Adaptive changes in the kinetochore architecture facilitate proper spindle assembly

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1 A RT I C L E S Adptive chnges in the kinetochore rchitecture fcilitte proper spindle ssemly Vlentin Mgidson,5,6, Rj Pul,3,5, Nchen Yng, Jeffrey G. Ault, Christopher B. O Connell,6, Irin Tikhonenko, Bruce F. McEwen, Alex Mogilner 3,7 nd Alexey Khodjkov,4,7 Mitotic spindle formtion relies on the stochstic cpture of microtuules t kinetochores. Kinetochore rchitecture ffects the efficiency nd fidelity of this process with lrge kinetochores expected to ccelerte ssemly t the expense of ccurcy, nd smller kinetochores to suppress errors t the expense of efficiency. We demonstrte tht on mitotic entry, kinetochores in cultured humn cells form lrge crescents tht susequently compct into discrete structures on opposite sides of the centromere. This compction occurs only fter the formtion of end-on microtuule ttchments. Live-cell microscopy revels tht centromere rottion medited y lterl kinetochore microtuule interctions precedes the formtion of end-on ttchments nd kinetochore compction. Computtionl nlyses of kinetochore expnsion compction in the context of lterl interctions correctly predict experimentlly oserved spindle ssemly times with resonle error rtes. The computtionl model suggests tht lrger kinetochores reduce oth errors nd ssemly times, which cn explin the roustness of spindle ssemly nd the functionl significnce of enlrged kinetochores. Chromosome segregtion during cell division is encted y the mitotic spindle. Chromosomes connect to the spindle through kinetochores tht cpture microtuules nd ttch to their plus ends, the principle descried s serch-nd-cpture (S&C; refs 5). A rmifiction of the S&C mechnism is tht kinetochore size nd shpe ply fundmentl role in determining the efficiency nd fidelity of chromosome segregtion. Intuitively, lrger kinetochores re expected to increse the proility of encounters etween kinetochores nd microtuules, which would lso promote errors such s ttchment of sister kinetochores to the sme spindle pole (syntelic) or ttchment of single kinetochore to oth poles (merotelic). Cellulr regultions tht minimize erroneous ttchments while expediting spindle ssemly remin unknown. Here we demonstrte tht the shpe of the kinetochore s outer lyer chnges mrkedly nd rpidly during the norml course of mitosis. At the onset of spindle ssemly, sister kinetochores expnd to lmost completely encircle the centromere. After the formtion of end-on ttchments to microtuules the enlrged kinetochores downsize into smll discs on opposite sides of the centromere. Computtionl nlyses suggest tht the oserved reorgniztion of the kinetochore rchitecture simultneously enhnces the efficiency of microtuule cpture nd suppresses the numer of erroneous ttchments. Error reduction is due to improvements in the ngulr orienttion of enlrged kinetochores tht result from lterl interctions with microtuules efore the formtion of end-on ttchments. If these lterl interctions re impeded, the numer of errors increses significntly. RESULTS The outer lyer of unttched kinetochores encircles the centromere Properly ttched kinetochores pper s nerly diffrction-limited spots in fluorescence light microscopy 6 (LM) nd s -nm discs positioned on opposite sides of the centromere in electron microscopy 7,8 (EM). However, the kinetochore outer lyer is enlrged when cells re rrested in mitosis owing to lck of microtuules 9. Enlrged kinetochores hve een oserved lso during prometphse in HeL cells. To test the ide tht kinetochore size nd shpe chnge during norml spindle ssemly we detiled the kinetochore rchitecture t vrious mitotic stges. In non-trnsformed humn cells RPE, the outer-kinetochore protein CenpF forms compct spots during lte prophse nd metphse, ut prtilly encircles the centromere shortly fter nucler Wdsworth Center, New York Stte Deprtment of Helth, Alny, New York, USA. Indin Assocition for the Cultivtion of Science, Jdvpur, Kolkt 73, Indi. 3 Cournt Institute nd Deprtment of Biology, New York University, New York, New York, USA. 4 Rensseler Polytechnic Institute, Troy, New York 8, USA. 5 These uthors contriuted eqully to this work. 6 Present ddresses: Ntionl Cncer Institute, Frederick, Mrylnd 7, USA (V.M.); Nikon Instruments, Melville, New York 747, USA (C.B.O C.). 7 Correspondence should e ddressed to A.M. or A.K. (e-mil: mogilner@cims.nyu.edu or lexey.khodjkov@helth.ny.gov) Received 6 Novemer 4; ccepted 3 July 5; pulished online August 5; DOI:.38/nc33 34 NATURE CELL BIOLOGY VOLUME 7 NUMBER 9 SEPTEMBER 5 5 Mcmilln Pulishers Limited. All rights reserved

