Journal of Cell Science o:10.1242/jcs.110387 doi: 4720–4727 125, ß Science Cell of Journal 2012 June 7 Accepted ieohrsmyrva hc rtisrsoddrcl othe to directly respond proteins which reveal is may kinetochores kinetochores unattached unclear. largely at at is proteins signaling enriched SAC SAC for are required all the whether proteins and of recruited are enrichment they SAC how Burke but 2008), Most 2002; Stukenberg, Hardwick, and and 2004). (Musacchio kinetochores al., unattached 2003; al., et et McCleland 2001; al., Meraldi 2001; et al., Nabetani et 2001; (Fraschini al., signal et SAC Gardner a are of kinetochores production functional the for and unattached essential 1995) from al., originates et signal (Rieder kinetochores checkpoint in The (Slp1 anaphase- Cdc20 yeast). APC/C-activator the the SAC fission to Mad3 The of and Mad2 through 2011). activity of 2006) binding (Peters, al., (APC/C) the cyclosome et or inhibiting complex Saurin promoting by 2011; Kapoor, anaphase al., and (Kallio prevents Maldonado SAC et 2003; functional Santaguida Hagan, a and 2011; Bub3 for Petersen required 2002; and al., is kinase et Bub1, B yeast) Aurora fission Mad1, organisms), the in addition, yeast), some (Ark1 In network 2007). fission in Salmon, signaling in and (Musacchio (BubR1 (Mph1 SAC Mad3 Mps1 conserved surveys Mad2, proteins The the that 2007). (Musacchio comprises unattached pathway remains Salmon, chromosomes as the and signaling anaphase of prevents any and as a spindle long the to is called attachment also chromosome (SAC; checkpoint) (Walczak checkpoint two spindle assembly spindle mitotic mitotic the The the to 2010). of al., segregated microtubules et are by cells chromosomes daughter division, cell During Introduction words: Key yeast, enrichment fission kinetochore in eliminates In that that, signaling. mutant evidence SAC Mph1 restores provide An kinetochores we arrest. to Here, SAC mutant unclear. stable this a is of for recruitment signaling spindle importance forced SAC mitotic vital whereas for the signaling, of crucial SAC is to proteins. is abolishes Mph1 SAC attached of proteins kinase stably enrichment the SAC an have of is all chromosomes recruitment there of all where kinetochores, enrichment until unattached the from anaphase originates Whether into signal checkpoint entry The blocks kinetochores. their (SAC) through checkpoint assembly spindle The Summary ( correspondence for *Author 3 2 1 Heinrich Stephanie kinetochores checkpoint to assembly recruitment spindle protein of upstream hierarchy and the crucial in is localization kinetochore Mph1 4720 u3i h ide n a3a ela h a1Md ope ttelwredo h irrh.I p1i riiilyrcutdto signaling. recruited SAC artificially functional is that for Mph1 suggest kinetochores If and at kinetochores hierarchy. enriched at the be Mph1 of to of end need role lower crucial necessarily the the not at highlight does results complex and complex Our Mad1–Mad2 Bub1 Mad1–Mad2 activity. top, the the SAC on as Mph1 for well and dispensable network as Ark1 becomes the with Mad3 Ark1 analyzed hierarchy kinetochores, We and three-layered kinetochores. a middle, at identify enriched the and are in kinetochores proteins, to Bub3 SAC localization other protein no SAC but for Ark1, dependencies kinase of Aurora the and Mph1 only when functional rsn drs:FidihMece nttt,Mubesrse6,C-08Bsl Switzerland Basel, CH-4058 66, Germany Germany Maulbeerstrasse Jena, Tuebingen, Institute, D-07745 D-72076 Miescher 10, 39, Friedrich Albert-Einstein-Strasse Spemannstrasse address: Centre, Society, Present Research Planck Septomics Max address: the Present of