lxnr .Nue,Aad .Gniel,AmdZ abua,VbaSrvsaa ao higand Ohring Jacob Shrivastava, Vibha Balboula*, Z. Ahmed Gentilello, S. Amanda is oocytes Nguyen, L. kinase mouse Alexandra haspin in by I H3 meiosis histone for on required 3 threonine of Phosphorylation ARTICLE RESEARCH 5066 2014 October 2 Accepted 2014; June 27 Received ` Egypt. Medicine, 35516, Veterinary Mansoura of University, Faculty Mansoura Department, Jersey, Theriogenology New address: of *Present USA. University 08854, State male NJ The Piscataway, in Rutgers, Genetics, expressed of to Department albeit highly cells, somatic is in expressed it also is Although mRNA meiosis-specific haspin 1999). gametes, discover al., to et screen (Tanaka cDNA (Higgins, subtracted humans a spermatocytes mouse to in in discovered first from was mRNA conserved Haspin 2003). is that kinase interest. great of unique is this segregation control that Therefore, in underpinnings women. errors molecular in mistakes These the miscarriage (Hunt of determining (MI). of cause females I genetic percentage leading meiosis in the during are significant errors arise gametes A to homologs haploid segregating 2002). prone generates Hassold, is that and precursors, process diploid cellular from the Meiosis, INTRODUCTION Oocyte maturation, Meiotic I, Histone Meiosis kinase, phosphorylation, , Haspin WORDS: KEY the . regulated of highlighting and is MI at while segregation regulator between chromosome MI, a how than during in as differences rather segregation important haspin chromosome arms inhibited, and implicate chromosome CPC was data along Our during AURKC attachments AURKC . where requires of oocytes correction MI the in that observed suggesting similar were those inhibition at haspin to following defects was meiotic along The CPC remained and absent. the localization of distribution I the whereas mitosis, intact, in metaphase Unlike II. at metaphase attachments kinetochore– and improper alignment, microtubule inhibitors and MI morphology abnormalities Using during chromosome display haspin oocytes in understood. for Haspin-perturbed role oocytes. fully mouse a not in show we is approaches, significance their overexpression MI yet cells, regulating complex germ an in in AURKC, passenger enriched and are Haspin chromosomal homolog, mitosis. AURKB regulate the to kinetochores and that to (CPC) kinase (AURKB) to a B Aurora prone is recruiting thereby Haspin is kinase 3, females. threonine haploids, on in H3 generates aneuploidy histone phosphorylates to that lead division that the errors (MI), I Meiosis ABSTRACT Schindler Karen uhrfrcrepnec ([email protected]) correspondence for Author 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,56–08doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. apn(lokona S2 sa tpclserine/threonine atypical an is GSG2) as known (also Haspin ` hoooa asne ope CC Die l,20;Wang 2005; al., et (Dai is (CPC) the of H3T3 complex recruitment passenger of signals chromosomal chromosome mark phosphorylation this metaphase, and of kinetochores, mitotic (Dai to regulation spindle restricted In and bipolar 2009). a 2005), (Dai establishing al., and al., et plate 2009) the al., et metaphase et for (Dai cohesion Dai the essential 2005; is cell at H3T3 culture Higgins, chromosomes of tissue of phosphorylation dividing alignment that mitotically or reveal Knockdown in 2014). lines haspin al., et of (Maiolica inhibition CENP-T and macroH2A of of substrates 137 57 haspin serine threonine (H3T3), known H3 only histone of the 3 date, threonine are To required oocytes. is in haspin whether meiosis known for not is it However, extent. lesser a pcfclyaltdIAlclzdARCCC oee,we However, but AURKC-CPC. AURKC-CPC ICA-localized kinetochore-localized ablated affect specifically not haspin did of perturbation surprise, activity our To cohesin. of not regulation and condensin, through by structure MI of chromosome completion bivalent proper for maintaining required is haspin is that and loading activity, AURKC how known not is Balboula by regulated. it specifically and 2014; however, to correct 2014); activity Schindler, localization al., catalytic and to et AURKC own (Balboula its eggs. deacetylation required generate by histone II) governed uniquely (Met is to chromosomes, chromosomes II is attachments metaphase to AURKC euploid kinetochore–microtubule localization that its wild-type improper found in with MI we during Consistent they functions that oocytes. model non-overlapping the whereas functionally some supporting I), spindle, (Met have can the I to (Schindler metaphase localizes AURKB the during AURKB oocytes and (ICA) kinetochores mouse that to axis in localizes interchromatid AURKC and 2014; AURKC However, 2012). of Schindler, al., 2009), et loss and for al., (Balboula compensate express et AURKC oocytes that and Shuda demonstrated AURKB (Sasai previously defects We both 2004). the al., rescue ectopic et al., depleted, can et been AURKC Tseng has 2010; of AURKB al., expression where et cells Sharif called 2004; mitotic al., In CPC homolog et 1998). the Sasai AURKB within 2004; al., functions an et also (Li which contain (AURKC), kinase cells C Aurora Meiotic mitosis. in Therefore, cycle. cell 2011). mitotic the al., for localized essential dictates et activity that midzone mark CPC a spindle (Qian is haspin by the cytokinesis H3T3 of phosphorylation to regulates is moves it H3T3 CPC , where (Carmena During the 2009). plate and Funabiki, metaphase and dephosphorylated the Kelly 2012; at al., chromosomes et of navigates alignment then the CPC controlling correcting thereby The by attachments, transition kinetochore–microtubule 2010). metaphase–anaphase improper al., the through et cell Yamagishi the 2010; al., et ee edmntaeta os oye oti apnkinase haspin contain oocytes mouse that demonstrate we Here, CPC the of subunit catalytic the is (AURKB) kinase B Aurora

Journal of Cell Science RESULTS mitosis. in localization AURKB-CPC does AURKC-CPC indicate it meiotic they than and regulates differently ICA, activity the haspin along activity that during AURKC attachments these requires of I correction activity. Met the AURKC that suggest of data loss These with consistent aneuploidy, and subsequent attachments kinetochore–microtubule improper observed still ARTICLE RESEARCH ci eoyeiigaet(i.1) apni mlctdin implicated is Haspin 1F). (Fig. an This A, agent 1E). latrunculin depolymerizing (Fig. with ectopically treatment cap actin upon actin expected, disrupted the was As with localization phalloidin colocalized GFP–haspin. TRITC-tagged haspin with expressing expressed actin oocytes detected with colocalizes we in haspin cap, whether actin the determine is the locale To subcellular Actin cap’. in this 1E). ‘actin giving is (Fig. name region, that spindle cortical cortex and this oocyte to chromatin the restricted the of GFP–haspin to at region I, proximity discrete haspin Met close a at detected to starting localized we that, conducting also found In I, 1C). we of Met H3T3 (Fig. experiments, axes these At the addition chromatid sister 1D). is the at (Fig. and haspin with oocytes that twofold in supporting kinase increased further Notably, H3pT3 haspin, 1C,D). and exogenous of (Fig. chromatin assessed levels stages with the meiotic all colocalized during GFP–haspin H3pT3 oocytes. with H3pT3 expressed not mouse correlate we were To haspin, available shown). of haspin not localization commercially (data endogenous system with our that in react specific found to We indicated antibodies oocytes. in 1999). expressed al., et (Tanaka haspin cells contain mRNA. maturation somatic meiotic undergoing in oocytes Therefore, zygotic levels levels, ( expression destruction abundant expression low as its maternal not to is with prior expression persists embryos Compared that data one-cell 1B). in The (Fig. expressed and maternally embryos. meiosis a preimplantation during is haspin and that oocytes indicate generated libraries mouse cDNA on from RT-PCR quantitative the performed from we I Met mitosis. in of differs metaphase H3pT3 during observed of demonstrate pattern and pattern haspin, 2009). localization al., contain the et oocytes that Markaki that 2006; suggest al., data et restricted These (Dai is mitosis phosphorylation in its centromeres because the to specific, along I). phosphorylation meiosis centromeres (Telo H3T3 is at Notably, I ICA only ICA. not the mark along during histone also this absent but found was we I, but Met I), During and in persisted (Ana I) Phosphorylation that I 1A). (Met found (Fig. anaphase MI II) We of (Met MII maturation. metaphase recognizes of metaphase during meiotic phosphorylated of that was stages H3T3 on different antibody at (H3pT3) fixed an H3T3 performed we oocytes, phosphorylated using determine in mouse first in specifically exists To immunocytochemistry and kinase known. 