Ato o orsodne([email protected]) correspondence for *Author Sweden. va Eulers Stockholm, von 77 Institute, Karolinska Biology, Molecular and eia etrUrct nvrietwg10 54C teh,The Utrecht, CG 3584 100, Netherlands. Universiteitsweg Utrecht, Center Medical oaadArr- otnet ciaePk nmitosis in Plk1 Bruinsma activate Wytse to continue Aurora-A and Bora ARTICLE RESEARCH ß eevd2 ue21;Acpe 1Nvme 2013 November 11 Accepted 2013; June 20 Received domain polo-box C-terminal is conserved function 1 the Plk1 of by control Golsteyn mediated Spatial 1998; 2006). part al., al., in poles et spindle et Kang to 1995; (Arnaud as al., mitosis et well as to during G2, described kinetochores in been and centrosomes has the Plk1 to 2004). recruited Pines, (Fang be and (APC/C) Lindon degraded 1998; complex/cyclosome is al., Plk1 promoting et 2012). mitosis, mitosis anaphase from of the exit stages al., early Upon by the expression 1997). during al., its et peaks et regulated; and (Uchiumi G2 cycle Bruinsma in cell induced highly first 2009; is is Plk1 Glover, of activity multiple Expression and at and localization controlled expression, are its (Archambault of Plk1 al., control of through et and functions levels, (Bruinsma distinct cytokinesis arrest The DNA-damage-induced cohesion, 2012). a chromatid from sister recovery important maturation, several assembly, centrosome of as regulator spindle key such a processes, is cell-cycle-associated (Plk1) 1 kinase Polo-like INTRODUCTION Plk1 Mitosis, to Bora, committed Aurora-A, WORDS: has KEY it to once This refractory activity activated. Plk1 but fully robust is mitosis. activation, with Plk1 to cell initial once a sensitive activity provides its highly Aurora-A in switch; by in Plk1 bistable fluctuations Aurora-A of a of activation as the inhibition function Thus, are may activity. complex Plk1 Aurora-A Bora–Aurora-A sustain the to of Plk1 sufficient amounts once minimal that to find activated, Aurora- We sufficient continued is Plk1. for of is essential phosphorylation is T210 of activity which fraction A-dependent mitosis, a Aurora-A in addition, retained In of is mitosis. Bora in amount Plk1 activate small and Bora– phosphorylate a We the mitosis. that that in Plk1 show of show we activator how major Here be the unclear mitosis. remains currently to complex in Aurora-A is shown sustained it is and was activity mitosis, in Bora Plk1 into Aurora-A, However, entry kinase to first Bora. prior the conserved is cofactor degraded by Plk1 its a phase (T210). division. with G2 domain concert on in kinase cell T210 the phosphorylation proper on of phosphorylated T-loop requires for the in required Plk1 threonine is of (Plk1) Activation kinase-1 Polo-like ABSTRACT nttt eTcooisBoeia,330Tnrf,Spain. Tenerife, 38320 Biomedicas, Tecnologias de Instituto ilg,Isiueo oeua eeisvvi,Vdnk 03 Z40 Prague, CZ14200 1083, Videnska v.v.i., Republic. Genetics Czech Molecular of Institute Biology, lsala 2,16 X mtra,Netherlands. Amsterdam, CX, 1066 121, Plesmanlaan eateto eia nooyadCne eoisCne,University Center, Genomics Cancer and Oncology Medical of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,8181doi:10.1242/jcs.137216 801–811 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2 eateto elBooy h ehrad acrInstitute, Cancer Netherlands The Biology, Cell of Department 4 nddd Investigacio de Unidad 1,2 io Macu Libor , ´ ,Hsia nvriai eCanarias, de Universitario Hospital n, ˚ rek 1,3 3 aoaoyo acrCell Cancer of Laboratory ¨ amnoFreire Raimundo , ,P o 8,S 171 SE 285, Box PO 3, g 5 eateto Cell of Department 4 reLindqvist Arne , eiu T1)i h -opo t iaedmi Jn tal., et (Jang domain kinase its of threonine T-loop conserved the a in of (T210) phosphorylation residue orderly on dependent an is in event division cell Plk1 during 2012). allow roles al., to manner. essential multiple et is execute (Bruinsma control mitosis to spatiotemporal of late and level mitosis in early This midzone in kinetochores spindle the the r and as such directs recognition sites, subcellular PBD for the substrates substrate controlling specificity, Plk1 Besides Plk1. distinct by kinases phosphorylation other eventual prime of spatiotemporal influence can such, the As under that is 2003b). functions al., Plk1 et of Cdk1 Elia control as 2003a; such kinases al., other et by (Elia created are which residues rec serine can domain This (PBD). ensonta nte ebro h uoakns family, kinase aurora in the Polo of recently phosphorylate T210 has member can it another Aurora-B However, to that continued 2005). corresponding shown al., been residue been et the T-loop has (Mortensen T210 yeast) T214 a Cdk1 budding human in how of for yeast, homolog Polo phosphorylation In and sole unclear through (the responsible mitosis. Cdc5 degraded, activate is in to is shown Plk1 is it of Bora phosphorylation kinase However, once maintained 2008b). which is al., phosphorylation et Macu 2002; al., al., Seki et et continued (Jang on Seki dependent T210 2003; strictly of al., is phosphorylation et Plk1 of Eyers activity Plk1- 2008; the Mitotic al., to 2008a). a et Aurora-A (Chan directs in spindle which with degraded TPX2, mitotic interact as is Macu subsequently such can co-activators, 2006; Bora other Aurora-A al., mitosis, and et manner enter Aurora-A, dependent (Hutterer cells of Plk1 Once co-activator to 2008). a (also bind Bora borealis), to on aurora known depends protein crucially as G2 known in T210 of A) phosphorylation is kinase aurora G2 as in known T210 (also (Macu Aurora-A of kinase when phosphorylation the activity Plk1 by initial full mediated The when its mitosis. G2, reaches enter in it cells until starts increases residue gradually this activity of Phosphorylation 2002). cie elto fbt oaadArr- srqie to required is Aurora-A and that Bora Plk1 keep evidence both to of is sufficient provide is Depletion mitosis Bora–Aurora-A amount in active. small of we a activity Plk1 that residual show of contrast, of We activation Bora–Aurora-A. phosphorylation on In and dependent still T210 T210 a cells. of mitotic have phosphorylation not overall human does Aurora-B to in that contribution found We major mitosis. during suspect Plk1 likely very a mitosis. and during kinase localization activity Its this Plk1 2012). makes controlling al., Aurora-A et kinetochores to Waal the similarity der at (van attachments cytokinesis microtubule and of including mitosis, correction during chromosomal roles error the several of has part and is complex Aurora-B passenger 2012). mammalian al., et in (Carmena mitosis cells during phosphorylation T210 to contribute ato hsitiaerglto steatvto fPk.This Plk1. of activation the is regulation intricate this of Part ehv netgtdtecnrlo 20popoyainof phosphorylation T210 of control the investigated have We ˚ e ta. 08 eie l,20b.Efficient 2008b). al., et Seki 2008; al., et rek 1,5 n Rene and giepopoyae henn or threonine phosphorylated ognize .Medema H. ´ cuteto l1t distinct to Plk1 of ecruitment Drosophila 1,2, ˚ e ta. 2008; al., et rek * n a also can and ˚ e tal., et rek 801

