Journal of Cell Science 93,moi I(apnadPlad 96,fri Goeand (Goode formin 1986), Pollard, (Schroeder, and requires F-actin In (Maupin function II as septum. myosin and 1973), such division molecules assembly the conserved ring with and evolutionarily actomyosin coupled membranes and yeast, new tightly cytokinetic fission mitosis of is the assembly into constriction of the yeast, ring entry completion upon Actomyosin fission upon breaksdown mitosis. spindle assembled In mitotic produce when is to constricts size. cell ring the equal of actomyosin region medial of the at daughters fission easily by shape in are divide cylindrical are and and cells methods yeast ring Fission and organism. contractile this in approaches applied based cytokinesis, experimental actomyosin of various an study the using since for divides model it attractive since an as emerged has oee,wehrtesaeo h elisl ly oei the in role 2010). a investigated. Wu, been plays not and itself has Pollard cell cytokinesis of the 2005; aspects of of Gould, shape regulation and the whether Wolfe However, (Balasubramanian 2004; detail al., in investigated cell et been the the have on and and of and execution ring cytokinesis successful ring the positioning actomyosin in steps actomyosin sequential contractile stable necessary are the surface the assembly, of of The and constriction cytoplasm. closure proper F-actin the the between of to division interaction leads upon II al., et myosin generated (Balasubramanian ring Forces actomyosin 2004). the contractile involves a human, of to yeasts function from organisms, many in Cytokinesis Introduction contexts. cellular of variety interplay a intimate in an mechanical apply reveals simple study to but a words: Our slip, by likely Key model. described not are the be do which of can uniform conditions predictions slippage dynamics, same and quantitative ring actomyosin the the the of positioning and towards of dynamics under stable the slip some geometry cell and the verify and the between rings we unstable in of the imaging, are portions role of fluorescence cells cylindrical stability Using crucial of The locally model. actomyosin a regions surface. on the spherical cell play assemble the locally of mechanics that constrict in positioning rings uniformly and assemble and actomyosin geometry that assembly contrast, proper rings cell the By and Contractile of that poles. ring. surface regulators show contractile cell and we the the components of on Here molecular constriction positioning elucidated. key stable been the its have of ring, many ring actomyosin years, contractile the the Over of constriction. assembly proper requires cytokinesis Successful Summary work this to equally ` contributed authors *These 6 5 4 3 2 1 yeast Mishra fission Mithilesh in placement of site fidelity division ensures geometry cellular Cylindrical 3850 o:10.1242/jcs.103788 doi: 3850–3857 125, Science Cell of Journal 2012 March 25 Accepted oeShlomovitz Roie uhrfrcrepnec ( correspondence for Author eateto ilgclSine,Ntoa nvriyo igpr,174,Singapore 117543, Singapore, of University India National 560065, Sciences, Bangalore Biological (TIFR), of Sciences Israel Department Biological 76100, for Rehovot, Centre Institute, National Weizmann Physics, India of 560080, Singapore Department Bangalore 117411, Institute, Singapore, Research of Raman University National Singapore Institute, 117604, Mechanobiology Laboratory, Sciences Life Temasek 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. nrcn er h iso yeast fission the years recent In cioyi ig elgoer,Cl division Cell geometry, Cell ring, Actinomysin 4 1, i Gov Nir , [email protected] ,YniHuang Yinyi *, 4 aa Rao Madan , ; [email protected] cioacaoye pombe Schizosaccharomyces 2, ,Pay Srivastava Pragya *, 3,5, ) ` n oa Balasubramanian Mohan and 3, ,RmnjmSrinivasan Ramanujam *, Mrse l,18;CagadNre 96 eGf tal., et Goff Le 1996; Nurse, and ring Chang actomyosin proper 1986; the the al., of through et contraction medially (Marks and and divide positioning shapes and cylindrical assembly, have ends, cells hemispherical yeast fission (WT) Wild-type implications. Results biological its the discuss verify and ring, We actomyosin images model. the fluorescence from of mechanical model this can interplay of simple geometry predictions and a cell assumptions This and using rings understood septation. actomyosin be of proper dynamics the support between geometry a undergo a with rather cylindrical regions cellular but in locally assembled constriction rings is whereas normal behavior, geometry sliding that undergo the not find where do We regions stable spherical ring. in the actomyosin located on rings the mechanics actomyosin of and constriction geometry and cell positioning of role the investigate 2011). al., et from (Minc inferred urchins been the sea also has in of geometry work dependence cellular Recently, on site 2005). division al., cell et (Ge to investigated between leads not been interplay have cells mechanisms an the although of cytokinesis, underscoring and morphogenesis placement morphology septum cylindrical in the defects of that compromise shown have yeast a fission in Aspenstro studies Previous 2009). 