Laboratory Miescher Friedrich 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. h eednisaogSCpoen o hi erimn to recruitment their for proteins SAC among dependencies The p1 ioi,Sideasml hcpit Kinetochore checkpoint, assembly Spindle Mitosis, Mph1, [email protected] 1 an Windecker Hanna , ) 1,2 ioeHustedt Nicole , eut n Discussion and Results APC/C, the kinetochores. of from inhibition as away for happen Mph1 required can ultimately of This and activation is that only kinetochores. which Aurora suggests and Mad2, at the kinetochore of the at enriched being importance factors upstream visibly central Mph1 the are and demonstrates that Ark1 proteins with SAC functional is signaling function In crucial signaling. SAC only in SAC the Ark1 proteins. of seems SAC for kinetochores other vital to of Mph1 is enrichment Recruiting kinetochore Mph1 at for of Mad2 and and enrichment signaling top, Kinetochore on Mph1 bottom. kinase the the and the Ark1 kinase with Aurora organization the hierarchical in a find We consequences. dependencies functional recruitment the pombe analyzed eukaryote in kinetochore unicellular We inhibition amenable 2004). genetically or of al., depletion et protein network (Meraldi available of a experiments degrees the as different possibly varying However, controversial, of often kinetochore. result and incomplete the is of information state attachment sesteiprac ffsinyatMh oaiainto localization Mph1 yeast fission of To 2010). importance al., et the (Maciejowski recent view assess more this but 2011), challenged al., SAC have et results Hached the 2006; Chen, of al., and et localization (Liu Zhao vertebrates 2003; in kinetochore signaling SAC for that essential is proposed Mps1 kinase been has It signaling SAC kinetochore and abolishes enrichment Mph1 of truncation N-terminal 1,3 n ik Hauf Silke and hr A ee a eetrl eee osuythe study to deleted entirely be can genes SAC where , 1, * bub3 dltdfsinyatcls SAC cells, yeast fission -deleted bub3 Schizosaccharomyces D el,teSCis SAC the cells, hr Report Short Journal of Cell Science xrse rmtethiamine-repressible the from expressed ( rmtpaewsdtrie ytepeec fPo–Cer tSB.Smosaea nD. in as are Symbols SPBs. at Plo1–mCherry of presence the by determined was prometaphase eegono ihmdu wt thiamine, (with medium rich on grown were ftetuctdvrin rmteedgnu ou eutdin resulted locus endogenous N- the from protein versions truncated Expression Mph1 1A). the (Fig. the of intact domain kinase truncated the leaving terminally, we kinetochores, unattached codn osidelength: spindle ( mitosis. to in according died whic that in cells cells indicate indicate circles triangles recorded, filled was recorded, nmt81 mitosis was entire mitosis the from which exit in cells not indicate Circles but SPBs. at Plo1–mCherry of presence the by determined ( control. loading a as serves Cdc2 immunoblotting. by analyzed were strains activity. indicated SAC the for required of is kinetochores to localization Mph1 1. Fig. tsidepl ois(Ps a sda akrfrmttccls(uvhl ta. 99.Rpeettv el n h ecnaeo ioi cells mitotic of percentage the and cells Representative 1999). al., et (Mulvihill cells mitotic for ( marker a shown as are used signal was (SPBs) bodies pole spindle for temperature at restrictive the at imaging live-cell by followed were alleles G ersnaieclsfo h xeietsoni .GPsgaswr eoddudrietclcniin.( conditions. identical under recorded were signals GFP E. in shown experiment the from cells Representative ) rmtr(aie l,19)wsidcdb h elto ftimn.Mttcclswr dniidb h rsneo l1mhrya Psadclassi and SPBs at Plo1–mCherry of presence the by identified were cells Mitotic thiamine. of depletion the by induced was 1993) al., et (Basi promoter n . 