External forces control mitotic spindle positioning

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1 A R T I C L E S Externl forces control mitotic spindle positioning Jenny Fink 1, Nicols Crpi 1, Timo Betz 2, Angelique Bétrd 2, Meriem Chebh 1, Ammr Azioune 1, Michel Bornens 1, Cecile Sykes 2, Luc Fetler 2, Dmien Cuvelier 2 nd Mtthieu Piel 1,3 The response of cells to forces is essentil for tissue morphogenesis nd homeostsis. This response hs been extensively investigted in interphse cells, but it remins uncler how forces ffect dividing cells. We used combintion of micro-mnipultion tools on humn dividing cells to ddress the role of physicl prmeters of the micro-environment in controlling the cell division xis, key element of tissue morphogenesis. We found tht forces pplied on the cell body direct spindle orienttion during mitosis. We further show tht externl constrints induce polriztion of dynmic subcorticl ctin structures tht correlte with spindle movements. We propose tht cells divide ccording to cues provided by their mechnicl micro-environment, ligning dughter cells with the externl force field. Proper control of the cell division xis is crucil for cell fte, development nd tissue orgniztion. In niml cells, it depends on externl stimuli tht polrize the cell cortex 1,2. This polriztion is trnsmitted to the mitotic spindle nd regultes its position, which determines the xis of cell division. In vivo, n symmetricl distribution of corticl cues usully results from cell s contcts with its dhesive micro-environment extrcellulr mtrix nd neighbouring cells 3 5. In vitro cultured cells, which lso exhibit regulted spindle orienttion 6,7, provide the opportunity to experimentlly ddress the contribution of physicl environmentl prmeters, such s force, cell shpe nd cell substrte dhesion, which re difficult to mesure nd modulte independently in vivo. Such prmeters re relevnt for the physiology nd morphogenesis of individul cells 8,9 nd recent studies hve strted to ddress their role in tissue morphogenesis nd polrity 1,11. However, most studies hve been conducted on interphse cells, leding to limited understnding of how mechnicl constrints cn trnslte into specific cell response process clled mechnotrnsduction 8,12. In previous work 7,13, we plced cells on dhesive microptterns, nd showed tht dhesion geometry lone cn dictte the xis of cell division in cultured cells. Here, we investigted how these geometricl cues control mitotic spindle orienttion. We show tht dhesion geometry imposes force field, which is signlling throughout mitosis to dynmiclly polrize the mitotic cell body, eventully leding to spindle orienttion. RESULTS The distribution of retrction fibres during mitosis dicttes spindle orienttion While cells round up in mitosis, they remin connected to the dhesive substrte through retrction fibres (Fig. 1), membrne tubes filled with ctin filments 14. These fibres hve been proposed to recruit polrizing fctors to the cell cortex, leding to spindle orienttion 13. However, retrction fibres my simply be historicl mrks of interphse cell polrity nd hve no role in spindle orienttion. To directly ddress this question we crried out lser bltion of retrction fibres during lte prometphse nd followed spindle orienttion by visuliztion of the chromosome plte. Cells were plted on two different dhesive micropttern shpes to control the sptil distribution of retrction fibres 15 : br, generting two opposite sets of retrction fibres, nd n symmetricl cross, generting four sets of retrction fibres (Fig. 1b,c). Without lser bltion, most mitotic spindles ligned with the long xis of the respective micropttern (Supplementry Fig. S1) showing only slight rottion between lte prometphse nd metphse (1 15, Fig. 1d). We observed no significnt spindle rottion when retrction fibres were blted on both sides of cells dividing on br ptterns, leving no retrction fibres on the cell periphery (16 ±12, Fig. 1b d nd Supplementry Fig. S2). Strong rottion of the mitotic spindle occurred only in cells dividing on cross ptterns, when retrction fibres fcing mitotic spindle poles were blted (76 ±21, Fig. 1b d nd Supplementry Fig. S2), leding to spindle lignment with the remining two sets of retrction fibres. Additionl effects of retrction fibre bltion were metphse dely probbly due to destbiliztion of the metphse plte (Fig. 2, second row t 24 min) nd significnt but trnsient deformtion of the cell body (Fig. 2,b). Those behviours occurred on both br nd cross ptterns, excluding their impct on the strong spindle rottion observed on cross ptterns. Thus, bltion experiments directly demonstrted tht retrction fibres provide externl cues during mitosis to orient the mitotic spindle. Retrction fibres exert strong forces on the mitotic cell body Cell deformtion on retrction fibre bltion indicted tht retrction fibres exert pulling forces on the cell body (Fig. 2,b). Furthermore, 1 Institut Curie, CNRS UMR 144, 26 rue d Ulm, Pris Cedex 5, Frnce. 2 Institut Curie, CNRS UMR 168, 26 rue d Ulm, Pris Cedex 5, Frnce. 3 Correspondence should be ddressed to M.P. (e-mil: mtthieu.piel@curie.fr) Received 16 July 21; ccepted 26 April 211; published online 12 June 211; DOI: 1.138/ncb2269 NATURE CELL BIOLOGY VOLUME 13 NUMBER 7 JULY Mcmilln Publishers Limited. All rights reserved.

2 A R T I C L E S MyrPlm GFP c Lser cut Lser cut Fibronectin Top view Side view n = 11 Lser cut Lser cut b n = 1 2' ' 2' 45" 6' 91' 111' d Totl rottion ( ) ' Figure 1 Retrction fibre distribution dicttes mitotic spindle orienttion. () HeL cell dividing on br-shped fibronectin micropttern (left) nd expressing MyrPlm GFP to visulize the plsm membrne (middle nd right). A mximum intensity projection (top view of the cell, middle) nd three-dimensionl reconstruction (side view, right) of 85 deconvoluted Z -stck plnes t.3 µm intervls re presented. (b) Mitotic HeL cells on br- (top row) or cross-shped (bottom row) microptterns (outlined by the ornge dshed line). Cells re expressing MyrPlm GFP (green) nd histone2b mcherry (red) to visulize plsm membrne/retrction fibres nd chromosomes. Retrction fibres orthogonl to the chromosome plte ' ' 2" 2' 94' 123' MyrPlm GFP Histone2B mcherry Br Cross Br Cross Control Abltion were lser blted (white rrows t min). To show both cell body nd retrction fibres, two Z plnes were superposed in the green chnnel for the first imge in the top row. Other imges re single confocl slices. Time is in min ( ) nd s ( ). (c) Observed chromosome plte orienttion (red lines) on br- nd cross-shped microptterns (ornge) before nd fter cutting. Retrction fibres/plsm membrne, green. (d) Totl rottion (finl initil ngle) of the metphse plte in control cells nd in cells fter retrction fibre bltion. n = 27 (br control), 69 (cross control), 11 (br bltion) nd 1 (cross bltion). Error brs represent stndrd error., P vlue <.1 (Student s t -test). Scle brs, 1 µm. it hs been shown tht cells spred long retrction fibres during telophse 14, implying tht retrction fibres cn ber the forces involved in post-mitotic cell respreding 16. Forces exerted by retrction fibres my thus be key polrizing signls during mitosis, similr to forces triggering mechnotrnsduction processes during interphse 8,12. Opticl tweezers mesurements crried out on retrction fibres confirmed tht they exerted strong pulling forces (Fig. 2c e; see Methods for detils). Fibres ccessible to tension mesurement hd n verge length of 13 ±3 µm (n = 2) from the substrtum to the cell body. They hd tension of 245±42 pn (n = 116 on 2 fibres), leding to totl force of bout 7 nn exerted on ech side of cell dividing on br-shped pttern (there is n verge of 27±3 retrction fibres per cell side (n = 26 on 13 cells)). Such forces cn significntly deform cells 17 nd re consistent with the cell body deformtion observed in metphse cells dividing on br-shped ptterns (6±.9%, n = 3, Fig. 2f nd Supplementry Fig. S3). Mitotic cells ttched to br ptterns were treted with drugs to inhibit ctin filment ssembly or myosin ii motor ctivity. This tretment led to n elongtion of the cell body in the xis of the retrction fibres. Conversely, treting cells with clyculin A, which increses myosin ctivity 