1995; (D’Avino, al., (Fankhauser proteins of et family F-BAR the of members 2007), Eck, nti td,w s h iso yeast fission the use we study, this In 1,2,6, m 09,adailnrltdproteins anillin-related and 2009), ¨m, ` 2, ,Mylg Sevugan Mayalagu *, .pombe S. eerhArticle Research samdlto model a as 1 , Journal of Cell Science ahrta peia uat,bcueptnildfcsin geometry. possible defects of to instead attributed potential function be protein because could osmotically spheroplasts of mutants loss with an mutants, spherical work in in spherical cytokinesis to wall than chose cell rather We as the to environment. of (referred altered stabilized removal cells of WT by cells spherical in spheroplasts) generated behavior first ring we actomyosin geometry, assess To 1999b). nyeccti htdsut h elwl,admitie na in maintained an and with wall, cells cell of the Wild- disrupts treatment cycle. that by cocktail cell prepared enzyme the were to with spheroplasts mCherry-tubulin dynamics type of instances ring some behavior actomyosin In the correlate 2005). imaged spheroplasts al., of et simultaneously generation Karagiannis we the 2000; for al., used et were (Naqvi that cells Rng2p WT protein the or IQGAP-related in Rlc1p–GFP, of chain, domain light calponin-homology regulatory the II myosin of version fused ovsaieteatmoi ig efrtepesdaGFP- a expressed first we ring, actomyosin the visualize To eeomtclytne h ulttv n uniaie(swe (as quantitative spheroplasts and these qualitative because The The rings. been S1B). tense. have Fig. or osmotically could material were arcs fission (supplementary cells of early sliding intact absence and mature the in in as detected to rings not anillin- were localized The signals Mid1p radius. Mid1p However, in protein shrinking material related supplementary simultaneously 1A,B; 1–8), (Fig. they poles Movies along the slid while towards systematically they cortex unstable; ring, the a mechanically as actomyosin integrity were the their maintained assembled, spheroplasts once in that observed rings S1A). we Fig. cases material most (supplementary after In spindle anaphase the in 1A,B). late of (Fig. assembled breakdown rings the not rings these data cells, intact staining; actomyosin in blue Unlike aniline assembled by We successfully judged (as osmolysis. shown) wall undetectable with cell prevent (or a without of) to spheroplasts levels cases, sorbitol all in M that found 0.8 containing medium elgoer n eldvso 3851 division cell and geometry Cell h elisedo osrcigcnrptly cl bar: Scale centripetally. of constricting pole 5 the of to instead slipped cell observed rings the 68 the of (0.6 39 section shown. 3D the of intensity projections Maximum microscopy. spinning confocal using disc imaged with and pads sorbitol and agar medium on minimal M mounted 0.8 were with Spheroplasts and supplemented sorbitol. Methods medium and minimal Materials in the grown in described as made ( Atb2–mCherry and GFP of contraction. of initiation instead upon surface centripetally the ingressing or along spherical slide in protoplasts formed conical rings Actomyosin 1. Fig. m m. peolsso el xrsigRlc1p– expressing cells of Spheroplasts A rcd-F ( chd1-GFP or ) m tpsz)are size) step m B were ) Journal of Cell Science 2 h ako elwl nshrpat ih edt the to lead might spheroplasts in ring. wall actomyosin constricting cell the of a position the or of of ingression; destabilization lack with the coupled constriction (2) cells) surfaces conical undergoing 1Bi; along or than (Fig. (spherical slide rather radius rings rings sectional apex i.e. cross actomyosin decreasing geometry, the of with of dynamics consequence towards sliding a from the were cone proceed suggested (1) observations possibilities: the to These two 6). observed of Movie was material region supplementary sliding with basal those ring in the shape, spherical; exactly even conical not occurred a instability were sliding al., spheroplasts et The the Liu 2004). when 1999a; al., al., et et Goff Mishra (Le 1999; division checkpoint cell cytokinesis of the completion of which until maintenance 6), the Movie from results material and likely assembly supplementary ring 1Bi; of (Fig. cycles the additional disassembly in to sliding led and septation of constriction for absence ring force spheroplasts, driving 1Bi–iii), many the In (Fig. as sliding. F-actin contractility and actomyosin 1Ai–v) in identifying (Fig. replicated thus II are myosin dynamics both sliding of these images of features later) see will 3852 ora fCl cec 2 (16) 125 Science Cell of Journal lofudta neeyisac hr ipae rings misplaced where instance every sliding in a underwent that and found ( ends cells also cylindrical cell these in the Notably, behavior. near actomyosin 9–11) assembled Movies rings material supplementary 2Bi–iii; (Fig. eipeia nso h el newn ldn behavior sliding as introduction such by underwent formins exacerbated in was on mutations cells behavior of assembled sliding the This that 2A). of rings (Fig. actomyosin ends that hemispherical 1996; showed al., is cells et mutant which (Sohrmann protein, positioning anillin-related site Ba division the for Mid1p, important in the in behavior defective then ring actomyosin cells would defects, imaged end-caps We wall constriction. hemispherical during the cell slide of at than cause assembled misplace the rings rather was to contractile geometry instability sought cell ring If we resemble morphology. cortical end, that their cells in this of cells ends To WT hemispherical the wall. to that rings cell cells actomyosin normal yeast fission a in assembled have rings contractile of behavior he ta. 98.Imaging 1998). al., et ¨hler odsigihbtenteepsiiiis eivsiae the investigated we possibilities, these between distinguish To edn odvso ftecl.Tearw in arrows The cell. these the contraction of min) successful division 36 a to (time underwent leading cell and the stabilized of were part rings ring cylindrical actomyosin the the at these when assembled along However, slipped ends. rings cell cells such hemispherical hemispherical min) the 0–28 at (time assembled ends rings actomyosin In the the cells. when at the assembled of when ends constrict hemispherical actomyosin than cell showed rather the cells slip at these rings rings of actomyosin microscopy showed Time-lapse often ends. cells Such hours. 