Journal of Cell Science n nitga oeo elccecnrla h ne fmitosis of onset the at 2011). control cycle Lindqvist, cell and cyclin-B–Cdk1 of (Medema kinase mode bistable mitotic integral a the an for as and described functions that feature Bora–Aurora-A suggests a by This switch, levels Plk1 complex. reduced of Bora–Aurora-A severely activation with the the levels of low activity and very robust and at extremely the sustained is to Plk1 be contrast of can in activation that mitotic show G2, in data situation These mitosis. in Plk1 inactivate ARTICLE RESEARCH htaohrkns a osbypopoyaeT1 tlater 802 at T210 phosphorylate cycle. cell possibly implying the can of Aurora-A, stages kinase on Aurora-A dependent another of exclusively that inhibition not to is mitosis activation refractory in activity relatively Plk1 (Macu be Nonetheless, (Macu to 2008b). Bora al., and appears Aurora-A et on Seki of dependent 2008; activation is initial G2 the that in shown Plk1 previously mitosis have in others Plk1 and of We phosphorylation T210 regulates Aurora-A phosphorylation this signal mitosis. studying off-target in when an specifically event caution possibly demands in is hence bd558400 seen antibodies and staining both kinetochore that with centrosomes. the cells that human at mitotic and suggests appropriate data at signal our the blots observations However, Plk1 the recognize T210-phosphorylated western These recognizes in antibodies on depleted. increase both was epitope reduced an that Plk1 clearly of demonstrate observed case when was the even kinetochores In kinetochores S1A,B). we at Fig. Plk1 ab39068 material supplementary of and 1D,F; RNAi, level the (Fig. by Plk1 total stained of depletion clearly the both after with persisted antibodies staining antibodies kinetochore both pT210 However, 1D). addition, (Fig. In In kinetochores or 1D,E). weak (Fig. absent. very signal was ab39068 the even with of staining reduction centrosomal by the Plk1 clear contrast, of a depletion clear in and a cells resulted Consistent mitotic showed siRNA in antibody mitosis. centrosomes bd558400 the in at the signal located report, previous is centrosomes to our at Plk1 proceeded with staining where next phospho-T210 We kinetochores, of 1C). and specificity (Fig. did the blots at western antibodies phosphorylated look on T210A towards Plk1 both the specificity of recognize the T210 expected to confirming failed As thus but mutant, Plk1 antibodies. wild-type reactivity the pT210 the recognize and tested the Plk1. and wild-type cells of by endogenous mitotic myc-tagged from recognized of Plk1 RNAi immunopurified also T210A-mutated by immunoprecipitates was we band Plk1 in at Furthermore, same of antibodies band the depletion addition, both clear after In a disappeared 1B). (Fig. and identified Plk1 We over total well 1A). (Fig. contained that cells , mitotic samples mitotic a in to 2012; 90% resulted cells al., which osteosarcoma U2OS et shake-off, of (Carmena cultures Abcam Biosciences synchronized peptide from BD ab39068 phosphorylated from Macu Plk1; of a bd558400 region and against T210 phospho- two the raised of encompassing use antibodies made in we phosphorylation specific phosphorylation, activity T210 T210 maximal of of pattern reaches kinetics and the G2 (Macu with in coincident activated mitosis, first is mitosis in Plk1 phosphorylation T210 of Analysis RESULTS 0kawt ohatbde htmgae ttesm egtas height same the at migrated that antibodies both with kDa 70 ˚ ˚ ˚ e ta. 09.T banmttccls esubjected we cells, mitotic obtain To 2009). al., et rek e ta. 09.T ute hrceietespatiotemporal the characterize further To 2009). al., et rek e ta. 08.Teeosrain ugs htPlk1 that suggest observations These 2008). al., et rek ˚ e tal., et rek el,wiePk eeswr o fetd(i.2A,C). (Fig. affected not were levels Plk1 mitotic in phosphorylation while the Aurora-B. T210 of we using cells, or inhibition 2007) inhibited partial al., kinases a Aurora-A et selectively observed (Manfredi aurora MLN8054 either was inhibitor different activity pharmacological for the Aurora-A subsequently selectivity of and When distinct inhibitors mitosis in with with cells them synchronized treated first we cells, and Aurora-A by Plk1 investigated of we activation Therefore, Aurora-B. sequential 2012). of in T-loop al., possibility phosphorylation et the T210 for (Carmena to cells responsible contribute indeed human also kinase could report in Aurora-B the kinetochores recent that at mitosis kinase is a Polo in of Aurora-B Interestingly, phosphorylation kinetochores that 2007). at logical al., showed proteins T210 A et known several entry. of (Ruchaud Aurora-B, member kinetochore- phosphorylate mitotic phase family kinase a of to second Aurora that the time the is reasoned the candidate promote we around might therefore phosphorylation Kang kinase and 1998; al., addition, 2006) localized et In (Arnaud al., 2012). kinetochores al., to et et recruited also (Bruinsma Plk1 is G2 where Plk1 site during the activated are centrosomes first the is that suggested been has it inlfo htse ihtetoat-hsh-20antibodies anti-phospho-T210 the two anti- reduced the with clearly two seen inhibitors the that kinase from of aurora signal one S1C–F). the any Fig. with material or Treatment (supplementary anti-Plk1 antibodies an phospho-T210 clearly CREST- using with could overlapped when that we kinetochores staining the staining at signal Aurora- immunofluorescent a of observe confirm inhibition upon to could sensitive Indeed, we is signal B. whether this of wondered presence therefore the that We Plk1 to 1). T210-phosphorylated be Fig. find not (see we level might be the that which should epitope determine kinetochores, an to it recognize at study However, Plk1 that in T210-phosphorylated 2012). used of al., was et ab39068 that (Carmena noted cells mitosis a human in kinetochores to line in at lead in Plk1 can not T210-phosphorylated depletion is in This Aurora-B reduction minimal. that very observations is previous of cells with phosphorylation human in T210 mitosis overall that in to Plk1 indicate Aurora-B observations of selectively contribution to These the seem phosphorylation. not did T210 Aurora-B Aurora- we reduce of of inhibitors, Depletion depletion 2B,C). upon molecule (Fig. phospho-T210 A small in selective decrease a the observed with the with and obtained Consistent Aurora-A mitosis. data of in contribution phosphorylation the T210 to examined Aurora-B and kinase combination RNAi a aurora by or the both Aurora-B of with Aurora-A, mitotic observed depleted human next we in we effects inhibitors phosphorylation the T210 confirm in does To role Aurora-B cells. major of that a inhibition T210 evidence play after further not seen reduced that providing cells to alone, mitotic similar Aurora-A in level a Aurora-B inhibition to Dual and Aurora-B phosphorylation cells. Aurora-A that these arrest both in indicating mitotic of inhibited effectively Taxol-induced S3A), was a Fig. ZM447439 function on of of material addition override phosphorylation (supplementary Moreover, complete 2A). a substrate a (Fig. by S10 caused kinase and at Aurora-A H3 Aurora-B on histone T288 in site at reduction reduction autophosphorylation a by the demonstrated as in inhibitors targets, both respective their Nonetheless, inhibit mitosis. the did in in reduction T210 significant pharmacological of a in the phosphorylation result with not did Aurora-B ZM447439 inhibitor of inhibition Interestingly, osuyterqieet o 20popoyaini mitotic in phosphorylation T210 for requirements the study To and G2, in centrosomes the to recruited are Plk1 and Aurora-A ora fCl cec 21)17 0–1 doi:10.1242/jcs.137216 801–811 127, (2014) Science Cell of Journal Drosophila and