2012; meiosis H3T3 not al., an in still et whether is role Wang maturation and its 2012; meiotic 2005; (Dai al., 2010), oocyte al., et al., cells et Huertas Dai et 2012; somatic 2006; Yamagishi al., al., in et et Dai cycle Antoni 2009; De al., cell et gene Dai key mitotic a 2005; germ-cell-specific as Higgins, the noted and a of 1999) al., as et regulator (Tanaka testes identified mouse in was expressed haspin oocytes Although mouse localization in and meiosis activity during expression, kinase protein Haspin et ewne odtriewehrhsi rti was protein haspin whether determine to wanted we Next, oocytes, in expressed is mRNA haspin whether determine To , 0) ossetwt observed with consistent 30%), Gfp in-vitro tge apncN in cRNA haspin -tagged mtrdoocytes -matured ta. 03 n in and (Panigada 2013) yeast budding al., in et divisions cell asymmetric regulating 0 Mo -t n eenal betwe oye were oocytes when in haspin of incubated absent Overexpression 2A). oocytes nearly (Fig. in 5-Itu were of nM I and 500 H3pT3 Met in 5-Itu incubated ethanol), 90% of (100% by nM 100 vehicle reduced were in signals incubated oocytes with haspin, Compared control inhibited immunocytochemistry. 5-Itu using 2012; that H3pT3 al., confirmed detecting first et by We Antoni small-molecule 2012). we (De al., whether haspin a et maturation, for Wang determine (5-Itu), specificity meiotic high 5-iodotubercidin To with oocyte Wang inhibitor in 2010). for 2009; oocytes al., al., required et matured et is Markaki activity Antoni Yamagishi 2012; De haspin 2012; al., 2005; al., et al., et Huertas et Dai 2012; 2006; al., al., et et Dai anaphase 2005; (Dai to Higgins, chromosomes and time lagging phenotypic and of of misalignment host chromosome length onset, a prolonged causes cells including somatic consequences, in progression haspin meiotic of for Inhibition required is activity Haspin n efre muoyohmsr odtrietefinal the determine – 2D) to (Fig. which II immunocytochemistry at Met time at the performed arrested – and h body and 16 polar at a cells extruded al., treated controls the et fixed (Dai we maturation, haspin of depleted the cells in HeLa delay 2006). reported in a with transition Fig. consistent G2-M are material because delays of (supplementary these 5-Itu Perturbations delays and S2B), to caused incubation. also specific of treatment not h CHR 5 were By oocytes after of kinetics 5-Itu. 40% even and meiotic-resumption when meiosis nM resume resume 100 to to controls longer nM, failed of h 500 with to 1 presence 5-Itu took of oocytes compared concentration all the the increased in oocytes we when meiotic-resumption contrast, matured of reduced were 20% slightly oocytes underwent in observed maturation oocytes meiotic kinetics of Control We oocytes induction after I. 2C). h when nuclear (Fig. 2 Met first and occurs 1.5 enter is The between NEBD and which maturation meiosis. meiosis (NEBD), meiotic resume resume breakdown and during envelope follicles event from morphological removed are Antoni 2012). (De al., cells et mitotic in Wang haspin 2012; inhibit al., to in than et used and less doses oocytes tenfold in 5-Itu approximately of haspin the was inhibit addition experiments to subsequent required the dose our by the that oocytes note 2012), in we al., inhibited et to Wang cells inhibitors be 2012; small-molecule cycling al., can mitotic et activity Huertas to 2012; haspin similar al., et that, Antoni indicate (De Fig. another data material 2012), These (supplementary al., signals S2A). haspin et the of (Huertas H3pT3 with inhibitor (CHR) treated small-molecule CHR-6494 drug, were of oocytes S1). doses the when Fig. same results material adding similar (supplementary observed in return after We kinase not H3T3 did h and the 1 diminished is haspin Within a that loss because oocytes. supporting rescue haspin, not further could of H3pT3, (K466R) of pocket activity ATP-binding catalytic the This in the 2B). mutant on (Fig. oocytes depended 5-Itu-treated in rescue H3pT3 of loss the rescued iiigcls ae oehr hs aasgetta haspin ICA. that the along suggest pattern data MI-specific a these in together, H3T3 phosphorylates Taken asymmetrically also to least cells. specific is at perhaps dividing having is oocytes that haspin in substrate with additional MI consistent one are during data cytokinesis these Because asymmetric, 2011). al., et odtrietecneune fhsi niiinfrmeiotic for inhibition haspin of consequences the determine To they until MI of prophase the at arrested remain Oocytes ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal rbdpi thaliana Arabidopsis nvitro in utr eim Moreover, medium. culture mro (Ashtiyani embryos 5067

Journal of Cell Science emnlvsce e ,mtpaeI n ,aahs ;Tl ,tlpaeI I rMtI,mtpaeI.Saebr:10 bars: Scale II. metaphase II, Met blastoc or BL, embryo; MII 8-cell I, 8C, telophase embryo; I, 2-cell Telo 2C, I; embryo; anaphase 1-cell 1C, I, oocyte; Ana incompetent mean I; INC, the metaphase group. as I, each shown, in Met also oocytes vesicle; 15 are germinal with levels twice, phosphorylation least H3T3 at of conducted Quantification microscopy. confocal using oteidctdsae ro ofxto n rcsigt eetGPhsi gen,DA(le,Hp3(e)()o ci rd EF.Atrs,chr Asterisk, (E,F). (red) actin or (D) (red) H3pT3 10 (blue), in DNA incubated (green), were GFP–haspin eggs detect II to Met processing (F) and bridge. fixation anaphase an to prior stages indicated the to mean EERHARTICLE RESEARCH h aepretg foctswr tMtIa bevdin resume observed to failed as more 5068 I but Met dose, at lower the were treated with oocytes When treated I. of samples Telo percentage reach in were same to 5% failed the only and 5-Itu, that I nM oocytes Met 500 at those with were dose, Of 10% 2D). lower II, (Fig. Met the II At Met reached reached. stage meiotic f exogenous microscopy against confocal normalized by were obtained and slices RT-PCR optical quantitative are by Shown DNA. determined with and were (H3pT3) times levels 3 three Expression histone repeated embryos. on was preimplantation 3 experiment threonine The phosphorylated experiment. of kinase. representative detection protein and haspin fixation active to contain prior oocytes Mouse 1. Fig. 6 ...(he needn xeiet) CF emnlvsceitc oye eeioae,mconetdwt f-apncN n matured and cRNA Gfp-haspin with microinjected isolated, were oocytes Germinal-vesicle-intact (C–F) experiments). independent (three s.e.m. , 0 foctsrahdMtI.About II. Met reached oocytes of 60% , 0 foocytes of 80% m fltuclnA(a )fr1 i ro ofxto.Sonaerepresentative are Shown fixation. to prior min 10 for A) (Lat A latrunculin of M A emnlvsceitc oye eeioae n matured and isolated were oocytes Germinal-vesicle-intact (A) § 5octsprsae B eaiemN eeso apni oye and oocytes in haspin of levels mRNA Relative (B) stage. per oocytes 15 oyehdetrditrhs dsrbda nepae.We interphase). as the (described if as interphase 2E). nucleolus entered (Fig. a had reformed CHR sometimes oocyte (described of which bodies structure collapsed), ball-like polar when presence a as with into obtained collapsed eggs the DNA contained those observed that in occasionally to we matured similar Furthermore, were were results oocytes These meiosis. ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal 6 ...au,abtayuis h xeiet were experiments The units. arbitrary a.u., s.e.m. m ACF,2 (A,C–F), m nvitro in Gfp aaaesona the as shown are Data . oteidctdstages indicated the to m Z isti A). in (inset m poetosobtained -projections msmsin omosomes s;GV, yst; nvitro in o a rom

Journal of Cell Science eemndb iecl mgn hnoctswr aue nteidctdds f5Iu h aawr orce o ea nNB ro oaayi.T analysis. to prior NEBD as in (PBE) determine delay extrusion as for body h, corrected polar 16 were of for data Timing The drug (F) 5-Itu. indicated key. of the the dose in below indicated with II. maturation the shown times after metaphase in are three reached matured oocytes repeated stage were (‘C’) were oocytes meiotic collapsed experiments when the and imaging of (‘I’) live-cell Analysis interphase by (D,E,G) of determined min. Examples 30 microscopy. every confocal captured by were Images 5-Itu. of *** EERHARTICLE RESEARCH lsl xmn h ieiswt hc h oye were oocytes the which with more kinetics to imaging the live-cell used examine also closely attempt We failed cytokinesis. to owing undergo arose to phenotypes rare these that speculate mean the as shown are data A,B, For 5-Itu. of nM 500 are or of Shown (EtOH) detection 5-Itu. ethanol and of of fixation addition presence to the the prior after in h) h (blue) (7 1 DNA envelop I microinjected and nuclear Met was (red) intact to material anti-serum] an indicated matured CREST with The were [(A) oocytes (B) Oocytes kinetochores containing panel. (A,B) (green), medium G. (H3pT3) culture in 3 to (NEBD) histone representative added breakdown on were envelope 3 drugs nuclear threonine The underwent phosphorylated analysis. that to oocytes prior or (CHR) A–F CHR-6494 MI. or perturbs (5-Itu) haspin iodotubercidin of Inhibition 2. Fig. P , .0 oewyAOA.Saebr:10 bars: Scale ANOVA). (one-way 0.001 Z poetosotie ycnoa irsoyfo n xeiet uniiaino h 3T inl r hw otergto ahimage each of right the to shown are signals H3pT3 the of Quantification experiment. one from microscopy confocal by obtained -projections rpaeIarse oye eeioae n matured and isolated were oocytes Prophase-I-arrested § m 5octsprgop V emnlvsce e ,mtpaeI n ,aahs ;Tl ,tlpaeI e II, Met I, telophase I, Telo I; anaphase I, Ana I; metaphase I, Met vesicle; germinal GV, group. per oocytes 15 .()Tetmn fNB sdtrie ylv-eliaigwe oye eemtrdi h niae dose indicated the in matured were oocytes when imaging live-cell by determined as NEBD of timing The (C) m. eoi eupin oto oye n hs rae with treated in those delay the and by for oocytes defined correcting control was After body. MI resumption, polar of meiotic the Completion of MI. extrusion through progressing ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal nvitro in ntepeec fteidctdcnetaino 5- of concentration indicated the of presence the in 6 s.e.m.; 5069 ein hese d