2 A RT I C L E S envelope rekdown (NEB, 93 of 74 kinetochores in 3 cells) nd during prometphse (67 of 55 kinetochores in 6 cells; Fig. ). Despite the pprent chnge in morphology, the mount of CenpF t the kinetochore remins constnt from prophse through prometphse (Fig. c). To detil chnges within the outer lyer we co-visulized CenpF (ref. 3) nd CenpE (refs 4,5), the two most peripherl kinetochore proteins cple of direct interctions with microtuules. CenpE ppers t the kinetochores only fter NEB (Fig. ). We hve previously demonstrted tht the centrl region of forming spindles ecomes devoid of chromosomes min fter NEB in RPE cells 6 (see Supplementry Videos nd 3). This feture llows one to identify cells tht re in the first minute of spindle ssemly. In these cells, CenpE is detected only t some of the kinetochores. Intriguingly, CenpF forms compct spots in CenpEnegtive kinetochores ut prtilly encircles the centromere in CenpEpositive kinetochores (Fig. d,e). As the mount of CenpF remins constnt during prophse prometphse (Fig. c), reltive volumes occupied y this protein cn e compred. Volumetric nlysis (Fig. f) suggests tht the outer lyer expnds during spindle ssemly. Unlike CenpF, proteins tht reside deeper inside the kinetochore (Hec, Mis; ref. 6) pper s compct spots throughout mitosis (Fig. ) even though the mount of Hec increses during prometphse (Fig. c) wheres the mount of Mis remins constnt (Fig. c). The volume occupied y Mis lso remins constnt (Fig. g). Thus, chnges in the kinetochore rchitecture occur primrily in the outer lyers of the orgnelle. To evlute the shpe of the outer lyer t higher resolution, we employed correltive LM/EM. Seril-section nlyses of three cells t NEB revel highly vrile kinetochore morphology. Although the exct loction of kinetochores is determined y mens of correltion of LM nd EM imges (Fig. 3), most of the kinetochores lck distinct pltes. In one cell ll kinetochores pper s ill-defined spots of firous mteril (Fig. 3). In the other two cells, 3% of kinetochores (53/85) exhiit trilminr pltes tht lrgely encircle the centromere. The gp etween the pltes of sister kinetochores is typiclly nm on one side nd lrger on the other side of the centromere (Fig. 3d). The rest of the kinetochores pper s ill-defined spots or prtilly ssemled pltes emedded in smll cloud of firous mteril (Fig. 3c). These oservtions suggest tht t the onset of spindle ssemly, the outer plte rpidly expnds from compct cloud to lrge crescent on the surfce of the centromere. Microtuules in the proximity of prtilly ssemled pltes indicte tht microtuule-medited forces my ply role in shping the kinetochore outer lyer 7 (Fig. 3c). To chrcterize the rchitecture of virgin kinetochores tht hve not intercted with microtuules, we treted cells in lte prophse with nocodzole nd fixed them immeditely fter detection of NEB (Supplementry Fig. ). As nocodzole completely depolymerizes microtuules within min (ref. 7), this experimentl pproch produces kinetochores whose rchitecture is unffected y interctions with microtuules or prolonged mitotic rrest. CenpF immunofluorescence suggests tht the outer lyers of virgin kinetochores consistently form crescents tht encircle the centromere (367/5 kinetochores in cells). In most cses, sister kinetochores seem to fuse together on one side of the centromere with gp on the other side of the centromere, where sister kinetochores re seprted y chromtin (Supplementry Fig. d). Seril-section EM confirms tht the outer lyers of sister kinetochores prtilly encircle the centromere (>8 kinetochores in the three cells). Three-dimensionl (3D) reconstructions of six typicl centromeres suggest tht the outer lyers of virgin sister kinetochores cover >5% of the centromere circumference ut remin seprted y nm (Fig. 3f). The outer lyer compcts fter formtion of end-on microtuule ttchment The oservtion tht the kinetochore outer lyers trnsition from encircling lrge prt of the centromere fter NEB to -nm discs on the opposite sides of the centromere t metphse, prompted us to serch for the event tht triggers kinetochore compction. Visul inspection of fluorescently lelled prometphse kinetochores nd microtuules suggests tht most compct kinetochores re ttched to the ends of microtuule undles. In contrst, enlrged kinetochores either lck microtuule ttchments or interct with microtuules lterlly (Supplementry Fig. ). As unmiguous discrimintion of end-on versus lterl interctions is difficult in LM, we treted RPE cells with the CenpE (kinesin-7) inhiitor GSK (ref. 8). CenpE drives congression of chromosomes from the vicinity of the pole to the spindle equtor 5,9, nd medites conversion from lterl to end-on ttchments. Prtil inhiition of CenpE with 5-nM GSK-9395 results in the ccumultion of mono-oriented chromosomes nd prolongtion of mitosis to > h in >8% of cells. Immunofluorescence demonstrtes tht outer (CenpF), core (Hec, Mis), nd inner (CenpA GFP) kinetochore proteins pper s compct spots on congressed chromosomes in GSK treted cells. In contrst, oth CenpF nd Hec form crescents on chromosomes tht reside ner the spindle poles (Fig. 4,). In GSK-9395-treted RPE cells with one llele of Md replced with Md Venus, t lest one kinetochore on ech mono-oriented chromosome remins Md-positive. Correltive LM/EM nlysis of 3 Md-positive kinetochores in 4 cells demonstrtes tht these kinetochores lck end-on microtuule ttchments ut interct lterlly with the wlls of djcent microtuules. The outer lyer of these kinetochores is enlrged to 4 5 nm nd is either spred longside n djcent microtuule undle (Fig. 4d) or prtilly encircles the centromere (see Fig. 3d). In contrst, end-on ttched kinetochores in the sme cells re -nm discs (Fig. 4d ). This difference in the rchitecture of end-on ttched versus lterlly intercting kinetochores suggests tht kinetochore compction occurs only fter the formtion of end-on microtuule ttchment nd independently in sister kinetochores. Intensity mesurements demonstrte tht kinetochores of congressed chromosomes in GSK-9395-treted cells contin s much Hec s in untreted metphse cells (compre Figs 4c nd f) wheres the mount of CenpF decreses to 5% of the norml metphse level (compre Figs 4c nd c, P <.). In contrst, the mount of oth CenpF nd Hec on polr chromosomes is higher thn seen during ny stge of norml mitosis (compre Figs 4c nd c,f, ll P <. in ech comprison). This difference suggests tht kinetochores enlrge if end-on ttchment is delyed. Similr increses in fluorescence intensity nd morphologicl enlrgement of the outer lyer re oserved when cells re rrested in the sence of microtuules (Supplementry Fig. 3). NATURE CELL BIOLOGY VOLUME 7 NUMBER 9 SEPTEMBER Mcmilln Pulishers Limited. All rights reserved

3 A RT I C L E S Prophse NEB Prometphse Metphse c CenpF CenpA DNA Prophse NEB Prometphse Metphse. Reltive fluorescence intensity Pro NEB Promet Met Ks = Cs = d Prophse NEB Erly prometphse Prometphse e CenpE DNA f Reltive volume CenpF. Prophse NEB Erly prometphse Prometphse. Pro NEB Promet Ks =,36,3,9 Cs = Figure Chnges in the outer kinetochore rchitecture t vrious stges of mitosis. ( c) The outer lyer enlrges t the onset of spindle ssemly nd susequently downsizes. () Mximl-intensity projections (including ll kinetochores) depicting RPE cells t vrious stges of mitosis. () Exmples of individul kinetochores from the outlined res in, shown t higher mgnifiction. The outer lyers (red, CenpF) pper s compct spots during prophse nd metphse ut expnd into crescents tht prtilly encircle the centromere during NEB nd prometphse. (c) Intensity of CenpF fluorescence remins constnt during outer lyer expnsion nd decreses during metphse. (d f) The outer lyer oth recruits dditionl proteins (CenpE, green) nd expnds (CenpF, red). (d) Mximl-intensity projections (including ll kinetochores) depicting RPE cells. (e) Exmples of individul kinetochores from the outlined res in d, shown t higher mgnifiction. (f) The volume occupied y CenpF increses from lte prophse to prometphse. Blue rs in c nd f re clculted s the men of the men vlues for multiple kinetochores in individul cells (n vlues listed elow the rs, Cs; cells). Error rs represent stndrd error of the men (s.e.m.). Yellow rs re men vlues clculted for ll kinetochores, pooled from ll cells in tht clss (n vlues listed elow the rs, Ks; kinetochores). Error rs represent stndrd devition (s.d.). Triple sterisks denote differences with P <.5 (two-tiled Student s t-test) for oth lue versus lue nd yellow versus yellow rs. Lrge compctle kinetochores support rpid nd low-error microtuule cpture Our oservtions tht the outer lyers of sister kinetochores rpidly expnd to encircle the centromere t the onset of spindle ssemly nd susequently compct into smll discs on formtion of end-on microtuule ttchments re consistent with previous reports in vrious types of mmmlin 9,,3 nd fly cells 4,5. However, in the context of the S&C mechnism, kinetochore enlrgement t the onset of mitosis 36 NATURE CELL BIOLOGY VOLUME 7 NUMBER 9 SEPTEMBER 5 5 Mcmilln Pulishers Limited. All rights reserved

4 A RT I C L E S Prophse NEB Prometphse Metphse Hec CenpA DNA Prophse NEB Prometphse Metphse. c Reltive fluorescence intensity Ks = Cs = Pro NEB Promet Met d d Prophse NEB Prometphse Metphse e Mis CenpA DNA. Prophse NEB Prometphse Metphse f g Rel. fluor. intensity Reltive volume..8.4 Pro NEB Promet Met Ks = Cs = Pro NEB Promet Ks = Cs = Figure The kinetochore core remins reltively compct throughout mitosis. ( c) The distriution of the core kinetochore component Hec is similr throughout mitosis (,, red) in spite of significnt increse in the mount of this protein during NEB nd prometphse (c). (d f) The distriution of Mis (d,e, red), the mount (f) nd the volume occupied y this protein (g) remin constnt throughout mitosis. Also note tht the shpe of the inner kinetochore remins compct throughout mitosis (green, CenpA GFP). (,d) Mximlintensity projections (including ll kinetochores) depicting RPE cells t vrious stges of mitosis. (,e) Exmples of individul kinetochores from the outlined res in nd d shown t higher mgnifiction. Blue rs in c,f nd g re clculted s the men of the men vlues for multiple kinetochores in individul cells (n vlues listed elow the rs; Cs, cells). Error rs represent s.e.m. Yellow rs re men vlues clculted for ll kinetochores pooled from ll cells in tht clss (n vlues listed elow the rs; Ks, kinetochores). Triple sterisks denote differences with P <.5 (two-tiled Student s t-test) for oth lue versus lue nd yellow versus yellow rs. seems counterproductive ecuse lrge kinetochores re expected to increse ttchment errors. Indeed, kinetochore enlrgement during mitotic rrest is postulted to drive the pronounced increse in ttchment errors in nocodzole wshout experiments 6. To explore potentil effects of kinetochore enlrgement on the numer of erroneous ttchments we employed computtionl modelling. In minimlistic stochstic models of spindle ssemly, rndomly locted chromosomes re expected to form stle NATURE CELL BIOLOGY VOLUME 7 NUMBER 9 SEPTEMBER Mcmilln Pulishers Limited. All rights reserved