Journal of Cell Science EERHARTICLE RESEARCH ytyiieadncdzl,smlswr tie o oa eeso l1adpopoyae 20 cl as 5 bars: Scale T210. lysates phosphorylated sync from After and immunoprecipitated Plk1. Plk1 were targeting of treated siRNAs T210A levels cell with and total treated U2OS wild-type were for of Plk1 Cells stained lysates Myc-tagged (D) were from antibodies. (C) samples immunoprecipitated indicated T210. nocodazole, the was phosphorylated with and Plk1 for blotted thymidine (B) and and by shake-off. nocodazole Plk1 and mitotic of thymidine by levels with harvested total synchronized were for cells blotted mitotic and mitosis. The A in nocodazole. Plk1 of on presence T210 the of Phosphorylation 1. Fig. ieohr tiig fclsdpce nD h loecneitniyi eitdrltv oCETsann ocretfrkntcoesize. kinetochore for correct to staining CREST to relative depicted is the intensity to fluorescence relative The depicted D. is in intensity depicted fluorescence cells The of D. stainings in kinetochore depicted cells of staining A 2Sclswr rae o 4huswt hmdn n usqetyrlae o 6husin hours 16 for released subsequently and thymidine with hours 24 for treated were cells U2OS (A) c tblnsann ocretfrcnrsm ie F uniiainof Quantification (F) size. centrosome for correct to staining -tubulin ora fCl cec 21)17 0–1 doi:10.1242/jcs.137216 801–811 127, (2014) Science Cell of Journal m .()Qatfcto fcentrosomal of Quantification (E) m. hronization sin as 803

Journal of Cell Science EERHARTICLE RESEARCH 20i soitdwt ciaino l1i sntadrc readout of 804 direct a phosphorylation not is that it Plk1 established of activation well with associated been is T210 has mitosis it in activity Although Plk1 of a regulation Aurora-A-dependent cells. Nonetheless, mitotic in T210 Aurora-B. MLN8054 the with inhibited on not is phosphorylated Aurora-A remains when but site Plk1 Aurora-A, of amount of significant activity overall on depends that primarily the conclude cells We of human in 2D). level phosphorylation (Fig. T210 the mitotic same while the centrosomes remained our at Plk1 phospho-T210 with total of accordance decrease a led In Aurora-A to of inhibition kinases. that observed by we aurora results affected we blotting western was different does this centrosomes, how T210 of bd558400 see the Plk1 inhibition to in signal Because at centrosomal reduction the a quantified Plk1 blots. see T210-phosphorylated not western do recognize we on T210-phosphorylated this be with phosphorylation not line might In aurora which Plk1. to sensitive but is inhibition, that antibodies kinetochores kinase these the that at indicates epitope with 1F, an recognize Fig. together in taken shown This, observations our S1C–F). Fig. material (supplementary 1 were used concentrations The exit. mitotic premature prevent to MG132 mitosis. inhibitor in proteasomal phosphorylation the with T210 together affects Aurora-A 2. Fig. h tnaddvaino he needn xeiet.()Qatfcto fcnrsmlsannso el rae si .Tefursec int fluorescence The A. 1A,B in Fig. as from treated Plk1 cells total bar of Error and stainings plotted. Plk1 was centrosomal T210-phosphorylated experiments of independent of Quantification three Bands of (D) the average (C) experiments. an to 1A. and independent relative Fig. calculated three depicted was in of Plk1 as total deviation treated over standard T210 then the phosphorylated genes of indicated ratio A the quantified. targeting were siRNAs with treated were c tblnsann ocretfrcnrsm size. centrosome for correct to staining -tubulin A el eetetda nFg A h niae niioswr de o 0minutes 90 for added were inhibitors indicated The 1A. Fig. in as treated were Cells (A) aeilFg 2 Due l,21) nodrt xld that during exclude activation to Plk1 order on In effect an 2011). for has al., site Aurora-B et of consensus Mps1 (Dou inhibition S2) a change that (supplementary Fig. contain latter consensus observations material Plk1 the this that overlaps with that that targets phosphorylation consistent demonstrate al., phosphorylate Mps1, to et can on (Santaguida able reversine were depends the inhibitor we Using 3A). kinase small 2010), (Fig. a mitosis Mps1 for enter except cells selective when ratio, occurs FRET that in change shift the abolishes completely l1wt h eetv niio I23 (Le 2536 BI inhibitor selective the with (Macu starts U2OS Plk1 mitosis activation emission of before Plk1 imaging CFP/YFP hours biosensor. time-lapse 6 the the by expressing in monitored stably change be cells a can which causes ratio, Plk1 by cells biosensor living (Macu in biosensor activity based Fo Plk1 Plk1-specific global a monitored using we directly mitosis far, in we this thus activity address issue To obtained Plk1 mechanism. affect data phospho-T210-independent a can the through Aurora-B on that Based exclude itself. cannot activity Plk1 of ora fCl cec 21)17 0–1 doi:10.1242/jcs.137216 801–811 127, (2014) Science Cell of Journal ˚ e ta. 08.Popoyaino this of Phosphorylation 2008). al., et rek rtrrsnneeeg rnfr(FRET)- transfer energy resonance ¨rster m L85 n 2 and MLN8054 M ˚ e ta. 08 n niiinof inhibition and 2008) al., et rek m M449 B Cells (B) ZM447439. M ´ na ´ te l,2007) al., et rt niyis ensity indicate s , 5–

Journal of Cell Science EERHARTICLE RESEARCH oto Fg B.Tecmiaino uoaAadAurora-B and DMSO Aurora-A the of of combination that The follows 3B). completely (Fig. Aurora-A Plk1 control on of dependent curve is activation Plk1 shown of was activation As (Macu inhibitors. initial entering indicated the cells the of of before, presence ratios the CFP/YFP in the mitosis by quantified mitosis we in entry synchronized inh then mitotic the and of genes addition indicated indicates the arrow targeting premature The siRNAs 2536. prevent nocodazole. with BI to by treated FRET-ba of added mitosis cells the addition was in of expressing indicates MG132 synchronized ratios cells arrow treatment. cells indica emission 2536-treated The nocodazole of the CFP/YFP BI nocodazole. and ratios targeting of and thymidine emission siRNAs Quantifications control subsequent CFP/YFP with (F) show by bar of treated MG132. stills mitosis Scale Quantifications mitosis The in nM. (E) entering ratios. synchronized 100 exit. cells emission while of mitotic of CFP/YFP activity concentration ratios false-color-coded Plk1 a emission showing for at CFP/YFP movie biosensor used of a was Quantifications from 2536 (C) Stills BI cells. (D) mitosis. individual entering 10 activity of Plk1 deviation for biosensor FRET-based the 10 mitosis. expressing in cells activity 2536-treated Plk1 BI affects Aurora-A 3. Fig. m .()Qatfctoso F/F msinrto fclsetrn ioi ntepeec fteidctdihbtr.Errbr niaetestan the indicate bars Error inhibitors. indicated the of presence the in mitosis entering cells of ratios emission CFP/YFP of Quantifications (B) m. ˚ e ta. 08.Hwvr hnArr- sihbtd the inhibited, is Aurora-B when However, 2008). al., et rek A tlsfo oi hwn as-oo-oe F/F msinrto.Tesil hwcnrland control show stills The ratios. emission CFP/YFP false-color-coded showing movie a from Stills (A) ea nteiiilPk ciain nefc eddntobserve not did we effect an a activation, we to Plk1 led Next depletion initial activation Aurora-A the that transition. in the found delay we not monitored G2–M Again and 3C). does the (Fig. Aurora-B curve Aurora-B and at that Aurora-A Aurora-A activation as depleted show activity Plk1 results Plk1 influence These on effect alone. similar inhibition a shows inhibition ora fCl cec 21)17 0–1 doi:10.1242/jcs.137216 801–811 127, (2014) Science Cell of Journal e genes. ted btr and ibitors dard sed 805 s:

