Ato o orsodne([email protected]) correspondence for *Author Singapore Drive, Medical 28 Engineering, Singapore. and 117456, Sciences Integrative for School igpr,8CleeRa,Snaoe195,Singapore. 169857, Singapore Road, College 8 Singapore, egXu Peng mitotic for alignment dynamics motor regulates B56-PP2A REPORT SHORT ß eevd7Arl21;Acpe 2Ags 2014 August 12 Accepted 2014; April 7 Received requires 1 process Upon This 2012). form. plus-end K-fibers al., the stable et (or microtubules), Wandke plate of metaphase 2012; tip the al., (Kapoor to et plate Stumpff delivering metaphase 2006; the motors al., 2007), towards plus-end-directed al., et the chromosomes et by Yang guide counteracted 2007; that al., is et motion Tanaka laterally this 2007; and motor al., The et microtubule-associated 2008). (Li minus-end-directed motion Desai, poleward requires rapid and that a Foley often the undergo Tanaka 2009; initially and chromosomes Walczak, is 2009; the attached and Kapoor, (Cai the at attachment microtubules and between of chromosomes kinetochore interaction surface lateral an duplicated initial by of mediated plate, alignment metaphase proper For INTRODUCTION Chromosome PP2A, congression , Kinesin, BUBR1, WORDS: KEY and stable exit. mitotic of and establishment attachments, subsequent kinetochore-microtubule the functional for essential is plate, metaphase which the towards movement alignment we chromosome chromosome balancing mitotic Thus, by motor-driven in plate. functions B56-PP2A promoting metaphase that the propose for towards required movement chromosome is the interaction Furthermore, the cells. to B56-depleted B56–BUBR1 in movement rescued chromosome is if plate restored metaphase Strikingly, be . can motor formation minus-end-directed K-fiber kinesin- major as a known KIFC1), B56 (also HSET or the the of 14 of depletion Notably, by depleted rescued K-fibers. cells be can in stable family chromosomes establishing of movement directly by plate, poleward by metaphase primarily than the alignment towards rather movement chromosome chromosome promotes balancing show B56-PP2A we Here, elusive. that remained but has checkpoint alignment, mechanism(s) chromosome underlying mitotic proper the for the required with are subunits BUBR1, interaction regulatory protein The (PP2A) their microtubules. 2A through formation) of phosphatase (B56-PP2A), protein plus-end (K-fiber of the family connection and B56 kinetochore end-on the the physical between and metaphase movement and the chromosome functional motor-driven at both chromosomes involves duplicated plate of alignment Proper ABSTRACT rga nCne tmCl ilg,Dk-U rdaeMdclSchool, Medical Graduate Duke-NUS Biology, Cell Stem & Cancer in Program 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,46–53doi:10.1242/jcs.154609 4567–4573 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 1,2 ai .Virshup M. David , 1 n agHu Lee Hyun Sang and 2 U Graduate NUS 1, * oal,teB6sbnt B6P2)rcutP2 ofr a form to the PP2A at BUBR1 2009). protein recruit checkpoint mitotic essential (B56-PP2A) Shenolikar, the with subunits complex and B56 (Virshup the holoenzyme substrate Notably, and the localization of subcellular specificity the determine subunits regulatory‘B’ which in heterotrimer a forms that phosphatase 2010). threonine al., components et (Welburn multiple network upper KMN 1A, of the by (Fig. within Ndc80/Hec1) destabilized manner phosphorylation are bind end-on K-fibers and Aurora-B-mediated directly an formed that erroneously in Mis12 whereas kinetochore K-fibers panel), the stabilize KNL1, at and 2006), components to al., et key (Cheeseman the (named network KMN for the factors, microtubule-binding essential irtbl tahetadmttcei,adtu o the for kinetochore– thus stable and exit, of stability. mitotic chromosome establishment of and maintenance subsequent attachment microtubule the essential movement is movement chromosome balanced for This plate. balancing metaphase the by towards alignment chromosome promoting the plate. in metaphase that defect the in the towards possible movement components to chromosome secondary is network be it might KMN cells Furthermore, the B56-depleted al., 2010). et of Liu phosphorylation al., 2011; increased al., et et Posch Lesage network 2011; 2010; KMN al., the et including (DeLuca B, components Aurora the directly of on phosphorylation substrates reverse kinetochore to is shown been has PP1 B56-PP2A PP2A However, than components. rather network that KMN the proposed Aurora-B-mediated of reversing phosphorylation by been formation K-fiber for has responsible 2013). it al., et Xu KMNTherefore, rescues 2011; al., partly et (Foley the misalignment B chromosome this Aurora by inhibiting of accompanied whereas components, phosphorylation misalignment network subunits