after carrying we h lentivirus (localizes 48 with At infected CPAP 2007). were al., and et centrioles) (Kleylein-Sohn 2007) centrioles) (CEP135, (Kleylein-Sohn distal centrioles) of KDa al., of the 135 end et at ends proximal of the (localizes at protein distal localizes 2 centrosomal which centrioles), centrin the of 2007), ends at al., et localizes (Kleylein-Sohn kDa which 110 of protein (CP110, coiled centriolar including proteins, centriolar n PPfc eeas oml(w e el nPIPKI in cell) per (two normal also CEP135 were of numbers foci The cell). CPAP per (two and normal was cells these in eltdcls(i.5,) hs eut ugs htthe that PIPKI suggest in foci results CP110 These and 2 5A,C). centrin (Fig. additional cells depleted xesv E15 PPand occurred, CPAP centrioles CEP135, lentivirus nascent excessive after of h disengagement the 72 when Indeed, still infection centrioles. were parental the that with procentrioles engaged over-duplicated to correspond might yepeso fete idtp rkns-edHA–PIPKI kinase-dead PIPKI or suppressed that function. suggesting wild-type further significantly either 5F), (Fig. was of cells OS expression overexpression U-2 by in Myc–PLK4 induced amplification S3N). by centriole Fig. the material addition, (supplementary In overexpression resulted S3M), that PLK4 amplification Fig. from centriole centriole material the (supplementary of enhanced PLK4 stage synergistically PIPKI of of level early depletion the the this, affecting with at agreement HsSAS-6 In PIPKI biogenesis. and/or that PLK4 suggesting against 5E), (Fig. rescued completely the PIPKI supporting of further lack the distal panel), the to the attached by right that (identifiable panels) hypothesis 5D, centriole middle (Fig. mother and a left appendages) of 5D, wall Fig. proximal P2, and serial- (P1 of PIPKI analysis PIPKI microscopy in sectioned electron transmission observed Moreover, were cell) ulcto hntp asdb PIPKI by caused phenotype specific or PIPKI PLK4 designed with duplication When along we S3L). co-depleted was Fig. this, HsSAS-6 material S3K) Fig. (supplementary test material HsSAS-6 (supplementary To or PLK4 down HsSAS-6. knock connection to or a siRNAs suggesting pre- PLK4 centriole, one parental with per around procentriole procentrioles PIPKI 2007) one al., multiple centriole. et of Strnad 2005; existing biogenesis al., et to (Leidel HsSAS-6 leads or 2005) al., et amplification. ofrhrcaatrz hs xesv eti oiin foci 2 centrin excessive these characterize further To thsbe eotdta vrxrsino L4(Habedanck PLK4 of overexpression that reported been has It c dpee el,w hce h oaiaino several of localization the checked we cells, -depleted ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal c L4(h e euao fcnroeduplication) centriole of regulator key (the PLK4 , c dpee eaclssoe w procentrioles two showed cells HeLa -depleted c c n L4 efrttse hte they whether tested first we PLK4, and elto loyeddmr than more yielded also depletion c tblnfc mr hntoper two than (more foci -tubulin c dpee el Fg 5B,C). (Fig. cells -depleted c c ih oneatPLK4 counteract might ed oprocentriole to leads c c h etil over- centriole the , xasinwas exhaustion c c dpee cells. -depleted dpee cells -depleted c tblnfoci) -tubulin c c c functions without , shRNA, 1297 c c c -

Journal of Cell Science EERHARTICLE RESEARCH 1298 This antibody. not PLK4 but MBP–PLK4 MBP–CEP152 monoclonal purified or MBP mouse recognized specifically a PIPKI antibody complex between developed correlation a we spatial form the they understand PIPKI that with co-immunoprecipitated indicating GFP–PLK4 PLK4. tagged PIPKI endogenous whether i.SC,frhredrigteseiiiyo hsantibody. this of specificity the PIPKI endogenous material endorsing endogenous of (supplementary the for Colocalization further PLK4 seen stained S4C), of that nicely partner Fig. to binding antibody similar the pattern PLK4 CEP152, a our endogenous with siRNAs visualize that by centrosome to obtained was showed Images used PLK4-specific microscopy. 3D-SIM be staining that immunofluorescence can indicate by This antibody independent results PLK4 PLK4 centrosome. These new S4B). two the this Fig. at material by (supplementary PLK4 stains abolished it and 01.Ti si iewt h ointa L4adCEP152 and al., PLK4 et that (Sir notion 6B) the Fig. with and line and 3C in shape (Fig. is similar CEP152 This a of the 2011). with that structure that PIPKI to Both ring-like diameter suggesting overlap. a proteins 6B), displayed two (Fig. PLK4 these of observed spaces was working centrosome the 1-748 c soitswt vrxrse GFP- overexpressed with associates splmnaymtra i.S4A) Fig. material (supplementary c ihedgnu L4at PLK4 endogenous with nvivo in Fg A.To 6A). (Fig. c n PLK4, and c and c , rti rget eehgl erddwe uiidfrom purified the when and degraded CEP152 highly coli length were full fragments the protein Because PLK4. PIPKI with interaction whether tested PIPKI we and PLK4, CEP152 both Because PIPKI oitreit eino h C Sne ta. 2012). al., et with (Sonnen PCM PIPKI the position of and Dzhindzhev region 2010) 2010; al., intermediate et al., to Hatch et 2010; al., (Cizmecioglu et colocalize and interact efudta h rpi oobx(P)dmi fPK is PIPKI of PLK4 terminus of PIPKI within domain region (CPB) PIPKI PIPKI polo-box for truncated sufficient cryptic and the 6F), (Fig. necessary that fragments PLK4 found recombinant truncated we of series a Using PIPKI n L4adsgetafntoa osqec fti interaction. this of consequence functional a PIPKI suggest between and association PLK4 physical a and results of These notion 6E,F). the (Fig. support binding strongly PLK4 for necessary not is terminus etw etdtedrc neato ewe PIPKI between interaction direct the tested we Next epoue the produced we , nvitro in c c mar L4activity PLK4 impairs .PIPKI ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal uldw sasuigprfe B–L4adHis– and MBP–PLK4 purified using assays pull-down ro asidct s.d. indicate difference. bars significant Error no N.S., experiments. independent ed(D.U2O el eeperae ih1 MH for HU mM 16 with pretreated were cells OS U-2 (KD). dead eeqatfe o el xiiigmr hntwo than (E,G) more (G). exhibiting foci cells for quantified anti- were and anti-HA with stained were c group each in cells and Next, endogenous of Expression PIPKI cells control. exogenous OS a U-2 as Non-treated used harvest. were before HA– h indicated 44 the additional or EV with transfection PIPKI by followed h, 24 Sclswssgiiatysprse yteexpression the HA–PIPKI by either suppressed of significantly was U-2 in cells OS amplification centrosome HU-induced (F,G) the plotted. in centrioles four HA–PIPKI than or more control with DAPI. cells and of antibodies percentage were 2 The group anti-centrin each anti-HA, in with Cells stained (E) antibodies. indicated the PIPKI exogenous HA– RNAi-resistant or (EV) PIPKI vector empty siPIPKI with or transfected siNC with treated being wild- HA-tagged of (PIPKI form type RNAi-resistant the PIPKI of of expression depletion the amplification by centriole caused The (D,E) PIPKI experiments. or independent control in centrioles cells. four depleted than more 5 with bar: cells Scale inserts. as images shown centrosome are Enlarged numbers. centriole increased onedgnu PIPKI shPIPKI endogenous (A) down (B). DAPI and 2 antibody anti-centrin using indirect microscopy to immunofluorescence subjected or (A) immunoblotting or (shNC) control PIPKI non-specific either carrying lentivirus cells. in PIPKI foci of Depletion 4. Fig. tblnatbde swl sDP,adte h samples the then and DAPI, as well as antibodies -tubulin c c lal hw