5 A RT I C L E S 5 nm. c 5 nm d 5 nm e 5 nm f Firous coron, 3D Figure 3 Kinetochore morphology t the onset of spindle ssemly. () Correltive LM/EM nlysis of kinetochore morphology t NEB. Mximlintensity projection of the entire cell nd n individul focl plne re shown. Through correltion of LM ( ) nd EM ( ) imges, positions of individul kinetochores re identified. Outlined res re shown t higher mgnifiction. Kinetochore lcks the distinct trilminr plte. Blck rrows mrk prtilly disssemled nucler envelope. ( d) Exmples of kinetochore morphology in RPE cells t NEB. Ech chromosome is shown in LM (left) nd seril EM sections. Approximtely 5% of kinetochores re morphologiclly indistinct (). The other 5% exhiit 38 prtilly (c), or fully ssemled pltes tht lrgely encircle the centromere (d). Arrowheds denote firous coron; rrows point t distinct trilminr pltes. (e,f) Morphology of virgin kinetochores in cell fixed less thn min fter NEB in the sence of microtuules (see Supplementry Fig. for whole-cell view nd preprtion detils). (e) Complete series of sections through sister kinetochores. Yellow rrows mrk trilminr pltes; red rrows point t microtuules; lck rrows mrk remnnts of nucler envelope. (f) Exmples of 3D reconstructions of the outer lyer (first imge corresponds to the kinetochores shown in e). The outer lyers lrgely encircle the centromere. NATURE CELL BIOLOGY VOLUME 7 NUMBER 9 SEPTEMBER 5 5 Mcmilln Pulishers Limited. All rights reserved

6 A RT I C L E S CenpA GFP, CenpF, DNA CenpA GFP CenpF, DNA CenpA GFP CenpF CenpA GFP CenpF, DNA CenpA GFP CenpF CenpA GFP, Hec, DNA d d DIC CenpA GFP Hec, DNA CenpA GFP Hec. c Reltive fluorescence intensity Congressed Polr Congressed Polr Ks = Cs = Hec CenpF Md venus DIC Md venus 5 nm Figure 4 Kinetochore outer lyer compction occurs on the formtion of end-on microtuule ttchments. (,) Kinetochores re enlrged on polr, ut compct on congressed, chromosomes. Whole-cell imges re mximlintensity projections tht include ll kinetochores in the cell. Individul kinetochores re shown s mximl-intensity projections. CenpF delinetes the outer lyer () nd Hec delinetes the kinetochore cores (). (c) Amounts of oth CenpF nd Hec re incresed on polr chromosomes. Blue rs re the men kinetochore intensity clculted s the men of the men vlues for multiple kinetochores in individul cells (n vlues re listed elow the rs. Cs: cells). Error rs represent s.e.m. Yellow rs re men vlues clculted for ll Ks kinetochores pooled from ll cells in tht clss (n vlues listed elow the rs. Ks; kinetochores). Error rs represent s.d. Triple sterisks denote differences with P <.5 (two-tiled Student s t-test) for oth lue versus lue nd yellow versus yellow rs. (d,d ) Sister kinetochores on congressing chromosomes exhiit distinctly different morphologies. (d) A single-plne differentil interference contrst (DIC) imge of the cell ner the centre of the mitotic spindle. Arrows denote congressing chromosomes. The centromere region of one congressing chromosome (yellow outlined) is lso shown t higher mgnifiction in DIC nd Md Venus fluorescence (r = µm). Arrows point t the pproximte positions of the leding (yellow) nd triling (red) kinetochores. (d ) Consecutive 8-nm electron microscopy sections through the centromere of the chromosome shown in d. Note the size difference etween the triling end-on ttched kinetochore (red rrows) versus the leding kinetochore (yellow rrows) tht lcks endon ttchment ut intercts with microtuules lterlly. The cells re treted with 5-nM GSK 9395, cell-permele inhiitor of CenpE to slow down chromosome congression nd conversion from lterl to end-on microtuule ttchments. end-on microtuule ttchments instntneously when growing microtuule plus end runs into kinetochore. Microtuule cpture y sister kinetochores is uncorrelted nd the kinetochore tht hs lredy ttched to microtuule cn cquire n dditionl connection if it is hit y nother microtuule. Thus, fter the initil formtion of monotelic ttchment, the next cpture is y chnce correct or erroneous (results in syntelic or merotelic ttchments) 7 3. A mjor limittion of these simplistic models is tht they predict unrelisticlly long times for spindle ssemly due to low proility of encounters etween microtuules nd -nm smll kinetochores 7. Our experimentl oservtions suggest tht the size of the cpture trget ws underestimted in these models, which prompted us to replce smll discoid kinetochores with lrge crescents (Supplementry Fig. 4) tht ecome compct fter microtuule ttchment. The durtion of compction (τ comp, Supplementry Fig. 4) is not directly reveled in our experimentl nlyses. Therefore, we explored vrious compction times nd found tht this prmeter does not significntly ffect the time of spindle ssemly or the numer of errors (Supplementry Fig. 4d,d ). Enlrged kinetochores ccelerte the time of spindle ssemly to 5 min (Supplementry Fig. 4e), which is in greement with the oservtion tht the metphse plte forms in 8 min in RPE cells 6. However, the numer of erroneous ttchments predicted y this simultion is lrge (>3%, Supplementry Fig. 4e). We hve previously suggested tht the numer of erroneous ttchments is reduced y the rottion of the chromosome tht occurs immeditely on the initil cpture 8. This rottion orients the centromere so tht its xis (the line connecting the centres of sister kinetochores) ecomes roughly prllel to the cptured microtuule (Fig. 5). As result, the unttched sister kinetochore is less likely to cpture microtuules from the sme spindle pole s its sister, which suppresses ttchment errors 8. NATURE CELL BIOLOGY VOLUME 7 NUMBER 9 SEPTEMBER Mcmilln Pulishers Limited. All rights reserved

7 A RT I C L E S First cpture Centromere rottion First compction, second cpture Second compction Percentge of error per cell μm. μm.3.67 μm τ rot = 3 s τ comp = 6 s τ rot (s) τ comp (s) size (μm) c Percentge of error per cell Optiml centromere τ cpt (min) Figure 5 Effects of kinetochore enlrgement compction on the efficiency nd fidelity of cpture-driven spindle ssemly. () Crtoon of the events suggested in this model. Kinetochores re shown s red crescents. Green lines represent properly ttched microtuules; red lines represent potentil erroneous ttchments. () Error frequencies predicted t vrious gp sizes for vrious rottion nd compction times. (c) The frequency of errors nd durtion of spindle ssemly for different gp sizes t specific rottion nd compction times. Shded re represents the rnge of prmeters tht result in ll chromosomes ttching in <8 min with <% of errors. The predicted optiml geometry of the centromere is shown s n inset in c. Note tht the predicted optiml geometry is not similr to the centromere rchitecture oserved in prometphse cells (Fig. 3). Simultions tht consider oth rottion nd compction of initilly lrge crescent-shped kinetochores suggest tht rpid rottions significntly reduce the errors. Such rpid rottions ( 3 s) hve een directly oserved in live cells 6. Similrly, the errors re suppressed if kinetochores compct in <6 s (Fig. 5). At the conservtively pessimistic prmeters of τ rot = 3 s nd τ comp = 6 s, the model predicts similr cpture times ut lower error rtes ( 3%, Fig. 5c) thn simultions without the rottion. However, owing to the inverse reltionship etween the speed of spindle ssemly nd the numer of errors, efficient mitosis seems to require specific nd highly uniform geometry of the centromere (Fig. 5c). For exmple, spindle ssemly in 8 min t the error rte % is predicted for nrrow rnge of gps etween sister kinetochores ( µm, Fig. 5c). This prediction is ill comptile with the roustness of cell division 3 nd the oserved vriility in the centromere rchitecture (Fig. 3f). Specificlly, the.. µm gps oserved on mny virgin centromeres (Fig. 3f) would led to >5% errors. Lterl interctions tht precede formtion of end-on ttchments increse roustness of spindle ssemly Difficulties in reconciling simultions with experimentl oservtions prompted us to re-evlute fundmentl ssumption emedded in ll existing computtionl models of spindle ssemly tht the formtion of microtuule ttchment is single-step process. In vivo oservtions revel tht formtion of end-on ttchments is preceded y lterl interctions etween the kinetochores nd the wlls of microtuules 4,6,3,33. These interctions dominte during erly prometphse when centromere xes ecome prtilly ligned with the spindle xis 6,33. The extent of this ngulr lignment is similr in cells rendered incple of forming end-on ttchments 34 y depletions of the NDC8 component Nuf (ref. 6). Thus, the ngulr lignment is driven primrily, if not exclusively, y lterl interctions etween kinetochores nd microtuules. To gin further insights into the role of lterl interctions during incorportion of chromosome into the spindle, we used flttened RPE cells tht express fluorescent fusion of the checkpoint protein Md (ref. ). Correltive LM/EM demonstrtes tht lterl interctions do not remove Md from the kinetochores (Fig. 4), which provides redout for the formtion of end-on ttchment. The prolonged mitosis in flttened cells, with some chromosomes ttching to microtuules only during lte prometphse 35, enles us to follow the ehviour of individul chromosomes during their incorportion into the spindle. Time-lpse recordings of 6 chromosomes in 7 cells demonstrte tht centromeres rotte to roughly lign with the spindle xis while Md Venus is still present on oth sister kinetochores (Fig. 6). The disppernce of Md 4 NATURE CELL BIOLOGY VOLUME 7 NUMBER 9 SEPTEMBER 5 5 Mcmilln Pulishers Limited. All rights reserved