Aurora-B-dependent chromosome B56 massive increased of knockdown to siRNA-mediated Xu The 2012; leads al., 2013). et Suijkerbuijk al., 2013; al., et et Kruse alignment 2011; al., chromosome et for (Foley redundantly function and kinetochore ae;DLc ta. 02.A U2i seta o K-fiber misaligned for increased essential markedly is NUF2 NUF2 of As 2002). lower depletion 1A, al., formation, Fig. et NDC80; reduced the as also DeLuca known in cells (also panel; Hec1 HeLa component of in levels essential protein NUF2 the of the siRNA- Depletion NUF2, by complex. formation Ndc80 chromosome of K-fiber depletion to disrupted To selectively mediated lead subunits. B56 we of would this, depletion as test network degree network, similar KMN KMN a to the misalignment the to K-fibers of attach stably was disruption to cells inability B56-depleted an in to alignment due chromosome in defect the of If inhibition by caused formation that K-fiber from B56 different of is depletion subunits by caused misalignment Chromosome DISCUSSION AND RESULTS rti hshts A(PA samjrclua serine- cellular major a is (PP2A) 2A phosphatase Protein ee eso htB6P2 ucin nmitotic in functions B56-PP2A that show we Here, 4567

Journal of Cell Science infcn (Student’s significant nlddi h eodtaseto.E,epyvco;M,mtrdfcet T idtp.(,,)Dt hwtemean the show Data (C,D,F) cell type. HeLa wild GFP-positive WT, in deficient; alignment motor chromosome MD, of vector; Quantification empty EV, (F) HSET. transfection. of second depletion the verify in to included analysis immunoblot Right, ( nM)]. (20 siRNA HSET or a:b ( which cells for HeLa of percentages mean HR REPORT SHORT eeotnfudna otesideplso ewe h spindle the chromosomes 4568 between or misaligned 2013), poles spindle al., those the to et However, near Xu found c). often 2011; were panel al., misalignment chromosome 1B, et caused (Fig. also (Foley subunits B56 manner of depletion redundant alignment a opposite chromosome reports promote in previous the with B56 Consistent all between b). that panel demonstrating found 1B, (Fig. majority poles spindle the with chromosomes, a: An panel). (left ‘a’ as designated 0 is of of line) ratio ratio (yellow a:b i A 2 plate an cortex. the metaphase bars: cell whereas using the Scale the from plate, interval) shown. C, and (KT) h metaphase are pole in kinetochore 24 the images spindle each graph at a of representative the aligned (with distance and For the chromosomes transfections fixation B. and siRNA-mediat two indicates before from ‘b’, verify after h as analysis to cells designated 3 immunofluorescence analysis is HeLa for using immunoblot distance of K-fibers. MG132 misalignment panel, analysis of with chromosome Lower disruption Immunofluorescence treated of microtubules. by (B) were severity to caused control. Cells kinetochore that Ctl, nM). the from cells. (100 linking different HeLa siRNAs directly is in network subunits NUF2 KMN of B56 human of depletion the depletion of by model caused schematic misalignment . Fig. n . 0 el e odto) h niae iNswr rnfce noclsa o ,ecp 0n fpamdecdn h niae F-agdHE was HSET GFP-tagged indicated the encoding plasmid of ng 10 except E, for as cells into transfected were siRNAs indicated The condition). per cells 100 n . 0 el e odto) el eetasetdtiewt h niae iNswt 4hitra B6sRA 8 M oehrwt control with together nM) (80 siRNAs [B56 interval 24-h a with siRNAs indicated the with twice transfected were Cells condition). per cells 100 t -test). , a:b , § . niae idymslge hoooe ewe h pnl oeadtemtpaepae o ,the D, For plate. metaphase the and pole spindle the between chromosomes misaligned mildly indicates 0.5 . r hw ( shown are 0.5 n . 0 ieohrsprcniin.()Lf,Qatfcto fcrmsm lgmn in alignment chromosome of Quantification Left, (E) condition). per kinetochores 200 § eie stertoo h itneo ahkntcoefo the from was kinetochore each distance of relative distance the The of panel). plate ratio the left metaphase as the 1C, defined (Fig. from kinetochore measured each was of distance relative disrupting of NUF2. of more case depletion the indicating by in cells, K-fibers than B56-depleted misalignment in chromosome cortex severe cell the and poles . niae eeeymslge hoooe oaie ewe the between localized chromosomes misaligned severely indicates 0.5 oaayetesvrt fcrmsm iainet the misalignment, chromosome of severity the analyze To ora fCl cec 21)17 5747 doi:10.1242/jcs.154609 4567–4573 127, (2014) Science Cell of Journal n . 9clsprec odto.Lf,teinterpolar the Left, condition. each per cells 19 6 .. * s.d.; m P .(,)Qatfcto fthe of Quantification (C,D) m. , .5 ** 0.05; A pe panel, Upper (A) P , .1 S not NS, 0.01; ai f0 of ratio b ndicated ed s