ietbnigt L4(i.6C). (Fig. PLK4 to binding direct shows clearly c rtis enroe ontePLK4-binding the down narrowed we proteins, c c c ln ol o idt L4 n h PIPKI the and PLK4, to bind not could alone c seii (shPIPKI -specific osrcsadwr eicbtdwt Ufran for HU with re-incubated were and constructs osrcs D h eeso noeosand endogenous of levels The (D) constructs. otekns oan(i.6,) h -o C- or N- The 6E,F). (Fig. domain kinase the to 35 c W)o iaeda (PIPKI kinase-dead or -WT) -aee E12Ntria fragment N-terminal CEP152 S-labeled AC eaclsifce o 8hwith h 48 for infected cells HeLa (A-C) n n . . c c 0,rslsaedrvdfo tlatthree least at from derived are results 100, 0,rslsaedrvdfo tlatthree least at from derived are results 200, c eedtrie yimnbotn with immunoblotting by determined were a eemndb muoltig(F). immunoblotting by determined was c idtp W)o HA–PIPKI or (WT) type wild epesn ru a uniidand quantified was group -expressing c c c c c n E12cmeefrthe for compete CEP152 and hNsfrwr olce for collected were for shRNAs ) B oso PIPKI of Loss (B) . idt h P oanof domain CPB the to bind c neato Fg D.Using 6D). (Fig. interaction nue xesv eti 2 centrin excessive induces c c m a ece by rescued was eaclswere cells HeLa , c .()Qatfcto of Quantification (C) M. fiinl knocked efficiently c K)PIPKI -KD) c c c tteinner the at eut in results n PLK4 and c c kinase -tubulin c After . c c - N- E. c

Journal of Cell Science 00 oln ta. 00 oese l,20)adispotential al., its and et 2009) al., Guderian et 2009; Rogers vivo 2010; (Brownlee al., al., and et et itself Nigg Holland Cunha-Ferreira 2010; both 2007; 2012; phosphorylate Nigg, Rogers, can 2007; and al., PLK4 Eckerdt et 2011). 2005; Kleylein-Sohn Stearns, al., 2011; et al., (Bettencourt-Dias et fidelity and duplication of PLK4. of amounts region PIPKI increasing that of suggesting addition the PIPKI MBP–PLK4, by down EERHARTICLE RESEARCH 35 ( ac ta. 00,MPtge ullnt L4ple down 2010; pulled al., PLK4 full-length et MBP-tagged Dzhindzhev 2010), 2010; al., et al., Hatch et (Cizmecioglu literature 35 S–CEP152 elcnrle ciiyo L4i seta o centriole for essential is PLK4 of activity Well-controlled S–CEP152 usrt E12(imcol ta. 00 Dzhindzhev 2010; al., et (Cizmecioglu CEP152 substrate c i o fettebnigo E12t L4(i.7A), (Fig. PLK4 to CEP152 of binding the affect not did 1-748 1-748 )by el lhuhPIPKI Although well. c nvitro in n E12d o idt h same the to bind not do CEP152 and rnlto.Cnitn ihthe with Consistent translation. c ol tl epulled be still could in ta. 00 ac ta. 00.Teeoe eoverexpressed we Therefore, 2010). al., et CEP152 Hatch FLAG-tagged 2010; al., et ftewl-ye(T rkns-ed(D PIPKI (KD) kinase-dead amounts or varying (WT) without wild-type or the with ATP, of activity for PLK4 competing the by compared another one we PIPKI inhibit might Because PIPKI they and with 7D). kinases (Fig. interaction the activity whether PLK4 tested next We PIPKI that indicating osntbn oAP(uze l,20)btbnst PLK4 to binds but 2000) al., et (Kunz ATP vitro to bind not does PIPKI not but 7B) (Fig. CEP152 from with and Results phosphorylate incubated could assay. itself PLK4 were kinase both that a demonstrated antibody experiments perform anti-FLAG these to normal by MBP–PLK4 by or purified obtained IgG precipitates mouse The immunoprecipitation. by tacmaal ee oiswl-yecounterpart wild-type its to level comparable a at ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal infcn ifrne ro asidct s.d. indicate bars Error no difference. N.S., significant experiments. independent three least 1 with treatment h 16 a before mlfcto nU2O el a significantly was cells OS U-2 in Myc– amplification (F) plotted. and centriole quantified PLK4-overexpression-induced foci was 2 group centrin four each than in more with The cells DAPI. of and percentage stained antibody were 2 group anti-centrin each with in Cells siRNA. HsSAS-6 PIPKI (shPIPKI or lentivirus (shNC) with control infected carrying were cells HeLa (A,B) amplification. centriole HsSAS6-dependent oto.(E,F) control. a ( Cells treatment group. doxycycline four each without than in more quantified with were Cells centrioles DAPI. as with antibodies as 2 well anti-centrin then and were anti-HA Cells with expression. stained PLK4 induce to (+Dox) PIPKI HA-tagged or (EV) with vector transfected empty were PLK4 Myc-tagged expressing or (WT) PIPKI wild-type (KD) of kinase-dead expression the by suppressed el eetasetdwt eaiecnrl(iC or (siNC) PIPKI control negative with transfected were PIPKI cells from centriole results the that rescues duplication HsSAS-6 0.2 or bar: PLK4 Scale of +4). depletion and are +1 serial-sections (0, of shown mother numbers one The around (M). P2) centriole and (P1 procentrioles two PIPKI serial-sectioned from images (TEM) microscopy electron Transmission with (D) treated were PIPKI that or cells control four of than samples more in with centrioles cells of Quantification 1 (C) bars: Scale centrosomes. normally duplicated at the proteins of centrosomal positions indicated the illustrate panels) diagrams (middle indicated schematic the The visualize proteins. to centrosomal stained and fixed then PIPKI 5. Fig. c 1-748 ol o opt ihPK substrates. PLK4 with compete not would c iN (siPIPKI siRNA nHL el n uiidti protein this purified and cells HeLa in c hN o 8h()o 2h()adwere and (B) h 72 or (A) h 48 for shRNA ) c elto assPK-and PLK4- causes depletion n c . hN o 8ho 2h. 72 or h 48 for shRNA 0,rslsaedrvdfo at from derived are results 100, c ,wt rwtotPK or PLK4 without or with ), c h e-nU2O cells OS U-2 Tet-On The . c c dpee el showed cells -depleted n L4aeboth are PLK4 and 2 m c /ldoxycycline g/ml o)wr sdas used were Dox) -specific c c elto.HeLa depletion. PIPKI . c m c c .()Co- (E) M. m influences M. constructs Fg 7C), (Fig. c 1299 -KD in

Journal of Cell Science EERHARTICLE RESEARCH ooeduhe e ohri igecl yl r associated are cycle cell 1300 single a in mother limited per is assembly daughter centriole one the how to and built are centrioles How DISCUSSION PIPKI Both data). (unpublished idn oadihbtn L4kns activity. kinase PLK4 inhibiting and to binding PIPKI our that self-regulation, suggest achieve results and PLK4 for biogenesis crucial procentriole is initiate activity to PLK4 that exploration. context the future in require Nevertheless, will that mechanism complicated more PIPKI purified recombinant of of using self- addition amounts the increasing by by interrupted 7G, the not down Fig. was interaction in This for pulled MBP–PLK4. shown were As important 2010). Myc–PLK4 PIPKI al., is overexpressed et of (Guderian which PLK4 binding of dimerization, the activation whether PLK4 determined affects we activity, PIPKI how explore PLK4 To PLK4. PIPKI to binding that by suggest activity PLK4 results These by ATP. caused not for was competition inhibition this that suggesting level, comparable PIPKI L4atvt nads-eedn anr(i.7-) When PIPKI 7D-F). (Fig. of manner dose-dependent amounts a equal in activity PLK4 To PIPKI or WT , PLK4, of that 7D-F).threefold PIPKI (Fig. of amount phosphorylation the When CEP152 and autophosphorylation 0 fCEP152 of 50% c W n PIPKI and -WT c K eutdi a in resulted -KD 1-748 hshrlto eeihbtd(i.7D-F). (Fig. inhibited were phosphorylation c . K ossetyrpesdPK ta at PLK4 repressed consistently -KD c 0 fPK uohshrlto and autophosphorylation PLK4 of 40% c c ol etancnroedpiainby duplication centriole restrain could W rPIPKI or -WT n L4wr de,PIPKI added, were PLK4 and c W n PIPKI and -WT , 0 niiino PLK4 of inhibition 20% c c Fg G,idctn a indicating 7G), (Fig. K a nrae to increased was -KD c K inhibited -KD