Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore microtubule attachment

Transcription

1 A R T I C L E S Reruitment of the humn replition liensing protein y the loop omin of is require for stle kinetohore mirotuule tthment Dileep Vrm 1, Srikrip Chnrsekrn 2, Lynsie J. R. Sunin 3, Kren T. Reiy 4, Xiohu Wn 1, Dwn A. D. Chsse 5, Kthleen R. Nevis 6, Jennifer G. DeLu 3, E. D. Slmon 1,7 n Jenette Gowen Cook 2,4,7, protein ritil for replition origin liensing in G1 phse, is egre uring S phse ut re-umultes in G2 phse. We now emonstrte tht humn hs seprle essentil mitoti funtion. lolizes to kinetohores uring mitosis through intertion with the omponent of the N8 omplex. G2-speifi epletion of rrests ells in lte prometphse owing to normlly unstle kinetohore mirotuule (kmt) tthments n M1-epenent spinle-ssemly-hekpoint tivity. ins unique loop extening from the ro omin of tht we show is lso require for kmt tthment. Muttion of the loop omin prevents kinetohore loliztion n rrests ells in prometphse. Super-resolution fluoresene mirosopy inites tht ining to the loop omin promotes mirotuule-epenent onformtionl hnge in the N8 omplex in vivo. These results support the onlusion tht ining to is essentil for n extene N8 onfigurtion n stle kmt tthment. The ell-ivision yle is the proess of omplete n preise uplition of the entire genome in S phse followe y urte hromosome segregtion in mitosis. The formtion n stility of kmt tthments uring mitosis epens on the N8 omplex, N8 (hs), Nuf2, Sp24 n Sp25 (refs 1,2). /Nuf2 n Sp24/25 form imers respetively tht re tethere together t the roxy termini of /Nuf2 n the mino termini of Sp24/Sp25 y long α- helil oile-oil ro omins. In the mile of the ro omin is hinge site proue y 4-mino-i loop in tht is thought to ply key role in mirotuule-ining ynmis n tthment 3. Bining prtners for the loop region of yest /N8 proteins hve een ientifie 4,5, ut the prtners n funtion of the unique loop omin t metzon kinetohores remins unknown. In this stuy, we ientify the protein s n essentil prtner for N8 ynmi funtion through intertion with the loop omin. is require for DNA replition origin liensing, the initil step in genome uplition, whih ours in G1 phse 6,7. Proteins involve in origin liensing re not stritly DNA replition ftors, however; some liensing proteins hve funtions in trnsription 8,9, the DNA-mge response 1,11, entrosome uplition 12 n mitosis 13,14. We report here the previously unisovere mitoti role for the replition liensing protein tht is istint from its role in DNA replition. RESULTS Inhiition of funtion inues mitoti rrest Both C6 n proteins re require for replition liensing in humn ells, ut we foun n intriguing ifferene in the phenotypes of C6-eplete ells when ompre with -eplete ells. C6- eplete ells rrest primrily in G1 s expete 15,16, ut -eplete ells rreste in oth G1 n G2/M, espite the ft tht oth proteins ooperte in the sme DNA replition step, MCM loing (Fig. 1). The umultion of G2 ells inite possile unique mitoti role for tht we set out to test. To eliminte the possiility tht mitoti phenotype simply reflete replition errors in the preeing S phse, we took vntge of the ft tht is tively egre uring S phse fter its G1 origin liensing role is omplete 17 (Fig. 1). We synhronize ells in erly S phse, t whih time origins re fully liense, n relese them into meium 1 Deprtments of Biology, University of North Crolin, Chpel Hill, North Crolin 27599, USA. 2 Biohemistry n Biophysis, University of North Crolin, Chpel Hill, North Crolin 27599, USA. 3 Deprtment of Biohemistry n Moleulr Biology, Coloro Stte University, Fort Collins, Coloro 8523, USA. 4 Curriulum in Genetis n Moleulr Biology, University of North Crolin, Chpel Hill, North Crolin 27599, USA. 5 Duke Institute for Genome Sienes n Poliy, Duke University Meil Center, Durhm, North Crolin 2771, USA. 6 Criovsulr Reserh Center, Msshusetts Generl Hospitl, Chrlestown, Msshusetts 2129, USA. 7 Corresponene shoul e resse to E.D.S. or J.G.C. (e-mil: tslmon@emil.un.eu or jen_ook@me.un.eu) Reeive 18 Otoer 211; epte 26 Mrh 212; pulishe online 13 My 212; DOI: 1.138/n2489 NATURE CELL BIOLOGY VOLUME 14 NUMBER 6 JUNE

2 A R T I C L E S BrU Control 6 t1 S 5% 14% 25% G2 G1 5% 9% 46% 74% 29% 42% DNA ontent 2C 4C 2C 4C 2C 4C Control t1 6 C6 Tuulin protein Thymiine rrest n relese t1 in S ( egre) P Thymiine relese (h) Control t1 A A G1 S G2 M G1 Effiient liensing No new liensing Lne: e No. relese (h): (G2) 2 (G1) No. relese (h): Control + vetor Vetor res Control t1 t1 + vetor t1 + res 2C 4C 2C 4C Figure 1 Cells eplete of fter S phse o not omplete ell ivision. () Norml humn firolsts (NHF1-htert) were trnsfete with s trgeting 6, t1 or GFP (ontrol) messenger RNAs for 72 h. Cells were lelle with BrU for the finl hour n then nlyse y flow ytometry for ell-yle position (left) n y immunolotting for enogenous C6, n tuulin (right). () Digrm of experimentl esign. Enogenous protein levels normlly rop uring S phse owing to uiquitin-meite proteolysis, n reovery egins in G2 (otte line). Relese from n erly S lok into t1 trnsfetion meium loks protein re-umultion (soli grey line). () Immunolot nlysis of enogenous ontining ontrol short interfering RNA () or t1. As expete, ontrol ells h very low mounts of in S phse, n re-umulte in G2 (Fig. 1, lnes 1 5). Synhronize ells trete with t1 oul not re-umulte in G2, however (Fig. 1, lnes 7 1). ( is phosphorylte in G2 y stress MAP kinses 17,18.) epletion uring S phse use no ely in S-phse progression (Supplementry Fig. S1,), n y 9 h fter relese, oth ontrol n -eplete ells exhiite norml hromosome onenstion n/or nuler envelope rekown (for exmple, Fig. 2 n t not shown). This unique experimentl pproh llowe us to generte ells tht unerwent norml G1 n S phse ut lke uring G2 n M phse. We then investigte the ility of -eplete ells to trnsit mitosis to G1 fter noozole rrest n relese (Fig. 1,e). Control ells omplete ell ivision, ut -eplete ells remine rreste with G2 DNA ontent (Fig. 1e, ontrol versus t1 ). A ell line stly expressing norml with synonymous muttions in protein in ells synhronize s shown in ; M phse ours etween 9 n 1 h post-relese from the seon thymiine rrest. () Stle HeL ell lines trnsue with empty vetor (lnes 1 4) or n -resistnt form of ( res lnes 5 n 6) were synhronize s in n trnsfete with either ontrol trgeting GFP (lnes 1 n 2) or with t1 (lnes 3 6). Cells were relese from erly S phse into noozole (No.) for 1 h n then either ollete ( h) or relese for 2 h into G1 phse, n protein levels were nlyse y immunolotting. (e) Flow ytometri nlysis of DNA ontent of the ells in. Unroppe imges of lots re shown in Supplementry Fig. S9. the trget site ws fully ple of mitoti progression (Fig. 1,e). Unphosphoryltle (ref. 18) lso omplemente the ell-ivision phenotype of -eplete ells (Supplementry Fig. S1,). Cells eplete of uring S phse i not umulte phosphorylte H2AX y the susequent G2, mrker of DNA mge, wheres trnsfetion of synhronous ultures to eplete either or nother liensing protein, Or6, resulte in the umultion of phospho-h2ax-positive foi, presumly from inomplete replition n fork ollpse (Supplementry Fig. S1e). Most of the synhronize -eplete ells rreste just efore metphse with most hromosomes positione ner the spinle equtor. A smller frtion of ells rreste in prometphse (Fig. 2, n Supplementry Fig. S2,). In ition to epletion in synhronize ells, we lso miroinjete purifie nti- ntioy (Supplementry Fig. S2) into HeL ells expressing GFP-tgge histone H2B uring prophse or erly prometphse n onute live-ell imging (n = 27). Anti- ntioy inue oth severe 594 NATURE CELL BIOLOGY VOLUME 14 NUMBER 6 JUNE 212