4 for hw.Easdtm ssoni iue.A es i el were 5 cells bars: six Scale least category. At each minutes. in in imaged shown is time 3D Elapsed perform shown. to used was (0.6 microscopy time-lapse hemispherical confocal at 510 assembled LSM rings ends. actomyosin the indicate (Ci,ii) eegoni E oepnnilpaea 25 at phase exponential to YES in grown were the mid1 at cells. cylindrical assembled of rings ends actomyosin hemispherical of Slippage 2. Fig. 1 n ( and -18 C m ) tpsz) n aiu rjcin are projections maximum and size), step m tea1 D mid1 for3 mid1 1 el xrsigRlc1p–GFP expressing cells -18 D .In 1 temperature-sensitive -18 for3 mid1 D m m. ( mid1 mid1 A -18 ) tea1 mid1 ˚ 1 el (Biii), cells -18 ,sitdt 36 to shifted C, -18 for3 D 1,( -18, mid1 for3 D mutants B 1 cells -18 D ) ,we ), for3 ˚ C D Journal of Cell Science ldn nspherical in sliding supplementary 13). arrows; 12, with Movies cross- marked material smaller 2C the of (Fig. region at of diameter the region sectional attachment the to cell from this diameter down the cross-sectional to slid maximal of rings prior actomyosin ends However, ends, cell extreme 2007). extreme the the al., to to et led rings consistently (Huang this actomyosin that of found We anchoring shown). not (data cells coysnrnsa h eled Hage l,20) oensure To 2007). al., et (Huang ends the cell of the and anchoring at Tea4p prevent rings to Tea1p, known actomyosin of is which comprising complex), complex (tip Pom1p protein a of presence ( ( cylindrical regions 2Biii). (Fig. cylindrical ensued septation the and constriction in assembled netgtdtepsto ftedvso etmi spherical in septum lower division the is of we Then, position which caps. the end spherical S2Ai,ii), investigated as well Fig. as spheroplasts WT material in that out (supplementary than 3 cells in slipped 20 rings of actomyosin that showed experiments imaging actomyosin the imaged and we shape tip-complex, hemispherical in of the (Rlc1p-GFP) ring to presence due to is due ring not the of instability that ioi uliwr lcdo h aesd fteseptum the as, (such mutants of of variety a site. side division the spherical same of the placement the that the establishing affects on geometry S2B), Fig. placed post- material the were that (supplementary such nuclei septum division the mitotic misplaced cells spherical of el eda h etitv eprtr f36 of temperature restrictive the at held cells ugs ht()teisatnospsto ftern fradius of observations ring the these of concentration, position instantaneous the myosin (1) that to density suggest force proportional contractile turnover the be Taking must to section). filaments later and myosin a ring the in contracting the (discussed 3B), a for (Fig. along roughly that width II be implies fixed This to myosin uniform of found 3B). the was (Fig. ring time is addition, the in of In constant myosin length 3A). unit per (Fig. of intensity ring mean images it intensity the II until of myosin the integrity circumference labeled their the that maintain of showed Analysis and poles. coplanar the are approaches 9– rings 1–5, Movies the the material of stress 13) (supplementary most In sliding contractile poles. ring the the of towards instances slide of to ring components contractile the the tangential On cause occur. the to hand, ingression for other rigid tense too osmotically possibly The contractile are stress. spheroplasts the elastic membrane of the stresses. component exceeds viscous normal stresses, stress by the elastic balanced when is membrane occurs stress Ingression by tangential balanced active the The is surface. while cell stress the normal to normal active and have tangent mechanical which the by ring, along actomyosin driven components contractile ring the cortical by the generated the describing stresses of model shrinking theoretical of a dynamics these developed the within and of assembled the dynamics measurements rings ring quantitative understand made of to We dynamics geometries. sought the spherical of next basis we physical cylindrical instability, ring in regions actomyosin hemispherical assembled in septation. those and assembled constriction normal whereas underwent that regions sliding, rings cell, underwent single within even that, confirmed a further We ring. determining cortical the in factor of important stability an is geometry cell that established enx netgtdwehratmoi ig underwent rings actomyosin whether investigated next We h ntblt ftern ntehmshrcled fthe of ends hemispherical the on ring the of instability The u xeiet nshrpat n yidia es el using cells yeast cylindrical and spheroplasts on experiments Our aigmd hscreainbtenshrclgoer and geometry spherical between correlation this made Having mid1 1)clscud oee eatiue to attributed be however could, cells -18) orb6 mid1 2 el Vree l,19) Time-lapse 1998). al., et (Verde cells -25 -18 mid1 tea1 D and Fg C and 2C) (Fig. tea1 ˚ , .Apoiaey60% Approximately C. tea4 n . ) omlring normal 5), uat)together mutants) mid1 -18 orb6 tea4 r -25 ( D t ) lcda qao n ttepls respectively. poles, the at and equator at placed is width radius ring of the cell and ( ring active time. the the in hence of constant (and length I unit These intensity per lines). fluorescence force) black II contractile by Myosin horizontal the plotted the that is by show series (represented images time mean The its (i–iv). to cells normalizing spheroplast four width for ring (min) average the and (blue) circumference igo peeadcntk ausbetween values take can and sphere on ring otatl oc density force contractile ln h ig oeta h ihritniya h w nsi eutof result a is ends two the at intensity higher ( uniform the effects. is that projection II force) Note Myosin contractile ring. the active the that the along show hence images (and along These intensity normalize pixels ring. fluorescence we 5 the convenience over along the binning For value which then ring. maximum over and the scale maximum) of the its circumference