eerhIsiue iclsInFed,Lno CA3Y UK. 3LY, WC2A London Fields, Inn Lincolns Institute, Research ae Bancroft* James CENP-E- and pathways CENP-Q- dependent by promoted is congression Chromosome ARTICLE RESEARCH eevd2 etme 04 cetd3Nvme 2014 November 3 Accepted 2014; September 25 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This " Oxford, UK. of § 3RF, University OX1 Pathology, Oxford, of UK. Road, School 7AL, Parks Dunn CV4 South are William Coventry Sir Warwick, Biology, address: of Cell *Present attachments University Biomedical School, of Medical Division Warwick Building, amphitelic Biology from Cell Mechanochemical emanating Such the microtubules poles. is to which spindle that in attach attachments opposite biorientation, bipolar) kinetochores chromosome (or sister coincident amphitelic of is of process Congression establishment anaphase. the to segregated in are with poles chromatids sister spindle a before for opposite time metaphase of in remain period Chromosomes similar both plate. human metaphase In from the 2010). of equidistant al., takes et position process Walczak this 2010; a cells, al., et equator, (Kops spindle poles chromosomes spindle the which by at process align the is congression Chromosome INTRODUCTION Kinetochore, in Mitosis Congression, CENP-Q, function CENP-E, WORDS: central KEY a plays mechanisms. CENP-O congression CENP-Q evidence chromosome and coordinating provide of recruitment data phosphoregulation Plk1 our that in together, Taken role of stabilisation. its functions complex from the pulling independent Thus, depolymerisation-coupled and are intact. loading complex CENP-E kinetochores in CENP-O onto CENP-Q Plk1 the CENP-Q of loading leaves in the and affect mutation not phosphorylated point does is mutant that single S50A residue a a – by (S50A) abolished these of are Both to pulling. functions depolymerisation-coupled CENP-E to in of CENP-Q (Plk1) for recruitment recruitment-independent role CENP-E the kinase a for reveal polo-like further required We kinetochores. also targets is CENP-P, that – CENP-O, kinetochores CENP-U) (comprising and complex CENP-O CENP-Q a the – CENP-Q of that subunit show microtubule we through kinetochores Here, equator pulling. the (2) to depolymerisation-coupled and move distinct pole motor, lattice the two CENP-E near microtubule biorientating directed least the plus-end the at along the to by slide using chromosomes kinetochores driven of (1) congression is mechanisms: the Congression is equator. mitosis spindle of step key A ABSTRACT hs uhr otiue qal oti work this to equally contributed authors These uhrfrcrepnec ([email protected]) correspondence for Author 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,1114doi:10.1242/jcs.163659 171–184 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. ,§ hlpAuckland Philip , , 52 i n eut nteformation the in results and min 15-20 ` rsn drs:CU London CRUK address: Present nvivo in § aaiaP Samora P. Catarina , motnl,the Importantly, . ` ude hog h eod(edn)sse,adte hnslide then they and microtubule sister, (leading) stabilized second other the and through bundles (K-fibres) kinetochore– pre-existing fibres of lattice the microtubule with engage to also called shown can been Congression have kinetochores mono-orientated 1992). biorientation: on before Salmon, place and take Koshland Mitchison 1991; 1988; here al., al., et et (from (Coue pulling) depolymerisation-coupled depolymerising plus-ends to attached kinetochore–microtubule remaining states poleward- kinetochore the sister from spindle movement comes moving movements the these towards for pole congress force movements The switch to equator. sustained able from undergo are sisters chromosomes they away These because 1993). because al., and et motion (Skibbens poleward oscillatory between with compatible nla.I hsrgr,orpeiu okhssonta the that shown has required is work is (CCAN) previous pulling network our centromere-associated depolymerisation-coupled regard, constitutive this of In mechanism unclear. the from 2009; are al., within complexes sites et Ska attachment microtubule kinetochore. and Gaitanos end-on the Ndc80 the the 2009; form a Thus, to al., proposed 2010). form al., to et et inability (Daum Welburn the severe plate and a al., congression in metaphase results et chromosome complex in either (Schmidt failure of Ska plus-ends depletion autonomously Extensive microtubule to of 2012). able and complex) depolymerising control Ndc80 the track microtubules Ndc80 not through (but to complex state bind K-fibre Ska The directly depolymerising the can complexes dynamics. ensure net (3) end-on a kinetochore–microtuble and mediate in microtubules (1) remains for attachment depolymerising that maintain to required (2) proteins attachments, a involve microtubule–kinetochore molecules Such these must unknown. to largely mechanism the contrast are In pulling 2002). depolymerisation-coupled mechanisms, al., 2010). et termed lateral-sliding are (Putkey these al., – chromosomes a poles of spindle et however, polar the majority plate; around Kim trapped the metaphase remain a CENP-E, subset 2006; forming of congress, al., inhibition chromosomes or et depletion (Kapoor Following minus-end- plus-end-directed the CENP-E the or 2007) by al., kinesin et mediated (Yang dynein is motor directed kinetochores by chromosomes attached of sliding laterally The mechanisms. congression these position in to mechanisms multiple plate. have metaphase cells the on Thus, chromosomes 2011). al., Magidson 2011; et al., in et (Kitajima equator early spindle the conformation at kinetochores sister ring-like of biorientation ‘instantaneous’ a the favours that in prometaphase that reveals arranged oocytes mouse are biorientate. and chromosomes then cells kinetochores human in these 2009). work plate, al., Furthermore, metaphase et Cai the 2006; at al., Once et (Kapoor equator spindle the towards n nrwD McAinsh D. Andrew and hte h rcs fedo tahetcnb separated be can attachment end-on of process the Whether involved are that molecules the identify to is challenge key The " nvitro in ihthe with , 171

Journal of Cell Science eiw,sePreec n uaaa 01 cis and CCAN core McAinsh The 2011; 2013). Straight, Fukagawa, and Westhorpe and (for 2011; Perpelescu Meraldi, CENP-X) (H3) and 3 see Histone directly CENP-S reviews, to that CENP-W, 2010; and factors 17- CENP-N) (CENP-T, a core al., and nucleosomes is of (CENP-C CCAN set CENP-A et The a to 2009). bind contains (Amaro al., that et attachments complex Toso subunit 2007; end-on al., et of for McClelland required not formation is it that the but congression, chromosome normal for ARTICLE RESEARCH 172 Fig. material at (supplementary Plk1 Plk1 affect of not of localisation did S1B,C, S1B,C, polar depletion levels Fig. CENP-Q the material effect, the kinetochore-specific (supplementary reduced from 84% Moreover, n CENP-O CENP-Q by S1A). of kinetochores loss Fig. of as the material observations depletion in CENP-Q these (supplementary resulted of confirm kinetochores CENP-Q to to depletion subunits of able complex Kang that depletion were CENP-O 2008; other We demonstrated al., 86% of kinetochores. has et binding by the (Hori 2006) destabilises reduced work al., Previous was et 1B). protein (Fig. quantitative kinetochore-bound depletion CENP-Q the the Moreover, ( of that 1A). (Fig. population confirmed against levels reduced immunofluorescence were antibodies non-detectable levels protein total to (siRNA) with the depleted RNA that we demonstrated interfering CENP-Q small Immunoblotting CENP-Q, using human cells oligonucleotides. HeLa of in function protein the the investigate To of chromosomes accumulation polar causes potentially, CENP-Q of Depletion and, in RESULTS roles microtubule direct have kinetochore might pulling. they depolymerisation-coupled the that suggesting regulating to 2011), 2011). directly al., key al., bind et other subunits et