Journal of Cell Science uaieACsbtae,arr n oolk iae,as hwn vdneo bqiiain hsi h is xml fmtssnot regulation. mitosis cycle of cell example of motif. first form degradation the ancient yeast is fission evolutionary This a ubiquitination. an of of reflect evidence addition might no the and show by APC also suppressed the kinases, is by polo-like of toxicity and regulated Expression this aurora and conjugation. substrates, APC arrest, in ubiquitin mitosis putative prophase and during a APC degraded is to the B leading by Cyclin toxic, mediated is degradation yeast timely fission for required motif N-terminal ihol adu fpoen dniid(awe ta. 2007; al., et (Lauwaet identified proteins of handful a only with 2006). al., et a several (Ciccarelli into if group split POD or then the first including 2010) diverged groups al., Excavates, the et termed last (Fritz-Laylin group Oxymonads the ancestor from the directly eukaryotic diverged and that common are group), Parabasalids POD Diplomonads the the the as including to if group, (referred unclear a remains study of the It for part biology. interest considerable cell of basic thus of and Fritz- 2010) 2006; al., al., et et basal Laylin and (Ciccarelli most eukaryotes the of distant maladsorption one evolutionary considered 2007).and Diplomonads, in the al., of et member resulting a Morrison is 2001; mammals (Adam, disease of diarrhoeal intestine (Eme small lineages study). Parabasalid this and and 2011 in Diplomonad al., present or the et is recognizable in APC not and are lacking an necessary components Although are its 2001). be lineages al., eukaryotic to et most Geley (Yamano shown 1998; ubiquitination for been al., APC et the have N- by the recognition B for in sufficient cyclin D-boxes as al., of et such Yu terminus sequences is 1995; al., conserved B anaphase-promoting et Loosely King the Cyclin 1994; 1996). of al., onset by et 1990). (Hershko the conjugation (APC) al., to complex ubiquitin prior et proteasome by Whitfield the anaphase by 1989; degradation al., for targeted et cyclin of (Draetta degradation is 1989; including Cdk1 B al., a of mechanisms Regulation several et form 1989). by al., (Brizuela B, et achieved Riabowol mitosis cyclin 1989; into al., partner, et entry Draetta binding by for its part essential and in complex 1) (cyclin- mediated Cdk1 kinase is kinases. dependent protein cycle by cell orchestrated cascades eukaryotic signaling the of Regulation Introduction words: Key study, present the In components. (APC) in complex cyclin anaphase-promoting The mitotic all domain. single lacks eukaryotic a the genome that of its show part components, we small regulatory very cycle a cell representing many organisms has model pathogen in human conducted been divergent has highly research regulation cycle cell Most Summary 10.1242/jcs.121632 doi: 2246–2255 126, ß Science Cell of Journal USA 2013 March 94720, 4 CA Accepted Berkeley, California, of ( University correspondence Biology, for Cell *Author and Molecular of Department Ste complex anaphase-promoting The 2246 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. The intestinalis Giardia paeGugehn,La .Hl n .ZcesCande Zacheus W. and Holt J. Liam Gourguechon*, ´phane Giardia Giardia npaepooigcmlx P,ClclnA ylnB, Cyclin A, Calyculin APC, complex, Anaphase-promoting elccei orydfnda h oeua level molecular the at defined poorly is cycle cell [email protected] saprstcpooonta ooie the colonizes that protozoon parasitic a is elcceporse neednl fthe of independently progresses cycle cell iri intestinalis Giardia Giardia Giardia el,btti erdto per ob needn fteuiutnto aha.Other pathway. ubiquitination the of independent be to appears degradation this but cells, sesnilfrporsinit ioi.Strikingly, mitosis. into progression for essential is ) sielfrsuyn h osraino uaytcptwy.Although pathways. eukaryotic of conservation the studying for ideal is Giardia Giardia Results regulatory different eukaryote. divergent uses highly that this in cycle an mechanisms cell highlight cell findings distinct other Our with phosphorylation. evolutionarily interactions by and by proteins regulated cycle presumably is In progresses B APC. naturally an cyclin without that the cycle organism