Journal of Cell Science fNF splmnaymtra i.SAB.B otat the contrast, By S1A,B). Fig. of depletion material Depletion to (supplementary results NUF2 graph). similar of showed lower also components S1A, was Ndc80 other Fig. siRNAs) material relative different of supplementary (reproduced average cells two NUF2-depleted the in Notably, by kinetochores 0.5. at the be found of kinetochore would distance a pole of (b). spindle (a/b) poles distance the spindle relative the the between instance, distance For the to (a) plate metaphase REPORT SHORT el Fg D.Atog hoooemslgmn ol also could misalignment NUF2-depleted chromosome to of Although compared 1D). distance as (Fig. relative cells cells B56-depleted a in with B56-depleted observed kinetochores in of kinetochores number the of was cells distance relative average . . Fg C ih ae) utemr,a increased an Furthermore, panel). right 1C, (Fig. 0.3 , . Fg C ih panel; right 1C, (Fig. 0.3 . . was 0.5 rmt hoooeainettruhaptwydifferent network. pathway KMN the cells a to might K-fibers NUF2-depleted through stabilizing B56-PP2A alignment or from Thus, control chromosome S1C,D). B56- promote either Fig. in in shorter material substantially than (supplementary also cells was in monopole depleted spindle 2007), center the the S- al., from of inhibitor distance Eg5 kinetochore et the the with (STLC), (McClelland trityl-L-cysteine treatment by defects generated cells spindle monopolar bipolar induce seta nclslcigKfbr Cie l,20a ose al., et Kops 2009a; al., et (Cai becomes K-fibers pathway lacking cells congression in essential chromosome motor-driven cells B56-depleted The in HSET alignment of chromosome activity promotes motor minus-end-directed the Suppressing ora fCl cec 21)17 5747 doi:10.1242/jcs.154609 4567–4573 127, (2014) Science Cell of Journal niae iNs(0 M.Saebr:5 bars: the using Scale interval) nM). 24-h (100 a siRNAs (with indicated twice transfected cells HeLa of (Student’s significant etooe a eie sae .** 1. area the as between defined area was of centrosomes size show comparable Data a panel. microtubules, upper the in mean shown the in as intensity (cytoplasm), mean 2 1 the area area by in divided intensity microtubules) mean for (cold-stable the chosen indicates were ratio degree The similar quantification. a to aligned chromosomes cells HeLa in microtubules ( cold-stable of Quantification 5 (C) bars: Scale experiments). independent irtblsaeidctd( indicated are microtubules (Ctl) control with together nM), [B56 (80 HSET-3 interval NUF2 or 24-h or a nM) with (80 siRNAs siRNAs indicated the with plate. twice chromosome metaphase in the B56 defect to of the movement depletion to by secondary caused is K-fibers subunits of Loss 2. Fig. h enpretgs( percentages microtubules. mean The cold-stable of proteins. analysis indicated Immunofluorescence the (B) of depletion confirmed siRNA-mediated cells the metaphase nocodazole-arrested of analysis n . 0clsprec odto)fo .Clswith Cells B. from condition) each per cells 20 6 9 ..Frtecniin htlce cold-stable lacked that conditions the For s.d. T-pcfcsRA(0n).()Immunoblot (A) nM)]. (20 siRNA UTR-specific t ts) D muoloecneanalysis Immunofluorescence (D) -test). 6 .. fclspsesn cold-stable possessing cells of s.d.) n . 0 el e odto;three condition; per cells 100 el eetransfected were Cells P , m m .1 S not NS, 0.01; m. m. 4569

Journal of Cell Science HR REPORT SHORT ST oevr h eceefc a pcfct HSET, 4570 S2B; to Fig. material (supplementary specific IC2 loss chain concomitant was in intermediate resulted the effect also of that rescue dynein of of depletion activity the because motor metaphase Moreover, minus-end-directed of the the HSET. 1F). antagonizing towards (Fig. the by movement promotes movement cells plate B56-PP2A chromosome co-depleted poleward that suggest HSET motor-driven results promote and motor-defective these the Collectively, B56 of on not in re-expression dependent chromosomes as did was HSET, HSET-N593K of cells activity chromosome B56-depleted motor on Furthermore, in effect effect. an misalignment rescue of possibility off-target and the the B56 excluding siRNA-mediated reversed 1F), in (Fig. partly alignment S2A) chromosome cells Fig. co-depleted material GFP- HSET of These (supplementary Re-expression 2009a). HSET plate. chromosome tagged metaphase al., promotes the et normally towards cells B56-PP2A movement co-depleted Cai that HSET S1E; suggest and results Fig. NUF2 in material seen 1E), (supplementary was (Fig. what plate metaphase to B56-depleted the similar at of aligned population chromosomes with independent the cells two were subunits increased using HSET substantially B56 cells of siRNAs depletion poles, of Depleted spindle kinetochores the depletion the to near cells. with Whereas misalignment, MG132 chromosome HeLa massive inhibitor arrest. caused proteasome in metaphase the cause subunits with time treated same B56 the subsequently at we (also them the