8 A RT I C L E S : :3 5: 5:3 8: 9: : μm XY XZ Phse contrst Md Phse contrst Md Figure 6 Centromere rottion on the surfce of the spindle precedes formtion of end-on microtuule ttchment. () Selected frmes from multi-mode time-lpse recording of RPE cell flttened to 3-µm (see Supplementry Video for full recording). The top row shows phse contrst (medil slice) nd the ottom row shows Md Venus fluorescence (mximlintensity projections). Both kinetochores on mono-oriented chromosomes re Md-positive (rrows) indicting the sence of end-on ttchments. Rpid rottion tht orients centromere xes roughly prllel to the spindle xis precedes chromosome congression nd relese of Md from the kinetochores. () Higher-mgnifiction view of the centromere mrked (red rrows) nd (yellow rrows). Note tht the centromeres ecome stretched only during congression shortly efore the relese of Md from the kinetochores. The cell is in chmer tht restricts mitotic rounding to 3 µm. from the kinetochores occurs 5 5 min fter the centromere ecomes roughly ligned with the spindle xis nd initites congression to the spindle equtor (Fig. 6 nd Supplementry Video ). This ehviour supports the ide tht centromere rottion precedes formtion of endon ttchments. On the sis of these oservtions, we modified the model to incorporte two-step process in which centromere rottion, medited y lterl kinetochore microtuule interctions, precedes microtuule end-on cpture (Fig. 7). We postulte tht the centromere rottes until the intercting microtuule reches the edge of the kinetochore t the gp (Supplementry Fig. 5). Lterl interctions re expected to initite within seconds fter NEB (ref. 6). Simultions tht consider centromere rottions efore end-on ttchment predict tht lrger kinetochores increse oth the speed nd ccurcy of the spindle ssemly. Unlike in ll previous models, the reltionship etween the speed nd ccurcy of spindle ssemly is not inverse nd the <% of error rte is predicted for much wider rnge of gp sizes thn in the ttchment rottion scenrio (compre Figs 7c nd 5c). Incorportion of oth types of centromere rottion (one driven y lterl interctions nd one resulting from end-on ttchment) into the model decreses the numer of errors to 5%, nd the error rte ecomes lrgely insensitive to the kinetochore size. With ny gp size <.5 µm, the ssemly time is <8 min, nd the error rte is <5% (Fig. 7e). Interestingly, the lowest error rte (Fig. 7e) is predicted when the gp etween sister kinetochores is the commonly oserved.-µm (Figs nd 3). Thus, lterl interctions tht precede formtion of end-on ttchments increse roustness of spindle ssemly. Devint geometry of the nscent spindle increses chromosome mis-segregtion Our model suggests tht the kinetochore rchitecture is dpted for spindle ssemly tht involves prtil ngulr lignment of centromeres tht normlly occurs on the surfce of nscent spindle efore the formtion of end-on ttchments 6. If pre-lignment is impeded, enlrged kinetochores would led to n increse in the numer of errors (Fig. 5c). This prompted us to evlute the orgniztion of the erly prometphse spindle in cells tht missegregte chromosomes. The lrgest frequency of centromere rottions is oserved during erly prometphse 6 when the centromeres reside on the surfce of nscent spindle (Fig. 8). This pttern forms in most RPE cells (>9% (47/5)). We find tht those untreted RPE cells tht fil to form the cler zone (3/5) re prone to exhiit lgging chromosomes during nphse (Fig. 8). To experimentlly impede formtion of the cler zone, we treted RPE cells with 3-µM nocodzole (for <3 min), locted cells tht just entered mitosis, wshed out the drug, nd followed formtion of the spindle y 3D time-lpse microscopy. Under these conditions, the cler zone consistently fils to form (n =) nd the centromeres re intermixed with microtuules, which impedes ngulr lignment of the centromeres. Forty-three per cent of the nocodzole-treted cells (9/) exhiit lgging chromosomes during nphse telophse (Fig. 8c). Therefore, lck of cler zone in the centre of the nscent spindle correltes with n incresed frequency of errors. DISCUSSION Direct oservtions of microtuule cpture y kinetochores 4,3 hve estlished S&C (ref. 5) s the sic principle of spindle ssemly. Multiple mechnisms such s the sptilly selective stiliztion of microtuules y RnGTP (ref. 36), the rottion of chromosomes 8, the formtion of specific sptil rrngement during erly prometphse 6, nd the sweeping movements of growing microtuules 37 promote microtuule cpture nd thus ccelerte spindle ssemly. However, the inverse reltionship etween the efficiency nd ccurcy of S&C-driven spindle formtion inherent NATURE CELL BIOLOGY VOLUME 7 NUMBER 9 SEPTEMBER Mcmilln Pulishers Limited. All rights reserved

9 A RT I C L E S Centromere rottion First cpture First compction, second cpture Second compction d Percentge of error per cell Percentge of error per cell 5 5 c 4 3. μm. μm.3.67 μm τ rot (s) μm. μm.3.67 μm τexpnd = 3 s τ rot (s) τ comp (s) τexpnd = 3 s τ comp (s) e Percentge of error per cell Percentge of error per cell τrot = 3 s τcomp = 6 s size (μm) Optiml centromere Optiml centromere τexpnd = 3 s τrot = 3 s τcomp = 6 s size (μm) τ cpt (min) τ cpt (min) Figure 7 Computtionl models tht consider centromere rottion due to lterl interctions with microtuules predict experimentlly oserved prmeters of spindle ssemly. () Crtoon of the events suggested in the model. Blue lines represent the centrl prt of the nscent spindle with high density of microtuules nd devoid of chromosomes. Kinetochores cn glide longside these microtuules resulting in rottion of the centromere. Green lines represent properly end-on ttched microtuules; red lines represent potentil erroneous ttchments. (,c) Results of the simultion tht considers single centromere rottion tht tkes plce efore endon microtuule ttchment nd is driven y lterl interctions. () Error frequencies predicted for vrious gp sizes t vrious rottion nd compction times. (c) Error frequencies nd durtions of spindle ssemly for vrious gp sizes t conservtively estimted rottion nd compction times. (d,e) Results of the ll-inclusive simultion tht considers expnsion nd compction of the kinetochore s well s two susequent rottions: one driven y lterl interctions (s in,c) nd one tht results from the end-on microtuule ttchment (s in Fig. 5,c). (d) Error frequencies predicted for vrious gp sizes t 3-s expnsion time nd vrious rottion nd compction times. (e) Error frequencies nd durtions of spindle ssemly for vrious gp sizes t specific expnsion, rottion nd compction times. Shded res in c nd e mrk the rnge of prmeters tht result in ll chromosomes ttching in <8 min with <% of errors. Predicted optiml geometry of the centromere is shown s insets in c nd e. Note the similrity of the predicted optiml geometry to the experimentl oservtions (Fig. 3). in ll previous computtionl models is incomptile with the well-estlished roustness of mitotic regultions. Our oservtion tht the kinetochore outer lyer expnds t the onset of mitosis demonstrtes tht previous computtionl models underestimted the size of microtuule-cpturing trget. By introducing dptle kinetochore geometry nd pre-lignment of centromeres due to rpid lterl kinetochore microtuule interctions efore the formtion of end-on microtuule ttchments 4,6,3,33,38, we hve constructed the first model tht predicts relisticlly rpid spindle ssemly with error rtes tht re sufficiently low to e hndled y error-correction mechnisms 6,39,4. A non-trivil prediction of our model is tht kinetochore expnsion during the phse of spindle ssemly when lterl interctions dominte cretes synergistic reltionship etween the efficiency nd fidelity of spindle ssemly. 4 NATURE CELL BIOLOGY VOLUME 7 NUMBER 9 SEPTEMBER 5 5 Mcmilln Pulishers Limited. All rights reserved