3 A R T I C L E S Control t1 7 9 h 9 h 6 9 h 9 h Perentge of ll mitoti ells Luiferse t1 Erly/mi Lte Anphse n prometphse prometphse/metphse telophse 1 h 1 h e Perentge of injete ells Arreste in mitosis Complete mitosis Buffer Anti- Buffer-injete Anti--ntioy-injete Figure 2 G2-speifi inhiition inues mitoti rrest. () HeL ells synhronize in erly S phse were relese into ontrol (luiferse) or t1 for either 9 h (top n mile pnels) or 1 h (ottom pnels), followe y fixtion n stining with DAPI to lel hromosomes (lue), nti-tuulin ntioy to lel mirotuules (re) n nti-knl1 ntioy to lel kinetohores (green). () Quntifition of the results t 1 h in ely in hromosome ongression n n rrest ner metphse for the entire 3 h urtion of imging (Fig. 2,e). Control ufferinjete ells (n = 15) or ontrol nti-mouse IgG-injete ells (n = 8) exeute mitosis normlly (Fig. 2,e n Supplementry Movies S1 n S2). Anti- injetion uring lte prometphse or metphse (n = 1) resulte in 7% of the ells remining rreste in tht stge for up to 8 h. During this perio, ells progressively lost hromosome lignment ut i not ivie (Supplementry Fig. S2 n Movie S4). y mitoti stge; n = 1,5 ells. ( e) HeL ells stly expressing GFP histone H2B were injete with ontrol uffer (, n = 15) or nti- ntioy (, n = 27). Selete frmes of GFP histone (top pnels) n phse-ontrst imges (ottom pnels) re shown. (e) Quntifition of the results from the miroinjetion experiments in n. Sle rs, 5 µm. Time is h.min.s. See lso Supplementry Movies S1 S4. lolizes to mitoti kinetohores through intertion with the omponent of N8 The lte-prometphse rrest of -eplete ells prompte us to exmine loliztion in mitosis. Remrkly, we etete enogenous o-loliztion with known kinetohore protein,, in prometphse in oth noozole-trete PTK2 n HeL ells (Fig. 3, n t not shown). epletion y tretment in S phse eliminte etetle nti- kinetohore stining (Supplementry Fig. S3). We oserve no kinetohore loliztion of NATURE CELL BIOLOGY VOLUME 14 NUMBER 6 JUNE

4 A R T I C L E S + Noozole Prophse Prometphse Metphse e Anphse f Telophse Figure 3 trnsiently lolizes to kinetohores uring prometphse n metphse. () Noozole-trete PTK2 ells were immunostine with nti- ntioy n nti- the inhiitor geminin 19,2 t ny stge of mitosis (Supplementry Fig. S3). A seon nti- ntioy lso etete enogenous t kinetohores in LLCPK1 ells (Fig. 3 f). Thus, lolizes to kinetohores in ontrol ells t time oinient with the stge t whih -eplete ells rrest. In serh for -interting proteins y two-hyri sreening in yest, we ientifie multiple inepenent lones ontining portions of humn (Supplementry Fig. S4); the shortest ontine mino is (Supplementry Fig. S4,). To etermine whether n intert iohemilly, we inute immoilize terilly expresse GST with lystes of synhronous HeL ells. Enogenous ssoite with GST ntioy. ( f) LLCPK1 ells t ifferent stges of mitosis were immunostine with nti- ntioy n nti- ntioy. Sle rs, 5 µm. ut not GST lone (Fig. 4). Nuf2 ws lso retrieve from ell lystes y GST (Fig. 4), initing tht n ssoite with the N8 omplex, possily through iret intertion with (se on intertion of the fusions in yest). We further onfirme the intertion etween n y reiprol o-immunopreipittion of the enogenous proteins (Fig. 4,). To etermine whether reruits to kinetohores in mitosis, we stine for enogenous in -eplete PTK2 ells. epletion resulte in profoun loss of t kinetohores in noozole-trete prometphse (Fig. 4, ottom pnel n Fig. 4e) n metphse ells (Supplementry Fig. S4, ottom pnel) when ompre with ontrols (Fig. 4 n Supplementry Fig. S4, 596 NATURE CELL BIOLOGY VOLUME 14 NUMBER 6 JUNE 212

5 A R T I C L E S GST Input GST Luiferse ACA e 1.4 Nuf IP Input + he1 fluoresene intensity Luiferse he1 f Or6 Luiferse Or6 ACA (ND) g 2.5 IP Input + he1 Or6 fluoresene intensity Luiferse he1 Figure 4 is require for kinetohore loliztion. () A lyste of T98G (humn gliolstom) ells ws inute with es ote with terilly proue GST or GST, n the enogenous n Nuf2 were etete in the input or oun frtions y immunolotting. () Whole-ell lystes of HeL ells were inute with pre-immune serum or nti- ntioy, n enogenous ws etete in the input n immune omplexes. (ND: o-migrtes too losely to IgG hevy hin for etetion in this immunopreipitte, IP.) () Whole-ell lystes of HeL ells were inute with ontrol serum or nti- ntioy; enogenous n were etete in the input n immune omplexes. () Noozole-trete PTK2 top pnels). We oserve no erese in extrts of - eplete ells (Supplementry Fig. S4), n epletion h no effet on expression or kinetohore loliztion (Supplementry Fig. S4e). Other kinetohore proteins, inluing omponents of the Mis12 omplex, Knl1 n the Zwint1 RZZ omplex, were lso lolize normlly in -eplete ells (Fig. 2 n Supplementry Figs S4e n S8,). Similrly to, ORC is essentil for replition origin liensing, n the Or2 n Or6 suunits in prtiulr hve lso een reporte to lolize to entromeres or kinetohores 13,14. ORC is require for hromtin loliztion in G1 (ref. 21), n the Or6 suunit ssoites with (Supplementry Fig. S5; ref. 22). We foun tht, s for, Or6 immunofluoresene intensity t kinetohores ws lso sustntilly reue in -eplete ells (Fig. 4f,g). Or6 epletion h no effet on or kinetohore loliztion, however (Supplementry Fig. S5,e). Interestingly, epletion of resulte in 5% erese in the intensity of Or6 kinetohore stining (Supplementry Fig. S5,). To ompre the reporte mitoti ells sujete to he1 RNAi were fixe n stine with nti- ntioy n nti-aca ntioy to mrk kinetohores. (e) Quntifition of kinetohore fluoresene intensity in reltive to ontrol luiferse--trnsfete ells; n = 8 kinetohores; P <.1. (f) HeL ells were trete with ontrol luiferse or he1, fixe n stine using nti-or6 ntioy, nti- ntioy to monitor the knokown n nti-aca ntioy to lel kinetohores. (g) Quntifition of Or6 kinetohore fluoresene intensity in f reltive to ontrol luiferse--trnsfete ells; n = 125 kinetohores; P <.1. Dt re presente s men ± s.. Sle rs, 5 µm. Unroppe imges of lots re shown in Supplementry Fig. S9. efets inue y Or6 inhiition 14 to those proue y epleting, we first lulte the mitoti inies of synhronous ells eplete of Or6. Or6 is not egre in S phse like, so we oul not employ the sme synhroniztion strtegy to remove Or6 only efore mitosis. Or6 epletion use 5% inrese in the numer of mitoti ells when ompre with ontrols (Supplementry Fig. S5f) y prouing fourfol inrese in the numer of telophse ells (Supplementry Fig. S5g). In ition, ells eplete of Or6 exhiite norml ol-stle kmts (Supplementry Fig. S5h). These oservtions support the onlusion 14,23 tht Or6 hs role lte in mitosis uring ytokinesis istint from the role of uring prometphse or metphse. The loop omin is require for loliztion n nphse onset Our two-hyri nlysis inite tht the -ining region of inlues short interruption in the oile-oil pttern postulte to e flexile loop 3 (Supplementry Fig. S4,). To etermine whether NATURE CELL BIOLOGY VOLUME 14 NUMBER 6 JUNE

