Journal of Cell Science opnn ftemte etil n orpr oeo ohrcnroepoeni h euaino etooa centrosomal of regulation the in protein mother a of role a report to and centriole mother the composition. of component a pcfcsae ftecl yl r nybgnigt being to beginning only are cycle cell 2012). Glover, the and (Fu of understood stages duringspecific centrosomal between (http://centrosome.dacya. interactions However, 2009) ucm.es). al., (Andersen et the Nogales-Cadenas at 2003; proteins al., 383 et as many as suggests of mining presence the database proteins, has centrosomal 100 interphase about purified identified of analysis While spectrometry 2005). mass al., a et Casenghi 1999; and Rieder, and recruitment (Khodjakov centriolar protein removal through and modified duplicated being composition being protein with t remodeled through actively material progress pericentriolar cells the As mass, (PCM). electron-dense an this in division, embedded tw cell are of after composed Soon is cycle. organelle cell complex the during change dynamic 2011). Stearns, and of Nigg (Cuschieri fluids 2007; base cellular al., and the particles et transduction cells, form signal of for movement centrosomes critical the are and cycle, the which cilia, cell motile control cells and the In primary that cells. exited daughter poles two have the spindle that shape, into it DNA of cell , of formation segregation During for accurate the transport. important to protein is the contributes and that arranges positioning During organelle organelle of functions. bound array cellular centrosome non-membrane radial critical the of this center, number interphase, organizing large a regulates major the As Introduction words: Key Par6 including proteins, of Par6 number gamma complex. large homolog polarity a 6 PAR of defective the loss partitioning of the protein components with composition, the polarity protein is protein and on the centrosomal complex depend organelle identified dynein/dynactin roles we bound the specific study, non-membrane microtubules, These this This intact cells. In quiescent function. centriole. in and mother, ciliogenesis composition or (Par6 and older, protein cells the dividing age, at in in appendages organization differ microtubule that regulate to centrioles known two contains centrosome The Summary 10.1242/jcs.121186 doi: 860–870 126, ß Science Cell of USA Journal 2012 92697-2300, December CA 3 Irvine, Accepted Irvine, California, of ( University correspondence Biology, for Cell *Author and Developmental of Department Vale a Par6 through composition protein centrosomal Par6 860 osqec,teewr eet ncloeei,mcouueognzto n etooeroinaindrn irto.Par6 Par6 composition. migration. protein centrosomal during of reorientation regulation the centrosome for required and not were organization proteins microtubule these Par6 but with ciliogenesis, aPKC, associated and in Par3 defects with interacted were there consequence, 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. etooeognzto n rti opsto undergo composition protein and organization Centrosome ra omy aiTrae n hitn Su Christine and Tormanen Kati Dormoy, ´rian c sanvlcmoeto h ohrcnroe hsseii oaiainrqie h Par6 the required localization specific This centriole. mother the of component novel a as ) a c a6 p150 Par6, dpnetpathway -dependent sa h ohrcnroeadcontrols and centriole mother the at is a hc otospoenrcutett h etooetruhp150 through centrosome the to recruitment protein controls which , [email protected] Glued A complex PAR , ecl yl,tecnrsm is centrosome the cycle, cell he ) arlsae etilsthat centrioles barrel-shaped o tterlin* ¨ ooo lh (Par6 have 6 microtubule- alpha defective We partitioning a protein homolog 2000). polarity in the Munro, centrosome that In shown and the previously (Gillingham to 2002). manner proteins dynein/dynactin- independent Merdes, these domains a target localization and contain that pericentrin by and (Dammermann AKAP450 microtubules contrast, process along to delivered dependent are centrosome , and the as such Proteins, understood. al., is proteins et appendage-associated