is cell the eukaryotic this in through 2003) a telophase Toczyski, of and to example (Thornton first APC progress an and of absence anaphase the complete to engineered erdto ncnrs oalmttccciscaatrzdto characterized cyclins mitotic all Though to date. contrast in al., degradation et (Gallego system conjugation 2007), ubiquitin active progression an for and 2003), required is Although divergent, mitosis. into highly although B, eukaryotes. cyclin then other to in and similar cycle phases cell 2006), M the and Thus, two G2 al., 2006). by (Nohy al., cell cytokinesis et et the Sagolla to concurrently of prior Sagolla poles spindles, replicated opposite separate 2001; are to cell segregated Each al., that simultaneously similarity. et nuclei sequence of diploid (Bernander basis two the for candidates on contains as cyclin identified been mitotic have a genes several By 2008), 2008). al., al., et et Reiner 2008; al., the et mining Davids 2007; al., et Morrison n oanhmlg oBtp ioi yln Fg 1A; (Fig. cyclins mitotic B-type to domains) homology box cyclin domain the in in similarity and (53% cyclins sequence candidates, to limited these has homologs of one cyclin shows organisms putative other of alignment Sequence Giardia ntepeetsuy eso htoeo hs cyclins, these of one that show we study, present the In ioi,Staurosporine Mitosis, , Giardia ylnBlcsadgaainmotif degradation a lacks B cyclin Giardia ylnBi o euae yubiquitin-mediated by regulated not is B cyclin acooye cerevisiae Sacchoromyces eoedtbs o ylnhmlg (Reiner homologs cyclin for database genome Giardia Giardia a rtaoe(agme al., et (Paugam proteasome a has ylnBlcsteconserved the lacks B cyclin Giardia eerhArticle Research ´ Giardia tan aebeen have strains kv ta. 2000; al., et nkova Giardia Giardia erdto of degradation , ylnBin B cyclin a 1 S, G1, has ylnB, cyclin Giardia Giardia Journal of Cell Science ylnBcudntspotfsinyatgot tterestrictive the at 1986). growth yeast al., fission et support not (Marks single could the cdc13p B cyclin of cyclin mutant mitotic sensitive essential temperature a complement yeast could expression fission in 3HA function cyclin. B mitotic a cyclin is suggest and of data Cdk1 these with absence associates together, the Taken 2B). in (Fig. activity observed histone immunoprecipitated was No 2B). (Fig. the activity kinase ATP, purified and with H1 incubation histone and immunoprecipitation after addition Cc)wr lsiidit h aeoisotie nDadtefeunyof frequency the and D in plotted. outlined category categories each the into or classified (control) were protein (CycB) exogenous no expressing ( experiment) subcategories. mitotic into 2 categorized bars: Scale staining. DAPI oimnpeiiae ihCk in Cdk1 that with demonstrated co-immunoprecipitates We epitope. B Myc triple a with yeyatadyatoverexpressing yeast and yeast (+ type cdc13 yeast of terminus yeast), (Wt protein exogenous (+ no expressing yeast of assay ihatil A(ealtnneioe a and tag epitope) (hemaglutinin HA tagged triple We a S1). with Fig. material supplementary ( indicated. are motifs degradation and boxes Cyclin eukaryotes. ( yeast). wild-type degradation. of Toxicity 1. Fig. Gi odtriei hsgn a ioi ylnw sae its assayed we cyclin mitotic a was gene this if determine To yB or cycB) A ceai ftesqec lgmn fcci rmvarious from B cyclin of alignment sequence the of Schematic ) .pombe S. Giardia Giardia Giardia el.Epyvco a sda eaiecnrl(Wt control negative as used was vector Empty cells. ylnB(ceso,G58337)wsepesdin expressed was GL50803_3977) (Accession, B cyclin ylnBfsdwt h erdto inlfo h N- the from signal degradation the with fused B cyclin Gi ylnBi es slne oislc of lack its to linked is yeast in B cyclin m yBNd1) ( cycB/Ncdc13). .