6 A R T I C L E S Wil type : Loop MUT : QLAEYHKLAR KLKLIPKGAE NSKGYDFEIK FNPEAGANCL VKYRAQVYVP LKELLNETEE QLAEYHKLAA SQGQGQGAAQ GASSSGQQQG SASAASGAGQ SASASAGGQQ LKELLNETEE Sp25 Sp24 Nuf2 GFP Wil type Supe. Loop MUT Ctrl IP Wil type IP Loop MUT Anti-GFP Wil-type GFP GFP Or6 Wil-type GFP Or6 GFP he1 he1 Loop MUT GFP Loop MUT GFP he1 he1 e GFP Wil-type GFP he1 Loop MUT GFP GFP MT f Perentge of ll mitoti ells Erly/mi prometphse Lte prometphse/metphse Wil-type resue Loop MUT resue Anphse n telophse he1 Figure 5 trgeting to kinetohores epens on the flexile loop region of. () Digrm of the N8 omplex showing the loop region n the onstrution of the loop replement mutnt, Loop MUT (pte from ref. 38). () Enogenous ws immunopreipitte from lystes of synhronously growing HeL ells trnsfete with GFP plsmis; Ctrl IP inites the use of norml mouse serum s ontrol. GFP in the oun ( IP) n unoun (Supe.) frtions ws etete with nti-gfp ntioy. () PTK2 ells were trete with he1 followe y trnsfetion with either wil-type GFP or Loop MUT GFP onstruts. The ells were trete this omin is require for ining, we reple the norml loop sequene with sequene of lterntive mino is of similr length, whih we term Loop MUT (Fig. 5). A similr sequene provies flexile linker in well-stuie GFP PCNA fusion 24. Both wil-type n Loop MUT were tgge with GFP t their C termini. Strikingly, Loop MUT GFP file to o-immunopreipitte with, wheres wil-type GFP oun reily (Fig. 5, ompre lnes 4 n 5). Both GFP-tgge wil-type n Loop MUT with noozole, then fixe n stine using nti- ntioy n nti-gfp ntioy. () The sme s in, exept tht HeL ells were stine with nti-or6 n nti-gfp ntioies. (e) The sme s in, exept tht HeL ells were stine with nti-tuulin ntioy to mrk mirotuules n nti-gfp ntioy to mrk GFP t kinetohores. (f) Quntifition of mitoti stges in -eplete ells expressing etopi wil-type GFP or Loop MUT GFP stine with DAPI; n = 125 GFP-expressing ells. Sle rs, 5 µm. See lso Supplementry Fig. S8 n Movies S5 n S6. Unroppe imges of lots re shown in Supplementry Fig. S9. lolize normlly to kinetohores in PTK2 n HeL ells (Fig. 5,e). We then teste loliztion in PTK2 ells eplete of enogenous ut expressing -resistnt versions of wil-type GFP or Loop MUT GFP. lolize to kinetohores normlly in ells expressing wil-type GFP (Fig. 5, top pnel), ut ws strikingly unetetle t kinetohores in ells expressing Loop MUT GFP (Fig. 5, ottom pnel). Or6 kinetohore trgeting ws lso epenent on the loop (Fig. 5). 598 NATURE CELL BIOLOGY VOLUME 14 NUMBER 6 JUNE 212

7 A R T I C L E S M1 M1 Knl Control t1 M1 M1 Knl1 M1 fluoresene intensity M1 fluoresene intensity M1 M1 ACA 1 1 Luiferse t1 Wil-type Loop MUT Loop MUT GFP Luiferse 9 h Luiferse f 1 h m1 9 h m1 1 h e t1 9 h t1 1 h g t1 +m1 9 h t1 +m1 1 h Figure 6 n the loop omin re require to stisfy the spinle-ssemly hekpoint. () Top n mile pnels: -eplete or ontrol ells s in Fig. 2 (1 h fter thymiine relese); inset shows Knl1 stining. Bottom pnels: -eplete ells expressing -resistnt Loop MUT GFP. Cells were fixe n stine with ntioies to M1 to ssess spinle-ssemly-hekpoint tivity n Knl1 to mrk kinetohores; inset shows ACA stining. () Quntifition of M1 kinetohore levels in mitoti ells trete with ontrol or t1 ; n = 125 kinetohores; HeL ells expressing only Loop MUT GFP ore striking similrities to -eplete ells. Only 14% rehe lte prometphse n 86% remine in erly to mi-prometphse (Fig. 5e,f n Supplementry Movie S6 n Fig. S6), unlike ells expressing wil-type GFP (12/13 ells, Supplementry Movie S5 n Fig. S6). Live-ell imging of ells expressing only Loop MUT GFP lso revele 1% mitoti rrest phenotype (n = 29). The phenotypes of Loop MUT GFP ells were more severe thn -eplete ells, whih oul e ue to quntittive ifferenes in the effetiveness of the ifferent s in synhronize versus synhronous ultures, efets in spinle struture from perturtion or to other funtions of the loop prt from ining. The rnge of phenotypes in P <.1. () Quntifition of M1 levels in mitoti ells expressing wil-type or Loop MUT onstruts inste of enogenous ; n = 15 kinetohores; P <.1. Dt re presente s men ± s.. ( g) Doule-thymiine synhronize HeL ells were trnsfete with ontrol luiferse (), t1 (e), m1 (f) n omintion of oth t1 n m1 (g) n then stine with DAPI (pseuo-oloure re) n nti-tuulin ntioy (green) t 9 h or 1 h fter S-phse relese. Sle rs, 5 µm oth irumstnes ws similr, however (Supplementry Fig. S6,), initing tht the Loop MUT phenotype might e lrgely expline y filure to reruit to kinetohores. The sene of t kinetohores uses efetive kmt tthment n M1-epenent rrest These phenotypes prompte us to proe the spinle-ssemlyhekpoint sttus of rreste ells. In -eplete ells, kinetohore loliztion of the hekpoint protein, M1, on ligne hromosomes ws 5.2-fol higher thn in ontrol ells (Fig. 6, top n mile pnels, n Fig. 6). We mesure similr 4.5-fol M1 inrese t ligne kinetohores in ells expressing Loop MUT GFP NATURE CELL BIOLOGY VOLUME 14 NUMBER 6 JUNE

8 A R T I C L E S 4 C Control 4 C t1 4 C MT MT MT MT Bu1 MT Bu1 MT GFP Perentge of tthe kinetohores K K streth (µm) Luiferse Luiferse t1 t1 Wil-type Loop MUT Wil-type Loop MUT he1 he1 ZM e he1 + Loop MUT 4 C MT GFP Auror B LoopMUT GFP Atthment 4 C MT Knl1 4 C MT Knl1 4 C MT GFP 4 C MT GFP Luiferse +MG132+ZM t1 +MG132+ZM he1 +-9A +MG132+luiferse he1 +-9A +MG132+t1 Figure 7 n the loop omin re require for stle kmt tthments. () Control ells in metphse, -eplete ells n ells expressing Loop MUT (in ple of enogenous ) in lte prometphse were inute with ie-ol PBS (whih llows retention of only stle kmts), followe y fixtion n stining with nti-tuulin ntioy (mirotuule, MT) n either nti-bu1 ntioy to mrk the kinetohores or nti-gfp ntioy for etopi, s inite. () Quntifition of the frtion of kinetohores mking suessful ontt with kinetohore fires in -eplete or Loop MUT ells; n = 25 kinetohores () Inter-kinetohore (K K) istnes in the inite ells; n = 1 kinetohore pirs in eh tegory. P <.1. Dt re presente versus wil-type GFP (Fig. 6, ottom pnel, n Fig. 6). Kinetohore loliztion of nother hekpoint protein, BuR1, ws unltere y epletion (t not shown). To etermine whether M1 reruitment fully ounts for the mitoti rrest proue y inhiition, we o-eplete M1 with. As emonstrte previously 25, ontrol ells eplete of M1 lone unerwent premture nphse onset (Fig. 6, ompre with f). Importntly, o-epletion of M1 with ypsse the mitoti rrest of -eplete ells (Fig. 6, ompre e with g). We thus onlue tht epletion inues persistent M1-epenent s men ± s.. () Synhronize HeL ells trete with either ontrol luiferse or t1, s inite, were trete with oth ZM (Auror B inhiitor, igrmme) n MG132 (nphse inhiitor) for 3 min t 8.5 h fter S-phse relese, followe y ol tretment n stining with nti-tuulin ntioy n nti-knl1 ntioy. (e) Synhronize HeL ells were o-trnsfete with oth he1 n either plsmi enoing unphosphoryltle 9A- GFP (top pnels) followe y MG132 tretment for 1/2 h t 8.5 h fter S-phse relese or plsmi enoing Loop MUT GFP (ottom pnel). The ells were then sujete to ol tretment t 9 h fter S-phse relese n stine with nti-tuulin ntioy n nti-gfp ntioy. Sle rs, 5 µm. spinle-ssemly-hekpoint signlling. Of further note, synhronize ells o-eplete of oth M1 n i not proue mny nphse hromosome riges reltive to M1-eplete ells. We resone tht unreplite DNA segments woul not e segregte properly, leing to the proution of nphse hromosome riges. The sene of inrese nphse riges reinfores our ssertion tht is not require uring S phse for omplete DNA replition. On the other hn, synhronous ells eplete of either or Or6 proue mny nphse hromosome riges (Supplementry Fig. S7, n t not shown). 6 NATURE CELL BIOLOGY VOLUME 14 NUMBER 6 JUNE 212