10 A RT I C L E S Axil Trnsverse Axil Trnsverse : :3 4:3 7:3 :3 :3 :3 5: : :3 5: 5:3 5:3 8:3 3:3 3: c Axil Trnsverse : :3 4:3 7:3 :3 :3 :3 5: Figure 8 Anorml geometry of the nscent spindle during erly prometphse correltes with erroneous chromosome segregtion. () Typicl pttern of spindle formtion (RPE cell). Note tht ll centromeres (CenpA GFP) reside on the surfce of the nscent spindle for the first 6 8 min of prometphse. () Exmple of n untreted RPE cell where the formtion of the cler centrl zone fils for unknown resons. The centromeres re interspersed from the onset of spindle ssemly. Although ll chromosomes congress onto the metphse plte with only minor dely, severl chromosomes lg ehind during nphse (rrows), which is indictive of erroneous kinetochore ttchments. (c) Spindle ssemly fter nocodzole wshout. Formtion of the cler centrl zone does not occur nd chromosomes re lgging during nphse (rrows). Asterisks mrk mother centrioles (lelled with centrin GFP). To ensure the detection of the cler zone ech time point is rotted nd shown s mximl-intensity projections in precisely xil nd trnsverse orienttions. See Supplementry Fig. 6 nd Supplementry Videos 3 5 for conventionl views of these cells. Time in minutes:seconds from NEB (,) or completion of nocodzole wshout (c). This cn explin the roustness of the process in spite of the inevitle size vrition of individul kinetochores in rel cells. A corollry of this prediction is tht conditions tht impede domintion of lterl interctions during the initil stges of spindle ssemly increse the numer of erroneous ttchments. Centromere lignment y mens of lterl interctions primrily tkes plce when centromeres reside on the surfce of the nscent spindle 6,33. Therefore, conditions tht ffect the formtion of the hollow spindle during erly prometphse re deleterious for chromosome segregtion. Consistent with this notion, ttchment errors re consistently oserved in cells depleted for chromokinesins 4,4, condition tht hs een shown to disrupt formtion of the ring 6. Further, chromosomes tend to mis-segregte when microtuules nd centromeres ecome intermixed during erly prometphse owing to reversile drug tretments (Fig. 8) or trnsient devitions from the proper geometry in cells with norml centrosoml ctivity Sutle nd trnsient chnges in the geometric constrints during the initil stges of spindle ssemly my still hve devstting consequences for genomic stility even when the rchitecture of the mture metphse spindles is not directly ffected. NATURE CELL BIOLOGY VOLUME 7 NUMBER 9 SEPTEMBER Mcmilln Pulishers Limited. All rights reserved

11 A RT I C L E S METHODS Methods nd ny ssocited references re ville in the online version of the pper. Note: Supplementry Informtion is ville in the online version of the pper ACKNOWLEDGEMENTS This work ws supported y NIH grnt GM59363 to A.K. nd NSF grnt DMS- 86 to A.M. The Electron Microscopy ws enled y the use of the Wdsworth Center s Electron Microscopy Core Fcility. We thnk J. Pines (University of Cmridge, UK) for his generous dontion of Md Venus cells nd S. Li (Air Worldwide, USA) for ssistnce with the intensity quntifictions. AUTHOR CONTRIBUTIONS A.K. nd B.F.M. designed the experiments, V.M., N.Y., C.B.O C. nd I.T. performed the experiments. J.G.A., B.F.M., A.K. nd I.T. conducted correltive LM/EM. A.M. nd R.P. developed the computtionl models. R.P. designed computer code nd performed simultions. The mnuscript ws written y A.K., B.F.M. nd A.M. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. 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CENP-F is novel microtuule-inding protein tht is essentil for kinetochore ttchments nd ffects the durtion of the mitotic checkpoint dely. Chromosom 5, 3 39 (6). 4. Cooke, C. A., Schr, B., Yen, T. J. & Ernshw, W. C. Locliztion of CENP-E in the firous coron nd outer plte of mmmlin kinetochores from prometphse through nphse. Chromosom 6, (997). 5. Kim, Y., Heuser, J. E., Wtermn-Storer, C. M. & Clevelnd, D. W. CENP-E comines slow, processive motor nd flexile coiled coil to produce n essentil motile kinetochore tether. J. Cell Biol. 8, 4 49 (8). 6. Mgidson, V. et l. The sptil rrngement of chromosomes during prometphse fcilittes spindle ssemly. Cell 46, (). 7. Loncrek, J. et l. The centromere geometry essentil for keeping mitosis error free is controlled y spindle forces. Nture 45, (7). 8. Wood, K. W. et l. Antitumor ctivity of n llosteric inhiitor of centromeressocited protein-e. Proc. Ntl Acd. Sci. USA 7, (). 9. Kpoor, T. M. et l. Chromosomes cn congress to the metphse plte efore iorienttion. Science 3, (6).. Ci, S., O Connell, C. B., Khodjkov, A. & Wlczk, C. E. Chromosome congression in the sence of kinetochore fiers. Nt. Cell Biol., (9).. Shresth, R. L. & Drvim, V. M. Lterl to end-on conversion of chromosomemicrotuule ttchment requires kinesins CENP-E nd MCAK. Curr. Biol. 3, (3).. Collin, P., Nshchekin, O., Wlker, R. & Pines, J. The spindle ssemly checkpoint works like rheostt rther thn toggle switch. Nt. Cell Biol. 5, (3). 3. Roos, U. P. Light nd electron microscopy of rt kngroo cells in mitosis. II. Kinetochore structure nd function. Chromosom 4, 95 (973). 4. Goldstein, L. S. Kinetochore structure nd its role in chromosome orienttion during the first meiotic division in mle D. melnogster. Cell 5, 59 6 (98). 5. Church, K. & Lin, H. P. Kinetochore microtuules nd chromosome movement during prometphse in Drosophil melnogster spermtocytes studied in life nd with the electron microscope. 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L. & Alexnder, S. P. Kinetochores re trnsported polewrd long single strl microtuule during chromosome ttchment to the spindle in newt lung cells. J. Cell Biol., 8 95 (99). 33. Kitjim, T. S., Ohsugi, M. & Ellenerg, J. Complete kinetochore trcking revels error-prone homologous chromosome iorienttion in mmmlin oocytes. Cell 46, (). 34. DeLuc, J. G. et l. Hec nd Nuf re core components of the kinetochore outer plte essentil for orgnizing microtuule ttchment sites. Mol. Biol. Cell 6, (5). 35. Lncster, O. M. et l. Mitotic rounding lters cell geometry to ensure efficient ipolr spindle formtion. Dev. Cell 5, 7 83 (3). 36. O Connell, C. B., Loncrek, J., Kl, P. & Khodjkov, A. Reltive contriutions of chromtin nd kinetochores to mitotic spindle ssemly. J. Cell Biol. 87, 43 5 (9). 37. Klinin, I. et l. Pivoting of microtuules round the spindle pole ccelertes kinetochore cpture. Nt. Cell Biol. 5, 8 87 (3). 38. Merdes, A. & De My, J. The mechnism of kinetochore-spindle ttchment nd polewrds movement nlyzed in PtK cells t the prophse-prometphse trnsition. Eur. J. Cell Biol. 53, (99). 39. Nicholson, J. M. & Cimini, D. How mitotic errors contriute to kryotypic diversity in cncer. Adv. Cncer Res., (). 4. Silkworth, W. T. & Cimini, D. Trnsient defects of mitotic spindle geometry nd chromosome segregtion errors. Cell Div. 7, 9 (). 4. Mzumdr, M., Sundreshn, S. & Misteli, T. Humn chromokinesin KIF4A functions in chromosome condenstion nd segregtion. J. Cell Biol. 66, 63 6 (4). 4. Wndke, C. et l. Humn chromokinesins promote chromosome congression nd spindle microtuule dynmics during mitosis. J. Cell Biol. 98, (). 43. Bkhoum, S. F., Genovese, G. & Compton, D. A. Devint kinetochore-microtuule dynmics underlie chromosoml instility. Curr. Biol. 9, (9). 44. Gnem, N. J., Godinho, S. A. & Pellmn, D. A mechnism linking extr centrosomes to chromosoml instility. Nture 46, 78 8 (9). 45. Silkworth, W. T., Nrdi, I. K., Scholl, L. M. & Cimini, D. Multipolr spindle pole colescence is mjor source of kinetochore mis-ttchment nd chromosome missegregtion in cncer cells. PLoS ONE 4, e6564 (9). 46. Kleylein-Sohn, J. et l. Acentrosoml spindle orgniztion renders cncer cells dependent on the kinesin HSET. J. Cell Sci. 5, (). 44 NATURE CELL BIOLOGY VOLUME 7 NUMBER 9 SEPTEMBER 5 5 Mcmilln Pulishers Limited. All rights reserved