( m. Giardia .pombe S. D Giardia es xrsigGPtblnwere GFP-tubulin expressing Yeast ) E ylnBCk hw histone shows B/Cdk1 cyclin es el ( cells Yeast ) ydtriigwehrit whether determining by ylnBeaie yDCand DIC by examined B cyclin C opooyo h wild- the of Morphology ) Giardia Giardia Giardia , 0 cells/ 500 Giardia Giardia Giardia Giardia Fg A.In 2A). (Fig. B ylnB cyclin ylnB cyclin Growth ) ylnB cyclin Giardia ylnB cyclin cyclin Cdk1 oyatcc ( cdc2 of binding yeast detect to able to not were may We differences between cyclins. These differences to metaphase. in due before different be slightly arrested are are yeast cells fission progression that in anaphase when overexpressed observe of is we B inhibition phenotypes cyclin The to 1996). inhibiting leads al., yeast et but (Yamano form in non-degradable B replication a cyclin of DNA of Expression anaphase. allowing into yeast, progression postulate in we the 1C) expressed (Fig. days in 2–3 after that of increase segregate expression not but upon marked replicate observed each was A and cells Giardia 1D) prophase 1E). Fig. (Fig. of in shown percentage quantified as visible, and was category (where anaphase spindle or spindles no mitotic metaphase and (where the seen) cytokinesis mitotic be or could spindle prophase visualize mitotic interphase, into strain to cells a chromosomes classify used GFP–tubulin, We suggest segregate. properly results expressing to failed these but replicated 1C); Yeast were (Fig. 1B). nuclei (Fig. strains enlarged yeast wild-type expressing in cells growth cell affected of expression However temperature. ugsigta h ylnCkitrcini eko that nor or as weak such brucei eukaryotes is divergent Though interaction experiment. cyclin–Cdk the Giardia that suggesting nefr ihisfnto,tu asn h ioi lc we co- block mitotic the by and causing the Rum1, thus inhibitor regulates proteins function, Cdk1 that its detect the protein with as two cycle not interfere such cell do complex, yeast these Cdc2/Cdc13 we another cyclin–Cdc2 to since between the binding affinity Alternatively, However, interaction immunoprecipitation. low degraded. be but be an must progression in cannot mitotic decrease interaction for a B because required arrest cyclin is would activity cells scenario Cdc2 this In Cdc2. Giardia that hypothesis the test To 1A). (Fig. to group rwho as n hne nyatcl morphology cell the yeast for account in to possibilities changes by several caused effects are any toxic There cause cell 1B,C). yeast (Fig. inhibit or not did motif yeast, growth yeast, fission degradation in the expressed N-terminal fission when of protein, cdc13p upstream in the inserted was expressed toxicity, to when leading improperly accumulating oivsiaetefnto of function the investigate To in progression B cyclin of Depletion conclusion, In observe. hspplto vrg ersnsasbe fclswt very depletion. with no cells with cells of some and subset 3D) a (Fig. depletion represents significant average population this levels the reduced Morpholino efficiently mRNA of 2009). B cyclin Cande, the against and directed (Carpenter gene knockdown morpholino-mediated and specific 2011) Cande, and (Gourguechon Giardia in expressed when progression mitotic yeast. inhibit fission to it causes motif Giardia rcooa vaginalis Trichomonas Giardia eana -emnldgaainmtf neither motif, degradation N-terminal an retain ylnB ic u eut ugs hoooe can chromosomes suggest results our Since B. cyclin ylnBcudb idn n reesbyactivating irreversibly and binding be could B cyclin ylnBi prtn neednl fcc nthis in cdc2 of independently operating is B cyclin elln ariga3Atge leeo ylnB cyclin of allele tagged 3HA a carrying line cell Giardia P-needn ioi in mitosis APC-independent ylnBto B cyclin ylnBat sadmnn eaiemtn when mutant negative dominant a as acts B cyclin Giardia pert aeNtria erdto motifs degradation N-terminal have to appear , .pombe S. , Giardia Giardia 0 fe 4hus(i.3,) where 3A,B), (Fig. hours 24 after 40% ylnBwr lnae,wt notably with elongated, were B cyclin ebro h aaaais sister a Parabasalids, the of member a , Giardia Giardia d1 yimmunoprecipitation by Cdk1) Giardia ylnBepeso in expression B cyclin ylnBslc fadegradation a of lack B’s cyclin Giardia mar elcycle cell impairs Giardia Giardia n iso es mitotic yeast fission and ylnB hschimeric This B. cyclin iri ylnB cyclin Giardia ylnBw sda used we B cyclin Giardia ylnBadversely B cyclin ylnBcudbe could B cyclin Giardia Trypanosoma .pombe S. ylnB cyclin Giardia Giardia Giardia 2247 was . Journal of Cell Science uiidb muorcptto sbfr,te nuae ihhsoeH1 histone with incubated then [ before, and as immunoprecipitation by purified ape eeas tie ihCoaseBu ovrf qa odn of loading equal verify to Blue Coomassie with histones. stained also were Samples grs Sga o or t4 at hours anti-HA with 2 for incubated (Sigma) were were agarose Lysates promoters conditions. native non-denaturing their under under lysed indicated) cells (as or only GL50803_8037) Cdk1-3Myc (Accession, expressing Cdk1-3Myc and B-3HA cyclin on otebas P muorcpttdfato.