Journal of Cell Science ieSine,Pkn nvriy ejn 081 China 100871, Beijing University, Peking Sciences, Life rmt rmr ii iasml eoemttcentry Wang mitotic Gang to before disassembly matrix cilia pericentriolar primary the promote to Plk1 recruits PCM1 Article Research yaisacmaywt h elcce oee,tedetailed the disassembly However, and cycle. cell assembly and the decades, cilia with past the Ishikawa accompany the that dynamics Pedersen Over 2006; shown 2007; 2005). al., al., have al., et et evidences Qin Nachury as 2008; et 2010; (IFT), Rosenbaum, al., such and et (Follit transport proteins, Jin 2011; involved associated Marshall, intraflagellar are cilia that and BBSome, trafficking shown numerous membrane and have controlled, Uetake strikingly and 2010; is Quarmby evidences Nachury, Ciliogenesis 1971; and 2007). al., Seeley al., et still et 1979; (Fonte centriole Rieder, body mother 2005; basal the Parker, ciliary while the centriole place as although take acts assembly, also spindle of can bipolar duplication duplication cilia for undergo the to separation fact centrioles while and the liberates to quiescence, disassembly due cilia probably enter that dynamic exhibit and retinal often disorders cycle cells proliferating cell (BBS), other cells from when and assemble Syndrome exit cilia Primary disease 2011). Marshall, Bardet-Biedl and kidney (Ishikawa human of polycystic as cilium-related defects degeneration, numerous The such to 2006). lead Reiter, diseases, function and Singla cilia membrane 2009; primary ciliary al., on channels et and (Berbari receptors owning plays of transduction assembles which enrichment signal the surface, cellular that to and cell sensation organelle from in projects cell roles and critical body important basal an on based is cilium Primary Introduction the whereby mechanism a shRNA mitotic before suggest by regulators. disassembly data common cilia PCM1 some primary Our of share of interaction. regulation proliferation depletion the cell words: the in as and Key phosphorylation role facilitate dynamics mitosis, pivotal is ciliary to a Plk1 of of plays kinase and regulation PCM1 onset the PCM1 by priming the matrix Thus, between the pericentriolar entry. interaction at to as the Plk1 dynamics functions Notably, of manner. of disassembly recruitment activates Plk1. complex-dependent CDK1 activity dynein-dynactin and of and cilia a and kinase accumulation with assembly in primary 1 the dependent, pericentriolar (PCM) interacts material spindle the matrix that the pericentriolar Plk1 pericentriolar bipolar disrupted that found with wherein to showed dramatically kinase We maturation, entry, coincides we the regulation. Furthermore, mitotic centrosome recruits process resorption. cycle and before regulate and This Plk1 cell disassembly deacetylation of which and ciliary upstream cilia promote acts regulators, dynamics primary to (PCM1) ciliary cycle (HDAC6) for 6 Polo- connects required cell that deacetylase that is report histone key we player Plk1 study, the cycle. pivotal present enriched cell the of a the centrosome In of as elusive. one progression remains acts the (Plk1), regulation along cycle cytokinesis, dynamics 1 cell disassembly and kinase and dynamics assembly ciliary like exhibit links that surface, mechanism cell the the However, from emanate which cilia, Primary Summary work this to equally ` contributed authors *These 2 1 o:10.1242/jcs.114918 doi: 1355–1365 126, Science Cell of Journal 2012 December 11 Accepted hamoZhang Chuanmao uhrfrcrepnec ( correspondence for Author eateto ilgclSine,Clfri tt oyehi nvriy ooa A978 USA of 91768, College CA Biotechnology, Pomona, Membrane University, and Polytechnic Biomembrane State of California Laboratory Sciences, Key Biological State of and Department Differentiation and Proliferation Cell of Laboratory Key MOE 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. C1 l1 rmr cilium Primary Plk1, PCM1, 1, ,QagChen Qiang *, [email protected] 1, ` 1, ,XaynZhang Xiaoyan *, ) 1 oa Zhang Boyan , etooe n h ipaeeti motn o mitotic maturing for important from is displacement displacement its the promote and to centrosome, (Nlp) Ninein-like Plk1 phosphorylate could protein and Plk1 activated Also, CDK1 2009). Lu al., by et 2006; phosphorylated al., needs et sequentially process (Haren this be and to transition, phase Nedd1 and G2/M the maturation targeting at by for centrosome centrosome required the of is nucleation microtubule additional Nedd1 of the materials, recruitment the the phosphorylating pericentriolar of in by addition, the of centrosome In number proteins. of are centrosomal maturation CDK1, of A and capacity the A for 2008). Aurora increase crucial Plk1, anchoring including Schatten, kinases, protein dramatic 2002; of mitotic and (Bornens, the phosphorylation centrosome nucleation and the microtubule proteins in material, characterized centrosomal of been pericentriolar re-organization and has recruitment the additional process of features the this by principal and assembly spindle al., et Pugacheva 2010; which al., regulate et activity, 2007). to (Kinzel resorption A HDAC6 cilia cell primary Aurora activates the and regulating phosphorylates by for between responsible subsequently disassembly be both to cilia recently relationship though revealed the elusive, were HEF1 remain and the dynamics Pitchfork ciliary and underlying proliferation mechanisms etooemtrto sacuilse o h ioa mitotic bipolar the for step crucial a is maturation Centrosome 1 ioogZhuo Xiaolong , dr ta. 06 catn 08 Zhang 2008; Schatten, 2006; al., et ¨ders 1 ujnLiu Junjun , 2 igJiang Qing , c TR to -TuRC 1 1355 and Journal of Cell Science eerlae rmsrmsavto o 4hus ssonin G2 in shown occurred as wave resorption hours, cilia 24 primary second for the cells starvation 1A, kinase inhibitor NIH3T3 serum Fig. Plk1 When Plk1 from 2007). of using released al., by effect et were (Steegmaier resorption 1A) the nM) (Fig. cilia tested (100 transition primary BI2536 then the M We 1A; the on to (Fig. 1998). overlapping activity G2 from al., stimulation, undergo expressed et entry serum cells highly (Ferris after the were mitotic hours when A 22 analysis duration Aurora to before blotting and hours Western 16 Plk1 Plk1 S1B). that of stimulation Fig. showed time of the the material by after supplementary occurred activation hours wave 18 second serum the the about after Strikingly, and happened 1A). cilia 2007) hours (Fig. one al., 2 et other of about primary (Pugacheva the previously occurred stimulation reported Two One serum as stimulation cells. 