9 A R T I C L E S DC31 Sp24 9G3 Sp24 Merge Mis12 omplex Sp25 Sp24 ~41 nm Nuf2 Sp24 ~45 nm Distne (nm) Sp24 CH omin (9G3) P<.1 Luiferse t1 ro Distne (nm) Nsl1 CH omin (9G3) P< Luiferse t1 ro e Stle tthment Avg. ~41 nm Unstle tthment Avg. ~ 19 nm?? Avg. ~41 nm Mirotuule-ssoite protein(s) loop N8 omplex Proteins linking N8 to hromtin Centromere Figure 8 n the loop omin re require for proper N8 onformtion in vivo. () Digrm of the N8 omplex epiting the ntioy epitopes use for the Delt nlysis. () Representtive imge of norml mitoti metphse ell lelle with ntioies to the CH omin n the Sp24 he omin. The imges to the right show mgnifition of the re outline in the left imge. Sle rs, 1 µm. () Delt vlues (orrete for tilt 28 ) of men seprtion etween Sp24 n the CH omin (9G3 ntioy) in synhronize ontrol or -eplete mitoti ells (9 h fter S-phse The presene of high levels of M1 t kinetohores inite tht loss of or muttion of the loop proues kmt tthments tht re insuffiiently roust. We therefore investigte the ol stility of kmts y hilling ells t lte prometphse or metphse efore fixing them for immunofluoresene nlysis. The fluoresene intensity of kmts fter ol tretment for -eplete ells or Loop MUT GFP ells ws 5% of ontrols (Fig. 7, top n mile pnels, n Supplementry Fig. S7). Quntifition of kinetohores tht me ontt with ol-stle spinle mirotuules revele 77% relese), or in Ro-eplete ells (mitoti metphse ells selete from n synhronous popultion); n = 84, 47 n 54 kinetohore pirs respetively; (ontrol versus t1 ) P <.1, (ontrol versus ro ) P =.31 (not signifint). () The sme s in, exept tht Delt vlues of seprtion etween Nsl1 (Mis12 omplex suunit) n the CH omin were etermine; n = 74, 41 n 6 kinetohore pirs respetively; (ontrol versus t1 ) P <.1, (ontrol versus ro ) P =.86 (not signifint). Dt re presente s men ± s.. (e) Sle moel for the Loop omin intertion. erese in the numer of kinetohore fires in -eplete ells n n 84% erese in Loop MUT GFP ells reltive to orresponing ontrols (Fig. 7). As nother initor of kmt tthment, we mesure the inter-kinetohore (kinetohore kinetohore) istne of ligne sister kinetohore pirs; 1.4 µm for -trnsfete ontrols n 1.25 µm for -eplete ells expressing wil-type GFP. In ontrst, the kinetohore kinetohore istne ws just.96 µm in -eplete ells n 1.5 µm in ells expressing Loop MUT GFP (Fig. 7). These vlues orrespon to 5 6% loss of NATURE CELL BIOLOGY VOLUME 14 NUMBER 6 JUNE

10 A R T I C L E S kinetohore streth euse the unstrethe kinetohore kinetohore entromere length is.7 µm in ells trete with noozole 26. As in -eplete ells, there were no isernile hnges in the kinetohore loliztion of most other proteins exmine in ells expressing Loop MUT GFP (Supplementry Fig. S8). To etermine whether rtifiilly strengthening mirotuule tthments n ompenste for loss or muttions in the loop omin, we loke Auror-B-kinse-meite phosphoryltion. Phrmologil Auror B inhiition or muttion of the 9 phosphoryltion sites in the N-terminl til enhnes mirotuule-ining ffinity t this en of the N8 omplex 1,2,27 3. Treting -eplete mitoti ells with the Auror B inhiitor, ZM447439, use premture nphse onset similr to ontrol rug-trete ells or M1-eplete ells, emonstrting effetive Auror B inhiition (Supplementry Fig. S7f,g). We lso ouple our synhroniztion n epletion protool with expression of the Auror-B-resistnt (9A- GFP) omine with enogenous epletion. The result of this mnipultion ws ientil to tht of Auror B inhiition (t not shown). To etermine whether progression to nphse in these experiments involve stiliztion of kmt tthments, we trete ells with the protesome inhiitor MG132 to prevent nphse onset n sujete them to the olstility ssy. ZM trete or 9A- GFP-expressing ells lking retine stle kmts (Fig. 7,e). These results inite tht the sene of t kinetohores results in filure to estlish n/or mintin suffiiently strong kmt tthments. Intertion of with the loop is require for the norml onformtion of the N8 omplex t kinetohores of i-oriente hromosomes The loop region of llows onformtionl hnges within the N8 omplex 3. To etermine whether influenes this onformtion, we employe n in vivo two-olour super-resolution fluoresene mirosopy tehnique y lelling the two ens of the N8 omplex (Delt nlysis illustrte in Fig. 8; ref. 26). We use the 9G3 ntioy, whose epitope in resies next to the N-terminl CH omin, n nti-sp24 ntioy to mrk the C-terminl region of the N8 omplex n Nsl1 ntioy to mrk the Mis12 omplex, whih is out 4 nm insie Sp24 (ref. 26; Fig. 8). Mesurements of the verge seprtion, Delt, etween 9G3 n the two other ntioies yiele vlues of 41 n 45 nm for kinetohores of ligne hromosomes in ontrol ells t metphse (Fig. 8,). After epletion, the orresponing Delt vlues were 35 nm n 37 nm, respetively (Fig. 8,), vlues signifintly ifferent from ontrols (P <.1). To etermine whether these reutions were generl onsequene of unstle kmt tthments, we mesure Delt following epletion of Ro ( omponent of the RZZ omplex), whih, similrly to epletion, proues 5% reution in kmt stility 31. Unlike epletion, Delt vlues for Ro epletion ( 39 nm n 45 nm; Fig. 8,) were not signifintly ifferent from ontrols (P =.31 n P =.86, respetively); Ro epletion reue kinetohore kinetohore streth from 1.4 µm to 1.15 µm, emonstrting the effetiveness of the. In noozoletrete ells tht lk kmts, the istne etween the ens of the N8 omplex long the kinetohore kinetohore xis is short with Delt vlue of 17 nm (ref. 28). We mesure Delt vlues for -eplete n noozole-trete ells t 19 nm ± 7 nm for Sp24 to 9G3 n 2 nm±8 nm for Nsl to 9G3 (n = 1 sister kinetohore pirs, >5 ells). Tken together, these results inite tht the essentil mitoti funtion of is to in to the N8 omplex t the loop n mintin n extene onformtion long the lttie of kmts to stilize en-on tthments of spinle mirotuules to kinetohores. DISCUSSION Distint essentil funtions for in oth G1 n M phses Using unique pproh to eplete speifilly fter its replition funtion is omplete, we unequivolly emonstrte n essentil n previously unknown mitoti funtion for humn in kmt tthment. This isovery rises the question of why suh ul roles woul hve evolve. One possiility is to ensure orerly progression from S phse to M phse. is egre while replition is ongoing to voi re-liensing origins tht hve lrey fire 32,33. We reently reporte mehnism of stiliztion fter S phse through MAP-kinse-meite phosphoryltion 18. We now suggest tht stiliztion in G2 ells evolve to filitte its rpi re-umultion efore mitosis. Although yest is lerly not require for nphse 34,35, metzon my hve quire its mitoti role s mens of reinforing the epenene of key mitoti event on the ompletion of genome uplition. The quisition of funtions in two ifferent ell-yle phses hs the onsequene tht e-regultion of hs the potentil to ffet oth origin liensing n mitosis, n thus genome stility 36,37. The role of the loop omin The presene of high levels of M1 t kinetohores of -eplete ells or -Loop MUT -expressing ells inites tht n the loop re require for tthment ut not for M1 retention, unlike ells lking entirely, in whih oth tthment n M1 retention re efetive 28,29 (t not shown). The gloulr CH omin n N-terminl til of mke iret ontts with mirotuules, n the loop omin is not require for high-ffinity ining of N8 onsi omplexes to mirotuules, onstrut lking the region tht inlues the loop 38. It ws therefore surprising tht muttion of the loop omin, site 18 nm long the ro omin from these iret mirotuule ontts, h suh profoun negtive effets on the formtion of stle mirotuule tthments. Clerly the formtion of roust tthments in vivo requires not only the CH omin n N-terminl region, ut lso intertion with the loop omin. Reent stuies in yest hve shown requirement for the loop in yest /N8 for ining mirotuule-ining proteins (MAPs), tht is, the Dm1 omplex in uing yest or Dis1/TOG in fission yest 4,5. The mino-i sequene of the loop is not highly onserve 3, n humn hs no etetle sequene homology to Dm1 omplex memers or to Dis1/TOG. Nevertheless, oul serve n nlogous funtion y interting with mirotuules or n s yet, unientifie mirotuule-ssoite protein (Fig. 8e) or lterntively y inhiiting Auror B-meite kinetohore-fire estiliztion. A moel for intertion in mitosis The loop proues flexile hinge in the otherwise reltively rigi N8 omplex. In the omplete sene of mirotuules, the verge seprtion, Delt, etween the two ens of the N8 omplex is only 19 nm (ref. 26) in oth ontrols n -eplete ells. This 62 NATURE CELL BIOLOGY VOLUME 14 NUMBER 6 JUNE 212