Journal of Cell Science nr,w ol eraeteCPYPrtob digB 2536, BI adding by ratio CFP/YFP the with mitotic decrease during could mitosis activity we Plk1 in time- monitoring entry, to by cells Similar determined imaging. were synchronized lapse ratios emission we CFP/YFP mitosis, nocodazole. in activation of activation initial the G2. controls is during it that Plk1 that Aurora-B show not results and these Aurora-A Overall depleted. was Aurora-B when ARTICLE RESEARCH aiso el rae ihsRA agtn h niae ee n hnsnhoie nmtssb ooaoe h ro niae diino the of addition indicates arrow The nocodazole. CFP/Y by of mitosis 806 Quantifications in (C) synchronized CFP/YFP inhibitors. then of indicated and Quantifications and genes (B) genes indicated Plk1. indicated the and the targeting 1 T210 targeting siRNAs inhibitors. with phosphorylated siRNAs with treated indicated Aurora-A, with treated were of treated cells cells presence mitosis of 2A, the entering ratios Fig. for cells in analyzed of as were ratios mitosis activation. Lysates emission in Plk1 shake-off. synchronized and mitotic were phosphorylation by cells T210 harvested After abolishes Aurora-A. Aurora-A targeting of siRNAs inhibition Complete 4. Fig. of inhibition pharmacological or 2011). depletion Lindqvist, and RNAi-mediated (Medema Since in mitosis increase huge in a phosphorylation induce and protein kinases to mitotic lead of can activation loops rapid feedback positive very that established activation well been Plk1 has prevents It Aurora-A of inhibition mitosis. Complete in activation in a for Plk1 provide role activation global not a to does Plk1 exclude Aurora-B contribution not that major to initial do indicate clearly data for contributes they our responsible Aurora-B, also although addition, only but These In not 3F). mitosis. Plk1 complete is (Fig. of observe condition Aurora-A not activation any that did in show In also activation results activity. were Plk1 we cells of Plk1 kinases when observed inhibition addition, aurora total In not never of activity. did the Plk1 depleted inhibition we in of Aurora-B change results, any However, 30% induce earlier our than activity. with 10– more accordance initial from of by ranging the inhibition induced variable, of somewhat activity was mitosis 30% Plk1 inhibition in the although of in activity reproducible, highly extent Plk1 was decrease kinases in aurora of partial of reduction inhibition inhibition CFP/YFP This small contrast, 3E). The a In (Fig. to 3E). 2003). the (Fig. led for experiment al., Aurora-A constant the to remained et of cells In added duration DMSO-treated (Ditchfield in inhibition was by ratio 3E). activity induced (Fig. emission be MG132 aurora can imaging that inhibitors inhibitor of exit mitotic during Aurora-B-specific proteasomal premature prevent and the ZM447439 Aurora-A- addition, and to mitosis the tested in MLN8054 biosensor we activation adding Plk1 Next FRET-based to 3D). kinases by (Fig. the different cells of use mitotic contribution the in also activity can Plk1 monitor we that showing omntrtecnrbto fArr- n/r- oPlk1 to -B and/or Aurora-A of contribution the monitor To l1atvto nmtssi Aurora-A. for is responsible mitosis kinase in major activation the Plk1 that by indicating achieved strategy, be similar can mitosis a in Plk1 of inactivation of addition, activation In prevent Plk1. fully can Aurora-A indicate of observations inhibition effective These that combined. and depletion are when Aurora-A near mitosis of a in inhibition indicating activity cells, Plk1 -inhibited of shutdown or After complete Plk1-depleted levels activity. in to the reduced that Plk1 was of to activity of close Plk1 most treatment decrease MLN8054 of of marked hours depleted a 4 already in cells resulted to Aurora-A MLN8054 of addition diino L85.A eosre eoe elto of of of depletion reduction activity before, a Plk1 produced observed MLN8054 the mitotic we with by inhibition Plk1 As Aurora-A or inhibit of itself Aurora-A MLN8054. fully depleted Plk1 of could already of we cells addition whether inhibition mitotic tested in or and activation we depletion depletion Next, Plk1 to after 4B). similar of in (Fig. observed activation, Plk1 combination delay we of inhibition a a what full However, in caused of resulted inhibition G2. Aurora-A inhibition CFP/YFP full in of before, if the seen depletion activation As see extent. monitored or greater to a we inhibition to conditions activity Next Plk1 these affects Aurora-A 4A). in (Fig. ratio samples unaffected emission largely were all Plk1 of in levels Total in 4A). with not Plk1 (Fig. treated simultaneously MLN8054 T210-phosphorylated could and Aurora-A we to of depleted MLN8054, corresponding cells mitotic with band treated the or detect Aurora-A cells in of Plk1 T210-phosphorylated depleted residual Indeed, detect mitosis. did in we the activity although inhibit Plk1 to monitored to MLN8054 and added protein, attempt then remaining and an depleted RNAi through first In protein we 4A). the activity Aurora-A (Fig. of Aurora-A inhibition complete residual achieve of a detected amount after we Aurora-A activation small depleting Plk1 when of sustain Indeed, pool mitosis. to small into enough entry a be that might reasoned Aurora-A we active complete 100% never is kinases uoaArqie eea ifrn oatvtr oeetits exert Macu to 2002; co-activators al., and et Plk1 different of (Kufer activator several functions main the requires is Aurora-A Aurora-A that mitosis find in we activity Because regulate Bora and Aurora-A ora fCl cec 21)17 0–1 doi:10.1242/jcs.137216 801–811 127, (2014) Science Cell of Journal A el eetetdwt ihrcnrlsRA or siRNAs control either with treated were Cells (A) m L85 ntepeec fM12and MG132 of presence the in MLN8054 M , 03%(i.4) Interestingly, 4C). (Fig. 10–30% ˚ e ta. 08 eie al., et Seki 2008; al., et rek Pemission FP

Journal of Cell Science hf nBr eree rmmttccls swl sardcinof reduction a as well as mobility cells, 5A). we clear mitotic (Fig. from a Bora retrieved mitosis observed This Bora study and in we protein. shift to literature G2 previous endogenous in order with the Bora In Consistent recognized detect proteins. clearly to two antibody antibody these look an closer of a generated take role to decided the the we so mediate at co- TPX2, characterized and would best Bora The are co-activator activators Aurora-A. by which Plk1 of wondered phosphorylation we 2008b), ARTICLE RESEARCH ihsRA agtn h niae ee n hnsnhoie nmtssb hmdn n ooaoe h ro niae diino h inhibito the of addition indicates tre arrow cells The of nocodazole. ratios and CFP/YFP Quantification emission thymidine of (E) CFP/YFP by Quantifications inhibitors. of mitosis (D) indicated Quantification in 2B. the (F) synchronized Fig. of siRNAs. then in presence indicated and as the the nocodaz genes MG132. treated in with with indicated were and and transfected treatment the Cells siRNAs mitosis targeting overnight (C) indicated entering after siRNAs band. the cells shake-off with non-specific with of mitotic a transfected ratios by indicates mitosis emission obtained Asterisk entering CFP/YFP were 4A. cells (M) Fig. of samples in ratios Mitotic as emission release. treated thymidine were after Cells mitosis. hours in (B) 7 activity harvested regulate were Bora cells and Aurora-A 5. Fig. leads phosphorylation Plk1 with T210 Aurora-A- cope reduced to Because mechanism activity. the of a Aurora-A provide consequence reduced in could This a G2. increased as during activity possibly were samples, 5C). levels phosphorylated (Fig. depleted cells as Bora knockdown effect, co-depleting double similar the Interestingly, in a When undetectable that saw was 5B). we T210 indicating (Fig. Bora perturbs and MLN8054, phosphorylation