0cls.( cells). 30 , 2 D m ,poeahs;bten2ad2.5 and 2 between prometaphase; m, el eefloe ylv-eliaiga h etitv eprtr for temperature restrictive the at imaging live-cell by followed were Cells ) nmt81 nmt81 rmtradclswr olwdb iecl mgn ttersrcietmeauefor temperature restrictive the at imaging live-cell by followed were cells and promoter rmtrrpesd rmnmlmdu akn haie(ihu thiamine, (without thiamine lacking medium minimal or repressed) promoter m ,metaphase; m, nda3-KM311 ( A ceai fteNtria p1tuctos ( truncations. Mph1 N-terminal the of Schematic ) oaiainadSCsgaig hra h ogrtruncation longer the The whereas 1B). signaling, (Fig. SAC (Mph1- Mph1 and type localization truncation wild to shorter similar abundance protein E Hroae l,18) hc rvnsmcouuefrain l1localization Plo1 formation. microtubule prevents which 1984), al., et (Hiraoka . xrsino h niae p1cntut rmtethiamine-regulatable the from constructs Mph1 indicated the of Expression ) 2.5 C A eursMh ttekntcoe4721 kinetochore the at Mph1 requires SAC el expressing Cells ) m ,anaphase; m, n . nda3-KM311 nda3-KM311 0 el.( cells. 200 H h indicated The ) D F h uaino rmtpaewas prometaphase of duration The . -5)mitie kinetochore maintained 1-150) , eildltoso h tan sdi E in used strains the of dilutions Serial ) plo1+–mCherry B xrcsfo snhooscultures asynchronous from Extracts ) nmt81 mis12–mph1 nda3-KM311 rmtrinduced). promoter n h indicated the and uin eeweakly were fusions ihlclzdMph1 localized with nr nomitosis into entry h h uainof duration The . mph1 fied Journal of Cell Science rti tkntcoe oprdt idtp p1(i.1G). (Fig. Mph1 wild-type to compared kinetochores at protein function. recruiting when kinetochore phenotype weaker Mph1- impair slightly The 1E,F). not (Fig. did Mph1 and Mis12 Mph1- signaling of to recruitment SAC Forced were fusion promoted phenotypes the artificially The that 2012). Mph1 of al., of deletion et recruitment as (Ito by 1E,F), (Fig. before rescued defect seen growth a a been to and has lead mitosis kinetochores in to delay Mph1 pronounced wild-type truncated of Mis12. protein recruitment kinetochore the Forced the to recruited fusion by kinetochores artificially to protein we dysfunctional, generally Mph1- the whether or localization kinetochore is activity. localization SAC kinetochore for that crucial suggesting 1C,D), (Fig. signaling (Mph1- 4722 odtriewehrteSCdfc eutdfo impaired from resulted defect SAC the whether determine To D D -0 a eepandb h eue rsneo this of presence reduced the by explained be can 1-302 -0)aoihdbt ieohr oaiainadSAC and localization kinetochore both abolished 1-302) ora fCl cec 2 (20) 125 Science Cell of Journal mad2 D -0 iikdtoeo wild-type of those mimicked 1-302 hc niae htforced that indicates which , D -0 rti was protein 1-302 fMh sacuilpeeust o A inln nfsinyeast. fission in signaling SAC for prerequisite crucial to a enrichment is Mph1 kinetochore response of Hence, Mph1. in of enrichment kinetochore delay mitotic in failure SAC a that indicate support data Mph1- These 1H). to (Fig. depolymerisation able microtubule they levels, both low very at were expressed were proteins fusion both When hte h niheto hsSCpoena ieohrsis kinetochores at protein SAC analyzed this we of of 1), 2009; localization enrichment (Fig. kinetochore signaling the al., SAC whether the for et Since obligatory seems 2009). Vanoosthuyse Mph1 al., 2002; et Hardwick, Windecker kinetochores unattached and at Bub1 (Millband and in Mad3, functional Mad2, is Mad1, enrich SAC the that reported bub3 previously we and Others in kinetochores unattached