Journal of Cell Science ioiHasegawa Hitoki II furrowing myosin cytokinetic in and SVIL activation PLK1-phosphorylated of role The Article Research ioi hs,PK hshrltstecnrsm-and centrosome- the early the phosphorylates In 2003b). PLK1 al., et Elia phase, the 2003a; at al., is mitotic et phosphorylated structures (Elia and subcellular sites PBD different specific 2004). of al., the binding et by to (Barr mediated PLK1 mitosis of of regulates stages Localization and multiple midbody, of the progression and the spindle centrosomes, central is as the such PLK1 kinetochores, structures, sequences. subcellular different phosphorylated to with localized associates that domain polo-box C-terminal (PBD) the and domain kinase 2008). N-terminal the Peters, and and Taylor furrow, 2008; cleavage al., et of (Salaun ingression abscission and formation microtubules/kinetochore mitosis interactions, formation, during spindle bipolar kinases structures regulate subcellular These and the kinases. to Aurora-B 1 localize and kinase dynamically Aurora-A polo-like the as such and kinases, (PLK1) mitotic processes by these of phosphorylation regulatory progression is major accurate the the multi- ensure of by to One mechanisms abscised cytokinesis. is complete are ring to cells the complexes complete, daughter is furrow and furrowing cleavage disassembled, the the 2007; of Once ingression 2005). Gruneberg, activity the plasma (Matsumura, motor drive and the myosin actomyosin-based that by (Barr forces of ring generates the plane face an of Constriction inner division of 2009). and the Glotzer, the on separation at organized, assembled membrane the is are the ring physically called After contractile that spindle, microtubules, division cells. antiparallel central cell of daughter of bundles , step two final disconnects the is Cytokinesis Introduction words: Key inhibited the in S238A-SVIL, regions mutant, myosin-II-binding site and actin/myosin-II- actin- phosphorylation an both PLK1 has that SVIL a show furrowing. of we aberrant Expression of induced report, of localization and PRC1. mitosis. the this cortex Expression promote with regulates of equatorial In can N-terminus. PLK1 association the which furrow. stages at how SVIL, and cleavage activation of multiple known spindle Ser238 II the not phosphorylates of myosin central of still PLK1 progression the PLK1. is ingression of to it regulates and substrate performed, a SVIL cortex that is been (SVIL), equatorial kinase have supervillin the division protein, serine/threonine binding at cell conserved during activation functions widely II PLK1 myosin a about studies is extensive (PLK1) Although 1 kinase Polo-like Summary 10.1242/jcs.124818 doi: 3627–3637 126, ß Science Cell of Journal 2013 May 18 Accepted ( correspondence for *Author 2 1 oeua ikbtentecnrlsideadtecnrciern ocodnt h ciaino ysnI o h nrsino the of ingression the for II myosin of a activation as the SVIL coordinate phosphorylated to of role ring possible contractile a the indicates and study spindle Our furrow. furrowing. central cleavage in the defects between caused link and molecular activation II myosin reduced region), tuh Natsume Atsushi eateto ersrey aoaUiest,Gaut colo eiie 5Tuua-h,Soak,Ngy,Jpn 466-8550 Japan, Nagoya, 466-8550 Showa-ku, Japan, Tsurumai-cho, Nagoya, 65 Showa-ku, Medicine, Tsurumai-cho, of 65 School Medicine, Graduate of University, School Nagoya Graduate Neurosurgery, University, of Nagoya Department Biology, Cancer of Division 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. L1i osre nawd ag fseisadi opsdof composed is and species of range wide a in conserved is PLK1 yoiei,Cnrlside L1 VL Phosphorylation SVIL, PLK1, spindle, Central Cytokinesis, 1 2 [email protected] ohnr Hyodo Toshinori , ohhk Wakabayashi Toshihiko , D y-VL(eee ftemoi-Ibnigrgo) u o of not but region), myosin-II-binding the of (deleted Myo-SVIL ) 1 r Asano Eri , 2 ihnr Hamaguchi Michinari , 1 aooIto Satoko , lhuhteefnig aeavne u current our PLK1 advanced by seems mediated networks there regulatory have cytokinesis, complicated controls more the 2010). be PLK1 findings to Barr, phosphorylating how of and by these understanding In PLK1 (Bastos abscission that furrowing. Cep55 showed Although of inhibited report protein, timing recent and F- midbody-localized a the and cortex furrowing, II mediates the equatorial myosin to as the MgcRacGAP well addition at as of RhoA Yu mutant actin of 2009; furrowing non-phosphorylated localization al., initiate a suppressed et to the Wolfe of cortex 2009; Expression equatorial al., and then et the which spindle, (Burkard at Neef phosphorylates central PLK1, 2003; PLK1 the RhoA al., spindle, at activates et central ECT2 of Neef the recruit to 2004; at PLK1 MgcRacGAP al., Once targets recruit 2007). et al., to (Liu et spindle are PBD central with the to associate PRC1, MKLP1, 2007; proteins al., including al., phosphorylated and et proteins, et Santamaria 2007; (Brennan spindle-localized MKLP2 al., et cytokinesis Central Petronczki of 2007). 2007; al., initiation et the essential however, the an Burkard mitosis; demonstrated hampered specific for and of the anaphase role previously stage enabled at inhibitors PLK1 later had of chemical the inactivation of in mitosis discovery in recent role plays the of PLK1 fashion its that of phase circular role spindle, elucidation critical a early monopolar The in 2008). the a al., arranged the a et assemble chromosomes establish to (Petronczki, to instead by chromosomes fail and cells surrounded of PLK1, spindle of separation absence bipolar the the the In poles. of and opposite formation spindle the regulate mitotic to proteins kinetochore-localized 1 aa Maeda Masao , 1 n aeh Senga Takeshi and 1 ioauKuribayashi Hirokazu , D c-VL(eee factin-binding of (deleted Act-SVIL 1, * c ta. 2005). al., et ¨ce 1 , 3627 Journal of Cell Science ueosctknssrltdpoen n a eurdfor with required associates SVIL was that and shown proteins has cytokinesis-related study 2010; al., numerous al., et recent Takizawa et 2006; A Fang al., 2009; et 2007). al., Takizawa 1997; et al., Crowley et 2003; Pestonjamasp al., et Chen al., 2012; et (Bhuwania degradation spreading, matrix cell and in attachment region, cell–substrate involved C-terminal is and a proteins, / to and N-terminalsimilar regions an myosin-II-binding of consists and SVIL F-actin- cytokinesis. of required completion the was for (SVIL), an that supervillin found protein, we II-binding screen, the this actin/myosin In realize screen. siRNA limited fully a performed to essential cytokinesis. is of substrates mechanisms PLK1 physiological regulatory other the of of elucidation role Thus, cytokinesis. coordinate to 3628 osac o oe L1sbtae seta o yoiei,we cytokinesis, for essential substrates PLK1 novel for search To ora fCl cec 2 (16) 126 Science Cell of Journal n rmtsteatvto fmoi It ud h cleavage Results the plane. guide division the to at II zone restricted myosin the ring of in contractile furrow activation the the and promotes spindle central and a the as connects functions SVIL SVIL that that of show scaffold loading we addition, the In for spindle. central PRC1 the with to association induce to SVIL Ser238 phosphorylates controls PLK1 of that show SVIL we report, this how In unknown. largely However, remain involved mechanisms 2010). molecular the al., and cytokinesis et (Smith cytokinesis dniynvlPK usrtsesnilfrtecmlto of completion the to the for screen essential in siRNA substrates an role PLK1 performed novel crucial We identify cytokinesis. a of plays progression phosphorylation cytokinesis PLK1-mediated compromises SVIL of Depletion r ifrn tgsi anaphase. in stages different are SVIL, for ( el eeeautdfrec xeiet(o hsadalsubsequent all and means this are (for data experiment the was each figures cells 200 for multinucleated and evaluated of performed, were ratio were cells the experiments and independent ( C, Three siRNA#2. in evaluated. SVIL as to treated GFP-SVIL-Res#2 were and cells resistance #1 confer siRNA indicated to SVIL the mutations to using silent immunoblotted has and GFP-SVIL-Res#1 hours lysed antibodies. 