depleting HSET Thus, by as proteins dynein shown). and motor not KIFC1) as minus-end-directed (data known the cells on of B56-depleted focused levels did in kinetochores the change the motor-driven Moreover, at not CENP-E in minus-end- cells. kinesin defect in motor B56-depleted plus-end-directed increase a in relative force) was K- a directed there (e.g. lacking whether movement the cells chromosome determine of in prompted to S1A–D) accumulation Fig. than us material supplementary poleward cells 1C,D; (Fig. of B56-depleted fibers extent in greater kinetochores The 2010). amre l,20)ddntsgiiatyrsu chromosome rescue significantly Fig. material not (supplementary S2C). cells did B56-depleted 2009) in misalignment al., et Palmer el tbyepesn F-agdhsoeHB(sa (as time-lapse envelope H2B nuclear live-cell from by Timed histone 3A,B). followed (Fig. GFP-tagged analysis were microscopy expressing marker) metaphase, through chromosome progress stably to cells chromosomes cells of B56-depleted dynamics motor allow the could of rescue whether assess exit To mitotic is trigger cells to B56-depleted sufficient in movement chromosome chromosome in Promoting defect a to chromosome secondary K-fibers for is of movement. loss cells required that these B56-depleted and is plate, in together, metaphase B56-PP2A Taken the towards that 2013). movement Draviam, SPAG5; indicate as and results known Shrestha (also astrin 2D; Fig. against HSET immunofluorescence and antibodies contrast, by B56 using K- confirmed in analysis further K-fibers By restore 2B, of was (Fig. presence cells not 2C). depleted The co-depleted was Fig. 2C). did NUF2 Fig. protein d,e; 2A) c; network panels (Fig. KMN the HSET panel if fibers K- of 2B, restored rescued depletion (Fig. and efficiently comparable plate formation subunits metaphase Importantly, the B56 fiber b). to and (panel alignment it HSET chromosome abolished of subunits whereas co-depletion a), B56 Cold-exposed panel of 2B, 1981). (Fig. depletion formation (Rieder, completed. K-fiber stable retained end-on- cells is cold HeLa whereas are cold, alignment K-fibers the attached in chromosome K-fiber depolymerize rescue K-fibers not after should Unattached 2A) co- even (Fig. plate, in HSET formation event metaphase and primary B56 the a of is on depletion formation alignment K-fiber chromosome in B56 promoting of function the the to If secondary movement is chromosome cells in B56-depleted defect in K-fibers of Loss ora fCl cec 21)17 5747 doi:10.1242/jcs.154609 4567–4573 127, (2014) Science Cell of Journal oto.Saebr:5 bars: Ctl, Scale shown. control. are anaphase to from NEBD chromosomes intervals. of 10-min images at Selected h 14 for was performed analysis imaging time- live-cell transfection, lapse second the At after interval. h 24-h 12 a with twice indicated siRNAs the with transfected were (green) GFP–H2B expressing stably exit. mitotic to trigger sufficient is in cells plate B56-depleted metaphase the to movement chromosome Promoting 3. Fig. h,chromosomes. Chr, of (purple). timecourse observations the during died (red) or metaphase in arrested prolonged underwent (green), from metaphase exited either cells are indicated; cells bars). tracked (blue individually of plotted fates is The h) (0 spent NEBD cells after selected randomly ( that A time from cells mitotic of of progression timing the of Quantification (B) A eacells HeLa (A) m m. n . 0.The 50).

Journal of Cell Science PAmgtas atycnrbt oefcetKfbrformation. K-fiber efficient B56- to that contribute and partly suggesting B56 also 3A,B), might of (Fig. the PP2A respectively) co-depleted of ( exit, cells cells formation metaphase control in overall in longer than note, HSET took the and Of plate HSET 2B–D). with of metaphase (Fig. depletion consistent subunits simultaneous again by B56 K-fibers 3), 3A,B; of (Fig. Movie restoration mitosis material exited successfully supplementary cells these Remarkably, HR REPORT SHORT elto fHE ihteB6sbnt etrdproper restored subunits co- B56 above, the reported in with As alignment 3A,B; HSET (Fig. chromosome 2). h of 15 Movie depletion in material mitosis exited misaligned supplementary and massively alignment with chromosome state Only prometaphase-like chromosomes. a in arrest than less in , mitosis exited cells control , all (NEBD), down break 0 fHL el eltdo 5 uuishdaprolonged a had subunits B56 of contrast, By depleted 1). cells Movie HeLa material of supplementary 80% 3B; (Fig. h 0.7 , 5 fB6dpee el completed cells B56-depleted of 15% , , 0 fHL el Fg 3A,B). (Fig. cells HeLa of 60% versus h 2 , . rmNB to NEBD from h 0.7 oehls,w ocueta 5-PAi seta for exit. mitotic essential the trigger towards to is sufficient movement is B56-PP2A which promoting plate, metaphase that by alignment conclude chromosome we