18, resulted in significnt rounding of the cell body (Fig. 2f nd Supplementry Fig. S3). Mitotic cell shpe thus depends on internl contrctility cting ginst resisting forces exerted by retrction fibres ( detiled model of cell rounding hs recently been proposed 19 ). Retrction fibre bltion perturbed this force blnce, leding to cell contrction in the xis of the blted fibres nd/or expnsion in the perpendiculr direction (Fig. 2g). Cells then slowly dpted to the new force field, eventully becoming round gin, with timescle similr to interphse cells djusting their contrctility to externl lods 2. On the br pttern, no externl force remined fter bltion, wheres on the cross pttern cells contrcted ginst the remining retrction fibres perpendiculr to the initil xis, which ws followed by spindle rottion. We conclude tht given dhesion geometry, by imposing the distribution of retrction fibres during cell rounding, genertes specific force field on the mitotic cell body, which in turn leds to polrized contrction of the mitotic cell: the rounding cell pulls stronger in the xis where more retrction fibres re present. Externl forces exerted on mitotic cells cn induce spindle rottion To directly test the role of externl forces cting through retrction fibres, we crried out uni-xil stretch of 25% on mitotic cells dividing on fibronectin-coted silicon thin films (Fig. 3 nd Supplementry Fig. S4). Using ring-shped ptterns of fibronectin llowed pulling directly on retrction fibres. As the min drwbck of cell stretching is its direct effect on cell shpe, we used ellipticl ptterns designed to produce perfectly circulr ring fter the stretch, voiding the well-known effects of elongted cell shpe on spindle orienttion 21 (see Methods for detils). During uni-xil stretching, the ends of the retrction fibres in contct with the substrte followed the deformtion of the pttern, leding to n elongtion of retrction fibres ligned with the stretch nd shortening of orthogonl retrction fibres ( 25% stretching of the silicon substrte in one xis induces retrction of 1% in the perpendiculr direction). Other fibres tilted towrds the direction of the stretch (Fig. 3b) without moving their contct point on the mitotic cell body. Such observtions llowed us to estimte the force field resulting from the stretching process (Fig. 3c): we ssumed tht the ends of the retrction fibres bound to the substrte moved with the pttern deformtion wheres fibre ends on the cell cortex 772 NATURE CELL BIOLOGY VOLUME 13 NUMBER 7 JULY Mcmilln Publishers Limited. All rights reserved.

3 A R T I C L E S 2' 3' 16' 24' 56' 69' 96' 89' MyrPlm GFP Histone2B mcherry b c x d w l F t L F p 3 1. Br Cross Angle ( ) Time (min) Width/length rtio (w/l) Opticl tweezer e f g Tension (pn) Ctrl CytoD Bleb ClyA Frequency Width/length rtio (w/l) Figure 2 Retrction fibres exert strong forces on the mitotic cell body. () Mitotic HeL cells on br- nd cross-shped microptterns (outlined by ornge dshed lines) before nd fter bltion (white rrows) of retrction fibres t min. Cells expressing MyrPlm GFP (green) nd histone2b mcherry (red) to visulize membrne/retrction fibres nd chromosomes. The first imges show superposition of two Z plnes in the green chnnel. The other imges re single confocl slices. The first row shows the sme cell s in Fig. 1. Time is in min ( ). (b) Chromosome plte ngle (red) versus cell shpe (width/length rtio, green) before nd fter retrction fibre bltion. The plots correspond to cells shown in (br, dotted line; cross, solid line). (c) Forces in retrction fibres mesured with opticl tweezers. L, retrction fibre length; x, bed displcement; ~7 nn ~7 nn Lser bltion Contrction Adpttion F p, pulling force of opticl tweezers; F t, tension of retrction fibre. For x L, F t = F p L/(4x ). Green, membrne; violet, bed. (d) Phse-contrst imge showing cell body nd retrction fibres with n ttched bed. Opticl tweezers pulling direction (rrow). (e) Mesured tension in retrction fibres; n = 116 on 2 fibres. (f) Shpe fctor of drug-treted cells dividing on br-shped microptterns. n = 3 (control, Ctrl), 54 (cytochlsin D, CytoD), 55 (blebbisttin, Bleb) nd 43 (clyculin A, ClyA). Error brs represent stndrd error., P vlue <.1 (Student s t -test). (g) Model for cell shpe chnges fter lser bltion (blck rrows). Intercellulr contrctility (blue rrows). Resisting retrction fibres (green rrows). Membrne/retrction fibres, green; chromosome plte, red; micropttern, ornge. Scle brs, 1 µm. did not move, leding to well-defined tilt of the fibres. We ssigned to ech fibre tension equl to the mesured verge tension nd we clculted the resulting force in ech xis, tking into ccount the contct ngle of the retrction fibres with the cell body: fter stretching, the min force xis ws found to be prllel to the stretching direction. Mitotic spindles turned towrds the stretching xis, wheres in cells dividing on un-stretched ovl rings spindles ligned with the long xis of the pttern nd cells plted on un-stretched circulr rings exhibited rndom spindle orienttion (Fig. 3d f nd Supplementry Fig. S4b,c nd Movie S1). Importntly, when cells were treted with low doses of the microtubule-depolymerizing drug nocodzole, perturbing strl microtubules but llowing nphse to proceed, no rottion of the spindle ws induced by stretching (Fig. 3f nd Supplementry Fig. S4d). These results demonstrte tht the mitotic spindle rottes to lign with the forces produced by retrction fibres nd tht this rottion requires norml strl microtubule rry. Mitotic cells exhibit dynmic subcorticl ctin network bised by dhesion geometry Forces exerted on mitotic Dictyostelium discoideum cells induce recruitment of myosin ii nd of the ctin crosslinker cortexillin, followed by locl contrction 22. Moreover, in mitotic PtK2 cells myosin ii nd ctin ccumulte t the bse of retrction fibres 14. Forces exerted by retrction fibres my thus induce recruitment of ctomyosin structures, leding to cell polriztion nd spindle orienttion. Lbelling ctin filments in fixed mitotic cells dividing on br ptterns (Fig. 4,b) reveled polrized subcorticl ctin structures fcing retrction fibres. Surprisingly, these structures were often found only t one side of the cell (Fig. 4,b). Expression of Lifect mcherry 23 or GFP Utr-CH (the clponin homology domin of utrophin tgged with green fluorescent protein 24 ) both binding to ctin filments without ffecting their dynmics visulized these subcorticl structures nd showed tht they were highly dynmic in HeL nd RPE1 cells (Fig. 4c nd Supplementry Figs S5, S6 nd Movies S2 S6). Similr dynmic ctin structures hve recently been observed in vrious cell types nd hve been shown to specificlly pper in mitosis 25. Their pprent movement is the result of fst polymeriztion/depolymeriztion cycles 25. We observed tht cells dividing on disc ptterns, which exhibit homogeneous distribution of retrction fibres, showed circulting ctin subcorticl structures throughout mitosis, s reported previously 25 (Fig. 4c nd Supplementry Movie S2). On ptterns imposing n inhomogeneous distribution of retrction fibres, such s cross or br ptterns, ctin structures were lso dynmic, but ppered more intense when fcing retrction fibres (Fig. 4c nd Supplementry Movies S3 nd S4). To quntify this polriztion process, we nlysed kymogrphs of time-lpse imges of ctin structures, tken in the min xis of the pttern (or in the xis of the mitotic spindle for the disc), nd in the orthogonl xis (Fig. 5). We mesured the time ctin structures spent in ech sector of the cell (mrked S1 S4 in Fig. 5, quntified in Fig. 5b nd Supplementry Tble S1), their totl intensity in ech sector integrted in spce nd time (Fig. 5c nd Supplementry Tble S1) nd the depth t which they penetrte into the cytoplsm (Fig. 5d nd Supplementry Tble S1). These dt showed tht subcorticl ctin structures rotted below the cell cortex pproximtely t the sme speed (6 7 min 1, Supplementry Tble S1) for ll exmined pttern shpes. For cells dividing on disc ptterns, structures were s strong nd went s deep inside the cell t 9 of the spindle xis s they did in the xis of the spindle, thus showing no bis. However, in NATURE CELL BIOLOGY VOLUME 13 NUMBER 7 JULY Mcmilln Publishers Limited. All rights reserved.