as of the (defined half width by ring falls the intensity over radians) intensity (in II intensity angle myosin The azimuthal cells. the spheroplast by four parameterized for II ring myosin the from of model circumference mechanical the of images. assumptions fluorescence the Testing 3. Fig. ln h icmeec ftern;ad()teactomyosin the viscous the by (3) balanced is during and stress Since contractile 3C). actomyosin ring; the (Fig. sliding time in the constant is of density force contractile circumference the along a erpeetdb oa angle polar a by represented be can f r 5 cos h 0 ( t ,where ), R h agnilcmoeto otatl oc e ntlength unit per force contractile of component tangential The . C elgoer n eldvso 3853 division cell and geometry Cell B ceai fcnrciern ntesraeo spherical of surface the on ring contractile of Schematic ) ysnI nest ( intensity II Myosin ) h ( 0 A ( t lto ysnI loecneitniyaogthe along intensity fluorescence II Myosin of Plot ) eoe h ntnaeu nua oiino the of position angular instantaneous the denotes ) f r freprui egho ig suniform is ring) of length unit per (force I scmue yfrtitgaigthe integrating first by computed is I vrgdoe h ring the over averaged ) p 2 o0crepnigt ring to corresponding 0 to h w 0 t rd safnto ftime of function a as (red) 2 h actomyosin the (2) ; I w yits by r roughly are Journal of Cell Science oiin,btdet h oa peia emtyo h cell, medial the experiments of at earlier the geometry from assembled spherical predicted as local indeed and the unstable was were to they due ring is but possibility actomyosin positions, another at the assembled yet misshaped directed rings However, that actomyosin the sites. the rings, in located because non-medially be mid-cell actomyosin could the This that cells. from implied away in septation This cells the of 5A–C). 36% (Fig. almost septa, misplaced only have myo52 cells, cells WT the the in of While 3% position. septum on consequence any o oc aac a eesl ovdt xlctyoti the ring, obtain the explicitly of to position angular solved the easily equation of be The dependence can time ring. balance the of force length for unit per friction) (inverse mobility and h hrceitcvelocity characteristic the estimating htti a eynro itiuincnee bu 0.12 about centered distribution narrow very a has this that tes ehave we stress, hp,spaaeawy oiindi h ideo h cell. the of middle in the defect shape in this if positioned determined first always we Therefore, are 2001; septa al., WT shape, et In of (Motegi 2001). abnormalities al., cortex et shape type behavior cell Win such non-essential is the to a the Myo52p, lead of morphology lacking myosin, Cells sections V studied sphere. cell that a as extent which we out an bulge in such end, conditions to compromised in this rings To actomyosin fidelity the cytokinesis. in role of physiological a played anchoring maintenance 4vi,viii). (Fig. size cell with linearly increases 3854 ytesiigtime scaled radii time sliding spheroplasts against same the and plotted the by when ends curve cell roughly master the a for of along data collapsed cells resulting ring the from the 4i,iii); of (Fig. collected position (angular time) data the use versus reorganize made to We feature ring position. the this angular of of instantaneous velocity its angular on the balance only radius, force cell depends for fixed making a equation In for the as that that II. implies noted ends, myosin first cell of we sliding comparison images and this fluorescence the spheroplasts the on in from data rings obtained experimental contractile with of model dynamics theoretical the of h ist h xeietldt Fg vvi.Knowing 4v,vii). (Fig. data by seen experimental as the ends cell to and fits spheroplasts both the for 1 Eqn by described iedrvtv)siigaogacl fradius sliding, of for scale cell time a gives a immediately along sliding derivative) time el eri h aenme fmoi e ntlength unit per myosin velocity characteristic of the of since number independent Further, is size. the same the the its that in of that the means independent constant this suggests particular, recruit roughly In and is size. this cells ring friction their of the different, ring independent in cells, the very density of force are origins contractile the force Since contractile inset). 4v,vii, (Fig. 0.17 and min where ete drse fteclnrclmrhlg ae ring based morphology cylindrical the if addressed then We sn hs emd ealdcmaio ftepredictions the of comparison detailed a made we this, Using n h ~ D h 0 R 0 steiiilaglrpsto ftering. the of position angular initial the is (0) h ora fCl cec 2 (16) 125 Science Cell of Journal a h et oiindaa rmtemiddle the from away positioned septa the had h _ 0 R 0 ( steaglrvlct ftern oedtrepresents (overdot ring the of velocity angular the is , t ) rmtecl mgs eotie itiuinof distribution a obtained we images, cell the from m ~ /i o peolssadcl ns respectively ends, cell and spheroplasts for m/min cn p 2 h R ~ { h esrdsipn time slipping measured the , f t 2 r s arc cos ~ .pombe S. L cot h R L 0 f r where , f 4 2 r Fg i,v.Tecre r well are curves The 4ii,iv). (Fig. e cosdfeetcls efound We cells. different across { t t s el ihuiomcylindrical uniform with cells c cot sarn rcincoefficient friction ring a is p 4 t s { ~ h L 0 R 2 f ( R r T 0 where , hsrelation This . ) rltdto (related myo52 5 3 , myo52 L t s D sthe is and , m had ð m/ t 1 D s Þ ) el,atog h coysnrnsasmlda eilsites, medial at assembled rings actomyosin the although cells, tbeadcntitda h aelcto Fg 5Di,ii; (Fig. location the same in the However, 14). at remained Movie and constricted material site supplementary cells, mid-cell and WT the in expected, at stable As assembled cells. WT rings the actomyosin to it compared and cells h cell the time slipping eutn aast a hnb itdt h xrsinfrteaglrposition angular the for expression with the 1) to (Eqn fitted