at Mph1 of Enrichment el sncsayfrSCsignaling SAC for necessary is cells dltdfsinyatclsdsietefiueo hs el to cells these of failure the despite cells yeast fission -deleted D -0 ean h blt o A inln n htthe that and signaling SAC for ability the retains 1-302 mph1- n omlzto si A). in as normalization and eea iepit nerymtss( for mitosis determined early was in points axis time spindle several the intensity on signal Ark1–GFP total of The shown. are region mitosis spindle throughout marker. the SPB of an kymographs as Representative Sid4–mCherry with from imaging exit cell not ( but recorded. mitosis was into mitosis entry indicate which triangles in recorded, cells in was cells mitosis indicate entire Circles the which SPBs. at the Plo1–GFP by of determined presence was prometaphase of duration at for imaging temperature live-cell restrictive by the followed were strains indicated ltwt hsesfo t o9t ecnie data percentile; of 95th median to to 5th normalized from whiskers ( with quantified plot was intensity Mph1– signal contrast. GFP main interference the differential to DIC, relative picture. magnified 1.4-times are Insets h niae akr eegona h restrictive the at for grown temperature were markers indicated the required in is activity kinetochores SAC to for localization Mph1 2. Fig. D 1-302 el sacneuneo eliminating of consequence a is cells nda3-KM311 bub3 D el eefloe ylive- by followed were Cells ) bub3+ D cells. n ie ihmethanol. with fixed and nda3-KM311 el) ( cells). ( A n . el expressing Cells ) n . 7cls o plot box cells; 17 B 8cls box cells; 48 , C The . The ) bub3 D Journal of Cell Science Fg D n hnmcouuefrainwsprevented was formation microtubule when and 3D) (Fig. oaiain p1i nyprilyadlreyindirectly largely by reduced of slightly is and material centromeres deletion at supplementary concentration partially 3D,E; Ark1 (Fig. S2). Fig. only Ark1 localizing for is required Mph1 SAC. the localization, (Jelluma the be in may cells Ark1 Mph1 human of of localization from function that crucial data suggests only this with 2010), and agreement al., the material et In supplementary directly to 3C; to S4). delay (Fig. mitotic Fig. recruited microtubules the is localization of shorten absence artificially not the did Mph1 Ark1 in Ark1 was of maintain inhibition that Mph1 kinetochore, When to suggests kinetochores. required S1, this Figs material continuously Together (supplementary inhibitor attributed the be S3). cannot of al., kinetochores the effects et from side by (Dischinger Mph1 to 3B) of indicated (Fig. loss as SPBs The at mitosis-specific 2008). activity, Plo1 preserved CDK1 of the was presence high continued SDS-PAGE although the maintained in cells Mph1–GFP 3B), minutes microtubules, and 5 (Fig. of within migration lacking inhibition slower disappeared cells Ark1 we largely When mitotic of signals 3A). Mph1 of (Fig. in in 1NM-PP1 localized inhibition Ark1 molecule mutation genetic small chromosome inhibited chemical the conditional by with proper a Ark1 abrogated was by When enrichment kinetochore prevented ( that Ark1 3A,B). kinesin-5 (Fig. on find was depends We crucially attachment activity 2011). Santaguida Mph1 on al., 2004; yeast kinase et depend al., fission Saurin of to et localization 2010; (Vigneron shown al., activity Mps1 been et kinase metazoan has B 2007), kinetochores Aurora al., to to et seem Mps1 localization (Maure and (Ipl1) independently Aurora yeast localize budding in Whereas cells. ntahdkntcoe Fg A n h rcino el,in cells, between of S1), Fig. similar fraction material (supplementary were detected the be could and signal a 2A) which (Fig. kinetochores unattached in kinetochores in preserved n p1aeteol A rtista tl display still that in proteins region SAC centromere/kinetochore only the Ark1 the Hence, at are S2). enrichment Fig. 2A), Mph1 material (Fig. supplementary and Mph1 2D; of (Fig. localization Ark1 the preserving bub3 Mph1 to and addition Ark1 between In is loop the feedback kinetochores localization that A at idea complex the 2009). 