known. of (Jakobsen not number identified overall the been but 2011), have proteins additional centriole Recently, 2007). is mother al., and the et membrane (Graser in plasma ciliogenesis role the for with a required centriole play mother to the proposed of in docking is appendages, which Distal 2006). Cep164, microtubules al., contain interphase et contrast, Lin of 2005; anchoring al., et its the (Guarguaglini and than control Cep170 which cycle as such ninein, cell proteins, Piel, recruit functionally and one appendages Bornens Subdistal with least 2002; 2002). decorated (Bornens, at appendages is by protein centriole important older older is This pair counterpart. a of Go a centriole elongates and as then (Strnad which serves matures procentriole, centriole a and of pre-existing formation the a for function process, platform and this composition During 2011). protein age, in (Chre differ that centrioles h erimn fpoen otecnrsm spoorly is centrosome the to proteins of recruitment The produces S-phase in duplication centrosome Semi-conservative ´ ine l,19;BresadPe,20;Ng n Stearns, and Nigg 2002; Piel, and Bornens 1997; al., et tien Glued a a otoscnrsmlpoendelivery protein centrosomal controls ) u td stefrtt dniyPar6 identify to first the is study Our . n p150 and c nz,20) sacneune one consequence, a As 2008). ¨nczy, -emns u a needn of independent was but C-terminus, Glued c elto eutdi altered in resulted depletion rmtecnrsm.A a As centrosome. the from , eerhArticle Research c also c as c Journal of Cell Science Kmhe ta. 98.Ti ope sasmldi response in assembled is complex This 1988). al., first et was (Kemphues and a aPkc and in Par3 Par6, identified proteins, three of composed is determined. It be to remains Par6 process alike, this behind of delivery mechanism protein the Loss but centrosomal -independent 2010). and al., dependent et (Kodani ua Par6 human Par6 individual human (the each Par6A of However, mouse homolog of likely of 2008). forms function HA-tagged (Etienne- expressing the al., by examined isoform et isoforms Par6 have Georgiou Macara individual and 2003; Gao have between Hall, but and polarity, distinguish cell Manneville in to proteins Par6 for failed role general approaches a negative support dominant using studies Several investigated. Par6 called are 2001). humans al., et Noda three 2000; and al., aPkc et two (Joberty isoforms Par3, Par6 two contain cells a mammalian example, with species, in other component Drosophila complex in PAR identified each of been cytoskeletal version single have polarity, protein of Homologs PAR Gao 2005). cell each 2000; al., et al., Etienne-Manneville controls et 2004; Macara, (Lin and assembly and junction tight Cdc42 and rearrangement activated to teshv sdioomseii nioisadhv detected have and and We antibodies Pals1. isoform-specific Par6 with used interactions have in there others and addition, formation in In proteins junction individual contact. these tight Par6A cell-cell for requirement of the cytosol, mouse in sites differences the were While at in Par6C was 2004). Par6B human mouse and Macara, junctions, tight and with associated (Gao cells MDCK h etooetruhefcso iignss ohPr and Par3 Both ciliogenesis. on effects through centrosome the function. and systems, both In 2010). al., Par6 et Kodani 2004; al., et (Solecki neurons h oe ftetremmainPr rtis hc in which proteins, Par6 mammalian three the of roles The polarity. cell of regulator known a is complex polarity PAR The h a6bnigprnr a3adak aebe ikdto linked been have aPkc and Par3 partners binding Par6 The a a a seta o h euaino etooeorganization centrosome of regulation the for essential was ttecnrsm fhmneihla el n mouse and cells epithelial human of centrosome the at u eea sfrsi amla el.For cells. mammalian in isoforms several but , b n ua a6 wihi Par6 is (which Par6C human and ) .elegans C. a Par6 , cenfrdfcsi elpolarity cell in defects for screen c n a6 telkl ooo of homolog likely (the Par6B and ) b n Par6 and a c fetdmicrotubule- affected aentbe fully been not have , a .elegans C. npolarized in ) and al 