time be versus then can sets data resulting nfrl yidia ein ftecl.T distinguish in microscopy To stable time-lapse al., by et constriction of cell. (Karagiannis more through CHD–GFP) process 2005) by assembly to the (labeled the rings imaged actomyosin down of the we possibilities, slipped regions these between model, cylindrical mechanical uniformly the and aawt epc otesae time scaled the to respect with data rdcin ftemcaia oe,En1(oi ie) rmteft othe to fits the From lines). (solid different 1 with Eqn data model, mechanical the of predictions ( ( spheroplasts ends cell for shown curve bu 0.17 about itiuino hrceitcvelocity characteristic of distribution h ntnaeu oiino h ig ecnmk eaiesit ln the along of shifts profile relative smooth make a can obtain size, we to same ring, axis the the time of because of cells iii, position and for instantaneous i ring in the the data of the the velocity reorganize from angular to obtained 1 the respectively, Eqn use each), We events images. (15 fluorescence spheroplasts and ends cell experiments. with model mechanical of ( predictions of Comparison 4. Fig. i , iii nua oiin i ain)o h igvru ie(nmnts for minutes) (in time versus ring the of radians) (in positions Angular ) R (in m /i o eled n 0.12 and ends cell for m/min vii T m m). i i)t rvlafxdaglrdsac ihterdu of radius the with distance angular fixed a travel to min) (in hwtecmaio fteeprmns(aapit)with points) (data experiments the of comparison the show ) R eetatteparameter the extract we , t s ii sprmtr eueti au of value this use We parameter. as n peolss( spheroplasts and ) t t s hsrslsi olpeot master a onto collapse a in results this , L f h r ~ 0 m versus /i.( m/min. t R s hc r arwypeaked narrowly are which , iv t s .Posfrcl ns( ends cell for Plots ). h nesso the show insets The . t o each for vi , viii R iersaigof scaling Linear ) R eed nyon only depends aho the of Each . t s orpo the replot to myo52 myo52 v and ) D D Journal of Cell Science nsmay ehv hw htatmoi ig in rings actomyosin on assemble they that as of soon regions as shown slide spherical spontaneously have cells ends/local cylindrical hemispherical we and spheroplasts summary, In Discussion fidelity process. the in cytokinetic the importance the of physiological of stability its the thus and on results, rings geometry These spherical actomyosin cell of division. actomyosin local influence cell the cytokinetic the faithful that demonstrate in compromise of instabilities conclude rings and to regions we rings lead cylindrical cells actomyosin and Thus, in reached spheroplasts constricted. geometry they Interestingly, in and as seen cell soon ends. that as to stabilized cell similar Movies process material geometry hemispherical a supplementary spherical with 5E–G; in (Fig. regions 15–17) down the in slipped unstable and were rings the eseta h otatl igsisweee tasmlso a on assembles it 2C), whenever (Fig. slips ends ring cell contractile in the rings that instances see contractile several we In of size. assembly cell multiple on of time slipping the with of dependence consistent is fluorescence rings of constricting 3A,B). analysis the (Fig. Our in assumptions time. these II along in mechanical myosin uniform constant of (1) simple (2) is images density and the force ring contractile cell in the the the that implicit mechanical at are ring simple assumptions model the by a The exerted of forces geometries using surface. contractile instability conical on understood based and sliding quantitatively model spherical be this locally can on experimentally, between rings verify the interactions actomyosin we complex assumptions, of certain of under dynamics which However, result myosin. subsequent and the filaments and actin is Assembly ring surface. contractile cell the evrf eea rdcin rmti oe,i atclrthe particular in model, this from predictions several verify We elgoer n eldvso 3855 division cell and geometry Cell eegoni E oepnnilpaeat phase exponential to 24 YES in Cultures grown (ii). were period constriction of and kymograph assembly and (i) Chd1p–GFP expressing in constriction and assembly ( (ii). period constriction and and assembly (i) of Chd1p–GFP kymograph expressing cells constriction WT and in assembly ring actomyosin of 4 bars: Scale B. in mgdb pnigds ofclmicroscopy 36 confocal at disk Spinning by imaged esrd h el ihartoo esta 0.8 than less of ratio was a cell with the cells of The axis measured. long the and along ends septa cell the two between distance The position. axis. cell ( the in along rings regions actomyosin non-cylindrical of slippage in and septa cells of Misplacement 5. Fig. and septa. misplaced ( with were cells daughter as long scored and short the between fcntn imtr cl as 4 bars: Scale diameter. regions constant the of at before ingress slipped and constrict then to and beginning middle cell close the regions to non-cylindrical the in rings assembled actomyosin Time-lapse that minutes. showed in microscopy shown is time Elapsed (0.5 14 section 3D A B uniiaino ipae et nWT in septa misplaced of Quantification ) ˚ ceai ersnaino septum of representation Schematic ) ,sitdt 36 to shifted C, myo52 ˚ E .Mxmmitniypoetoso the of projections intensity Maximum C. – G ielpeo coysnring actomyosin of Time-lapse ) D el.( cells. m ˚ m o or n then and hours 4 for C C .