4 A R T I C L E S Uni-xil stretch b Stretch c 5.7 e 4.7 GFP MyrPlm Before stretch14 min After stretch ' 14' Initil Initil No stretch Stretch Finl n = 13 Finl n = 12 d No stretch Stretch ' 3' 15' 18' 21' ' 3' 15' 18' 21' Figure 3 Stretching retrction fibres induces spindle rottion. () Cell stretching set-up. Membrne, green; micropttern, ornge. (b) Mitotic HeL cell expressing MyrPlm GFP to visulize membrnes. Mximum intensity projections of deconvoluted Z stcks. The insets show 1.5-fold mgnifiction. The dshed lines represent the orienttion of the retrction fibre before stretching. (c) Clculted force field before nd fter stretching. Blck rrows nd numbers represent totl forces (nn) in X nd Y xes. (d) RPE1 cells dividing on silicon substrtes with ovl ring microptterns (ornge). The pictures re superposition of phse-contrst imges nd the Hoechst signl (red) to visulize the DNA/chromosomes. Horizontl stretch (white rrow) just fter imge f α finl α initil Circulr ring Ovl ring No stretch Ovl ring to circulr ring Ovl to ovl Ovl ring to circulr ring, nocodzole Stretch cquisition t min. (e) Chromosome plte orienttion (red lines) in controls (upper row) nd stretched cells (lower row). The initil orienttion shows the orienttion in the first recorded imge nd the finl orienttion shows the orienttion t nphse onset. Membrne, green; pttern, ornge. (f) Sctter plot showing net rottions (α) of chromosome pltes for controls nd stretched cells. Negtive vlues represent rottion towrds (finl ngle for controls on ovl ring ptterns); positive vlues represent rottion towrds 9. n = 12 (circulr ring), 13 (ovl ring), 12 (ovl ring to circulr ring), 8 (ovl to ovl) nd 14 (ovl ring to circulr ring, nocodzole)., P vlue <.1 (Student s t -test). Time is in min ( ). Scle brs, 1 µm (b) nd 2 µm (d). the cse of the br nd the symmetric cross, they were both more intense nd went deeper inside the cell in the min xis of the pttern thn in the orthogonl xis. Results on disc ptterns proved tht the spindle lone did not induce the polriztion of the subcorticl ctin structures. Furthermore, when cells were treted with low doses of nocodzole leding to loss of spindle lignment with the br pttern, ctin polriztion remined in the xis of the retrction fibres nd did not follow the spindle poles (Fig. 6). Together our results show tht the intensity nd penetrtion depth of subcorticl ctin structures into the cytoplsm depend on the distribution of retrction fibres. Retrction fibres dynmiclly control subcorticl ctin polriztion in mitotic cells The impct of retrction fibres on subcorticl ctin cn be directly tested by lser bltion on cross shpes, s shown in Fig. 1. Indeed, fter bltion of retrction fibres fcing the spindle poles, ctin structures repolrized long the xis of the remining retrction fibres in 84% of cells dividing on cross ptterns (n = 19, Fig. 7 c nd Supplementry Movie S7). This ws lwys preceded by short period during which ctin structures ppered homogeneous long the cell periphery ( 11 min, Fig. 7d). When ll retrction fibres were blted (br pttern), ctin structures either lost their polriztion bis towrds the long xis of the br nd did not repolrize in the orthogonl xis (7/16 cells, Fig. 7,b nd Supplementry Movie S8) or kept bis in the originl xis (7/16 cells). We followed in prllel the mitotic spindle, lbelled by microtubule plus-end binding protein 3 (EB3) GFP. The spindle rotted by more thn 4 in 81% of cells tht showed repolriztion fter bltion on cross ptterns (verge rottion 56 ± 7, Fig. 7e), but only in 6% of cells fter bltion on br ptterns (verge rottion 15 ± 3). Importntly, spindle rottion lwys followed ctin repolriztion with n verge dely of 5 min (Fig. 7d), indicting tht ctin structure repolriztion my be responsible for spindle rottion. Subcorticl ctin structures exert pulling forces on the mitotic spindle In mny systems, spindle positioning depends both on ctin nd microtubules 1. Interestingly, more thn 2 yers go it ws observed tht centring forces cting on microtubule sters in snd dollr eggs were generted in the cytoplsm nd not t the cell cortex 26. This observtion hs recently been confirmed in other systems for mitotic spindle positioning 27,28. It is thus tempting to speculte tht motors bound to subcorticl or cytoplsmic ctin structures my be responsible for generting these cytoplsmic forces cting on strl microtubules in mitosis. Dynmic subcorticl/cytoplsmic ctin structures hve previously been described in mice meiotic oocytes 29,3 nd in mitotic cells in Xenopus embryos 31 nd they hve been proposed to ply role in spindle positioning nd elongtion, even if not necessrily cting through strl microtubules. Here we show tht in somtic mmmlin cells, such ctin structures lso 774 NATURE CELL BIOLOGY VOLUME 13 NUMBER 7 JULY Mcmilln Publishers Limited. All rights reserved.