12 DOI:.38/nc33 M E T H O D S METHODS Cell culture, chemicl tretments nd live-cell microscopy. The humn nontrnsformed htert-rpe cell line ws purchsed from Clontech in t pssge numer 8.5. Stocks of these cells t pssge numers were generted in the Khodjkov lortory nd kept in liquid nitrogen. A stle clone (RPE-8) tht co-expresses CenpA egfp nd centrin egfp (oth introduced y lentivirus) 6 ws used in most of the experiments descried here. Experiments tht required visuliztion of fluorescent Md were conducted in the RPE Md/Md Venus cell line provided y J. Pines, University of Cmridge. All cell lines were grown in ntiiotic-free DMEM supplemented with % FCS (Invitrogen) t 37 C, 5% CO. For live-cell imging, cells were grown on glss coverslips (no. /) nd mounted in Rose chmers contining CO -independent medi (Invitrogen) supplemented with % FCS. In-house tests for mycoplsm (high-concentrtion Hoechst stining) re negtive. Microtuule depolymeriztion ws induced y nocodzole (Sigm) t 3 µm. Motor ctivity of CenpE ws inhiited with GSK-9395 (ref. 8) purchsed from Hoyun Chemexpress. Multi-mode 3D time-lpse recordings were otined on Nikon TE-E PFS microscope with X Pln Apo, NA.4 oil immersion ojective lens. Fluorescence imges were cptured in spinning-disc confocl mode (GSU-, Yokogw) on ck-illuminted Cscde 5B EM CCD (chrge-coupled device) cmer (Photometrics). DIC imges were recorded on Photometrics CoolSnp CF cmer mounted on different port of the sme microscope. Full 3D volumes were recorded t ech time point t 5-nm Z-steps (48 6 plnes depending on cell thickness). To visulize formtion of the cler zone we first trcked 3D positions of mother centrioles nd then rotted the 3D volume t ech time point to fix the position of one mother centriole nd the orienttion of the spindle xis 47. This processing llowed us to oserve chromosome movement in the precisely trnsverse nd xil views. Mother centrioles were trcked using FIJI with the stndrd trcking plugin. 3D coordintes of the centrioles nd the imges were then imported into MtL. The imge volume ws pdded with lck ( vlue) voxels to prevent cropping during rottion. The rottion ws done in MtL through two sequentil steps, first in X Y nd then in Z. Rotted nd ligned imges were trnsferred ck to FIJI. Mximlintensity projections of the entire rotted volume were generted long the spindle xis (trnsverse view) nd orthogonlly to the spindle xis (xil view). Ech view presented in Fig. 8 contins oth centriole pirs nd ll kinetochores. Incorportion of individul chromosomes into the spindle ws oserved under conditions tht prevented cell rounding during mitosis. A coverslip with 3-µm microfricted feet ws plced on top of the coverslip with the growing cells. The contct etween coverslips ws mintined with negtive pressure using vcuum pump 48. Fixed-cell immunofluorescence. Cells were pre-extrcted in wrm PEM uffer (-mm PIPES, ph 6.9,.5-mM EGTA, 5-mM MgCl ) supplemented with.5% Triton X- for min nd fixed with % glutrldehyde for min in PEM. Microtuules were visulized with monoclonl nti-α-tuulin ntiody (DM, Sigm; : dilution). Kinetochores were delineted with the following ntiodies: rit αcenpf (Novus Biologicls, NB5-; :4 dilution), mouse αcenpe ntiody (Acm, 593; : dilution), mouse αhec (Acm, 363; : dilution), nd rit αmis (provided y I. Cheesemn, Msschusetts Institute of Technology, USA; :4 dilution) 49. Hoechst ( µg ml ) ws used to stin DNA (chromosomes). Inner kinetochores were visulized y mens of CenpA GFP fluorescence. Wide-field imges were recorded on DeltVision imging system (Applied Precision) with X NA.35 lens (Olympus). The imges were cptured with CH-35 CCD cmer (Photometrics) t 69-nm X Y pixel size nd -nm Z- steps. All imges were deconvolved with the SoftWoRx 5. deconvolution softwre (Applied Precision) nd ojective lens-specific point spred functions. Amir softwre (FEI) ws used for surfce rendering. The segmenttion threshold for fluorescence imges (Supplementry Fig. ) ws set t 5% of mximl intensity for ech dt set. Quntifiction of fluorescence intensity nd kinetochore volume. All mesurements were conducted in ImgeJ/FIJI nd clcultions were conducted in MS Excel. Integrted fluorescence intensity ws mesured within 3D volume centred on single kinetochore (whenever possile) or smll group of kinetochores (if their individul signls were not fully resolvle). The dimensions of the volume were set individully to include the entire oject of interest (3,5 voxels, volumes). Bckground intensity for ech mesurement ws mesured in the sme-dimensions volume positioned s close s possile to the oject of interest. Kinetochore intensity ws clculted y sutrcting ckground intensity nd dividing the result y the numer of kinetochores in the volume. Intensities of multiple kinetochores (usully ) were mesured in ech cell. Men fluorescence intensity per kinetochore ws clculted for individul cells nd then the men vlue of per-cell verges ws clculted. Alterntively, ll kinetochores mesured under prticulr experimentl condition were pooled together nd the men vlue ws clculted for this pooled popultion. Results of oth clcultions re presented in the figures (Ks: totl numer of kinetochores; Cs: totl numer of cells). All vlues re normlized so tht the men intensity t NEB equls. Kinetochore volumes were mesured with the 3D Oject Counter routine included in the stndrd distriution of FIJI. As the mount of CenpF nd Mis remins constnt during lte prophse prometphse, reltive volumes occupied y kinetochores cn e segmented t constnt threshold. Threshold vlues for segmenttion were set t % of mximl signl intensity for CenpF nd 5% for Hec dt sets. These thresholds were empiriclly determined to yield mximl numers of kinetochores per cell with miniml contmintion y flse ojects fter segmenttion. As in intensity clcultions, oth men vlues were clculted for per-cell verges nd for the pooled popultions (oth vlues re presented in the Figures). All vlues re normlized so tht the men volume t NEB equls. Men vlues were compred using two-tiled Student s t-test. Correltive electron microscopy. Cells were fixed in.5% glutrldehyde (Sigm) in PBS (ph ). Differentil interference contrst nd fluorescence imges were cquired t.-µm Z-steps through the entire cell volume shortly fter fixtion. Post-fixtion, emedding nd sectioning were done s previously descried 5. Seril 8-nm thin sections were imged t 8 kv on either Zeiss 9 (Crl Zeiss) or JEOL 4. Correltion of conspicuous morphologicl fetures etween differentil interference contrst nd EM imges ws used to mtch the orienttion nd Z positions for individul focl plnes nd determine exct kinetochore positions. Computtionl modelling. We consider tht spindle ssemly tkes plce in the sphericl volume tht ws occupied y the nucleus efore NEB. Implicitly, we ssume, following ref. 7, tht the RnGTP grdient focuses the microtuules into the nucler sphere. This ccelertes the serch few fold ut hs no effect on the error rte. Two centrosomes re plced t the opposite poles of the sphere t R cell nd +R cell positions. Ech centrosome nucletes N MT microtuules tht serch the spce isotropiclly. Ech microtuule is represented y rod with zero thickness tht undergoes dynmic instility. The plus end of microtuule grows stedily until ctstrophe occurs leding to microtuule shortening. The frequency of ctstrophe, s well s the growth nd shrinkge rtes, is constnt (results hve een found not to e sensitive to smll vritions of ctstrophe frequencies). We use the optiml zero rescue frequency 8. Microtuule dynmics re simulted y the Monte Crlo lgorithm: rndom numer is generted etween nd with equl proility. At ech computtionl step (with time increment t = s) the microtuule switches to shortening if this rndom numer is less thn [ exp( f ct t)]. New microtuules grow in rndom directions nd do not turn. In ll cses, if microtuule plus end extends eyond the nucler sphere s oundry or encounters chromosome rm, this microtuule undergoes ctstrophe nd shrinks ll the wy ck to the centrosome. The vlues for the numer of microtuules generted y ech centrosome (N MT ) nd the four prmeters of dynmic instility (v g, v s, f ct, f res ) used in the simultions re presented in Supplementry Tle. The rdius of the nucler/spindle sphere is set to mtch the geometry of mitosis in RPE cells 6. Effects of the microtuule dynmic instility prmeters hve een previously explored nd discussed 8. In the current simultions, conservtive vlues from the rnge explored in ref. 8 re used for v g nd v s. The numer of microtuules in the current simultions (6) is pproximtely twofold higher thn in previous models (5; ref. 8). This chnge is introduced to ccount for the difference in spindle ssemly time etween HT- 9 (5 min; ref. 8) nd RPE (8 min; ref. 6) cells. The model indeed predicts slower solute ssemly time if the numer of microtuules is lowered. However, the differences etween the predictions in the three considered scenrios (see elow) re not ffected y the numer of microtuules: reltive differences in the ssemly time s well s the predicted numer of errors remin the sme. A second set of stle microtuules runs long the spindle xis nd overlps in the centrl prt of the spindle. On the sis of microscopy dt, this dense microtuule rry forms shortly fter NEB ( min) nd persists through prometphse 6. The centromeres ecome positioned on the surfce of the nscent spindle shortly ( min) fter NEB (Fig. 7 nd ref. 45) nd lterlly interct with microtuule wlls. In contrst to end-on ttchments, lterl interctions cn occur long the entire length of the microtuule nd there is no evidence tht these interctions require microtuules to undergo plus-end dynmic instility. Therefore, the nscent spindle in our simultions comprises stle microtuules whose plus ends do not contriute to cpture. Geometry of the nscent spindle is derived from previously pulished dt 6. A microtuule plus end is instntly cptured nd stilized on encountering kinetochore. On cpture, new dynmic microtuule is nucleted t the sme pole to replce the stilized one. Chromosomes re modelled s solid 3D cylinders with R CH rdius nd length (Supplementry Tle ). The initil distriution of chromosomes I ch NATURE CELL BIOLOGY 5 Mcmilln Pulishers Limited. All rights reserved