10% cilia S1A). ciliated after visible these occurred clearly Fig. ciliary showed waves study cells disassembly starvation, material NIH3T3 to serum of of system (supplementary hours 70% 24 a than After established more cells. NIH3T3 and in to cilium dynamics regulation of marker cycle entry. linker mitotic cell potential before disassembly a and cilia as and primary dynamics act promote phosphorylated might ciliary Plk1 is that orchestrating Plk1 speculated we when (Macu Hence, A 2007), Aurora by 2011; al., and resorption activated Marshall, ciliary and et assembly (Ishikawa the entry and Pugacheva cilia mitotic cycle, before cell primary occurs to wave that related are indicated disassembly studies cilia Previous primary entry promote mitotic to before required disassembly is activity kinase Plk1 data Results cilia Our primary regulation. between assembly. cycle relationship spindle cell the state and on the mitotic dynamics insight into new bipolar centriole a the provide body/mother microtubules of basal for the ciliary disassembly required release Subsequently, cilia inducing and disassembly. the with thereby and interacting by deacetylation mitotic HDAC6, resorption cilia before primary activating Plk1 matrix for matrix-localized pericentriolar pericentriolar required of is the in activity which kinase to CDK1, The by entry. al., Plk1 phosphorylated et recruits is Hames 1999), 2010; turn al., al., et et Kubo Ge to 2005; 1994; 2002; proteins al., Merdes, of et and number on (Balczon a Dammermann maturation target Plk1 centrosome to during scaffold of a centrosome as enrichment serves that the protein for 2006). al., role et (Soung important mitosis during centrosome, an centrosome mitotic may play centrosome the maturing also on 2009). from component(s) al., other Plk1 that et suggesting to formation delocalizes hCenexin1 Soung modestly of cilia 2006; Plk1 depletion only al., centrosome, primary of interphase et unlike to (Soung recruitment However, Plk1 contributes the of also independent in and indicated participates research Lowery centrosome Previous 2007; hCenexin1 2008). al., al., et et that phosphorylation-dependent Lowery Petronczki 2003; 2004; a al., al., et localization as et (Elia serves Its domain polo-box binding which cytokinesis. as termed (PBD), portion, to and C-terminal domain its localizations entry on diverse depends mitotic capability exhibits 2005; al., Plk1 from et 2003). (Casenghi functions al., mitosis of et onset Casenghi the at formation spindle 1356 ovrf hshptei,w tie acetylated stained we hypothesis, this verify To satellite centriolar a PCM1, that identified we work, this In ora fCl cec 2 (6) 126 Science Cell of Journal ˚ e ta. 08 eie l,2008). al., et Seki 2008; al., et rek a tblna a as -tubulin n iaeda l1(Plk1 Plk1 dead kinase and curdi ycrnzto ihtepiaycladisassembly S1D). Fig. cilia material supplementary primary 1D,F; showed (Fig. that the entry cilia mitotic Plk1 with before without synchronization of localization in cells matrix occurred the pericentriolar whereas the preferentially Plk1; centrosomal of GFP effect such the no had However, S1C). Plk1K82R Fig. type. mutant material ciliary dead (supplementary wild kinase promoting the the in Plk1T210D or increase control with mutant efficiency compared active folds resorption kinase 2 the about showed that (Plk1K82R) GFP–Plk1 dead confirmed kinase GFP, or mutants overexpressing (Plk1T210D) active cells kinase type, T210 NIH3T3 wild at in A cilia Aurora primary of by activated wave and shown resorption (Macu have phosphorylated Evidences second is 1C). we Plk1 the (Fig. interference that too suppression, that disturbed RNA was Plk1 found cilia with primary and to treated Plk1 in due cells targeting of that is phenotype NIH3T3 the than that resorption immunostained confirm stimulation To ciliary with 1B). serum treated (Fig. defective after cells well with as in treated hours cells longer 18 control cells was cilia about the cilia the in primary BI2536 of second delayed length the The largely contrast, BI2536. was In wave cells. control resorption the in phase rtis nldn osiuieyatv l1(Plk1 fusion Plk1 active (Plk1–PCNTB) Plk1 constitutively Plk1–pericentrin including pericentriolar of proteins, by of examined expression was accumulation hypothesis ectopic This disassembly. the ciliary in that resulted speculated We iaeatvt srsosbefrtersrto fpiaycilia primary of resorption Plk1 the entry. for mitotic active before responsible the matrix-localized is support pericentriolar activity data kinase the these that Together, 1E). hypothesis (Fig. disassembly ciliary hs,wienihrPNBaoenrPlk1 nor alone PCNTB neither while phase, l1pooe rmr ii iasml vni h bec of absence the in activity. even A disassembly Aurora cilia primary activated promotes that Plk1 indicate data 2A; These S2). (Fig. Fig. control material GFP supplementary the but the promote with Plk1T210D, efficiently comparing could resorption that mutants, cilia found T210A primary or We type hours. wild the 18 not about of for presence inhibitor the in the mutant non-phosphorylation) (mimic Plk1T210D T201A Plk1, type or wild GFP-fused starvation GFP, serum with transfected from from were released released cells the cells 0.5 Simultaneously, To activity. NIH3T3 with A. treated starvation Aurora we serum of hypothesis, independent this process verify resorption the in cilia participate disassembly also primary cilia may Plk1 primary activated that of proposed wave We occurs. second the before when Plk1 entry activates key mitotic A the Aurora of proliferation, one cellular As of 2007). that regulators al., et phosphorylating (Pugacheva revealed HDAC6 by activating disassembly colleagues and cilia primary and promotes A Aurora Pugacheva HDAC6 Multiple activity. regulating 2007). in al., involved deacetylase be et to Zhang found 2007; been al., have mechanisms et Pugacheva 2002; (Hubbert resorption al., cilia cell et primary modulating as well in as functions motility, that and spreading deacetylase tubulin a is HDAC6 activating by resorption HDAC6 cilia primary promotes Plk1 eietilrmti.A xetd ii eesignificantly were cilia expected, at Plk1 localize the stably As in disassembled could proteins matrix. fusion pericentriolar These 2011). Rhee, and eakby nG hs el,w ol adydtc h signal the detect hardly could we cells, phase G0 in Remarkably, ˚ e ta. 08 eie l,20) muotiigof Immunostaining 2008). al., et Seki 2008; al., et rek CA KD PNBepesn el vni G0 in even cells –PCNTB-expressing m –CT nNHT Fg E (Lee 1E) (Fig. NIH3T3 in )–PCNTB L83 oihbtArr A Aurora inhibit to MLN8237 M KD PNBpromote –PCNTB CA )–PCNTB Journal of Cell Science l1 h roha hw h cuuaino eietilrPk ae uruddb ht otdln) e upeetr aeilFg 1 o im for S1D Fig. material supplementary perice See of dynamics line). the dotted show white to by selected were surrounded 1 points (area bar: time Plk1 Scale different pericentriolar movie. at (histogram of images full cilia magnified accumulation the primary Selected the s representing with captured. show shows then cells bars arrowhead were Error of cells experiments. The percentage independent the Plk1. the two of determine in images counted to Movie were counted phase. cells G2 were 200 of cells Averages The left. hours. 