5 A R T I C L E S Z projection Actin Actin Equtoril plne Microtubules Actin DNA 3.5 Microtubules c Equtoril plne Z projection Br ' 2' 4' 6' 15' b Percentge 5 25 Unipolr Bipolr Homogen Not ligned No structures Lifect mcherry Disc Cross Figure 4 Adhesion geometry cn bis dynmic subcorticl ctin structures in mitotic cells. () HeL cells dividing on 12 µm fibronectin lines were fixed, stined for ctin (phlloidin), microtubules nd DNA nd imged using spinning-disc confocl microscope. (b) Grph representing the distribution of subcorticl ctin structures in fixed HeL cells dividing on 12 µm lines (n = 7 from two independent experiments). Not ligned mens unipolr structure, but not ligned with the retrction fibre xis. Error brs represent stndrd error. ' 2' 4' 8' 15' ' 2' 4' 6' 28' (c) Mitotic HeL cells on br- (first row), cross- (second row) nd disc-shped (third row) microptterns (dshed ornge lines). Cells re stbly expressing Lifect mcherry to visulize filmentous ctin. The equtoril plne of cells ws imged during mitotic cell rounding. Imges for Z projections, on the right, were cquired t 1 µm intervls, fter complete cell rounding. Imges for cells dividing on br nd disc shpes were mirrored to lign with the cell on the cross shpe. Scle brs, 1 µm. Time is in min ( ). exist nd tht they re oriented by the dhesive geometry of the micro-environment, probbly due to externl forces cting on the cell body. When polrized, such structures my induce spindle movement nd lignment. As consequence, the mitotic spindle should move towrds newly formed ctin structures, leding to oscilltions in phse with oscillting ctin structures for cells dividing on br ptterns. We thus looked closely t time-lpse movies of cells expressing both n ctin mrker (Lifect mcherry) nd microtubule mrker (EB3 GFP). As expected the mitotic spindle often moved towrds subcorticl ctin structures (Fig. 8). To quntify this behviour, we nlysed kymogrphs of both Lifect mcherry nd EB3 GFP signls (see Methods, Fig. 8b e). We clculted, for ech occurrence of clerly polrized ctin structure, whether the net movement of the spindle ws towrds or wy from the structures. We found tht spindles moved towrds ctin structures in 66% nd wy from them in only 23% of ll cses (Fig. 8f,g). When strl microtubules were specificlly depolymerized with low doses of nocodzole, leding to mislignment of the spindle with the br pttern, this correltion ws lost (Fig. 8f,g). These results indicte tht subcorticl ctin structures, or ssocited protein complexes, probbly provide pulling forces ligning the mitotic spindle with the pttern xis. Cytoplsmic ctin structures ssembled nd disssembled on very short timescles, rotting t speed of bout 7 min 1 in cells plted on disc ptterns (or bout 12 µm min 1 long the cell periphery), wheres the mximum speed recorded for the spindle ws bout 8 min 1 (not shown). Cytoplsmic ctin structures thus moved t much higher speed thn the mitotic spindle. When ctin structures exhibit no bis s they trvel round the cell, short-timescle microtubule/ctin interctions would led to null verge force on the spindle, explining the reduced spindle rottion on disc ptterns 7. On the other hnd, when ctin structures re more dense, nd penetrte further inside the cell t given loctions, s observed t cell sides fcing retrction fibres on the br pttern, interction with strl microtubules would be more likely in these directions, resulting in net force ligning the spindle. DISCUSSION Here, we provide direct experimentl proof tht externl forces cn induce mitotic spindle rottion in humn cells. We propose tht these forces bis dynmic subcorticl ctin structures tht interct with strl microtubules, leding to mitotic spindle lignment with the externl force field. The effects of externl forces on polriztion of ctin structures hve been extensively studied in interphse cells 12,32, nd similr mechnisms my lso be ctive during mitosis to orient the mitotic spindle. Indeed, Src tyrosine kinses, known mechnosensors 8, hve been shown to control mitotic spindle orienttion 7, lthough the exct mechnism remins to be elucidted. Our results open directions for future reserch. Cytoplsmic ctin structures cting on the meiotic/mitotic spindle hve been identified in mice oocytes nd in Xenopus embryos 29,3. Our work, in greement with previous study 25, indictes tht these structures my be universl nd not restricted to erly stges of development. These structures, which hve been shown to depend on formins in mice oocytes, re dependent on ctin-relted protein 2/3 (Arp2/3) in somtic cells (our unpublished results nd ref. 25). It is possible tht both types of structure exist t different degrees in different cell types, with distinct functions in mitosis, similrly to wht is known for interphse cells. These Arp2/3-dependent structures were often more intense during the rounding process (dt not shown), indicting tht they depend on forces exerted on cells. Further experiments will be required to unrvel the moleculr links between these structures nd strl microtubules. Interestingly, in HeL cells it hs been observed tht wheres G protein α i1 (Gα i1 ) seems strictly corticl, Gα i2 lso exhibits strong cytoplsmic stining. In Fig. 3 of ref. 33, this stining ppers polrized, strongly resembling cytoplsmic ctin structures tht we hve described. This is indictive of n interesting dul corticl nd cytoplsmic recruitment of the clssicl Gα i LGN (G-protein signlling modultor 2) NuM (microtubule-binding nucler mitotic pprtus protein) pthwy on specific corticl nd subcorticl ctin structures, the second depending on externl forces cting on the mitotic cell. The dynmic NATURE CELL BIOLOGY VOLUME 13 NUMBER 7 JULY Mcmilln Publishers Limited. All rights reserved.

6 A R T I C L E S Lifect mcherry Time (15 min) Residence time (min) S1 S4 9 S3 S2 S1 S2 S3 9 S4 Distnce (28 μm) b 3 Residence time c Intensity d Penetrtion depth Distnce (μm) 7 Time (min) Reltive intensity (.u.) Penetrtion depth (μm) NS NS NS 2. 5 NS Br Cross Disc Br Cross Disc Br Cross Disc Figure 5 Quntifiction of subcorticl ctin polriztion for different dhesion geometries. () Kymogrphs of 7 pixels in width were creted from time-lpse movies (15 s) of cells expressing Lifect mcherry (top left picture). Cells were dividing on br-, cross- nd disc-shped ptterns (scheme: fibronectin pttern, ornge; ctin (retrction fibres, cortex), red; spindle, green; chromosome plte, blck). Kymogrphs were generted in the min xis of the retrction fibres (br nd cross shpe; ) or in the xis of the mitotic spindle (disc shpe; ) nd orthogonl to it (9 ) s indicted by the rrows (top left picture). The second row shows representtive exmple of the resulting kymogrphs for cell dividing on br-shped pttern. (b d) Residence time (b), totl intensity (c) nd penetrtion depth (d) of ctin structures mesured using kymogrphs generted long nd 9. The first row shows twofold mgnifiction of the white frme in. Blck lines symbolize the mesured height (residence time), re (intensity) nd width (penetrtion depth) of the Lifect mcherry signl. Grphs represent vlues for cells dividing on br-(n = 14), cross-(n = 13) nd disc-(n = 12) shped ptterns in nd 9 xes. Error brs represent stndrd error. NS, not significnt., P =.18 in c;, P <.1 (br) nd P =.1 (cross) in d (Student s t -test). nture of these structures lso remins to be understood. Dynmic structures rther thn sttic ones my improve spindle centring: if ctin structures recruit microtubule nchoring nd stbilizing fctors, sttic polrized ctin meshwork is likely to move the spindle off centre, which my induce symmetric cell division. It is lso possible tht spindle rottion is more efficient when strl microtubules from both spindle poles re lterntively being pulled on, wheres single sttic polrized structure would move the spindle rther thn rotte it efficiently. A full understnding of the interction of dynmic strl microtubules with dynmic ctin structures bering pulling forces will require both mthemticl modelling nd further more-direct experiments. It is well known tht growth nd movements in tissues generte locl constrints on cells 1,11. Our results indicte tht these constrints my guide the cell division xis nd thus be importnt for morphogenesis. In plnts, constrints generted by growing cells hve been shown to influence the cell growth xis, such feedbck being enough to explin lrge prt of the morphogenetic process 34. In growing niml tissues, cells in certin regions (most likely peripherl regions) my be subjected to extension forces, wheres others (most likely centrl regions) my be under compression. Aligning the cell division xis with the locl force