CENP-U microtubules of (Kang and taxol-stabilised CENP-U 2008; recruitment CENP-Q of the al., the Moreover, T78 et in with residue interaction Hori role through phosphorylated Plk1 2006; a including components, al., plays kinetochore CENP-O The et also 2012). al., chromosome Foltz et flux complex Mchedlishvili 2007; microtubule 2010; al., timely et poleward al., McClelland and et plus- with stability poorly kinetochore–microtubule (Amaro K-fibre altering interfere is turnover, as such end CCAN well can of as extended depletion congression, subunits that comprises this shows which 2008). CCAN of analysis al., complex, Phenotypic function et understood. CENP-O (Hori the CENP-U complex the and However, CENP-Q the and CENP-P, 2014), includes CENP-O, (Basilico al., CENP-M which and et CENP-K CCAN, CENP-I, CENP-H, extended comprising the recruits EPQeF rngn sdfrteersu experiments the the rescue of depleted these expression for CENP-Q the used against affecting transgene without siRNA CENP-Q-eGFP protein the endogenous that been Fig. had material confirmed (supplementary that of vector cells empty 50% S1B,C, the to control-siRNA-treated with Plk1 tagged in transfected transgene of The observed eGFP levels vector. those kinetochore-bound protein empty siRNA- rescued an an fluorescent partially or with a (CENP-Q-eGFP) green with transfected CENP-Q treated were enhanced cells siRNA where resistant CENP-Q experiment or rescue control a performed we 6 5 .)(eaiet EPA olwn siRNA-mediated following CENP-A) to (relative 6.2) ,30kntcoe,3 el) ossetwt a with Consistent cells). 30 kinetochores, 300 3, n n 5 5 ,10pls 0cls.T ueotoftre effects, target off out rule To cells). 60 poles, 120 3, ,60kntcoe,6 el) Immunoblotting cells). 60 kinetochores, 600 3, nvitro in Aaoe l,21;Hua 2010; al., et (Amaro xeiet,anme fcrmsmsrmie rpe around trapped remained chromosomes of number a experiments, T plate Only by metaphase . chromosomes congression. a their formed all chromosome aligned had and cells of CENP-Q-depleted timing of 10.3% the determined and we (mRFP)-tagged Histone2B–eGFP T Taking protein 2). co-expressing fluorescent cells (mRFP– red HeLa movies monomer time-lapse living collected we 2006). phenotype, in (Hori al., this et cells assess (Kang further cells DT40 human To CENP-Q-knockout CENP-U-depleted or of 2008) al., images also et can in phenotype This observed 1C). here be (from Fig. chromosomes; poles polar spindle termed number the on a around spindle; trapped the were within the kinetochores chromosomes on of of effect striking subset that a a is had note of a kinetochores position however, from form conclusion did, CENP-Q to of We loss 1C,D). This cells the (Fig. CENP-Q-depleted plate architecture. of metaphase ability normal kinetochore the (supplementary with global MCAK major consistent a play in or not does Ska3 CENP-Q role that indicating Kif18A, S1A), Fig. Ndc80, material of kinetochores. binding to key Plk1 a of is complex recruitment CENP-O the the for that confirm platform data These 1A). (Fig. ieycnrsino otcrmsmsadta hyaealso are the they in that defective and chromosomes are most complex) of CENP-O congression the timely been thus (supplementary (and has CENP-Q kinetochores 2006). did al., which to et (Foltz CENP-P, as CENP-Q S2A–C) subunit Fig. recruit S2A–C), material CCAN to congression Fig. the and shown material of chromosome polar depletion targets (supplementary that same with siRNA the phenotype second anaphase a alone), Histone-2B–GFP- gave entering Finally, of CENP-Q 1E). imaging vector cells (Fig. live-cell cells polar of using chromosome of expressing by number of assessed that with the rate when twice with increased cells CENP-Q an approximately (compared the in of with resulted congression contrast, transfection also transgene proportion following transgene In ( chromosomes CENP-Q pole. the 48% polar siRNA-resistant to the reduced chromosomes at an vector situated greatly with empty were with transfection that transfection calculated. ( be kinetochores the and 84% to allowed in CENP-Q chromosomes resulted This polar of and position). with Depletion kinetochores) chromosome cells of mark the proportion (to mark (to CENP-A staining and DAPI against fixation before anaphase antibodies 2009) variable prevent al., with with et (to associated siRNA- (Klebig min stage) differences cycle 90 out an cell rule for therefore either MG132 next and with We onset with vector. cells empty them the an treated or transfected transgene CENP-Q–eGFP and resistant siRNA using (Hori defects congression and 2008). delay chicken al., mitotic in et a CENP-Q of with causes knockout consistent cells the DT40 is min, that This cells 114 showing chromosomes. within control work all anaphase previous congress of underwent to undergoing that 99% failed cells 19% with cells the Of compared CENP-Q-depleted 1E). min, mitotic (Fig. of 96 prolonged 26% within a anaphase only with chromosome with These associated 1D). delay, were Fig. in defects arrows congression (yellow pole spindle the 5 ncnrlcls(i.1,) ossetwt u fixed-cell our with Consistent 1D,E). (Fig. cells control in 95% elto fCN- i o,hwvr fettekinetochore the affect however, not, did CENP-Q of Depletion ae oehr hs aaso htkntcoe lacking kinetochores that show data these together, Taken CENP-Q of cells depleted we effects, off-target out rule To a ora fCl cec 21)18 7–8 doi:10.1242/jcs.163659 171–184 128, (2015) Science Cell of Journal tbln i.1;splmnaymtra ois1, Movies material supplementary 1D; Fig. -tubulin, 5 ob h on fncerevlp breakdown, envelope nuclear of point the be to 0 6 .% fmtpaeclshvn unaligned having cells metaphase of 3.5%) 6 % Fg FG.Ti euto in reduction This 1F,G). (Fig. 8%) 5 4mn oprdwith compared min, 24 a -tubulin

Journal of Cell Science EERHARTICLE RESEARCH aiymme EPEi ua el Pte ta. 2002; al., et CENP-Q whether (Putkey investigated therefore cells We 2003). human al., 7 in et kinesin Weaver the CENP-E of depletion is member the cells for family reported CENP-Q-depleted that of in reminiscent phenotype very kinetochores chromosome onto polar CENP-E load The to required is plate. CENP-Q metaphase positioned the are to that pole chromosomes spindle of the subset near a transport to unable 1. Fig. e etpg o legend. for page next See hntpsascae ihCN- elto utbe must CENP-E. depletion and Thus, CENP-Q both CENP-Q kinetochores). losing of composite 100 on with a CENP-E as 2A,C; considered of (Fig. associated levels 80% the phenotypes reduced by also kinetochores). kinetochores CENP-Q 150 2A,C, of motor (Fig. the Depletion 95.6% of by levels cells kinetochores the on reduced of protein CENP-E We treatment targeting kinetochores. siRNA the an onto that with CENP-E immunofluorescence of with loading confirmed the for required was ora fCl cec 21)18 7–8 doi:10.1242/jcs.163659 171–184 128, (2015) Science Cell of Journal 173