severely Aurora-A T210 complex with Bora–Aurora-A marked mitotic treatment the a reduce with by could observed interfering we We further which depletion. Plk1, even Bora T210-phosphorylated T210 in after phosphorylated and at decrease cells cells, looked next mitotic mitotic of We in reduction in 5A). further present a (Fig. there in Bora Bora resulted However, mitotic of RNAi 2008a). through amount Bora al., of small et a depletion a Seki in 2008; still degraded al., was being et is (Chan Bora manner as levels, total b -TrCP-dependent A 2Sclswr rnfce o 8huswt iNs ooti 2smlsU2OS samples G2 obtain To siRNAs. with hours 48 for transfected were cells U2OS (A) uigmtssi ufcett eit h Aurora-A-dependent the mediate left to is a that sufficient Bora or of is Plk1 pool small mitosis T210-phosphorylated the that during of suggest results S4). loss (supplementary Fig. these material Together in (supplementary further activity Plk1 result any in reduction co-factor substantial not activity Aurora-A other did the the of TPX2 lower depletion Also, S3). not Fig. material did Aurora-A of -B of pool inhibition Aurora- further or small and However, cells. Aurora-A this Bora-depleted both in for inhibited B responsible we complete. activity is not Plk1 also Aurora-B albeit remaining almost Bora whether activity, Plk1 and test levels of To Aurora-A reduction cells to of clear addition, a co-depletion displayed reduced In after cells. was mitosis Bora- mitotic with entering these activity Plk1-depleted to compared with Plk1 MLN8054 overlapping when adding cells, When reduced in in 5F). depleted activity we (Fig. also activity Plk1 cells Plk1 was mitotic Next, control that down cells 5E). observed shut (Fig. we Bora-depleted also Indeed, co-depleted could cells. mitotic were we which in whether Plk1-depleted cells Bora in examined activity of the was and Plk1 on that effect MLN8054 Aurora-A depletion similar a mimicked through was There Bora completely cells. inhibition Plk1 curve when Aurora-A of activation inhibition However, with partial 5D). combined to (Fig. led FRET-based Bora activation our of with activity Depletion monitored bio-sensor. we Plk1 of activation to ora fCl cec 21)17 0–1 doi:10.1242/jcs.137216 801–811 127, (2014) Science Cell of Journal sof ated ole. rs 807

Journal of Cell Science oiatvto fPk n oso hshrlto fT1 we T210 of phosphorylation of loss and led Plk1 Bora of and Aurora-A inactivation of to co-depletion that observed loss we to Because similar T210 of phenotypes produces phosphorylation Bora of and Aurora-A of Depletion and phosphorylation T210 lost. Plk1 small mitotic is this that activity when absent only is Bora is of It pool Plk1. of activation and phosphorylation ARTICLE RESEARCH E nuil y–l1T1DUT el eetetdadfxda nC ro asrpeettesadr eito ftreidpnetexperiments independent three shake-of of mitotic deviation by standard harvested the finally represent in and bars to nocodazole Error tetracycline with C. with treated in 808 treated were as or cells fixed untreated hours and left 16 treated and i last were three hours the cells of 48 For U2TR deviation for ( T210D. myc–Plk1-T210D standard siRNAs spindle Plk1 the Inducible with monopolar myc-tagged indicate (E) transfected a bars exogenous were Error and the microscopy. cells panel) of through U2TR (upper expression determined myc–Plk1-T210D spindle was 5 Inducible ind bipolar spindles bar: (D) bars a monopolar Scale Error experiments. displaying showing panel. staining. cells cells lower MPM-2 mitotic mitotic on the of of based in percentage cells Examples indicated mitotic (B) are percentage experiments. Stainings the independent determine panel). Plk1. three to (FACS) of of sorting phenotypes deviation cell T210-dependent standard fluorescence-activated mediate by depletion analyzed and Bora siRNAs and Aurora-A 6. Fig. prominent mitotic a the to in 5%. spindles leads monopolar than scored Bora next more and We indicating not arrest. Aurora-A cells, mitotic was of mitotic co-depletion this 20% in that but an to resulted to indices, co-depletion this lead did However, mitotic elevates Bora and in Plk1 Aurora-A of of increase depletions depletion of Single 6A). cells, after (Fig. formation around mitotic hours have 48 the of cells mitotic therefore 2% control to were We growing that due Asynchronously cells 1988). transfection. of Glover, arrest number and the mitotic phenotypes determined (Sunkel observe a spindles of to is monopolar phenotype able prominent depletion most be The Plk1 also Plk1. inactive should with associated we that reasoned m .()Clswr ie n tie,a nB 8husatrtaseto ihsRA.The siRNAs. with transfection after hours 48 B, in as stained, and fixed were Cells (C) m. , 30% uat norcnrlsmlsw a lgtices in mutant active increase constitutively slight a that a This fact mutant. the saw T210D to attributed the we be of might samples expression upon control spindles Plk1 monopolar our active constitutively phenotype this In of spindle presence mutant. monopolar and a absence the had the both that scored in then cells We mitotic 6D). of active (Fig. fraction was activator Plk1, that upstream endogenous mutant its deplete Plk1 of to a regardless able reconstitute a are and we Bora of and way phospho-mimicking Aurora-A this use (Macu In siRNA 2009). made Plk1 a al., to et We resistant line with mutant. cell tetracycline-inducible Plk1-T210D this phenotype rescue active to proceeded constitutively spindle we phosphorylation T210 monopolar of loss the a to with depletion. cells Plk1 arrested with Aurora-A the of occurred of proportion as elevated similar spindle co-depletion a monopolar in mildly but resulted spindles, Bora depletions and monopolar Single of this displaying number 6C). cells (Fig. mitotic Monopolar the of phenotype 6B). 60% but in (Fig. population, resulted control depletion conditions the Plk1 in different occasionally occurred the only spindles in present cells ots hte oooa pnl omto a neddue indeed was formation spindle monopolar whether test To A 2Sclswr avse 8husatrtaseto with transfection after hours 48 harvested were cells U2OS (A) ora fCl cec 21)17 0–1 doi:10.1242/jcs.137216 801–811 127, (2014) Science Cell of Journal ndependent ct the icate duce lower ˚ . f. rek

Journal of Cell Science pnlsosre nBr–uoaAdfcetclsaea es in least at are monopolar cells the deficient Bora–Aurora-A that in Bora– shows observed of observation spindles functions additional this to However, of due the Aurora-A. rescue be fraction completely might to this the unable but were phenotype, We reduce 2005). 