1.TerCtriiwr escnevd ihol 25% only with Par6 conserved, less between were identity C-termini Their S1). Table Par6 between 71% eeldahg ereo osrainbtenterN-termini, Par6 their between between identity conservation 65% of with degree high the a examine revealed Par6 proteins, to Par6 Par6 human us other and prompted the of 2010) function and al., localization et (Kodani Par6 centrosome that finding Our centrosome Par6 nioisrie gis hi iegn -emn.W detected isoform-specific We of C-termini. divergent advantage Par6 their took against we raised antibodies members, family Par6 S1). Table Par6 between etooe hr tclclzdwith Par6 colocalized it Intriguingly, (supplementary where centrosome, protein S1A,B). the Fig. of material lost depletion was it Par6 as siRNA-mediated specific 2010). was upon al., staining this et and nucleus, (Kodani the to results localized previous our with consistent Results centrosome. the this control which by may pathway protein Par6 molecular polarity a which describe and by centriole mother mechanism We the function. and establish organization centrosome of regulator important not have ciliogenesis in aPkc and addressed. been Par3 mammalian the of Prulie between differences 2007; isoforms functional al., proteins, Par6 et to (Sfakianos Similar known 2011). polarized this yet to in contributions not specific are ciliogenesis their process but for urchin, sea required and 2004). cells also MDCK al., kidney were et in proteins (Fan cilia tubulin primary These acetylated of axoneme with the colocalizing stain epithelia, to found were aPkc oudrtn h ucinldfeecsbtenteethree these between differences functional the understand To nti td,w eosrt httePr rti Par6 protein Par6 the that demonstrate we study, this In a c sanvlcmoeto h ohrcentriole mother the of component novel a is ttecnrsm n etooa aelts hc is which satellites, centrosomal and centrosome the at c Par6 Fg A.Sqec lgmn ftetreproteins three the of alignment Sequence 1A). (Fig. hw nbakadga,respectively. gray, are and residues black related in closely shown and and Identical II highlighted. type are PB1 PDZ domains interaction Protein–protein proteins. proteins. family Par6 Par6 The 1. Fig. eius ( residues. a c b n Par6 and n Par6 and samte etil akr861 marker centriole mother a as b n Par6 and B euneaineto h he ua Par6 human three the of alignment Sequence ) b a c n ihrPar6 either and sacmoetadrgltro the of regulator and component a is h ubr ee opstoso mn acid amino of positions to refer numbers the ; c c Fg B upeetr material supplementary 1B; (Fig. Fg B upeetr material supplementary 1B; (Fig. a n ihrPar6 either and c tblna n ftetwo the of one at -tubulin c a ( c A oanmpo Par6 of map Domain ) soitdwt the with associated rPar6 or soitswt the with associates b rPar6 or c n 30% and ` ee al., et re c c san is and a b b , Journal of Cell Science TR-P- el eesandwt nioist Par6 to antibodies with stained were cells hTERT-RPE-1 h egdcanlt h u fpxldniisi h aeae nec niiulcanl e el eeanalyzed/experiment; were cells Ten channel. individual each in area same the in densities pixel histo of The sum material. pericentriolar the PCM, to centriole; channel daughter merged D, centriole; the mother M, Par6 proteins; of centrosomal colocalization these percentage of localization specific the show and (red) eeutetd(oto) rtetda nA. in as treated or (Control), untreated were siPar6 i.2 Par6 2. Fig. results Our the S1C). Fig. confirming material supplementary 2C; further Par6 human (Fig. antibody U2-OS, two of the and localization specific in was centrosomal hTERT-RPE-1 detected signal was lines this staining cell similar that A indicating 2A,B). cells, (Fig. HeLa in Par6 siRNAs depleted we Par6 for when staining present This 2A). (Fig. foci centrosomal 862 c 2 o 4husadsandwt nioisto antibodies with stained and hours 24 for #2) c tbln(re) ( (green). -tubulin ora fCl cec 2 (3) 126 Science Cell of Journal c oaie otemte centriole. mother the to localizes D eaclswr tie ihatbde oPar6 to antibodies with stained were cells HeLa ) c ihteidctdpoen.Tepretg ooaiainwsdtrie ycmaigtepxldniya