c c regulates inhibits c c - vdnespotn h hsclascaino endogenous of association solid provide physical 2) the 1, PIPKI Nevertheless, (Figs 2011). supporting al., experiments et multiple evidence Zellner from 2010; al., results et our Ren 2009; al., al., et et Keck 2003; al., Mu et 2011; (Andersen PIPKI studies PIPKI proteome why centrosome which of regulated, explain carefully and regulator association sensitive could be negative the to likely a is biogenesis, centrosome As activity. centriole PLK4 of limiting formation and centriole potentially Nigg restricts by 2012; that al., machinery duplication key et PIPKI centriole report the (Brito we to years here addition 2011), recent In Stearns, resolved. in fully identified been players not have that puzzles ioi u nrae ail meitl fe ioi.In mitosis. after immediately PIPKI during of rapidly downregulated association is the increases addition, it but direction: PIPKI exit, opposite mitotic mitosis at which the sharply of in drop and levels regulated phase this the M in SAS6 (STIL), highest however, the PLK4, protein are as with locus proteins, such Compared SCL-interrupting biogenesis, way. and centriolar centriole unique of other rather regulators a positive many in operates for regulation case the PIPKI of association ocnroe nG/ hs ospotpoetil assembly procentriole support to phase recruited G1/S are which in CPAP, is and centrioles timing SAS-6 2, This to centrin S1G). of that Fig. to from material similar emerge (supplementary to starts centriole procentriole the the when boundary G1–S the at c ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal ihtecnrsm.Teoeallvl n centrosome and levels overall The centrosome. the with le ta. 01 Mu 2011; al., et ¨ller emns(PIPKI terminus oan T -emns P,cytcpl o;P,pl box. polo PB, box; polo kinase cryptic KD, N-terminus; CPB, NT, C-terminus; length; CT, regions full domain; FL, binding red. identified in The highlighted C-E. are in shown assays pull-down (PIPKI truncated PIPKI His-tagged with incubated was GST–PLK-CT PIPKI or box of GST (KD) polo domain or kinase The (PLK4-CPB) (E) box (PLK4-PB). polo C- cryptic (PLK4-NT), (PLK4-CT), N-terminus terminus the including polypeptides, PLK4 His–PIPKI PLK4. of domain by analyzed PIPKI and (D) resin immunoblotting. His–PIPKI amylose with using incubated down was pulled MBP–PLK4 or MBP Purified 0.5 anti- bar: with Scale stained were cells PIPKI HeLa PIPKI (B) centrosome. lysate. the cell at Lys., colocalize cells. HeLa in PLK4 n L4 ae nterslsfo the from results PIPKI the between on sites based interaction PLK4, (CBB) the and Blue of Brilliant Schematic Coomassie (F) by staining. GST-fused shown and is GST fragments MBP–PLK4, MBP, PLK4 of loading The (C-E) PLK4. i.6 PIPKI 6. Fig. c A noeosPIPKI Endogenous (A) n niPK nioisadte nlzdb 3D-SIM. by analyzed then and antibodies anti-PLK4 and c c m ietyitrcswt h P oanof domain CPB the with interacts directly r euae hog h elcce sis as cycle, cell the through regulated are c .()PK ietyitrcswt PIPKI with interacts directly PLK4 (C) M. c N) -emns(PIPKI C-terminus -NT), - D )adaaye yimmunoblotting. by analyzed and N) c le ta. 00 Nogales-Cadenas 2010; al., et ¨ller a o ecie nterecent the in described not was c c id otecytcpl o (CPB) box polo cryptic the to binds c a nuae ihGTo GST– or GST with incubated was c ihacnroei is seen first is centriole a with c opeiiae ihGFP-tagged with co-precipitates sanwcmoeto the of component new a as c nvitro in C)o N-terminus or -CT) c c n PLK4 and S rMBP or GST id oPLK4. to binds c ihthe with c . c c , c N- c is

Journal of Cell Science EERHARTICLE RESEARCH a eemnduigtePK iaeasywt rwtotprfe idtp W)o iaeda K)PIPKI (KD) kinase-dead or (WT) wild-type CEP152 purified or without PLK4 or phosphorylated with of assay autoradiography kinase of PLK4 results the using determined was PIPKI of binding The 7. Fig. hshrltdPK E rCEP152 or (E) PLK4 phosphorylated osrc eesbetdt muorcptto sn nat-LGatbd.Apl-onasywsue odtrietec-rcptto ftere the of co-precipitation the His determine of to amount used increasing was an assay without pull-down or A with antibodies. antibody. MBP–PLK4 indicated anti-FLAG soluble an purified using with immunoprecipitation immunoprecipitates to subjected were construct P tiig B L4popoyae ohisl n E12 LGtge E12famn (CEP152 fragment CEP152 FLAG-tagged A CEP152. and itself His–PIPKI both of phosphorylates amount PLK4 indicated (B) the staining. of presence the without hwn h odn fprfe B–L4 ae n ,I ihat-LGatbd oaayeteimnpeiiae ulddw ymueIG(mIgG) IgG mouse by (IB) down immunoblotting pulled CEP152 1, immunoprecipitates and the Lane PLK4 analyze MBP–PLK4. of to purified phosphorylation antibody for the anti-FLAG substrate showing with a (Autoradio.) IB as autoradiographs 3, assay and 4–6, kinase 2 Lanes PLK4 antibody. Lanes a anti-FLAG MBP–PLK4. in purified or used of then loading was the It showing antibody. anti-FLAG with immunoprecipitated iho ihu B–L4i h L4kns sa.()PK ol o hshrlt PIPKI phosphorylate not His–PIPKI could and PLK4 MBP–PLK4 (C) of assay. loading kinase His–PIPKI PLK4 purified the when in assay MBP–PLK4 without or with emnso E12(CEP152 CEP152 of terminus , .1 .. osgiiatdfeec.()PIPKI (G) difference. significant no N.S., 0.01; c 1-748 c mar L4kns activity. kinase PLK4 impairs a nuae iho ihu B–L4 h ih ae hw omsi rlin le(B)sann niaigthe indicating staining (CBB) Blue Brilliant Coomassie shows panel right The MBP–PLK4. without or with incubated was a xrse sn an using expressed was ) 1-748 c D PIPKI (D) . F rmtreidpneteprmns igeatrs * represents (*) asterisk single A experiments. independent three from (F) c c osntafc h ieiaino L4 eaclsepesn ihrcnrlcntuto Myc–PLK4 or construct control either expressing cells HeLa PLK4. of dimerization the affect not does niisPK ciiy L4atpopoyaino L4mdttdpopoyaino CEP152 of phosphorylation PLK4-meditated or autophosphorylation PLK4 activity. PLK4 inhibits c sn B-uldw sa.Telaigo B n B–L4i hw yPneuS Ponceau by shown is MBP–PLK4 and MBP of loading The assay. MBP-pull-down a using , nvitro in A PIPKI (A) 1-748 h oe ae hw h odn fPIPKI of loading the shows panel lower The . rncito/rnlto ytm t neato ihMPPK a eemndwt or with determined was MBP–PLK4 with interaction Its system. transcription/translation c osntafc h neato ewe E12adPK.Te[ The PLK4. and CEP152 between interaction the affect not does ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal c h etpnlsosatrdorpso h L4kinase PLK4 the of autoradiographs shows panel left The . 2 PIPKI 1-748 c h ia rcpttswr hnbotdwt the with blotted then were precipitates final The . a vrxrse nHL el and cells HeLa in overexpressed was ) c c sidctd h pe ae hw the shows panel upper The indicated. as rtis EF uniiainof Quantification (E,F) proteins. P 1-748 , .5 obeatrs *)represents (**) asterisk double a 0.05; hnCEP152 when 1-748 35 ]e-aee N- S]Met-labeled a incubated was 1-748 sulting 1301