13 M E T H O D S DOI:.38/nc33 in the nucler sphere nd their orienttion re rndom. Before cpture, sister kinetochores re modelled s crescent-shped ojects (see Supplementry Tle nd Supplementry Fig. 3 for dimensions) wrpped round the centrl prt (equtor) of the chromosome. The width w KT nd length h KT of kinetochores re kept constnt. On cpturing microtuule, the kinetochore crescent condenses into smll cylindricl oject in τ comp time. This kinetochore geometry reflects our experimentl oservtions (Figs 4). To complete computtions with resonle time nd void difficulty in trcking steric inter-chromosoml interctions, single chromosomes positioned t fixed distnce wy from the pole pole xis re considered in individul simultions. The simultions re then repeted for multiple chromosome positions nd rndom orienttions. To otin the verge vlue of the cpture time (τ cpt ), we multiplied the cpture time of single chromosome y the logrithm of the totl numer of chromosomes N CH (Supplementry Tle ). Four different scenrios re considered. In the first scenrio, completely rndom distriution nd orienttion of chromosomes remins unltered on microtuule cpture. Kinetochores re shped s crescents t the onset of spindle ssemly nd compct from crescents to discs in τ comp fter cpture. In the second scenrio, microtuule cpture leds to rottion of the chromosome nd lignment of the centromere xis (line connecting centres of sister kinetochores) long the cptured microtuule in time τ rot. Kinetochores re shped s crescents t the onset of spindle ssemly nd compct from crescents to discs in τ comp fter cpture. As the chromosome rms re lrgely norml to the pole pole xis, the rottion primrily occurs round their longitudinl xis. In the third scenrio, rpid lterl interctions with the stle microtuules of the nscent spindle result in rpid decrese of the ngle etween the centromere xis nd spindle xis (line connecting the centrosomes) in time τ rot. The ngle of chromosome rottion is limited y the ility of kinetochores to mintin direct contct with stle microtuules, which in turn depends on the size of the kinetochore crescent. Kinetochores re shped s crescents t the onset of spindle ssemly nd compct from crescents to discs in τ comp fter cpture. Compction initites only fter end-on microtuule cpture nd completes in τ comp. Finlly, in the fourth scenrio the crescents re smll t the onset of the serch, with the initil gp size of.76 µm. In the next 3 s, the crescents grow linerly so tht the gp decreses to its finl size (. to.76 µm in vrious simultions). The time of crescent growth is constnt, irrespective of the finl gp size. Lterl interctions decrese the ngle etween the centromere xis nd the spindle xis s in scenrio iii; respective rte of rottion is very fst, few seconds, in this cse. Then, the end-on microtuule cpture leds to dditionl rottion of the chromosome nd lignment of the centromere xis (line connecting centres of sister kinetochores) long the cptured microtuule in time τ rot. Kinetochores compct from crescents to discs in τ comp fter end-on microtuule cpture. In the most simplistic simultions (scenrio i), microtuule cpture is not expected to chnge the position or orienttion of the chromosome 7,9,3. Rottion of the centromere considered in the second, third nd fourth scenrios inevitly shifts the chromosome from its originl position. However, in 8% of the cses the men vlue of the displcement cused y the rief rpid movement during the initil interction etween kinetochores nd microtuules is < µm in RPE cells 6. Such smll trnsltion does not significntly ffect the proility of susequent microtuule cpture. Lrger trnsltions re rre nd therefore not considered in the current model. All simultions re crried out for vrious gp sizes etween the crescent sister kinetochores (Supplementry Tle ). Computtionl dt presented in the mnuscript re otined from running simultions for ech set of the prmeters t lest, times. The numericl codes re implemented with C progrmming lnguge. Numericl experiments re performed on n IBM qud core Intel CPU server. The code of the simultion is ville s Supplementry Dt. 47. Sikirzhytski, V. et l. Direct kinetochore-spindle pole connections re not required for chromosome segregtion. J. Cell Biol. 6, 3 43 (4). 48. Le Berre, M., Auertin, J. & Piel, M. Fine control of nucler confinement identifies threshold deformtion leding to lmin rupture nd induction of specific genes. Integr. Biol. 4, (). 49. Kline, S. L., Cheesemn, I. M., Hori, T., Fukgw, T. & Desi, A. The humn Mis complex is required for kinetochore ssemly nd proper chromosome segregtion. J. Cell Biol. 73, 9 7 (6). 5. Rieder, C. L. & Cssels, G. Correltive light nd electron microscopy of mitotic cells in monolyer cultures. Methods Cell Biol. 6, (999). NATURE CELL BIOLOGY 5 Mcmilln Pulishers Limited. All rights reserved