Nonetheless, ramn.Ide,c-elto fHE n BUBR1 and severely with HSET cells MG132 of by of population metaphase the co-depletion in decreased substantially Indeed, arrested the subsequently depleted and treatment. panels), was left were 4A, HSET (Fig. purpose, cells cells HeLa this minus-end-directed in For BUBR1 the with HSET. together to in of due seen activity also misalignment motor was chromosome B56-depleted cells the in whether BUBR1-depleted misalignment HSET tested of chromosome we depletion rescue As cells, 2013). to al., able et 2013; Xu was al., 2012; et al., (Kruse et alignment Suijkerbuijk chromosome proper kinetochore to the contributes onto B56-PP2A of recruitment plate BUBR1-mediated metaphase chromosome the for towards required movement is interaction B56–BUBR1 The ora fCl cec 21)17 5747 doi:10.1242/jcs.154609 4567–4573 127, (2014) Science Cell of Journal ioi hoooeainetb balancing by in alignment (KT) chromosome kinetochores mitotic the at functions B56-PP2A 5 bars: Scale substitution. Ala-Ala-Ala-Ala to substitution; Phe Glu-Ala-Thr-His Ala-Ala EATH/AAAA, to to Ile Ile-Ile I/F, II/AA, acids; substitution; amino a.a., type; wild co-transfected. WT, were BUBR1 LAP-tagged siRNA- insensitive encoding vectors the expression transfection, indicated second the During condition). ( BUBR1 LAP-tagged the indicated re-expressing cells in HeLa alignment BUBR1-depleted chromosome of Quantification (B) h ieohrstruhHc,i nAurora-B- an manner. in dependent Hec1, to through HSET kinetochores of the recruitment the antagonizing be by might promoted movement chromosome This metaphase plate. the towards movement chromosome mean co- HSET ( without depletion or with cells in HeLa alignment BUBR1-depleted chromosome of HSET. quantification and Right, BUBR1 of depletion confirming siRNA-mediated analysis immunoblot Left, chromosomes, (A) and respectively. spindles mitotic the visualize against analysis antibody immunofluorescence an to using for subjected MG132 and after with h h treated 3 48 were At cells interval. transfection, 24-h initial a with were twice Cells transfected nM). with (80 together siRNA transfected BUBR1-specific was nM) (20 siRNA specific hoooemvmn oad h metaphase plate. the for towards required movement is interaction chromosome B56–BUBR1 The 4. Fig. 6 o-iecn oto Cl rHSET-3 or (Ctl) control Non-silencing .. * s.d.; n . P 0 e ahcniin.Dt hwthe show Data condition). each per 100 , .5(Student’s 0.05 m a .()Apooe model. proposed A (C) m. tblnadDP to DAPI and -tubulin n . t -test). 0 el e each per cells 100 9 UTR- 4571

Journal of Cell Science eeual orsu hoooeainetdfcsin defects alignment chromosome rescue B56 to (Xu to binding in previously defective unable mutants shown point were BUBR1 As 2013), B56–BUBR1 4B). al., (Fig. et the (LAP)-tagged BUBR1 of GFP-S-tag with RNAi-resistant importance replaced and the depleted using Endogenous was movement. BUBR1 tested chromosome By (Xu motor-driven we subunits in B56 panel). interaction 2013), to al., binding right in et defective 4A, mutants (Fig. point BUBR1 chromosomes misaligned REPORT SHORT iei oos(..HE)a h ieohrs ned the Indeed, kinetochores. the 4572 at HSET) control (e.g. might Hec1 motors that modified indicating N-terminally kinesin 2011), al., an et expressing (Mattiuzzo cells Hec1 in reported forces been al., pulling have et kinetochore-generated (Wong abnormal Kar3 Moreover, with Ndc80 2007). of 2012; of phosphorylation interaction al., Aurora-B-mediated the et promotes and Ndc80 2005), (Jin al., kinetochores et the Tanaka to localizes Kar3 homolog) yeast, budding motor- (HSET in the Interestingly, promotes point pathway. congression kinetochore driven BUBR1 the at of the also interaction Thus, kinetochore. HSet B56–BUBR1 defects the to of B56-PP2A recruit alignment depletion by cannot that (3) chromosome mutants caused and the defects depletion of co-depletion BUBR1 rescued alignment or (2) chromosome B56 2013), lines al., either the cell et rescued Xu MVA 2012; HSET al., BUBR1-mutant et the and (Suijkerbuijk, outer in with HeLa to alignment chromosome B56-BB2A BUBR1-depleted promote alignment to targeting sufficient was artificially chromosome kinetochores (1) motor-driven results: following in BUBR1 PP1- promotes both establishing from B56-PP2A emanating of where poles. microtubules which probability spindle with plate, higher connections in metaphase dependent a model the have towards chromosomes a movement favor chromosome results Thus, 2010). the al., et our (Liu to congression cold-stable chromosome not PP1 of but formation K-fibers, of the compromises recruitment (DeLuca markedly 2010). kinetochore al., kinetochores KNL1-mediated et network Posch 2010; the blocking KMN al., et Liu the Notably, on 2011; al., including et Lesage phosphorylation 