h etooein centrosome the at density pixel the comparing by determined was colocalization percentage The proteins. indicated the with c ihete ftoindependent two of either with c n h pcfct four of specificity the and a tblnsre stelaigcnrl ( control. loading the as served -tubulin ( A eaclswr rnfce ihcnrlsRA(ioto)o pcfcsRAt Par6 to siRNA specific or (siControl) siRNA control with transfected were cells HeLa ) c tbln(re)adPar6 and (green) -tubulin c c rd n acetylated- and (red) a olonger no was a c c tbln(re) cl as 10 bars: Scale (green). -tubulin rd n n fcnrn,Cp6,nni rHSS6(re) h cartoons The (green). HsSAS-6 or ninein Cep164, centrin2, of one and (red) h etooe osann ihatbde ocnrn,wihis which centrin2, to antibodies with Co-staining centrosome. the proteins be Par6 cells. mammalian three may in the there between that differences indicate this functional they at prominent role addition, a have In may organelle. family protein important Par6 the of member second a Par6 establish rd.( (red). enx eemndtepeielclzto fPar6 of localization precise the determined next We C TR-P- n 2O el eesandwt nioist Par6 to antibodies with stained were cells U2-OS and hTERT-RPE-1 ) B etr ltaayi fttlpoenlstsfo eaclsthat cells HeLa from lysates protein total of analysis blot Western ) c sacmoeto h etooeadsgetthat suggest and centrosome the of component a as m m. n 5 .( 3. E Serum-starved ) c rm rsn the present grams (siPar6 c c within and c Journal of Cell Science h oaiaino hs Par6 these of localization the osrcsdsrbdi rd eesandwt nioist eti2(re) egdiae r loson cl a:10 bar: Scale shown. also are images Merged (green). centrin2 to antibodies with stained were (red) A in described constructs rsn tbt etils(aibr ta. 02,cnimdour Par6 confirmed 2002), that al., observation et (Salisbury earlier centrioles both at present T GFP-Par6 WT, Par6 The 3. Fig. localization centrosomal the that Par6 for hypothesize signal to Par6 three us the led of C-termini proteins the between differences sequence The C-terminus Par6 yohss egnrtdfl eghaddlto osrcsof constructs deletion and length full generated we hypothesis, Par6 The 2A,D). (Fig. centrosome interphase a xesv ooaiaino Par6 There for of distal respectively. colocalization the stained appendages, extensive of centriole it components was mother known because subdistal are centriole which and ninein, mother and the Cep164 to corresponded aa oydrn iignssi TR-P- el Fg 2E). 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HeLa in constructs c tr(5–7) GFP-Par6 (259–376), Cter c nysan n etil fan of centriole one stains only c sanvlcmoeto the of component novel a is c ihnni n another and ninein with c pstv centriole -positive c trA(5–1)adGFP-Par6 and (259–317) A Cter bomlnce t7 or fe nc-on(supplementary knock-down Hoechst after dye hours 82 72 DNA with at the nuclei time, deformed with abnormal over or staining fragmented increased by with 33342, visualized In cells cells. as of siRNA-treated nuclei, control frequency or the untreated addition, parallel was in effect a This seen Trypan in S2A). by not Fig. resulted assessed material hours (supplementary as 72 staining viability, and Blue cell 48 in 24, decrease for time-dependent cells HeLa Par6 from of role depletion the determined next We Par6 otemte centriole. mother the to of form the GFP-tagged of a While Par6 the localization 3B,C). the Par6 of (Fig. centriole-specific protein length Par6 centriole mother endogenous length full one the full exogenously-expressed at reproduces expressed that detected we demonstrating was when centrosome GFP interphase 3A). (Fig. cells Par6 mle -emnlfamns(i.3–)ddntadwere entire and the that not indicate results did These Par6 or cytosol. 3A–C) the (Fig. 1–258) in found fragments instead acids C-terminal (amino smaller N-terminus GFP-tagged centrosome, c c c -emnsi eesr n ufcett agtti protein this target to sufficient and necessary is C-terminus iha -emnlGPtgadepesdte nHeLa in them expressed and tag GFP N-terminal an with sesnilfrcl growth cell for essential is c c Par6 ( -emns(mn cd 5–7)lclzdt the to localized 259–376) acids (amino C-terminus A trB(1–7) ( (312–376). B Cter ceai ersnain fPar6 of representations Schematic ) c samte etil akr863 marker centriole mother a as B eaclsepesn F-agdPar6 GFP-tagged expressing cells HeLa ) c m ncl oesai.Par6 homeostasis. cell in 6 .