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SAC is Mph1 that confirming bnac fMh splmnaymtra i.S) eeinof Deletion S1). mph1 Fig. material (supplementary Mph1 of abundance ncnrs otepoone feto r1o Mph1 on Ark1 of effect pronounced the to contrast In D blse h A-eitddlyin delay SAC-mediated the abolished el agl rsrelclzto fteArr kinase Aurora the of localization preserve largely cells cut7-446 mph1 bub3 bub3 mph1- bub3 D D ohi nohrieuprubdmitosis unperturbed otherwise an in both ,Mh oaie okntcoe,btthe but kinetochores, to localized Mph1 ), -0,teSCrsos in response SAC the 1-302, el.Ide,Mh oaie ounattached to localizes Mph1 Indeed, cells. D D bub3 D bub3+ el Fg A.Teitniyo p1at Mph1 of intensity The 2A). (Fig. cells 1-150 el ssfiin otasi h SAC the transmit to sufficient is cells D el,atog h ioi ea was delay mitotic the although cells, and or bub3 bub3 bub3 D bub3 D D el Fg C.I the In 2C). (Fig. cells el,a a h total the was as cells, bub3 el ognrt the generate to cells ihteMh N- Mph1 the with bub3 D D bub3 -5,teSAC the 1-150, el Fg 2B), (Fig. cells D D el.This cells. el was cells bub3 D fMh enocslclzto fAk ocentromeres, to Ark1 of localization reinforces Mph1 recruitment and localization Mph1, (supplementaryof of localization weak is kinetochore Ark1 Ark1 the that is for suggest required promote results effect these together, Taken this also S2). Fig. but material may Bub1, slightly, of localization Mph1 independently centromere that Ark1 suggesting diminishes further cells edaklo sboe,btsm r1sillclzsto unattached localizes to still Mph1 recruit Ark1 to enhanced some sufficient 2). but (Fig. and kinetochores is broken, and Bub1 is centromeres loop In of feedback phosphorylation. localization H2A-S121 centromeric through presumably ugsigta u1prilypooe r1localization Ark1 of promotes deletion Additional partially delocalized. when Bub1 even that suggesting of deletion 2,idctn htMh n u3ati h aepathway. same the in act Bub3 and of Mph1 Deletion that indicating S2), seen of is deletion enhancement the further in as localization No S2). Ark1 Fig. in material 2004), defect (supplementary similar al., a et (Vanoosthuyse to Bub1 leads of enrichment kinetochore the kinetochore of deletion Indeed, the Bub1. promoting of enrichment through localization al., indirectly Ark1 is et Mph1 for that Kawashima required suggests This 2007; 2010). al., al., of et Tsukahara phosphorylation 2010; et through (Kawashima centromeres H2A-S121 to histone Ark1 efficient of aid 2004) to recruitment known al., is for Bub1 et and required (Vanoosthuyse S5) Fig. material Bub1 is (supplementary of Mph1 localization S2). kinetochore Fig. the material supplementary 3E; (Fig. nihet omtescn ae fteheacy(i.4A). (Fig. kinetochore hierarchy their the Bub1 for of that layer other of find second each Deletion the and on form proteins depend enrichment, which SAC Bub3, other and all for dependencies of deletion upon because defective equally important, is (supplementary Ark1 S10) is and Fig. 4B,C) material localization (Fig. S5–S10). Mad1 other kinetochore Bub1, Figs of or all recruitment its material kinetochore of fully supplementary Mph1, the enrichment 4A; are For of (Fig. kinetochore Mph1 top proteins the