Journal of Cell Science agtdt h etooewe vrxrse,teuiu insert unique PIPKI the in overexpressed, sequence when centrosome the to targeted machinery procentrioles. excessive assembly of centriole formation the the inhibits counteracts and it well: function akrud(aantson.Atbd gis PI(4,5)P cytosolic against and Antibody membrane shown). strong not the (data to owing background centrosome the at pleckstrin delta (PLC C homology phospholipase GFP-tagged Using duplication. aldt ihih n pcfcsbellrlctos including locations, PI(4,5)P subcellular where specific ones any the highlight to failed ET Sgn ta. 00 oaiea h etooeand centrosome suggesting the – at 2012) al., localize et 2010) (Foraker integrity al., centrosome regulate et (Sagona al., et CENT) (FYVE- protein (Ford that centrosomal FYVE-domain-containing and clathrin and the 2011) as 2001) al., around such et vesicles Westlake proteins, membrane 2012; phosphoinositide-binding al., are et there (Foraker it that centrosome However, reported shown). not been (data has pits clathrin-coated and adhesions h usino hte PI(4,5)P whether of question the xrsinadcnrsm soito fPIPKI cell-cycle-dependent of the association completed, centrosome has and procentriole budding expression the why when the al., understood phase fully M et in not of Strnad accumulate is STIL it 1996; and Although SAS-6 al., 2009). PLK4, et al., et Paoletti Tang 2007; 2007; al., et (Kleylein-Sohn ARTICLE RESEARCH 1302 centrosome- the shows isoforms that PIPKI other in suggests the signal This of targeting none localization. other however, centrosomal in members; present PIPK commonly I is be that to sequence domain seems PIPKI kinase centrosome-targeting signal the centrosome-targeting in a the located contain Instead, 1E). not (Fig. does isoforms, xoueo h etooetreigmtfi PIPKI in motif centrosome-targeting the by of exposure exhausted been have PIPKI overexpressed motif. could which mechanisms, centrosome-targeting modification PIPKI of the fraction PIPKI a expose only PIPKI Because and of C- conformation unique insertion The plausible domain. is kinase terminal It the PIPKI exposed. in be motif of to centrosome-targeting order dimerization in way that certain a in modified ytreigt pcfcsbellrlclsadproviding and locales subcellular specific to PI(4,5)P the targeting governs that by code signal the PIPKI decipher of association to cell-cycle-dependent order to in sufficient However, required be not PIPKI 2012). might CEP152 recruit that al., suggesting present, et still is (Sonnen protein PIPKI PIPKI that PCM indicating intermediate PIPKI toroid, between PIPKI be CEP152 interaction the to likely physical Indeed, is CEP152. a association stable through This achieved CEP192. not but was between CEP152 association This stable the proteins. that PIPKI centrosomal observation the other by reinforced as mechanism the common PIPKI that suggesting of cells, most association in both centriole 2012), the around al., distribution et PIPKI overexpressed Sonnen and 2012; al., endogenous Yamashita, et and Mennella 2012; Pelletier Glover, 2012; and (Fu proteins centrosomal other lhuhol PIPKI only Although rdtoal,PIPKI Traditionally, c c c c i ttecnrsm,i sas osbeta other that possible also is it centrosome, the at _i3 piigioom n shgl osre mn l type all among conserved highly is and isoforms splicing sasn rmtecnrsm nMpaewe CEP152 when phase M in centrosome the from absent is 2 n h etooesrcl eiso h xsec of existence the on relies strictly centrosome the and otepoiaedwsra fetr hslasto leads This effector. downstream proximate the to c d otecnrsm.Frhrivsiain are investigations Further centrosome. the to H oan efie osecerGPsignal GFP clear see to failed we domain, PH) c i,wihdsigihsi rmohrPIPKI other from it distinguishes which _i3, c c i,aencsayt civ oeefficient more a achieve to necessary are _i3, c c ihtecnrsm olw h same the follows centrosome the with euae ait fclua processes cellular of variety a regulates i adntohrPIPKI other not (and _i3 2 c ly motn oe,sc sfocal as such roles, important plays snral idnadnest be to needs and hidden normally is c c oodoelpe elwt the with well overlapped toroid c Roe l,19)cvr the covers 1998) al., et (Rao i ih le h protein the alter might _i3 c i-xrsigclsshowed cells _i3-expressing 2 c a oei centriole in role a has xiie toroid-shaped a exhibited c c ieCP5,i an is CEP152, like , ihtecentrosome. the with c c c sfrs is isoforms) iia to Similar . evsits serves c 2 also and c h etil rcnrsm mlfcto asdb PIPKI by that caused suggesting overexpression, amplification PI(4,5)P PLK4 or centrosome treatment HU or depletion, centriole the htpoponstds nldn PI(4,5)P including phosphoinositides, that neednl fPI(4,5)P of independently PIPKI that previously reported ilbun ta. 00.I a ensonta h erdto of degradation the that shown 2010; been al., has et It Holland 2010). al., 2010; et al., Sillibourne et (Guderian PLK4 of degradation progression. agreement cycle in cell is affecting PIPKI without it of amplification 2010), loss centriole that al., observation et our Sillibourne with 2010; 2010; weakened. al., al., et is et (Guderian Holland activity PLK4 endogenous of PLK4 expression restricted tested that been not has such model this or Although substrates ATP of PLK4 access the and/or other PIPKI protein the of of binding conformation the the impair that propose We crucial. is abtmncoa nioyaantPIPKI against antibody monoclonal Rabbit constructs Nigg and Antibodies Erich the by by cell-line induced provided was tetracycline-inducible 1 expression of kindly Myc–PLK4 addition The 2007). was al., et h. PLK4 mM (Kleylein-Sohn 24 16 Myc-tagged with synchronized for were expressing cells OS (Sigma) treatments U-2 mM) release]. hydroxyurea 2 h (Sigma, 9 a thymidine standard by h a separated 16 with [two synchronized block were double-thymidine cells Lipofectamine V using HeLa Kit cells (Invitrogen). Nucleofector into RNAiMAX introduced Line were Cell duplexes Amaxa siRNA or (Lonza). (Roche) and 9 HeLa transfected X-tremeGENE were cells using (FBS). OS U-2 (Promega). serum Reagent Transfection or were 6 (Invitrogen) bovine FuGENE 2000 Lipofectamine cells fetal using cultured transfected were 10% were cells HEK293T HEK293T cells containing IMCD3 and DMEM:F12 FBS. NIH3T3 10% in containing MDA-MB-231, DMEM in OS, maintained U-2 HeLa, transfection and culture Cell METHODS AND MATERIALS PIPKI of overexpression duplication and centriole of PIPKI procentrioles the of level multiple of loss another to activity because crucial providing machinery appropriate seems This the activity, 2010; phosphorylation. maintain to its al., addition et in inhibits regulation PIPKI Holland that certain and suggest 2010; a results PLK4 Our al., reaches 2010). activity al., et et its Sillibourne (Guderian once level destroyed is threshold PLK4 model a which to leads in which phosphorylation, multi-site requires PLK4 Ln ta. 07.I hspoes h hsclitrcinof interaction physical the process, this In 2007). PIPKI endosome clathrin- al., recycling the et the E- to (Ling of network and trans-Golgi transport the the E-cadherin facilitates from thereby cadherin between and AP1B scaffold complex adaptor a as functions ucina h etooe eetees norhnsbt the both hands PIPKI our of in forms kinase-dead Nevertheless, and wild-type centrosome. the at function ucinlrl nohrcnrsm-eae ellrprocesses. cellular centrosome-related other in role functional yimnzn abt ihrcmiatHstge PIPKI His-tagged recombinant with rabbits immunizing by rti euae yPIPKI centrosomal only by the is regulated PLK4 whether protein the PIPKI investigate of to consequences between to important functional necessary interaction the be into physical will insight studies centriole greater of Future negative provide control homeostasis. accurate its and the and for assembly platform PLK4 physical a regulator provides positive PIPKI the regulator by both CEP152, that engaging suggest results Our overduplication. centrosome h uopopoyaino L4tigr ohatvto and activation both triggers PLK4 of auto-phosphorylation The c ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal ihEcdei,btnttePI(4,5)P the not but E-cadherin, with 2 sntesnili iiigcnroedpiain We duplication. centriole limiting in essential not is m /ldxccie(Clontech). doxycycline g/ml c ttepoia nso aetlcentrioles, parental of ends proximal the at 2 rdcin o xml,PIPKI example, For production. c c a euaesm ellrprocess cellular some regulate can n hte PI(4,5)P whether and c ed otegnrto of generation the to leads c c nvivo in c a eeae tEpitomics at generated was n L4 ti also is It PLK4. and assPLK4-dependent causes c 2 ol iial rescue similarly could 2 eeae yPIPKI by generated ih cu and occur might , wn otehighly the to owing c oPK might PLK4 to c c c 2 id to binds Before . blocks a a has c c c ,