10 the bar: 24 on shown for are starved images then immunofluorescence blue Representative and RFP–H2B; protein, red, fusion (Ac-tub); indicated acetyl-tubulin show the Green, are 2008). hours al., 18 et for (Chen released 20:1 cells rel of of o After images ratio 10 percentage immunofluorescence hours. a bar: The representative Scale 48 at s.d. and cilia. co-transfected for show (histogram), for were starvation bars panel antibody Error RFP–H2B serum BI2536. upper anti-acetyl-tubulin and with with the using p-Super-Plk1 treated treated in microscopy cells panel. shown simultaneously represent immunofluorescence lower are and bars for and control, green collected counted and as we were were control, particular-length p-Super A cells cilia the with or Aurora represent the cells p-Super-Plk1 of bars and points, percentage Violet ( and Plk1 time the A. control. RFP–H2B show different that in loading histograms with showing shown The a transfected starvation starvation. analysis as serum serum were blotting blotted from from cells western release was release after panel: Actin after hours points at release. Lower 18 time after occurred (red s.d. about the abolished hours waves at show was cells resorption 18 bars wave control ciliary at resorption of Error second cells two those experiments. the than control cells, BI2536, independent the control by two inhibited in in in was expressed panel: activity counted highly Upper kinase were Plk1 BI2536. 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F I33clswr rnfce ihGPPk n F–2,snhoie tG/ hs ydul-hmdn lc,adrlae o or to hours 9 for released and block, double-thymidine by phase G1/S at synchronized RFP–H2B, and GFP–Plk1 with transfected were cells NIH3T3 ) m .( m. D cuuaino eietilrPk orltswt h iir iasml.NHT el eearse nG hs by phase G0 in arrested were cells NIH3T3 disassembly. ciliary the with correlates Plk1 pericentriolar of Accumulation ) m m. ( l1rgltspiaycladssebybfr ioi 1357 mitosis before disassembly cilia primary regulates Plk1 A iecus hwn h ecnaeo h I33clswt ii fe ees rmserum from release after cilia with cells NIH3T3 the of percentage the showing course Time ) oaie ihPk ntecnrsm 8husatrreleased after hours 18 centrosome the co- on HDAC6 Plk1 cilia Furthermore, basal with primary 2B). when the localized (Fig. centrosomes or disassembled the already also centrioles We and were the cilium, antibodies. the on acetyl-tubulin of localized body and HDAC6 HDAC6 that with observed hours 18 and B h ii nB23-rae I33clswr longer were cells NIH3T3 BI2536-treated in cilia The ) m .( m. E I33clswr rnfce with transfected were cells NIH3T3 ) h el with cells the f ii tsm of some at cilia ( bu and 2 about C .Averages ). NIH3T3 ) nright). on d Scale .d. nthe in n aeat ease DNA. , ntriolar acetyl- ages re Journal of Cell Science the DC n F oehr rFa–DC n GFP–Plk1 the and of tubulin Flag–HDAC6 acetylated of or level the together, Then, respectively. GFP together, and HDAC6 Flag– alone, Flag–HDAC6 To alone, GFP–Plk1 alone, deacetylase deacetylation. transfected GFP either were the with tubulin cells HEK293T stimulate effect, promoting deacetylation could tubulin thereby Plk1 Plk1 access HDAC6 that and of found Flag–HDAC6 we activity 2005), of al., co-IP et the by Intriguingly, interaction by 2D). this (Fig. and supported 2C) performed further (Fig. HDAC6 found was We with and antibody. interact anti-Plk1 could with Plk1 probed HDAC6 that then and was antibody complex anti-GFP Flag-tagged IP the by overexpressing immunoprecipitated cells or of was lysate GFP–HDAC6 The HEK293T GFP- assay. transfected (IP) we immunoprecipitation with Next, 2B). (Fig. cells starvation b analyzed serum was tubulin from of level deacetylation the and deacetylation. minutes tubulin 15 were promote for GFP–Plk1 significantly temperature plus could ( room Flag–HDAC6 combination at antibodies. or lysed HDAC6 indicated GFP, were and the plus cells Plk1 with Flag–HDAC6 co-locali the The The probed Flag–HDAC6, Plk1 assay. and blotting. and GFP–Plk1, that deacetylase cells blotting GFP, show tubulin with western G0 a images transfected by in to The cells analyzed subjected cilium Plk1. HEK293T were primary and HDAC6. complexes the of HDAC6 IP activity of against The deacetylase antibodies body assay. ( with (IP) basal panel). immunostained immunoprecipitation the (bottom to were co on centrosome subjected was Cells localized the cells panels). HDAC6 on ciliated middle that of HDAC6 and show percentage with (top the images cells and The G2 microscopy, (Ac)-tubulin. in immunofluorescence ( acetyl centrosomes by s.d. and analyzed show HDAC6 were 0.5 bars cells against with the Error antibodies treated later, bars. and hours starvation, green 18 serum as from At deacetylation. showed released activity. tubulin were A T210A promote Aurora mutant to inhibit Plk1 to HDAC6 nonphosphorylatable or activates T210D and mutant to Plk1 phosphorylation-mimic binds Plk1 2. Fig. 1358 nvitro in ora fCl cec 2 (6) 126 Science Cell of Journal uui ectlto sa rm( from assay deacetylation tubulin nvitro in ectlto sa (Destaing assay deacetylation B E I33clsrlae rmsrmsavto o or G)o 8hus(2 eec-muotie with co-immunostained were (G2) hours 18 or (G0) hours 0 for starvation serum from released cells NIH3T3 ) ydnioer.Tepretg ftblnaeyaino h oto a ae s10(..100%). (i.e. 1.0 as taken was control the of acetylation tubulin of percentage The densitometry. by ) C , D l1itrcswt DC.HK9Tclswr rnfce ihGP rFa-agdHA6and HDAC6 Flag-tagged or GFP- with transfected were cells HEK293T HDAC6. with interacts Plk1 ) uigti rcs,Pk a ergltdb uoaAand/or A Aurora disassembly. cilia by primary regulated promote be to Aurora-A resorption. may with cilia Plk1 coordinates primary process, the this we in During resulting together, activity, stimulate to Taken deacetylation HDAC6 with its interacts 2E,F). Plk1 tubulin activated (Fig. the the that deacetylated concluded cells, in of dramatically expressed both was were when Flag– Flag–HDAC6 Remarkably, slightly and tubulin. or could GFP–Plk1 of GFP–Plk1 Flag–HDAC6 deacetylation that, with alone, the together showed GFP stimulate GFP Results and or alone, blotting. control HDAC6 western the with by compared analyzed was cells l1acmltdaon eietilrmti tG phase G2 at matrix cilia, primary pericentriolar of around resorption the accumulated with Plk1 along PCM1 above, by matrix described pericentriolar As to recruited is Plk1 ( A I33clsoeepesn F,GPtge idtp W)Plk1, (WT) type wild GFP-tagged GFP, overexpressing cells NIH3T3 ) a Tblnwspoe salaigcnrl ( control. loading a as probed was -Tubulin F uniiainof Quantification ) E l1stimulates Plk1 ) m MLN8237 M ne and unted western y zes Journal of Cell Science 4hus usqety eimnsandadcutdtepretg fclswt ii tdfeettm onsatrsrmr-tmlto.Toind Two re-stimulation. serum after points time different at cilia with cells s.d. of show percentage bars the Error counted performed. and were immunostained experiments we Subsequently, hours. 24 rnae l1140 l1PDo l1PD(A,adimnsandwt nioisaantPk rMc nyedgnu l1()adisPDdomain PBD its and (D) Plk1 endogenous Only Myc. or Plk1 against antibodies with immunostained and (2A), GFP–PCM1- PBD with Plk1 co-localize or (F) Plk1–PBD 1–400, Plk1 truncates F–l1wr ie ihcl ehnladimnsandwt niPM nioy cl a:10 bar: Scale antibody. anti-PCM1 with immunostained and methanol cold with fixed were GFP–Plk1 lte ihat-C1atbd.