and on SAC for are Ark1 required and Indeed, partially proteins hierarchy. independently SAC recruited localization other be in can all Mph1 proteins of and two these Ark1 that of indicates localization dependencies preserved localization protein The SAC of network The rnia irrh em osre.Vrert uoaBis B and Aurora Vertebrate fragmentary conserved. are seems the S11), hierarchy Fig. organisms principal material localization (supplementary other contradictory kinetochore sometimes from the dependencies in S7, lowest Figs material Mad2 (supplementary hierarchy. versa putting vice not S8), depends but Mad2 Mad1, of enrichment on the Kinetochore (Millband 2002). S9) on Hardwick, Fig. and material effect of (supplementary slight Mad3 deletion of very localization neither S7, a Figs whereas material has (supplementary third S8), Mad2 the Mad3 and In Mad1 hierarchy, of S2). localization the S1, but Figs of material 2009), layer supplementary al., 2; (Fig. et intact S8) Windecker S7, 2002; of Figs deletion Hardwick, material (supplementary and Mad3 (Millband and Mad2, Mad1, of expression esseaial nlzdtekntcoelocalization kinetochore the analyzed systematically We lhuhtedt nSCpoenkntcoelocalization kinetochore protein SAC on data the Although A eursMh ttekntcoe4723 kinetochore the at Mph1 requires SAC mph1 D bub3 bub1 bub1 mph1 bub3 mph1- evslclzto fMh n r1largely Ark1 and Mph1 of localization leaves mar r1lclzto oesrnl than strongly more localization Ark1 impairs or D or D obedlto splmnaymtra Fig. material (supplementary deletion double bub3 1-302 bub3 blse ieohr nihetof enrichment kinetochore abolishes . splmnaymtra i.S2), Fig. material (supplementary mad1 bub3 nor hc loabrogates also which , bub3 mad2 mph1 D bub3 fet the affects el,this cells, in mph1 D bub1 mph1 cells or D Journal of Cell Science vrtm sclsetrdmtss( mitosis entered cells as time over olwdb iecl mgn ttersrcietmeauefor temperature restrictive the at imaging live-cell by followed mgn ihSd–Cer steSBmre.Rpeettv yorpso h pnl einaeson h oa inlitniyo r1GPo the on Ark1–GFP of intensity signal total The ( shown. mitosis are early region ( in spindle 2B. points the Fig. time in of several as kymographs for are Representative Symbols determined SPBs. marker. was at SPB axis Plo1–mCherry the spindle of presence as the Sid4–mCherry by with determined imaging was prometaphase of duration The DMSO. bnac fMh–F nccigcls(y)admttclyarse el rae ih()o ihu ( for without temperature or restrictive intensi (+) the signal with Mph1–GFP at treated Maximal imaging cells ( picture. cell antibodies. arrested main control) mitotically the (loading and to anti-Cdc2 (cyc) relative and magnified cells anti-GFP 2.5-times cycling are in Insets Mph1–GFP DAPI. of with ( abundance stained quantified was was DNA min. cells 5 mitotic and min 0 after methanol ie anfe eaiet h anpcue h rsneo oaie l1sga niae htclsaei ioi.Tepretg fclswith cells of percentage The mitosis. in are cells that indicates signal Plo1 localized a of ( presence determined The was picture. signal main Mph1 the and to relative magnified times 5 of presence the cut7-446 in mitosis into released and 1989) a3adBb1 hc onie ihtestronger proteins the SAC with other coincides are to 2007). hierarchy Salmon, which compared the and (Musacchio in divergence BubR1, variable Most evolutionary and 2009). al., al., et Mad2 Mad3 Meraldi et 2004; and al., Essex Mad1 et of 2004; Gillett In 2001; upstream Chen, proteins. and is and SAC B Bub1 (Sharp-Baker other Aurora examined, of and localization organisms 2011), the all al., for et required are Saurin Mps1 2004; 2010; al., al., et et (Vigneron Mps1 Santaguida of localization efficient for required Ark1. and Mph1 of localization kinetochore Interdependent 3. Fig. 4724 leelast oooa pnls(aa n aaia 90.Mttcclswr ie ihmtao,adDAwssandwt AI nesae2.5- are Insets DAPI. with stained was DNA and methanol, with fixed were cells Mitotic 1990). Yanagida, and (Hagan spindles monopolar to leads allele ora fCl cec 2 (20) 125 Science Cell of Journal n . 