Journal of Cell Science tat NiNgtv oto iN Ivtoe)wsue as shown. siRNA are (O1) used both 1 HsPIPKI oligonucleotide using of was HsPIPKI by depletion or (Invitrogen) obtained for CEP192 O2) were siRNA (O1, results oligonucleotides Control Similar control. Negative negative RNAi Stealth HsPIPKI 2008); al., et HsPIPKI HsPIPKI 2008); TAAA-3 ATCTCT-3 GCAACTCAA-3 ATGGCTCTGGAA-3 CAA-3 AGCTTGTAAA-3 GGAAGTTGCAATCAAA-3 AGAGAGAGGATGTGCAGTATGA-3 CCTACAGGTTCATCAAGAAACTGGA-3 were cells was HeLa supernatant of Subconfluent The 8 of (Promega). Institute transfection. presence 6 the after FuGENE in Shanghai infected using h China) 48-72 2:1:1 Sciences, Ge, of of collected ratio Academy Gaoxiang a Chinese and Biology, at co- Cell from pCMV–VSVG and were shRNA, Biochemistry (gifts cells carrying HEK293T pLKO.1–puro pCMV-R8.91 sequencing. with by transfected confirmed were constructs 1 5 O1, (5 TGAACAACA-3 n.Rbi oylnlatbde gis h olwn rtiswere proteins following the against Abmart, antibodies at polyclonal generated was Rabbit antibody Inc. anti-PLK4 monoclonal Mouse 540). PIPKI polyclonal an obtain against to purified 469-668) PIPKI acids and MBP-tagged with harvested conjugated were column affinity anti-sera spleens, the collecting ARTICLE RESEARCH p .)1 MKl . MMgCl HEPES-KOH mM mM 1.5 [20 KCl, buffer mM 10 (IP) 7.2) immunoprecipitation (pH al., et using (Ling described 2002) previously as performed was Immunoprecipitation microscopy and Immunoprecipitation HsPIPKI Invitrogen. from obtained were oligonucleotides siRNA All siRNAs PIPKI shRNA The human cloning (AddGene). targeting vector by sequence pLKO.1 constructed the shRNA into were (Invitrogen) shRNA oligonucleotides carrying Lentiviruses Lentiviruses were constructs All sequencing. Technologies). by (Agilent verified Kit Mutagenesis Directed HsPIPKI the to or full-length encoding protein cDNAs binding maltose Sigma. HsSAS-6 and from (9E10), PIPKI Anti-ninein Cruz human was Cruz. Myc truncated Santa Santa (7G4) Sigma; from from antibody were was from (GNS1) antibody were B Jeffrey (Y-16) cyclin (HA-7) from and HA gift (91.390.21) (HIS.H8, and generous His following a Biosciences), (M2) the (BD (20H5, (Roche); GFP C-Nap1 against 2 Millipore), (Abnova), centrin antibodies ODF2 used: Salisbury), Mouse 2009) were al., A302- (Proteintech). et proteins (Schmidt Laboratories, Nigg) CP110 Erich (Bethyl from and gift CEP152 generous (a (Abcam), CEP192 (Proteintech), 480A), GFP CPAP CEP135, (Millipore), (Ser10) actin, H3 phospho-histone used: GX4-,pT2,pT4 rpA–2 osrcs pcDNA3- constructs. pcDNA3–FLAG, HA–PIPKI HA–PIPKI RNAi-resistant pMAL–C2X Addgene. pCMV–HA, and from or into obtained was pET-42 subcloned Myc–PLK4 and pET-28, PCR pGEX–4T-1, by obtained C- MBP-tagged the with conjugated PIPKI of column terminus affinity with purified PIPKI were GST–His-tagged recombinant with rabbits PIPKI against antibodies 9 -GTACCTGTACTTCATGCAG-3 9 -GCGTGGTCAAGATGCACCTCAAGTT-3 9 a 9 Sra ta. 07;CP5-1 5 CEP152-O1, 2007); al., et (Strnad O,5 -O2, GmzFrei ta. 07 onne l,21;Zue al., et Zhu 2013; al., et Sonnen 2007; al., et (Gomez-Ferreria 9 E12O,5 CEP192-O2, ; c 9 a -TGTTAAGAAGTTGGAGCACTCTTGG-3 9 O iN,wr eeae sn ukhneSite- QuikChange using generated were siRNA, -O1 9 E12O,5 CEP192-O1, ; a O,5 -O1, nitreigRAsqec agtn luciferase targeting sequence RNA interfering An . 9 sA-,5 HsSAS-6, ; aioais4152 rPIPKI or 451–562) acids (amino a b epciey nessae tews,rslsfor results otherwise, stated Unless respectively. , 9 E12O,5 CEP152-O2, ; ,5 c 9 a a 9 -TTGAAAGGTGCCATCCAGTTAGGCA-3 Ln ta. 02,PK n E12were CEP152 and PLK4 2002), al., et (Ling -CAGCAAAGGGTTACCTTCCAGTTCA-3 tbln(DM1A), -tubulin n PIPKI and 9 m L4O,5 PLK4-O2, ; c /lplbee(Sigma). polybrene g/ml W r–D hc oti mismatches contain which –KD, or -WT 9 -AGAAAAGCACGTTAATCAGCTA- 9 -ATCAGACAGAGGAATCAATAA- 9 a sda eaiecnrl All control. negative as used was ) 9 b -CCCAAAGGAAGACATTTTC- 2 9 MET,1m EGTA, mM 1 EDTA, mM 1 , L4O,5 PLK4-O1, ; eeotie yimmunizing by obtained were c c 9 9 nioy abtpolyclonal Rabbit antibody. os PIPKI mouse ; 9 c -AGGAGGTGTGTGTGG- -CAGCGTTTGCTGGGT- tbln(T-8,FLAG (GTU-88), -tubulin a 5 was 9 -CAGAACAACTGAA- a 9 rPIPKI or c HsPIPKI ; b c c 9 -emns(amino C-terminus -GTCGTGGTCA- aioais397- acids (amino L4 CEP152, PLK4, , iaeda (KD) dead kinase 9 -CACTGGTTT- b c 9 ,5 Anti-sera . (Mellman c O,5 -O2, 9 -GCG- b c 9 9 9 - - - ; . efre naEYASpreouinMcocp ytm(Zeiss) system Microscopy Superresolution ‘Plan-Apochromat’ alpha ELYRA an was with equipped a (3D-SIM) on microscopy performed illumination structured Three-dimensional a efre ihteZNsfwr akg Zis.Fnlimage Final (Zeiss). package software Photoshop. Adobe ZEN using performed the was processing with performed processing was image and reconstruction illumination Structured 0.5 (Zeiss). beads with measurements EMCCD control 885 mm 0.125 iXon at Andor acquired z were and Sections protocol. objective standard following immersion camera, oil (DIC) contrast al., with et 2000-U Devices). TE (Ling (Molecular Nikon for previously a methanol Metamorph using described acquired ice-cold were as images with stained Fluorescence fixed 2002). and coverslips, permeabilized glass immunofluorescence, min, Cocktail on 10 indirect grown Inhibitor For were Protease cells protocols]. Complete standard the following manufacturer’s NP-40, (Roche) Cocktail Inhibitor 0.5% Phosphatase PhosSTOP (Roche), NaCl, mM 150 n/ra prpit muto idtp W)o iaeda (KD) kinase-dead or (WT) wild-type N-terminus of CEP152 amount His–PIPKI recombinant appropriate without an or and/or with incubated was PLK4 yautoradiography. by rncito/rnlto ytm(rmg) ntepeec of presence the in (Promega), N-terminus Coupled CEP152 Quick System TNT the The using generated Transcription/Translation immunoblotting. was protein by 1-748) acids (amino analyzed being before [ nln ilb)i idn ufr[5m rs(H76,10mM 150 7.6), (pH for glycerol] Tris 10% 4 X-100, mM at Triton [25 h 0.5% (New 2 (DTT), buffer dithiothreitol resin mM binding amylose 1 NaCl, in or Biolabs) Healthcare) England and (GE proteins His-tagged beads with incubated glutathione-sepharose were proteins MBP-fused or GST vitro In h uhr elr ocmeiginterests. competing no declare authors The interests discussion. Competing stimulating their for McNiven Gina Mark thank and assistance We Schroeder technical microscopy. Barbara for fluorescence Razidlo, Clinic and Mayo centrifugation at ultra-speed Jason Hommerding with and Cynthia system, and lentivirus Biology, the Bakeberg sharing Cell L. kindly and for Biochemistry Sciences) of of microscope Academy Institute electron Chinese with (Shanghai assistance Ge Microscopy Gaoxiang technical (Electron for studies, Christensen Clinic) Trace Mayo and Facility, Huang Core Bing thank sincerely We Acknowledgements significant. statistically three least Student’s at by from calculated (SD) was deviation Significance standard experiments. and independent mean the represent Data analyses Statistical 2008). a Kaplan, on based and kidneys, (Meigs mouse approach or cells