2011; B, al., et Aurora reverse major of the has to be substrates PP1 to as shown K-fibers, thought was stabilizing is been directly subunits PP2A, in in function than involved B56 their rather phosphatase and PP1, and formation exit. K-fiber HSET mitotic both chromosome restore of rescuing to 2009a), co-depletion sufficient al., by et (Cai movement NUF2 of depletion network by KMN the disrupting directly poleward unlike chromosome Moreover, movement. antagonizing by the towards possibly motility plate, misaligned chromosome metaphase balancing in B56-PP2A massively for essential role an chromosome indicating HSET, of the the activity the motor suppressing minus-end-directed by Indeed, rescued was for cells cells. B56-depleted in misalignment B56-depleted account by in chromosomes formation cannot K-fiber of K-fiber inhibition simple formation an promote that revealed analysis However, to quantitative in kinetochores. our the B56- at proposed phosphorylation components B 2011), Aurora been reversing al., network has et KMN PP2A (Foley fashion the Aurora-B-kinase-dependent of right phosphorylation promotes 4B, (Fig. interaction plate. metaphase B56–BUBR1 mutants the towards the point movement chromosome that BUBR1 indicating of chromosome graph), the (left defects rescued kinetochores they HSET the alignment although of to depletion panel), properly Strikingly, localize right panels). to 4B, ability (Fig. the BUBR1 retained of depleted cells eas elto fB6sbnt assincreased causes subunits B56 of depletion Because u aaas niaeta 5-PAi e eitrof mediator key a is B56-PP2A that indicate also data Our agtn oto iN eeprhsdfo Dharmacon. from purchased were siRNA control targeting 5 ATTAACATA-3 5 ATTA-3 TGCCGTGAAACGTATA-3 STCS 5 HSET-CDS, eune r sflos BUBR1-3 follows: as CTGCCT-3 are sequences AAA-3 lgmn yblnigcrmsm oeettwrsthe towards chromosome movement exit, mitotic 4C). chromosome mitotic (Fig. plate in balancing trigger metaphase functions to by B56-PP2A and alignment that K-fibers functional suggesting of the allow to formation sufficient was cells the minus-end-directed B56-depleted by in to movement knockdown HSET chromosome recruitment the promoting HSET Nonetheless, antagonize inhibiting kinetochore. Aurora-B- by might of movement chromosome levels Fig. B56-PP2A material the (supplementary S3D), increased Hec1 of subunits phosphorylation mediated B56 HSET As S3A–C). of of Fig. domain material stalk depletion (supplementary the cells mammalian to the bound in Hec1 of domain N-terminal xrsigHBGPwr rce o 4ha 0mnintervals. min 10 at h 14 for tracked were H2B–GFP expressing Ivtoe) h olo 5-pcfcsRAtree orB56 four targeted siRNA B56-specific AAGTAGTCCATATGTT-3 of B56 2000 pool Lipofectamine isoforms: with 2012; The performed al., was et Transfection (Invitrogen). (Suijkerbuijk 2009b). al., previously pEGFP- et described into and Cai as cloned pLAP-BUBR1 were PCR plasmids respectively. were HSET fragments pcDNA-HA, HSET and and pCS2-Myc 1–409) acids (amino Hec1 siRNAs and Plasmids METHODS AND MATERIALS AE rmr nioisaanttefloigpoen eeused; were proteins following SDS- for the buffer against lysis B56 cell antibodies SDS Primary 4% using PAGE. prepared were extracts Protein analysis Immunoblotting Resolution Super iXon a an using with 3D-SIM used. equipped with were were (Nikon) temperature Images Probes) Microscope Scientific). room (Molecular (Thermo at 594 DAPI acquired with Fluor to counterstained Alexa were Inc. conjugated and Cells Antibodies (1:2000) 488 Fluor from antibodies Alexa secondary was and Isotype-specific (ACA) 1:100) (1:500). antibody Cruz; Anti- bovine X-100; Santa Triton 1:2000). following 4% 0.1% the and (sc-98605, BSA containing against 1% antibodies containing astrin PBS PBS Primary in to h. diluted were 1 exposed proteins for permeabilized and (BSA) were albumin X-100 cells serum Triton fixed The min 0.5% methanol. 10 for with cold methanol. ice on with cold Tek) placed fixation or were cells (Lab before 4) assay, (Fig. microtubule slides (PFA) cold-stable the chamber paraformaldehyde For 4% coverglass-bottomed with on fixed were grown cells imaging HeLa live-cell time-lapse and Immunofluorescence (DMEM) medium Biochemicals). mM, Selleck this (3 (2 mM, in Eagle’s S-trityl-L-cysteine (20 used ZM447439 Sigma), Drugs ng/ml, modified (200 Sigma), Scientific). nocodazole (Thermo were FBS Dulbecco’s study 10% were with H2B, in supplemented histone GFP-tagged expressing cultured stably those including treatment Cells, drug and culture Cell lmnsA otae o ielpelv-elaayi,astage-top a analysis, CO live-cell digital time-lapse with For a incubation with software. microscope AR inverted (100 Elements Ti-E TIRF Eclipse Apo Nikon CFI a on mounted (Andor) 9 9 -GATCAAACATGACGGAATT-3 -CTCAACTTTACCACCAAGTTA-3 d Frse ta. 07,NF a126,Acm,Hc (9G3, Hec1 Abcam), (ab122962, NUF2 2007), al., et (Forester 9 n B56 and ; ora fCl cec 21)17 5747 doi:10.1242/jcs.154609 4567–4573 127, (2014) Science Cell of Journal 9 e1 5 Hec1, ; 9 HSET-3 ; a 9 -TCAGAAGCAGCCCTGTCAA-3 ,5 9 n yolsi yenhaycan(DYNC1H1), chain heavy dynein cytoplasmic and ; e 9 ,5 -CAATACAAGTGCCGAATAA-3 9 6 -AAGTTCAAAAGCTGGAUGATCTT-3 9 -TTAATGAACTGGTGGACTA-3 14 i)ojcieadpoesdwt h NIS- the with processed and objective oil) /1.40 9 T,5 UTR, m ,BOO nentoa)adMG132 and International) BIOMOL M, 9 9 2 B56 ; U22 5 NUF2-2, ; ie Tki a sd el stably Cells used. was (Tokai) mixer 9 -CATGTCCCAGGGCTATCAAAT-3 d ,5 9 YCH mr oladnon- and pool smart DYNC1H1 . 