Journal of Cell Science rae el hthdbe rnfce ihteepyvector load empty to the required is with CENP-Q kinetochores. that onto transfected indicate CENP-E data been These had 2D,E). (Fig. that cells treated bcrn h rti ntespraat xrsino h rngn and transgene the staining of nonspecific Expression as supernatant. shown the is in fraction protein pellet the The obscuring panels. two and top CENP-Q corresponding the against the antibodies and with eGFP probed CENP-Q with was Endogenous empty tagged blot either The CENP-Q h. with expressing 48 transfected vector for then siRNA-resistant and against an h siRNA or an 12 vector or for siRNA siRNA) control with (CENP-Q transfected CENP-Q been had that causes lysates CENP-Q E1 protein chromosomes. outer-plate polar of the accumulation of Depletion 1. Fig. ARTICLE RESEARCH 174 are were that we above, congression reported makes We recruitment. chromosome CENP-Q CENP-E of whether independent to or contributions kinetochores, additional from CENP-E of proteins in unbinding phenotype motor the chromosome reflects polar simply the cells whether CENP-Q-depleted is question key CENP-E- The and CENP-Q- in cells pairs depleted kinetochore unaligned of Fate of kinetochores to to binding the CENP-E rescued partially transgene Transfection kinetochores. the on with CENP-E of either vector levels to the empty with quantified an CENP-Q transfected and or then transgene of and CENP-Q–eGFP siRNA-resistant siRNA binding the CENP-Q or cells that affect treated control we found results, not with these confirm and To did 2B,C). experiment (Fig. kinetochores CENP-E reciprocal of the depletion performed next We 3 bars: F. an Scale in polar with described cells. with probed experiment cells rescue and the of DAPI in Quantification with chromosomes (G) stained were CENP-A. 1 the Cells against with reduce fixation. treated antibody To were before h. cells min 48 alignment, 90 further on for stage a mitotic for the plasmid of for expression construct effect siRNA CENP-Q–eGFP eGFP control siRNA-resistant control an or a with siRNA or transfected CENP-Q then with and treated h were 14 Cells cells. E1 HeLa 0.2 ( at images planes eGFP microscopy focal and Immunofluorescence siRNA (F) CENP-Q (red). and vector siRNA- (green) and vector siRNA CENP-Q–eGFP CENP-Q resistant siRNA- (black), and siRNA vector control cells CENP-Q–eGFP with from resistant treated data been timing had represent that lines H2B–mRFP red expressing co-expressing and cells green E1 Black respectively. HeLa cells, of timings mRFP– onset. the anaphase and of indicate H2B–eGFP time lines the chromosome orange to last and NEB the and Blue when plate, from metaphase time the time the to the to congressed of (NEB) t Quantification breakdown respectively. (E) envelope 2, breakdown. nuclear and envelope 1 nuclear Movies of material are point cells supplementary CENP-Q-depleted in and in control point available of arrows Videos Yellow kinetochores. row). unaligned (second to cells of CENP-Q-siRNA-treated movies and mRFP– live-cell and row) from unaligned H2B–eGFP to co-expressing Frames point cells (D) E1 arrows poles. HeLa yellow spindle and the poles, spindle around the kinetochores on centred are 2 and against a antibody is an stained image and cell (green) E1 antisera HeLa CREST metaphase with CENP-Q-depleted a of images microscopy and to (red) correspond antisera cells CREST and metaphase with (green) stained CENP-Q siRNA then against control were antibodies a that with siRNA transfected CENP-Q cells or in and pairs cells kinetochore metaphase (zoom) of magnified images microscopy Immunofluorescence (B) corresponding orsodt h eaievle fCN- tiigi oto n CENP- and panels control two in bottom staining the CENP-Q ( of of cells single base values Q-depleted a the relative from at the are Values to kinetochores stack. correspond single the the of of plane images focal zoom the and spacing) z poeto 1 oa lnsa 0.2 at planes focal (10 -projection , a tblnlaigcnrlaesoni h oe w panels. two lower the in shown are control loading -tubulin 0 fteitniymaue ncontrol-siRNA- in measured intensity the of 50% n 5 m m pcn)o EPQsRArsu xeietin experiment rescue CENP-Q-siRNA a of spacing) m ,10kntcoe,3 el) C Immunofluorescence (C) cells). 30 kinetochores, 150 3, BCF;10 (B,C,F); m a tblni oto n CENP-Q-siRNA-treated and control in -tubulin z poetos(0fclpae t0.2 at planes focal (10 -projections m (D). m A muolt fwoecl HeLa whole-cell of Immunoblots (A) a tbln(le.Teiae of images The (blue). -tubulin a m tblncnrlaesonin shown are control -tubulin pcn) ombxs1 boxes Zoom spacing). m a tblni oto (top control in -tubulin n a 5 tbln(e) The (red). -tubulin 2, z poetos 10 -projections, § 0 oe,50 poles, 100 m MG132 M a -tubulin. m 5 m 0, needn)mcaimi eurdfrgnrtn sustained generating for in pulling depolymerisation-coupled required through in movement plateward is (CENP-E- 3 CENP-Q-dependent mechanism a cells. and that CENP-Q-depleted independent) suggest 2 in data 50.4% 1, these only Overall, (chromosomes with compared cells the 3A), behind Fig. CENP-E-depleted were in kinetochores unaligned pole of spindle 69.7% behind Indeed, trapped poles. kinetochores unaligned higher prediction a their have One of should proportion cells S2E). CENP-E-depleted that is Fig. able data non- these kinetochores material from orientated of (supplementary of 100% congress 50% (Wood over almost to and state with S2D) rigor result, Fig. rema material similar a kinetochores a nM into 300 gave biorientated motor 2010), with al., the CENP-E et puts of which from CENP-E inhibition GSK925293, of loss Moreover, CENP-Q-depleted efficient more in a kinetochores. to phenotype attributed be stronger cannot the cells Thus, 2A–C). Fig. irettd(sjde ni h is rm ftemovie; the of frame was first axis sister–sister 90 the approximately the by if in rotated 3–6) in Movies material judged supplementary CENP-Q-depleted both (as non- in as assigned biorientated min were pairs 5 Sister time- cells. CENP-E-depleted of collected and course cells and the marker), over movies pole marker)lapse spindle kinetochore (a (a eGFP–centrin1 eGFP–CENP-A and used expressing we cells this, K For microscopy. HeLa live-cell the using uncongressed by affected of pairs fates kinetochore states the tracking kinetochore therefore by chromosomes differing We of congression CENP-E. these with removed how compared also kinetochores, determined that to CENP-Q binding of CENP-Q CENP-Edepletion of affect depletion because not question this did answer to position a in aeal tahd(aore l,20;shmtci i.3) The 3A). Fig. in schematic 2006; is al., second et the (Kapoor and mono-orientated attached are is laterally from sister sisters state one this which these distinguish in to pairs that possible sister sure not is be it cannot because biorientated We orientated. as classified htuo elto fCN- (Student’s CENP-Q of depletion with compared upon CENP-E of that depletion following less significantly depolymerisation-coupled to utilising due certainly events pulling. kinetochores almost congression are the cells biorientated sliding, CENP-E-depleted lateral in for Because observed example). essential see further also is a CENP-Q 3D,E; see for pairs CENP-E example). (Fig. 5 plate sister Movie metaphase (where also orientated further material the supplementary to of congress cells a 80% 3D,E; to able (Fig. kinetochores), were for to CENP-E-depleted bound stalled 4 remains in remained Movie Surprisingly, material and unable supplementary also congress were equator pairs spindle to kinetochore orientated the the kinetochores), towards progress to 3C,E). unable (Fig. ( majority and the with stalled similar, very also were CENP-E- kinetochorepairs unaligned non-biorientated these and of fates CENP-Q- The state 3B). non-biorientated a (Fig. in occupying 80% similar around with very cells, depleted was pairs kinetochore uncongressed non-biorientated and orientated of proportion relative emtial mosbe ncnrs,kntcoepiswt a with be pairs would kinetochore of biorientation contrast, axis sister–sister state, In impossible. this geometrically in because, as classed non-biorientated were pole Similarly, spindle the poles. behind positioned spindle pairs kinetochore opposite from coming microtubules attachmentswith end-on make not this could with kinetochores improbable because be geometry would Biorientation 3A). Fig. in schematic ent httelvlo ieohr-on EPEwas CENP-E kinetochore-bound of level the that note We from absent also is CENP-E (where cells CENP-Q-depleted In ora fCl cec 21)18 7–8 doi:10.1242/jcs.163659 171–184 128, (2015) Science Cell of Journal , 45 ˚ rls eaiet h oepl xswere axis pole–pole the to relative less or ˚ eaiet h oet-oeai (see axis pole-to-pole the to relative nn tle (supplementary stalled ining . t -test 0)remaining 90%) P 5 0.0148;