published HeLa from isolated were Centrosomes isolation Centrosome FLAG-tagged a Briefly, 2010). al., et Holland (CEP152 2010; fragment CEP152 al., approach et published previously a (Hatch following determined was activity PLK4 assay 1400 kinase kV. PLK4 JEOL 80 a at in operating was observed (JEOL) samples were microscope Microscopy electron Specimens cell Electron transmission Clinic. the the Mayo at Facility, of procedures Core standard sectioning to serial Microscopy according (Electron performed fixative and Trump’s using Embedding h 1 Sciences). for fixed were cells The microscopy electron Transmission muorcpttdwt niFA nioy 0.2 antibody. anti-FLAG with immunoprecipitated 33 sn xe otae(irsf) A (Microsoft). software Excel using mgJ(I,Bted,M)wsue oqatf autoradiographs. quantify to used was MD) Bethesda, (NIH, ImageJ 35 ses oo hneswr lge sn h lgmn aaee from parameter alignment the using aligned were channels Color -steps. ]ehoie(eknEmr,ue o uldw sa n analyzed and assay pull-down for used (Perkin-Elmer), S]methionine m T NwEgadBoas o 0mna omtemperature. room at min 30 for Biolabs) England (New ATP M rti uldw assay pull-down protein ˚ ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal .Tepeiiae eewse ietmswt idn buffer binding with times five washed were precipitates The C. c nkns ufrwt 5 with buffer kinase in 1-748 a vrxrse nHL el and cells HeLa in overexpressed was ) m imtrmlipcrlfluorescent multispectral diameter m Z Sre eetkna 0.1- at taken were -Series m P i[ Ci vleof -value 6 32 0/.6dgtlinterference digital 100/1.46 ]T Pri le)and Elmer) (Perkin P]ATP , m .5wsconsidered was 0.05 fprfe MBP– purified of g m steps. m 1303 t -test

Journal of Cell Science od .G,Pas,B . ign,M . als . wn .J,Gbo,A., Gibson, J., D. Owen, Y., Vallis, K., M. Higgins, M., B. Pearse, G., M. Taner, Ford, Y., C. Chen, R., S. Majeed, A., M., T. P. Evans, Janmey, M., S. C., Camus, B., Laschinger, A. Foraker, K., Ling, D., L. P. J. Arora, Maller, Y., and T. L. Sayegh, A. El Lewellyn, M., T. Yamamoto, F., Eckerdt, I., Cunha-Ferreira, G., Tzolovsky, K., Weiskopf, D., Q. Yu, S., N. Dzhindzhev, Haselmann- L., Ehret, F., Settele, R., Bahtz, M., Arnold, C. G. O., Rogers, Cizmecioglu, and W. D. Buster, E., J. Klebba, W., C. Brownlee, C. G. Rogers, and W. C. Brownlee, Funding authors manuscript. All the on manuscript. commented the and prepared K.L. results and and the data J.H. the discussed J.L.S., analyzed Q.X., study, columns. the affinity designed constructed and proteins PIPKI and PIPKI purified purified and and constructed performed constructed Y.Z. and experiments; proteins the recombinant of most performed Q.X. contributions Author ARTICLE RESEARCH 1304 S., Voronov, R., Zoncu, G., Cestra, K., Letinic, L., Pellegrini, G., M. Paolo, Bettencourt-Dias, Di and W. Sullivan, A., Debec, W., Zhang, M., Riparbelli, I., Bento, A., Rodrigues-Martins, I., Cunha-Ferreira, M. Bettencourt-Dias, and M. S. Gouveia, A., D. Brito, M., and Riparbelli, C. L., Carbonara, Carpenter, A., J., Rodrigues-Martins, A. M., Firestone, Bettencourt-Dias, X., Su, K., Ling, and F., A. E. S. Nigg, P., Bairstow, Mortensen, T., Mayor, J., C. Wilkinson, S., J. Andersen, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.141465/-/DC1 online available material Supplementary material Supplementary months. 12 after R01-DK090038]. release number for [grant PMC Health in by of Deposited funded Institutes are and Y.Z. National and Digestive and J.H. and DK90728]. NIDDKD Diabetes number [grant of (NIDDKD) Cure Institute Diseases the National Kidney for and Komen KG100902] G. Cancer number National Susan [grant the 1R01CA149039-01A1], from number grants [grant research by Institute supported were co-workers and K.L. hdm,B . akt,D . iiipnoi,M . Schro J., M. Difilippantonio, L., D. Sackett, M., B. Ghadimi, M. D. Glover, and J. Fu, ae,N . oih,S .adPlmn D. Pellman, and A. S. Godinho, J., N. Ganem, rmtscnrsm nert nerymtsstruhsaiiainof stabilization through mitosis early in ch-TOG. integrity centrosomal centrosome promotes Corre B., S. assembly, A. junctions. actin N-cadherin gelsolin, C. of regulates strength McCulloch, adhesion PIP5KIgamma and by and produced A. bisphosphate R. centriole novo Anderson, de for pathway 4-dependent kinase polo-like formation. a of Identification and Nature G. Callaini, M. M., D. Bettencourt-Dias, Glover, A., Rodrigues-Martins, M., Riparbelli, centrosome. the to CPAP and I. Plk4 Hoffmann, of induce and recruitment C. to Antony, U., Plk4 Weiss, stabilizes Twins subunit regulatory amplification. centriole 2A Phosphatase Protein euaino hshtdlnstlpopaekns ye1gmab h FERM P. the by Camilli, talin. gamma De 1 of type and domain kinase R. phosphate M. phosphatidylinositol of Wenk, regulation J., Guo, S., Chang, M. degradation Bettencourt-Dias, through and amplification SAK/PLK4. centrosome M. of limits D. ligase Glover, ubiquitin G., SCF/Slimb Callaini, E., Laue, amplification. promote that Sci. mechanisms Life duplication Mol. Cell. centriole of deregulation control. number and structure Glover, centriole: and G. Callaini, F., Balloux, N., development. Carmo, M. K., M. D. Gatt, L., Lehmann, endocytosis. regulates and AP2 with 281 interacts directly A. kinase R. Anderson, profiling. correlation protein M. Mann, ellns n orltswt ueia hoooa aberrations. chromosomal numerical cancer colorectal with diploid correlates in Cancer not and but T. lines, aneuploid, Ried, in cell and exclusively G. occurs Auer, instability A., Jauho, T., clathrin of material. nucleation peri-centriolar the and T. centriole in H. AP180 McMahon, by and clathrin membranes. on R. and lattices P. PtdIns(4,5)P2 Evans, of binding R., C. Hopkins, lyr rpsegr uigmitosis? during passengers or players etooe ocrmsmlinstability. chromosomal to centrosomes c 20632-20642. , n efre iaeasy;YH uiidfl-eghadCtriu of C-terminus and full-length purified Y.H. assays; kinase performed and 20) A/L4i eurdfrcnroedpiainadflagella and duplication centriole for required is SAK/PLK4 (2005). 467 ur Biol. Curr. 714-718. , 20) rtoi hrceiaino h ua etooeby centrosome human the of characterization Proteomic (2003). a .R,J,Dxe,S .adBosy .M. F. Brodsky, and J. S. Doxsey, Jr, R., I. ˆa, 21) sels sasafl o h ne fcnroeassembly. centriole of onset the for scaffold a is Asterless (2010). ur Biol. Curr. ur Biol. Curr. Nature 70 20) yeIam61popaiyioio phosphate phosphatidylinositol Igamma661 Type (2006). 1021-1034. , 21 .Cl Biol. Cell J. .Cl Biol. Cell J. 27 21) tutrdilmnto fteitraebetween interface the of illumination Structured (2012). 420 428-432. , cec NwYr) NY York), (New Science 15 19 183-190. , 85-89. , 2199-2207. , Nature 43-49. , c rtis uiidatbde gis PIPKI against antibodies purified proteins, 198 nvitro in 195 426 pnBiol. Open 21) hwm orlcne please: license, your me Show (2012). 591-605. , 231-243. , el o.Lf Sci. Life Mol. Cell. 570-574. , 20) etooeapiiainand amplification Centrosome (2000). ur pn elBiol. Cell Opin. Curr. Nature 21) e12at sasafl for scaffold a as acts Cep152 (2010). rti idn sas X.X. assays; binding protein 20) ehns ikn extra linking mechanism A (2009). 20) Phosphatidylinositol-4,5 (2007). o.Bo.Cell Biol. Mol. 460 2 291 120104. , .Cl Biol. Cell J. 278-282. , 21) eosrcigthe Deconstructing (2012). 21) etils active Centrioles: (2010). 1051-1055. , 20) erimn and Recruitment (2002). 20) Simultaneous (2001). 67 k . Neumann, E., ck, ¨ 2173-2194. , 24 18 191 21) Clathrin (2012). .Bo.Chem. Biol. J. 4-13. , 3026-3038. , 731-739. , 20) The (2009). 