11 A R T I C L E S verge istne extens to 41 nm for ontrol metphse ells when kinetohores hieve full mirotuule omplement. In the sene of, N8 oes not mke this full extension, prouing Delt mesurements 68 76% etween the two extremes. In ontrst, Ro epletion removes the min reruitment ftor for the ynein yntin omplex n isrupts other kmt-ining sites ontriute y this mirotuule motor 31,39,4. The ft tht N8 n opt the full extene shpe in Ro-eplete ells, whih, similrly to -eplete ells, hve unstle kmt tthments 31, provies eviene ginst generl iniret effet of tthment on N8 onformtion. We propose tht physilly links the loop to the mirotuule or mirotuule-ssoite protein, n this extr tthment stilizes the extene onformtion n enhnes the mirotuule ffinity of the N8 omplex provie y the CH omin n N-terminl til. METHODS Methos n ny ssoite referenes re ville in the online version of the pper t Note: Supplementry Informtion is ville on the Nture Cell Biology wesite ACKNOWLEDGEMENTS We thnk A. Desi for proviing Knl1, Nsl1, Dsn1 n Spinly ntioies, A. Mushio (Mx Plnk Institute of Moleulr Physiology, Dortmun, Germny) for nti-m1, Zwint1 n ZW1 ntioies, B. Stillmn (Col Spring Hror Lortory, New York, USA) for nti-or6 ntioy, S. Tylor (University of Mnhester, UK) for nti-bu1 n BuR1 ntioies, T. Stukenerg (University of Virgini t Chrlottesville, Virgini, USA) for nti-sp24 ntioy, T. Yen (Fox Chse Cner Center, Philelphi, Pennsylvni, USA) for nti-cenp-e ntioy n I. Cheesemn (Whitehe Institute of Biomeil Reserh n MIT, Cmrige, Msshusetts, USA) for nti-sk3 ntioy. We re grteful to A. Desi (University of Cliforni Sn Diego, L Joll, CA, USA), D. Cheermthur, T. Stukenerg, K. Slep n T. Mres for helpful isussions n to J. Mik for generting onstruts. We woul lso like to thnk other memers of the Slmon, Cook, A. Desi n J. Nevins lortories for their support uring this projet. J.G.C. ws supporte y NIH K1 CA9497 n NIH GM8324, E.D.S. ws supporte y NIH GM24364 n J.G.D. ws supporte y NIH GM88371 n grnt from the Pew Sholrs Progrm in the Biomeil Sienes. AUTHOR CONTRIBUTIONS D.V. n J.G.C. esigne n rrie out experiments, nlyse t n wrote the mnusript. S.C., L.J.R.S. n K.T.R. rrie out experiments n nlyse t. D.A.D.C. n J.G.C. onute the two-hyri sreen. K.R.N. n S.C. hrterize the rrest of C6- n -eplete norml firolsts. X.W. n D.V. onute the Delt nlyses. J.G.D., E.D.S. n J.G.C. esigne experiments, nlyse t n wrote the mnusript. All uthors proofre the mnusript. COMPETING FINANCIAL INTERESTS The uthors elre no ompeting finnil interests. Pulishe online t Reprints n permissions informtion is ville online t 1. DeLu, J. G. et l. Kinetohore mirotuule ynmis n tthment stility re regulte y. Cell 127, (26). 2. Cheesemn, I. M., Chppie, J. S., Wilson-Kulek, E. M. & Desi, A. The onserve KMN network onstitutes the ore mirotuule-ining site of the kinetohore. Cell 127, (26). 3. Alushin, G. M. et l. The N8 kinetohore omplex forms oligomeri rrys long mirotuules. Nture 467, (21). 4. Hsu, K. S. & To, T. N8 internl loop interts with Dis1/TOG to ensure proper kinetohore-spinle tthment in fission yest. Curr. Biol. 21, (211). 5. Mure, J. F. et l. The N8 loop region filittes formtion of kinetohore tthment to the ynmi mirotuule plus en. Curr. Biol. 21, (211). 6. Mhi, Y. J., Hmlin, J. L. & Dutt, A. Right ple, right time, n only one: replition initition in metzons. Cell 123, (25). 7. Slfni, R. A. & Holzen, T. M. Cell yle regultion of DNA replition. Annu. Rev. Genet. 41, (27). 8. Snyer, M., He, W. & Zhng, J. J. The DNA replition ftor MCM5 is essentil for Stt1-meite trnsriptionl tivtion. Pro. Ntl A. Si. USA 12, (25). 9. Fith, M. J., Donto, J. J. & Tye, B. K. Mm7, suunit of the presumptive MCM helise, moultes its own expression in onjuntion with Mm1. J. Biol. Chem. 278, (23). 1. Cly-Frre, L., Pelizon, C., Sntmri, D., Pines, J. & Lskey, R. A. Humn replition protein C6 prevents mitosis through hekpoint mehnism tht implites Chk1. EMBO J. 22, (23). 11. Yoshi, K. et l. CDC6 intertion with ATR regultes tivtion of replition hekpoint in higher eukryoti ells. J. Cell Si. 123, (21). 12. Thin, K. E., Gonzlez, M. A., Gurguglini, G., Nigg, E. A. & Lskey, R. A. Depletion of liensing inhiitor geminin uses entrosome overuplition n mitoti efets. EMBO Rep. 6, (25). 13. Prsnth, S. G., Prsnth, K. V., Siiqui, K., Spetor, D. L. & Stillmn, B. Humn Or2 lolizes to entrosomes, entromeres n heterohromtin uring hromosome inheritne. EMBO J. 23, (24). 14. Prsnth, S. G., Prsnth, K. V. & Stillmn, B. Or6 involve in DNA replition, hromosome segregtion, n ytokinesis. Siene 297, (22). 15. Feng, D., Tu, Z., Wu, W. & Ling, C. Inhiiting the expression of DNA replitioninitition proteins inues poptosis in humn ner ells. Cner Res 63, (23). 16. Nevis, K. R., Coreiro-Stone, M. & Cook, J. G. Origin liensing n p53 sttus regulte Ck2 tivity uring G1. Cell Cyle 8, (29). 17. Nishitni, H., Trvirs, S., Lygerou, Z. & Nishimoto, T. The humn liensing ftor for DNA replition umultes in G1 n is estilize fter initition of S-phse. J. Biol. Chem. 276, (21). 18. Chnrsekrn, S., Tn, T. X., Hll, J. R. & Cook, J. G. Stress-stimulte mitogentivte protein kinses ontrol the stility n tivity of the DNA replition liensing ftor. Mol. Cell. Biol. 31, (211). 19. Wohlshlegel, J. A. et l. Inhiition of eukryoti DNA replition y geminin ining to. Siene 29, (2). 2. MGrry, T. J. & Kirshner, M. W. Geminin, n inhiitor of DNA replition, is egre uring mitosis. Cell 93, (1998). 21. Miorno, D., Moreu, J. & Mehli, M. XCDT1 is require for the ssemly of pre-replitive omplexes in Xenopus levis. Nture 44, (2). 22. Chen, S., e Vries, M. A. & Bell, S. P. Or6 is require for ynmi reruitment of uring repete Mm2-7 loing. Genes Dev. 21, (27). 23. Bernl, J. A. & Venkitrmn, A. R. A verterte N-en rule egron revels tht Or6 is require in mitosis for ughter ell sission. J. Cell Biol. 192, (211). 24. Leonhrt, H. et l. Dynmis of DNA replition ftories in living ells. J. Cell Biol. 149, (2). 25. Merli, P., Drvim, V. M. & Sorger, P. K. Timing n hekpoints in the regultion of mitoti progression. Dev. Cell 7, 45 6 (24). 26. Wn, X. et l. Protein rhiteture of the humn kinetohore mirotuule tthment site. Cell 137, (29). 27. Wei, R. R., Al-Bssm, J. & Hrrison, S. C. The N8/HEC1 omplex is ontt point for kinetohore-mirotuule tthment. Nt. Strut. Mol. Biol. 14, (27). 28. Guimres, G. J., Dong, Y., MEwen, B. F. & Delu, J. G. Kinetohore-mirotuule tthment relies on the isorere N-terminl til omin of. Curr. Biol. 18, (28). 29. Miller, S. A., Johnson, M. L. & Stukenerg, P. T. Kinetohore tthments require n intertion etween unstruture tils on mirotuules n N8(). Curr. Biol. 18, (28). 3. Cimini, D., Wn, X., Hirel, C. B. & Slmon, E. D. Auror kinse promotes turnover of kinetohore mirotuules to reue hromosome segregtion errors. Curr. Biol. 16, (26). 31. Yng, Z., Tulu, U. S., Wsworth, P. & Rieer, C. L. Kinetohore ynein is require for hromosome motion n ongression inepenent of the spinle hekpoint. Curr. Biol. 17, (27). 32. Aris, E. E. & Wlter, J. C. Replition-epenent estrution of limits DNA replition to single roun per ell yle in Xenopus egg extrts. Genes Dev. 19, (25). 33. Nishitni, H. et l. Two E3 uiquitin ligses, SCF-Skp2 n DDB1-Cul4, trget humn for proteolysis. EMBO J. 25, (26). 34. Devult, A. et l. Ientifition of Th11/Si2 s the ortholog of the replition liensing ftor in Shromyes erevisie. Curr. Biol. 12, (22). 35. Hofmnn, J. F. & Beh, D. t1 is n essentil trget of the C1/St1 trnsription ftor: requirement for DNA replition n inhiition of mitosis. EMBO J. 13, (1994). 36. Arentson, E. et l. Onogeni potentil of the DNA replition liensing protein CDT1. Onogene 21, (22). 37. Liontos, M. et l. Deregulte overexpression of h n hc6 promotes mlignnt ehvior. Cner Res. 67, (27). 38. Ciferri, C. et l. Implitions for kinetohore-mirotuule tthment from the struture of n engineere N8 omplex. Cell 133, (28). 39. Strr, D. A., Willims, B. C., Hys, T. S. & Golerg, M. L. ZW1 helps reruit yntin n ynein to the kinetohore. J. Cell Biol. 142, (1998). 4. Vrm, D., Monzo, P., Stehmn, S. A. & Vllee, R. B. Diret role of ynein motor in stle kinetohore-mirotuule tthment, orienttion, n lignment. J. Cell Biol. 182, (28). NATURE CELL BIOLOGY VOLUME 14 NUMBER 6 JUNE