7 el,bxpo ihwikr rm5ht 5hpretl;dt omlzdt eino el rae o i) Protein min). 0 for treated cells of median to normalized data percentile; 95th to 5th from whiskers with plot box cells, 170 n n . . 0 el) ( cells). 100 3cells; 23 nda3-KM311 6 s.d.). B m el eegona h etitv eprtr for temperature restrictive the at grown were Cells ) fteAk-s niio N-P Hu ta. 07 ra qiaetaon ftesletDS.The DMSO. solvent the of amount equivalent an or 2007) al., et (Hauf 1NM-PP1 inhibitor Ark1-as3 the of M C el expressing Cells ) ntepeec f10 of presence the in nda3-KM311 n . 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(Aurora (Fig. kinetochore Ark1 the to recruitment protein SAC for model u xeiet n ulse eut o ugs general a suggest now results published and experiments Our mph1+ rthe or mis12-mph1- 2 nda3-KM311 N-P a eemndb muoltigusing immunoblotting by determined was 1NM-PP1 ) D 1-302 rae ih1MP1adfxdwith fixed and 1NM-PP1 with treated , uincntutwr olwdb live- by followed were construct fusion D cdc25-22 el eefloe ylive-cell by followed were Cells ) mph1+ uain(oeoe al., et (Moreno mutation el) ( cells). E el were Cells ) Plo1 yin ty Journal of Cell Science ceefrSCpoenlclzto eednisa ntahdkntcoe D,kntcoe o ne eso E rkntcoe of kinetochores or (E) tension under not kinetochores (D), kinetochores unattached details. at dependencies for localization protein SAC for scheme erimn fBb n u3 rbbyt h ue kinetochore outer 1996). the al., to probably et Bub3, (Hardwick and proteins Bub1 SAC 2012; of al., Recruitment or et 2012) Shepperd al., 2012; et al., et Yamagishi London 2009; al., et (Kemmler 3; 2, (Figs study this in ( determined determined. were ( not Dependencies symbols n.d., dependency. by 2002). KM311 of indicated Hardwick, those absence and for the Millband except arrows *, S5–S9), gray S2, and Figs dependency dependencies. material partial supplementary localization a protein arrows SAC dashed of dependency, hierarchy The 4. Fig. h F inlwsmaue vrtm sclsetrdmtss( mitosis entered cells as time over measured was signal GFP The . P hshrlto,OMd,Md nte‘pn ofrain -a2 a2i h coe’cnomto MpliadMscho 2007). Musacchio, and (Mapelli conformation ‘closed’ the in Mad2 C-Mad2, conformation; ‘open’ the in Mad2 O-Mad2, phosphorylation, , B , C h niae tan eefloe ylv-eliaiga h etitv eprtr for temperature restrictive the at imaging live-cell by followed were strains indicated The ) ( A ceai fSCpoenlclzto eednis lc rosidct a indicate arrows Black dependencies. localization protein SAC of Schematic ) n . cells; 7 { idce ta. 09 ,Vnotus ta. 04 ,Kdr ta. 2005; al., et Kadura #, 2004; al., et Vanoosthuyse §, 2009; al., et Windecker , ntaeafebc opwt r1(uoaB osrnte the strengthen to B) (Aurora Ark1 with loop may 2012), feedback al., et a Krenn 2011; initiate Bolanos- al., et 2007; Kiyomitsu 2011; al., al., et et Garcia (Kiyomitsu Blinkin) (KNL-1, Spc7 protein 6 ...Rpeettv ulii al ioi r hw.