( antibody. anti-PCM1 with blotted etilrPk n h eietilrmtra l1(aanot (data Plk1 material pericentriolar the the subpopulations: and the two prophase, into Plk1 to divided centriolar G2 be During with could al., style. centrosome Plk1 dot-staining the centrosomal et bright on and concentrated two G1 (Kishi Plk1 during or Fig. of that, one reports portion observed also a material we phases, previous 2011), S supplementary al., with et Mahen 1F; accordance 2009; (Fig. In entry S1B,D). mitotic before muorcptto I)eprmnswt niPk nioyadIGcnrl n h Pcmlxswr hnaaye ywsenbotn ihtei the with blotting western 4 by at analyzed hours then 3 were for complexes IP 4B the glutathione–Sepharose and PBD2A-coated control, IgG and ( antibody antibodies. anti-Plk1 with experiments (IP) immunoprecipitation PCM1. with interacts and co-localizes Plk1 3. Fig. causes PCM1 C-terminus-deleted mitosis in both of arrested Overexpression cells that in 3A,B). other (Fig. phase confirmed each co- with G2 interact we Plk1 Plk1 in and assay, PCM1 material immunoprecipitation shown). pericentriolar through not around and PCM1 (data proteins with the Plk1 localizes among is with PCM1 that co-immunoprecipitated found mass and by analysis followed performed spectrometry antibody We anti-Plk1 processes. using two immunoprecipitation these an coupling look component(s) to us the inspired for Plk1 localized pericentriolar of accumulation and of to level Fig. material phase on the supplementary 1F; G2 whereas accumulated S1D). (Fig. steady from breakdown), kept Plk1 Plk1 period envelope centriolar that the (nuclear during showed NEBD material time- also pericentriolar by microscopy followed expression lapse GFP–Plk1 Accordingly, shown). h pta n eprlcreainbtencladisassembly cilia between correlation temporal and spatial The C l1PDdmi neat ihPM.MttcHL ellstswr ujce oaGTpl-onasywt S- S–B-o GST– or GST–PBD- GST-, with assay pull-down GST a to subjected were lysates cell HeLa Mitotic PCM1. with interacts domain PBD Plk1 ) D .Saebr:10 bars: Scale C. D – G el eetasetdwt GFP–PCM1- with transfected were Cells ) m ( .( m. A l1c-oaie ihPM nteprcnroa arxo 2cls 2paeHL el expressing cells HeLa phase G2 cells. G2 of matrix pericentriolar the in PCM1 with co-localizes Plk1 ) H ˚ .Tebaswr sltdadtepoen on otebaswr eaae ySSPG and SDS-PAGE by separated were beads the to bound proteins the and isolated were beads The C. I33clswr rnfce ihGPo GFP–PCM1- or GFP with transfected were cells NIH3T3 ) l1rgltspiaycladssebybfr ioi 1359 mitosis before disassembly cilia primary regulates Plk1 upce,PDAnihrlclzdo etooe o co- nor centrosomes GFP–PCM1- on with localized neither localized was PBD2A K540A) (H538A, suspected, GFP–PCM1- PBD2A with co-expressed mutant PBD 2002), and PBD al., generated of et Plk1 (Seong need function-loss centrosome on the a Plk1 of demonstrated localization that for kinase domain the work aggregates. not previous cytoplasmic but in domain, interaction Considering the PBD for Plk1 responsible is the domain, that showed results GFP–PCM1- proteins The with Plk1 truncated co-expressed of and number generated with a were interaction cells, the the by in for PCM1 required truncated is disrupted the To Plk1 PCM1. of truncated also domain the 3D). which with (Fig. interacts determine granules was Plk1 endogenous that the suggests the of result Plk1 aggregation This Instead, the co- PCM1. of with co-localized truncated Plk1 By localization on the of localize the S3A,B). overexpression to that GFP–PCM1- Fig. than in found cytoplasm Plk1 material of in immunostaining (supplementary aggregate to centrosome (GFP– likely PCM1 C-terminus-deleted more a expressed we PCM1- study, 2003). endogenous Tsukita, this as and its Kubo In well of 2002; as Merdes, subset pericentrin, and a (Dammermann centrin, also PCM1 as but such proteins mutant proteins, the cargo only not of aggregation D ln rc-rnfce ihGFP–PCM1- with co-transfected or alone C D )i h el n ofre htti rnae C1was PCM1 truncated this that confirmed and cells the in C) m .( m. B h el retdi 2Mpaewr ujce to subjected were phase G2/M in arrested cells The ) D D Fg G.Frhroe the Furthermore, 3G). (Fig. C ,adte utrdwtotsrmfor serum without cultured then and C, D n y-agdPlk1 Myc-tagged and C D xrsigcls we cells, expressing C D nHL el.As cells. HeLa in C D C(Fig.3E,F). ependent ndicated Journal of Cell Science opopoyaal B Amtn,adteitrcino C1wt l1PDwsdsutdb ocvtn.CoaseBu C)sann hwdthe showed staining (CB) by Blue not Coomassie but roscovitine. domain by PBD Plk1 disrupted control. type was con, wild PBD (E). by Plk1 down GST–PBD2A pulled with and be PCM1 GST–PBD could of of PCM1 interaction antibody. proteins anti-PCM1 the loading with and total blotting mutant, western 2A by analyzed PBD were nonphosphorylatable beads the to bound proteins D iaeihbtrrsoiie(10 roscovitine inhibitor kinase CDK ( control. loading protein total h Pcmlxswr nlzdb etr ltigwt h niae niois() rtelststetdwt rwtotrsoiiewr subject were roscovitine without 4 or at with 4B treated glutathione–Sepharose lysates GST–PBD2A-coated the or and (D); GST–PBD- antibodies GST-, indicated with the incubating with by blotting western assay by pull-down analyzed were complexes IP The el xtdfo eahs.Cci 1adSr0popoyae itn 3wr rbda h niaoso h elccephases. cycle cell the of indicators the as probed were H3 9 histone with Ser10-phosphorylated treatment after and histon down B1 Ser10-phosphorylated shifted Cyclin was disappeared. metaphase. band mitosis up-shifted from during The exited antibody. occurred cells anti-PCM1 that with band blotting and western up-shifted mitosis by the analyzed of RO3306, indicator inhibitor an CDK1 as the probed of with presence treated the When in down antibody. came and mitosis, in up-shifted i.4 idn fPM ihPk tmtssi d1kns ciiydependent. activity kinase Cdk1 is mitosis 30 at at Plk1 minutes with 30 PCM1 of Binding 4. Fig. the detected we localization, PCM PCM1- Plk1 of and effect PCM1 PCM1- of overexpressed both and disassembly ciliary for PBD. its via PCM1 that with indicate interacting by results material pericentriolar these to recruited Together, is 3C). Plk1 co-precipitated (Fig. specifically one former was the PCM1 with and GST–PBD2A, GST– or using assay PBD pull-down a to subjected were lysates cell mitotic 1360 rnfce ihGPo GFP–PCM1- or GFP with transfected eopin n losgetta h rsneo C1greatly PCM1 disassembly. ciliary of observation cilia presence to Pl the inhibit the that contributes suggest active with also dramatically and localized consistent resorption, could PCM are control, data that These GFP resorption. GFP–PCM1- not we interference, RNA expected, but Plk1 As to S3D). similar that, Fig. re-stimulation cilium found material serum with supplementary after cells points 3H; of time (Fig. percentage different at the counted Then, was hours. 