72 were and cells siRNAs, the indicated later, the with together antibody. GFP-SVIL-Res#2 anti-SVIL with immunoblotted and ( lysed were cells second the and first the Two ( siRNA. for screen. each performed shows with were graph treatment experiments after The independent cells . multinucleated indicated of the ratio four for screen, the second used the were In genes. siRNAs screen, 88 first different for the used In were evaluated. siRNAs hours was different cells two 72 multinucleated and of siRNAs, ratio with the transfected later, were plates 24-well on cultured i.1 VLi yoiei-soitdprotein. cytokinesis-associated a is SVIL 1. Fig. E C eaclswr ie ihmtao/ctn n immunostained and methanol/acetone with fixed were cells HeLa ) h el eetasetdwt F-VL F-VLRs1 or GFP-SVIL-Res#1, GFP-SVIL, with transfected were cells The ) B h el eetasetdwt iNs n 2huslater, hours 72 and siRNAs, with transfected were cells The ) a tblnadtences(ocs) n1 n2adAna3 and Ana2 Ana1, (Hoechst). nucleus the and -tubulin 6 .. ** s.d.; P , .1 .;ntsignificant). not n.s; 0.01, ( A eacells HeLa ) D The ) Journal of Cell Science ihec iN,ad7 or ae h el eefxdand fixed were cells the later hours 72 and transfected were siRNA, plates each 24-well in with cultured material cells (supplementary HeLa sites S1). consensus Table. whose PLK1 phosphorylation genes the have to 88 similar and selected sites mitosis we in results, phosphorylated their are of on products number Based 2008). large (Dephoure al., mitosis a in et increased identified is phosphorylation has whose study proteins previous A cytokinesis. rnfce el splmnaymtra i.S1E). Fig. GFP- non- material in a (supplementary observed be cells the was transfected to localization appears along similar this because GFP–SVIL but signal metaphase, specific of in localization GFP–SVIL observed was Faint SVIL, spindle S1E). (supplementary endogenous midbody Fig. the at of material and spindle central localization the 1). at the localized (isoform GFP–SVIL to expressed the Similar checked exogenously also of We S1D). localization central Fig. the material to localized (supplementary is of spindle 1 isoform depleted that cells indicating alone, in 4 observed isoform clearly was localization Fig. SVIL material S1C). (supplementary siRNA SVIL observed with transfected not cells were in localizations the specific at These the concentrated 1E). As became (Fig. midbody spindle. SVIL cytokinesis, In central the to the of metaphase. progressed called midzone cells cells, in the dividing of observed of region microtubules the was in accumulated SVIL SVIL antibody. anaphase, of anti-SVIL we localization the cytokinesis, using Diffuse in analysis cytokinesis during function immunofluorescence SVIL SVIL by results of localization into subcellular These the insight cytokinesis. examined 1D). compromises further (Fig. function gain SVIL SVIL of To loss wild-type siRNA-resistant the was that cells with to indicate multinucleated transfected compared of cells ratio SVIL in The reduced 1C). (Fig. significantly siRNA affected not the was SVIL by SVIL, siRNA-resistant of wild-type expression siRNA. expressed the whereas exogenously the and to endogenously ablated effectively resistance both siRNA which SVIL confer that showed SVIL, that analysis Immunoblot siRNA-resistant mutations encoding silent or plasmid contains wild-type a with either together wereGFP-tagged siRNA cells HeLa SVIL experiment. rescue with the S1B), transfected for 1 Fig. isoform used material not we did (supplementary thus alone 4 cells in isoform of multinucleated expressed the depletion isoform the induce and 1 Major by isoform that experiment. was rescue cells depletion, lost confirm HeLa a SVIL To out by were induced carried 1B). was we bands (Fig. cells multinucleated siRNAs both of increase SVIL and of respectively, transfection 4, of bands isoform immunoreactive of two Pro245 Luna, detected after and , and antibody acids 1 The (Fang amino 395 1. isoform cells additional isoform non-muscle are has 4 in which Isoform expressed SVIL, 2013). are of 4, isoforms the isoform two study examine recent that A protein. to the showed knockdown SVIL to siRNAs targeting We the against of cells S1A). efficiency siRNAs antibodies Fig. multinucleated material polyclonal different supplementary of 1A; generated four (Fig. ratio 10% the than all additionalmore found increased of we two (SVIL) and used transfection validation, supervillin further we the for screen, genes that second six these the for In siRNAs 1A). (Fig. ( cells genes six screen, SEC61B of of round first depletion the is In cytokinesis the cells. for multinucleated required induce proteins to of known multinucleated depletion of the number because the cells on We tubulin. analysis for quantitative paclitaxel performed FITC-labeled and Hoechst with stained 2 D n 5 D,wihcrepn oioom1and 1 isoform to correspond which kDa, 250 and kDa 220 , SVIL , UBAP2L nue oeta 0 multinucleated 10% than more induced ) SQSTM1 , CALCOCO1 , TBC1D15 , VLwr rninl xrse iho ihu wild-type without or with expressed of of mutants presence transiently deletion the or were in length SVIL the SVIL full of in HA-tagged shift expression. SVIL mobility PLK1 of the further checked Ser238 To we phosphorylates 2008). cells, PLK1 al., analysis whether et by proteomics (Dephoure confirm phosphorylation the analysis S238 detected spectrometry addition, al. mass In et Dephoure 2003). can by performed 2B). consensus al., (Fig. PLK1 the et in (Nakajima that residue Ser238 threonine ([E/D] demonstrated or sequence site, serine has the phosphorylate phosphorylation evidence one is there Accumulating PLK1 that revealed has Analysis SVIL 2A). of potential sequence (Fig. B1/ acid phosphorylation cyclin amino the the than of for other responsible kinase is mitotic to Cdk1 a SVIL started has that of level thickness suggesting expression the B1 decrease, in cyclin the when increase determine observed clear to was A band band shift. SVIL mobility each electrophoretic of of level the phosphorylated. which thickness are the Phos-tag, and proteins measured when using We shift cycle, investigated mobility the cell was enhance SVIL can the of of lysed mitosis. shift were mobility stages during block thymidine different SVIL double at of by synchronized phosphorylation cells HeLa examined next Ser238 We at SVIL phosphorylates PLK1 oaiaino ioi-eae rtis fPLK1-mediated If the proteins. in mitosis-related roles important of plays kinases localization SVIL mitotic of by localization the Phosphorylation for essential is PLK1 we PLK1, by confirm phosphorylated To directly 2E). an was aa1–345 (Fig. performed SVIL PLK1 S238A, of wild-type not Ser238 of analysis but that presence wild-type, Immunoblot the of an in PLK1. shift SVIL or mobility kinase-dead Wild-type a of or showed presence Ser238. the wild-type in of expressed SVIL was either phosphorylation aa1–345 SVIL aa1–345 of the of shift mutant mobility S238A to the due if tested was by next the We eliminated 2D). to clearly (Fig. was due kinase-dead PLK1 or type was is fragment wild-type SVIL with shift immunoprecipitated with treated the the and together (K82M), expressed PLK1 that was confirm SVIL used any To we show not phosphorylation, 2C). did (Fig. mutants presence deletion the other in shift but SVIL analysis. PLK1, aa1–345 immunoblot wild-type of to shift of mobility subjected the were observed lysates We cell and PLK1, ne faahs otecmlto fcytokinesis of completion the to anaphase S2). Fig. material the (supplementary of from and midbody SVIL and spindle Both central onset mitosis. the to during localized PLK1 were PLK1 and phosphorylated