field my relese the stress stored during the b 2 nm nocodzole; spindle misligned Residence time (min) Time (15 min) 9 Lifect mcherry EB3 GFP NS ' 2' 4' 6' 8' (Lifect mcherry) Ctrl NZ Distnce (28 μm) NS c Reltive intensity (.u.) NS 9 9 Ctrl NZ d Penetrtion depth (μm) Chnge in focus 9 9 Ctrl NZ Figure 6 Polriztion of dynmic subcorticl ctin structures persists when strl microtubules re depolymerized nd the spindle is misoriented. () HeL cell stbly expressing Lifect mcherrry nd EB3 GFP to visulize filmentous ctin nd spindle poles, respectively. The cell is dividing on br-shped pttern s shown in the scheme (top, fibronectin pttern, ornge; ctin (retrction fibres, cortex), red; spindle, green; chromosome plte, blck). As result of tretment with low doses of nocodzole (2 nm), the spindle is misligned during mitosis (middle). Kymogrphs were generted in the Lifect mcherry chnnel long the retrction fibre xis ( ) of the br pttern nd orthogonl to it (9 ). Although the spindle poles re ligned with the 9 xis, polriztion of subcorticl ctin structures remins in the xis of the retrction fibres (, bottom). (b d) Grphs representing residence time (b), totl intensity (c) nd penetrtion depth (d) of ctin structures long nd 9 xes in HeL cells dividing on br-shped pttern (see Fig. 5 for mesurement). Cells were treted with either dimethylsulphoxide (Ctrl,.2%, n = 14) or nocodzole (NZ, 2 nm, n = 15). Error brs represent stndrd error. NS, not significnt., P <.1 for Ctrl nd P =.6 for NZ (Student s t -test). Scle br, 1 µm. Time is in min ( ). growth process in regions in which cells re in extension. This would fluidize the tissues, llowing it to undergo extensive deformtion while growing 35. Such process is predicted to be especilly importnt for fst-growing tissues with short cell division times, undergoing lrge deformtions, typicl of erly development of vertebrte embryos, for which internl constrints due to growth cnnot be compensted by cell cell rerrngements. We propose tht cells hve evolved to use ll possible sources of informtion, including mechnicl signls, leding to more robust determintion of their division xis. METHODS Methods nd ny ssocited references re vilble in the online version of the pper t Note: Supplementry Informtion is vilble on the Nture Cell Biology website ACKNOWLEDGEMENTS We re grteful to J. Colombelli for helpful discussions nd preliminry experiments on retrction fibre cutting; L. Mhdevn for helpful discussions concerning mechnicl cortex nisotropy; D. Gerlich (ETH, Zurich, Switzerlnd) nd R. Tsien (UCSD, Sn Diego, USA) for the pires-puro3 MyrPlm GFP plsmid; V. Doye (IJM, Pris, Frnce) for the pires-neo histone2b mcherry plsmid; W. Bement (University of Wisconsin, Mdison, USA) for the GFP Utr-CH plsmid; R. Wedlich- Soldner (IMPRS, Mrtinsried, Germny) nd G. Montgnc (Institut Curie, Pris, Frnce) for the Lifect mcherry plsmid; M. Heuze (Institut Curie, Pris, Frnce) nd A. M. Lennon-Dumesnil (Institut Curie, Pris, Frnce) for the Lifect mcherry lentivirus; V. Frisier, the Nikon Imging Center nd the PICT IBiSA of the 776 NATURE CELL BIOLOGY VOLUME 13 NUMBER 7 JULY Mcmilln Publishers Limited. All rights reserved.

7 A R T I C L E S c Retrction fibre bltion on br 1" +5" Retrction fibre bltion on cross Percentge of cells showing ctin repolriztion " +5" Br Cross d e Totl rottion ( ) Time (min) t 1(repol) t 2(rot) Br Cross t 2 t 1 b d Lifect mcherry EB3 GFP Ctrl 2 Lifect signl (.u.) Left side Equtoril plne " " 3" 75" 15" 135" c Time (min) Right side Length (μm) e Spindle pole displcement (μm) Left pole Right pole b EB3 Lifect EB3 Lifect After bltion br 1' 21' 25' 29' 37' 45' After bltion cross 1' 21' 25' 29' 37' 45' Figure 7 Mitotic spindle rottion nd movement strongly correlte with polriztion of dynmic subcorticl ctin structures. () Abltion of retrction fibres (white rrows) in cells dividing on br (top pnel) nd cross ptterns (bottom pnel). Retrction fibres re visulized by Lifect mcherry expression nd re cut t s. Ptterns, dshed ornge lines. (b) Evolution of subcorticl ctin structures (Lifect mcherry) nd the mitotic spindle (EB3 GFP) fter retrction fibre bltion of the cells shown in. Cell dividing on br pttern (top pnel); cell dividing on cross pttern (bottom pnel). White rrows highlight regions with higher density of subcorticl ctin structures. (c) Quntifiction of ctin structure behviour fter bltion of retrction fibres. Actin repolriztion mens polriztion of subcorticl structures in the xis orthogonl to the blted retrction fibres. Repolriztion ws tken into ccount only when lsting longer thn 1 consecutive minutes. n = 16 for br shpe; n = 19 for cross shpe., P = (Chi-squre test). (d) Grph representing the time when ctin strts to repolrize in the xis orthogonl to retrction fibre cutting (t 1(repol) ), time when the spindle strts to rotte (t 2(rot) ) nd the dely between the strt of ctin repolriztion nd the strt of spindle turning (t 2 t 1 ). Abltion is t t =. (e) Quntifiction of spindle behviour fter bltion of retrction fibres. Totl rottion mens mximum spindle rottion towrds the xis orthogonl to retrction fibre cutting. n = 16 for br shpe; n = 19 for cross shpe. Error brs represent stndrd error., P <.1 (Student s t -test). Scle brs, 1 µm. Time is in s (, ) nd min (, b). Institut Curie for technicl support in microscopy; J. Boulnger for imge tretment using ndsfir; nd Z. Mcirowski, C. Guérin nd A. Viguier for FACS sorting of stble cell lines. We thnk M. Thery nd J. Aubertin for helpful discussions throughout the course of this work nd A. W. Murry, E. Pluch, A. M. Lennon- Dumesnil, A. Tddei, M. Thery, S. Misery-Lenkei nd members of the Piel lbortory for criticl reding of the mnuscript. This work ws supported by the Centre Ntionl de l Recherche Scientifique, the Institut Curie nd by ANR (ANR-6-PCVI-1) nd HFSP grnts to M.P. J.F. ws supported by pre-doctorl fellowships from Boehringer Ingelheim Fonds nd the Assocition pour l Recherche sur le Cncer. f Time (min) Time (1 min) 5 1 Length (25 μm) Ctrl NZ Time (min) g 5 1 Percentge Ctrl NZ Anti-correlted movement No movement Correlted movement Figure 8 Subcorticl ctin structures exert pulling forces on the mitotic spindle. () Time-lpse sequence of HeL cell stbly expressing Lifect mcherrry nd EB3 GFP. The white sterisk mrks the position of the spindle pole t time zero. Scle brs, 1 µm (first imge) nd 2 µm (second imge). Time is in s ( ). (b) A HeL cell dividing on br pttern (s shown in the scheme: fibronectin pttern, ornge; ctin (retrction fibres, cortex), red; spindle, green; chromosome plte, blck) nd stbly expressing Lifect mcherry nd EB3 GFP to visulize ctin filments nd spindle poles. Equtoril plnes were imged every 15 s using spinning-disc confocl microscope. Kymogrphs of 7 pixel in width were creted prllel to the spindle s indicted by the white br nd rrows in the picture. Scle br, 1 µm. (c) Kymogrph resulting from b. The dshed white res highlight the ctin signl; the white lines highlight spindle boundries. (d) Lifect mcherry temporl profile corresponding to the kymogrph shown in c (red signl) for the right nd the left side of the cell. (e) Spindle pole displcement corresponding to the kymogrph shown in c s determined by utomted detection of the spindle boundry in the kymogrph (green signl). Note tht the spindle lwys chnges direction with dely, to eventully move towrds the structures. (f) A HeL cell dividing on br-shped pttern s shown in b. Kymogrphs were generted long the retrction fibre xis in the Lifect mcherry nd EB3 GFP chnnel ccording to b. Resulting kymogrphs of control cells (Ctrl, first row) nd nocodzole-treted (NZ, 2 nm, second row) cells. Note tht the spindle does not oscillte nymore in NZ-treted cells. (g) Quntifiction of spindle pole movements with respect to subcorticl ctin structures of control- (Ctrl, n = 47 for 12 cells) nd nocodzole- (NZ, 2 nm; n = 42 for 11 cells) treted cells; correlted movement mens spindle movement towrds ctin structures; nti-correlted mens movement wy from ctin structures., P vlue is.14 (Chi-squre test). AUTHOR CONTRIBUTIONS J.F. designed, crried out nd nlysed most experiments nd wrote the rticle, N.C. crried out most cell stretching experiments s well s experiments shown in Fig. 2f nd Supplementry Fig. S3, T.B. crried out nd nlysed opticl trp experiments (Fig. 2c e), A.B. crried out some cell stretching experiments, M.C. crried out the experiments shown in Fig. 2f nd Supplementry Fig. S3, A.A. developed the method to produce microptterns on stretchble substrtes, M.B. contributed ides, NATURE CELL BIOLOGY VOLUME 13 NUMBER 7 JULY Mcmilln Publishers Limited. All rights reserved.