( m. tpsz)aeshown. are size) step m mgso el counted cells of Images ) D ielpeanalysis Time-lapse ) myo52 myo52 m D (D–G). m cells D Journal of Cell Science oetmt h unvrrt fmoi Ii lpigrings slipping a in with dynamic II highly myosin be to of found t rate was II turnover Myosin out spheroplasts. the carried We estimate experiments high. (FRAP) is to photobleaching rate after happen turnover recovery only myosin can fluorescence and This filament constriction. ring II the the myosin if for of limiting kinetics rate turnover not the is be that slipping indicates same. to experiments and the a molecular are need constriction from the and changes that sliding goes in suggest these involved would ring processes While appears this the dissected, dynamics behavior. quantitatively the when constriction and smoothly constant to appears change and The ring stabilizes surface. to the cylindrical and local surface of a cell encounters width it the assembly as of soon following part as constricts slips spherical ring locally contractile a on the 5E–G), (Fig. cells sebe nalclyclnrclsrae iial,in Similarly, surface. it cylindrical when locally constricts a and on stabilizes assembles and geometry, spherical locally 3856 hti hsstain h nhrn ftern u othe to due sliding ring the the counterbalance of can in anchoring deposition observed the wall suggests situation, frequently cell spheroplasts) this enhanced not WT in with is that (compared sliding mutants fact The ring ring. spherical the of actomyosin sliding than leads rather that constriction cortex, the ring a of actomyosin ingression (having that to stabilizing such geometry in ring role actomyosin cylindrical a the play locally might the diameter) of The cross-sectional position constant the site. of division maintenance in with cell role concomitant a new assembled play the constriction are that ring likely which is septum, It and 2003). membranes Nurse, with and treated (Pardo cells poisons cylindrical tubulin septation-defective in observed that diameter. anchored/ constant the would unless a in assembled has be region, which ring to region require spherical cylindrical would cytokinetic or a septation ensuing the in by fixed unstable that be on imply inherently dynamics actomyosin geometries the yeast of spherical cylindrical description physical spheroplasts, the with and experiments cells The driving ring. circumference, the homogeneity ring of the re-establish the quickly that along currents circumferential forces contractile in inhomogeneities that active to fact lead the recruitment local of on myosin of molecular based context in inhomogeneities is the uniformity the there explanation in likely unlikely after more seems Are by The this spheroplasts. only – II? established assembled constriction is density myosin force is and trigger are and established that ring images F-actin uniformity checkpoints the the of this from that recruitment is II given How angular myosin different maintained this. of with with distribution slipped consistent of uniform measurements have Our ring. would the the of disruption ring a different to leading not, the velocities, it were of for circumference, densityparts ring force the contractile along the uniform that implies is This ring. cytokinesis the of investigated integrity have we septation. studies with our uncoupled and attributable in is be that that (Wu may fact differences cells the the if to in unclear is It constriction 2005). II Pollard, during constant myosin that increases remains shown concentration has concentration II analysis work myosin previous our constriction, that although throughout fact Interestingly, sec the rate. 30 to much sliding to points is sec the which 12.9 2009), than al., from et faster range Sladewski 2002; that Chang, cells and (Pelham intact in reported those 1/2 h odareetbtentesml ehnclmodel mechanical simple the between agreement good The h ldn eairw bev smr ai oprdto compared rapid more is observe we behavior sliding The the of maintenance the is revisit we that aspect crucial A f16 of 6 ora fCl cec 2 (16) 125 Science Cell of Journal e ( sec 3 n 5 8.Ti unvrrt a oprbeto comparable was rate turnover This 18). myo52 D rngtv uvtr ih lopa oei h ieiyof fidelity the in role a types. play cell diameter other also sectional in cross might cytokinesis constant curvature long of a negative region of a or may generation with factors axis the short and These these and breaking in yeast. rings symmetry as metazoan actomyosin Thus, of such in stability systems. in types cortex difference cell the cell for ring other account the actomyosin in with the 1000 than the that strongly eggs possible least more also that at 2009; is interact al., It is et might noteworthy 2011). Minc 1997; types al., al., negative is et et cell (Kelly Stewart yeast these of It fission a than in lower or (with fold cytokinesis. pressure axis diameter turgor to short estimated cross-sectional and prior long constant morphology, a curvature) generate of spherical maximal facilitates types a region cell despite for cell to other and division position Xenopus many type undergo enough of eggs ring urchins wild fertilized strong sea Although actomyosin the cytokinesis. not of the fidelity of is that of geometry suggests wall maintenance This cylindrical cell sliding. ring the the actomyosin the to the at here counterbalance assemble that due is rings above where the with cases anchoring consistent all explanation nearly an actomyosin ends; in hand, cell other seen the is On slippage 2005). al., ring et (Ge (supplementary S2) geometry Fig. cell material spherical the by induced instability xrsigyatsri n rDne uvhl o the for Mulvihill Daniel Dr mCherry-tubulin the and for strain Sato Masamitsu yeast Dr to expressing due are thanks Many Acknowledgements al., et a (Tran under temperature sealed experimental microscopy, pads, of the agarose centrifugation at Cultures 2% imaged time-lapse containing speed 2004). and media VALAP low EMM2 using For 1 or medium. coverslip by YES growth with in microscope. concentrated resuspended slides on and on min) were or confocal 3 software r.p.m., cells (2000 7.6) Meta (v growing MetaMorph exponentially by LSM510 (Plan driven microscope and Zeiss 200M camera Axiovest Orca-ER Zeiss al., Hamamatsu et on (Moreno either X- 100 wall) imaged (cell Triton Blue Apo were 1% Aniline cells (DNA), with DAPI Live permeabilized with 1991). and stained and formaldehyde PBS 3.7% in 100 with fixed were Cells Microscopy of Cultures 1991). al., et (Moreno medium minimal or YES mid1 in grown were Cells spheroplasting and media Yeast The strains Yeast Methods and Materials leu1 H60 n esseddi -ufrcnann . obtl h cell The sorbitol. M 1.2 containing 5 E-buffer to in phosphate adjusted sodium re-suspended mM was 100 and citrate, suspension sodium 6.0) mM (50 pH E-buffer in once washed were nye(-42 im)a 36 at Sigma) (L-1412; enzyme OD orb6 3XGFP::kan mgJsoftware. ImageJ o icsin n .Rmsayfrcmet ntemanuscript. the on groups comments research for M.R. Ramaswamy and S. M.B. and of discussions members for thank to wish We strain. ih08Msrio n rw ihgnl hkn 8–0rpm)(sm tal., et spheroplasting. (Osumi after r.p.m.) hours (80–90 MM four shaking in imaged gentle E-buffer suspended typically with then were grown with were Cells and Cells 1998). sorbitol a washed sorbitol. by M M followed were 0.8 0.6 spheroplasted, with were containing Cells cells E-buffer of with microscopy. 80–90% wash once sorbitol contrast M 1.2 phase containing by monitored h+ ade6-210 18 MBY6432 for3 eoeiaig o peolsigclswr rw nmnmlmdu M)for (MM) medium OD minimal The in hours. grown were 24 cells spheroplasting For imaging. before and Rlc1p-GFP 595 D -32 2 l1-F and Rlc1p-GFP -25 .pombe S. 1 Rlc1p-GFP, -18 mid1 f0305 utrswr hnsitdt 36 to shifted then were Cultures 0.3–0.5. of 6 ade6 1 l1-F:ua;MBY2699 Rlc1p-GFP::ura4; -18 orb6 14 qipdwt ooaaCU2 pnigds system, disk spinning CSU-21 Yokogawa a with equipped /1.4) r r4- ura4 tan sdi hssuywr MBY5985 were study this in used strains 26h;MBY1287 h-; -216 tea1 2 l1-F:ua;MBY7159 Rlc1p-GFP::ura4; -25 agf rmD ailMliil;MBY7294 Mulvihill); Daniel Dr from gift (a 595 myo52 1h;MBY2309 D18h-; D ftecluewsawy anandbten0206 Cells 0.2–0.6. between maintained always was culture the of for3 mid1 myo52 D D 1 l1-F)o o or ( hours 4 for or Rlc1p-GFP) -18 eeiae t36 at imaged were mid1 6 D ˚ 10 o 03 iue.Pools omto was formation Protoplast minutes. 10–30 for C eegoni E t24 at YES in grown were 1 Rlc1p-GFP, -18 7 el/l el eeicbtdwt lysing with incubated were Cells cells/ml. K4-m4-F-H:lu+ura4 JK148-nmt41-GFP-CHD::leu1+ mid1 1 l1-F:ua;MBY3580 Rlc1p-GFP::ura4; -18 tea1 ˚ ˚ .Iae eeaaye with analyzed were Images C. o – or ( hours 2–3 for C myo52 tea1 D mid1 D cer-t2:p rlc1- mcherry-atb2::hph D ˚ :r4lu-2ura4D- leu1-32 ::ura4 1 Rlc1p-GFP::ura4; -18 ni hyrahdan reached they until C m orb6 mid1 fclswr placed were cells of l myo52 2 and -25 1 Rlc1p-GFP, -18 for3 D CHD-GFP. D myo52 myo52 mid1 -D18 -18 D D Journal of Cell Science i,J,Wn,H,MClu,D n aaurmna,M K. M. Balasubramanian, and D. McCollum, H., Wang, J., Liu, eGf,X,Uzg .adSmns V. Simanis, and S. Utzig, X., Goff, Le eGf,X,Wolr,A n iai,V. Simanis, and A. Woollard, X., T. Goff, P. Le Macklem, and L. Y. Jia, M., S. Kelly, od,B .adEk .J. M. Eck, and L. B. Goode, un,Y,Ce,T . e .adBlsbaain .K. M. Bimbo Balasubramanian, J., and Karagiannis, W. Ge, G., T. Chew, Y., K. Huang, M. Balasubramanian, and N. S. Naqvi, V., Wachtler, G., T. Chew, W., Ge, M. Glotzer, and E. Bi, K., M. Balasubramanian, hnsteAvnadGrrd eieCre eeomn Chair; N.S.G. Development a Career Foundation. by Levine Gertrude part Millennium and in Singapore Alvin supported the Mechanobiology the were thanks the R.S. from and and fellowship M.M. Foundation from Singapore. support Millennium Institute, Avance research Singapore Recherche acknowledges la M.B. the and de 3504-2]. grant Promotion number Program la Science [grant Frontier pour Human Franco-Indien an Centre by funded was M.R. Funding akasr . emn,A,Crti . ti,S,Hfan .adSmns V. Simanis, and K. Hofmann, S., Utzig, L., Cerutti, A., Reymond, C., Fankhauser, P. Nurse, and F. Chang, Ba Aspenstro References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.103788/-/DC1 online available material and Supplementary 2006285]; support. their number for 710589], [grant number Foundation [grant Minerva Science Binational the ’vn,P P. P. D’Avino, he,J,Sevr .B,Wete,S,Wn,Y,Pige .R,Gud .L and L. K. Gould, R., J. Pringle, Y., Wang, S., Wheatley, B., A. Steever, J., ¨hler, ,-eagua ytaesbnt sesnilfrdvso etmasml in assembly septum division for essential is subunit, pombe. Schizosaccharomyces synthase 1,3-beta-glucan a idn h ide n iigi right. it timing and middle, the finding vdnefraspaincekon nShzschrmcspombe. Schizosaccharomyces in checkpoint septation Genet. a for evidence fXnpsoocytes. Xenopus of n rmtstescesu opeino yoiei nrsos oprubto of perturbation pombe. to Schizosaccharomyces response in in cytokinesis ring network of actomyosin initiation completion the septation successful the the regulates promotes positively and Lsk1p kinase nuclear The (2005). assembly. actin of morphogenesis. Sla2p/End4p cellular Hip1-related in with involved interacts Pal1p is protein and yeast fission novel The (2005). eemnnsTap e4,adPmpihbtdvso-etmasml tcl ends cell at assembly yeast. division-septum fission inhibit in Pom1p and Tea4p, Tea1p, determinants at F-actin of reorganization the in element key a is gene cdc15 mitosis. pombe S. The (1995). cells. animal and yeast fission yeast, R818. budding in cytokinesis yeast. fission in division D. McCollum, esn rmAilnrltdproteins. Anillin-related from lessons 194. reorganization. actin and dynamics m P. ¨m, 262 Cell 163-172. , 82 20) o osafl h otatl igfrasf yoiei - cytokinesis safe a for ring contractile the scaffold to How (2009). 20) oe fFBRPHpoen nterglto fmembrane of regulation the in proteins F-BAR/PCH of Roles (2009). 19) oeo ookns n i1 ndtriigtest fcell of site the determining in Mid1p and kinase polo of Role (1998). 