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Journal of Cell Science EERHARTICLE RESEARCH otecl otx hra -irshv lsed rxmlto is proximal 176 plus-ends microtubules have proximal of K-fibres are whereas microtubules geometry cortex, astral cell of the the plus-ends to pole, the i.e. spindle reversed, the Behind on forces chromosomes counter polar generate CENP-E and CENP-Q and pole the plate. between metaphase reside the that to pairs equator kinetochore move to order 3. Fig. e etpg o legend. for page next See elto fec rti ol le h oiin of positions spindle polar of the the position high-resolution the fixed-cell to measure used alter to we relative imaging hypothesis, fluorescence would pole wide-field this test the protein To behind pole. poleward, each located them kinetochores chromosomes of that move predict would would move we depletion depolymerisation- Thus, sliding (anti-poleward). plus-end lateral would directs the towards CENP-E-driven CENP-Q it the whereas If pulling, plate. coupled metaphase the ora fCl cec 21)18 7–8 doi:10.1242/jcs.163659 171–184 128, (2015) Science Cell of Journal

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K h E HeLa 48 orientated after live of eGFP–centrin1) in and fates min CENP-A The 5 (D) over of siRNA. pairs h CENP-E kinetochore K 48 or HeLa after CENP-Q live eGFP–centrin1) with in and min treatment eGFP–CENP-A 5 over expressing pairs (stably kinetochore cells non-biorientated of fates orange 6). The and and (C) 4), 5 and (classes 3 kinetochores 2, orientated 1, represents (classes Pink kinetochores eGFP–centrin1. non-biorientated and eGFP–CENP-A expressing stably cells opl xsidctsnnboinain h eue nl ( pole- angle spindle reduced the The and non-biorientation. 4 indicates chromosome axis of the to-pole axis axis, sister–sister on sister–sister the lines kinetochore between the green angle represent dotted 5 axis, The and pole-to-pole spindle. 4 the mitotic chromosomes represents the line within pairs dotted kinetochore black unaligned pairs. of kinetochore orientation unaligned the of Fates 3. Fig. ARTICLE RESEARCH ie el eeiae,adtecnr fms o the for mass of centre the CENP-A on the based and against identified imaged, antibodies was were monopole with cells stained fixed and were and marker) Cells (kinetochore kinetochores. fixed of attached syntelic assessment end-on of then the prophase with in for frequency enabling in changes high 2004), KIF11), a al., positional treated separation et induces as (Lampson This known centrosome attachments 1999). were al., To (also prevents et Eg5 (Mayer CENP-Q. cells which monopolar of or min, inhibitor in 90 spindles, CENP-E an pole of monastrol, monopolar spindle depletion the generate following to spindles kinetochores of individual position the pole. the affects to differentially relative chromosomes CENP-E polar depletion and that CENP-Q prediction our of supporting Thus kinetochores). 107 astral average and on pole were spindle 4B,C; (Fig. Kinetochores the 4A,B). to 1.29 (Fig. relative microtubules pairs kinetochore ieohrs n EPEsRA ( CENP-E-siRNA- and kinetochores) ieohr-opl itne o aheprmn ( experiment each for and The staining 4D). distances (Fig. CENP-A monopole the the to kinetochore-to-pole on distance Euclidian based the found calculated positions automatically then kinetochore We details). the for Methods and Materials 2.62 of occupying pole, distance the average from further an were cells CENP-Q-depleted in retto tt fuaindkntcoepisi EPQ ( The CENP-Q- (B) in assay. pairs our kinetochore in unaligned possible of not state was orientation distal 5) pole number the biorientated (e.g. by from chromosomes K-fibre chromosome adjacent this an Discriminating proximal to kinetochore. pole attached the laterally therefore by and are mono-orientated and kinetochore pole is the 6 behind indicates Chromosome are 5 3 non-biorientated. chromosome and 2 of 1, axis Chromosomes spindle biorientation. to relative axis sister–sister ofrhrvldt hsfnig emaue h itneof distance the measured we finding, this validate further To m m( 6 n 0.43 5 5 ieohrs.I otat ieohr pairs kinetochore contrast, In kinetochores). 159 2 m )fo h oei EPEdpee cells CENP-E-depleted in pole the from m) c tbln(pnl oemre) The marker). pole (spindle -tubulin m 5 m( n ,frtfaeo h oi.Tedashed The movie. the of frame first 0, 5 1 ieohrs rae eaK HeLa treated kinetochores) 211 6 0.99 n § needn experiments. independent 3 A ceai representing Schematic (A) m c )(i.4B,C; (Fig. m) tblnsga (see signal -tubulin # n 45 5 217 ˚ fthe of ) n n , 5 § 90 2, 4 ˚ nepyvco eosrtda 4 euto nPk at Plk1 CENP-Q in with reduction transfection contrast, 84% In cells). an kinetochores. with ( demonstrated material transfected to kinetochores vector been (supplementary had Plk1 empty that result an cells of previous CENP-Q-depleted S2), recruitment our Fig. with the Consistent affected mutant htmvscrmsmstwrso wyfo h pole the from away or geometry. microtubule towards the on chromosomes depending moves pulling that that depolymerisation-coupled plus-ends, independent drive model recruitment) are microtubule CENP-E our (that of mechanisms towards support CENP-Q-dependent data kinetochores whereas these control-siRNA- slides together, deviation in CENP-E standard Taken narrower kinetochores much 4F). its a (Fig. with and to albeit activity cells, due distance monopole transfected mean force similar the motor CENP-E a between occupied anti-poleward CENP-E kinetochores) cells the from CENP-Q-depleted unbinding lack inhibit in with also kinetochores cells (which to treating Conversely, control by 4E). GSK925293 result of (Fig. this that distances confirmed with nM We 300 kinetochore-to-pole compared 4E). monopole of (Fig. the cells to cumulative distribution CENP-E of closer the depletion moving the then that in was show plots calculate frequencies resulted These distribution 4E,F). (Fig. to the cumulative plotted distances, these used kinetochore-to-pole of the average of then frequency distribution were condition) Snaai ta. 01;teeoe CENP-Q site therefore, phosphorylation 2011); Plk1 al., a the as et reported (Santamaria that not is kinetochore. 50 the indicating indicates serine at Moreover, assembled bound, therefore successfully CENP-Q had kinetochore complex and CENP-O remains Plk1 al., CENP-U et of (Kang that presence CENP-U upon The dependent is 2006). kinetochores recruitment to that shown Plk1 has ( of work Previous 62% cells). 60 to kinetochores, levels Plk1 kinetochore eiu (CENP-Q residue h eaaino h oeo EPQi chromosome Plk1 in in CENP-Q and assembly of complex role CENP-O the recruitment. in of congression, separation the needn ftercuteto l1adCENP-O and Plk1 subunits of complex are recruitment congression the chromosome of in independent CENP-Q of roles The euaoyeet eteeoemttdsrn 0t alanine to 50 serine 2006). mutated key al., a therefore represent phosphorylated might et (CENP-Q We is this event. that Nishino CENP-Q suggesting is regulatory 2011), 2005; in al., et 50 CENP-Q al., (Rigbolt the serine to et CENP-E that Moreover, of localisation (Ahonen for required demonstrated kinetochore is Plk1 that and Plk1 have 2011) CENP-U-bound by reports phosphorylated Previous oprbewt hto idtp EPQeF Fg 6A,B, (Fig. ( CENP-Q–eGFP 43% wild-type to of recruitment that CENP-E with comparable rescued partially This eceteCN- elto hntp.Taseto with Transfection phenotype. depletion CENP-Q CENP-Q the rescue oprdwt hto oto el rnfce iha empty an with transfected cells 6A,B, control (Fig. of vector that with compared needn experiments, independent el) ofrhrvldt h oeta motneof importance potential CENP-Q the CENP-Q-depleted phospho-mimetic transfected the validate we with 50, cells serine further at phosphorylation To 6A,B, (Fig. pole kinetochore cells). spindle six each approximately at with trapped pairs defect, alignment an had still etw netgtdwehrCENP-Q whether investigated we Next S50A ora fCl cec 21)18 7–8 doi:10.1242/jcs.163659 171–184 128, (2015) Science Cell of Journal S50A eF aldt eceCN- erimn when recruitment CENP-E rescue to failed –eGFP eF)o oapopommcigapri acid aspartic phospho-mimicking a to or –eGFP) 6 .% i.5A,B, Fig. 3.7%; S50D n 5 eF) efrttse hte h S50A the whether tested first We –eGFP). ,60kntcoe,6 el) hs cells These cells). 60 kinetochores, 600 3, § el e xeietper experiment per cells 8 6 n 2;Fg 5A,B, Fig. 12%; 5 ,30kntcoe,30 kinetochores, 300 3, nvitro in S50D S50A S50A n S50A eF transgene. –eGFP 5 ,10pls 60 poles, 120 3, eF rescued –eGFP eF enables –eGFP Kn tal., et (Kang , eF could –eGFP n 5 6 nvivo in ,600 3, 10%), 177