SVIL of for phosphorylates localization antibody directly Ser238 PLK1 at specific that SVIL indicate a results generate these Ser238, to Although failed PLK1. by sites we phosphorylation of additional that be indicates phosphorylation wild-type may S238A-SVIL there of 2G, full-length that also of Fig. to Phosphorylation compared in SVIL. We reduced clearly shown was 2F). As S238A-SVIL (Fig. S238A-SVIL. phosphorylation and the an performed abolished at substitution whereas Wild-type Ser238 PLK1, autoradiography. by to phosphorylated separated was subjected were aa228–256 and mixtures gel reaction SDS-PAGE The by PLK1. from recombinant purified was with SVIL of aa228–256 S238A l popaae h oiiysitidcdb wild- by induced shift mobility The -phosphatase. nvitro in nvitro in 6 nvivo in [S/T] L1popoyae VL3629 SVIL phosphorylates PLK1 W iaeasyuigfl-eghwild-type full-length using assay kinase and 6 iaeasy S-ue idtp or wild-type GST-fused assay. kinase [E/D]; l nvitro in popaae Atge aa1–345 HA-tagged -phosphatase. W yrpoi mn acid) amino hydrophobic a , eas xmndteco- the examined also We . l popaaetreatment -phosphatase .coli E. n mixed and Journal of Cell Science h eta pnl a lal iiihd(i.3) hs results These 3B). at (Fig. diminished localization clearly SVIL was ST4AA-PRC1, the spindle of central the to presence the the localization ST4AA-PRC1 with In of PLK1 3B). overexpression (Fig. the 2007), Consistent by reduced al., was Thr602). spindle et central Ser601, (Neef phosphorylation To finding PLK1 Thr578, 2007). previous the al., for (Ser577, et substituted sites were (Neef alanines spindle which central in ST4AA-PRC1 used by the we PLK1, of the phosphorylated localization at the for inhibit specifically is PLK1 crucial 2002), is of spindle phosphorylation al., the loading central et and of cytokinesis Mollinari during organization 1998; PLK1 the localization. al., SVIL for et of for essential localization (Jiang required protein if was a tested spindle PRC1, next central the We at of 3A). presence (Fig. PLK1 the inhibitor in reduced PLK1 was spindle the central accumulation the however, at proteins anaphase; both in were of the spindle PLK1 without central and or the SVIL with at immunostained. localized released and then were BI2536 Cells inhibitor hour. PLK1 1 presence for the in MG132 released of and treatment in nocodazole arrested by were cells prometaphase HeLa the cytokinesis, during to PLK1 an of activation SVIL in the inhibit specifically result To anaphase. of should in activity SVIL of localization localization PLK1 aberrant of the suppression the for spindle, central required is phosphorylation 3630 ora fCl cec 2 (16) 126 Science Cell of Journal h erimn fSI otecnrlspindle. central the to SVIL of for recruitment required is the localization, as well as activation, PLK1 that indicate oehrwt APC n hnimnpeiiae ihanti- with immunoprecipitated cells then HEK293T the and in deletion HA–PRC1 expressed with GFP-tagged transiently together interaction. were the SVIL for of mutants responsible was SVIL antibody of anti-GFP region which examined using next We immunoprecipitation S3B). Fig. material (supplementary was cells by association the detected specific in The proteins also S3B). two Fig. the material HA– of (supplementary of association anti-HA presence the the indicating by in immunoprecipitated PRC1, only were precipitated was lysates GFP–SVIL cell antibody. the and HEK293T in cells, HA–PRC1 proteins, without two or with these expressed between was association GFP–SVIL the confirm To S3A). material (supplementary Fig. cytokinesis from of completion colocalize the PRC1 a to and anaphase as SVIL PRC1 partner. identified binding we analysis, these SVIL extensive candidate central of of the result to interaction a SVIL of As recruitment direct spindle. the for the required were interact SVIL detect directly with that factors not additional that speculated could We proteins. we PLK1, dependenton was spindle central the to localization SVIL the Although PRC1 with associates SVIL niae hshrltdSVIL. phosphorylated an indicates to anti- subjected with and immunoprecipitated antibody and HA cells HEK293T in expressed erddfrso S–VL(a2–5) n h arrowhead the ( and GST. (aa228–256), indicates GST–SVIL indicate of asterisks forms The degraded lower (aa228–256). the GST–SVIL in indicates the arrow panel indicates The panel (aa228–256). upper GST-SVIL the phosphorylated recombinant in the arrow of The staining proteins. panel (CBB) lower Blue the Coomassie and shows the proteins, shows recombinant panel of upper phosphorylation The SDS-PAGE. by separated n h oiiysitwseaie yimnbotn.( immunoblotting. by examined was shift mobility without the or and with The treated antibody. were anti-HA immunoprecipitates (K82M) with kinase-dead immunoprecipitated or and wild-type PLK1 with together by cells examined HEK293T was shift with mobility ( cells the immunoblotting. HEK293T and in GFP–PLK1, expressed without were or SVIL of mutants deletion ( phosphorylation. PLK1 of sequence L1fr3 iue t30 at minutes with 30 incubated for was PLK1 S238A-SVIL or wild-type ( of immunoblotting. 228–256 the by residues and examined PLK1, was kinase-dead shift in or mobility expressed wild-type was with S238A-SVIL cells or HEK293T wild-type of aa1–345 tagged AEwt hstg h rp hw h eaietikesof thickness ( relative SVIL. the of SDS- shows band by graph each separated The were Phos-tag. the lysates with at cell PAGE lysed The and points. block time thymidine indicated double SVIL. by of synchronized Ser238 were phosphorylates PLK1 2. Fig. G D Atge ullnt VLwstransiently was SVIL full-length HA-tagged ) Atge a–4 VLwsepesdin expressed was SVIL aa1–345 HA-tagged ) B e28o VLi nteconsensus the in is SVIL of Ser238 ) ˚ ntepeec f[ of presence the in C nvitro in C Atge ullnt and full-length HA-tagged ) iaeasy h arrow The assay. kinase ( l c A F -phosphatase, - 32 GST-fused ) h cells The ) ]T and P]ATP E HA- ) Journal of Cell Science hshrltdSI.W is xrse ASI (wild-type HA–SVIL expressed first this We and that PRC1 To SVIL. of speculate PRC1. association phosphorylated the with to examined the we association hypothesis, us for this its with confirm led affect phosphorylation SVIL may spindle SVIL event central of phosphorylation of the interaction requirement at the localization the indicating and findings PRC1, above The PRC1 with Ser238 interaction at regulates SVIL of phosphorylation PLK1-mediated mitosis. during SVIL with complex a in were enter PLK1 to and started cells the anti-SVIL B1, that mitosis cyclin indicates an of PLK1 Expression and PLK1. with AuroraB and PRC1, immunoprecipitated PRC1 for were blotted and lysates antibody cell points. time different by The at of arrested lysed association and were released block, cells the thymidine double HeLa examined mitosis. we during Finally, proteins endogenous 4C). these (Fig. of interaction proteins direct HA.two the for showed probe clearly to assay immunoblot pull-down to The subjected were beads beads, the agarose then glutathione and to bound PRC1 aa1–200 GST-fused with HA-tagged by performed. produced was was assay SVIL pull-down aa676–1009 a proteins, PLK1- al., two of the interaction direct for these et the confirm site microtubule- To critical N-terminus, Mollinari 4B). the 2009; (Fig. was SVIL a PRC1 with interaction al., of the aa1–200 et and that found (Liu We C-terminus, in 2002). between a in the has region site PRC1 associated in SVIL. targeting sites to was binding phosphorylation spindle the SVIL for of required central aa676–1009 PRC1 the determined 4A, of also Fig. We region PRC1. with in interaction the shown for responsible As antibody. HA , or fe h ees.A hw nFg D ohPRC1 both 4D, Fig. in shown As release. the after hours 8 nvitro in rnlto n mixed and translation Fg D.T ute ofr h oaiain emeasured in shown we diminished As localization, anaphase. significantly in