8 A R T I C L E S discussion nd supervised prt of the work of J.F., C.S. contributed ides nd discussion nd supervised the work on opticl trp experiments, L.F. crried out lser bltion experiments (Fig. 1), D.C. set up the cell stretching device, supervised the work of A.B. nd contributed ides nd discussion, nd M.P. supervised the work, crried out experiments nd wrote the pper. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Published online t Reprints nd permissions informtion is vilble online t reprints 1. Gönczy, P. Mechnisms of symmetric cell division: flies nd worms pve the wy. Nt. Rev. Mol. Cell Biol. 9, (28). 2. Ahringer, J. Control of cell polrity nd mitotic spindle positioning in niml cells. Curr. Opin. Cell Biol. 15, (23). 3. Lechler, T. & Fuchs, E. Asymmetric cell divisions promote strtifiction nd differentition of mmmlin skin. Nture 437, (25). 4. Lu, B., Roegiers, F., Jn, L. Y. & Jn, Y. N. Adherens junctions inhibit symmetric division in the Drosophil epithelium. Nture 49, (21). 5. Fernndez-Minn, A., Mrtin-Bermudo, M. D. & Gonzlez-Reyes, A. Integrin signling regultes spindle orienttion in Drosophil to preserve the folliculrepithelium monolyer. Curr. Biol. 17, (27). 6. Toyoshim, F. & Nishid, E. Integrin-medited dhesion orients the spindle prllel to the substrtum in n EB1- nd myosin X-dependent mnner. EMBO J. 26, (27). 7. Thery, M. et l. The extrcellulr mtrix guides the orienttion of the cell division xis. Nt. Cell Biol. 7, (25). 8. Vogel, V. & Sheetz, M. Locl force nd geometry sensing regulte cell functions. Nt. Rev. Mol. Cell Biol. 7, (26). 9. Terenn, C. R. et l. Physicl mechnisms redirecting cell polrity nd cell shpe in fission yest. Curr. Biol. 18, (28). 1. Frhdifr, R., Roper, J. C., Aigouy, B., Eton, S. & Julicher, F. The influence of cell mechnics, cell cell interctions, nd prolifertion on epithelil pcking. Curr. Biol. 17, (27). 11. Ruzi, M., Vernt, P., Lecuit, T. & Lenne, P. F. Nture nd nisotropy of corticl forces orienting Drosophil tissue morphogenesis. Nt. Cell Biol. 1, (28). 12. Woznik, M. A. & Chen, C. S. Mechnotrnsduction in development: growing role for contrctility. Nt. Rev. Mol. Cell Biol. 1, (29). 13. Thery, M., Jimenez-Dlmroni, A., Rcine, V., Bornens, M. & Julicher, F. Experimentl nd theoreticl study of mitotic spindle orienttion. Nture 447, (27). 14. Crmer, L. P. & Mitchison, T. J. Myosin is involved in postmitotic cell spreding. J. Cell Biol. 131, (1995). 15. Azioune, A., Storch, M., Bornens, M., Thery, M. & Piel, M. Simple nd rpid process for single cell micro-ptterning. Lb. on chip 9, (29). 16. Burton, K. & Tylor, D. L. Trction forces of cytokinesis mesured with opticlly modified elstic substrt. Nture 385, (1997). 17. Guck, J. et l. The opticl stretcher: novel lser tool to micromnipulte cells. Biophys. J. 81, (21). 18. Chrtier, L. et l. Clyculin-A increses the level of protein phosphoryltion nd chnges the shpe of 3T3 fibroblsts. Cell Motil. Cytoskeleton 18, 26 4 (1991). 19. Stewrt, M. P. et l. Hydrosttic pressure nd the ctomyosin cortex drive mitotic cell rounding. Nture 469, (211). 2. Jungbuer, S., Go, H., Sptz, J. P. & Kemkemer, R. Two chrcteristic regimes in frequency-dependent dynmic reorienttion of fibroblsts on cycliclly stretched substrtes. Biophys. J. 95, (28). 21. O Connell, C. B. & Wng, Y. L. Mmmlin spindle orienttion nd position respond to chnges in cell shpe in dynein-dependent fshion. Mol. Biol. Cell 11, (2). 22. Effler, J. C. et l. Mitosis-specific mechnosensing nd contrctile-protein redistribution control cell shpe. Curr. Biol. 16, (26). 23. Riedl, J. et l. Lifect: verstile mrker to visulize F-ctin. Nt. Methods 5, (28). 24. Burkel, B. M., von Dssow, G. & Bement, W. M. Verstile fluorescent probes for ctin filments bsed on the ctin-binding domin of utrophin. Cell Motil. Cytoskeleton 64, (27). 25. Mitsushim, M. et l. Revolving movement of dynmic cluster of ctin filments during mitosis. J. Cell Biol. 191, (21). 26. Hmguchi, M. S. & Hirmoto, Y. Anlysis of the role of strl rys in pronucler migrtion in snd dollr eggs by the colcemid-uv method. Dev. Growth Differ. 28, (1986). 27. Minc, N., Burgess, D. & Chng, F. Influence of cell geometry on division-plne positioning. Cell 144, (211). 28. Wuhr, M., Tn, E. S., Prker, S. K., Detrich, H. W. 3rd & Mitchison, T. J. A model for clevge plne determintion in erly mphibin nd fish embryos. Curr. Biol. 2, (21). 29. Azoury, J. et l. Spindle positioning in mouse oocytes relies on dynmic meshwork of ctin filments. Curr. Biol. 18, (28). 3. Schuh, M. & Ellenberg, J. A new model for symmetric spindle positioning in mouse oocytes. Curr. Biol. 18, (28). 31. Woolner, S., O Brien, L. L., Wiese, C. & Bement, W. M. Myosin-1 nd ctin filments re essentil for mitotic spindle function. J. Cell Biol. 182, (28). 32. Yoshigi, M., Hoffmn, L. M., Jensen, C. C., Yost, H. J. & Beckerle, M. C. Mechnicl force mobilizes zyxin from focl dhesions to ctin filments nd regultes cytoskeletl reinforcement. J. Cell Biol. 171, (25). 33. Woodrd, G. E. et l. Ric-8A nd Gi lph recruit LGN, NuMA, nd dynein to the cell cortex to help orient the mitotic spindle. Mol. Cell Biol. 3, (21). 34. Hmnt, O. et l. Developmentl ptterning by mechnicl signls in Arbidopsis. Science 322, (28). 35. Rnft, J. et l. Fluidiztion of tissues by cell division nd poptosis. Proc. Ntl Acd. Sci. USA 17, (21). 778 NATURE CELL BIOLOGY VOLUME 13 NUMBER 7 JULY Mcmilln Publishers Limited. All rights reserved.