Journal of Cell Science hssga n ofre httelvl eeeuvln o the for quantified equivalent first were levels We the kinetochores. that confirmed on and detected signal this be performed could thus signal a CENP-Q where We cells to analysis kinetochores. CENP-Q–eGFP, non- our or restricted However, on low and experiments a 6A). signal additional had (Fig. cells eGFP some kinetochores transfection, detectable transient to the to recruited owing were proteins EERHARTICLE RESEARCH 178 ( 2.4 average 6A,B, (Fig. on pole per having pairs kinetochore phenotype, congression severe n 5 motnl,bt CENP-Q both Importantly, ,60kntcoe,6 el) hs el lodslydaless a displayed also cells These cells). 60 kinetochores, 600 3, S50A S50A eF rCENP-Q or –eGFP eF n CENP-Q and –eGFP n 5 ,10pls 0cells). 60 poles, 120 3, S50D S50D –eGFP –eGFP 6 0.8) ieohrsprcniin.Nx,w one h ubrof CENP- the number that found the CENP-Q and and cells counted 6C, these Q–eGFP in (Fig. we pole per Next, transgenes pairs kinetochore condition). CENP-Q per mutant kinetochores and wild-type efudta h EPQeF n CENP-Q and CENP-Q–eGFP the that for 6.5 found with we compared 6D, constructs, Fig. the mutant for S50A; zero S50D was pole and per pairs wild-type kinetochore of number median (the rngnsrsoe h idn fCN- okntcoe to kinetochores to CENP-E of binding , the restored transgenes Q S50A 5 ftelvl esrdi oto el Fg 6D; (Fig. cells control in measured levels the of 75% eF rngn,ams opeeyrsudtephenotype the rescued completely almost transgene, –eGFP ora fCl cec 21)18 7–8 doi:10.1242/jcs.163659 171–184 128, (2015) Science Cell of Journal n § 5 ieohrsprcniin.Likewise, condition). per kinetochores 150 S50D oato oidc oooaiy h mg sa is z image The 1 monopolarity. with induce treatment to of monastrol min 90 CENP-E or by CENP-Q followed control, siRNA with h 48 for treated against and antibodies (green) with CENP-A stained cell a E1 of HeLa image microscopy Immunofluorescence (D) 5 areas. bar: boxed Scale the of single images enlarged a show is Insets image The (red). pericentrin against (green) antibodies antisera CENP-E CREST and or with CENP-Q stained with and h siRNA had 48 that for cells treated K been HeLa of images Immunofluorescence microscopy (B) CENP-E. of and depletion CENP-Q during ‘d’ pole-to- distances the kinetochore of measurement the representing chromosomes. polar counter on generate forces CENP-E and CENP-Q 4. Fig. hddaesrepresent areas Shaded lines, (red siRNA CENP-Q control with lines, treated (green been siRNA had that centre cells monopole in the from distances kinetochore E uuaiedsrbto fkinetochore of distribution pole. Cumulative the (E) to kinetochore each from distance measurement by and followed identification identification pole kinetochore of process automated the show overlays analysis successive The spacing). represent bars Error (green, treated (blue n CENP-Q-siRNA- in distances kinetochore represent line, (purple lines, (red lines, (red siRNA control n that with cells treated in been centre had monopole the from distances poeto alfclpae,15 planes, focal (all -projection 5 5 5 ieohrs n CENP-E-siRNA- and kinetochores) 159 2 xeiet with experiments 5 eF rngns u o h CENP- the not but transgenes, –eGFP 6 n ..()Tecmltv itiuinof distribution cumulative The (F) s.d. 5 n m 5 ,with 4, .()Qatfcto fpole-to- of Quantification (C) m. n ,1 el) hddareas Shaded cells). 10 1, 5 ,17kntcoe)cells. kinetochores) 107 2, n 6 § 5 c § tbln(e) el were Cells (red). -tubulin s.d. el)adGSK923295 and cells) 8 a ,with 5, el) EPEsiRNA CENP-E cells), 8 tbln(le and (blue) -tubulin 6 s.d. n 5 ,with 3, § m el)and cells) 8 A Schematic (A) t0.2 at m S50D § z cells). 8 n n -slice. –eGFP § § m m 150 150 m M

Journal of Cell Science oefrCN- nCN- odn nokntcoe and kinetochores onto and complex. Plk1 loading CENP-O of the independent CENP-E is that in pulling a depolymerisation-coupled CENP-Q support data for 6E, these (Fig. together, role Taken levels condition). transgene per high kinetochores at observed recruitment EERHARTICLE RESEARCH ehnssta rv h ogeso fsc polar such of distinct congression sliding two lateral least the well-documented at the (1) are drive 7A): there (Fig. which chromosomes that in Our model anaphase. mechanisms a in chromatids thereby support and sister congress data of can mitosis disjunction in accurate early pole allow chromosomes the conformation to ensure close that 2011). are ring-like mechanisms that al., have amphitelic et must a of (Magidson cells result However, breakdown to into a envelope of nuclear as organised lateral majority following equator being the spindle from the that kinetochores at convert indicate (biorientation) cells attachment rapidly human in chromosomes studies Recent DISCUSSION a revealed This and kinetochores. CENP-Q transgene and the individual CENP-Q–eGFP of for on level correlation positive CENP-E the plotted of also are transgene. that we the 6A,B) data, expressing Fig. not these cells (see From some of experiments consequence a rescue simply ( partial population-based the our Thus, condition). per kinetochores CENP-Q 5. Fig. ieohr.A xetd hsrltosi a otupon lost was each is CENP-Q at relationship molecules molecules this with CENP-E CENP-Q expected, of transfection of As number recruitment the kinetochore. upon the dependent that indicating cl a:5 1 bar: with Scale metaphase in arrested were el.Errbr indicate subtraction, bars background Error after CREST cells. of that to relative iN eceeprmn nHL el.Clswr rae ihCN- iN rcnrlsRAfr1 n hntasetdwt nsiRNA-resistant an with transfected then and h 12 for siRNA control or siRNA CENP-Q with treated were Cells cells. CENP-Q K expressing HeLa plasmid in experiment rescue siRNA m .()Qatfcto fPk eesi h CENP-Q the in levels Plk1 of Quantification (B) m. S50A eF ece ieohr erimn fPlk1. of recruitment kinetochore rescues –eGFP 6 s.d. S50A eF racnrleF xrsinpamdfrafrhr4 .T eueteefc ftemttcsaeo lgmn,cells alignment, on stage mitotic the of effect the reduce To h. 48 further a for plasmid expression eGFP control a or –eGFP S50A m G3 o 0mnbfr iain el eesandwt RS niea(le n natbd gis l1(red). Plk1 against antibody an and (blue) antisera CREST with stained were Cells fixation. before min 90 for MG132 M eF,wt oCENP-E no with –eGFP, , n 5 0)rsu in rescue 50%) 3, § S50D S50A 0 ieohrs 0cls h ahdln niae EPQlvl ncontrol-siRNA-treated in levels CENP-Q indicates line dashed The cells. 30 kinetochores, 300 –eGFP, n eF iN eceeprmn.Sann neste eedtrie tec kinetochore each at determined were intensities Staining experiment. rescue siRNA –eGFP § 150 A muoloecnemcocp mgs( images microscopy Immunofluorescence (A) rbblt fboinaindet h ihrdniyof density higher chromosomes the move CENP-E-dependent will kinetochores that to attached the laterally show increase of data due sliding to our order biorientation However, in microtubules. necessary of is state pole CENP-Q 1993) probability the al., et to how Skibbens movement 2008; al., that et understand Kim (see models to Existing below). be (see process An this will anti- congress. to to or contributes failure question poleward a and their stalling important sustain in other resulting to that state, unable the kinetochores This metaphase poleward Sister appear while the state. 1993). CENP-Q pole state, (facing from lack poleward-moving away al., sister an a maintains one et sister in of (Skibbens remaining consequence motion plate) a equator which is long-duration spindle in motion undergo ago, the years can 20 towards kinetochores colleagues and biorientated Skibbens by described in CENP-Q for sisters. role biorientated a of reveals movement as in and the sliding congression kinetochores lateral of out from rule mechanism to CENP-E the us of allows unbound). that absence also cells is the CENP-Q-depleted CENP-E is (where it absence CENP-Q the Importantly, not in but congress CENP-E, to that of 7A, able of observation Fig. still our movement study; are on kinetochores based (this the biorientated is plate conclusion in This metaphase 2). recruitment the that chromosome CENP-Q to CENP-E for sisters role of biorientated a al., (2) independent et and 1), (Kapoor is chromosome CENP-E 7A, (Fig. by 2006) kinetochores (non-biorientated) of o oclsiiilymv hoooe osidepoles? spindle to chromosomes move initially cells do How those to analogous are movements CENP-Q-dependent These ora fCl cec 21)18 7–8 doi:10.1242/jcs.163659 171–184 128, (2015) Science Cell of Journal z sie faCENP-Q a of -slice) S50A –eGFP 179