( shown. are mitosis early in nuclei Representative s.d.). A eursMh ttekntcoe4725 kinetochore the at Mph1 requires SAC bub3 D el F.Setext See (F). cells D – F General ) nda3- Journal of Cell Science 91 t25 at 1991) ai cwezrfreclettcnclspot ui Kamenz, Julia support, technical excellent Eva- Andre and for Langegger Schwoerzer Maria Illgen, Maria Eva-Maria Binder, Julia thank We Acknowledgements antibodies. anti- primary as (Roche, rabbit used anti-GFP or were Mouse SC-53) GTX10873) (Koch described. Cruz, extraction (GeneTex/Acris, (Santa previously Cdc2 protein anti-Cdc13 as and performed mouse 2009) were 11814460001), al., 2012) et al., (Windecker et cells of Synchronization Immunoblotting image. single device a was to charged-coupled cells projected a subsequently fixed a with 3 of Typically, of Corporation). microscope Imaging Devices stack (Molecular AxioImager 2009). software MetaMorph Zeiss al., and camera a et on (Windecker performed Precision) described (Applied microscope previously DeltaVision a as on performed was imaging Live-cell Microscopy the harbouring Strains conditions Culture replacing by modified locus the 2 endogenous and and PCR the (SKRN…NKTP) by 150 into deleted to integrated 2 were acids (SKRN…TPIP) amino 302 to to corresponding performed bases was the (Ba tagging mutants, GFP targeting S1. gene Table PCR-based material by supplementary in listed are Strains pombe S. at Methods and signaling Materials Mph1 SAC how determine of to to aspects interesting localization be recruited. all is will Mph1 it for Additional and 4D). central kinetochores, 4E). 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(Fig. 2001; al., al., et et (Skoufias Garcia kinetochores hierarchy ‘tension-less’ localization protein to SAC recruited the are of layers note signal two We upper SAC 2010). the Salmon, a that and create Maresca tension 2009; Musacchio, under and come (Nezi not do but 4D). microtubules (Fig. 2005) al., et Antoni (De Cdc20 the inhibit and then which Mad2 molecules, Mad2 co-recruits additional Mad1 activates complex microtubule 2002). 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Cladie E., Buffin, Z., S. Xie, K., Hached, ilt,E . sei,C .adSre,P K. P. Sorger, and W. C. Espelin, S., E. Gillett, and C. M. Monteagudo, A., P. Tavormina, C., Yellman, A., Poddar, D., R. Gardner, aca .A,Korga .adTd,T. Toda, and N. Koonrugsa, A., M. Garcia, rshn,R,Brta . ucii .adPat,S. Piatti, and G. Lucchini, A., Beretta, R., Fraschini, se,A,Dmemn,A,Lwly,L,Ogm,K n ea,A. Desai, and K. Oegema, L., Lewellyn, A., Dammermann, A., Essex, aua . e . aoshye . adik .G n ae,S. Sazer, and G. K. Hardwick, V., Vanoosthuyse, X., He, S., Kadura, icigr . rp,A,Xe . alo,J .adSmns V. Simanis, and R. J. Paulson, L., Xie, A., Krapp, S., Dischinger, Mapelli, L., Nezi, V., Sala, C., J. Canman, D., Cimini, G., C. Pearson, A., Antoni, De elm,N,Dne,T . lerct . wakwk,N .adKp,G J. G. Kops, and P. N. Kwiatkowski, T., Sliedrecht, B., T. Dansen, N., Jelluma, hn .H. R. Chen, ai . cmd .adMudel K. Maundrell, and E. Schmid, G., Basi, Ba uk,D .adSuebr,P T. P. Stukenberg, and J. D. Burke, t,D,Sio .adMtuoo T. 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KNL1/Spc7 Cell protein kinetochore the phosphorylates hcpitfnto u o o l hoooesgeainrlso Bub1p. of roles segregation spindle for chromosome essential all is for proteins Biol. not Bub Cell. and but Mad function yeast checkpoint fission of targeting Kinetochore spindle the maintains kinase Mph1 by Spc7/KNL1 to B. J. Bub3 checkpoint. Millar, and and Bub1 G. of K. recruitment Hardwick, J., Rappsilber, J., G. rmt h ovrinfo oooa obplrcrmsm attachment chromosome bipolar to monopolar from shugoshin. of conversion independently the promote uoaBptnitsMs ciaint nuerpdcekon salsmn tthe at establishment checkpoint rapid ensure to activation Mps1 potentiates B Aurora o ieohr oaiaino a1 a2 u3 n EPE neednl fits of independently CENP-E, and Bub3, activity. 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