24 for serum ic eietilrmtra oaie l1i responsible is Plk1 localized material pericentriolar Since ora fCl cec 2 (6) 126 Science Cell of Journal ˚ )o nrae sacnrlwr nlzdb etr ltigwt niPM nioy oprdwt h nepaelst,tepstv band positive the lysate, interphase the with Compared antibody. anti-PCM1 with blotting western by analyzed were control a as untreated or C) D nclayrsrto.NHT el were cells NIH3T3 resorption. ciliary on C D , E niiino h iaeatvt fCK irpstePM–l1itrcin ioi eacl yae rae iho without or with treated lysates cell HeLa Mitotic interaction. PCM1–Plk1 the disrupts CDKs of activity kinase the of Inhibition ) m b )fr1hu t37 at hour 1 for M) Atnwspoe sattlpoenlaigcnrl ( control. loading protein total a as probed was -Actin 1pooe rmr ciliary primary promotes k1 D n utrdwithout cultured and C l Pae ( -PPase. ˚ eesbetdt muorcptto sa yrbi niPk nioyo abtIGa control. a as IgG rabbit or antibody anti-Plk1 rabbit by assay immunoprecipitation to subjected were C D disrupts C B nepaeadmttcHL ellstswr nlzdb etr ltigwt anti-PCM1 with blotting western by analyzed were lysates cell HeLa mitotic and Interphase ) D C, o hw) ugsigta D1cudb h iaethat kinase the be could CDK1 data that and 4B al., suggesting (Fig. et again shown), Vassilev occurred 1997; PCM1 not al., of band et lower Azevedo the (De 2006), present was CDKs for n srsosbefrtepopoyaino C1 efound We PCM1. which of (9 determine phosphorylation RO3306 to the when shown) that, for not responsible (data is lysate introduced one were cell inhibitors mitotic kinase the protein into of phosphorylated number was A PCM1 was 4A). mitosis that (Fig. lysate in suggesting mitotic occurred, PCM1 band the lower of When band with 4A). (Fig. the anti-PCM1 treated up-shifted that with clearly observed was the immunoblotted was regulating It were and antibody. treatment kinase and nocodazole respectively, interphase priming by of separated synchronized the Lysate cells for Plk1. HeLa and 2008). mitotic seek PCM1 al., to between et substrates (Petronczki interaction set its kinase to we priming binds of Next, Plk1 assistance model, the canonical with the to its According for essential Plk1 is with CDK1 interaction by PCM1 of Phosphorylation ( A nepaeadmttcHL ellststetdwith treated lysates cell HeLa mitotic and Interphase ) C nepae ioi n ioi-eesdHL ellstswere lysates cell HeLa mitosis-released and mitosis Interphase, ) l Pae h psitdbn iapae n a and disappeared band up-shifted the -PPase, m )o ocvtn (10 roscovitine or M) ˚ o or.Tebaswr sltdadthe and isolated were beads The hours. 3 for C m O36fr2 iue,atm hnthe when time a minutes, 25 for RO3306 M b Atnwspoe sthe as probed was -Actin m ) h inhibitors the M), l Pae(0 for U (400 -PPase dt GST a to ed 3was H3 e Journal of Cell Science hl ntepeec frsoiie h neato fPk with the Plk1 of about that interaction the found Plk1; roscovitine, in of We with presence the 4C). co-immunoprecipitated rapidly in (Fig. while was treatment level PCM1 the phosphorylated dramatically after phosphorylation could minutes RO3306 25 PCM1 because affect Plk1, would decrease with PCM1 to interaction of roscovitine phosphorylation its chose the We whether mitosis. investigate during PCM1 phosphorylates idefamn F)o C1c-oaie ihPk and Plk1 with cells the co-localized HeLa only PCM1 that in of revealed expressed (F2) microscopy fragment and middle Fluorescence constructed 5A). were (Fig. fused fragments GFP PCM1 three required purpose, with this is For phosphorylation interaction. its the as for the PCM1 map on to set site we phosphorylation manner, phosphorylation-dependent PCM1 a in regulating kinase the priming to the by us interaction. as prompted reduced PCM1–Plk1 acts results the may largely These CDK1 was 4E). type that (Fig. speculate PCM1 wild roscovitine the down of loss-of- of addition pull the capacity to to the similar GST–PBD assay, GST–PBD-2A, pull-down mutant the fusion function GST–PBD in When used 4D). was (Fig. protein abolished clearly was PCM1 nwn htteitrcinbtenPk n C1mybe may PCM1 and Plk1 between interaction the that Knowing l1rgltspiaycladssebybfr ioi 1361 mitosis before disassembly cilia primary regulates Plk1 noHL el n h rtiswr muorcpttdby immunoprecipitated were proteins the transfected and were binding cells mutant protein HeLa (T703A)–GFP into F2 performed PCM1– and for subsequently F2–GFP important assays. we is T703 interaction, at Plk1 PCM1 of phosphorylation the hwdta h idefamn fPM F)cnbe can (F2) PCM1 of fragment middle CDK1 by the phosphorylated proteins that the showed and for constructed purified were and were this vectors mutant verify site non-phosphorylation To expression this 5D). its (T703A) (Fig. and phosphorylates PBD F2 to GST-tagged that PCM1 speculation, kinase of binding priming the a promotes CDK1 the also that is be suggesting T703 a CDK1, the may as of Furthermore, serve site 5D). may phosphorylation (Fig. and conserved PBD Plk1, for for site SS/TP conserved binding the motif a binding in T703, the sequence that 5B; resemble from noticed (Fig. acid acid we amino PCM1, amino the cytoplasm of analyzing fragment with interact By middle could the 5C). PCM1 co-IP of (Fig. with fragment Plk1 middle this in the confirmed only We that assay S4). dispersed Fig. material supplementary were fragments c tblna h pnl oei ioi,wieteohrtwo other the while mitosis, in pole spindle the at -tubulin C12adPMF 73 rtiswr ujce oa kinase to CDK1 subjected with were assay proteins phosphorylation T703A GST-tagged PCM1F2 purified and The PCM1F2 human. chicken, and in mouse the fragment (XENLA), among PCM1F2 motif of T703) sequences or protein 703, (threonine SS/TP conserved bar: Scale cytoplasm. the centrosomes, into 10 on dispersed Plk1 others with the co-localize whereas PCM1 could three F2 were these only A Among mutants, in antibody. showed anti-Plk1 mutants with truncated probed the with ( transfected vector. pEGFP-N3 cells into cloned The were respectively. mutants 1461–end, truncated amino and represent 591–1460 F3 and 1–590, F2 acids F1, PCM1. of Plk1. mutants and truncated required PCM1 three is between CDK1 interaction by the T703 PCM1 for of Phosphorylation 5. Fig. B n S–B2 rtis(F). proteins GST– of GST–PBD2A loading and total PBD the showed were staining antibody. (CB) beads anti-GFP Blue the with Coomassie to blotting bound western proteins by the analyzed and isolated The were 4B. beads glutathione–Sepharose GST– with GST–PBD2A-coated incubated or were (E). PBD- lysates antibody the anti-Plk1 assay, pull-down with GST blotting For western complexes by IP analyzed antibody. were anti-GFP rabbit were by lysates immunoprecipitated the immunoprecipitation, and For assay. immunoprecipitation pull-down to GST and subjected F2–GFP were expressing (T703A)–GFP lysates F2 cell HeLa interaction. Plk1 blot. ( western marks WB, was asterisk PCM1F2. The assay phosphorylated panel). this (bottom antibody in anti-GST proteins by GST-tagged probed of loading The panel). blotting western ( by antibody. analyzed anti-Plk1 were with proteins anti-GFP IP by The (IP) antibody. immunoprecipitated and lysed were F3–GFP or E , F m hshrlto fPM 73i epnil o PCM1– for responsible is T703 PCM1 of Phosphorylation ) .