Journal of Cell Science EERHARTICLE RESEARCH rsaepse wyfo h oeb h oa jcinforce ejection polar the 180 by pole the from towards away and pushed pole are the chromosome time, from arms same the away At astral-microtubules. further of plus-end pole the the behind are that 6. Fig. e etpg o legend. for page next See h oe(age l,20) epooeta hr sasecond a is there that propose We 2007). towards al., forces, et kinetochores anti-poleward (Yang attached these pole laterally counter the slide can To motors 2012). dynein al., et (Stumpff ora fCl cec 21)18 7–8 doi:10.1242/jcs.163659 171–184 128, (2015) Science Cell of Journal

Journal of Cell Science ahkntcoerltv ota fCN- fe akrudsubtraction, background after CENP-A of that n to relative kinetochore each i.6 EPQsrn 0i eurdfrCN- erimn and recruitment CENP-Q CENP-E of for required is congression. 50 orderly serine CENP-Q 6. Fig. ARTICLE RESEARCH nkntcoemcouuednmc.Spotfrti idea this we for 50, to Support serine effect recruited direct on dynamics. a is mediates dependent kinetochore–microtubule for CENP-Q protein that is on idea required it alternative unknown that the not manner favour an Although is in that above). CENP-E kinetochores possible (see that In remains show pulling depletion). data CENP-Q 7C). depolymerisation-coupled Fig. or phenocopies our expressing complex (which in cells CENP-O addition, mutant in the schematic S50A of kinetochore-bound loss remain the (see the both out which affects kinetochores rule Plk1, of indirectly can binding to we CENP-Q However, the factors affecting that other by is pulling possibility depolymerisation-coupled One pulling? biorientation. from promote to distance to optimum pole spindle the proposed the at themselves position anti-poleward to and is poleward mechanisms utilise kinetochores that MCAK suggests data thus current kinetochore, Overall, kinetochore. 2013). the the of mono-orientation to the promoting back MCAK microtubule Draviam, astral depolymerase the depolymerise and microtubule the (Shrestha the that requires revealed has end-on work configuration an recent to regard, attachment lateral this CENP-E-dependent In from transition 7B). how 1 Fig. chromosomes determine in 7A, 2 Fig. and to in kinetochore and 1–4 important mono-orientated (chromosomes pole states lateral, biorientated be between from converted will away are kinetochores It an movement. than (pushing) rather a pulling), of (depolymerisation- coupled movement failure kinetochore poleward defective the due also a likely, to that most therefore, is follows congress to pair It sister biorientated motility. a kinetochore mean and poleward would pulling depolymerisation-coupled this in CENP-Q direct, CENP- for If of requirement effect kinetochores. the mono-orientated reflect on to likely Q is process this poleward. that chromosomes suggest We moves that process CENP-Q-dependent 2hadte rnfce iha iN-eitn lsi expressing plasmid for siRNA-resistant siRNA an control with or CENP-Q transfected siRNA CENP-Q–eGFP, CENP-Q then with and treated h were 12 Cells cells. K HeLa in uniiaino h vrg ubro ieohr ar e oei cells in pole per pairs kinetochore of CENP-Q number expressing average indicate the bars of Error quantification cells. control-siRNA-treated in levels inli el ece ihCN-–GP CENP-Q–eGFP eGFP CENP-Q–eGFP, kinetochore with the rescued between cells relationship in outliers. the signal represent demonstrating dots plot whiskers outliers, A line range, excluding (E) The interquartile values 6B). the maximum Fig. represents the from box represent (taken the cells median, control the in represents levels the indicate levels, lines CENP-E Dotted kinetochore pole, of per quantification pairs and the kinetochore of of with quantification number cells showing average of plots Analysis Box (D) kinetochores. values outliers. transgene-positive maximum represent the dots represent outliers, whiskers excluding range, interquartile the represents CENP-Q each for Box levels (C) transgene variant, kinetochore cells). of 60 quantification condition, showing per plot poles (120 experiments rescue independent Q–eGFP G3 o 0mnbfr iain el eesandwt nioisagainst antibodies with stained 1 were with Cells the fixation. metaphase of before in effect min arrested the 90 were reduce for To cells MG132 h. alignment, 48 on further stage a mitotic for plasmid expression eGFP uniiaino EPElvl nteCENP-Q the in 5 levels bar: CENP-E Scale of (red). quantification CENP-E and (blue) CENP-A Q 5 S50D o ol EPQcnrbt odepolymerisation-coupled to contribute CENP-Q could How 3, § eF iN eceeprmns neste eedtrie at determined were Intensities experiments. rescue siRNA –eGFP 0 kinetochores, 300 n S50D 5 3, S50A n h ieohr EPEsignal, CENP-E kinetochore the and § eF n CENP-Q and –eGFP 5 ieohrs h ierpeet h ein h box the median, the represents line The kinetochores. 150 S50A A muoloecnemcocp mgs( images microscopy Immunofluorescence (A) eF n CENP-Q and –eGFP S50A § 0cls h ahdln niae CENP-E indicates line dashed The cells. 30 eF rCENP-Q or –eGFP S50D eF iN eceexperiments rescue siRNA –eGFP S50D n S50A m 5 S50D n three from –eGFP .()Lf panel, Left (B) m. n 3, 5 5 eF n CENP- and –eGFP 3, § 3, eF racontrol a or –eGFP 0pls(etpanel) (left poles 60 § § 6 5 rgtpanel). (right 150 5 kinetochores. 150 ..Rgtpanel, Right s.d. S50A rCENP- or z m -slice) M uiidCN- rti a ietybn otaxol-stabilised to bind directly can microtubules protein CENP-Q purified from comes C4)adlgtdit EF-1(mt etr lneh using Clontech) vector; (empty pEGFP-N1 into and CENP-Q Bam MC246 ligated primers the (using and PCR vector, using MC248) expression by amplified CENP-Q–eGFP was sequence human coding a generate experiments rescue To siRNA and biology Molecular penicillin U/ml Eagle’s 100 modified serum, 100 calf Dulbecco’s and foetal in 10% grown containing were (DMEM) cells medium K treatments HeLa drug and and HeLa-E1 transfection siRNA culture, Cell METHODS AND MATERIALS AGAAA-3 aya hmxrs)1 t30n,txl(ors t10 at (Tocris) taxol nM, 300 at h 14 Chemexpress) Haoyuan nuao.TeHsoeBmF elln a anandin maintained was line cell Histone2B–mRFP 500 The incubator. a anandi 500 in maintained was ehns em niey natraiepsiiiyi that is direct possibility a such a alternative in dynamics Thus, kinetochore–microtubule An modulates unlikely. S4). CENP-Q seems Fig. mechanism material (supplementary is with nocodazole microtubules cells Moreover, of CENP-Q-depleted depolymerisation 2012). following in rescued kinetochores al., partially to et CENP-E Mchedlishvili of 2007; binding are 2010; al., al., that et et proteins this (Amaro McClelland kinetochores CCAN to controlling binding in – that CENP-Q for kinetochores cells in required to CENP-L-depleted to is CENP-E bound or step of remains CENP-H- idea binding CENP-E regulatory direct However, attractive the CENP-Q. important allows An event an phosphorylation congression. is chromosome 50 serine these cells. observed CENP-Q-depleted attribute in the movement yet to chromosome cannot in dynamics defects we kinetochore–microtubule of depolymerisation-coupled in Therefore, congression changes the study). through allowing (this still pulling kinetochores while effect kinetochore same 2009), the biorientated al., have of to et reported turnover (Maffini is depletion the CENP-E However, reduces CENP-Q microtubules of depletion oietf h iaersosbefrterglto fCENP-Q of regulation the for responsible function. kinase human required the in be Plk1 clearly identify will of work to inhibition Future 2011). or al., depletion et the (Santamaria cells to cell sensitive serine in not of is acids phosphorylation 50 amino that show by Plk1 experiments labelling (SILAC) for culture isotope substrate stable a CENP-Q However, be 2006). 