the cells was the confirm across of spindle further intensity accumulation fluorescence central To the 5D). the whereas of (Fig. spindle, localization to central the S238A the examined were to S238D-SVIL next and localized wild-type We Both S238D-SVIL. 5C). and wild- S238A- (Fig. to levels PRC1 similar at type SVIL ST4AA-PRC1 with Both interacted examined. ST4DD-PRC1 was (ST4DD-PRC1),and SVIL acid with interaction aspartic the and or PLK1-mediated (ST4AA-PRC1) if alanine with tested either replaced were proteins. PRC1 also two of of sites binding We phosphorylation PLK1-mediated the affect PRC1. could PRC1 to of phosphorylation SVIL the of enhances Ser238 binding were at Immunoblot phosphorylation 5B). that (Fig. indicate lysates S238A-SVIL results and These S238D- wild-type antibody. to of cell compared binding PRC1 increased to the SVIL revealed anti-HA precipitates the and of analysis HeLa with GFP–PRC1, in with expressed immunoprecipitated were HA-tagged together examined. S238D-SVIL was cells and acid with aspartic SVIL S238A- to to wild-type, PRC1 Ser238 of at binding mutation the co- treatment.a result, to PLK1 this of Ser238 validate by precipitation further amount for PRC1 To alanine in the of increase increased substitution the abolished the significantly but PLK1 phosphorylation PRC1, the precipitated by 5A, to Fig. SVIL in analysis shown of immunoblot As precipitated. to GFP–PRC1. affinity subjected for GFP–PRC1, were probe then HA–SVIL were expressed to precipitates transiently bound The that proteins the cells lysates and HeLa with from mixed were made precipitates by The PLK1 HA–SVIL immunoprecipitation. kinase-dead non-phosphorylated or or phosphorylated wild-type purified either and with together S238A) and elcdwt lnns h rp hw h ai fclswt VLat SVIL with ( cells of spindle ratio central the were the shows Thr602 graph The and alanines. Ser601 with Thr578, replaced indicated Ser577, the ST4AA-PRC, for In immunostained proteins. were and cells methanol/acetone The with released. fixed and nocodazole-arrested were ST4AA-PRC1 i.3 L1i eurdfrtelclzto fSI otecentral 25 the of to SVIL presence of localization the for required spindle. is PLK1 3. Fig. ** ihDS rPK niio o or h el eefxdwith fixed The were proteins. cells indicated The the hour. for 1 immunostained for and inhibitor methanol/acetone PLK1 or DMSO with rp hw h ai fclswt VLa h eta pnl ( spindle central the at SVIL with cells of ratio the shows graph P , .1.( 0.01). ( A ooaoearse eaclswr eesdi the in released were cells HeLa Nocodazole-arrested ) B eaclstasetdwt F-agdwl-yeor wild-type GFP-tagged with transfected cells HeLa ) m fM12fr1hu.Clswr hnincubated then were Cells hour. 1 for MG132 of M L1popoyae VL3631 SVIL phosphorylates PLK1 n 5 0 ** 30, P , 0.01). n 5 30, Journal of Cell Science niae iepit.Tecl yae eeimnpeiiae ihat-VLatbd,adteimnpeiiae eebotdwt h indicat the with blotted were immunoprecipitates the wi and immunoblotting antibody, anti-SVIL to with subjected ( immunoprecipitated were proteins. were precipitates recombinant lysates The of cell beads. The staining The glutathione–agarose Blue antibody. points. to Coomassie time anti-HA bound shows indicated with panel PRC1 immunoprecipitated lower ( of The and 1–200 antibodies. antibody. HA–SVIL residues anti-GFP HA GST-fused with and or together anti-HA GST cells with with HEK293T immunoblotting precipitated ( in to antibodies. expressed subjected indicated were the were with PRC1 immunoprecipitates immunoblotting of to mutants subjected deletion were immunoprecipitates and The antibody. anti-HA with immunoprecipitated F-agdmoi euaoylgtcan(L)and (RLC) chain light lapse time regulatory expressed by constitutively myosin SVIL mammary which human cells, of GFP-tagged used examined MCF10A we absence cells, we furrowing, epithelial the thus, monitor To in furrowing; microscopy. of ingression the in progression furrow functions and critical play initiation proteins the spindle-localized in Central furrow cleavage PLK1-mediated the zone confine limited to required that is SVIL SVIL spindle. of central the association at indicate localization SVIL the stabilizes and promotes PRC1 results with Ser238 at clearly These phosphorylation was spindle central the to disrupted. S238A of accumulation 5E, Fig. PRC1. with associates SVIL 4. Fig. 3632 ora fCl cec 2 (16) 126 Science Cell of Journal ( A F-agdfl-eghaddlto uat fSI eeepesdi E23 el oehrwt APC and HA–PRC1 with together cells HEK293T in expressed were SVIL of mutants deletion and full-length GFP-tagged ) otxso h w eaaigduhe el eeldaclear a polar revealed the between cells length daughter the separating to two length the furrow during of the of cortexes broadened of Measurement the 6A). ratio significantly In (Fig. of the 6A). cells became (Fig. SVIL-knockdown region in ingression furrow narrow ingression of cells the the completion the the to contrast, MCF10A until cells, restricted plane siRNA-transfected in was division control furrow spindle In cleavage and control noticed cells. between central we process furrowing SVIL-depleted analysis, the in this the difference In marked a S4B). to Fig. SVIL material (supplementary depleted localized SVIL addition, efficiently In is S4A). transfection Fig. material siRNA (supplementary only expression and expressed 1 cells MCF10A isoform (H2B). 2B histone DsRed-tagged D obetyiiebokdHL el eerlae n ye tthe at lysed and released were cells HeLa thymidine-blocked Double ) C ) nvitro In rnltdH–VL(a7–09 a affinity was (aa676–1009) HA–SVIL translated B F-agdfull-length GFP-tagged ) dantibodies. ed hanti- th Journal of Cell Science VLt ofn h urwi h etitdzn ttedivision the at zone for restricted the required in is furrow plane. II the confine myosin to PLK1-mediated with SVIL that interaction GFP– indicate results and in These observed the phosphorylation 6F). clearly cells, of (Fig. was control cells absence to furrow SVIL similar the the was of in that broadening observed ingression normal was GFP– showed Although division SVIL. cell endogenous and 6E), (Fig. either GFP– expressed constitutively material GFP– that (supplementary siRNA-resistant cells respectively MCF10A II, binding S5B,C). for myosin Fig. critical and were (supplementary F-actin aa1–174 and cells to aa346–675 demonstrated of the analysis regions Immunoprecipitation in that but S5A). actin, actin, Fig. polymerized non-polymerized material with II associate with myosin to the and able not confirmed F-actin was to first SVIL binding cells. We for We in furrow. responsible 2003). the S2 SVIL required of of al., the was region ingression et F-actin and proper or (Chen II the F-actin chain myosin and for with heavy with association ring, SVIL II interact if contractile myosin tested to the the and reported of of domain been F-actin components has major 6C,D). SVIL (Fig. are II cells wild-type myosin in became S238D-SVIL-expressing not furrow but division and cells The cell S238A-SVIL-expressing microscopy. and in time-lapse elongated protein, by endogenous monitored deplete was to with transfected siRNA were SVIL GFP-tagged Cells S238D-SVIL. siRNA-resistant and MCF10A S238A- expressed generated wild-type, we constitutively region, that limited the cells in To to furrow required 6B). was the (Fig. Ser238 confine of cells phosphorylation SVIL-knockdown the in whether determine furrow the of widening D y-VL(eee fa114 eegenerated were aa1–174) of (deleted Myo-SVIL D c-VL(eee fa3665 or aa346–675) of (deleted Act-SVIL D c-VLepesn cells Act-SVIL-expressing D Myo- noeosSI a upesdb iN rnfcin Both transfection. siRNA by suppressed GFP– was SVIL endogenous with siRNA-resistant expressed association constitutively cleavage GFP-tagged SVIL that the at cells activation if SVIL II HeLa myosin the tested furrow. the for by next required affected was II We not myosin clearly was 7B). Anillin, (Fig. and was depletion F-actin control as contractile furrow the the such of components ring, to other cleavage of Localization compared 7B). (Fig. cells the cells of SVIL-knockdown Accumulation at in anaphase. of diminished in activation RLC furrow the cleavage during phosphorylated affected the phosphorylation at depletion II analysis SVIL RLC myosin immunofluorescence whether and suggests with examine performed which to delayed then associated 7A), We was is (Fig. cytokinesis. phosphorylation cells SVIL SVIL-depleted that RLC in contrast, control prolonged in mitosis In after of hour progression the cells. 