( not fraction. fraction immunoprecipitated Sup, IP, to lysate; beads; cleared blotting the CL, western respectively. to anti-HA B, bound and cyclin or anti-Myc Cdk1 by for analyzed probe and buffer sample in aeilFg 2 ioi el.Orrslsin results Our cell). (supplementary they mitotic cell as S2, the nuclei of Fig. poles material of opposite pairs towards two segregated the are surrounding revealed spindles immunostaining mitotic tubulin two anaphase, In formed through cells). control never proceeding in in 2% cells spindles to no depleted compared 3E). control S2, cells, (Fig. B Fig. B material to cyclin cyclin (supplementary morpholino Finally spindles of compared 3E). devoid mismatch (Fig. entirely 3D) signal (Fig. were the cells the enlarged large-sized with 10 Additionally, of were transfected count sets B cells could two cyclin we to of cells, corresponding more certain nuclei, were replicated cells per In depleted morphological chromosomes 3D). B (Fig. notable cyclin condensed in caused chromosomes changes; signal) immunofluorescence mitotic 1994). of al., indicator et an (Westendorf thus is progression and mitosis (phosphoT-P-L-K/Q) during substrates phosphorylated recognizes bands antibody MPM2 positive The MPM2-antibody by mitosis-specific, 3B) (Fig. of levels normal at the on growing effect no cells had oligos unaffected Giardia morpholino and mismatched contrast, grow, cyclin In of not rates. depleted do totally 3C); cells which (Fig. of 60% B, population by mixed number a cell reflects the this reduced B cyclin of Depletion in interact Cdk1 and B Cyclin 2. Fig. 2248 elto fcci a ugdb osof loss by judged (as B cyclin of Depletion 32 ]AP aeigo itn a iulzdb uoaigah ( autoradiography by visualized was histone of Labeling P]-ATP. ylnBlvl.Dpeino ylnBlvl loreduced also levels B cyclin of Depletion levels. B cyclin ora fCl cec 2 (10) 126 Science Cell of Journal Giardia , 0 oprdwt h imthoiocontrol. oligo mismatch the with compared 60% hoooe.Scnl,clsdepleted cells Secondly, chromosomes. ˚ ,wse xesvl ihPSte boiled then PBS with extensively washed C, Giardia cells. Giardia B Giardia ylnB3H was 3-HA B Cyclin ) ( A el expressing Cells ) el htwere that cells r similar are . 1,000 32 P). uigtecl yl na snhooscl ouain As population. levels cell B asynchronous cyclin an track in cycle to 4B). (Fig. cell us staining content the allowed 3HA DNA during B analysis nuclear cyclin of single-cell basis for the This quantified on then G2/ cycle or 4A), cell S (Fig. the G1, program into of categorized CellProfiler phases B were M Cells the cyclin 2006). al., and individual for et each (Carpenter immunofluorescence cycle, assay for levels using cell cytometry DNA and the cell, cell protein during measuring a by levels levels, performed B we were cyclin We therefore the behavior. release in measure cyclin and entry accurately typical synchronize mitotic showing to thus for unable cycle, essential cell is the B cyclin Giardia that shown the during Having vary levels B Cyclin rmtr(lpyB3A,cci eesoclae during oscillated tetracycline-inducible levels B exogenous, cyclin an 3HA), contrast, (NlopCycB using In activity. promoter promoter expressed B cyclin or in when S change G1, that little difference in suggesting was quantified levels S4A,D), notable there Fig. GFP no material in (supplementary G2/M) content observed cells and transcriptional G2/M we G1 the 2008), between GFP change at al., (8.4% regulated previous et their was a (Reiner B to level contrast cyclin and In suggesting to S4A,C). according report and content Fig. G2/M and material (GFP) S (supplementary DNA G1, protein open cytometry. into by classified their B fluorescent content were DNA cells cyclin green versus Again, fluorescence the with GFP analyzed replaced frame we reading differences transcriptional degradation and/or cyclin a synthesis suggests which protein and in growth of cell rates. model control to a due temporal