Journal of Cell Science eoi tg rtedu htw sdt nii apn we structures state. haspin, DNA interphase-like collapsed inhibit an in and re-enter to eventually extrusion, delays used that body – polar we consequences a and that phenotypic NEBD identical drug not to nearly respect the with observed was or addition drug was stage of maturation haspin in time meiotic the when than of resumption meiotic Regardless I meiotic inhibited. Telo impeding completing and of consequence I in Met 100 in deficiency or delayed still controls were oocytes more ieymne.Teeoe iia oadlyo npaeostin onset a anaphase in of bodies delay polar a extruded to oocytes similar 5-Itu these Therefore, of manner. nM results 60% timely 500 the only by to 2D, Similar Fig. with MI 3). in completing Movie treated material in (supplementary oocytes controls delay the 2C), a (Fig. for exhibited correcting delay Despite 2). NEBD 1, Movies material starting supplementary kinetics, similar with bodies at polar extruded 5-Itu nM 100 ARTICLE RESEARCH rao ahsse hoai Fg DE.Bt perturbations Both 3D,E). (Fig. 5070 chromatid the sister measured and each spreads chromosome of performed area we overexpression chromosome II, the Met the compare at eggs To haspin-overexpressing and I. in 5-Itu-treated Met between morphology from morphology we exit that after abnormal note experiments We 5). observed 3C,D). 4, (Fig. Movies only material II chromosomes chromosomes supplementary segregating contained 1C; Met between (Fig. always bridges to anaphase I formed matured Ana that in when oocytes if unraveling Furthermore, as elongated were appeared chromosomes they the the – observed we phenotype haspin, control- opposite catalytic overexpressed to compared we the the and when of contrast, oocytes, upon By injected 75% 3B). depended (Fig. in haspin rescue of bivalents activity this normal-shaped detect Furthermore, because to oocytes. oocytes, able 5-Itu-treated were in we haspin overexpressed ( we when overly compact and unusually ( bivalent extended and ends obvious the appeared at that with ‘pointy’ 3A). regions oocytes (Fig. with I 5- chromosomes time Met of ( dose fewer structures the higher chromosome observed the identifiable of with we inhibited Itu, 95% clearly was haspin frequently nearly contained when and and However, 5-Itu chromosomes oocytes spindles Control nM 500 bivalent 5-Itu-treated of morphology. in these detection nM chromosomal 100 matured and abnormal assess in oocytes matured fixation displayed 5-Itu, oocytes closely to and nM controls 100 prior more with haspin Compared I oocytes when chromosomes. To haspin-inhibited Met I and Met control to inhibited. matured in we delayed abnormalities, was oocytes the activity several observed in we experiments, abnormalities maturation meiotic the During condensin delocalizing chromosome by perturbs morphology activity haspin of Alteration activity 1 at haspin Starting in S1). treating increase Fig. material when apparent (supplementary the NEBD than haspin or after of dose timing 2G) later but (Fig. the higher either inhibition MI, reflecting twofold NEBD, of to prior roughly oocytes completion to a compromised Similar to in inhibitor. required still later oocytes haspin the of maturation inhibition allowed of 2D, meiotic Fig. addition in we presented to observations our NEBD, prior NEBD perturbing complete of consequence in MI of completion timely oocytes. for mouse required is activity haspin mitosis, ocnimta alr opors oMtI a o a not was II Met to progress to failure that confirm To , 0hatridcino eoi auain(i.2F; (Fig. maturation meiotic of induction after h 10 m -t-rae oye.Teeoe the Therefore, oocytes. 5-Itu-treated M , % rcrmsmsta eerounded were that chromosomes or 5%) , , 0)admr htcnandeither contained that more and 60%) 5) hspeoyewsrescued was phenotype This 35%). , oprdwith compared h 2 m M, h muto M2o e hoooe Fg HI.In 3H,I). (Fig. centromeres at chromosomes cohesin I of loss Met causes on in inhibition oocytes SMC2 increase haspin in twofold mitosis, of following a data, with found amount these treatment we 20% with the after haspin, Consistent overexpressed 60% by 3G). we by (Fig. where and SMC2 5-Itu 5-Itu nM detectable nM 500 100 with decreased treatment inhibition contained haspin contrast, oocytes By performed 3F,G). Control of (Fig. inhibitor. previously we in reported haspin I be as Met the condensin, SMC2, to of could matured localized presence were the that morphology of oocytes determine on loss To chromosome immunocytochemistry 2011). with al., abnormal et (Lee correlated I the Met chromatid at sister whether oocytes at Similar and mouse complexes. centromeres in both at to axes present common is is SMC2 that haspin, protein to condensin a and is and I SMC2 multi-protein condensin mitosis II. complexes, a in two is contain compaction Cells Condensin chromosome meiosis. 2011). regulates al., that in et complex altered (Lee were oocytes condensin of mouse subunits when observed phenotypes MI. during morphology chromosome alters perturbation activity Therefore, haspin 5-Itu-treated 3D). of (Fig. thinner is In are area they more the 3E). because oocytes are smaller (Fig. haspin-overexpressing they the because chromatid in area whereas in rounded, the smaller are of chromatids the oocytes, area the reduced h odn fcnesnt eoi hoooe n osnot does and cohesin. chromosomes of regulates meiotic removal overexpressed haspin to the that regulate was condensin suggest of haspin data loading when the these in sister Therefore, levels changes 4D,E). detect that or (Fig. not premature did localization from indicating we protected REC8 the Furthermore, perturb and centromeres, 4C) by intact (Fig. not still at supported proteolysis did is further SGOL2 cohesion haspin are chromatid of of data inhibition These presence I, 4A,B). (Fig. Met cohesin in subunit cohesin that, REC8 meiosis-specific revealed the I of 5- Met Detection in of cohesin oocytes. immunocytochemistry chromatid conducting sister by assessed oocytes also Itu-treated We 2006). al., et (Dai o hw) hrfr,Hp3i rrqiiefrdriving for prerequisite a is (data 5-Itu- H3pT3 defect localization in Therefore, ICA AURKC the shown). rescue of not not did Overexpression to oocytes We added S3A–D). treated (supplementary was kinetochores. NEBD Fig. CHR completed at or ICA material oocytes not 5-Itu after the either medium but qualitative when along culture controls, results reduced our of identical was found that confirmed signal with end analyses AURKC compared and start These that the as assessment 5C). pairs (Fig. signal kinetochore lost using the sister points oocyte the only plotted each mark we presence to oocytes in CREST the centromeres, chromosomes I individual quantify at along Met To AURKC intensities 5A,B). of 2A,B), (Fig. (Fig. amount ICA and 5-Itu the along nM our 500 AURKC the To and in 5A,B). centromeres (Fig. at ICA both ICA H3pT3 lost the contained oocytes still along although oocytes surprise, of and detected 40% 5-Itu, centromeres of we at nM whether 100 AURKC manner, In determine I. Met same To to 5-Itu in the matured 2010). oocytes in immunocytochemistry in al., by AURKC Wang endogenous regulated et 2010; al., is Yamagishi et Wang AURKC-CPC 2012; 2012; al., al., et 2006; Antoni et al., De 2005; et al., Dai et 2005; Dai Higgins, to and (Dai kinetochore– AURKB-CPC attachment and recruits microtubule alignment chromosome H3T3 regulate to of centromeres phosphorylation mitosis, In ICA MI from AURKC-CPC to prevents localizing levels H3pT3 of Alteration hs hoooemrhlg hntpsaeietclt the to identical are phenotypes morphology chromosome These ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal

Journal of Cell Science * EERHARTICLE RESEARCH ru a o auae.Teeeprmnswr eetda es he ie ihama f1 oye e ru.Dt r xrse stemean the as expressed are Data group. per oocytes C, 15 In of staining. mean DAPI a we sister with by (blue) each times detected DNA of three was and least taken DNA (red) at D. were SMC2 repeated in (F,H) measurements were eggs phenotypes. Area experiments the morphology These (E) from the saturated. chromatid chromosomes. demonstrating not single panels of was a image group spreading microinj representative of magnified and or are area the 5-Itu, Shown lysis the immunocytochemistry. to nM is to by correspond 500 detected point prior asterisks or Each (EtOH) and h) ImageJ. ethanol labels (18 in in number II tool matured the were line Met Oocytes freehand to (D) the maturation (I). using abundance before chromatids and o microin GFP–haspin H) Prophase-I-arrested were (red, or (C,H,I) matured oocytes localization A. PBS SMC2 maturation, and panel with or in to (green) (C) ‘normal’ Prior cRNA staining to (B) DAPI equivalent indicated (5-Itu). by were the 5-iodotubercidin morphology bivalents with Resolved of nM. concentration microinjected 500 indicated isolated, was the were dose of 5-Itu presence The the GFP–haspin-K466R. or in morphology. GFP–haspin (5-Itu)] chromosome h I 9 Met alters and haspin (control) of Perturbation 3. Fig. P , .5(n-a NV,G Student’s G; ANOVA, (one-way 0.05 t ts,I.Saebr:10 bars: Scale I). -test, Z poetosotie ycnoa irsoy nFH h ae oe a e ota h brightest the that so set was power laser the F,H, In microscopy. confocal by obtained -projections m m. ABFG rpaeIarse oye eeioae n matured and isolated were oocytes Prophase-I-arrested (A,B,F,G) nvitro in oMtI 1 )()o e 7h HI ro oaayi fchromosome of analysis to prior (H,I) h) (7 I Met or (C) h) (16 II Met to ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal nvitro in etdwt PBS, with jected oMtI[ h [7 I Met to 6 s.e.m.; ocytes 5071 ected D, re

Journal of Cell Science EERHARTICLE RESEARCH oeARCmgtlclz oteIAidpneto the of independent ICA 5072 the Alternatively, to this. localize reflect might might AURKC-specific AURKC the differences as antibody some the robust the in as and However, not AURKC. severe antibody, is of more that recognizes was than that group survivin 5D–F). nM This (Fig. of 100 S3E,F). the pattern kinetochores Fig. localization after material at administered perturbed (supplementary was retention 5-Itu resumption survivin when of meiotic obtained but loss were the observed results Niedzialkowska ICA, we Similar docking 2010; AURKC, the for to al., Similar along responsible et 2012). is (Kelly al., et and kinetochores survivin mark mitosis, at In H3pT3 complex (also oocytes. survivin the 5-Itu-treated of in detects localization BIRC5) the kinetochores as detected known to also not we delocalized, but ICA the I. to Met AURKC during of localization ocnimta h P,adntjs UK,is AURKC, just not and CPC, the that confirm To lhuhHp3getyicesdaloe h chromosomes the GFP–haspin. over overexpressing all by increased chromosomes greatly I H3pT3 Although Met to CPC ICA I. the Met to during AURKC-CPC kinetochores localize to to not but required Similar is activity 5G–I). meiotic together, of (Fig. after Taken haspin S3G,H). absence administered dose Fig. was material complete high (supplementary 5-Itu resumption and the when obtained 5-Itu, in ICA were ICA the of results along the INCENP dose pINCENP from results low of pINCENP the of loss the in a to observed beginning form we we Similar AURKC, AURKC, immunocytochemistry. with AURKC-phosphorylated of by activity (pINCENP) the localized assess detected To INCENP. called haspin when perturbed is inhibited. CPC is ICA-localized Regardless, CPC. eas odce neprmn odieecs AURKC- excess drive to experiment an conducted also We CPC, the of component a phosphorylate AURKC and AURKB ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal infcn oewyAOA()o Student’s statistically or not (B) ANOVA were [one-way data significant 10–15 The with group. twice per least oocytes at repeated were experiments 10 bars: the Scale from experiment. microscopy representative same confocal are Shown by D). obtained in projections blue and C, (red) in REC8 (red maturation for DNA the stained to were added Samples was medium. 5-Itu or (EtOH) ethanol and cRNA indicated matured the with microinjected were isolated, oocytes Germinal-vesicle-intact DNA (C–E) and (blue). red) REC8 anti-sera, for (CREST stained concentrations kinetochores were indicated Oocytes (green), the fixation. to of prior presence 5-Itu the of in h) (7 matured I and isolated were oocytes I. intact sister Met affect at not does cohesion haspin chromatid of Perturbation 4. Fig. hoooe.Dt hwtemean the along show signal Data REC8 chromosomes. the of Quantification (B,E) nvitro in oMtI( )pirt iain nC, In fixation. to prior h) (7 I Met to AB Germinal-vesicle- (A,B) m m. 6 ...These s.e.m. nvitro in t ts (E)]. -test oMet to Z -

Journal of Cell Science EERHARTICLE RESEARCH oaiaini losniiet ihlvl fHp3o fthe if haspin- or H3pT3 Because of in levels controls. high altered to of AURKC-CPC sensitive whether also grossly know those is not localization do is we with oocytes, overexpressing morphology although compared lower, ICA slightly chromosome absent, were AURKC-CPC signals S4A–C). kinetochore perturbed not Fig. that material observed note (supplementary We activity still and localization we 1D), (Fig. before slice optical an from chromosome selected d a were show ( merge) images kinetochore in the zoomed (green at The subunits remaining ICA. CPC subunit the CPC indicated along the The localized of 10 (A,D,G) presence diffusely bars: (5-Itu). the but 5-iodotubercidin highlight merge) panels of in ‘zoom’ concentrations red The indicated antisera; images. the representative of are shown presence and the immunocytochemistry localization. in ICA 5-Itu) AURKC-CPC for perturbs h haspin 9 of Inhibition 5. Fig. RS nieat aktekntcoe.Teeeprmnswr eetda es he ie ih1 oye e ru.EO,ethanol. EtOH, group. per oocytes 15 with the times following three chromosomes, least on localization at as subunit repeated CPC quantitative were show of experiments analyses Examples The These (C,F,I) ImageJ. kinetochores. method. in the qualitative function mark profiles’ a ‘plot to using the antisera localization using CREST axis zoom, the interchromatid in or image kinetochore chromosome no diffuse, axis; interchromatid m .(,,)Qatfcto fCCsbntlclzto.Kdfue ieohr n ifs inlo necrmtdai;KIA ieohr and kinetochore K+ICA, axis; interchromatid on signal diffuse and kinetochore K+diffuse, localization. subunit CPC of Quantification (B,E,H) m. rpaeIarse oye eeioae n matured and isolated were oocytes Prophase-I-arrested oaiainta tes hs aamgtrfettene o a for need the reflect same might the data ICA These oocytes within AURKC others. clear is, than 5-Itu-treated more localization That displayed in chromosomes S4D). some oocytes, Fig. overexpressed material was (supplementary catalytically not haspin but wild-type inactive when rescue localization partial ICA a by AURKC observed in diffuse we Furthermore, appear methods. localization detection our the makes morphology elongated ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal nvitro in oMtI( o control, for h (7 I Met to Z poeto.Scale -projection. eseto the of sessment tce by etected CREST s of use 5073

Journal of Cell Science re us fcl eim efxdoctsadperformed and and and oocytes attached fixed sensitive the we detect is medium, better to a cold allows immunocytochemistry After spindle of microtubules. treatment pulse kinetochore-attached the brief This the of of 1981). visualization to (Rieder, remainder treatment, attached cold depolymerizes fibers as the such Spindle conditions whereas kinetochores. under stable to are kinetochores made spindle plate. the I haspin chromosome Met of the in inhibition at Therefore, increase misalignment shown). chromosome significant not causes (data a aberrant were had 5-Itu ( misalignment nM incubated 500 Oocytes shown). in not (data misalignment chromosome tahet,laigt iaineta h e lt and, plate of kinetochore–microtubule I Met ability improper the the at correct misalignment alters to to leading overexpression attachments, oocytes either or by ICA the inhibition In the I. at Ana during localization haspin pull the AURKC to causing begins perturbing spindle somehow of the summary, is once chromosomes apart presence the fall Met at of to after sisters rigidity SGO2L The protected the being of and that not REC8 1C). is or presence of cohesin (Fig. I either presence that the but I indicates that I II Met with Met indicating Ana at consistent sisters 42, in separated the aberrant, to bridges Furthermore, 36 severely chromosome 6F). from Fig was 3C; ranged MI count (Fig. content chromosome observed 40) the chromosome (totaling frequently when normal we even was and chromatids haspin sister 3C), excess (Fig. separated containing eggs hypercondensed in univalent. were chromosomes fewer one the or 3C), contrast, extra (Fig. one By rounded contained different typically more eggs was were the the II and aneuploidy haspin, Met inhibited at of we morphology types When chromosome