9 DOI: 1.138/ncb2269 M E T H O D S METHODS Cell culture, plsmid trnsfections nd DNA lbelling. HeL cells were cultured in DMEM/Glutmx (Gibco) with 1% fetl clf serum (FCS) nd ntibiotics (penicillin/streptomycin; Gibco). htert-rpe1 cells were cultured in DMEM-F12 (Gibco) with 1% FCS, 2 mm glutmine nd ntibiotics (penicillin/streptomycin). Lser bltion experiments, requiring stble expression of fluorescent mrkers, were crried out in HeL cells. Stretching experiments were crried out in both HeL (Fig. 3b) nd RPE1 cells (Fig. 3d f nd Supplementry Fig. S4). HeL nd RPE1 cells were cultured in Leibovitz s CO 2 -independent medium without phenol red during live-cell imging nd micro-mnipultion experiments. Plsmid trnsfection ws crried out using Lipofectmine LTX nd Plus regent (Invitrogen). pbos-histone2b mcherry IRES neo, pires-puro MyrPlm GFP, pires-puro Lifect mcherry nd pegfp-n3 EB3 plsmids were used. Positive cells were selected with G418 (.5 mg ml 1 ) or puromycin (.33 µg ml 1 ) nd subsequent fluorescence-ctivted cell sorting (FACS). The pcs2 GFP Utr-CH vector ws trnsiently trnsfected. To generte stble RPE1 cell line, we infected RPE1 cells with vesiculr stomtitis virus G (VSVG)-pseudotyped humn immunodeficiency virus (HIV)- derived lentivirus contining the Lifect mcherry DNA. Positive cells were selected for medium expression of Lifect mcherry using FACS sorting. DNA in living RPE1 cells ws stined with.1 µg ml 1 Hoechst for 15 min. Cell fixtion nd stining. All solutions were mde using BRB8 buffer. Cells were pre-extrcted nd fixed for 1 min using 4% prformldehyde,.25% glutrldehyde nd.5% NP-4, then fixed for 1 min in 4% prformldehyde nd.25% glutrldehyde nd finlly treted with.1 M NH 4 Cl for 1 min. Actin nd DNA were stined using AlexFluor594-phlloidin (Invitrogen) nd DAPI (4,6-dimidino-2-phenylindole). Microtubules were immunolbelled using nti-α-tubulin (1:2,, mouse, clone B-5-1-2, Sigm) nd secondry nti-mouse- Alex488 ntibody (Jckson ImmunoReserch Lbortories). Drug tretments. Cells were incubted with 5 µm blebbisttin, 1 µg ml 1 cytochlsin D nd 2 nm nocodzole, for 3 min, or 1 nm clyculin A for 15 min before fixtion. For specific depolymeriztion of strl microtubules, HeL nd RPE1 cells were incubted with 2 nm nocodzole for 1 h before imge cquisition. This concentrtion, lthough severely ffecting strl microtubules (Supplementry Fig. S4d), did not prevent RPE1 cells from entering nphse with norml timing. Microptterning on glss nd flexible substrtes. Adhesive fibronectin microptterns were produced using deep-ultrviolet illumintion through photomsk s previously described 15. To bind PLL-g-PEG (poly(l-lysine)-grftedpoly(ethylene glycol)), silicon membrnes were illuminted for 5 min using deep ultrviolet nd incubted with solution of N -hydroxysuccinimide (16 mm) nd N -(3-dimethylminopropyl)-N -ethyl-crbodiimide-hydrochloride (35 mm) for 3 min. After quick rinsing, PLL-g-PEG (1 mg ml 1 in 1 mm HEPES, ph 8.5) ws incubted for 1 h (ref. 36). Abltion experiments. Abltion experiments (Figs 1, 2 nd Supplementry Fig. S2) were crried out on mitotic cells during lte prometphse, s soon s cler chromosome plte could be detected. The two-photon lser scnning microscope set-up used ws n LSM51 Met (Zeiss) equipped with 4.8 wter immersion objective nd coupled to Miti DeepSee femtosecond lser (69 1,2 nm; Spectr-Physics) nd 633 nm HeNe lser. Abltion ws crried out in the cell dhesion plne. Noise ws removed from the cquired pictures using the despeckle function of ImgeJ. For bltion in cells co-expressing Lifect mcherry nd EB3 GFP (Fig. 7 nd Supplementry Movies S7, S8), we used Yokgw CSU-X1 spinning hed mounted on n Eclipse Ti inverted microscope (Nikon), coupled to n EMCCD (electron-multiplying chrge-coupled device) cmer (Evolve; Photometrics), nd Oil Iris objective (S Fluor, Nikon). Abltion ws crried out 2 3 µm bove the cell dhesion plne using n ils system interfced with pulsed 355 nm ultrviolet lser (Roper Scientific). The pulse ws 4 ps t 1 mw, 1 µj per pulse, scnning t 25 µs per pixel. Mesurement of retrction fibre tension using opticl tweezers. Crboxylted polystyrene beds with dimeter of.958 µm (Polyscience) were trpped nd clibrted using the power spectrl density method 37. The typicl trp stiffness ws 5 fn nm 1. The bed ws plced on fibre for bout 5 min. After the connection ws tested, the bed ws displced 1 nm perpendiculr to the length of the fibre, following step protocol. This protocol gives the pulling force (F p ) nd the totl distortion (x) of the fibre. The tension (F t ) depends, in the first order, on the displcement x following the reltion F t = F p L/(4x). In the regime of smll extension, the elsticity of the fibre does not contribute to the pulling force. Cell stretching on flexible substrtes. Fibronectin microptterns were generted on thin silicon membrnes (Gel pk PF-6-X4; thickness: 15 µm; Teltek). Membrnes were clmped in custom-mde device llowing membrne stretching using micrometric screw, with mximl extension of 25% in 3 s. A rectngulr polydimethylsiloxne chmber ws ttched onto the membrne using vcuum grese, nd cells were cultivted for t lest 4 h before stretching. Stretching ws crried out in lte prometphse, s soon s cler chromosome plte could be detected. Only cells tht hd been stretched more thn 1 min before nphse were considered for nlysis. For microptterning, membrnes were stretched to mximum extension nd circulr ring-shped microptterns were creted through deep-ultrviolet illumintion, using smll photomsk. The membrne ws then un-stretched, leding to ovl ring-shped microptterns on which cells were seeded. Stretching to mximum extension during the experiment resulted gin in circulr ringshped microptterns, excluding ny effect of the dhesive pttern geometry nd subsequent cell elongtion on the observed spindle rottion 21,38,39. Using ringshped microptterns furthermore prevented cell dhesion below the cell body, so stretching ws trnsmitted to the mitotic cell body only through retrction fibres. Force estimtion for stretching experiments. To estimte the chnge in the force field generted by retrction fibres on stretching, simple mthemticl model of the cell s three-dimensionl (3D) sphere connected by fibres to 2D ellipticl pttern ws built using MATLAB softwre. Attchment points of the fibres to the cell body were plced round the cell equtor, s observed in Fig. 1. The cell ws ssumed to hve 1 retrction fibres with homogeneous density on the pttern, ttched t the closest point on the cell body equtor (s in ref. 13). The pttern ws deformed to circulr ring nd the fibre position clculted ssuming no gliding of the fibre ttchment point on the cell body, s shown in Fig. 3b. Ech fibre ws ssigned tension corresponding to the men vlue mesured in Fig. 2e, nd the projected forces on the X nd Y xes were clculted. Live-cell imging. Cell recordings were done using Yokgw CSU-X1 spinning hed mounted on n Eclipse Ti inverted microscope (Nikon) for high-resolution imging ( 1 objective: Figs 1, 4 nd Supplementry Figs S3 nd S4d; 6 objective: Figs 