dilution with tight by inconsistent reduced decrease are is fivefold levels of to levels four- amounts sudden cyclin visible This shows S3), in cytoplasm. which Fig. the G2, in material in B cell (supplementary cyclin cytokinesis a staining to or B compared cyclin when anaphase showed no generally in or population) cell little cells total the model, of 1–2% mitosis. (representing this during low degraded to with is returning B we Consistent cyclin and suggesting Thus, G2/M, G1, cycle, in before cells. cell levels persisting the G1 during S-phase, oscillate in in B cyclin increasing observed of levels levels B the that levels cyclin to conclude for cells cells a compared staining B showed phase in cells) 4D) G1 total respectively, cyclin (Fig. S of increase, (42% fivefold Both 4D). low cells and (Fig. G2/M 4B,C). four- and had (Fig. cells) cells total levels cells) of (43% background untransfected total to expressing of as similar 3HA 15% B well cyclin wild-type, (representing G2/M as and was in of staining S levels B staining cells, G1, cyclin background for of HA determined value define mean 3 then The to 4C). B al., used (Fig. et cyclin fluorescence were Reiner of 2008; cells al., phase levels untransfected et G2/M (Poxleitner The the cycle in cell 2008). the were of cells 4A) the (Fig. of most described, previously 99.Tu ecnld htdpeino ylnBprevents B cyclin enlargement. cell of and that depletion al., condensation et that is Riabowol conclude 1988; Giardia exception we Beach, and Thus only (Booher 1989). depleted in is the occurs B cyclin anaphase; still condensation and chromosome progression and metaphase mitotic spindle mitotic of into the depletion of where formation organisms, prevents most cyclins in observed those to odsrmnt ewe rncitoa n post- and transcriptional between discriminate To ewne oko hte ylnBlvl ayduring vary levels B cyclin whether know to wanted we , el rmetrn ioi n asschromosome causes and mitosis entering from cells Giardia Giardia elcycle cell Giardia el ufcetyto sufficiently cells vnwhen even Journal of Cell Science P opnnsi utpeekroi iegsso that show lineages of eukaryotic analysis multiple phylogenomic in Giardia and 1999; Apc1-11, components al., as et APC (Fang such APC 2006) the Exhaustive of Peters, Securin. components conserved and highly polo-like the (AUK) the the kinase as of mining aurora the such for components (PLK), responsible regulatory kinase is other APC of the turnover degradation, Geley B as 1996; cyclin such al., mediating protozoa and et 1998), (Yu al., primary of et brucei animals (Yamano in yeast degradation in 2001), observed al., been et and observed has B ubiquitination divergence cyclin APC-mediated considerable sequence, the Despite is B Giardia cyclin of regulation material that (supplementary suggest 3 post-transcriptional. results Fig. predominantly G1 The in in levels S4B,D). observed B higher threefold cyclin that Fig. and of to cells levels cells) S (low in G2/M similar levels pattern higher 2.5-fold a cells, in cycle cell the ewr nyal oietf w P uuis(o1adCdh1) and (Doc1 Moreover, subunits 2011). APC two al., identify in et to able (Eme only unrecognizable were we be to as diverged rcooa vaginalis Trichomonas Hmatne l,20;L ta. 03.I diinto addition In 2003). al., et Li 2003; al., et (Hammarton and ylnBi o ubiquitinated not is B cyclin Trichomonas Giardia eoedtbs aldt dniyayof any identify to failed database genome ihrlc nACo ti ohighly so is it or APC an lack either u ecudietf l the all identify could we but , T. fe diino G6 n ol o eetayhigher any also detect we not could experiment, and AUK MG262 separate and of PLK a B, addition cyclin after In 3HA-tagged the 5A). immunoprecipitated (Fig. poly-ubiquitinated or of PLK enrichment proteins B, considerable cyclin the of despite versions not AUK, weight our did molecular also of higher We enrichment AUK). any PLK, detect any B, observe (cyclin substrates not APC candidate did we proteins, conjugated which aphidicolin, cell of two cell 5B). halted addition (about (Fig. persistently growth and contrast, hours immediately 24 replication, In DNA after blocks 5B). of halt toxic Fig. immediately became accumulation matrix not cycles, but did the