21) The (2011). (2011). a , b Mu enla . ezhli . coad .L,Chn . a,F,Rogers, F., Kan, B., Chhun, L., K. McDonald, B., Keszthelyi, V., Mennella, Go and K. Baumer, L., Cerutti, M., Delattre, L. S., Pelletier, Leidel, and D. G. Gupta, M., Hasegan, S., Lawo, aeac,R,Siro,Y . ikno,C .adNg,E A. E. Nigg, and J. C. Wilkinson, D., Y. Stierhof, R., Habedanck, A. E. Nigg, and A. Uldschmid, J., Westendorf, S., G., J. Guderian, Caldwell, K., S. Chanda, W., D. Buster, U., Rath, A., M. Gomez-Ferreria, iaaa . Flu D., Kitagawa, ela,D . ozls .L,Sn,C,Bro,C . ag . Kendziorski, P., Wang, A., C. Barlow, C., Song, L., M. Gonzales, L., D. D. Mellman, D. Kaplan, and E. T. Meigs, and G. D. Huntsman, A., D. Turbin, C., Carbonara, F., S. Bairstow, K., A. Ling, R. Anderson, and W. M. Bunce, J., A. Firestone, L., R. Doughman, K., Ling, and W. P. Majerus, H., J. Hurley, M., Kisseleva, P., M. Wilson, J., Kunz, D. Y. Stierhof, R., Habedanck, M., Clech, Le J., Westendorf, J., Kleylein-Sohn, ek .M,Jns .H,Wn,C . ike,J,Ce,D,Jsesn .L., S. Jaspersen, D., Chen, J., Binkley, C., C. Wong, H., M. Jones, M., J. Keck, J. P. Casey, and P. Kelly, E., T. Meigs, D., D. Kaplan, Oka, and T. Asano, Y., Yazaki, T., Wada, N., Kizuki, Y., W. Shibasaki, D. H., Ishihara, Cleveland, and H. Hoover, S., Niessen, W., Lan, J., A. Holland, T. B. Stearns, Stillman, and K. and Siddiqui, G., W. S. Prasanth, D. S., A. Hemerly, Cleveland, J., A. Holland, A., Kulukian, M., E. Hatch, F. R. Irvine, and R. Lim, K., Lee, L., M. Giudici, F. R. Irvine, and C. P. Emson, L., M. Giudici, aaaa . aae . knu,T,Tuia .adTuia S. Tsukita, and S. Tsukita, T., Okanoue, Y., Yamane, Y., Nakagawa, Mu erw .L,Saky .T n oaosi .S. R. Nowakowski, and T. J. Sharkey, A. L., E. T. Nigg, and Megraw, M. A. Fry, K., Tanaka, D., Y. Stierhof, T., Mayor, Liu, K., Boeneman, B., A. D’Assoro, C., V. Negron, L., S. Barrett, L., W. Lingle, lr . cmd,D,Senrn,S,Mroosaa . emn,V., Lehmann, E., Mirgorodskaya, S., Steinbrink, D., Schmidt, H., ller, ¨ lr . cmd,D,Dee,F,Hri,R,Pobdu .adLne .M. B. Lange, and A. Ploubidou, R., Herwig, F., Dreher, D., Schmidt, H., ller, ¨ aemn,K,Dee,F,Gsaso,N,Kslr . erc,H tal. centrosome. et Drosophila H. mitotic the Lehrach, J. of T., analysis EMBO Kessler, functional N., and Proteomic Gustavsson, (2010). F., Dreher, K., Habermann, loecnemcocp eel oano h etooeciia for critical centrosome the of domain a organization. material reveals pericentriolar microscopy fluorescence G.C.,Huang,B.andAgard,D.A. of features organizational higher-order reveals material. centrosomes pericentriolar of imaging ookns l4fntosi etil duplication. centriole in functions betaTrCP- Plk4 kinase Polo controlling by number degradation. centriole mediated regulates autophosphorylation J. assembly. D. spindle Sharp, and and centrosome R. D. Rines, 1561. oyeaecnrl xrsino eetmRNAs. A. select of R. expression controls Anderson, polymerase and C. cells. Mammalian 470-480. interaction direct a via adaptin. trafficking 1B mu E-cadherin with and junction and adherens modulates targets A. kinase R. Anderson, phosphate phosphatidylinositol gamma adhesions. focal I regulates Type (2002). in and elegans C. in duplication centrosome cells. for human required family protein a defines specificity. signaling determines kinases A. R. Anderson, Cell A. E. Nigg, and embryos. elegans C. P. oigr .P,X,T,Nee,M,Ru,M .e al. et P. M. Rout, centrosome. M., yeast Niepel, the T., of Xu, phosphoproteome P., spindle. E. mitotic Holinger, bipolar a of establishment the in Chem. lipid beta-catenin novel for role this of members of analysis family. deletion/substitution kinase by and isoform overduplication third centrosome limits Y. activity stability. kinase own its 4 autoregulating kinase Polo-like cells. duplication. human in centriole number copy for centrosome and required centriole is and Plk4 Biol. with Cell J. interacts Cep152 (2010). splice 5-kinase 4P isoform phosphatidylinositol Igamma variants. Type 5-kinase of mobility and 4-phosphate localisation phosphatidylinositol I Type of gamma. variant splice ue es ie sawdsra etooesafl component scaffold centrosome isolated widespread from identification a its centrioles: is mother centrosomes. 2 with associated fiber preferentially dense Outer and Drosophila of proteomes centrosome the human. of analysis ontology Gene (2011). xoe h etooa oto ircpaysyndromes. microcephaly of root centrosome centrosomal the cycle-regulated exposes cell for required is cohesion. C-Nap1 protein centrosomal USA development. Sci. tumor L. breast Acad. J. in Natl. Salisbury, instability and chromosomal C. drives Reynolds, amplification M., C. Whitehead, W., 21) PApopaaeat pnSS5t nuecnroefrainin formation centriole ensure to SAS-5 upon acts phosphatase PP2A (2011). 19) yeIpopaiyioio--hsht -iae.Coigo the of Cloning 5-kinases. phosphatidylinositol-4-phosphate I Type (1998). 13 190-202. , ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal 279 omn ner Biol. Integr. Commun. ice.J. Biochem. ESLett. FEBS 29 .Cl Biol. Cell J. 10829-10832. , 191 3344-3357. , a.Cl Biol. Cell Nat. o.Bo.Cell Biol. Mol. .Bo.Chem. Biol. J. kgr . oaosa . elr . eol .adGo and J. Reboul, D., Keller, J., Polanowska, I., ckiger, ¨ 721-729. , 20) l4idcdcnroeboeei nhmncells. human in biogenesis centriole Plk4-induced (2007). 20) yeIgmapopaiyioio hsht kinase phosphate phosphatidylinositol gamma I Type (2007). 20) h ciainlo fpopaiyioio phosphate phosphatidylinositol of loop activation The (2000). odSrn ab Protoc. Harb. Spring Cold .Cl Biol. Cell J. 580 379 e.Cell Dev. 99 151 a.Cl Biol. Cell Nat. .Cl Sci. Cell J. 1978-1983. , 6933-6937. , 489-496. , 837-846. , Nature 7 20) tIs,P-euae ula poly(A) nuclear PtdIns4,5P2-regulated A (2008). 12 273 115-125. , 4 .Cl Biol. Cell J. 1687-1697. , 20) slto fcnrsmsfo cultured from centrosomes of Isolation (2008). 20 308-311. , 20) ua e12i eurdfrmitotic for required is Cep192 Human (2007). 8741-8748. , 176 550-562. , 420 123 ur Biol. Curr. a.Cl Biol. Cell Nat. 343-353. , 14 89-93. , 2163-2169. , 1148-1158. , o.Cell Mol. 188 21) Subdiffraction-resolution (2012). 2008 20) oe neuronal-specific novel A (2004). cec NwYork) (New Science 17 191-198. , 1960-1966. , Nature prot5039. pdb , 5 14 a.Cl Biol. Cell Nat. 1-11. , 1159-1168. , 20) dniiaino a of Identification (2004). 20) h intracellular The (2006). Science cy P. nczy, ¨ 21) Subdiffraction (2012). 451 21) elcycle cell A (2011). 20) r1controls Orc1 (2009). rnsCl Biol. Cell Trends 20) Centrosome (2002). 21) l4trans- Plk4 (2010). 21) Cdk5rap2 (2011). 1013-1017. , 323 20) SAS-6 (2005). 7 1140-1146. , 789-793. , 20) The (2005). 20) The (2000). 332 1557- , .Biol. J. (2001). (2010). nczy, ¨ Proc. Dev. 21 ,