435-444. , e.Cell Dev. nu e.Biochem. Rev. Annu. ,A,Rjgpln . i,J n aaurmna,M K. M. Balasubramanian, and J. Liu, S., Rajagopalan, A., ´, op ice.Pyil Physiol. A Physiol. Biochem. Comp. 19) o iso es iso ntemiddle. the in fission yeast fission How (1996). .Cl Biol. Cell J. 12 20) ehns n ucino omn ntecontrol the in formins of function and Mechanism (2007). 987-996. , Genetics n.Rv elMl Biol. Mol. Cell Rev. Int. 19b.Cnrligspaini iso yeast: fission in septation Controlling (1999b). .Cl Sci. Cell J. 143 ur Genet. Curr. 153 76 19a.Aayi ftecs eeprovides gene cps1 the of Analysis (1999a). 1603-1616. , 19) esrmn featcproperties elastic of Measurement (1997). 593-627. , o.Bo.Cell Biol. Mol. 1193-1203. , 122 20) oprtv nlssof analysis Comparative (2004). 35 1071-1079. , o.Bo.Cell Biol. Mol. 571-584. , 118 16 272 4124-4138. , 607-613. , ur Biol. Curr. 19) Drc1p/Cps1p, (1999). 1-31. , 20) Polarity (2007). 16 Cell 358-371. , o.Gen. Mol. 14 84 R806- , 191- , ´ e ed,F,Wly .J n us,P. Nurse, and J. D. Wiley, F., Verde, rn .T,Poet,A n hn,F. Chang, and A. Paoletti, T., P. Tran, twr,M . eeis . ooa . aaahn .P,Mle,D .and J. D. Muller, P., S. Ramanathan, Y., Toyoda, J., Helenius, P., M. Stewart, M. Lord, and J. M. Previs, E., T. Sladewski, apn .adPlad .D. T. Pollard, and P. Maupin, cree,T E. T. Schroeder, Q. J. Wu, and D. T. Pollard, oran . akasr . rdek .adSmns V. Simanis, and C. Brodbeck, C., Fankhauser, M., Sohrmann, ak,J,Hgn .M n ym,J S. J. Hyams, and M. I. Hagan, J., Marks, ehm .J n hn,F. Chang, and J. R. Pelham, P. Nurse, and M. Pardo, sm,M,St,M,Ihjm,S . ooi . aai .adYgci H. Yaguchi, and T. Takagi, M., Konomi, A., S. Ishijima, M., Sato, K. M., M. Osumi, Balasubramanian, and X. Tang, C., K. Wong, I., N. Naqvi, oei . ri .adMbci I. Mabuchi, and R. Arai, F., Motegi, oeo . lr .adNre P. Nurse, and A. Klar, S., Moreno, iha . aaini,J,Tatan . ag . colm .and D. McCollum, H., Wang, S., Trautmann, J., Karagiannis, M., Mishra, ic . ugs,D n hn,F. Chang, and D. Burgess, N., F. Minc, Chang, and A. Boudaoud, N., Minc, i,T . aht . uvhl,D . a,K .adHas .S. J. Hyams, and M. K. May, P., D. Mulvihill, Y., Gachet, Z., T. Win, u .Q n olr,T D. T. Pollard, and Q. L. J. Wu, K. Gould, and A. B. Wolfe, eae ommainrokns n ytncdsrpykns,i eurdfor required is kinase, dystrophy myotonic and kinase rho mammalian to related cells. yeast fission living in fusions elrounding. cell A. A. Hyman, iaet ntecnrciern n fatnlk iaet ntemttcsideof spindle mitotic the in filaments actin-like of and ring contractile the in filaments meromyosin. yeast. i1gn sesnilfrcretpstoigo h iiinspu nfsinyeast. fission in septum division the of positioning correct Dev. for Genes essential is gene mid1 heavy- and light-chain regulatory by phosphorylation. dynamics chain ring contractile and function myosin-II iso es:terl ftecytoskeleton. the of role the yeast: fission yoiei nfsinyeast. fission in cytokinesis 19) yaiso elwl omto nfsinyat Schizosaccharomyces yeast, cytokinesis. fission during microtubules in formation wall myosin-heavy-chain cell pombe. of of Dynamics auto-inhibition (1998). relieves chain light function. regulatory myosin iso es,oeo hc ucin nplrzdcl rwhadmvsrpdyi the in rapidly moves and growth cell polarized in functions cell. which of one yeast, fission cioacaoye pombe. Schizosaccharomyces fctknssi epnet io etraino h eldvso ahnr in machinery division cell the of perturbation minor to pombe. response Schizosaccharomyces in cytokinesis of K. M. Balasubramanian, ln positioning. plane growth. cells. HeLa dividing cioacaoye ob:Mo2i ocre ihgot oaiyand yeast polarity fission ring. actin growth the cytokinetic with in the of functions concerned component a non-overlapping 69-79. is is with Myo51 Myo52 cytokinesis, myosins pombe: V Schizosaccharomyces type cycle. Two cell the with USA morphogenesis Sci. cell Acad. coordinates Natl. and Proc. polarity cell of maintenance nfsinyeast. fission in yeast. o.Bo.Cell Biol. Mol. a.Rv o.Cl Biol. Cell Mol. Rev. Nat. rnsCl Biol. Cell Trends uglGnt Biol. Genet. Fungal ur Biol. Curr. a.Cl Biol. Cell Nat. 10 rc al cd c.USA Sci. Acad. Natl. Proc. Nature 2707-2719. , 21) yrsai rsueadteatmoi otxdiemitotic drive cortex actomyosin the and pressure Hydrostatic (2011). 17) ci ndvdn el:cnrciern iaet idheavy bind filaments ring contractile cells: dividing in Actin (1973). Science Cell elgoer n eldvso 3857 division cell and geometry Cell 19 12 .Utatut o.Src.Res. Struct. Mol. Ultrastruct. J. 469 1096-1101. , 1367-1380. , o.Bo.Cell Biol. Mol. 144 15 20) qaoilrtnino h otatl ci igby ring actin contractile the of retention Equatorial (2003). 310 2 226-230. , 855-858. , 20) h l1/l1 hshts nue completion ensures phosphatase Clp1p/Flp1p The (2004). 10-18. , 414-426. , 20) onigctknsspoen lblyadlocally and globally proteins cytokinesis Counting (2005). 95 Nature 310-314. , 21) nesadn yoiei:lsosfo fission from lessons cytokinesis: Understanding (2010). 24 18) ragmn fatnflmnsadmyosin-like and filaments actin of Arrangement (1986). 20) ci yaisi h otatl igduring ring contractile the in dynamics Actin (2002). ehd Enzymol. Methods .Cl Sci. Cell J. 7526-7531. , 178-206. , 20) pi eiin:codntn yoiei in cytokinesis coordinating decisions: Split (2005). 11 Science 19) oeua eei nlsso iso yeast fission of analysis genetic Molecular (1991). 149-155. , 19) iso es r6 e/h rti kinase protein ser/thr a orb6, yeast Fission (1998). 419 Methods 20) dniiaino w yeVmoisin myosins V type two of Identification (2001). 21) nlec fcl emtyo division- on geometry cell of Influence (2011). 20 82-86. , 20) ehnclfre ffsinyeast fission of forces Mechanical (2009). 20) mgn re loecn protein fluorescent green Imaging (2004). 300 70 3941-3952. , 18) rwhplrt n yoiei in cytokinesis and polarity Growth (1986). 117 .Cl Sci Cell J. 1688-1692. , 1569-1574. , 33 3897-3910. , 220-225. , 20) euaino iso yeast fission of Regulation (2009). 194 795-823. , . 94 up.5 Suppl. 92-103. , 229-241. , 19) h dmf1/ The (1996). .Cl Sci. Cell J. 20) yeII Type (2000). (2001). 114 ,