affinity growth, it promoted cell 5A), ubiquitin-specific (Fig. were MG262 bands a proteins poly-ubiquitinated Although the Poly-ubiquitinated using 5A). of (Fig. eukaryotes. substrates enriched are other kinase which further Aurora in (PLK) B, kinase cyclin APC Polo-like tagged poly-ubiquitinated 3HA or of carrying (AUK) enrichment cells promote in inhibitor to proteins proteasomal the used hypotheses, was these MG262 test To cyclin APC. that the and possibility the raised This Bin 2005). Wang, in and subunits (Kumar APC characterized previously pnadto fM22adprfcto fubiquitin- of purification and MG262 of addition Upon Giardia P-needn ioi in mitosis APC-independent sdt eiebcgon B)signal. (BK) a were background as proteins define plotted 3HA-tagged to categories any used cell carrying these not of Cells each histogram. in signal B cyclin ( size large of and size the cells (reflecting into size subdivided normal further two of were the Cells for shown. size is the cell populations using of determined Histogram were program. morpholino CellProfiler B cyclin with treated ioi;atnwsue slaigcnrl ( control. loading as used was into entry actin quantify mitosis; to used was mitosis, proteins during recognizes phosphorylated which indicated. antibody, as used, MPM2 antibodies The (Li-Cor); the software to and correspond scanner pseudocolors and Odissey visualized an was using signal quantified The by blotting. analyzed western after and quantitative hours harvested 24 were control. cells negative transfection, a mismatched as or (mismatch) morpholinos morpholino B anti-cyclin with transfected n iewsosre hncci eeswr reduced. were levels B 2 cyclin bars: morphology when Scale cell observed unusual was An size cells. and knockdown B cyclin time. and against plotted results and ( hours 8 plotted. every and monitored signal actin the against ( normalized were A from i.3 elto fcci in B cyclin of Depletion 3. Fig. srgltdidpnetyo bqii conjugation ubiquitin of independently regulated is D C oaiaino ylnBi oto utasetdcells) (untransfected control in B cyclin of Localization ) was cells cyclin-B-depleted and control of Growth ) m .( m. , %ocrec nnra el)adthe and cells) normal in occurrence 1% E ie ftecnrlclsadcells and cells control the of Sizes ) Giardia . rpnsm brucei Trypanosoma 9 futetdcells) untreated of 99% Giardia . ( A B el were Cells ) h signals The ) 2249 Journal of Cell Science hs ihannfursetlblt eemn h ea aeof rate decay the determine to label allow non-fluorescent to a 2005) with chase Hehl, and (Regoes tag vivo SNAP the with protein akrudlvl n aawsaaye si .( types. B. cell in indicated as all analyzed of was fluorescence data and levels background el sdlydatr2 or,teei oimdaemitotic 6A). 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Fig. 2250 oivsiaetesaiiyof stability the investigate To aeigo h rti ihfursen olwdb pulse by followed fluorescein, with protein the of labeling ora fCl cec 2 (10) 126 Science Cell of Journal C el o rnfce eeue odefine to used were transfected not Cells ) Giardia Giardia B h aepplto sAwas A as population same The ) vntog rwhof growth though even , Giardia ylnBw agdthis tagged we B cyclin D envle for values Mean ) elcycle. cell .brucei T. 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(Fig. A respectively to hours half-life calyculin B its 38.7 increasing and cyclin and degradation, B 23.4 staurosporine the cyclin we of Using rate both the Thus, exit. reduced described, normal. mitotic assay was inhibits stability mitosis, A size entering calyculin unlike from cell cells though, whereas 6A), the prevents (Fig. 3D), cells staurosporine cells, (Fig. that cells these conclude chromatin depleted depleted B Additionally, B condensed cyclin cyclin 6C). the (Fig. showed to similar specific blot (38%) MPM2 of western loss frequently their the with by and by 6B) treated 3% signal determined Fig. to compared cells, spindles, cells as DMSO-treated with cells in mitosis, (no contrast, cells spindles enter form of to In inability to (25% 6B). failed spindles observed cycle Fig. mitotic staurosporine were cell spindles, multiple cells on with showing some multinucleated or 6A), and many morphology (Fig. mitosis inhibit since cell to