Journal of Cell Science i,J . ar .R,Ncoa,A . avlo .P,Kusi,M,Sossick, M., Khurshid, P., O. Carvalho, K., A. Nicholas, R., A. Barr, H., J. Sir, Bonnet, S., Thambirajah, A., Boeckx, N., Vloemans, F., Tack, E., J. Sillibourne, cil .J n nesn .A. R. Anderson, and J. N. Rusten, Schill, J., Poulton, K., Liestøl, L. M., S. N. Rogers, Pedersen, P., and I. M. Nezis, Peifer, P., A. M., Sagona, D. Roberts, M., N. Rusan, and C., Y. Xue, G. Z., Zhang, Rogers, L., Wen, H., Zou, H. M., Ye, J. C., Hurley, Jin, X., and Gao, Z., A. Liu, R. J., Ren, Anderson, V., I. Boronenkov, S., U., Misra, Kossatz, D., V. S., Rao, Chauhan, M., May, D., Keller, Y., Homsi, P. A., Quesenberry, B., Puklowski, Woda, H., Knecht, J., Wallace, A., Purohit, A., G. M. Pihan, Bornens, and L. C. Rieder, A., Khodjakov, P., M. Meyer, M., Y. Piel, Yamashita, and M. L. Bornens, Pelletier, and L. J. Salisbury, M., Paintrand, M., Moudjou, A., Paoletti, W. J. Raff, and A. E. Nigg, A. E. Nigg, cmd,T . lyenSh,J,Wsedr,J,L lc,M,Lvi,S B., S. Lavoie, M., Clech, Le J., Westendorf, J., Kleylein-Sohn, I., T. Schmidt, oae-aea,R,Aacl . Dı F., Abascal, R., Nogales-Cadenas, T. Stearns, and A. E. Nigg, A. E. Nigg, ARTICLE RESEARCH ircpaypoencmlxfrsarn rudprna centrioles. parental around ring a forms F. Gergely, Genet. complex and G. protein C. Woods, microcephaly C., T. D’Santos, duplication. S., Reichelt, centriole Grand-Perret, A., in and role its M. and Cell 4 Biol. Bornens, kinase Mol. polo-like C., of F. Autophosphorylation Ramaekers, P., -iaetp gmaslc ainsepesdi ua el ipa distinctive display cells human targeting.cellular in expressed variants splice Igamma H. type 5-kinase to Stenmark, FYVE-CENT and of C. recruitment Raiborg, midbody. KIF13A-mediated the block I., through to cytokinesis R. controls levels Skotheim, Plk4/Sak E., T. regulates ligase ubiquitin Slimb reduplication. centriole SCF The (2009). kinetochore. and X. Yao, phosphorylation. protein interfacial a for kinase: flattened phosphate fold phosphatidylinositol kinase IIbeta type of Structure (1998). control to HsSAS-6 targets and PLK4 duplication. by centrosome regulated is ligase E3-ubiquitin FBXW5 Gru J. tumors. S. malignant Doxsey, centrosome and to centrioles cells. vertebrate daughter in and behavior mother and the activity of contributions respective and development. centrosome-associated animal during not inheritance is cells centrioles. of animal lumen distal the in 3089-3102. to confined centrin is centrin of centrosomal Most (1996). Res. Acids Nucleic A. Montano, tehf .D n ig .A. E. CP110. Nigg, and D. Y. Stierhof, ulcto n neetasymmetries. inherent and duplication disease. and Biol. cancers. human wl,V,Kbca . ih . an,M .e al. et P. M. Manns, A., Pich, S., Kubicka, V., nwald, ¨ 17 43 215-221. , ur Biol. Curr. 21) irKt30 nitgae aaaeo ibd,centrosome midbody, of database integrated an 3.0: MiCroKit (2010). 1147-1153. , 20) etooedpiain frlsadlicenses. and rules of duplication: Centrosome (2007). 20) rgn n osqecso etooeaertosin aberrations centrosome of consequences and Origins (2006). 20) etooeB ua etooa rtisdatabase. proteins centrosomal human a CentrosomeDB: (2009). a.Cl Biol. Cell Nat. Cell 21 n.J Cancer J. Int. ice.J. Biochem. 547-561. , uli cd Res. Acids Nucleic 139 acrRes. Cancer 19 37 1005-1011. , 19) etooedfcsadgntcisaiiyin instability genetic and defects Centrosome (1998). 663-678. , D175-D180. , .Cl Biol. Cell J. a.Cl Biol. Cell Nat. 21) h etooecce etil biogenesis, Centriole cycle: centrosome The (2011). 20) etils etooe,adclai health in cilia and centrosomes, Centrioles, (2009). 12 422 20) w oe phosphatidylinositol-4-phosphate novel Two (2009). 362-371. , 119 20) oto fcnroelnt yCA and CPAP by length centriole of Control (2009). 58 473-482. , 3974-3985. , 2717-2723. , 184 ´ez-Pe 38 a.Cl Biol. Cell Nat. 21) etooeaymtyand asymmetry Centrosome (2012). 13 225-239. , D155-D160. , ur pn elBiol. Cell Opin. Curr. .Cl Biol. Cell J. rz . aao .M n Pascual- and M. J. Carazo, J., ´rez, 1004-1009. , Cell 149 13 1154-1160. , 94 317-330. , 829-839. , 21) PtdIns(3)P (2010). 21) h SCF- The (2011). 21) primary A (2011). 24 .Cl Sci. Cell J. 541-546. , rnsCell Trends 20) The (2000). (2010). 109 Nat. , onn .F,Shrelh . enad,H n ig .A. E. Nigg, and H. Leonhardt, L., Schermelleh, F., K. Sonnen, hp,N,Sn . crm,M,Co,S,Ln,K n nesn .A. R. Anderson, and K. Ling, S., Choi, M., Schramp, Y., K. T. Sun, Tang, N., A. and Thapa, B. W. R. Hsu, S., Anderson, K. Wu, and H., R. J. Fu, J., N. C. Tang, Schill, X., Tan, C., A. Hedman, Y., Sun, A. R. Anderson, and P. M. Wagoner, K., Ling, and Y., Sun, A. Khodjakov, U., Euteneuer, T., Vinogradova, S., Leidel, P., A. Strnad, E. Nigg, and D. Y. Stierhof, E., Anselm, M., A. Gabryjonczyk, F., K. Sonnen, F. K. O’Connell, and J. W. Jahng, E., D. Anderson, Y., Liu, H., M. Song, h,F,Lw,S,Br,A,Pnhv . ap,A,Rctr . Mu C., Richter, A., Ralph, D., Pinchev, A., Bird, S., Lawo, F., Zhu, and J. Hu, E., Leof, R., Anderson, D., R. Singh, Y., Huang, Q., Xu, X., Xiong, L. M. Giudici, and F. R. Irvine, Y., Xia, ag .J,L,W . ag . u . og . u,X n i,H L. H. Yin, and X. Luo, P., Dong, K., Xu, J., Wang, H., W. Traub, Li, and J., Y. A. Wang, R. Anderson, B., Doray, K., Ling, K., S. Mishra, R., J. Thieman, ele,M,Bblk . aosn .H,Gre,C,Ulu,E,Vl,I,Roth, I., Volf, E., Umlauf, C., Gerner, H., L. Jakobsen, R., Babeluk, M., Zellner, M., Okuda, S., Yoshihiro, A., Oga, T., Furuya, H., Matsuyama, Y., Yamamoto, etae .J,By,L . ahr,M . rgt .J,Evn .E,Pu L., Phu, E., K. Ervin, J., K. Wright, V., M. Nachury, M., L. Baye, J., C. Westlake, tutrdilmnto irsoypoie oe nih noacietr of architecture into insight novel provides centrosomes. human microscopy illumination structured 563-571. yl euae rti htcnrl etil length. centriole controls that protein regulated cycle Cell I type Endosomal directional EGF-stimulated migration. for cell required is kinase phosphate phosphatidylinositol 203-213. Go centriole and recruitment Plk4 in cooperate duplication. Cep152 and Cep192 Human (2013). factors. C. assembly in centriole of duplication levels centriole the controlling regulates by 2A-SUR-6/B55 elegans phosphatase Protein . ite,R,Hmn .A n elte,L. biogenesis. Pelletier, centrosome and regulates A. Cep192 A. ortholog Hyman, R., Kittler, T., K. Ling, Commun. Res. Biophys. I 5-kinase dpo ea appendage. beta2 adaptor 2 M. L. cells. migrating integrin directionally polarized and in complex trafficking exocyst the regulates signaling Phosphoinositide cycle. duplication centrosome the during centriole per procentriole nMo xrsini ltlt fhatyvlner fe ihpoenmeat protein high after volunteers cycle. healthy methylation the of to platelets relationship in diet: expression MaoB in J. Kondrup, and E. cancer. bladder in 10 recurrence tumor and K. progression Naito, predicts and K. Sasaki, S., Kawauchi, polarization. epithelial 98. and clustering E-cadherin directs rmr ii ebaeasml siiitdb a1 n rnpr protein transport al. and et Rab11 C. by D. initiated Slusarski, is S., assembly D. membrane Kirkpatrick, S., cilia J. Primary Beck, C., Chalouni, signaling. Ca(2+) InsP3-mediated 167 in PIP5KIgamma87 of role Critical rc al cd c.USA centrosome. Sci. the Acad. to Natl. Rabin8 Proc. of trafficking complex-dependent (TRAPPII) II particle cy P. nczy, ¨ 6449-6455. , 1005-1010. , 25 20) ltrnrgltsteascaino IKgma6 ihteAP- the with PIPKIgamma661 of association the regulates Clathrin (2009). 144-155. , ora fCl cec 21)17 2310 doi:10.1242/jcs.141465 1293–1305 127, (2014) Science Cell of Journal 21) nascainbtentp I type between association An (2012). c v,anwslc ain on nrdnsadhumans. and rodents in found variant splice new a _v6, .Cl Sci. Cell J. 20) euae sA- eesesr omto fasingle a of formation ensure levels HsSAS-6 Regulated (2007). .Cl Biol. Cell J. c I -iaecnrl G eetrlssmlsorting. lysosomal receptor EGF controls 5-kinase PIP 21) rtoissuyrvasapeoiatchange predominant a reveals study proteomics A (2011). il Open Biol. 126 411 178 108 3223-3233. , 416-420. , .Bo.Chem. Biol. J. 297-308. , 2759-2764. , 1 965-976. , 21) hshtdlnstl4-phosphate Phosphatidylinositol (2011). 20) etooehyperamplification Centrosome (2004). e.Cell Dev. .Nua Transm. Neural J. 284 20) h amla SPD-2 mammalian The (2008). 13924-13939. , c 22 IP5kns n Exo70 and 5-kinase PI4P ur Biol. Curr. a.Cl Biol. Cell Nat. 116-130. , 20) yeIgamma I Type (2007). o.Bo.Cell Biol. Mol. 20) PPi cell- a is CPAP (2009). ln acrRes. Cancer Clin. 118 18 ller-Reichert, ¨ 653-662. , e.Cell Dev. 136-141. , e.Cell Dev. 11 21) 3D- (2012). .Cl Biol. Cell J. 825-831. , Biochem. 23 (2013). (2012). (2004). (2011). (2011). 1305 87- , Dev. 20 13 , ,

Journal of Cell Science