1 with with increased declined and was transfected for release RLC the during of cells specific different Phosphorylation at activation points. HeLa lysed antibody time and II Ser19. released nocodazole-arrested, an were at myosin siRNAs using RLC global immunoblot phosphorylated examined by first myosin cytokinesis of al., We SVIL- activity in et reduced furrowing with II. Komatsu associated aberrant 2006; was the cells Spudich, that knockdown speculated and is we ring Dean furrow thus, contractile 2009; 2000); cleavage the al., of the constriction et at of (Asano II promotion myosin (Takizawa the contraction of for for required Activation N- II 2007). myosin the al., activate phosphorylation to of et the Ser19 induce association at can RLC II the of myosin with that SVIL reported of terminus have furrow cleavage studies the at Previous II myosin of activation controls SVIL D y-VLadGFP– and Myo-SVIL D muorcpttswr lte ihat-Aadanti-GFP and anti-HA with blotted were and The immunoprecipitates lysed antibody. were anti-HA cells with The immunoprecipitated HA–SVIL. with in cells expressed HEK293T transiently were ST4DD-PRC1 ( ST4AA-, relative indicated. The are antibody. intensities anti-GFP band and anti-HA were with immunoprecipitates blotted The immunoprecipitated antibody. and anti-HA lysed with with were cells cells HEK293T The in GFP–PRC1. expressed transiently were SVIL intensities ( relative indicated. the are and software, were ImageJ intensities using Band measured and analysis. precipitated immunoblot affinity to were subjected HA–SVIL and to GFP–PRC1, bound of expressed proteins lysates transiently with that mixed cells were HeLa precipitates anti- The by antibody. precipitated HA affinity was non- HA–SVIL or phosphorylated or phosphorylated wild-type and GFP-tagged PLK1, with kinase-dead cells HEK293T in PRC1. expressed and the SVIL regulates of Ser238 interaction of Phosphorylation 5. Fig. el,a niae ytedse ht ie nD were D, in ( lines software white ImageJ dashed using the measured by indicated as cells, and GFP ( for PRC1. immunostained and methanol/acetone fixed were with S238D-SVIL and S238A- wild-type, GFP-tagged ( antibodies. c-VLand Act-SVIL L1popoyae VL3633 SVIL phosphorylates PLK1 E h loecneitniisars h anaphase the across intensities fluorescence The ) D eaclsta osiuieyexpressed constitutively that cells HeLa ) B Atge idtp,S3A n S238D- and S238A- wild-type, HA-tagged ) D D c-VLlclzdt h central the to localized Act-SVIL y-VLwr salse,and established, were Myo-SVIL C F-agdwild-type, GFP-tagged ) ( n A 5 ASI was HA–SVIL ) 10). Journal of Cell Science VLwsdpee ysRAtaseto.Tertoo urwttlwseautdi ahcl n lte ntegah h vrg aisaeindicate are ratios average The graph. the on plotted and cell each in evaluated was furrow/total of ratio The (** transfection. bars siRNA horizontal by depleted was SVIL D rnfce ihSI iN odpeeedgnu rti,adcl iiinwsmntrdb ielpemcocp.( microscopy. time-lapse (** indicated by are monitored ratios average was The division graph. cell the on and plotted protein, and endogenous cell each deplete for to evaluated siRNA SVIL with transfected itnebtenteplrcree ttl a esrd h ai ffro/oa fec elwspotd n h vrg aisaeidctdi t in indicated are ratios average the and plotted, was cell each of furrow/total of ratio The measured. was (total) cortexes (** polar the between distance xrsigclsbtnti GFP– GFP– in of in not Localization but suppressed 7C). cells (Fig. was expressing SVIL RLC wild-type phosphorylated to similar spindle ( microcopy. time-lapse by monitored zone. was restricted division the cell in later, furrow hours the Twenty-four confine siRNAs. with to transfected required were is SVIL 6. Fig. 3634 pnl yPK hshrlto rmtsteacmlto of accumulation central the the promotes at phosphorylation localized PLK1 SVIL that by suggest spindle in results material These (supplementary RLC prolonged S7). of and Fig. delayed phosphorylation was cells during 7A), S238A (Fig. RLC the to cells of Similar analysis. immunoblot SVIL-knockdown phosphorylation by cells examined S238A in also cells cytokinesis S238D-expressing We and 7D). wild-type the in (Fig. to diminished S6A,B), compared clearly Fig. cells was S238A material RLC RLC. (supplementary phosphorylated of II phosphorylated F- accumulation myosin with associate siRNA and could for S238D-SVIL actin by and immunostained S238A- SVIL both Although endogenous and of transfection depleted were cells HeLa S238D-SVIL activation. and S238A- II wild-type, myosin GFP-tagged siRNA-resistant for expressed that required of was phosphorylation PLK1 the by whether SVIL determined we Finally, 7C). (Fig. y-VL ( Ayo-SVIL. P , .1.( 0.01). ora fCl cec 2 (16) 126 Science Cell of Journal C F C1Aclsta osiuieyepesdsRArssatGPtge wild-type, GFP-tagged siRNA-resistant expressed constitutively that cells MCF10A ) C1Aclsta osiuieyepesdsRArssatGPtge idtp,S3A n 28-VLwr salse.Teclswere cells The established. were S238D-SVIL and S238A- wild-type, GFP-tagged siRNA-resistant expressed constitutively that cells MCF10A ) P , .1 .;ntsignificant). not n.s; 0.01, D c-VLepesn cells Act-SVIL-expressing D Myo-SVIL- ( A C1Aclsta osiuieyepesdbt F–L n DsRed–H2B and GFP–RLC both expressed constitutively that cells MCF10A ) o h nrsino h urwta a eitdb PLK1 by mediated was that furrow al., the et mechanism of (Smith regulatory ingression novel cytokinesis a the of presented we for completion study, the this ingression In the for 2010). for furrow required the was SVIL, of MgcRacGAP protein, how scaffold but showed large study furrowing, al., Recent the of of elusive. furrow remains initiation et ingression the maintains continuous PLK1 phosphorylation in (Santamaria activation the started RhoA induces PLK1-mediated for has also constriction 2007). PLK1 but once chemical the furrowing ingression for small essential of only using not is initiation studies activity PLK1 Recent that revealed progression inhibitors division. accurate cell the these ensure at of to proteins structures various subcellular phosphorylates distinct kinetochores and centrosomes, spindle the central to and localized dynamically is PLK1 Discussion furrowing coordinate to cortex equatorial 7E). the (Fig. at II myosin active P , .1 .;ntsgiiat.( significant). not n.s; 0.01, D c-and Act- B D h ai ftefro egh(urw othe to (furrow) length furrow the of ratio The ) E y-VLwr salse,adendogenous and established, were Myo-SVIL ceai ersnainof representation Schematic ) D h ai ffro/oa was furrow/total of ratio The ) D c-VLand Act-SVIL egraph he dby Journal of Cell Science laaefurrow. cleavage aaomleyeadimnsand oeta h eta pnl oaiaino VLi o iil fe %prfradhd iain h grap The fixation. 4% ( paraformaldehyde with cortex 4% fixed equatorial after were visible the cells not at is The RLC SVIL siRNA. phosphorylated of SVIL with localization with spindle cells transfected central of the and ratio that established Note were immunostained. S238D-SVIL and and paraformaldehyde S238A- wild-type, GFP-tagged siRNA-resistant el r ifrn.Tegahsostertoo el ihpopoyae L tteeutra otx( cortex equatorial the at RLC phosphorylated with paraformalde cells and of cells ratio methanol/acetone-fixed the the that shows Note graph immunostained. and The paraformaldehyde different. 