( m. %o el exhibiting cells of 8% c C (Par6 al summarizing Table ) c T,GFP-Par6 WT), c c c c c , Journal of Cell Science efrteaie etooa rti opsto by composition control protein in proteins centrosomal centrosomal various Par6 and of levels examined the centrosome. the comparing at first role a has protein We this whether investigate to us ta. 09,wr iia ncnrladPar6 and control in similar the were of 2009), components al., all et are which WD, Par6 of association prominent The Par6 specified unless point text. time the early in this all conducted at As we experiments nuclei. morphology, subsequent nuclear of and division morphology cell affecting normal and growth Par6 Par6 cell that show for results critical These data). (unpublished the results with same out carried was Par6 analysis second same The S2B,C). Fig. material 864 oprdt oto el Fg AB.W loosre a observed also We Par6 4A,B). (Fig. of cells absence control the to in and compared decreased centrin2) Odf2) centriole dramatically and and the ninein were Cep170, CPAP of (Cep164, proteins appendages Cep152, centriole of mother Cep192, levels STIL, the contrast, (HsSAS-6, In 4A,B). (Fig. c c a fiinl eltda 4husps-iecn without post-silencing hours 24 at depleted efficiently was otostepoencmoiino h centrosome the of composition protein the controls c ora fCl cec 2 (3) 126 Science Cell of Journal dpee el.Telvl of levels The cells. -depleted c seii iN (siPar6 siRNA -specific c ihtemte etil led centriole mother the with c 2 n eotie the obtained we and #2) c tbln C2o GCP- or GCP2 -tubulin, c TR ope (Fant complex -TuRC c dpee cells -depleted c when , c is fpoen ttecentrosome. the at proteins of ra fmcouue htetne oad h elperiphery, cell Par6 the of towards 77% extended radial but that a organization microtubules displayed cells of the Control array monitored cytoskeleton. in microtubule we serum 10% the Second, of about upon 5A). in (Fig. detected ciliogenesis cilia only the were primary Par6 cilia of of contained primary absence starvation, some the cells in on hTERT-RPE-1 mislocalized are depends Par6 that altered which proteins the centrosomal whether ciliogenesis, determine Par6 examined to in assays centrosome four performed We Par6 infcn euto ntelvl fcnroa aelt proteins, Par6 satellite Par6 centriolar PCM-1, of levels including the in reduction significant euao fmcouueacoig a otfo h centrosome the a from Par6 ninein, lost was C-terminal conditions, anchoring, these microtubule the of Under regulator 5C). overexpressed (Fig. we domain localization when defects observed organization microtubule were Similar 5B). (Fig. microtubules eacls uha hs fA ncmaio ocnrlcls * cells. control to comparison in A, of Par6 those in as proteins such centrosomal cells, of HeLa intensity 10 show fluorescence bar: Insets the Scale shown. of centrosome. are the dye of 33342 area enlarged Hoechst the by stained DNA with images oPar6 to ( i.4 Par6 4. Fig. A c c c c oto n Par6 and Control ) dpee el,idctn htPar6 that indicating cells, -depleted siRNA-transfected control or control of 80% about While . ly nipratrl ntercutetadmaintenance and recruitment the in role important an plays otostefnto ftecentrosome the of function the controls c rd n n of one and (red) c euae h rti opsto ftecentrosome. the of composition protein the regulates c dpee el otie admyorganized randomly contained cells -depleted c c dpee el sfntoa.Frt we First, functional. is cells -depleted dpee eaclswr tie ihantibodies with stained were cells HeLa -depleted a B4adCp9 Fg B.Thus, 4B). (Fig. Cep290 and BBS4 , c tbln e12o e10(re) Merged (green). Cep170 or Cep152 -tubulin, c m .