appears discernible cytokinesis, A on Calyculin their no 6A). hours characterize (Fig. had 15 progression further MG262 after to inhibitor effect proteasomal actin The and effects. tubulin the of or appreciable localization any these roscovitine have on inhibitor not both did effects specific acid Although okadaic Cdk/MAPK inhibitor inhibitors, phosphatase the addition. specific more as cell of other such specificity, affected broad hours adversely 24 have inhibitors A within calyculin and staurosporine growth inhibitor inhibitor phosphatase kinase The the phosphorylation. of role ots hte ayui n tuoprn stabilize staurosporine and A calyculin whether test To Giardia ylnBb leigispopoyain we phosphorylation, its altering by B cyclin Giardia Giardia ylnBi erdddrn ioi ya P and APC an by mitosis during degraded is B cyclin Giardia Giardia el.Du-rae el eesandfor stained were cells Drug-treated cells. ylnBi cieydgae,rte than rather degraded, actively is B cyclin ylnBi euae by regulated is B cyclin ylnB(i.5,) u aaalso data Our 5C,D). (Fig. B cyclin Giardia a oimdaeeffect immediate no has Journal of Cell Science -/ oi.I suceri hs rtisbn occi B cyclin to bind proteins these if unclear T-P- antibody is a MPM2 It to correspond the motif. not with using do x-K/R phosphopeptides stained are the correlates that be drug B suggesting could phosphorylation either cyclin bands interacting by with this of stability interact unaffected increased and that is proteins phosphorylated residues, Several threonine data treatment. of we Our on phosphorylation A degradation. the calyculin B least predict of that cyclin during would presence indicate inhibits the drug also we cells 8B). in this Fig. cycle stabilized that in arrests is conclude cell (depicted B complete the and cyclin be with Since in should B degradation treatment point B cyclin this cyclin cells cytokinesis At yet stabilizes or anaphase. anaphase S3), A in Fig. once calyculin B material cyclin no (supplementary have of formed cells Untreated levels partially cytokinesis. with detectable undergo high anaphase not late do at as yet in exit, maintained spindles, mitotic arrest prevents are then A proceed, levels calyculin cells contrast, B prior In cycle cyclin 8B). cell (Fig. the but where levels in se, onset stage a per mitotic at degradation to accumulate B to cells cyclin causes inhibit rather not does staurosporine Giardia indirect. suggests is itself, B interacting B cyclin cyclin B of of cyclin phosphoregulation phosphorylation that of the not phosphorylation and and the proteins staurosporine affect That degradation. 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Pulse Fisher Thermo reagent, anti-ubiquitin stripping Restore (using by were using blots blotting blots Bio-Rad; western analyzed between antibodies, anti-HA stripped secondary and or anti-rabbit, anti-actin and sciences) instructions anti-mouse kit), HRP-linked life UbiQapture manufacturer’s (Enzo the kit Ubiquitin-conjugated with the UbiQapture (accession (provided scientific). the to using Fisher AUK purified inhibitor according (Thermo affinity proteasomal of then inhibitors the were of proteins protease allele absence, then Halt or 3HA-tagged were with presence, the cells a (5 in These MG262 hours express GL50803_104150). 12 for (accession (Gourguechon to incubated PLK methods or 2011), described GL50803_5358) previously Cande, using constructed, and were lines Cell assays Ubiquitination B anti-cyclin with transfected cells 3HA analyze to and B used FITC was (both cyclin program with images CellProfiler transfected and The cells 102 3HA morpholino. population, B oligos cell cyclin untransfected, the the for mismatch in taken on were levels channels) B DAPI depending cyclin levels, quantify To 3HA assays B were cytometry Quantitative cyclin cells or histogram. cells, a size on depleted of plotted al., was B et distribution experiment (Carpenter and cyclin cell (www.cellprofiler.org) The entire program of the 2006). 