12 M E T H O D S DOI: 1.138/n2489 METHODS Cell ulture, trnsfetion n flow ytometry. HeL (norml n stly expressing histone H2B) n NHF1-htert ells were ulture in DMEM (Difo or Invitrogen) supplemente with 1% fetl ovine or lf serum (Sigm), 1 U ml 1 peniillin n 1 mg ml 1 streptomyin. LLCPK1 ells were grown in DMEM with 5% FBS n PTK2 ells were ulture in MEM-α meium supplemente with 15% FBS. Trnsfetions were performe with totl of 1 nm of eh uplex using Dhrmfet 1 regent (Dhrmon/Thermo Sientifi) oring to the mnufturer s guielines. Stle ell lines expressing hve een esrie previously 18. Flow ytometri nlysis for ell-yle position ws performe s esrie previously 16. Syntheti s: t1 n 6 (ref. 16) were synthesize y Invitrogen/Life Tehnologies, m1 ws purhse from Dhrmon (SMART pool tlogue no. L ), ro from Dhrmon (SMARTpool tlogue no. L ), he1(humn) (ref. 28) 5 -AAAAAGAACCGAAUCGUCUAGAA-3, he1 (rt kngroo) 5 -AAUGAGCCGAAUCGUCUAAUATT-3 n or6 (ref. 14) 5 - AAGAUUGGACAGCAGGUCGACUU-3 were synthesize y Dhrmon. HeL ells were synhronize y tretment with 2 mm thymiine for 18 h followe y relese for 9 h n then re-tretment with 2 mm thymiine for 18 h. Syntheti uplexe RNA oligonuleoties were trnsfete into HeL ells oring to the mnufturer s instrutions. Other ell mnipultions inlue 1 µm noozole tretment, 3 µm ZM tretment n ol tretment for 1 min with ie-ol PBS. Antioies. Guine pig nti- is esrie in ref. 41 (ilute 1:3, for immunolots n 1:1 for immunofluoresene stining); rit polylonl nti- ws purhse from Snt Cruz Biotehnology (tlogue no. s28262). Other primry ntioies use in this stuy inlue nti-tuulin monolonl (1:2, for immunolots n 1:5 for ell stining, Sigm-Alrih, no. 926, lone DM1A), nti-ynein IC monolonl ntioies (1:2 for stining Sigm-Alrih, no. D5167, lone 7.1), nti- (1:6, for immunolots n 1:5 for stining, Am, no. 3613, lone 9G3), Nuf2 (1:5, for immunolots n 1:2 for stining, Am no. 1758, lone 28-37), nti-gfp polylonl (1:2 for stining, Invitrogen, no. A6455), monolonl (1:1 for stining, Chemion/Millipore, no. MAB358) or monolonl (1:5, for immunolots, Clonteh, Mountin View, no , lone JL-8), nti-c6 monolonl (1:1, for immunolots Snt Cruz Biotehnology, no. s-9964 lone 18.2), nti-geminin polylonl (1:2 for stining, Snt Cruz Biotehnology, no. s-1315), nti-phospho-h2a.x(ser139) (1:1, for stining, Millipore, no , lone JBW31), nti-m1 polylonl (GeneTex, no. GTX19519). Alex 488-, Rhomine Re-X-, Cy5- or HRPlelle seonry ntioies were otine from Jkson ImmunoReserh. Or6 polylonl ntioy (1:2) for stining ws generous gift from B. Stillmn; nti-or6 for immunolots (1:1,) ws purhse from Snt Cruz Biotehnology (no. s-32735, lone 3A4). Immunolots were snne into Aoe Photoshop n ny mnipultions for rightness were pplie to the entire imge; finl figures show roppe regions n the unroppe lots re provie s Supplementry Fig. S9. Immunofluoresene mirosopy, live-ell imging n Delt nlysis. Cells were typilly fixe for 2 min using 4% prformlehye fter permeiliztion with.5% Triton X-1. For Sp24 stining, 2% prformlehye ws use. For ynein IC stining, the ells were fixe for 6 min t 2 C with ie-ol methnol. Cells were rinse in PHEM uffer (12 mm PIPES, 5 mm HEPES, 2 mm EGTA n 4 mm mgnesium ette, t ph 6.9) efore fixtion. All of the ntioy inutions n wshes were lso performe in PHEM uffer plus.5% BSA. DAPI stining (.1 µg ml 1 ) ws for 1 min, n ells were mounte using Prolong Antife (Moleulr Proes). All ntioy inutions were onute t 37 C for 1 h. For imge quisition, three-imensionl stks were otine sequentilly t 2 nm steps long the z xis through the ell using high-resolution Nikon onfol mirosopy equippe with Yokogw CSU1 spinning is with imge mgnifition yieling 65 nm pixel size from the mer 42 n 1/1.4 NA (Plnpo) DIC oil-immersion ojetive (Nikon). Delt nlysis to mesure seprtions t high (nnometre) ury etween protein epitopes lelle with seprte olours ws rrie out s esrie previously 26. For miroinjetion, polylonl nti- ntiserum 41 ws eplete of ntioies to non- proteins y repete rouns of inution with lystes of ultrviolettrete HeL ells (whih lk ). These ntioy preprtions were then onentrte y entrifugtion using miroonentrtors. Anti- ntioy, ontrol IgG or uffer were injete into HeL ells tht stly express GFP fusion to histone H2B either in prometphse or in metphse. Injetions were rrie out on Zeiss IM mirosope equippe with phse optis n 4/.75 NA (Plnflur) ojetive t 35 C using Sge ir urtin inutor. Injetions were 5% ell volume with Nrishige miromnipultor (Nrishige USA). Mitoti progression ws monitore y live-ell imging using oth phse-ontrst n GFP histone H2B fluoresene nlysis with imges quire every two minutes until the en of the urtion of imging. For live-ell imging of -eplete ells, HeL ells were plte to 5% onflueny in T25 flsk n trete with to. At 24 h post- trnsfetion, ells were trypsinize, ounte n the wil-type n mutnt onstruts were eletroporte long with mcherry histone H2B expression plsmi using the Lonz Nuleofetor pprtus. At 24 h post-eletroportion, ells were imge y time-lpse mirosopy every 4 min for 5 h using Deltvision PersonlDV Imging System (Applie Preision) equippe with Photometris CoolSnp HQ2 mer n 6/1.42 NA (Plnpo) DIC oil-immersion lens (Olympus). Protein protein intertion ssys. For two-hyri sreening, the full-length humn omplementry DNA ws inserte into pgbt9 (Clonteh). Cotrnsformnts of yest strin PJ69 (ref. 43) with two DNA fusion lirries (plentl DNA or thymus DNA, Clonteh) were selete on meium ontining 1 mm 3-mino-1,2,4 trizole (Sigm). More thn 8 million o-trnsformnts of eh lirry were sreene; lones were ientifie from oth lirries. GST pullowns n o-immunopreipittions were performe essentilly s esrie previously 41. GST ws proue in BL21(DE3) purifie on glutthione Sephrose (GE Helthre) n inute with ell lystes prepre in uffer 1 (5 mm HEPES t ph 7.2, 33 mm potssium ette,.5 mm EDTA,.5 mm EGTA,.1% Noniet P-4, 1% glyerol, protese n phosphtse inhiitors). Cell lystes were prepre in uffer 1 or 1:1 mix of uffer 1 n CSK uffer (1 mm PIPES, t ph 7., 1 mm NCl, 3 mm surose, 3 mm MgCl 2 plus protese n phosphtse inhiitors), igeste with mirool nulese efore lrifition y entrifugtion, n inute with GST-protein-ote es for 2 h, or with ntioies for o-immunopreipittion n protein A es, wshe three times n then relese y oiling in SDS PAGE loing uffer. 41. Cook, J. G., Chsse, D. A. & Nevins, J. R. The regulte ssoition of with minihromosome mintenne proteins n C6 in mmmlin ells. J. Biol. Chem. 279, (24). 42. Mox, P. S., Moree, B., Cnmn, J. C. & Slmon, E. D. Spinning isk onfol mirosope system for rpi high-resolution, multimoe, fluoresene spekle mirosopy n green fluoresent protein imging in living ells. Methos Enzymol. 36, (23). 43. Jmes, P., Hlly, J. & Crig, E. A. Genomi lirries n host strin esigne for highly effiient two- hyri seletion in yest. Genetis 144, (1996). NATURE CELL BIOLOGY