6E). al., (Fig. could Aurora-A–Bora- cells et and depleted we Plk1-depleted Weerdt the both de that in (van spindles which monopolar observed itself mitosis, by in we delay activity However, mitotic of a regulation in refined results its lacks Plk1 of ARTICLE RESEARCH iety(hne l,20;Sk ta. 08) nti a twould Plk1 it way bind this to In 2008a). known al., is et Seki Bora 2008; al., because et Plk1 (Chan directly be with mutually might contact complex not selective direct Bora–Aurora-A in two, the the of envision First, is scenarios. addition why We exclusive then the MLN8054. mitosis, to inhibitor in refractory Aurora-A Plk1 Bora– activity of if Plk1 activator But mitotic major phosphorylation. the T210 is of Aurora-A loss to complete leads almost also Aurora-A, an with residual combined the Aurora-A, of mitosis. of the inhibition depletion phosphorylation pharmacological Similarly, in of for T210. loss or at phosphorylation complete Plk1 responsible depletion to of T210 leads with is Aurora-A the combined of it Bora inhibition of of that all, depletion conclude siRNA-mediated not to if there reasons feel majority, we good this, prove unequivocally are cannot data mitosis. our in Although activation Plk1 is for Aurora-A responsible that kinase past, plausible primary most the the personal seems in Archambault, it Thus, (V. antibody communication). batches Indeed, its between antibody. variability discontinued the phospho-specific claiming temporarily from of has stem batches Abcam could different of observations immunoblot by use contrasting confirmed is not These RNAi a signal is after analysis. not that kinetochores this this possible at but does remains course phosphorylated, that that of highly cells Plk1 is indicating of It human amount epitope. depleted, we small different in al., a is to signal et Plk1 in corresponds Carmena kinetochore when unlike Polo indeed the disappear However, al. that 2012). of et found al., Carmena activation et by (Carmena Aurora-B-dependent work limited a recent Drosophila during that or The locations time. suggests specific small at of a mitosis phosphorylate period cannot in can data Plk1 Aurora-B Our of that best. pool possibility at the limited out very rule is a Plk1 of to phosphorylation Plk1, point of mitosis. activator in data primary and the interphase, these as in Aurora-A together, both and Bora Taken is of Plk1 mitosis. complex cells that once and entered inactivation cells, to and have mitotic sufficient dephosphorylation activation. in is to state Plk1 refractory activity active quite Aurora-A continued its little in for very Plk1 crucial maintain that cells, is show mitotic in we fraction retained are Also, is this Bora Aurora-A of that pool and show has total and We the Bora mitosis. cell of in fraction that activity a a Plk1 that once of show maintenance sustained the we raising for is required Here, 2008a), activation al., mitosis. upon et Plk1 degraded Seki entered how 2008; be al., question to et shown the (Chan was mitosis Bora (Macu into However, entry Bora Aurora- 2008b). cofactor by al., cycle its cell et the with of together phase G2 A the during activated is the Plk1 as act DISCUSSION Bora and mitosis. in Aurora-A Plk1 of that activators predominant Together demonstrate T210. at results phosphorylation Plk1 these aberrant of result the part u aaso httecnrbto fArr- oT210 to Aurora-B of contribution the that show data Our u steBr–uoaAcmlxtesl ciao fPlk1? of activator sole the complex Bora–Aurora-A the is But srsrce oavr hr eidi mitosis in period short very a to restricted is ˚ e ta. 08 Seki 2008; al., et rek osu onPk ciiywe oaArr- a inhibited was Bora–Aurora-A when activity Plk1 ( time more down much takes shut it that to found of we inhibition Indeed, kinase. to upstream refractory result, its relatively a be would As activity cells. T210 Plk1 mitotic Second, mitotic rapidly, in MLN8054. inhibited for very be again target might exchanged occur dephosphorylation is inhibit could ATP phosphorylation the in ATP-competitive completely before ATP T210 an for to complex, exchanged for this is MLN8054, MLN8054 impossible, Whenever as not phosphorylation. such if difficult, compound very be hsh-pcfcPk-T1 a bandfo ba n BD and Abcam from (Macu described obtained previously was Anti-Plk1 was biosciences. Plk1-pT210 Phospho-specific reagents and siRNAs Antibodies, METHODS AND MATERIALS ooo fPk Eisne l,20) siRNA-mediated 2004). al., et the Plx1, (Erikson with Plk1 loop feedback of positive LOK homolog a and in SLK the function kinases of these Both homologs Ste20-like 2003). are al., the et kinases Walter as 2000; al., such et (Ellinger-Ziegelbauer Plk1, on T210 is mitosis. dephosphorylation of T210 stages different how the resolve Further at to 2012). controlled required al., be et will (Kachaner work loop a recently feedback uncovering optineurin, was positive its through Plk1 potential activity 2008). MYPT1 and al., affect et to PP1 dephosphorylate (Yamashiro shown cells mitosis. to mitotic shown in in been T210 have active MYPT1 fully subunit T210 regulatory for not responsible phosphatase is the dephosphorylation that suggests This 4C). Fig. otealo-ohn eiino el ntebiko mitosis. of brink the on cells of decision Aurora-A– all-or-nothing the involved the contribution for important to also action an provide of is could mode loop itself activation a Bora–Plk1 Plk1 Such cyclin-B– 2009). which regulating al., in in et (Lindqvist role process a a play activation, This to 2009). Cdk1 known al., is et regulation (Lindqvist activation cycle of off own them type their switch cell to enforce systems threshold, difficult systems render it high these making Bistable a on, that at switched system. occurs once loops on but bistable switching feedback Plk1 as irreversible a interlinked transition Aurora-A–Bora-mediated as of the act consist that could suggest activation slowly occurs inactivation Plk1 very that and Aurora-A–Bora of Aurora-A. amounts by small kinetochores controlled is the Plk1 at specific, of activation very majority Plk1 the a controlling while has in Aurora-B it right role that Thus the localized possibility regulation. be can spatiotemporal distinct not restricted a biosensor therefore such remains might up FRET-based pick this to and Our tool early cells in in kinetochores. window diffuse time the rapidly distinct very at in a of at mitosis phosphorylation occurs partially that Aurora-B Aurora- Polo postulate by for al. mitotic least Polo role et human of in a Carmena activation at However, for and cells. support phosphorylation activation Plk1 any is global find in B not Aurora-B the do it we that presented Interestingly, for data plausible the From responsible the very 2012). in al., implicated et seems (Carmena was in cells both Aurora-B cells, human mitotic recently, in (Walter T210 Polo–Plk1 More lymphocytes of of activation of 2003). cycle loss in al., cell exerted the only in et as or marginal such result is situations effect not specific shown). their not that did (data indicate might mitosis LOK This or interphase and in either SLK phosphorylation, of depletion niPk F) niTX (H300), anti-TPX2 (F8), Anti-Plk1 , eea te iae aebe ugse ophosphorylate to suggested been have kinases other Several u idnsta utie l1atvto nmtssrle on relies mitosis in activation Plk1 sustained that findings Our or)ta hnw ietyihbtdPk ( Plk1 inhibited directly we when than hours) 4 ora fCl cec 21)17 0–1 doi:10.1242/jcs.137216 801–811 127, (2014) Science Cell of Journal Xenopus c tbln(13 n niatn(I-19) anti-actin and (H183) -tubulin lk,wihi nw to known is which Plkk1, Drosophila ˚ e ta. 2008). al., et rek , Drosophila 0minutes, 30 Xenopus n in and 809 .