2011). to al., et shown (Kang been Plk1 has of would 50 pool serine CENP-U-bound of the phosphorylation be the for most responsible be is to that likely kinase in the defects Finally, the explain pulling. depolymerisation-coupled also could same dynamics these microtubule above, in discussed changes As recruited. be can CENP-E that way ooaoe(ors 4ha 1 at h 14 (Tocris) nocodazole periods time – and concentrations following the at performed were treatments (5 CENP-E and UGGGAAUAUUCA-3 control (5 manufacturer’s were CENP-Q the used 2011), then to al., sequences (MEM) according oligofectamine et medium oligonucleotide h] (Samora 24 Eagle’s siRNA using further siRNA modified The a in instructions. for medium. transfected h DMEM [24 non-selective to h changed were in 48 for maintained nM) (Invitrogen) (53 were lines oligonucleotides cell other oato Tci)a 1 at (Tocris) monastrol u aasgetta hshrglto fCN- through CENP-Q of phosphoregulation that suggest data Our Iand HI m gml m gml 2 ora fCl cec 21)18 7–8 doi:10.1242/jcs.163659 171–184 128, (2015) Science Cell of Journal 9 1 Eco tat,Ivtoe) EPQ2(5 CENP-Q-2 Invitrogen), Stealth, 48adteeF–EPAeF–eti1cl line cell eGFP–centrin1 eGFP–CENP-A the and G418 nvitro in 2 9 nvitro in -ACUCUUACUGCUCUCCAGUdTdT-3 I(M26.pC7 a hnmttduigaquick a using mutated then was pMC276 (pMC276). RI 1 nvivo in tetmcna 37 at streptomycin 9 m m tat,Ivtoe) EPP(mr ta. 2010) al., et (Amaro CENP-P Invitrogen), Stealth, iceity hc eosrtsta the that demonstrates which biochemistry, o 0mnadM12a 1 at MG132 and min 90 for M gml Aaoe l,21) ed idthat find do We 2010). al., et (Amaro splmnaymtra i.S3). Fig. material (supplementary 2 1 m gml 48ad0.3 and G418 9 -GGUCUGGCAUUACUACAGGA- 2 ˚ ne %CO 5% under C 1 S939 CN- inhibitor; (CENP-E GSK923295 , 9 m -CAGAGUUAAUGAC- nvitro in gml 2 2 m 1 9 o 0min. 90 for M min.Drug Ambion). , nahumidified a in uoyi.All puromycin. m Kn tal., et (Kang o 0min, 60 for M 181

Journal of Cell Science CENP-Q TTTGTTTGTCCTTCAGAAGCCAGATCTTTCAGATGATTTTTAT-3 GATCTGGCTTCTGAAGGACAAACAAAGCAC-3 EERHARTICLE RESEARCH 182 the and (Stratagene) kit phospho-mutants CENP-Q CENP-Q mutagenesis site-directed primers: of generation following change the quick for a template using a as was used construct then CENP-Q siRNA-resistant This (pMC308). oligonucleotide TTGTC-3 AACGCTAGGCCTTCTTCATTAGCT-TATCTATTTCCTCTTGGAGC AAATAGATAAAATGGTAGAG-3 MC297 primers the and \(5 (Stratagene) kit mutagenesis site-directed change ihCN- rcnrlsRA n rw o 4hi E.The MEM. in h 14 for grown and siRNAs control or CENP-Q with TTATTTTTT-3 TGTGCTTTGTTTGTCCTTCAGAATCCAGATCTTTCAGATGATTT- GATTCTGAAGGACAAACAAAGCACACTAAC-3 9 -GACAAAGCTAATGAAGAAGGCCTAGCGTTGCTCCAAGAGG- S50D 9 ,t edrtetaseerssatt h EPQsiRNA CENP-Q the to resistant transgene the render to ), eF (5 -eGFP 9 .FrsRArsu xeiet,clswr transfected were cells experiments, rescue siRNA For ). 9 -AAAAAATAAAAATCATCTGAAAGATCTG- S50A eF (5 -eGFP 9 n C9 (5 MC298 and ) 9 -ATAAAAATCATCTGAAA- 9 9 9 n (5 and ) -CTCTACCATTT- n (5 and ) 9 -GTTAG- 9 -GTGC- 9 ), el eefxda omtmeauefr1 i n2 MPIPES mM 20 in min 10 MgCl mM for 1 temperature EGTA, mM room 10 containing at 6.8 pH fixed were Cells microscopy Immunofluorescence EPE[:,0;(ead ta. 04] abtat-k3(:5)(a (1:250) rabbit anti-Ska3 Switzerland), Basel, rabbit of University 2004)], Santamaria, Anna al., from gift et anti- kind rabbit (Meraldi primary Incorporated), [1:1,500; with Antibodies mouse CENP-E (1:250; h anti- 2006), antisera 1 mouse min. CREST al., for 30 Aldrich), Abcam), et for incubated McAinsh (1:500; PBS were (1:500; in anti-CENP-A cells anti-CENP-O BSA every rabbit 3% fixed PBS with antibodies: the with blocking washed blocking, before then After times were three then min were 10 Cells formaldehyde. 4% uee rnfcinraet(oh) el eete nuae o a for incubated using then analysis. transgene before were h CENP-Q cells 48 a (Roche); further or reagent 1 with Clontech) transfection transfected vector; FuGene6 cells (empty the and pEGFP-N1 DMEM of to changed then was medium ora fCl cec 21)18 7–8 doi:10.1242/jcs.163659 171–184 128, (2015) Science Cell of Journal eedn eoyeiainculdpligaddynein- and pulling depolymerisation-coupled dependent move CENP-Q- (PEFs) whereas forces anti-poleward, ejection chromosomes CENP-E polar forces. and generated sliding the lateral of direction Arrows the poles. indicate spindle the on behind acting positioned mechanisms chromosomes the showing movement. Schematic pole (B) from the away at AP, aligned plate. and metaphase biorientated is 4 congress pathways. Chromosome to CENP-Q-dependent order and/or in CENP-E- pole through microtubules opposite of the plus-end from either the emanating with or kinetochore lattice free microtubule its the engage mono- will is and 3 orientated Chromosome kinetochore. (P) the poleward at pulling CENP-Q-dependent depolymerisation-coupled by microtubule driven are plateward that the persistent movements to making congress by to plate able metaphase and 2006). biorientated to al., is able et 2 then Kapoor Chromosome is in it reported where (as through CENP-E, biorientate plate by the driven to sliding, this congress lateral CENP-E, to through able kinetochore is sister chromosome black attached the laterally at and ‘L’ mono-orientated is mechanisms. 1 combined CENP-E-dependent Chromosome the and through CENP-Q- pole of the action and plate the positioned between chromosomes chromosome unaligned of for model congression proposed a CENP-E. showing and Schematic CENP-Q (A) of through congression action chromosome combined for the model A 7. Fig. tagt(etop n tagt 2013). and Straight, Westhorpe and from core (Westhorpe taken into Straight CCAN is the components of extended separation 2007; and The al., 2006). et al., McClelland et 2006; Okada al., several et 2011). from (Foltz al., obtained references al., et were et (Kang CCAN (Kang [5] within [3] and Dependencies 2009), study al., this et [4] (Maffini 2006), (Maia [2], [1] 2012), references: al., following et the from dependencies obtained direct Percentage were make microtubule. to the shown with been contact have Blue arrows that direct). red proteins be and represent to events, shown phosphorylation been represent not lines have (or indirect which are dependencies, loading dependency). represent lines of dashed level Black the dependencies indicate loading values direct (percentage represent lines black dependencies. Solid phosphorylation showing and Schematic loading (C) kinetochore these arrow). between (grey cycle states and to attachment pole, able spindle probably the are from kinetochores chromosome the a dictates of mechanisms distance these amongst poleward. balance chromosomes Force move sliding lateral driven a tbln(:00 Sigma- (1:1000; -tubulin 2 .%Tio -0 and X-100 Triton 0.2% , m g