groups. and experimental chromosome the II overexpressed, the between Met However, showed or at 6E,F). data (Fig. inhibited morphology aneuploid The was were number. haspin oocytes chromosome when determine that an performed to and II spread Met to oocytes matured with 6C,D). (Fig. compared attached improperly assessed haspin we overexpressing of 10% when Gfp oocytes than Similarly, Less in 6A,B). defects. attachments (Fig. these kinetochores) all sister at contained both attached to controls not attach were to 30% pole one and that from 5-Itu, (fibers come of attachments not nM abnormal did 500 displayed in kinetochores incubated of when 30% that found We kinetochores. tahet Blol n cide,21) hnAURKC When 2014). Schindler, kinetochore–microtubule and improper during (Balboula alignment destabilizing attachments chromosome through regulate I to Met required our is in AURKC aneuploidy control causing kinetochore–microtubule attachments perturbs cannot haspin of we Inhibition that H3pT3 procedure. of experimental amount precise ARTICLE RESEARCH 5074 and plate I Met the on aligned properly 27.78% not chromosomes misalignment dose had chromosome the as of 18.56% that percentage increased. found also the We increased, in spindle. alignment 5-Itu bipolar chromosome a of assess had to that I oocytes Met in the to oocytes 5-Itu matured of first is presence we localization the defects, meiotic arm aneuploid. these are AURKC-CPC with II altered associated Met whether to determine mature that To oocytes inhibited, is activity ial,t sestecneune fteedfcs we defects, these of consequences the assess to Finally, attachments of types the assess to assay an performed we Next, ijce otos ery5%o ieohrswere kinetochores of 55% nearly controls, -injected 6 0 foctsicbtdi 0 M5Iuhad 5-Itu nM 100 in incubated oocytes of 10% P , .5 n-a NV) s49.78% as ANOVA), one-way 0.05, 6 .8 (mean 3.68% 6 ... fcnrloocytes control of s.e.m.) nsitu in chromosome , 6 0 of 60% 7.95% Blol n cide,21) h aasgetta ICA- MI. that these during suggest process attachment Because data the AURKC AURKC- regulate the kinetochore-localized eggs. of CPC, not inhibition 2014), and II global AURKC-CPC, Schindler, localized of Met and that to in (Balboula similar are aneuploidy phenotypes to ultimately, ta. 99.BcueM novskeigsse chromatids sister plays keeping haspin that involves possible is it MI (Tanaka segregate, homologs cells Because while mitotic together 1999). with al., compared cells et meiotic in expressed oocytes. of mouse improper regulator in crucial to segregation a of is chromosome leads haspin MI persistence Therefore, 6). ultimately a (Fig. have eggs that aneuploid is oocytes attachments there when AURKC kinetochore–microtubule activity, 2014), of loss haspin Schindler, to perturbed Similar and for 5). (Balboula (Fig. required localization ICA function meiotic the is the regulates promote to activity haspin to AURKC-CPC later, required its of and is that 2C), haspin (Fig. First, find a resumption MI. define We of to steps function. and several its kinase haspin confirm for in to active report or role contain first haploid-cell- meiosis oocytes the in is mouse a This function that determined. its yet as 1999), not was al., identified oocytes et (Tanaka originally gene was specific haspin Although DISCUSSION eotdpeoye soitdwt osadgi fcondensin the of with gain and consistent loss The are with 3). associated overexpression (Fig. phenotypes and reported overexpressed inhibition following is observed haspin we as to haspin that twice phenotypes fails morphology and when II chromosome inhibited, and localizes is I haspin condensin much 2003). when al., both chromosomes et to to Ono common localize 2002; al., subunit (Hirano, et the Losada segregation SMC2, 2001; chromosome al., chromosome et establishing Kimura in regulating 2012; role and essential architecture 4). an 3, (Figs play to perturbed appeared was Condensins cohesion condensation chromatid chromosome sister but intact that be found we 2006), al., et the meet to MI. segregation signaling of chromosome needs meiosis-specific mitotic that data modify suggest controlled. molecules our is data AURKC-CPC targeting Therefore, these not that localized how 2013). Furthermore, of and arms al., importance is chromosome at et data the activity into (Rattani AURKC recent insight I provide is Met kinetochore-localized at hypothesis activated that this these with regulates demonstrating that, 5). (Fig. AURKC-CPC suggest Consistent ICA-localized data kinetochore attachments. These mitosis, the 6A). in at (Fig. unlike not inhibition localization haspin and found the persist following ICA, attachments We affects kinetochore–microtubule the 2010). improper specifically However, al., at haspin et AURKC Yamagishi al., of of et 2011; Wang inhibition 2010; al., al., that et et Wang 2009; Wang at al., (Dai 2012; et H3T3 Markaki attachments 2006; of kinetochore–microtubule al., is et phosphorylation regulate it where to mitosis, locale, 2012; this required In to al., localization AURKB 2009). et are drives Schindler at centromeres al., 2009) 2014; localization highly et Schindler, MI-specific al., more and a Shuda et also (Balboula has and ICA is Shuda cells the AURKC 2008; meiotic hypothesis. in al., and expressed this et (Balboula with localize Lee consistent 1A), 2014; in (Fig. meiosis-specific does I Schindler, Met that it other at ICA findings than the where Our along MI extends segregation. phosphorylation regulating H3T3 chromosome in mitotic roles regulating different or additional ti nrgigta apnmN smc oehighly more much is mRNA haspin that intriguing is It ncnrs oarpr nterl fhsi nmtss(Dai mitosis in haspin of role the on report a to contrast In ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal

Journal of Cell Science EERHARTICLE RESEARCH fSC oaiaini ietcneuneo oso localized of loss loss of the consequence that direct feasible a AURKC- Nakazawa is is prevents localization 2007; it haspin SMC2 chromosomes, al., of of to et inhibition localizing Lipp from that 2011; (Bembenek systems, CPC Given al., 2011). I other et al., condensin Collette et In of 2013; al., localization S1). after et the phosphorylation Fig. Therefore, regulates H3T3 material in AURKB in meiosis. loading increase (supplementary condensin during an in NEBD delay in found NEBD a We NEBD is I. meiosis. to there after Ana mitosis, prior until with loads axes compared chromosomes chromosome but with onto associate mitosis, loads not through II does centromeres Condensin at and restricted I is it Met meiosis prometaphase, oocyte in 2011). chromosomes in al., onto whereas et loads I (Lee condensin meiosis mitosis, and In the mitosis of chromosome between dynamics and differ onset, complexes spatiotemporal include anaphase the during Interestingly, phenotypes cohesin misalignment. of These loss resolution 2011). to and prior al., compaction individualization, et bivalent controlling (Lee respectively II, hra UK oaie oteside(abuaand (Balboula spindle in AURKB ICA, AURKC although the for the that compensate showed to and can previously We kinetochores localizes 2014). to Schindler, AURKB localizes However, (Wang 2010). AURKC whereas al., kinetochores et Yamagishi meiosis, 2011; to al., in et AURKB-CPC of Wang phosphorylation 2010; drives function al., mitosis, et In haspin biochemical 2009). by al., in et H3T3 Slattery and 2012; al., sequence these et in how similar (Ferna on highly light are shed are aneuploidy. Aurora-CPC will prevent meiosis to and studies and MI control condensin future mitosis complexes and between of I functions intriguing differences Met and condensin The in preventing localizations is chromosomes inhibition loading. haspin on that SMC2 likely II is of it Moreover, 3), loss loading. (Fig. condensin observe on H3pT3 we out of rule because loss point of this effect at direct cannot a we However, activity. AURKC-CPC amla oye oti ohARBadARC which AURKC, and AURKB both contain oocytes Mammalian ´ dzMrnae l,21;Ssie l,20;Schindler 2004; al., et Sasai 2011; al., et ndez-Miranda ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal BD] tH ethanol. EtOH, (B,D)]. [Student’s irijce ihbfe rhsi cRNA haspin or were buffer but with E, microinjected in Oocytes as (F) treated 5-Itu. were nM 500 of presence and matured isolated were oocytes vesicle-intact 10 in bars: outlined Scale area white. the of Magn., view A,B. magnified in as conducted fixation. was and Analysis medium cold to exposure h uniaiedt hwthe mean show data quantitative times. The three repeated were experiments All treatments. overexpression and 5-Itu between chromosome morphology in spread difference typical the a and demonstrate right the on images The by microscopy. detected epifluorescence were merge) kinetochores in and (red merge) in (blue fixation, DNA After chromosomes. and the spindle spread the collapse to to monastrol exposed were eggs after E,F, maturation, For maturation. to prior irijce ihbfe rhsi cRNA matured haspin and or buffer with microinjected isolated, were oocytes Prophase-I- arrested (C,D) were group. oocytes per 40 analyzed and oocyte were per kinetochores analyzed 12 least At per oocyte. kinetochore each of of types attachments the of Quantification (B) slices. optical and projections representative are confocal Shown by microscopy. detected were (blue) DNA and (red) kinetochores fixation. (green), and Microtubules to medium prior cold 5-Itu to nM exposure 500 of presence the and matured isolated were oocytes I-arrested aneuploidy. causes and alters attachments haspin kinetochore–microtubule of Perturbation 6. Fig. Aurkc 6 ... * s.e.m.; nvitro in nvitro in t ts FG,towyANOVA two-way (F,G), -test 2 nvitro in / 2 P oMtI 1 )i the in h) (16 II Met to oMtI( n )in h) 9 and h (7 I Met to , oye,ARChas AURKC oocytes, .5 **** 0.05; oMtI( )pirto prior h) (7 I Met to m .()Germinal- (E) m. AB Prophase- (A,B) P , 0.0001 5075 Z -