Journal of Cell Science 20n;Rce n erccie(1 tetracycline and MLN8054 Roche) Pharma), nM; (250 Ingelheim (1 Boehringer MG132 nM, (100 2536 (1 BI used: n h eobnn rti ue oahsiietgwspurified was tag histidine instructions a manufacturer’s to the following fused (Qiagen) resin protein Ni-NTA The recombinant using (Novagen) 79–559. vector the pET-28a residues in and cloned acid was amino fragment recombinant cDNA comprising against corresponding Bora raised from was human 9E10) antibody (clone His-tagged anti-Bora anti-Myc rabbit anti-Aurora-B and The Biochem, Laboratories) Covance. Cortex Transduction from (BD CREST Anti- from Millipore. Sigma, from were from were was anti-Aurora-A-pT288 H3-pS10 and tubulin and MPM-2 Anti-Aurora-A Signaling. Cell Cruz. from Santa from were ARTICLE RESEARCH ihtepopoyae 20atbd htwspeaee ihAlexa 810 with prelabeled sequentially was that performed antibody was T210 T210 phosphorylated phosphorylated the (Macu and with were Plk1 previously analysis of described immunofluorescence staining for as staining performed antibody and Fixation analysis FRET and Immunofluorescence mM 1 7.4, pH HEPES, mM (50 buffer lysis in MgCl extracted were Cells blotting western and Immunoprecipitations 10 Cell counting cytometry, iodide. described. flow as by propidium ethanol events, determined with was ice-cold distribution counterstained in stage cycle fixed and Alexa-Fluor-488-conjugated and and antibodies MPM-2 into harvested with secondary stained were release determine were To Cells cells 16-hour mg/ml). (70%). index, (1 PLK1 tetracycline a mitotic of of the addition expression by by assays, induced thymidine followed reconstitution was T210D For with treatment ng/ml). synchronization (250 hours) nocodazole through 24 obtained mM, (2.5 were cells Mitotic synchronization Cell the to using according instructions. performed For with Technologies) manufacturer’s were times (Life siRNAs indicated reagent (Lonza). medium of RNAiMAX the Lipofectamine Transfections for serum in treated mg/ml). grown (1 were bovine tetracycline cells were expression, fetal clones of treatment induction Stable Tet-system-approved Invitrogen) selection. mg/ml, (400 containing clonal zeocin by by clones followed and stable transfection of by generated selection promoter were biosensor tetracycline-inducible FRET-based (Macu the a expressing of previously control described streptomycin. Plk1 the were and mg/ml under T210A 100 Plk1 mutants PLK1, and T210D wild-type penicillin myc-tagged expressing U/ml lines 100 (Lonza), Cell modified FCS L-glutamine, 10% Dulbecco’s mM with in supplemented 2 grown Gibco) (DMEM; were medium cells Eagle’s U2OS osteosarcoma Human transfections and culture Cell (5 2004) al., et pSuper the Vugt on (5 based was (van insensitive UGCAGATCAAC-3 is previously Short mutant T210D described Dharmacon. Plk1 the from which (5 were Ambion Bora and and targeting Probes Aurora-A RNAs negative Molecular a interfering (as and from GAPDH or were luciferase ON- control) targeting Dako. 633 pools Alexa from smart Fluor TARGETplus 488, antibodies Alexa Fluor secondary and Alexa horseradish-peroxidase-coupled 568 antibodies Secondary Fluor immunization. before moiie npoenA(i-a) muoopee were for sample Laemmli Samples Immunocomplexes or immunoblotting. buffer by immunoblotting. lysis analyzed (Bio-Rad). either and by in buffer prepared analyzed were A blotting and western protein washed extensively on immobilized vrih 1 or)a 4 incubated at and content hours) (15 protein total for overnight normalized inhibitors), protease ta. 08.Oiouloie targeting Oligonucleotides 2008). al., et 9 m -GGAAAGAAGGGAUCCCUAA-3 ,Mlenu hraetcl) M449(2 ZM447439 Pharmaceuticals), Millennium M, 2 MET,1 P4,1m a,1m Na mM 1 NaF, mM 1 NP-40, 1% EGTA, mM 1 , 9 m -GCUCUGUGAUAACAGCGUG-3 ;Sga,ncdzl 20n/l im) reversine Sigma), ng/ml; (250 nocodazole Sigma), M; 9 n iuoaBwsotie rmDharmacon from obtained was siAurora-B and ) TPX2 ˚ ihplcoa niPK antibody anti-PLK1 polyclonal with C ˚ e ta. 08.UO el stably cells U2OS 2008). al., et rek eedsrbdpeiul (Macu previously described were m 9 /l Sigma). g/ml; .Tefloigduswere drugs following The ). ˚ e ta. 08.Double 2008). al., et rek Plk1 9 n nsRAto siRNA an and ) eeotie from obtained were m ;AstraZeneca), M; 9 -CGGCAGCG- 3 VO 4 ˚ and rek a 4 - nest.TeFE-ae rb o oioigPK ciiyhas activity maximum PLK1 monitoring kinetochoral for and (Macu probe previously described been FRET-based centrosomal The the intensity. as measuring 40 performed using was immunofluorescence system described, of imaging Quantification RT deconvolved objectives. Deltavision NA of a projections on intensity acquired maximum Z-stacks, show Images 488. Fluor rvtto rga acreoisn t ..;teGatAec fteCzech the NWO of the Agency Grant W.B.], the and R.M.]; R.M. [to CancerGenomics.nl [to Program Research Initiative Gravitation Scientific Genomics for Netherlands Organisation the Netherlands the (NWO): by supported was work This Funding and W.B. paper. antibody. the an R.M. wrote generated and R.F. R.H.M. L.M. experiments. A.L., designed experiments. and performed conceived and designed conceived, W.B. contributions Author interests. competing no declare authors The siRNA. Aurora-B interests for Competing Netherlands) The on Utrecht, comments (UMC, and Lens discussions M. for S. laboratory and Medema paper the the of members the thank We Acknowledgements rhmal,V n lvr .M. D. Glover, and V. Archambault, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.137216/-/DC1 online available material Supplementary A.L.]. [to material Research Supplementary Cancer Cancer for Child Foundation Swedish Swedish the the A.L.];, and A.L.]; [to A.L.]; [to Research [to Council Strategic Foundation for Research Society Swedish Swedish the R.F.]; the to CDS2007-0015 2010 number Economı CONSOLIDER-Ingenio [grant de SAF2010-22357]; Ministerio number L.M.]; [grant to Competitividad 13-18392S number [Project Republic 0mnts h mgswr rcse ihIae sn h ai Plus Ratio the using ImageJ with (http://rsb.info.nih.gov/ij/). processed plug-in were system, images FRET- The imaging minutes. the Elite 20 expressing 20 Deltavision stably a a on cells using monitored U2OS was of biosensor, excitation based CFP after ratio risa . aimkr,J .adMdm,R H. R. Medema, and A. J. Raaijmakers, W., Bruinsma, A. E. Nigg, and J. Pines, L., Arnaud, yr,P . rko,E,Ce,L .adMle,J L. J. Maller, W. and M. G. Kirschner, and L. H. Yu, Chen, G., Fang, E., Erikson, A., P. Eyers, T., Johnson, C., Haworth, R., Ellston, A., Tighe, L., V. Johnson, C., Ditchfield, Sillje A., Santamaria, Y., H. E. Chan, Macisaac, A., Clark, Z., Xu, Z., Salloum, M., Platani, X., Pinson, M., Carmena, rko,E,Hyta,T .J,Qa,Y-.adMle,J L. J. Maller, and Y.-W. Qian, J., A. T. Haystead, E., Erikson, K., Tsujikawa, E., Yamada, H., K., Hoepker, Karasuyama, J., F. H., Ivins, Ellinger-Ziegelbauer, W., J. Chao, F., L. Haire, P., Rellos, H., E. A. B. Elia, M. Yaffe, and C. L. Cantley, H., E. A. Elia, Ko C., Schubert, von Z., Dou, iegnei hi ucin n regulation. and functions their in divergence iekns ttekntcoecnrmr eino ioi chromosomes. mitotic of region kinetochore/centromere the at Chromosoma 1 kinase like aiymmesatvtsteaahs-rmtn ope nmtssadG1. and mitosis in complex Cell anaphase-promoting Mol. the activates members family A. Aurora kinase protein the of activation for mechanism to Cenp-E and Mad2, BubR1, targeting by anaphase kinetochores. with S. S. Taylor, alignment and N. Keen, A., Mortlock, of degradation betaTrCP-dependent through hBora. function A Aurora mitotic centromeres. regulates al. at et kinase M. D. Polo Glover, activates e1001250. U., complex Eggert, passenger H., Ogawa, space. and F., time in off and on kinase-1 like iaefrpl-iekns Pk uigteG/ rniini oai cells. pathway. somatic activation kinase in polo-like the transition in loop G2/M the during (Plk) Cells kinase Genes polo-like E. for Nishida, kinase and of K. Todokoro, regulation and domain. targeting Polo-box substrate B. the M. by phosphodependent Yaffe, Plks and for J. S. basis Smerdon, molecular C., L. Cantley, D., Mohammad, substrates. mitotic to Plk1 1228-1231. localizing domain Plk1. pSer/pThr-binding and kinase Mps1 human for motif consensus A. E. 21) uniaiems pcrmtyaayi eel iia substrate similar reveals analysis spectrometry mass Quantitative (2011). Chromosoma 2 ora fCl cec 21)17 0–1 doi:10.1242/jcs.137216 801–811 127, (2014) Science Cell of Journal 6 163-171. , 5 .5N betv.Iae eeaqie vr 0or 10 every acquired were Images objective. NA 0.75 .Cl Biol. Cell J. 107 491-498. , 424-429. , 117 457-469. , 161 nr . atmra . lw,S n Nigg, and S. Elowe, A., Santamaria, R., rner, ¨ 267-280. , Cell 20) t2-iekns SK,aregulatory a (SLK), kinase Ste20-like (2000). ˚ e ta. 08.TeCPYPemission CFP/YFP The 2008). al., et rek 115 20) oolk iae:cnevto and conservation kinases: Polo-like (2009). ,H .W n ig .A. E. Nigg, and W. H. H. ´, 19) F agn eel ua Polo- human reveals tagging GFP (1998). 