Journal of Cell Science AI loeci sticaae(IC,Roaieo ea e,CY5 Red, Texas a or with Rhodamine (FITC), equipped isothiocyanate Three- LLC) fluorescein Precision, DAPI, (Applied (Invitrogen). microscope 0.2 Elite goat in DeltaVision 100 acquired were a in cells mitotic using raised of stacks image antibodies three dimensional cross- min highly secondary AlexaFluor-conjugated 10 with every min adsorbed PBS 30 with for incubated washed and then times were Cruz), Cells Abcam). Santa (1:1000; (1:250; anti-Plk1 anti- rabbit rabbit Rockland), (1:250; anti-CENP-Q ARTICLE RESEARCH hnn .J,Kli,M . am .R,Blo,M,Mne .A,Yfe .B., M. Yaffe, A., I. Manke, M., Bolton, R., J. Daum, J., M. Kallio, J., L. Ahonen, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.163659/-/DC1 online available material Supplementary material Supplementary release. D. immediate A. for to PMC BB/I021353/1 in Biological number Deposited and [grant McAinsh]. Biotechnology (BBSRC) number the Council [grant by Research Partnership supported Training Sciences was Doctoral work MRC This the MR/J003964/1]. by funded Fundac (MRC); the is Council from Auckland fellowship Research P. a Medical by the Samora P. from C. studentships and by funded is Bancroft J. Funding C.P.S. and J.B. P.A., was from manuscript contributions The P.A. with by A.D.M. performed siRNA by were CENP-P 6 written and in 5 chromosomes Figs polar in rescue Experiments of (C.P.S.). live-cell observations experiments, initial were photoactivation 1–4 and Figs except experiments in J.B., Experiments by A.D.M. out by supervised carried and initiated was project The contributions Author interests. competing no declare authors The manuscript. the interests on Competing comments for data Meadows, Smith with J. C. thank help and also for We Royle Fitton processing. S. image B. with Meraldi, antibody, P. help Ska3 for the Smith C. for and Santamaria analysis A. to you Thank Acknowledgements nitrogen 2007). al., liquid et al., anti- (McAinsh mouse a et previously Primary described 2006). (McClelland using as described performed prepared was previously Immunoblotting as were technique extracts grinding protein Whole-cell Immunoblotting 40 a using LLC), Precision, and (Applied H2B–eGFP co-expressing cells mRFP– of imaging time-lapse Fluorescence kinetochore. imaging same Live-cell the on CREST or CENP-A of intensity normalising the and to Photoshop values background SoftWorx with subtracting made SoftWorx, generated using were using manually measurements were Fluorescence-intensity deconvolved (Adobe). figures Illustrator and were and camera LLC), HQ stacks Precision, CoolSNAP a Image (Applied and source Scientific). light state (Roper solid (Chroma), set filter n eodr nioisfr1ha omtemperature. room at h 1 for antibodies secondary and etos eecletdeey3mnfrattltm f1 h. 10 of time total a for (7 min stacks 3 Image every a collected Scientific). and were source (Roper sections) light camera Xenon 409– HQ2 (Chroma), bands CoolSNAP 652–700) (reflection transmission 598–617, mirror 625–644, 500–553, dichroic 561–590, 456, 575/25, 464–492, Quad-mCherry 381–401, (excitation set, bands mCherry filter and 632/60) 525/50) emission emission 475/28, (excitation Mre) rmr nioiswr nuae o 4hoengta 4 at overnight h powder 14 milk for 3% incubated with were Tween20 (1:10,000; antibodies 0.1% Primary PBS (Marvel). in antibodies diluted were Amersham) secondary anti-rabbit or anti-mouse horseradish-peroxidase-conjugated secondary and Rockland), (1:500; anti-CENP-Q eso-esn F/ hsheioeadmdltsteascainof association the modulates kinetochores. and at proteins phosphoepitope spindle-checkpoint J. 3F3/2 G. Gorbsky, tension-sensing and T. P. Stukenberg, a tblnwspromdo esnlDlaiinmicroscope Deltavision Personal a on performed was -tubulin c tbln(:0;Acm,muemncoa anti-Ndc80/Hec1 monoclonal mouse Abcam), (1:200; -tubulin 6 i-meso . Aojcieln na Olympus an on lens objective NA 1.4 oil-immersion a tbln(:000 im-lrc)o rabbit or Sigma-Aldrich) (1:10,000; -tubulin 20) oolk iae1cetsthe creates 1 kinase Polo-like (2005). 6 ur Biol. Curr. A13ojcie ihGFP with objective, 1.3 NA ¸ a aaaCie a para o ˜ 15 1078-1089. , ni Tecnologia. e ˆncia 6 m 2 steps m m m ˚ z C - celsvl,N,Wee,S,Hlakr,R,Muse,J,Bla,M,Amaro, M., Belwal, J., Mouysset, R., Holtackers, S., Wieser, N., Mchedlishvili, cllad .E,Brs,S,Aao .C,Wne,J . ewl M., Belwal, R., J. Winter, C., A. Amaro, S., Borusu, E., S. McClelland, P. Meraldi, and D. A. McAinsh, ag .H,Pr,C . i,T . on,N . ag .K,Km .Y,Park, Y., B. Kim, K., J. Bang, K., N. Soung, S., T. Kim, H., C. Park, H., Y. Kang, i,Y,Hue,J . aemn .M n lvln,D W. D. Cleveland, and M. C. Waterman, E., J. Heuser, Salmon, Y., Kim, D., Cimini, L., Cameron, P., Hergert, A., M. Lampson, M., T. Kapoor, cis,A . ead,P,Daim .M,Ts,A,&Sre,P K. P. Sorger, & A., Toso, M., V. Draviam, P., Meraldi, D., A. McAinsh, 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, i,Y,Hlad .J,Ln .adCeead .W. D. Cleveland, and W. Lan, J., A. Holland, Y., Kim, ae,T . aor .M,Hgat,S . ig .W,Shebr .L and L. S. Schreiber, W., R. King, J., S. Haggarty, M., T. Kapoor, U., T. Mayer, Macedo-Ribeiro, S., Maffini, M., Barisic, L., Kabeche, Z., Garcia, R., A. Maia, P. Meraldi, and T. A. Saurin, J., G. Kops, P. Meraldi, and S. Borusu, A., Toso, C., Klebig, u,S,Wn,Z,Jag . un,Y,Wr,T,Za,L,Du .adYo X. Yao, and Z. Dou, L., Zhao, T., Ward, Y., Huang, K., Jiang, Z., Wang, T. S., Fukagawa, Hua, and K. Maenaka, M., Okada, T., Hori, R. J. McIntosh, and A. V. Lombillo, M., Coue, T., Zimniak, M., Cai,S.,O’Connell,C.B.,Khodjakov,A.,&Walczak,C.E. A. Rojas, D., Prumbaum, R., J. Weir, S., Maffini, F., Basilico, Alonso, J., I. Kingston, E., Wang, R., Holtackers, P., C. Samora, C., A. Amaro, aisn . ’onl,C . Lonc B., C. O’Connell, V., Magidson, Junqueira, L., A. Pereira, Z., Maliga, L., A. Manning, R., R. A. Maia, M. S., T. Maffini, Kapoor, and A. Khodjakov, K., Renduchitala, W. A., M. M. Kirschner, Lampson, and J. T. Mitchison, E., D. Koshland, J. Ellenberg, and M. Ohsugi, S., T. Kitajima, atns .N,Snaai,A,Jypaah .A,Wn,B,Cni .and E. Conti, B., and Wang, III A., R., A. J. Jeyaprakash, Yates, A., O., Santamaria, N., A. T. Bailey, Gaitanos, E., B. Black, T., E. L. Jansen, R., D. Foltz, Potapova, N., J. McAvoy, S., Sivakumar, J., J. Daniel, D., J. Wren, R., J. Daum, nuefihu hoooesegregation. chromosome faithful ensure P. Meraldi, and C. A. 5033-5047. ope otoscrmsm ogeso n pnl bipolarity. spindle and congression P. chromosome Meraldi, controls and complex D. A. McAinsh, dynamics. kinetochore-microtubule Biol. of control to specification .E n e,K S. K. Lee, and E. J. otemtpaepaebfr biorientation. before plate A. metaphase Khodjakov, the and F. to B. McEwen, D., E. kinetochores. at complex PBIP1-CENP-Q the h ua ieohr rtisNfRadMm1 r eurdfraccurate for required are Mcm21R and segregation. Nnf1R chromosome proteins kinetochore human The 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., rti hshts eit hoooecnrsintruhrglto of an regulation through produce congression to chromosome mediate coil 1 coiled phosphatase protein flexible a and tether. motor kinetochore motile processive essential slow, a combines dniidi hntp-ae screen. phenotype-based a in identified J. T. Mitchison, H. kinetochore- Maiato, stabilizes and that I. phospho-switch Kaverina, CLASP2 attachments. a A., microtubule mediate D. 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Journal of Cell Science cmd,J . rhnr,H,Bezemni . aheih .M,Wilson- M., N. Dashkevich, A., Boeszoermenyi, H., Arthanari, C., J. Schmidt, and Schmidt, M., A. Bauer, F., Straube, Zhang, R., L., Malik, S., J. Elowe, B., Ross, Wang, A., L., Santamaria, Conway, B., Mogessie, P., C. Samora, T., P. Johansen, J., Henningsen, V., Akimov, A., T. Prokhorova, T., K. Rigbolt, uky .R,Cae,T,Mrhw .K,Sl,A . ono,R S., R. Johnson, D., A. Silk, K., M. Morphew, T., Cramer, R., F. Putkey, T. Fukagawa, M., R., Perpelescu, J. Yates, X., I. McLeod, K., Okawa, T., Hori, M., I. Cheeseman, M., Okada, Y. L. Yu-Lee, and R. B. Brinkley, H., S. Lin, R., Evans, Y., Kurasawa, M., Nishino, icio,T .adSlo,E D. E. Salmon, and J. T. Mitchison, ead,P,Daim .M n ogr .K. P. Sorger, and M. V. Draviam, P., Meraldi, ARTICLE RESEARCH 184 irtblsadbnst uvdprotofilaments. Bathe, curved A., to R. binds Milligan, and M., microtubules Oberer, M., al. Markus, et N., M. Monnier, M., E. A. Kubalek, E. 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