Journal of Cell Science C-oaie UK,adteeoctshv similar have oocytes these delocalizes similar and inhibition a of haspin have AURKC, AURKC, H3pT3 correction ICA-localized to and pattern haspin the localization Because and 2014). (Balboula wild-type MI regulating Schindler, during segregation and correct alignment ensure in include chromosome to 2012). attachments al., kinetochore–microtubule AURKB et functions improper Schindler 2014; from Schindler, These and (Balboula functions oocytes non-overlapping ARTICLE RESEARCH ninpls N eehroal rmdb nintraperitoneal Billerica, Laboratories, Millipore, an (EMD Harlan 5076 gonadotropin by equine weeks, primed of (6–8 IU hormonally 5 mice of were injection CF-1 IN) Indianapolis, mature maturation Sexually and microinjection collection, Oocyte expression (Life gene (Mm00494767_s1) for used haspin C were transcription. comparative for NY) reverse the and by Island, detection specific generated Grand was Technologies, probes cDNA hexamers purified random II, Taqman Using was Island, SuperScript protocol. mixtures Grand manufacturer’s the Technologies, and the (Life from to kit according RNA isolation NY) isolated total RNA were PicoPure and stages the sample processing. using indicated each before the to frozen at and added embryos mice or CF-1 oocytes from 50 of total A RT-PCR and synthesis cDNA to METHODS AND and MATERIALS function mitosis. AURKC and MI segregation localized between chromosome the on regulated how is in light regulating differences shed important for highlight to specificity are studies first these is summary, the In there AURKC. meiosis of population oocyte whether kinetochore in determine pathway of regulatory be to this will a regulating It investigate in 2010). of to participates al., interest also et loading (Yamagishi and localization the centromeres, AURKB-CPC at regulating T120 histone at for phosphorylates H2A BUB1 mitosis, specificity In AURKC. the to subpopulation Most defects. appears is phenotypic the haspin rescue intriguing not or localization by does ICA overexpression its H3T3 restore because loading of AURKC-CPC localization. phosphorylation regulate AURKC can maintain contrast, to AURKC By required of be histone Therefore, might overexpression defects. acetylation phenotypic case, and localization a this the attachments rescue misalignment, In kinetochore–microtubule chromosome aneuploidy. – improper and activity of AURKC are persistence of associated of histones phenotypes component loss hallmark oocytes, a with the these RBBP7, display for In and hyperacetylated depleted complexes. of oocytes deacetylase force in histone kinetochores driving ICA from main lost the is the and AURKC-CPC be instance, For to 2014). regulation. appear al., in et modifications Balboula roles 2014; Histone Schindler, the potential and (Balboula complex and determine is AURKs to 2013). polarity. the al., embryonic warranted for or haspin et egg haspin are regulating (Panigada the of cycle yeast, studies specificity cell budding further the In Therefore, with 1E). coordinate (Fig. polarization to distribution eggs actin cell regulate Alk2 II and actin Alk1 Met polarized homologs, the AURKC-CPC in at know haspin regulating localized cap yet detected in not we role of meiosis-specific Furthermore, do a AURKB. loss We has activity, haspin 2014). to whether AURKC Schindler, formally of and defects loss (Balboula the function with oocytes attribute of we those to phenotypes nAIPim70 Lf ehoois rn sad NY). Island, Grand Technologies, using (Life acquired 7000 were Prism Data ABI stages. an between levels expression in difference u tde eelta euaino UK-P localization AURKC-CPC of regulation that reveal studies Our t ehdwsue odtriethe determine to used was method Gfp RA( g was ng) (2 mRNA eo ibr,TeNtelns.Hsi RAwsijce t60ng/ 600 at injected (Qiagen, was kit cRNA m RNeasy Haspin an Netherlands). using The purified Limburg, was Veno, product final The NY). Island, vitro ietdMtgnsskt(gln ehoois at lr,C) After CA). with Clara, linearization Santa DNA Technologies, (Agilent kit Mutagenesis Directed oa (5 R an to eunig apnwstge ihGPa h -emnsand N-terminus the at (5 GFP K466 with C-terminus. the tagged at was SGOL2 Haspin sequencing. rvn eoi auain(sfiie l,19) l oye were oocytes All (polyvinylpyrrolidone) 1996). al., MEM/PVP et 37 at (Tsafriri in matured maturation injection, meiotic prevent after 2.5 h containing follicles 44–46 antral from isolated at were oocytes Germinal-vesicle-intact MA). rmcmecal vial lns(EHatcr,Ltl Chalfont, Little Healthcare, (GE clones available commercially and from haspin GFP-tagged generate To and overlay. and recovery avoid Cloning oil to an microinjection, chamber into water drugs to surrounding the a of prior with partitioning humid the kept a h were 1 as that 5-Itu. dishes used for in was 5-Itu (1:2000) were maturation oocytes in DMSO experiments, rescue the and For incubated experiments. experiments a CHR all as 5-Itu in used control CZB all was in (1:1000) in diluted ethanol 100% (5-Itu) control UK) respectively). Oxon, 5-iodotubercidin 1:2000, Abingdon, and of (a (1:1000 Therapeutics, (CHR) Chroma CHR-6494 presence or from #10010375) the gift MI; Arbor, in Ann Chemical, incubated (Cayman were Oocytes inhibition Haspin nsmlrydltdscnayatbd nahmdfe hme.Cells chamber. humidified a 5 in in mounted antibody hour were secondary additional primary an diluted in for incubation similarly incubated After and in chamber. washed v/v appropriate were humidified cells 0.01% a in solution, BSA, in antibody incubated w/v h then 0.3% 1 were (PBS, for solution Tween-20) oocytes blocking The in for min. diluted 0.3% 20 antibodies X-100, (PBS) Triton for v/v saline BSA) 0.1% (PBS, w/v permeabilization phosphate-buffered by followed using in min fixed 20 (PFA) were oocytes paraformaldehyde detection, pINCENP 2% and H3pT3 AURKC, For Immunocytochemistry pIVT- respectively), into 6833875, and 40054435 ligated IDs clone UK; Buckinghamshire, ntttoa nmlueadcr omte n eecnitn with consistent were and committee care guidelines. the and NIH by use approved plus were animal MEM experiments institutional in animal All performed CA). were Novato, Instruments, Injections 7– cRNA. with indicated 2.5 microinjected the were of Oocytes pl 10 medium. (CZB) Bavister Ziomek, ieTcnlge,GadIln,N;#258 t110 h following The or 1:100. #5063S at #322588) MA; NY; Beverly, MA; Island, Technology, Grand anti- Signaling Technologies, Beverly, 1:1000, Life (Cell Technology, at 488 Fluor anti-SMC2 Signaling Alexa 1:500, (Cell at anti-survivin PA) #2808S) 1:500, Pennsylvania, of at 1:1000, University PA) Schultz, anti- at Pennsylvania, Richard from 1:100, of gift University (a at (a anti-REC8 Lampson, anti-pINCENP #15-234) Michael 1:100, at from CA; #39153) gift CA; Davis, at Carlsbad, Motif, Inc., #A300-BL1217) (Active H3pT3 (Antibodies TX; CREST Montgomery, 1:30, Laboratories, 2010). al., (Bethyl et Chiang AURKC 2011; Schindler, and Stein 2011; al., Kapoor, and et Lampson previously Lee 2009; al., described 2005; et from Duncan as 2014; Schindler, performed (gift and (Balboula were (1:1000) assays microtubule detection cold-stable SMC2 Japan), RIKEN, parameters. performed Hirano, Tatsuya were above mounting to and according staining Permeabilization, respectively. 0 Sga tLus O o 0mno %PAi B t4 at PBS in PFA 4% of or min detection 20 X- for For Triton MO) PBS+0.1% Louis, MO). in St PFA (Sigma, Louis, 2% 100 using fixed St were cells Sigma, survivin, and (1:333, REC8 DAPI containing CA) and l h olwn nioiswr sdfrimnfursec:anti- immunofluorescence: for used were antibodies following The m irnn sn eook iia irijco (Sutter microinjector digital Xenoworks a using milrinone M sn nmesg McieT i Lf ehoois Grand Technologies, (Life kit T7 mMachine mMessage an using Sgol2 ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal 9 -AGA-3 RAa 0 ng/ 500 at cRNA nvitro in ˚ nahmdfe topeeo %CO 5% of atmosphere humidified a in C m irnn Sga tLus O M69 to #M4659) MO; Louis, St (Sigma, milrinone M m Gfp fVcahed(etrLbrtre,Burlingame, Laboratories, (Vector Vectashield of l 9 sn h ukhneLgtigMliSite- Multi Lightning QuikChange the using ) ytei fcRNA of synthesis nvitro In Nde Iaah ta. 07 n ofre by confirmed and 2007) al., et (Igarashi n uiiain RAwsgenerated was cRNA purification, and I m l. auainocre nognculture organ in occurred maturation nsitu in Sgol2 9 -AAA-3 liy hoooesrasand spreads chromosome ploidy, DA eePCR-amplified were cDNAs , 9 fhsi a mutated was haspin of ) a tblncnuae to conjugated -tubulin ˚ 2 overnight, C nChatot, in in