4% are or methanol/acetone cells with fixed were cells The iNs eefxdwt %prfradhd n muotie.Tegahsostertoo el ihpopoyae L tteeutra otx( cortex equatorial the at RLC phosphorylated with cells of ratio the shows graph The immunostained. and paraformaldehyde ( 4% with fixed were siRNAs, fmoi Iadfrtecnieeto h urwi the in furrow the activation of confinement the the for for SVIL Our and utilizes furrow. II PLK1 cleavage myosin that the of of indicate broadening and results cortex equatorial SVIL significant the at nonphosphorylatable II induced central myosin the of of activation at the SVIL expression suppressed of Exogenous accumulation the spindle. by for Ser238 required at was SVIL PLK1 of Phosphorylation SVIL. of phosphorylation ( immunoblot. by examined was RLC of phosphorylation the and points, time furrow. indicated cleavage the the at at lysed II were myosin cells of The activation released. the and regulates SVIL 7. Fig. C eaclsta osiuieyepesdsRArssatGPtge wild-type, GFP-tagged siRNA-resistant expressed constitutively that cells HeLa ) n 5 0.( 30). E ceai ersnaino L1mdae ysnI ciaina the at activation II myosin PLK1-mediated of representation Schematic ) ( A eaclstasetdwt oto rSI iN eenocodazole-arrested were siRNA SVIL or control with transfected cells HeLa ) irpe,adteclsaen ogral oasml the assemble to able longer no spindle are is central cortex cells the equatorial the the of and at to absence disrupted, RhoA furrowing of the of accumulation progression organization, In the cytokinesis. for essential complete is ring contractile furrow of completion the for plane ingression. division the at zone restricted D h oriae cino h eta pnl n the and spindle central the of action coordinated The c-and Act- D y-VLwr eeae n rnfce ihSI siRNA. SVIL with transfected and generated were Myo-SVIL n 5 L1popoyae VL3635 SVIL phosphorylates PLK1 0.( 30). D eaclsta osiuieyexpressed constitutively that cells HeLa ) B eacls rnfce with transfected cells, HeLa ) hw the shows h hyde-fixed n 5 30). Journal of Cell Science a910o VLfsdwt S a rdcdi atra n recombinant and bacteria, in produced was GST antibody, with anti-SVIL fused an supplemented generate SVIL (WAKO) To mammary CA). of F12 Carlsbad, human DMEM aa99–160 (GIBCO, serum in The horse cultured Australia). 5% with were Hendra, MCF10A, (EQUITEC, cells, with serum supplemented epithelial and bovine Japan) Osaka fetal (WAKO, DMEM 10% in cultured were cells HeLa chemicals and antibodies Cells, Methods and Materials mechanisms progression. molecular cytokinesis of the in role into involved insight physiological ring- greater the provide contractile of will can SVIL and elucidation SVIL thus, spindle- region. proteins; mitotic limited localized the additional in with cleavage furrow the associate at the II confines myosin and of association activation furrow SVIL the at spindle, induces central localization II the myosin for with at PRC1 Once with spindle. central association the its promote to promote Ser238 may spindle phosphorylation. central RLC for for the kinases pathways cortex RhoA-mediated at Citron of SVIL equatorial activation and activation. ROCK the II as at myosin such kinases RhoA protein II myosin cytokinesis. activates induces SVIL during II phosphorylated Currently, the activation myosin furrow. how know cleavage promote not the do to of we constriction pathways the redundant for activation utilizes demonstrate PLK1 results These that myosin SVIL. to regulate phosphorylating addition can by In PLK1 activation that 2012). II showed al., study et our pathways, (Vazquez-Martin AMP- these of furrow phosphorylation the energy-sensing promote at to the RLC (AMPK) the kinase phosphorylates study protein recent PLK1 activated to A catalytic 2007). that promote al., contractile contribute to et showed (ROCK) (Lowery ROCK the cytokinesis phosphorylate during of to kinase can activity constriction PLK1 Rho-associated reported initiate and ring, to RLC. been RLC phosphorylates of has al., et Komatsu phosphorylation 2009; PLK1 al., by et a activation (Asano 2000). induced furrowing II distorted RLC myosin or non-phosphorylatable delay of of inhibition expression the Previous exogenous cytokinesis. that of reported execution the studies for essential and II myosin to protein hub a further progression. cytokinesis coordinate as Although to role components critical multiple connect 2010). a play al., may SVIL needed, et can is analysis (Smith SVIL EPLIN example, protein, cortex- For the localized and SVIL. KIF14, protein, spindle-localized with the with associate associate have to proteins cytokinesis-related reported of been coordinate number to A ring contractile molecular furrowing. and result a proper spindle also This central is the activation. II between myosin II link with myosin association SVIL of that suppression suggests the the and of broadening furrow significant exhibited II, We myosin furrowing. with association of of association completion expressing cells the The cytokinesis the for that 2008). showed crucial during al., is proteins et ring, two Gregory these contractile 2008; al., the et (D’Avino of a component RacGAP50C, cells. critical Drosophila in these Drosophila between structures link subcellular the molecular important two Recent an furrowing. the revealed because of progression and have the studies spindle for crucial central 2003). furrowing is al., the ring contractile induces et of the communication (Straight ingression continuous components Thus, furrow during maintain the of microtubules delocalization to of depolymerization required is also spindle central organized An furrowing. initiate to ring contractile 3636 nsmay esoe htPK hshrltsSI at SVIL phosphorylates PLK1 that showed we summary, In of activation the for prerequisite a is RLC of Phosphorylation ora fCl cec 2 (16) 126 Science Cell of Journal ooo fMcaGP soitdwt nli,a Anillin, with associated MgcRacGAP, of homolog D y-VL hc sdfciein defective is which Myo-SVIL, ocvtn rmSnaCu,M12fo noLf cecs(amndl,NY) (Farmingdale, Sigma-Aldrich. inhibitor Sciences from CDK1 thymidine Life and Enzo TX), nocodazole from (Houston, and MG132 Chemicals Cruz, Santa Selleck from from roscovitine purchased inhibitor PLK1 was CA). (Carlsbad, BI2536 Rhodamine- Invitrogen Japan). from obtained (Tokyo, Cruz was Tesque phalloidin Santa Nacalai conjugated antibodies, antibody, anti-GFP anti-PRC1 CA); MA); Cruz, (Danvers, (Santa Signaling Cell antibody, PLK1 rmtefloigmnfcues anti- manufacturers: following used purchased were the we antibodies antibody, from Other anti-SVIL (aa99–160). Sweden) GST-SVIL the Uppsala, recombinant BioScience, purify with Healthcare To coupled (GE weeks. columns 2 HP injected every NHS-activated Louis, and times HiTrap (Sigma-Aldrich) St. four adjuvant rabbit (Sigma-Aldrich, Freund’s a with beads into mixed agarose was protein glutathione The using MO). purified was protein irsoe(lmu,Tko aa) h ai fmliulae el was fluorescence cells IX71 multinucleated an of using ratio fields. the selected taken The randomly five to Japan). were from evaluated Tokyo, Images and with according (Olympus, (Invitrogen) fixed visualization. microscope paclitaxel were (Invitrogen) FITC-labeled for each cells with of the Hoechst stained nM RNAiMAX and later, 20 paraformaldehyde hours with 4% Lipofectamine Seventy-two transfected and instructions. plates manufacturer’s with and 24-well S1. genes in Table the material cultured siRNA of supplementary in were name shown The cells are Invitrogen. HeLa each from for purchased siRNAs of was sequences siRNAs of library N- A the on screen by tag siRNA introduced HA were or mutations GFP a Point mutagenesis. with CA). site-directed vector View, PCR-based retrovirus Mountain SVIL pQCXIP Full-length (Clontech, library. a terminus cDNA HeLa into a cloned from PCR was by amplified was SVIL Human constructs DNA auatrrspooo.Fv irltr ftasae rti a nuae with incubated was protein translated the of following microliters WI) Five protocol. Madison, manufacturer’s (Promega, system was Transcription/Translation coupled SVIL aa676–1009 HA-tagged vitro In cell The lysates. 