14 SUPPLEMENTARY INFORMATION DOI:.38/nc33 c 5 µm 5 µm 5 µm min 6 min 7 min (fixed) DIC CenpA-GFP, CenpF DNA 3 µm Nocodzole Single opticl plne CenpA-GFP, CenpF DNA Mx. intensity projection d µm CenpA-GFP, CenpF, DNA CenpA-GFP CenpF 3-D rendering e f 5 µm min 4 min min min Fixed 3 µm Nocodzole.5 µm Supplementry Figure Experimentl pproch used to chrcterize the virgin kinetochore rchitecture t spindle ssemly onset. () Selected differentil interference contrst (DIC) imges (individul plnes) illustrting prophse cell immeditely prior to the ddition of 3-mM nocodzole ( min) nd t nucler envelope rekdown (NEB, 6 min). The cell ws fixed with % glutrldehyde immeditely fter NEB. () A single opticl plne ner the middle of the cell shown fter fixtion in DIC nd fluorescence. Notice tht chromosomes (lue, Hoechst 33343) pper to e still ligned long the remnnts of the nucler envelope (rrows) indicting tht the cell ws fixed t the onset of mitosis. (c) Mximl intensity projection of the entire cell. (d) Higher mgnifiction view of kinetochores from the oxed re in (c). The outer lyer (red, CenpF) is enlrged nd lrgely encircles the centromere. Inner kinetochores (green, CenpA-GFP) remin compct. Mximl intensity projections of locl su-volume nd surfce-rendered reconstruction segmented t 5% of mximl intensity. (e, f) Tretment history of the cell shown in Figure 3e,f. (e) Selected DIC nd corresponding fluorescent imges (CenpA-GFP nd Centrin-GFP) depicting different cell prior to the ddition of 3-mM nocodzole ( min), during lte prophse (4 min), t (NEB, min), nd immeditely fter fixtion ( min). Asterisks indicte the loction of the centrioles. The oxed re in pnels D nd E is shown in Figure B t higher mgnifiction. (f) Electronmicroscopy imge of the sme cell. Remnnts of the nucler envelope re clerly visile (yellow rrows). The ox denotes the centromere presented in Figure. 5 Mcmilln Pulishers Limited. All rights reserved

15 SUPPLEMENTARY INFORMATION µm µm Erlier prometphse CenpF Mts CenpF Mts CenpF Mts CenpF Mts µm Lter prometphse µm CenpF Mts CenpF Mts CenpF Mts CenpF Mts Supplementry Figure Kinetochore outer lyer compction correltes with the formtion of end-on microtuule ttchment. () In erlier prometphse cells, kinetochores with n enlrged outer lyer re present throughout the cell (insets, ). However, few kinetochores tht disply prominent end-on ttchment to microtuule undles re compct (insets 3, 4). () In lter prometphse, t lest one kinetochore displys n enlrged outer lyer on ech monooriented chromosome nd these enlrged kinetochores lck end-on microtuule ttchments (insets, ). In contrst, oth sister kinetochores on ioriented chromosomes re compct (insets 3, 4). Wholecell imges re mximl-intensity projections tht include ll kinetochores in the cell. Individul kinetochores re shown s mximl-intensity projections of locl su-volumes. 5 Mcmilln Pulishers Limited. All rights reserved

16 SUPPLEMENTARY INFORMATION c Ks = 4 55 CenpF CenpA DNA CenpF CenpA DNA CenpF CenpA DNA Reltive fluor. intensity Cs = CenpF 5 min 8 min 5 min Ks = Hec CenpA DNA Hec CenpA DNA Hec CenpA DNA Reltive fluor. intensity Cs = Hec 5 min 8 min 5 min Ks = Mis CenpA DNA 5 µm Mis CenpA DNA Mis CenpA DNA 3 µm nocodzole, 5 min CenpA Red fluor Time in nocodzole.5 µm Reltive fluor. intensity Cs = Mis 5 min 8 min 5 min Supplementry Figure 3 Distriution nd mounts of vrious kinetochore proteins in the sence of microtuules. () Mximl intensity projections (include ll kinetochores) depicting RPE cells fter 5-min exposure to 3-mM nocodzole. Notice tht the nlyses were only on cells tht hd formed metphse plte prior to the ddition of nocodzole s evident from the pttern of chromosome distriution nd positions of centrosomes on the opposite sides of the pltes. () Exmples of individul kinetochores from the oxed res in (), shown t higher mgnifiction. CenpF forms lrge crescents tht cn completely encircle the centromere. The distriutions of Hec nd Mis lso pper to roden leit to lesser extents thn CenpF. (c) Reltive fluorescence intensities of kinetochores t vrious times fter ddition of nocodzole. The mount of the outer lyer protein CenpF (red) remins t the level typicl for untreted metphse (compre with. Fig.c, p >.3, two-tiled Student s t-test for oth lue vs. lue nd yellow vs. yellow rs.) nd then increses pproximtely threefold. The mount of Hec instntly increses pproximtely twofold over the levels typicl for kinetochores during metphse (compre with Fig.c, p <., twotiled Student s test for oth lue vs. lue nd yellow vs. yellow rs.). The mount of Mis in nocodzole-treted cells is not significntly different from the metphse level (compre with Fig.c, p >.9, two-tiled Student s test for oth lue vs. lue nd yellow vs. yellow rs). Blue rs in (c) re men kinetochore intensity clculted s men of men vlues for multiple kinetochores in individulcells (n vlues ove the rs, Cs; cells) Error rs represent s.e.m. Yellow rs re men vlues clculted for ll kinetochores pooled from ll cells in tht clss (n vlues ove the rs, Ks; kinetochores). Error rs represent s.d Mcmilln Pulishers Limited. All rights reserved

17 SUPPLEMENTARY INFORMATION wkt { dcen dkt wkt wkt { hkt τ cpt τ comp c { d 5 d 5 4 e τ cpt (min) τcomp (s) % of error per cell 3. μm. μm.3.67 μm τcomp (s) % of error per cell τcomp = 6 s size (μm) τ cpt (min) Supplementry Figure 4 Effects of kinetochore enlrgement-compction on the efficiency nd fidelity of cpture-driven spindle ssemly. (- ) Architecture of the virgin (unttched) centromere considered in the previous computtionl models of spindle ssemly () vs. the current model ( ). dkt, dimeter of the discoid kinetochore in previous models; wkt nd hkt, width nd height of the expnded crescent-like kinetochore;, segment of the centromere not covered y the kinetochore outer lyer. () Digrm showing the chnges in the centromere rchitecture considered in the minimlistic computtionl model. t cpt, time from the onset of spindle ssemly to end-on ttchment; t comp, durtion of the conversion from the expnded crescent to compct rchitecture of the kinetochore. (c) Sequence of events envisioned in the minimlistic model. Attchment triggers kinetochore compction ut does not ffect orienttion of the centromere. Green lines represent properly ttched microtuules, red lines - potentil erroneous ttchments. (d, d ) Durtion of spindle ssemly nd frequency of errors predicted for vrious finl gp sizes, nd vrious durtions of kinetochore compction. Notice tht oth efficiency nd ccurcy of spindle ssemly remin nerly constnt t t comp > 6 s. (e) Frequency of errors nd durtion of spindle ssemly predicted for centromeres with vrious finl gp sizes for specific vlues of t expnd nd t comp Mcmilln Pulishers Limited. All rights reserved