Journal of Cell Science emaiie o 0mn odplmrz h ci a,eg were eggs cap, actin and the fixed depolymerize previously To were that min. NY; 30 solution Island, Actin blocking for Grand of in Technologies, permeabilized drops/ml eggs (Life 2 anti-human-IgG reagent to adding Probes #R37112) by Ready detected goat 555 was Red cap Alexa-Fluor-633-conjugated actin anti-rabbit-IgG The (#A21091). and donkey anti- goat Alexa-Fluor-546-conjugated (#A10040) Alexa-Fluor-488-conjugated (#A11060), anti-mouse- dilution: rabbit IgG 1:200 Alexa-Fluor-546-conjugated NY) a (#A10680), Island, mouse-IgG (Grand at Technologies used Life and from were antibodies secondary ARTICLE RESEARCH eindadpromdeprmnsadwoetemnsrp.Alco-authors All manuscript. the K.S. manuscript. wrote experiments. the and analyzed corrected and experiments performed performed and J.O. and designed V.S. A.Z.B., A.S.G., A.L.N., contributions Author interests. competing no declare authors The interests Competing Fellmeth feedback. Jessica for thank antibody NJ) also SMC2 Piscataway, We University, the CHR-6494. (Rutgers for for Hirano antibody, Tatsuya Therapeutics pINCENP antibody, Chroma the REC8 and for the Lampson for Michael Schultz studies, Richard NEBD for the NJ) Piscataway, with University, H3pT3 assisting (Rutgers and Munoz RT-PCR Catalina the with immunofluorescence, Pennsylvania, assistance of for (University PA) Fram Philadelphia, Brianna acknowledge authors The Acknowledgements post-tests Tukey with legends, intervals. the confidence in 0.05% indicated and as ANOVA, Software two-way Graphpad or Prism CA). using Jolla, calculated (La was significance Statistical clear intensity analyses Statistical pixel no kinetochore. measure to to with kinetochore ImageJ from staining’, axes in chromatid function DNA the profiles’ along ‘diffuse ‘plot confirmed were the and assessments was using Qualitative localization. present; ICA ICA the or was at kinetochore along line clear and ICA no where kinetochores localization’, the in the ICA diffuse distribution’, at plus axis ‘kinetochore proteins localization as visible; plus clear characterized ‘kinetochore CPC-associated a were phenotypes: oocytes which of three I of without localization Met one pINCENP, having chromosomes the and pointy survivin dense assess distinctive AURKC, a and had To they short if appearance. ‘extended’ exhibited and distinguishable, bivalents, were they distinguishable bivalents if if analysis. ‘normal’ according ‘rounded’ parameters: scored alignment following qualitatively for the was Only used to 3A) 2012). (Fig. were al., morphology spindles et Chromosome (Lane the bipolar colleagues to exhibiting and according oocytes Lane scored by of was region described selected alignment parameters a Chromosome or from chromosomes chromosome. signal to the restricted intensity signal background pixel average average an an subtracting by determined Bethesda, (NIH, ImageJ Z individual using analysis, analyzed to Prior and MD). processed were images All analysis Image 20 a (Life System with Imaging NY) Auto Island, FL EVOS Grand an Technologies, NC) using Monroe, acquired One, were Bio images (Greiner and dish 96-well glass-bottomed a into 63 placed a with ploidy epifluorescencemicroscope 200M For Axiovert experiment. Zeiss an using obtained in were oocyte images each analyses, for constant kept was power laser Optical 1.5- 40 imaging. a for with used microscope was confocal objective laser-scanning Meta 510 Zeiss A Imaging fixation. to prior min 10 for #10010630) 10 with treated tg a etdt 37 to heated was stage poeto mg a sdfraayi.Pxlitniiswere intensities Pixel analysis. for used was image -projection m tp ihazo f2 hnsga neste eecompared, were intensities signal When 2. of zoom a with steps m P m vle eeotie sn Student’s using obtained were -values arnui Cya hmcl n ro,MI; Arbor, Ann Chemical, (Cayman A latrunculin M 6 ˚ n %CO 5% and C betv.Frlv-eliaig oye were oocytes imaging, live-cell For objective. Z lcswr is vradadtefinal the and overlaid first were slices 2 a maintained. was Z lcswr banduig1.0– using obtained were slices 6 betv.Temicroscope The objective. t ts rone-way or -test 6 r63 or 6 e,J,Ktjm,T . an,Y,Ysia . oia . iao . iae M. Miyake, T., Miyano, T., Morita, K., Yoshida, Y., Tanno, S., T. Kitajima, J., Lee, T. K. Jones, and Y. Yun, I., S. Lane, M. T. Kapoor, and A. M. Lampson, T. Hirano, and O. Cuvier, K., Kimura, el,A . hni,C,Xe .Z,Zeht . iua .adFnbk,H. Funabiki, and H. Kimura, C., Zierhut, Z., J. Xue, C., Ghenoiu, E., A. Kelly, H. Funabiki, and E. A. Kelly, J. C. Williams, and M. R. Schultz, G., J. Knott, H., Igarashi, ut .A n asl,T J. T. Hassold, and A. P. Hunt, ura,D,Slr . oeo . ilnea . atnz . ia,A., Vidal, A., Martinez, A., Villanueva, J., Moreto, M., Soler, D., Huertas, T. Hirano, amn,M,Welc,M,Fnbk,H n anhw .C. W. Earnshaw, and H. Funabiki, M., Wheelock, M., Carmena, K. J., Schindler, and Fuchs, Z. T., A. Balboula, Rutten, V., Schubert, M., A. Moghaddam, K., R. Ashtiyani, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.158840/-/DC1 online available material Supplementary material Supplementary months. 12 after [grant release funds. Health for start-up of PMC institutional Institutes in University National Deposited the Rutgers from and funding R00HD061657]; by number supported was work This Funding eAtn,A,Mfii . np,S,Mscho .adSnaud,S. Santaguida, and A. Musacchio, S., Knapp, S., Maffini, A., Antoni, De M. J. Higgins, and V. A. Kateneva, J., Dai, olte .S,Pty .L,Glneg . ebnk .N n Csankovszki, and N. J. Bembenek, N., Golenberg, L., A. E. M. Petty, S., Lampson, K. and Collette, M. R. Schultz, K., Schindler, E., F. Duncan, T., Chiang, Chan, and G. Csankovszki, J., Khanikar, J., K. Verbrugghe, N., J. Bembenek, K. Schindler, and M. R. Schultz, P., Stein, Z., A. Balboula, Ferna A. M. Lampson, and M. R. Schultz, T., Chiang, E., F. Duncan, M. J. Higgins, and A. B. Sullivan, J., Dai, M. J. Higgins, and S. S. Taylor, S., Sultan, J., Dai, M. J. Higgins, and J. Dai, ign,J M. J. Higgins, nmmainoctsadsmtccells. somatic and oocytes mammalian in Y. Watanabe, and tension. or microtubule-kinetochore alignment by bivalent predicted by 1955. is not oocytes but mouse attachment in activation complex attachments. chromosome-spindle regulates BubR1 extracts. egg Xenopus 5420. in complex condensin human 21) uvvnraspopoyae itn 3troie3t ciaethe activate to 3 threonine B. H3 Aurora histone kinase phosphorylated mitotic reads Survivin (2010). following view. behavior B-centric Aurora oscillatory an attachments: calcium change eggs mouse fertilization. in PLCbeta1 of 2183. 1408-1418. hrtn . oft . ae,S,MDrot .e al. et Haspin. J. kinase histone McDermott, the of S., inhibitor Patel, small-molecule a D., of activity Moffat, M., Charlton, e1002939. Sci. hoooa asne ope CC:fo ayrdrt h ofte of godfather the to rider easy from (CPC): mitosis. complex passenger chromosomal patterning embryonic to contributes and mitosis Arabidopsis. during in 3 threonine A. at Houben, H3 and histone R. F. Blattner, D., Demidov, el eelta pnl-oeitgiyi ioi eurscrmsm cohesion. chromosome Sci. requires mitosis Cell in J. integrity spindle-pole that reveal cells meiosis. of cause leading G. a is cohesion oocytes. in aneuploidy weakened age-related that Evidence (2010). embryos. elegans C. in bridges chromatin by induced in function C. CPC R. for deacetylation histone of requirement oocytes. a mouse unveils RBBP7 of oocytes. mouse in meiosis during Genet. kinase B PLoS aurora from functions distinct reveals htadfciesideasml hcpiti o h rmr as of Can cause S., primary Ortega, the B., not N. is Ghyselinck, checkpoint eggs. assembly mouse in spindle aneuploidy age-associated defective maternal a Aurora that of functions kinetochore the alters Haspin B. of inhibitor small-molecule A B. Aurora and Haspin metaphase by cohesion normal chromosome and phosphorylation 3 Thr H3 alignment. histone chromosome mitotic for required alignment. chromosome metaphase eae rtis itntv ru fekroi rti kinases. protein eukaryotic of group distinctive a proteins, development. related mammalian early during C 2661-2672. aurora for role M. Malumbres, ne-iad,G,Taaa . Martı M., Trakala, G., ´ndez-Miranda, .Cl Biol. Cell J. 21) ifrn oe o uoaBi odni agtn uigmtssand mitosis during targeting condensin in B Aurora for roles Different (2011). 