4 at cell minutes clear and 60 for ice obtain antibodies on primary to minutes indicated 15 minutes the for with Tris- 20 PMSF) mixed mM for were mM (50 lysates r.p.m. 1 buffer NP-40, 15,000 TNE 0.1% in at NaCl, lysed mM centrifuged and 150 PBS 7.4, cold PH with HCl once washed were cells The Immunoprecipitation (37 stage heat the were on placed Japan) were Tokyo, cells Twenty- (IWAKI, the (Invitrogen). transfection, dishes RNAiMAX after Lipofectamine glass-bottom hours using four mm siRNAs 35 with on transfected grown cells The imaging ng/ml Live-cell 40 with were and treated shaking medium. different by fresh were collected the into were cells at cells released arrested lysed the subsequently Mitotic and hours. into synchronization, medium mM 13 released mitotic for fresh 2 nocodazole and of into For presence PBS released the points. in hours, with incubated time with 15 times again for were incubated three cells thymidine were The washed of hours. cells hours, 8 the for 24 medium block, for fresh thymidine thymidine double mM by 2 cells synchronize To the synchronization Cell transfection, using 2 after with CA) cells Jolla, hours the the with to La Forty-eight added combination were (Stratagene, were (Invitrogen). in the supernatants protein vectors cDNA with 2000 each each pVPack-Ampho transfected Lipofectamine encoded were expressed and that cells constitutively pVPack-GP vector HEK293T that retroviral infection. pQCXIP retrovirus cells by MCF10A established and lines HeLa cell stable of Generation 5 were expression SVIL suppress to used siRNAs the of sequences The transfection siRNA n h netdclswr eetdwt 1 with selected were cells infected the and 5 was luciferase targeting siRNA UGAUGAAGCCAGAUGAUGAU-3 GAGAACAAGGGAAUGUUGAGAGAAU-3 eecletda h niae nevl n rcse sn eaimaging Meta using processed and intervals CA). Sunnyvale, indicated Devices, (Molecular the cells v6.1 live at software of images collected fluorescence and were Phase-contrast (Olympus). microscope IX81 eetasetdwt 0n iN sn ioetmn NiA (Invitrogen). RNAiMAX Lipofectamine using siRNA nM 20 with transfected were h ed eewse he ie ihTEbfe n upne nsml buffer. sample in suspended and buffer TNE with times three washed were beads protein-G The or protein-A– with then rnlto n S uldw assay pull-down GST and translation 9 -CUUACGCUGAGUACUUCGATT-3 2 ehrs ed o nadtoa 0minutes. 60 additional an for beads Sepharose 9 nvitro in sRA2.Tesqec ftecontrol the of sequence The (siRNA-2). a tblnatbde,SgaAdih anti- Sigma-Aldrich; antibodies, -tubulin m /lprmcnfr3days. 3 for puromycin g/ml 9 rnltduigTTS6Quick SP6 TNT using translated m /lplbee(Sigma-Aldrich), polybrene g/ml sRA1 n 5 and (siRNA-1) 9 9 -GGAGG- h cells The . ˚ )o an of C) ˚ and C 9 - Journal of Cell Science ehue . hu . Ville C., Zhou, N., Dephoure, en .O n pdc,J A. J. Spudich, and O. S. Dean, ’vn,P . aea . aab,L,Zag . ily .S,Lu,E .and D. E. Laue, S., J. K. Lilley, E. W., Zhang, Luna, L., and Capalbo, T., N. Takeda, Takizawa, P., P. Z., D’Avino, Fang, C., T. Smith, L., J. Crowley, hn . aiaa . rwe,J . h .W,Gto .L,Kmaa . Sato, T., Kambara, L., C. Gatto, W., S. Oh, L., J. Crowley, N., Takizawa, Y., Chen, ukr,M . aijwk,J,RdiuzBao . ek,M,Lwr,D M., D. Lowery, M., Repka, V., Rodriguez-Bravo, J., Maciejowski, E., M. Burkard, ukr,M . adl,C . aohle . hn,C,Soa,K . Fisher, M., K. Shokat, C., Zhang, S., Larochelle, L., C. Randall, E., F. M. A. Burkard, Straight, and M. T. Kapoor, U., Peters, M., I. Brennan, Kru Z., Fang, S., Cornfine, R., Bhuwania, ats .N n ar .A. F. Barr, and N. R. Bastos, Sillje A., F. Barr, eeicbtdwt ICcnuae nirbi nioy(nirgn or (Invitrogen) 4% antibody or cells anti-rabbit the (50:50) PBS, FITC-conjugated with minutes methanol/acetone 30 washing with for After PBS cold antibodies. incubated in primary FBS with with 7% were incubated with then fixed blocked were and poly-D-lysine-coated cells and the The on paraformaldehyde. coverslips seeded were cells glass released and Nocodazole-arrested microscopy Immunofluorescence 10 ar .A n rnbr,U. Gruneberg, and H. A. F. Hosoya, Barr, and K. Hamao, S., Asano, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.124818/-/DC1 online available material A.N.]. Supplementary to 23107010 and 22570182 H.H. numbers to [grant Culture, 23791591 Japan Education, T.S., of of to Technology Ministry and the Science by Sports, supported was research This Funding authors All project. M.H. manuscript. the the and supervised H.K. writing T.W. T.S. and to A.N., data. contributed M.M., the E.A. S.I., interpret T.H., to screen. helped siRNA experiments. perform performed to helped and designed H.H. contributions Author helpful for Biology assistance. Cancer of technical Division and the discussions of members the thank We Acknowledgements (3 SVIL aa228–256 GST-fused or GST Purified vitro Japan). In Tokyo, (OLYMPUS, FV1000 microscope acquired confocal were scanning laser Images a (Invitrogen). using antibody anti-mouse Rhodamine-conjugated H75 0m MgCl mM Hepes 10 mM 7.5, (50 buffer pH kinase in MA) Billerica, (Millipore, active-PLK1 recombinant [ eovdo D-AEgl.Tegl eedidadsbetdt autoradiography. to subjected and dried were gels The gels. SDS-PAGE on resolved ujce oSSPG n muoltiguigat-Aantibodies. anti-HA using immunoblotting and SDS–PAGE to subjected c - al cd c.USA Sci. P. Acad. S. Natl. Gygi, and chain J. light S. regulatory myosin for is cells S2 Drosophila phosphorylation. mitotic of cortex equatorial coysncnrciern ihsidemcouue ttecl iiinsite. division cell the at microtubules spindle Sci. with ring contractile actomyosin M. D. Glover, uevli eraie h ci yokltnadicessivdpda efficiency. invadopodial increases Cell and Biol. cytoskeleton Mol. actin the J. reorganizes Supervillin E. Luna, and M. supervillin. Ikebe, in D., domains X. Li, O., l1sl-raiainadpiigpopoyaino sY- ttespindle the at cells. al. human HsCYK-4 et in B. division of M. of Yaffe, phosphorylation onset A., the priming S. regulate Carr, midzone and M., K. self-organization Shokat, C., Plk1 Zhang, R., K. Clauser, oolk iae1atvt npstoigRo n rgeigctknssi human in cytokinesis triggering and RhoA positioning in cells. activity V. 1 P. kinase Jallepalli, Polo-like and P. R. otosvrert pnl lnainadcytokinesis. and elongation spindle vertebrate controls their enables and podosomes to contractility turnover. myosin-dependent couples Supervillin abscission. orderly ensure to midbody the fcl division. cell of during ingression furrow in Cell II myosin of cells. HeLa chain in cytokinesis light regulatory phosphorylated 32 m ]T.Terato itrswr nuae t30 at incubated were mixtures reaction The P]ATP. fGTpoeno S uinpoenfr1hu t4 at hour 1 for protein fusion GST or protein GST of g 121 131 rc al cd c.USA Sci. Acad. Natl. Proc. iaeassay kinase 1151-1158. , 847-860. , .Cl Sci. Cell J. ,H .adNg,E A. E. Nigg, and H. H. ´, 20 20) neato ewe nli n aGP0 oncsthe connects RacGAP50C and Anillin between Interaction (2008). a.Rv o.Cl Biol. Cell Mol. Rev. Nat. LSONE PLoS 948-962. , 2 125 MET,1m T,250 DTT, mM 1 EGTA, mM 1 , 105 20) uniaiealso ioi phosphorylation. mitotic of atlas quantitative A (2008). .Bo.Chem. Biol. J. 2300-2314. , 10762-10767. , ee Cells Genes n . euoel .A,Bklrk,C . Elledge, E., C. Bakalarski, A., S. Beausoleil, J., ´n, 20) hmclgntc eel h eurmn for requirement the reveals genetics Chemical (2007). 21) l1ngtvl euae e5 erimn to recruitment Cep55 regulates negatively Plk1 (2010). 1 20) h iaesrl nmoi erimn othe to recruitment myosin in role kinase’s Rho (2006). e131. , 20) yoiei:paigadmkn h ia cut. final the making and placing Cytokinesis: (2007). 104 4383-4388. , 20) oolk iae n h orchestration the and kinases Polo-like (2004). 278 14 gr . ua .J n idr S. Linder, and J. E. Luna, M., ¨ger, 555-568. , 5 46094-46106. , .Cl Biol. Cell J. 429-440. , 20) ieteiec o oe of roles for evidence Direct (2009). 