( m. srqie for required is B Quantification ) c -depleted P , 0.01. c Journal of Cell Science on.Tehsormsostepretg fclsi hc h etooei o eretd nAD egdiae ihDAsandb ocs 34 d 33342 Hoechst by stained DNA with images merged F A–D, In reoriented. not is 20 centrosome the bars: which Scale in cells shown. of are percentage the ( shows point. histogram time The each wound. for quantified was recovery to surface antibodies cell of percentage The wounding. after ioi el ihdfcieside t2 n 8husps-rnfcin ( post-transfection. hours 48 and 24 at and spindles red) defective (P-H3, with H3 cells phospho-histone mitotic to antibodies with stained were rtisfo h nepaecnrsm,mttcside of of spindles mislocalization mitotic the centrosome, detected interphase Par6 first and control the is we which from when transfection, proteins siRNA point the time after interphase the hours alignment 24 DNA mitosis. and of organization during spindle examined we organization function, Par6 of as domain normal defects C-terminal the that the Par6 indicate also They for microtubules. necessary ecnaeo el ihanra irtbl raiain ( organization. microtubule to abnormal antibodies with cells of percentage ae,w eetdovosdfcsi pnl omto,wt 83% Par6 with formation, mitotic spindle in of defects obvious detected we later, Par6 that suggest findings These data). 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Fig. ntepretg fmttcclsfo %i oto el o12% to cells control in 3% from increase cells an mitotic was of percentage there the observation, in this with Consistent spindles. c csa oiatngtv,pouigsmlrfunctional similar producing negative, dominant a as acts c c a tbln(e)and (red) -tubulin otostefnto ftecentrosome. the of function the controls tbln(re) h itga hw h ecnaeo el ihanra irtbl raiain ( organization. microtubule abnormal with cells of percentage the shows histogram The (green). -tubulin a tbln h itga hw h ecnaeo el ihpiaycla rosidct h oaiaino Par6 of localization the indicate Arrows cilia. primary with cells of percentage the shows histogram The -tubulin. c c c B dpee el ipaigaern multipolar aberrant displaying cells -depleted ncdw.A hr esr fcentrosome of measure third a As knockdown. dpee el eesmlr oee,2 hours 24 However, similar. were cells -depleted oto n Par6 and Control ) m AF;5 (A,F); m a tbln(re)1 or fe onig rosso h oaiaino h etooe h oe ie hwteeg fthe of edge the show lines doted the centrosome, the of localization the show Arrows wounding. after hours 10 (green) -tubulin m c BD;100 (B–D); m dpee eaclswr tie ihatbde oPar6 to antibodies with stained were cells HeLa -depleted m ( A E 0 el eeaaye e experiment; per analyzed were cells 100 (E) m eu-tre oto n Par6 and control Serum-starved ) C eaclsoeepesn GFP-Par6 overexpressing cells HeLa ) a tbln(re) h itgasso h ecnaeo ioi el n h ecnaeof percentage the and cells mitotic of percentage the show histograms The (green). -tubulin c E is on rai oto n Par6 and control in area Wound ) nPar6 in ra efudta oto el oee h on area wound the covered Par6 cells whereas control hours, 20 a that after into found completely introduced wound we the into we area, cells Par6 was of which movement or time-dependent motility the wound, Monitoring control Cell scratch of 2011). a monolayer Dawe, the in and towards centrosome measured (Vaughan the edge of motility, cell reorientation leading examined the we requires Finally, 5D). which (Fig. mitosis in arrested sse ntemjrt fcnrlcls(i.5) loehr these Par6 Altogether, of 5F). centrosome (Fig. the cells that control indicate of results edge cells majority wound these the the addition, in towards seen In centrosome as 5E). their (Fig. reorient area to unable wound were the in fill to unable ak ayipratcnrsmlpoen,i non-functional. is proteins, centrosomal important many lacks F c c dpee el,sgetn htPar6 that suggesting cells, -depleted dpee TR-P- el eesandwt nioist Par6 to antibodies with stained were cells hTERT-RPE-1 -depleted oto n Par6 and Control ) Par6 c c rd and (red) c samte etil akr865 marker centriole mother a as T(e)o GFP-Par6 or (red) WT n 5 c dpee TR-P- el t0 0ad2 hours 20 and 10 0, at cells hTERT-RPE-1 -depleted ;* 4; c a dpee TR-P- el eesandwith stained were cells hTERT-RPE-1 -depleted P tbln(re) h itga hw the shows histogram The (green). -tubulin , .5 ** 0.05, D oto n Par6 and Control ) c dpee TR-P- cells. hTERT-RPE-1 -depleted P , c c 0.01. mdl ae)adteprimary the and panel) (middle tr(e)wr tie with stained were (red) Cter c c dpee el,which cells, -depleted dpee el were cells -depleted c dpee el were cells -depleted c dpee eacells HeLa -depleted ye c Journal of Cell Science ysiRNA. by hne nfursec nest fvroscnrsmlpoen ttecnrsm nHL el,i h Par6 the in cells, HeLa in centrosome the at proteins centrosomal various of intensity fluorescence in changes ( o le h etooa soito fPar6 of association centrosomal did the nocodazole with alter microtubules not of Par6 Depolymerization as 2010). mechanism microtubule-dependent protein centrosomal Par3 on of for effects removal monitored interactions Interestingly, and composition. these cells Par3 of HeLa depleted from significance we the regulation, Pkc centrosome determine isoforms to aPkc two order the with and ope;tenmesrfrt oiin faioai eius ( residues. acid amino of positions Par6 to refer numbers the complex; hte Par6 Par6 whether between association The p150 Par6 complex. PAR the involve Par6 not centrosome does that interphase regulation the suggests centrosome from Par3 data) absent that are finding (unpublished the isoforms with proteins, aPKC together centrosomal result, and This any S3). of Fig. levels material the Par6 on centrosomal effect including no had RNAi Par6 addition, In 6B,C). that (Fig. 6A). revealed (Fig. cells experiments Par6 our complex in co-immunoprecipitation isoforms polarity aPkc PAR Reciprocal and Par3 the Par6, of the between component known a (Asse is Par6 As complex polarity PAR the Par6 Par6 Centrosomal 6. 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Fig. in co-wrote experiments experiments, the carried of Tormanen Kati some manuscript; out the co-wrote and experiments the antibodies sharing for Stearns Tim us. and with Salisbury Jeffrey Pelletier, a nlzdfrtepretg fiett n similarity. and and identity format of Clustal percentage in the using was for file performed analyzed sequence was The was (NCBI) Biosoft). Information (Lynnon software Biotechnology DNAman for Center National the onn,M n il M. Piel, and M. Bornens, M. Bornens, Asse nuae vrih ihpiayatbde t4 at antibodies primary with overnight incubated Acknowledgements test Student–Newman–Keul’s the means comparisons. by multiple followed as for variance expressed of analysis are multifactorial values All analysis Statistical Par6 and Q9BYG5.1) Par6 number of (accession alignment sequence multiple The analysis sequence Protein protease with 5 with supplemented incubated was NP-40 lysate 0.5% total of 4 and mM mg (20 at 1 NaCl, buffer reaction, lysis antibody in mM each min 50 For 30 for inhibitors). 7.4, ice on pH lysis direct Tris, by harvested were cells HeLa Co-immunoprecipitation nesn .S,Wlisn .J,Myr . otne,P,Ng,E .adMann, and A. E. Nigg, P., Mortensen, T., Mayor, J., C. Wilkinson, S., J. Andersen, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.121186/-/DC1 online available material Supplementary after release [grant for Health PMC in of months. Deposited Institutes 12 C.S.]. National to the R01GM089913 by number supported was work This Funding Vale contributions Author the Pierre of Guarguaglini, reading Gulia Drs critical to for grateful Tan Johnson, are Go Kirsten Ming Drs We and and manuscript. Herrington Morrissette Kari Lee, Naomi Jennifer thank We eaae ySSPG,taserdt ircluoeo VFmembranes PVDF or blotting. western nitrocellulose by to analyzed and transferred NJ), Clifton, buffer, SDS-PAGE, (Whatman, lysis with by washed were separated Immunocomplexes Sweden). Uppsala, Healthcare, as n. o ora omtmeaue rtiswr eetduigECL using and peroxide) detected 0.3% film. were luminal, X-ray Proteins acid, to coumaric temperature. exposure 8.5, by room pH visualized at Tris-HCl, mM hour (100 1 reagents ImmunoResearch for (Jackson Inc.) antibodies Labs, secondary HRP-conjugated appropriate the nz,Rie La Rainer ¨nczy, eaieyrgltsisdni-yatndpnettreigt h centrosome. the to targeting dynein-dynactin-dependent Sci. 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