( m. C nvitro in Gallus eaclstasetdwt F,F–F,F2–GFP, F1–GFP, GFP, with transfected cells HeLa ) ohmn n urudn residues surrounding and human, to iaeasy hshrlto assay Phosphorylation assay. kinase nvitro in D euneaayi hwn the showing analysis Sequence ) Fg D.T eemn if determine To 5D). (Fig. nvitro in ( A (middle iga of Diagram ) Xenopus B HeLa ) Journal of Cell Science etooe.HL el xrsigGPPk eetetdwt 1 with treated Plk1. were of GFP–Plk1 accumulation expressing pericentriolar cells for HeLa responsible centrosomes. is PCM1 6. Fig. the on Plk1 of accumulation the impairment centrosome, the with of on along that localization PCM1 found the We to of PCM1. nocodazole followed and with and Plk1 cells al., both the network et treated microtubule we Kamiya on 2004), 2006; disrupt PCM1 al., dynein- al., et of et and Kim (Gurling 2008; microtubules accumulation activity of the complex entirety dynactin that the report requires centrosomes the to According on PCM1 depends of matrix entirety pericentriolar the domain, in PBD Plk1 of Plk1 Plk1. Recruitment and for PCM1 between site interaction at binding the PCM1 for a responsible of is creates on phosphorylation which CDK1 Based the 5F). by that (Fig. was concluded T703 F2 PBD we type the data, wild with the these the mutants 5E, GST– with (T703A) Fig. comparing and F2 in abolished the showed GST–PBD of result with capacity the binding to incubating Similar by separately. to PBD2A subjected assay were the (T703A) pull-down of F2 lysates Plk1 a and the F2 results, of overexpressing our cells verify amount HeLa further To the 5E). with dramatically (Fig. that was reduced probed mutant non-phosphorylation showed then the with were Result associated complexes antibody. IP anti-Plk1 The antibody. anti-GFP 1362 otasetdwt C1RA n Nirssateoeoswl yeGPPM rGPvcosa ai f2: n muotie ihanti-Plk1 with 10 immunostained bars: and of Scale defects 20:1 areas. the of pericentriolar rescued ratio GFP–PCM1 the a exogenous indicate RNAi-resistant at were arrows expressed Cells vectors the Green GFP. GFP panel), panel). not (lower or but (upper control GFP–PCM1 GFP–PCM1 accumulation GFP type type exogenous pericentriolar wild wild expressed exogenous ( the RNAi-resistant exogenous with antibody. of RNAi-resistant Compared overexpression anti-PCM1 and antibody. by with rescued RNAi wa immunostained be PCM1 The PCM1 can and with antibody. RNAi of p-Super-PCM1 co-transfected anti-PCM1 PCM1 accumulation or by with pericentriolar Plk1 (control) immunostained the endogenous ( p-Super and of that arrow). with accumulation fixed show green transfected were to by were cells chosen (indicated GFP–Plk1 ov the was RNAi expressing p50 transfection, (MERGE) PCM1 without after cells by or hours untransfected disrupted with Forty and completely Plk1 20:1. transfected accumulated of with pericentriolar ratio photograph with a immunofluorescence w cells at immunostained the co-transfected and of were fixed percentages were H2B the p50–GFP with revealed ( transfected analysis panel). cells Statistical (lower HeLa panel). activity. (upper complex dynein-dynactin antibody requires Plk1 centrosomes on Plk1 of accumulation ora fCl cec 2 (6) 126 Science Cell of Journal C fiinyo C1RA ncdw nHL el.Wsenbotn hwdteefcec fPM nefrne -ue-C1adGFP– and p-Super-PCM1 interference. PCM1 of efficiency the showed blotting Western cells. HeLa in knockdown RNAi PCM1 of Efficiency ) D m ooaoefr4hus ie n muotie ihat-C1atbd.( antibody. anti-PCM1 with immunostained and fixed hours, 4 for nocodazole M C1RA blse eietilracmlto feoeosGPPk.HL cells HeLa GFP–Plk1. exogenous of accumulation pericentriolar abolishes RNAi PCM1 ) ( A irtbl iasml irpsteacmlto fPk n C1on PCM1 and Plk1 of accumulation the disrupts disassembly Microtubule ) oaiaino l1wsuafce Fg D supplementary the we field, 6D; effects, off-target same (Fig. potential the the unaffected eliminate in To was S5). cells Fig. material Plk1 control the of material in localization pericentriolar of while around depleted western a blocked, Plk1 cells by was of the as accumulation examined In in the 6C). was functioned PCM1, PCM1 (Fig. efficiency immunostaining deplete which and depletion to blotting The 2008), GFP–H2B, al., cells. et with the co-transfected (Chen were indicator cells that transfection HeLa sequence irrelevant along into or PCM1 1995). centrosome against Vallee, al., and complex Vaughan et to 2004; Kamiya al., dynein-dynactin 2002; et Kim Merdes, Plk1 PCM1 2008; the and mislocalize (Dammermann of with microtubule also would coinciding dependent-recruitment function 1995), dynactin 6B), Vallee, In (Fig. antagonizes 6A). and which GFP- (Fig. overexpressing (Vaughan dynamitin, manner by staining p-50 activity punctate dynein tagged disrupted a areas we the in random addition, in in cytoplasm material co-localize the could pericentriolar of they in nocodazole, enriched and of PCM1 become presence both not although did that, Plk1 observed Fig. we material Interestingly, supplementary 6A; S3C). (Fig. abolished was centrosome hn ecntutdpSprvco otiigshRNA containing vector p-Super constructed we Then, m m. E oso pericentriolar of Loss ) erexpression B t anti- ith The ) Plk1’s s Journal of Cell Science htteacmlto fPk nprcnroa arxdpnson PCM1. depends of suggest matrix entirety pericentriolar data on the These Plk1 of 6E). accumulation the (Fig. the rescue that Plk1 confirmed could of control and GFP localization not respectively, pericentriolar RNAi- but PCM1 control, exogenous exogenous GFP only that, with and PCM1 PCM1 resistant against shRNA co-transfected cfodo eietilrmtra n ptemo the of upstream and the material assembling an bipolar pericentriolar of be of and recruitment downstream may of maturation acts PCM1 centrosome scaffold which by the assembly, material of 2006; spindle pericentriolar al., step the recruitment et intermediate to the Therefore, (Gurling 2004). Plk1 al., et microtubules of Kim 2008; the al., by et dynein-dynactin with Kamiya centrosome by to along permanent transported recruited a complex complex not is protein is itself PCM1 DISC1-BBS4 and that protein the noticing its on to worthy pericentriolar of Plk1 Plk1 is of binding consequence of It simple the substrates. accumulation the than rather pericentriolar the is PCM1 by So, the recruitment material area. on pericentriolar this PCM1 Plk1 centrosome in of depletion of contrary, Plk1 the RNAi recruitment On abolished 2009). we by al., the substrate et depletion Plk1, (Zhang Plk1 Nedd1 affect material for previously, known not us docking-site the by did or a shown is PCM1 or As PCM1 deplete Nedd1. substrates whether to the determine RNAi To explored of on CDK1. one depends of Plk1 simply activity and kinase PCM1 between the 2005). interaction al., et the Hames 2002; Moreover, Merdes, pericentrin, centriolar and centrin, (Dammermann the ninein, etc. as recruitment Nek2, to such the proteins in recruited centrosomal implicated many been is of has which Plk1 matrix? PCM1, that by pericentriolar satellites cilia onto here primary relocated demonstrated promote