9 9 T,5 UTR, 9 p2,5 Spc25, ; -CAGGAGATTATTCTCACC- 9 -GAACGAGTAACCACA- EM a tbln(A3 Sigma; (AA13, -tubulin 9 8 MC camera EMCCD 885 + -GTCTCACAGATTG- 9 9 U2 5 NUF2, ; -CCACGGTTTATA- 9 B56 ; 9 te siRNA Other . c 9 ,5 -AAGCA- 9 Spc24, ; 9 -CAG- 9 ;

Journal of Cell Science Kops,G.J.P.L.,Saurin,A.T.andMeraldi,P. E. C. Walczak, and C. S. Ems-McClung, N., L. Weaver, S., Cai, h uhr elr ocmeiginterests. competing no declare authors The interests Competing reagents. valuable sharing for Jennifer CO) and University, Netherlands) J.P.L. State The Geert (Colorado Utrecht, IN), DeLuca Utrecht, Institute, Center Biology Medical Molecular (University (Indiana Kops Walczak Claire thank We Acknowledegements (M-63, HSET Cruz), Santa (9E10, Cruz), Myc Santa Cruz), Santa (Y-11, HA Abcam), REPORT SHORT aor .M,Lmsn .A,Hret . aeo,L,Cmn,D,Salmon, D., Cimini, L., Cameron, P., Hergert, A., M. Lampson, M., T. Kapoor, Y. Wang, and H.-G. Yu, P., Li, M. H., D. Liu, F., Virshup, Jin, and J. Goris, V., J. Louis, J., Maddox, M., M. C. Forester, T. Kapoor, and M. Maldonado, A., E. Foley, M. T. Kapoor, and A. G. E. Foley, J. DeLuca, and D. A. E. M. Salmon, S. and Lens, V. F., J. K. DeLuca, Kilmartin, M., J. Hickey, B., Moree, G., J. DeLuca, A. Desai, and M. E. Wilson-Kubalek, S., J. Chappie, M., I. Cheeseman, E. C. Walczak, and A. Khodjakov, B., C. O’Connell, S., Cai, E. C. Walczak, and S. Cai, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.154609/-/DC1 online available material Supplementary material Supplementary and programs; School. (S.H.L.) Medical Fellow Graduate Research Singapore Duke-NUS National of the and Foundation (D.M.V.) Research STAR National its the under by supported was research This Funding The S.H.L. and S.H.L. D.M.V. by by interpreted written and by was analyzed designed paper were and Data of S.H.L. conceived and D.M.V. were Experiments experiments. performed P.X. contributions Author ieohrspwrcrmsm congression. chromosome power kinetochores cross-linking by length microtubules. spindle sliding control and HSET/XCTK2 proteins family Kinesin-14 otemtpaepaebfr biorientation. before plate A. metaphase Khodjakov, the and F. to B. McEwen, D., are E. that attachment syntelic checkpoint. with tension the chromosomes by in sensed Cdk1. results complex and motor Cdc25C Kar3 of regulation PP2A by USA Sci. exit Acad. B56- mitotic the of Control on depends microtubules and phosphatase. PP2A kinetochores between attachments stability express. attachment kinetochore-microtubule mitosis. control during phosphorylation Hec1 cells. HeLa and in attachment death kinetochore-microtubule cell stable mitotic induces blocks inhibition hNuf2 (2002). the of site microtubule-binding core the kinetochore. constitutes network KMN conserved The fibres. kinetochore of absence the in congression Cycle Cell a.Cl Biol. Cell Nat. 8 3791-3793. , b Cell tbln(ba)and (Abcam) -tubulin .Cl Sci. Cell J. 104 127 19867-19872. , a.Cl Biol. Cell Nat. 983-997. , 11 o.Bo.Cell Biol. Mol. 20) h odls rvldt h pnl equator. spindle the to traveled less road The (2009). 787-789. , 124 20) hoooecnrsin ntebi-orient the on congression: Chromosome (2009). 622-634. , LSGenet. PLoS 13 1265-1271. , b 20 .Cl Biol. Cell J. atn(Sigma). -actin 21) idn h idegon:how ground: middle the Finding (2010). 20) hoooe a congress can Chromosomes (2006). 1348-1359. , 21) oso ucino h Cik1/ the of function of Loss (2012). Science el o.Lf Sci. Life Mol. Cell. a.Cl Biol. Cell Nat. 8 21) eprlcagsin changes Temporal (2011). e1002492. , 21) omto fstable of Formation (2011). 159 311 549-555. , 20a.Chromosome (2009a). 388-391. , 11 67 832-838. , 2145-2161. , rc Natl. Proc. (2009b). (2007). (2006). aaa . iaua . iaua .adTnk,T U. T. Tanaka, and Y. Kitamura, E., Kitamura, K., Tanaka, A. Desai, and U. T. Tanaka, i,D,Vegl . akr .B,Hr,T,Fkgw,T,Cesmn .M and M. I. Cheeseman, T., Fukagawa, T., Hori, B., C. Backer, M., Vleugel, D., Liu, ujebik .J . lue,M,Tier,A n os .J .L. P. J. G. Kops, and A. Teixeira, M., Vleugel, E., J. S. Suijkerbuijk, ade . aii,M,Sg,R,Ruh . of . mr,A . a,C H., C. Tan, C., A. Amaro, F., Wolf, V., Rauch, R., Sigl, M., Barisic, C., Wandke, S. Shenolikar, and M. D. Virshup, A. Prescott, K., E. James, M., Breugel, van H., Dewar, N., Mukae, K., Tanaka, i . u . in,Y n h,X. Zhu, and Y. Liang, W., Yu, Y., M. Li, Bollen, and J. Qian, B., Lesage, tmf,J,Wgnah . rnk . suy .L n odmn L. Wordeman, and L. C. Asbury, A., Franck, M., Wagenbach, J., Stumpff, M. V. Draviam, and L. R. Shrestha, L. C. Rieder, ebr,J .I,Vegl . i,D,Yts .R,II apo,M A., C., Goodner, M. J.