13 SUPPLEMENTARY INFORMATION DOI: 1.138/n2489 hr (thymiine) 3 hr (relese) 6 h 9 h si-ontrol time (h): si-ontrol tuulin lne: no. relese (h): (G2) 2 (G1) si-ontrol + vetor + vetor no. relese (h): vetor WT res siontrol 2 5A res + WT res A res e 2C 4C 2C 4C si-lu., synh. si-or6, synh., synh., synh. 8 h H2AX H2AX H2AX H2AX DAPI H2AX DAPI H2AX DAPI H2AX DAPI H2AX Figure S1 -epletion uring S phse oes not pertur replition. () HeL ells trnsfete with ontrol or t1 fter relese from oule thymiine synhroniztion esrie in Fig. 1 were hrveste t the inite time points n nlyze for DNA ontent y flow ytometry n () for protein y immunolotting. () HeL ells stly expressing norml (WT), or n unphosphoryltle erivtive with lnine sustitutions t positions S391, T42, T46, S411, n S491 (from Chnrsekrn, et. l., 211) were synhronize in erly S y oule thymiine lok n relese into s esrie in Fig.1. Noozole ws e uring S phse to hol ells riefly in erly mitosis ( =1 hrs fter thymiine relese into noozole) n then remove to llow ells to ivie (2 hrs). Flow ytometri nlysis of DNA ontent is plotte. () Immunolot nlysis of protein; the lnine sustitutions lok the phosphoryltion-inue gel moility shift. Vetor, ontrols, n WT t re the sme s Fig. 1. (e) Asynhronous HeL ells trete with ontrol luiferse, or6, or t1 s (s inite) or oule thymiine synhronize HeL ells trete with t1 in S n fixe t 1 hrs fter S phse relese (right-most pnels) were stine with DAPI (pseuo-olore green) n nti-phospho-h2a.x ntioy (re). 1

14 SUPPLEMENTARY INFORMATION si-lu. 9 h si-lu 1 h GP h 1 h :31:36 3:6:7 4:3:7 7::37 8:31: Figure S2 Inhiition of funtion inues mitoti rrest. Synhronize HeL ells trnsfete with ontrol luiferse () or t1 () were stine with DAPI (pseuo-olore re) n nti-tuulin ntioy (green) t 9 hrs (left pnels) or 1 hrs (right pnels) fter S phse relese. () Immunolot etetion of using the purifie nti- ntioy, GP47. () Metphse HeL ells stly expressing GFP-histone H2B were injete with the nti-ntioy followe y live-ell imging s in Fig. 2. Selete frmes of GFP-histone H2B (top pnels) n phse ontrst imges (ottom pnels) re shown. Sle rs = 5 µm. See lso Supplementry Movie S4. 2