20) -ci n ysnI binding II myosin and F-actin (2003). m )wsicbtdwt 0.5 with incubated was g) ˚ o 0mntsi 50 in minutes 30 for C LSONE PLoS LSBiol. PLoS 191 m 751-760. , T)ad1 and ATP) M 20) oolk kinase Polo-like (2007). ˚ .Tebaswere beads The C. 2 7 e409. , e1000111. , (2009). (2009). (2012). m .Cell J. m m iof Ci Proc. land gof etnaap .N,Pp,R . uful,J .adLn,E J. E. Luna, and D. J. Wulfkuhle, K., R. Pope, N., K. Pestonjamasp, ef . rnbr,U,Kpjih . i . ig .A,Slj,H n Barr, and H. Sillje, A., E. Nigg, X., Li, R., Kopajtich, U., Gruneberg, R., Neef, E. Nishida, and E. Taniguchi, F., Toyoshima-Morimoto, H., Nakajima, ef . riigr . ucif,J,Kpjih . ig .A,Myr .U and U. T. Mayer, A., E. Nigg, R., Kopajtich, J., Sutcliffe, C., Preisinger, R., Neef, Margolis, and T. Hunter, G., Schoehn, W., Jiang, P., J. Kleman, C., Mollinari, F. Matsumura, oey .M,Casr .R,Herl,M,Lm . lxne,J,Ksi . Ong, K., Kishi, J., Alexander, D., Lim, M., Hjerrild, R., K. Clauser, M., D. Lowery, Elia,A.E.,Cantley,L.C.andYaffe,M.B. i,J,Wn,Z,Jag . hn,L,Za,L,Ha . a,F,Yn,Y,Wang, Y., Yang, F., Yan, S., Hua, L., Zhao, L., Zhang, K., Jiang, Z., Wang, J., Liu, la .E,Rlo,P,Hie .F,Ca,J . vn,F . ope,K., Hoepker, J., F. Ivins, W., J. Chao, F., L. Haire, P., Rellos, E., A. Elia, i,X,Zo,T,Krym,R n rko,R L. R. Erikson, and R. Kuriyama, T., Zhou, X., Liu, Yu oas,S,Yn,T,Siaa . ut .A n kb,M. Ikebe, and A. R. Tuft, M., Shibata, T., Yano, S., Komatsu, of,B . aai . ernzi .adGozr M. Glotzer, and M. Petronczki, T., Takaki, A., B. Wolfe, Cufı A., Vazquez-Martin, M. J. Peters, and S. Taylor, J. E. Luna, and M. Ikebe, R., Ikebe, N., Takizawa, in,W,Jmnz . el,N . oe .J,Wh,G . utr .and T. Hunter, M., G. Wahl, J., T. Hope, J., N. Wells, G., Jimenez, W., Jiang, aiaa . mt,T . el . rwe,J . amei .J,Lfht,L M., L. Lifshitz, J., S. Palmieri, L., J. Crowley, T., Nebl, C., T. Smith, N., Takizawa, rgr,S . baii . ivro,J,Jns .M,Bjoe,A n Saint, and A. Bejsovec, M., W. Jones, J., Milverton, S., Ebrahimi, L., S. Gregory, M. Glotzer, tagt .F,Cen,A,Lmue . hn . etod .J,Sles .R and R. J. Sellers, J., N. Westwood, I., Chen, J., Limouze, A., Cheung, F., A. Straight, ag . aiaa . isn .A,Sih .C,Dlrt,A,Dvdo,M W., M. Davidson, A., Delprato, C., T. Smith, A., K. Wilson, N., Takizawa, Z., Fang, mt,T . ag .adLn,E J. E. Luna, and Z. Fang, C., T. Smith, Eberspa R., Neef, A., Santamaria, Le M., Petronczki, M. J. Peters, and N. Kraut, M., Glotzer, M., Petronczki, ag .adLn,E J. E. Luna, and Z. Fang, aan . anu .adPiet C. Prigent, and Y. Rannou, P., Salaun, c,O,Pen,A n lte,M. Glotzer, and A. Piekny, O., ¨ce, uevli p0) oe ebaeascae,Fatnbnigpoeni the in protein F-actin-binding membrane-associated, novel superfamily. villin/gelsolin A (p205): Supervillin Cdk1. of state activation the by controlled A. F. iae1i eurdfrcytokinesis. for required is 1 kinase A. F. Barr, reveals phosphorylation substrate. kinase) Plk1 (Polo-like a Plk as for Myt1 motif consensus a of Identification midzone. spindle mitotic the maintain L. R. .E,Gmetf,S,Cr,S .adYfe .B. M. Yaffe, and A. S. Carr, S., Gammeltoft, E., S. eie h oobxdmi neatm n dniisRc2a l1substrate. Biol. Plk1 Cell a Trends as Rock2 identifies and interactome J. domain EMBO Polo-box the defines lsiiyi mitosis. in al. plasticity et C. Fu, D., oeua ai o hshdpnetsbtaetreigadrglto fPk by Plks B. of M. regulation Yaffe, and and targeting domain. J. Polo-box substrate S. the phosphodependent Smerdon, for C., basis L. molecular Cantley, D., Mohammad, substrates. mitotic to Plk1 localizing domain binding oolk-iae1wt h ioi iei-iepoenCHO1/MKLP-1. protein kinesin-like mitotic 117 the with 1 Polo-like-kinase euaoylgtcanpopoyaino ysnI nmtssadctknssof cytokinesis and mitosis on II myosin of cells. phosphorylation mammalian chain light regulatory euae h oaiainadfnto fRhoA. of function and localization the regulates cleavage initiate to complex RhoGEF formation. RhoGAP/Ect2 furrow HsCyk-4 the of assembly directs periphery. cell the at activation II myosin facilitating by rti eurdfrcytokinesis. for required protein R. Fukunaga, iekns iet h MKmdae ciaino ysnrgltr ih hi tthe at chain light balance. regulatory energy myosin of independently of furrow activation cleavage cytokinetic AMPK-mediated the directs 1 kinase like biology. chemical TRIP6/ZRP-1. involving J. adhesions E. Luna, focal and of C. modulation M. Beckerle, M., L. Hoffman, G., A. Ehrhardt, n nli ntecnrciering. contractile the in Anillin and R. ysnI Inhibitor. II myosin J. T. Mitchison, MAPs. motility. cell and J. recycling E. integrin Traffic rapid Luna, F-actin-dependent and accelerates supervillin, G. D. Lambright, nacscl survival. cell enhances uevli nerycytokinesis. early in supervillin late and early al. in Plk1 et of mitosis. L. functions of elucidate Wortmann, stages to G., ZK-thiazolidinone inhibitor Siemeister, Plk1 V., novel Schulze, S., Prechtl, bodyguards. mitotic Plk1. with Cytokinesis the to of Entry recruitment promoting spindle. by central cells the human to in Ect2 cytokinesis RhoGEF of initiation the triggers 20) eldvso eursadrc ikbtenmcouuebudRacGAP microtubule-bound between link direct a requires division Cell (2008). 3233-3246. , 20) hieo l1dcigprnr uigmtssadctknssis cytokinesis and mitosis during partners docking Plk1 of Choice (2007). 20) R1i irtbl idn n udigpoenesnilto essential protein bundling and binding microtubule a is PRC1 (2002). 11 a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 782-799. , 26 20) h M fcnrlsideasml:mcouue,mtr and motors microtubules, assembly: spindle central of 3Ms The (2009). 2262-2273. , 20) hshrlto fmttcknsnlk rti ypolo-like by 2 protein kinesin-like mitotic of Phosphorylation (2003). 20) euaino ysnI uigctknssi ihreukaryotes. higher in cytokinesis during II myosin of Regulation (2005). 19) R1 ua ioi pnl-soitdCKsubstrate CDK spindle-associated mitotic human a PRC1: (1998). ´na 20) iscigtmoa n pta oto fctknsswt a with cytokinesis of control spatial and temporal Dissecting (2003). 20) R1coeae ihCAP oognz eta spindle central organize to CLASP1 with cooperates PRC1 (2009). 15 o.Bo.Cell Biol. Mol. .Bo.Chem. Biol. J. LSBiol. PLoS ur pn elBiol. Cell Opin. Curr. r,P n ees .M. J. Peters, and P. ´rt, 371-377. , Science d.Ep e.Biol. Med. Exp. Adv. .Bo.Chem. Biol. J. Cell ,S,Oiea-errs .adMnne,J A. J. Menendez, and C. Oliveras-Ferraros, S., ´, .Bo.Chem. Biol. J. L1popoyae VL3637 SVIL phosphorylates PLK1 .Bo.Chem. Biol. J. 21) uevli-eitdsprsino 5 protein p53 of suppression Supervillin-mediated (2013). 115 .Cl Biol. Cell J. 20) ooadArr iae:lsosdrvdfrom derived lessons kinases: Aurora and Polo (2008). 299 7 83-95. , e1000110. , yokltn(Hoboken) Cytoskeleton e.Cell Dev. 1743-1747. , ce,U,Es . uean . ubr,D., Mumberg, M., Husemann, K., Eis, U., ¨cher, o.Cell Mol. 275 10 18 ur Biol. Curr. 9-20. , 284 4024-4036. , .Cl Biol. Cell J. 34512-34520. , e.Cell Dev. 288 .Cl Biol. Cell J. 23059-23071. , 20) d1 ls uoa,adNk:the Neks: and Auroras, Plks, Cdk1, (2008). 21) h ebaeascae protein, membrane-associated The (2010). 139 20) nET-etasidi complex ECT2-centralspindlin An (2005). 278 20 7918-7929. , 21) oe neatr n oefor role a and interactors Novel (2010). 617 14 20a.Poemcsre id pSer/pThr- finds screen Proteomic (2003a). 2 a.Cl Biol. Cell Nat. 77-84. , 1255-1269. , 877-885. , 25277-25280. , 646-659. , 20) ooo h iefo Mitotic Rise-from the on Polo (2008). 41-56. , 18 20) uevli lw elspreading cell slows Supervillin (2007). 12 .Cl Biol. Cell J. 25-29. , 162 713-725. , Science 20) oeua neatosof interactions Molecular (2004). 157 863-875. , .Cl Sci. Cell J. .Cl Biol. Cell J. 1175-1186. , 67 299 20) oolk iae1 kinase Polo-like (2007). 20) oolk iae1 kinase Polo-like (2009). 20) rtoi screen Proteomic (2007). 9 elCycle Cell 346-364. , 436-444. , 1228-1231. , 170 20) fet fthe of Effects (2000). 20) s fthe of Use (2007). 120 571-582. , 20) Supervillin (2006). 174 3792-3803. , 11 447-458. , 20b.The (2003b). 21) Polo- (2012). 2422-2426. , .Cl Sci. Cell J. (1997). (2003).