to Plk1 We function is the how to As disassembly, cells 2006). interphase al., in centrosome et reported the and (Soung to was Plk1 Lee it recruits is although hCenexin1 2006; kinase scarce; that protein remains al., this centrosome spindle how to et of on recruited studies investigation Luca mitotic functional the abundant De Plk1, the the centrosomal 2004; with Comparing and al., 2011). centrosome Rhee, et maturation the (Barr on centrosome assembly function the the and regulate to during relocate negatively which pericentrin, these could of Plk1 All promote cilia. activate primary ciliary entry. that thereby of and formation mitotic delays activity imply before to activity deacetylase evidences disassembly bind its kinase cilia induce its could primary to of Plk1 HDAC6 inhibition dramatically Furthermore, depletion would and or resorption. cilia, phase primary Plk1 G0 of percentage at of and cells length the NIH3T3 of reduce deacetylate in overexpression al., as Plk1 disassembly, et responsible cilia active to also primary Pugacheva is of activity process 2002; kinase the Plk1 for HDAC6 that al., here found et We 2007). activating cell (Hubbert of tubulin regulators polymerized and key by resorption the cilia of primary phosphorylating inducing one in work A, participates Recent Aurora proliferation, be events. in that two to curious the identified were seem links has We that regulation component(s) other. the each cycle finding with cell correlated and yet independent dynamics cilia Primary Discussion eet(aantson.Peiu oksget htPM is PCM1 formation that ciliary suggests to work lead Previous would shown). not PCM1 (data of defect depletion the Plk1, l1i n fmn rtis nldn uoaA D1and CDK1 A, Aurora including proteins, many of one is Plk1 c TR o ulaino irtbls Unlike microtubules. of nucleation for -TuRC l1rgltspiaycladssebybfr ioi 1363 mitosis before disassembly cilia primary regulates Plk1 eodr nioisfr1hu tro eprtr.Frpiaycilia primary For at temperature. methanol with room permeabilized at and fixed hour were 1 2 cells the for immunofluorescence, antibodies secondary ea E23 n I33clswr utrdi MM(GIBCO) DMEM in cultured were cells NIH3T3 and HEK293T synchronization HeLa, and transfection culture, Cell Methods and Materials el ncvrlp eebifywse nPS n ie ih4% with fixed and at BSA) 3% on PBS, containing in minutes PBS washed 5 in for (diluted in being X-100 antibodies 4 Triton primary After with 0.2% washed incubated by temperature. and permeabilized room ice, were briefly cells at times, three were minutes PBS 15 coverslips for paraformaldehyde on Cells microscopy Immunofluorescence Plk1 type full-length wild of The construction mutation the like construct. point way a expression and PCM1- in truncated subcloned including were The PCM1-F2T703A, PCM1, assay. of for blotting with tools mutants western mouse powerful were immunizing and antibodies by also Both immunostaining PCM1 we (1665–2024). human and fragment 2002), C-terminal against Merdes, PCM1 antibody and polyclonal mouse of (Dammermann antibody the The Merdes and Dr raised Plk1. gene from The against gifts Millipore. were antibody from PCM1 purchased polyclonal was antibody the the Plk1 with obtain rabbit polyclonal pGEX-4T-1 immunized to (H538A/ we or protein protein, pET28a Plk12A Plk1 recombinant H538A/ His-tagged p-CMV-myc, Plk1T210A, the (358-end, p-EGFPC2, purifying into 1997), After Plk1PBD2A vectors. cloned and Erikson, then (358-end) were and Plk1PBD K540A) (Zhang (constitutively (Lee (1–400), Plk1T210D previously Plk1) Plk1 mutant), described dead K540A), of as (kinase form Plk1K82R obtained the active were and antibody 2009), al., and et gene Plk1 preparation Human antibody and construction Plasmid and penicillin U/ml 100 (Hyclone), serum bovine fetal 100 10% with supplemented 156wsprhsdfo xnMdhm L83 rmSlek RO3306 Selleck, inhibitor Sigma. from The from MLN8237 block. roscovitine Medchem, thymidine and the Axon Calbiochem into from from from added released purchased were hours transition. was (Sigma) 5 phase B12536 nocodazole about G1/S ng/ml after of in 100 medium treatment arrested the cells, the were With mitosis-arrested cells 2008). obtain Sigma), al., To et mM, (Chen (2.5 previously with and thymidine described cells double as into confluence 1:20 co-transfected the of were transfection, 80% ratio plasmids shRNA the targeting of about and indicator GFP–H2B the (Invitrogen) or to As 2000 RFP–H2B protocol. Lipofectamine dishes manufacturer’s utilizing to culture plasmids according grew indicated and diameter the use, with before 35-mm transfected day one in coverslips coverslips glass 80% No.1 mm to 22 overnight on planted were cells ciae eietilrPk ntr nue rmr cilia primary induces The HDAC6. turn activating A. and CDK1 in with Aurora interacting by by by Plk1 the disassembly matrix activated pericentriolar During pericentriolar then activated to and entry. recruited PCM1 mitotic regulating phosphorylated is before mechanism Plk1 a disassembly process, suggest co-workers and cilia results nephrocystin- and our of (Dvl2) primary together, phosphorylation Schermer 2 the Taken induces 1. and Dishevelled Plk1 that with disassembly; showed interacts cilia co-workers (Lee and Plk1 promotes Lee disassembly 2012). that cilia al., kinase primary et showed the Seeger-Nukpezah for that 2012; required al., showed resorption is et and ciliary Plk1 manuscript, online of induce this published activity of to were preparation reports Plk1 the cells, two During recruit entry. proliferating to mitotic in before needed whereas is Plk1; cilia primary PCM1 of for state independent steady and assembly cycle maintains cell PCM1 exit in quiescence, cells role enter When dual dynamics: play 2008; may cilia al., PCM1 primary that et regulating with indicating (Kim associating 2007), ciliogenesis al., by et the Nachury cilia during primary BBSome of and CEP290 formation the in important eefo im.Alaia xeiet eepromdacrigt approved to according performed were anti- experiments and animal guidelines. (p-10Ser) All H3 Sigma. from anti-Histone from were rabbit were PCM1 both and and B1 anti-cyclin Cruz Rabbit Santa Abgent. from was Seoul anti-HDAC6 Rabbit Sciences, against Flag, Biological antibodies GFP, of monoclonal Myc, Mouse tubulin, (Department Korea). South Rhee Seoul, Kunsoo University, from National gifts were cDNA ˚ vrih.Teclswr hnwse nPStretmsadicbtdwith incubated and times three PBS in washed then were cells The overnight. C 20 m ˚ ietybfr en nuae ihteatbde.Cvrlp were Coverslips antibodies. the with incubated being before directly C /lsrpoyi t37 at streptomycin g/ml , 0 ofune o rnfcin h el eegonon grown were cells the transfection, For confluence. 