15 SUPPLEMENTARY INFORMATION si-ontrol Merge Geminin Merge Prometphse Metphse Anphse Telophse Figure S3, ut not its inhiitor geminin, lolizes to kinetohores. () Doule thymiine synhronize HeL ells trete with ontrol luiferse (top pnels) or t1 (ottom pnels) were fixe 9 hrs fter S phse relese n stine with nti- ntioy n nti- ntioy. () HeL ells t the inite stges of mitosis were immunostine using nti-geminin ntioy n nti- ntioy to mrk kinetohores. Sle rs = 5 µm. 3

16 SUPPLEMENTARY INFORMATION Gl4DB Gl4AD geminin si-lu Vrm et l., Figure S4 loop 642 YPD SCD-UL 2.5 mm 3-AT 1 mm 3-AT Merge w/ ACA si-he1 e si-lu. Zwint1 tuulin Figure S4 n intert. () A yest-two-hyri lirry sreen of proteins interting with yiele multiple lones of the kinetohore protein. The igrm illustrtes the sequenes ontine in 5 inepenent lones isolte (some more thn one); the lotion of the loop omin is inite. () Yest strin PJ69 ws trnsforme with plsmis enoing fusions to the Gl4 DNA ining omin ( Gl4DB ) n fusions to the Gl4 trnsriptionl tivtion omin ( Gl4AD ). The protein sequenes inlue in the fusions re liste in supersript; Gl4AD fusion to the inhiitor geminin serves s positive ontrol, n negtive ontrols (empty vetors) re inite y. Seril five-fol ilutions of yest ultures were spotte onto gr pltes ontining the inite onentrtions of 3-mino-1,2,4 trizole ( 3-AT ) to mesure trnsriptionl tivtion of the GAL4pr-HIS3 intertion reporter n inute for 3 ys t 3 C efore photogrphing. YPD n SCD-UL ontrol pltes ontine no 3-AT. () Metphse PTK2 ells trete with he1 were stine with nti-, nti- n nti-aca ntioies. () Lystes from HeL ells trete with trgeting ontrol GFP, or were immunolotte for, n tuulin. (e) Synhronize HeL ells sujete to RNAi uring S phse for 9 hrs were stine using nti- n nti-zwint1 ntioies. Sle rs = 5 µm. 4

17 SUPPLEMENTARY INFORMATION Or6 input HeL lyste oun GST GST- Or6 Or6 si-ontrol Or6 Fluoresene Intensity Or6: Rtio siluiferse Or6 Or6 Crest e Or6 Or6 si-ontrol si-ontrol si-or6 si-or6 f g Mitoti Inex (%) % All Mitoti Cells h si-lu. ACA MT Bu1 MT si-or6 ACA MT 4 C 4 C 4 C Figure S5 filittes Or6 loliztion in mitosis; Or6 is not require for stle kmt tthments. () A lyste of HeL ells ws inute with es ote with terilly-proue GST or GST-, n the enogenous n Or6 were etete in the input or oun frtions y immunolotting. Reominnt proteins oun to the es were etete y Poneu S stining of the lot; the lot ws ut to proe the top for n the ottom for Or6. () Synhronize ontrol n t1 -trete ells were stine for Or6 n. () Quntifition of Or6 fluoresene intensity in ; n = 14 kinetohores. P <.1. () Asynhronously growing HeL ells trete with or6 for 3 ys s in Prsnth et. l., 22, were stine for Or6,, n ACA. (e) HeL ells s in were stine for Or6,, n. (f) HeL ells s in were stine using DAPI n nti-tuulin ntioy, n the perentge of iviing ells (mitoti inex) ws lulte for three ifferent overslips from three seprte experiments (n= 15). (g) The mitoti ells from f were lssifie into the inite stges. (h) Metphse ells from n lte prometphse ells from synhronize ultures eplete of were sujete to ol tretment n stine using nti-tuulin ntioy n either nti-aca ntioy or nti-bu1 ntioy s inite. Sle rs = 5 µm. 5

18 SUPPLEMENTARY INFORMATION wt : :8 :16 :36 :44 :76 : :8 :16 :36 :44 :76 Loop MUT : :48 1:36 2:28 3:24 5: : :48 1:36 2:28 3:24 5: Erly/Mi Promet Lte Promet Metphse Loop MUT Figure S6 Phenotypi omprison of mitoti rrest inue y epletion or Loop MUT expression. HeL ells trnsiently o-expressing mcherry-histone H2B n either wt -GFP () or Loop MUT -GFP () fter epleting enogenous were imge y time-lpse mirosopy every 4 min. Selete frmes of mcherry-histone H2B (top pnels) n phse ontrst imges (ottom pnels) re shown for eh se. () HeL ells were either synhronize in erly S phse n relese into t1 for 1 hrs (top pnels) or trete with he1 followe y trnsfetion with the Loop MUT onstrut (ottom pnels). Cells were fixe n stine with DAPI to lel the hromosomes, nti-tuulin ntioy to lel mirotuules, n either nti-knl1 ntioy (top) or nti-gfp ntioy (ottom pnels) to lel kinetohores. () Quntifition of the ells rreste in vrious stges of mitosis fter epletion (n = 15 ells) or Loop MUT expression (n = 125 ells). Sle rs = 5 µm. 6

19 SUPPLEMENTARY INFORMATION DAPI Knl1 DAPI Knl1 si-lu. 1 h si-m1 1 h si-m1 + m1 1 h + m1 1 h 1 h Asynh. Tuulin Fluoresene Intensity si-lu. Anphse Briges / 1 Anphses si-lu. synh. si-he1 WT Loop MUT si-m1 synh. si-m1 + synh. synh. si-lu. 9 h si-lu 1 h f si-lu + ZM 8.5 h si-lu + ZM 9 h e 9 h 1 h g + ZM 8.5 h + ZM 9 h Figure S7 is require to stisfy the spinle ssemly hekpoint. () Synhronize HeL ells trete with s trgeting ontrol luiferse,, M1, or oth n M1 s inite were fixe t 1 hrs fter S phse relese. For omprison, synhronously growing HeL ells were eplete of (ottom right pnel). All ells were stine with DAPI (pseuo-olore re) n nti-knl1 ntioy. () Perentge of nphse ells with t lest one nphse hromosome rige is plotte for the inite ell popultions. () Quntifition of totl tuulin fluoresene to evlute the loss of kinetohore-mirotuule stility in -eplete or Loop MUT- expressing HeL ells. n 1 ells, p <.1. (-g) Synhronize HeL ells trnsfete with either ontrol luiferse () or t1 (e) were mok trete or trete with 3 µm Auror B inhiitor ZM (s inite) t 8.5 hrs post S phse relese n fixe immeitely (f n g, left pnels) or fter 9 hrs (f n g, right pnels) followe y stining with DAPI (pseuo-olore re) n nti-tuulin ntioy. Pnels n e re reiterte from Figure 6. Sle r = 5 µm. 7

20 SUPPLEMENTARY INFORMATION si-lu. Nsl1 Nsl1 Protein si t1 loop MUT WT Sp Knl Zwint Nsl Dsn Or M1 (Met) Sk3 (Met) Dynein IC (Noo) ZW1 (Noo) Spinly (Noo) CENP-E (Noo) Figure S8 Neither epletion nor Loop MUT expression grossly perturs kinetohore struture. () Doule thymiine synhronize HeL ells sujete to epletion uring the 2 n thymiine relese for 1 hrs were fixe n stine with n ntioy to the Mis12 omplex suunit, Nsl1/ DC31 n with ntioy to. () HeL ells sujete to epletion or expressing resistnt GFP- Loop MUT fter the epletion of enogenous were stine with ntioies to kinetohore proteins involve in spinle ssemly hekpoint n kinetohore mirotuule tthment with or without noozole tretment s inite. Pluses n minuses inite reltive stining intensity ompre to ontrol ells. Sle rs = 5 µm. 8

21 SUPPLEMENTARY INFORMATION Fig 1 Fig 1 Fig 1 C6 Tuulin Fig 4 Fig. 4 n 4 Nuf * * Fig * * short exposure long exposure 52 * IgG hevy hin exise from lot efore proing. 45 Figure S9 Un-roppe immunolots orresponing to Figures 1-8. The regions shown in the figures re outline with she oxes. 9