-S., Kang, Lampson, C., Shang, III, S., Westermann, R., Y., Nakajima, J. J., Wong, Yates, D., Liu, M., Vleugel, I., P. J. Welburn, rs,T,Zag . asn .S . icet,T,Srihr . Kragh W., Streicher, T., Lischetti, Y., S. M. Larsen, G., Zhang, T., Kruse, oc,M,Kodl,G . wf,S,Kn,E . eua .G n Swedlow, and G. J. Deluca, M., E. King, S., Swift, A., G. Khoudoli, M., Posch, Palmer,K.J.,Hughes,H.andStephens,D.J. McAinsh, M., Belwal, R., J. Winter, C., A. Amaro, and S., A. Borusu, E., Musacchio, S. McClelland, C., Ciferri, M., Fiore, P., Totta, G., Vargiu, M., Mattiuzzo, ag . uu .S,Wdwrh .adRee,C L. C. Rieder, and P. Wadsworth, S., U. Tulu, Z., Yang, H. S. Lee, and M. D. Virshup, M., Kitagawa, A., E. Raetz, P., Xu, ue ieohr yKL poe uoaBkinase. B Aurora opposes KNL1 by kinetochore outer A. M. Lampson, nerto fkns n hshts ciiisb UR nue omto of formation ensures BUBR1 by activities phosphatase and kinase of Integration rmt hoooecnrsinadsidemcouuednmc during dynamics al. microtubule et spindle H. and Maiato, mitosis. congression U., chromosome Kutay, promote J., A. Pereira, spindle toward transport kinetochore poles. microtubule-dependent of mechanisms microtubules. spindle by U. T. capture Tanaka, and C. Antony, R., end. the to means attachments. kinetochore-microtubule stable oeadpligfret aiiaecnrsinadfl hoooealignment. chromosome full and congression Res. facilitate Cell to force pulling poleward balance. the tips irtbl rwhsprsinadsailcnrlo ieohr tension. kinetochore via of movements control spatial centromere and confine Cell Dev. suppression chromokinesins growth and microtubule Kif18A (2012). MCAK. and CENP-E kinesins Biol. Curr. requires attachment chromosome-microtubule cells. PtK1 metaphase osmn,P,Fed,S,Ca,C .M,Dui,D tal. et spindle. D. mitotic Drubin, the M., of S. map C. interaction Chan, S., Fields, P., Houshmand, interface. kinetochore-microtubule the regulate Cell differentially Mol. to targets M. distinct I. Cheeseman, and T. Fukagawa, makeover. a get phosphatases n 5-PApopaaecmlxsrglt ioi progression. mitotic regulate complexes Sci. phosphatase J. B56-PP2A Nilsson, and and P. S. Bjørn, T., Nielsen, neatosa mitosis. at interactions R. J. steps. membrane-trafficking discrete in subunits dynein P. bipolarity. spindle Meraldi, and congression chromosome controls and D. A. Hec1. modified N-terminally a expressing F. Degrassi, yeni eurdfrcrmsm oinadcnrsinidpneto the of independent congression and motion checkpoint. chromosome spindle for required is chromosome dynein promote to subunits targeting of family B56 congression. the via PP2A recruits 126 21) d2 euae uoaBatvt n microtubule-kinetochore and activity B aurora regulates Sds22 (2010). .Cl Biol. Cell J. ora fCl cec 21)17 5747 doi:10.1242/jcs.154609 4567–4573 127, (2014) Science Cell of Journal 1086-1092. , .Cl Biol. Cell J. 38 17 22 23 383-392. , 701-712. , 1017-1029. , 1514-1526. , il Open Biol. 18) h tutr ftecl-tbekntcoefbrin fiber kinetochore cold-stable the of structure The (1981). 21) bomlkntcoegnrtdpligfre from forces pulling kinetochore-generated Abnormal (2011). ur Biol. Curr. 21) euae agtn fpoenpopaae1t the to 1 phosphatase protein of targeting Regulated (2010). 178 ur pn elBiol. Cell Opin. Curr. ur Biol. Curr. 198 269-281. , .Cl Biol. Cell J. Chromosoma 20) h EPANCCDkntcoecomplex kinetochore NAC/CAD CENP-A The (2007). 847-863. , 2 479-486. , 21 20) ieohr-irtbl neatos the interactions: Kinetochore-microtubule (2008). R898-R903. , 17 o.Cell Mol. Nature 973-980. , 20) rmpoiciyt rcso:protein precision: to promiscuity From (2009). 20) oeua ehnsso kinetochore of mechanisms Molecular (2005). 191 21) pnl hcpitslnig PP1 silencing: checkpoint Spindle (2011). 84 21) uoaBpopoyae spatially phosphorylates B Aurora (2010). 21) aea oedo ovrinof conversion end-on to Lateral (2013). 20) ieohr yengnrtsa generates dynein Kinetochore (2007). o.Bo.Cell Biol. Mol. 61-74. , 145-158. , 20 21) ietbnigbtenBubR1 between binding Direct (2013). 434 33 LSONE PLoS 53-63. , 537-545. , 987-994. , e.Cell Dev. 21) ua chromokinesins Human (2012). 20) pcfct fcytoplasmic of Specificity (2009). o.Bo.Cell Biol. Mol. 18 .Cl Biol. Cell J. 6 MOJ. EMBO 23 e16307. , 3800-3809. , 745-755. , 20) Kinetochore (2007). 20) Molecular (2007). 20) protein A (2007). 21) BUBR1 (2013). 26 20 188 5033-5047. , 2885-2899. , 809-820. , (2012). .Cell J. 4573

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