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Wasserman, and M. Alvarado, E., Gallego, rt-aln .K,Pohi,S . igr .L,Dcs .B,Cretr .L., M. Carpenter, B., J. Dacks, L., M. Ginger, E., S. Prochnik, K., L. W. Fritz-Laylin, M. Kirschner, and H. Yu, G., Fang, D Moreira, Beach, A., and J. Trilles, Ruderman, L., L., Eme, Brizuela, J., Westendorf, F., Luca, G., Draetta, ais .J,Wlim,S,Luat . aac,T n iln .D. F. Gillin, and T. Palanca, T., Lauwaet, S., Williams, J., B. Davids, icrli .D,Dek,T,vnMrn,C,Cevy .J,Se,B n Bork, and B. Snel, J., C. Creevey, C., Mering, von T., Doerks, D., F. Ciccarelli, Friman, H., I. Kang, C., Clarke, R., M. Lamprecht, R., T. Jones, E., A. Carpenter, apne,M .adCne .Z. W. Cande, and L. M. Carpenter, D. Beach, and G. Draetta, L., Brizuela, Mg xeiet n nlsdtedt;SG,LJH n ...wrote W.Z.C. and L.J.H. performed S.G., L.J.H. data; and the S.G. analysed research; and the experiments designed Magdalena W.Z.C. and and S.G. antibody contributions Author anti-tubulin the strain, yeast advice. for FL52 technical of the for reading Gull for Uzlikova Li critical Fei Keith and Dr help to Prof. thanks technical Many for manuscript. colleagues this our thank We Acknowledgements (10 puromycin using terminator selected and promoter were B cells cyclin the Transfected into of 100 transfected described. control including was the construct region, under This sequences. was second GFP A Thus, promoter. GFP. the 3 replacing immediately nucleotides vector, pGFP-pac 5 the nucleotides 200 including region A assays gene Reporter ohr .adBah D. Beach, and R. Booher, Sva and E. J. Palm, R., Bernander, D. R. Adam, References or at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.121632/-/DC1 online publish, available material after Supplementary to release for decision PMC study in analysis, in Deposited role months. manuscript. and 12 no the had collection of funders preparation [grant data The Health of W.Z.C.]. Institutes design, to National the A1054693 by number supported was work This Funding manuscript. the lesann osanfrttlhsoeH1. histone Coomassie total or for autoradiography, stain by to followed staining PAGE SDS Blue by analyzed then boiling, el hnpotda itga o F tiiga niae.Clsnot Cells indicated. as subtracted staining were GFP which to levels, for G2/M background 3, or histogram define Fig. values. S to experimental G1, a in used from into as again as categorized proper, were cell, analyzed allow plotted transfected each then to for then were content fixation cells DNA Cells before and PBS GFP. GFP determine pre-warmed of in folding minutes oxygen-dependent 30 for incubated 642. ftepoenuiutnto ytmi iri intestinalis. Giardia in system ubiquitination al. protein et the of J. Pham, versatility. J., eukaryotic Chapman, early A., illuminates Paredez, gruberi Naegleria A., of Kuo, genome C., M. Field, of ancestor common last complex. the anaphase-promoting in cycle to points cell targets the its of and control eukaryotes. complex modern-like promoting and anaphase complex the of analysis phylogenomic MPF. of inactivation proteolytic D. abi uoakns:argltro ioi nabncet parasite. binucleate a in mitosis of 38 regulator a kinase: aurora lamblia 311 . uri,D . hn,J . idus,R . oft .e al. et J. Moffat, P. A., R. phenotypes. Lindquist, cell quantifying H., and Biol. identifying J. Genome for Chang, software analysis A., image CellProfiler: D. Guertin, O., iri intestinalis. Giardia itn 1kns sascae ihcmlxfrainwt h 6 subunit. p62 the with formation USA complex with Sci. with Acad. associated product Natl. is kinase H1 gene histone a the of interaction possible microtubules. pombe: Schizosaccharomyces fteGadalmlalf cycle. life lamblia Giardia the of 2+ 353-369. , 20) oadatmtcrcntuto fahgl eovdte flife. of tree resolved highly a of reconstruction automatic Toward (2006). 18) d2poenkns scmlxdwt ohcci n :eiec for evidence B: and A cyclin both with complexed is kinase protein Cdc2 (1989). 1283-1287. , n MDT ecin eesopdb digSSsml ufrand buffer sample SDS adding by stopped were Reactions DTT. mM 1 and 20) ilg fGadalamblia. Giardia of Biology (2001). M vl Biol. Evol. BMC MOJ. EMBO 7 R100. , uayt Cell Eukaryot. 86 4362-4366. , 7 9 2321-2327. , otecci tpcdnwsisre ontemof downstream inserted was codon stop B cyclin the to 18) novmn fcc3 nmttccnrlin control mitotic in cdc13+ of Involvement (1988). 11 hls rn.R o.B Soc. R. Trans. Philos. 265. , r,S G. 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