19) ietbnigo D2 protein CDC20 of binding Direct (1998). 83-95. , 20) uoaBculschromosome couples B Aurora (2003). a.Rv o.Cl Biol. Cell Mol. Rev. Nat. rnsBohm Sci. Biochem. Trends .Bo.Chem. Biol. J. 20a.Poemcsre finds screen Proteomic (2003a). LSONE PLoS 21) h chromosomal The (2012). 21) wthn Polo- Switching (2012). ur Biol. Curr. 20) feedback A (2004). 279 20) novel A (2003). LSBiol. PLoS 6 32219-32224. , e18793. , Science 20b.The (2003b). 37 13 ay ´a 20) Plk1 (2008). 10 534-542. , 691-697. , 265-275. , 6 0.95 299 10 , ,

Journal of Cell Science afei .G,Esd,J . ete .A,Bln,S . uekv,O., Burenkova, K., S. Balani, A., K. Meetze, A., J. Ecsedy, G., M. Manfredi, Macu Macu Rodrı A., Lindqvist, J. Pines, and C. Lindon, Le Sillje A., T. Kufer, ag .H,Pr,J-. u .R,Sug .K,Yn .M,Bn,J . Seong, K., J. Bang, S.-M., Yun, N.-K., Soung, L.-R., Yu, J.-E., Park, H., Y. Kang, ahnr . iie . alnie . ac,A,Bnet .L,Superti- L., K. Bennett, A., Bauch, E., Laplantine, J., Filipe, D., Kachaner, ag .J,M,S,Trd,Y n rko,R L. R. Erikson, and Y. Terada, S., Ma, Y.-J., Jang, utrr . edi,D,WrzPiz . imn . clifr .and A. Schleiffer, M., Zigman, F., Wirtz-Peitz, D., A. E. Berdnik, Nigg, A., and Hutterer, M. A. Fry, E., K. Mundt, M., R. Golsteyn, ARTICLE RESEARCH ´na niuo ciiyo L85,a rlyatv ml-oeueihbtrof inhibitor small-molecule al. et active J. orally P. LeRoy, an J., J. kinase. MLN8054, Huck, A M., of Aurora K. activity Hoar, M., Antitumor K. Galvin, W., Chen, Polo. of game the satvtdb uoaAt rmt hcpitrecovery. checkpoint promote to A aurora by activated H. R. is Medema, and 1 B. M. Yaffe, S., S. Taylor, C., Clouin, network. entry mitotic the in 185 redundancy and feedback mitosis: enter cells. human in exit mitotic 1. proper kinase to contribute polo-like to Plk1 of roles J.-M. mitotic Peters, into insights and novel Biol. N. Curr. reveals Kraut, 2536 BI J., inhibitor W. spindle. Rettig, the to M., kinase Aurora-A targeting for Biol. required Cell is J. TPX2 Human (2002). erimn okntcoe ytePk-BP neato sciia o proper for critical is al. interaction et Plk1-PBIP1 D. the segregation. T. by chromosome Veenstra, kinetochores S., to recruitment Garfield, H., Yu, Y.-S., piernpoie eaiefebc ehns o ioi progression. mitotic for mechanism feedback Cell negative Mol. a provides optineurin Israe G., Furga, henn 1 n h oeo eie17i h euaino amla polo-like mammalian of regulation the in 137 serine of kinase. role the and 210 threonine idn ate Bora. partner binding protein A. human J. a Knoblich, Plk1, function. of spindle localization mitotic subcellular in and implicated activity kinase the of regulation r,P,Ptock,M,Segae,M,D ir,B,Lp,J . Hoffmann, J., J. Lipp, B., Fiore, Di M., Steegmaier, M., Petronczki, P., ´rt, ˚ ˚ e,L,Lnqit . i,D,Lmsn .A,Kopae,R,Fer,R., Freire, R., Klompmaker, A., M. Lampson, D., Lim, A., Lindqvist, L., rek, e,L,Lnqit .adMdm,R H. R. Medema, and A. Lindqvist, L., rek, 193-202. , .Bo.Chem. Biol. J. 45 17 553-566. , 158 304-315. , ,H .W,Ko W., H. H. ´, l .adWi,R. Weil, and A. ¨l, 617-623. , 20) ioi ciaino h iaeArr- eursits requires Aurora-A kinase the of activation Mitotic (2006). rc al cd c.USA Sci. Acad. Natl. Proc. ge-rv,V n eea .H. R. Medema, and V. ´guez-Bravo, acrRes. Cancer e.Cell Dev. 277 20) ree rtoyi naahs inactivates anaphase in proteolysis Ordered (2004). 44115-44120. , o.Cell Mol. nr . rs,O . ead,P n ig .A. E. Nigg, and P. Meraldi, J., O. Gruss, R., rner, ¨ 11 69 147-157. , 4555-4558. , 21) l1dpnetpopoyainof phosphorylation Plk1-dependent (2012). 24 409-422. , .Cl Biol. Cell J. 104 20) uoaAadhoajoin hBora and Aurora-A (2009). 4106-4111. , 20) h small-molecule The (2007). 20) efrgltdPlk1 Self-regulated (2006). 20) hshrlto of Phosphorylation (2002). .Cl Biol. Cell J. 20) oolk kinase- Polo-like (2008). 20) h eiinto decision The (2009). 129 Nature 19) elcycle Cell (1995). 1617-1628. , 455 164 .Cl Biol. Cell J. 119-123. , 233-241. , (2007). otne,E . as . yi . yi .P n elg,D R. D. Kellogg, and P. S. Gygi, M., Gygi, W., Haas, M., A. E. Mortensen, Lindqvist, and H. R. Medema, atr .A,Cte,R . r atnz . ihzy .adHl,R J. R. Hill, and M. Gishizky, R., Martinez, Jr, E., R. Cutler, A., S. Walter, Bra M., T. A. M. Vugt, van aahr,S,Ymkt,Y,Ttuaa . oo . abci . t,M., Ito, K., Kaibuchi, H., Goto, G., Totsukawa, Y., Yamakita, S., Yamashiro, a e al .S,Hneed .C . a e os,A n es .M A. M. S. Lens, and A. Horst, der van C., C. R. Hengeveld, S., W., M. J. Waal, der J. van Kauw, C., Lindon, M., T. A. M. Vugt, van M., C. B. Weerdt, K. de D. van Ferris, and L. D. Longo, M. T., Uchiumi, D. Glover, and E. C. Sunkel, G. Fang, and R. G. J. Fang, Yates, C.-Y., and Jang, III A., J. R., Coppinger, J. A., Yates, Seki, C.-Y., Jang, H., Du, A., J. A. Coppinger, Musacchio, A., and Seki, S. S. Taylor, M., C. A. W. D’Alise, A., Earnshaw, Tighe, and S., Santaguida, M. Carmena, S., Ruchaud, d2-eedn euaino h d5Pl kinase. Cdc5/Polo the of regulation Cdc28-dependent phosphorylation. 787-797. otosrcvr rmaG N aaeidcdars nmmaincells. mammalian in arrest damage-induced DNA Cell G2 Mol. a from recovery controls uui euae ioi yatgnzn oolk iae1. kinase polo-like antagonizing by mitosis F. regulates Matsumura, 1 and subunit J. D. Hartshorne, t1,anwmme ftepl-iekns iaefml ihyepesdin expressed highly family kinase kinase polo-like the tissue. of hematopoietic member new a Stk10, Complex. Passenger Chromosomal the Res. by control division Cell 1. (2012). kinase polo-like H. from deregulating R. by activity Biol. Medema, control Cell. and complex/cyclosome checkpoint F. assembly M. anaphase-promoting R. spindle Wolthuis, Uncoupling R., Klompmaker, (2005). J., M. Rozendaal, promoter. (PLK) kinase polo-like human control and poles. Plk1 spindle kinase abnormal displaying the activate cooperatively a entry. Aurora mitotic kinase the and mitotic controls Bora of degradation beta-TrCP-dependent progression. the and and Plk1- (2008a). biorientation chromosome reversine. in inhibitor molecule MPS1 small 190 of the through role checkpoint spindle the Dissecting (2010). division. cell conducting passengers: 73-87. , 318 1407-1420. , 15 ora fCl cec 21)17 0–1 doi:10.1242/jcs.137216 801–811 127, (2014) Science Cell of Journal 25 799-811. , 2031-2044. , .Cl Biol. Cell J. Science rnsBohm Sci. Biochem. Trends .Bo.Chem. Biol. J. 320 s .adMdm,R H. R. Medema, and A. ´s, 181 1655-1658. , 65-78. , 18) oo ioi uato Drosophila of mutant mitotic a polo, (1988). .Cl Sci. Cell J. 21) osigadsprsigmitotic suppressing and Boosting (2011). 278 a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 36 20) ysnphosphatase-targeting Myosin (2008). .Bo.Chem. Biol. J. 18221-18228. , 578-584. , 19) elccerglto fthe of regulation cycle Cell (1997). 89 25-38. , 20) oolk kinase-1 Polo-like (2004). ur Biol. Curr. 272 20) Chromosomal (2007). 9166-9174. , 8 15 798-812. , 20b.Bora (2008b). e.Cell Dev. 2033-2037. , .Cl Biol. Cell J. x.Cell Exp. (2005). (2003). 811 Mol. 14 ,

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