60 21) odni n h pnl izn rvn yoiei failure cytokinesis prevent midzone spindle the and Condensin (2013). 446-462. , ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal a.Rv o.Cl Biol. Cell Mol. Rev. Nat. .Cl Sci. Cell J. 21) hoooetrioisme condensin. a meet territories Chromosome (2012). 122 e.Biol. Dev. 10 20) tutr,fnto n vlto fhsi n haspin- and haspin of evolution and function Structure, (2003). 199 4168-4176. , ln J. Plant e1004194. , elCycle Cell 21) eei irpino uoaBucvr nessential an uncovers B aurora of disruption Genetic (2011). 20) nfe oeo etoei rtcinb shugoshin by protection centromeric of mode Unified (2008). 269-284. , 124 312 68 3684-3694. , ee Dev. Genes Science 20) apn ioi itn iaerqie for required kinase histone mitotic a Haspin: (2005). 321-330. , 443-454. , 20) orcigaern ieohr microtubule kinetochore aberrant Correcting (2009). 13 20) e atr nmeiosis. in matters Sex (2002). 600-611. , 21) eetv irpino uoaCkinase C aurora of disruption Selective (2014). 13 330 20) h ua ioi hcpitprotein checkpoint mitotic human The (2005). ur Biol. Curr. 789-803. , elCycle Cell 235-239. , 19 mr,M,Pe M., amero, ˜ 20) hoooecnesto ya by condensation Chromosome (2001). 21) iigo anaphase-promoting of Timing (2012). ur pn elBiol. Cell Opin. Curr. 472-488. , a.Cl Biol. Cell Nat. n . soa,B,Gonza B., Escobar, J., ´n, 20) tde fhaspin-depleted of Studies (2009). 21) tapnphosphorylates AtHaspin (2011). 20 4 20) euaino mitotic of Regulation (2006). 1522-1528. , 665-668. , e.Cell Dev. 20) h iaehsi is haspin kinase The (2005). il Reprod. Biol. a.Cl Biol. Cell Nat. .Bo.Chem. Biol. J. Development rzd ato .and I. Castro, de ´rez ur Biol. Curr. 10 11 21) Knockdown (2014). 42-52. , Science Development 20) Alterations (2007). 21) Antitumor (2012). 21 741-750. , 20) Evidence (2009). LSGenet. PLoS 51-58. , Oncogene el o.Life Mol. Cell. 23 81 21) The (2012). 7 139 937-946. , 276 296 93-98. , 768-776. , lz A., ´lez, 1947- , (2012). 5417- , 2181- , 5077 138 31 8 , , ,

Journal of Cell Science aa,K,Ktym,H,Seoe,D . ui,S,Hna . iua M., Kimura, R., Honda, S., Fujii, L., D. Stenoien, H., Katayama, K., Sasai, L. B., C. Mayer, Rieder, S., Morrone, W., Helmhart, M., Ploquin, M., Wolna, A., M. Rattani, Bollen, and A. Eynde, Van M., Beullens, B., Lesage, J., Qian, aiaa . rat,P,Nsoi . ood,G,Csr,D . udi R., Quadri, G., D. Castro, G., Rotondo, A., Nespoli, P., Grianti, D., Panigada, n,T,Lsd,A,Hrn,M,Mes .P,Nuad .F n iao T. Hirano, and F. A. Neuwald, P., M. Myers, M., Hirano, A., Losada, T., Ono, and M. J. Higgins, W., Minor, J., P. Porebski, F., Wang, M. E., Yanagida, Niedzialkowska, and M. Ebe, R., D. Mehrotra, S. N., Georgatos, Nakazawa, and S. A. Politou, A., Christogianni, Y., Markaki, oaa . iao .adHrn,T. Hirano, and M. Hirano, A., Losada, M. J. Peters, and I. Poser, T., Hirota, J., J. F.,Lipp, Hanaoka, K., Kimura, K., Sugimoto, H., Matsuzaki, G., Sakashita, X., Li, T. Hirano, and M. Saitou, S., Ogushi, J., Lee, ARTICLE RESEARCH 5078 F., Villa, A., Chaikuad, M., E. Schoof, M., Medina-Redondo, de A., Maiolica, kn,Y,Ttua . uui . ig .A tal. et A. E. Nigg, F., Suzuki, M., Tatsuka, Y., Okano, during processes cells. cycle PtK1 metaphase cell essential oocytes. coordinates in I that al. meiosis et regulates A. platform Pendas, and O., regulatory Stemmann, W., T3 Xu, J., at Godwin, H3 targeting. histone B aurora mitotic chromosomal dephosphorylates Repo-man euae oaiyce eesr o ioi pnl oiinn n srequired is and M. arrest. positioning spindle mitotic Muzi-Falconi, mitotic tolerate for to and necessary P. cues polarity Plevani, regulates S., Piatti, 20) ifrnilcnrbtoso odni n odni It mitotic to II condensin and I cells. vertebrate condensin in of architecture chromosome contributions Differential (2003). centromeres. inner at paralogues Survivin 1457-1466. by tails H3 histone T. P. Stukenberg, until Top2. required from continuously distinct is mode Ark1 a that in kinase pattern telophase Aurora-B-like combinatorial the a by of phosphorylated part is chromatin. Thr3 mitotic configures at and H3 marks histone of Phosphorylation at,M,Jgnta,S,Lu .J,Nv,K,Hui .e al. et S. kinase. Hauri, protein K., Haspin Novy, the J., by Proteomics phosphoproteome H. Cell. chromatin Mol. Lou, the S., of Modulation Jeganathan, M., Gatti, the at compaction, condensin-mediated chromosomes. mitosis. for of not mitotic onset but with resolution, chromatid II sister condensin not but Sci. I Cell J. condensin of association passenger chromosomal T. novel Aurora-C. the Urano, protein, activates (INCENP) and protein centromere K. inner Furukawa, H., Taniguchi, oocytes. mouse in chromosomes Cell bivalent of construction for essential 22 3465-3477. , 120 18) h tutr ftecl-tbekntcoefbrin fiber kinetochore cold-stable the of structure The (1981). 1245-1255. , ee Dev. Genes .Bo.Chem. Biol. J. 21) oeua ai o hshseii eonto of recognition phosphospecific for basis Molecular (2012). eLife 13 Chromosoma 1724-1740. , e.Cell Dev. 2 e01133. , 16 3004-3016. , 279 ur Biol. Curr. 26 47201-47211. , 483-495. , 84 20) oei ees srqie for required is release Cohesin (2002). 145-158. , .Cl Sci. Cell J. .Cl Sci. Cell J. 21 Cell 21) odnisIadI are II and I Condensins (2011). 766-773. , 20) ietascainwith association Direct (2004). 21) es apnkinase haspin Yeast (2013). 20) uoaBcnrl the controls B Aurora (2007). 115 124 122 20) uoaCkns is kinase Aurora-C (2004). 109-121. , 21) gl rvdsa provides Sgol2 (2013). 1795-1807. , 2809-2819. , 21) Condensin (2011). o.Bo.Cell Biol. Mol. 21) PP1/ (2011). o.Biol. Mol. (2009). (2014). 23 , cide,K,Dvdno . rm . apo,M .adShlz .M. R. Schultz, and A. M. Lampson, B., Fram, O., Davydenko, K., Schindler, aaih,Y,Hna . an,Y n aaae Y. Watanabe, and Y. Tanno, T., Honda, Y., Gorbsky, Yamagishi, V., A. Kateneva, D., Patnaik, R., J. Daum, P., N. and Ulyanova, M. F., S. Wang, Lens, D., Patnaik, S., M. Waal, der van P., N. Ulyanova, F., Wang, ag . a,J,Du,J . idilosa . aeje . Stukenberg, B., Banerjee, E., Niedzialkowska, R., J. Daum, J., Dai, F., Wang, sn,T . hn .H,Hu .P n ag .K. T. Tang, and P. Y. Hsu, H., S. Chen, C., T. Tseng, M. Conti, and J. A. Hsueh, R., Zhang, Y., S. Chun, A., Tsafriri, Y., Tosaka, J., Tsuchida, K., Yomogida, M., Nozaki, Y., Yoshimura, H., Tanaka, ti,P n cide,K. Schindler, and P. M. Stein, R. Hall, and R. B. Brinkley, J. A., P. M. Donovan, Mancini, D., and S. M. Slattery, R. Schultz, J., Ma, K., Schindler, K., Shuda, Glover, E., Laue, H., S. McLaughlin, K., Lykke-Hartmann, J., Na, B., Sharif, 21) aenlyrcutdArr iaei oesal hnArr to B Aurora than stable A more development. S. early is U. and kinase maturation oocyte C mouse Aurora support recruited Maternally (2012). ak sals h ne etoeeadcrmsm bi-orientation. chromosome and centromere 330 inner the establish marks segregation. chromosome in B M. Aurora J. of Higgins, and J. G. mitosis. in centromeres at 1069. accumulation CPC promotes M. J. Higgins, oe hoooa asne rti htcncmlmn uoaBkinase Aurora-B complement cells. can mitotic that in protein function passenger chromosomal novel a hshrlto yHsi oiin uoaBa etoee nmitosis. in centromeres at B Aurora positions Haspin by phosphorylation P.T.,Gorbsky,G.J.andHiggins,J.M. Science fsemadeg:coigadcaatrzto ftonvltestis-specific segregation chromosome novel fly two and of yeast selective to characterization related and using regulators. AIE2) cloning (AIE1, studies kinases eggs: protein and cells: sperm germ of cAMP and of somatic changes inhibitors. follicular opposing phosphodiesterase in and compartmentalization levels involves maturation cells. somatic on (Haspin) effects kinase its protein 17057. and nuclear nuclei cell-specific spermatid germ al. in et haploid H. a Nojima, of M., characterization Okada, T., Nakanishi, T., Habu, liyassessment. ploidy 2986-2997. oocytes. mouse in alignment chromosome Dev. modulates Reprod. B kinase of Aurora meiosis in cytokinesis and transmission oocytes. chromosome mouse of fidelity for M. required Zernicka-Goetz, and M. D. iaespot ioi rgeso nteasneo Aurora-B. of absence the in progression mitotic supports kinase 239-243. , ora fCl cec 21)17 0657 doi:10.1242/jcs.158840 5066–5078 127, (2014) Science Cell of Journal . 330 109 N elBiol. Cell DNA 231-235. , E2215-E2222. , 76 21) oiiefebc opivligHsi n uoaB Aurora and Haspin involving loop feedback positive A (2011). .Cl Sci. Cell J. 1094-1105. , .Vs Exp. Vis. J. elMtl Cytoskeleton Motil. Cell 21) apnihbtr eelcnrmrcfunctions centromeric reveal inhibitors Haspin (2012). 21) os oyemconeto,mtrto and maturation microinjection, oocyte Mouse (2011). 17 123 823-833. , e.Biol. Dev. 21) h hoooepsegrcmlxis complex passenger chromosome The (2010). 4292-4300. , 53 2851. , 178 .Cl Biol. Cell J. 393-402. , 59 19) rti iaeprofile kinase Protein (1998). 21) itn 3Thr-3 H3 Histone (2010). 249-263. , .Bo.Chem. Biol. J. 199 19) dniiainand Identification (1999). rc al cd Sci. Acad. Natl. Proc. ur Biol. Curr. 251-268. , 21) w histone Two (2010). 20) Aurora-C (2009). 19) Oocyte (1996). elCycle Cell 274 21 17049- , Science 1061- , (2009). Mol. 8 ,

Journal of Cell Science