90% a tblnadaey-uui eeprhsdfo Sigma. from purchased were acetyl-tubulin and -tubulin ˚ Cat5%CO D ,PM-1 C1F,PM-3and PCM1-F3 PCM1-F2, PCM1-F1, C, PCNTB 2 topee o immunofluorescence, For atmosphere. , Plk CA -PCNTB b , Atnantibodies -Actin Plk1 KD -PCNTB c - Journal of Cell Science lotakD.Kno hefrpoiigu with us providing for Rhee Kunsoo Dr. thank also PCNTB iueitrasb olnpH2CDcmr,addfeetZscin then sections Z different and suite. camera, SoftWorx CCD by HQ2 projected CoolSnap were a by intervals minute ik tro eprtr o or fe ia ahi TS h itrwas filter the TTBS, in films. wash X-ray and final chemiluminescence After enhanced hour. by 1 visualization nonfat for for 3% temperature with developed TTBS room in at 1:5,000 diluted milk) (Jackson, antibody secondary conjugated .. ..adQC ocie h rjc n designed and project the Q.C., G.W., conceived experiments. performed Q.C. Q.C. and and G.W. experiments. G.W. C.Z., contributions Author for Merdes Andreas Dr thank We Acknowledgements chamber heated a within stage and sample hours heated 24 a cell for onto live RFP–H2B placed for (37 and was used GFP–Plk1 dish were culture with RFP–H2B the transfected and were GFP–Plk1 Cells with imaging. 5 transfected cells NIH3T3 was imaging cell Live PCM1 against oligonucleotide the of sequence The interference RNA at then overnight and milk) nonfat hour, 0.3% 3% 1 the and with for PCM1), TTBS NaCl temperature for in mM room 1:1,000 hours 4 500 at (diluted 3 7.4], antibody milk as 20% [pH with non-fat long Tris-HCl and probed 3% as mM glycine containing mA (20 20) 350 mM TTBS onto (while Tween 192 in V transferred blocked Tris, 100 was were mM at filter (25 hour samples 1 buffer protein for transfer the methanol) in SDS-PAGE, filters 10% nitrocellulose on resolved After immunoblotting and electrophoresis Gel in MgCl Biolabs) mM England 10 (New 7.5, CDK1 pH ng Tris 200 mM with 50 incubated were proteins F2T703A For vitro In mM 1 NaF, mM 8.0, 5 pH NP-40, Tris-HCl 0.5% mM EDTA, (20 mM buffer 0.5 lysis EGTA, in mM Na lysed 2 NaCl, and mM harvested 150 were immunoprecipitation cells and assay HeLa pull-down protein fusion GST 1 with added Mowiol with mounted 1364 ebr fti aoaoyfrhlflcmet n critical and comments helpful submission. to for prior paper laboratory the of this discussion of members a and microscope inverted IX-71 Olympus an 100 with equipped Precision) (Applied sdsrbdb etigadclege Dsan ta. 2005). al., et (Destaing colleagues and Destaing by described as 1 2009). autoradiography al., subsequently et and SDS-PAGE (Zhang by analyzed were samples and buffer, mrhm t30 at Amersham) ihHL ellstsa 4 at lysates cell HeLa 5 with 15 assay, and the proteins pull-down fused following GST protein or fusion GST by Healthcare) GST in For (GE expressed protocol. beads manufacturer’s were 4B proteins glutathione–Sepharose fusion using GST purified al., and et (Zhang previously described GST as supernatant the 2009). obtain to minutes 15 for g 15,000 G elhae eeaddadtemxue eerttda 4 at rotated were mixtures the and added were Healthcare) (GE ihsadr ICadTIC hdmn n AIfle eso ZEISS a CCD on cool sets with filter captured DAPI software. were image and Images Axvert rhodamine microscope. and TRITC, immunofluorescence M and 200 FITC standard with UCAGCUUCGUGAUUCUCAG-3 ufr o muorcptto sa,tespraatwsicbtdwt the with incubated was 4 at supernatant hour 1 the for antibodies assay, indicated immunoprecipitation sample gel For in suspended then buffer. and centrifugation brief by collected being before buffer ial,rslso ohteasy eeaaye ywsenblotting. western by analyzed were assays buffer. the sample both gel of in suspended results Finally, and harvested and washed were beads the incubation, .5mim [ mCi/ml 0.25 -ue-l1wr rnfce noHL rNHT el ihLipofectamine with cells NIH3T3 The or vector. and HeLa retro p-Super-PCM1 (Invitrogen). into p-Super The 2000 transfected Company. into Invitrogen were ligated by p-Super-Plk1 and sequenced were annealed clones then positive Company, Sunbio by CGGCACCGUGCAGAUUAACUU-3 ˚ .Temmrnswr hnbotdwt osrds eoiae(HRP)- peroxidase horseradish with blotted then were membranes The C. m For 3 6 ˚ lgHA6ado F–l1pamd,adtesbeun sa a done was assay subsequent the and plasmids, GFP–Plk1 and/or Flag–HDAC6 g VO ) ieiaigwspromduigaDlaiinlv eliaigsystem imaging cell live DeltaVision a using performed was Live-imaging C). nvitro in 14 i betv.Iae eecpue ih20m xouetmsi 10- in times exposure ms 200 with captured were Images objective. oil /1.40 nvitro in 4 MPS n 500 and PMSF mM 1 , rti hshrlto sa n uui ectls assay deacetylase tubulin and assay phosphorylation protein and ora fCl cec 2 (6) 126 Science Cell of Journal rti hshrlto sa,2 assay, phosphorylation protein c uui ectls sa,HK9Tclswr rnfce with transfected were cells HEK293T assay, deacetylase tubulin - 32 ˚ o 0mnts ecin eesopdb digglsample gel adding by stopped were Reactions minutes. 30 for C ]APad1 and ATP P] Plk1 KD ˚ o or.Tebaswr ahdfv ie ihlysis with times five washed were beads The hours. 3 for C -PCNTB 6 okal o 0mntso c n etiue at centrifuged and ice on minutes 30 for cocktail) m ˚ .Ffenmcoieso rti rGSepharose G or A protein of microliters Fifteen C. m lttin–ehrs Bbaswr incubated were beads glutathione–Sepharose 4B l 9 2 i[ Ci n h euneaantPk a 5 was Plk1 against sequence the and ; MET,5m T,10m ATP, mM 100 DTT, mM 5 EGTA, mM 2 , m /lDP o N tiig n analyzed and staining, DNA for DAPI g/ml 9 h lgncetdswr synthesized were oligonucleotides The . DA eakoldeother acknowledge We cDNA. c - 32 PCM1 ]AP(0mim,600Ci/mmol, 6,000 mCi/ml, (10 ATP P] m gofGST-taggedPCM1F2and DAadatbd.We antibody. and cDNA .coli E. ˚ o or.After hours. 2 for C PCNTB tanB2 and BL21 strain m gofsoluble , Plk CA 9 9 - - - e . rn,C . adrnd na .adTa,L H. L. Tsai, and F. Anda, de Calderon L., C. Frank, X., Ge, ot,V . ers .L n ifr .R. S. Hilfer, and L. R. Searls, G., J. V. G. Fonte, Pazour, and E. K. Fogarty, A., R. Tuft, A., J. Follit, ers .K,Mli,S .adL,C C. C. Li, and C. S. Maloid, K., D. Ferris, la .E,Cnly .C n af,M B. M. Yaffe, and C. L. Cantley, E., A. Elia, aia . a,P . uo . nehr,C,Ihzk,K,Kb,A,Tuia S., Tsukita, A., Kubo, K., Ishizuka, C., Engelhard, and K., Kubo, F. L., J. P. Bazan, Tan, P., A., S. Kamiya, Gygi, M., Aguiar, S., Schulz, T., Shida, R., S. White, H., Jin, etig . atl . iqi,B,Caae,A,Kohi,S,Oy .adJurdic, and S. Ory, S., Khochbin, A., Chabadel, B., Gilquin, F., Saltel, O., Destaing, G. Guarguaglini, and P. Lavia, H. M., S. Luca, Kim, and De M. Strnad, L., Havlicek, L., Meijer, S., Leclerc, F., W. Azevedo, De A. Merdes, and A. Dammermann, siaa .adMrhl,W F. W. Marshall, and H. Ishikawa, A. Merdes, and M. Wright, I., Callebaut, I., Bazin, H., M. Remy, L., Haren, aegi . ar .A n ig .A. E. Nigg, and A. F. Barr, M., Casenghi, Ko I., P. Duncan, U., Weinhart, P., K. Meraldi, B. M., Casenghi, Yoder, and J. C. Haycraft, M. Bornens, K., A. O’Connor, F., N. Berbari, E. Sillje W. A., F. Zimmer, Barr, and L. Bao, R., Balczon, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.114918/-/DC1 online available material Supplementary 31030044 Natural 30900726, C.Z.]. National numbers and to the [grant 90913021 Research China and and Basic of C.Z.]; China Foundation of Key to Technology Science 2010CB833705 State and Science number the of [grant by Ministry of supported Plan Development was work This Funding hn . hn,X,Jag . lre .R n hn,C. Zhang, and R. P. 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