4c, 5, 6, 8 nd Supplementry Figs S5, S6; or 2 objective: Fig. 3b), using n Eclipse TE 2-E inverted microscope (Nikon) for recording chromosome plte movements ( 1 objective: Supplementry Fig. S1) nd using n Axio Observer inverted microscope (Zeiss) for recording chromosome plte behviour fter cells stretching ( 2 objective; DNA stined with blue-fluorescing Hoechst stin, Fig. 3d). Microscopes were equipped with Coolsnp HQ2 cmer (Roper Scientific) nd were controlled either by the Metmorph softwre (Universl Imging) or by the Axio vision softwre (Zeiss). To llow visuliztion of retrction fibres nd subcorticl ctin structures tht re less bright thn the ctin cortex itself, the gmm vlue ws decresed uniformly for the following imges: Figs 1b, 4,c, 5 first row, 6 second row, Figs 7,b, 8,b, Supplementry Fig. S5, left column nd Fig. S6. Imge tretment using ndsfir softwre. Imges in Fig. 7,b, in Supplementry Movies S7, S8 nd imges showing Lifect stining in RPE1 cells (Supplementry Fig. S6) were denoised in 2D + time using ndsfir 4 (ptch size = 3, itertions = 8 nd Poisson Gussin vrince stbiliztion). A weighted verge between the originl nd the denoised imges ws obtined using 3% of the originl nd 7% of the denoised version. Mesure of subcorticl ctin structure residence time, intensity nd penetrtion depth into the cytoplsm. HeL cells expressing Lifect mcherry were recorded t single confocl plne t rte of one imge every 15 s. Kymogrphs (7 pixel width) were generted long the min xis of the retrction fibres (br nd cross shpe) or in the xis of the mitotic spindle (disc shpe) nd orthogonl to it (Fig. 5). Ech pir of resulting kymogrphs ws scled similrly, thresholded nd boundries of subcorticl ctin structures were detected mnully. The ctin cortex ws not tken into ccount. Assigned regions were then mesured for height (residence time), width (penetrtion depth into cytoplsm) nd integrted intensity. Mesure of spindle movements. Cells expressing both Lifect mcherry nd EB3 GFP were recorded t single confocl plne t rte of one imge in ech colour every 15 s. Cells with mitotic spindle tht ws well formed nd roughly ligned with the xis of the br pttern were nlysed. A kymogrph ws mde from line (7 pixels in width) ligned with the br pttern long xis (Fig. 8b,c). The vlues on the width of the line were verged. From this kymogrph, we extrcted both the temporl signl of ctin on ech side of the cell using line scn (Fig. 8d) nd the position of ech pole of the mitotic spindle fter NATURE CELL BIOLOGY 211 Mcmilln Publishers Limited. All rights reserved.

10 M E T H O D S DOI: 1.138/ncb2269 imge thresholding (Fig. 8e). A MATLAB routine clculted the slope of robust liner fit of the spindle position (verge of the position of both poles) in the time intervl corresponding to ech pek of the Lifect mcherry signl. Slopes bove 1% were considered significnt nd were counted either s nti-correlted when moving wy from, or correlted when moving towrds, ctin structures. Sttistics. Men±stndrd error ws presented, except in Supplementry Fig. S2c, where men±s.d. ws presented. To determine the significnce between two groups, indicted in figures by two (P vlues.2) or three sterisks (P vlues.1), Student s t-test or the Chi-squre test ws used, s indicted in the figure legends. P vlues.5 were considered nonsignificnt. 36. Azioune, A., Crpi, N., Tseng, Q., Thery, M. & Piel, M. Protein microptterns: direct printing protocol using deep UVs. Methods Cell Biol. 97, (21). 37. Tolic-Norrelykke, S. F. & Schffer, E. Clibrtion of opticl tweezers with positionl detection in the bck focl plne. Rev. Sci. Instrum. 77, (26). 38. Mniotis, A. J., Chen, C. S. & Ingber, D. E. Demonstrtion of mechnicl connections between integrins, cytoskeletl filments, nd nucleoplsm tht stbilize nucler structure. Proc. Ntl Acd. Sci. USA 94, (1997). 39. Hertwig, O. Ueber den Werth der ersten Furchungszellen für die Orgnbildung des Embryo. Experimentelle Studien m Frosch-und Tritonei. Arch. Mikrosk. Ant. 42, (1893). 4. Boulnger, J. et l. Ptch-bsed nonlocl functionl for denoising fluorescence microscopy imge sequences. IEEE Trns. Med. Imging 29, (21). 211 Mcmilln Publishers Limited. All rights reserved. NATURE CELL BIOLOGY

11 SUPPLEMENTARY INFORMATION DOI: 1.138/ncb2269 Br Initil orienttion (Lte prometphse) % 18 4 Finl Orienttion (Anphse) % n = 27 Cross % % n = 69 Figure S1 Chromosome plte orienttions of HeL cells dividing on br nd cross-shped micro ptterns. () HeL cells expressing Histone-2B-mCherry to visulize chromosomes were recorded every 3 min during division. Circulr grphs show the ngulr distribution of chromosome plte orienttions (red) of cells dividing on br or cross-shped micro ptterns (ornge). Chromosome plte orienttions in prometphse (initil orienttion) nd one time point before nphse (finl orienttion) re represented in ngulr sectors of 3 o Mcmilln Publishers Limited. All rights reserved.

12 SUPPLEMENTARY INFORMATION Width/Length rtio Br 5 time (min) 1 Angle ( ) Br 5 time (min) 1 b Width/Length rtio t mx Cross 5 time (min) 1 Angle ( ) t strt turn Cross 5 time (min) 1 c W/L initil W/L mx W/L strt turn t mx t strt turn br.89 +/ / /- 5 - n = 9 cross.99 +/ / / / /- 22 n = 7 Figure S2 Cell body shpe nd chromosome plte ngle fter bltion of retrction fibres during mitosis. (+b) Width/length rtio of the mitotic cell (left) nd chromosome plte orienttion (right) fter retrction fibers bltion (t time min), for single cells dividing on br () nd cross (b) shpe. Thick lines represent verge curves. (c) Tble shows men +/- stndrd devition vlues of initil width/length rtio of the mitotic cell before lser bltion (W/L initil ), mximum width/length rtio fter lser bltion (W/L mx ), width/ length rtio t beginning of spindle rottion (W/L turn ), time until mximum width/length rtio ws reched (t mx ), nd time until spindle strts turning (t turn ) Mcmilln Publishers Limited. All rights reserved.

13 SUPPLEMENTARY INFORMATION GFP-MyrPlm Histone-2B-mCherry Overly Clyculin A Blebbisttin Cytochlsin D Control Figure S3 Actin nd myosin 2 re required to mintin sphericl cell body shpe during metphse. HeL cells expressing MyrPlm-GFP nd Histone-2B-mCherry to visulize membrne nd chromosomes were llowed to round up during mitosis nd were subsequently treted for 15-3 min with different drugs cting on the cto-myosin system: control (first row), 1 µg/ml cytochlsin D (3 min, second row), 5 µm Blebbisttin (3 min, third row), 1 nm Clyculin A (15 min,fourth row). Scle br, 1 µm Mcmilln Publishers Limited. All rights reserved.

14 SUPPLEMENTARY INFORMATION Side view Top view Unixil stretch Unixil stretch Objectiv Cells on stretchble membrne 2 b No stretch No stretch Initil Finl (n=13) Initil Finl (n=12) c Stretch Stretch Initil Finl (n=12) Initil Finl (n=8) d Initil Stretch 2 nm Nocodzole Finl (n=14) Control NZ, 2 nm Figure S4 Stretching of retrction fibers induces spindle rottion dependent upon strl microtubules. () Schemtic drwing of cell stretcher. (b-d) Chromosome plte orienttion for RPE1 cells dividing on ovl ring, ring or ovl shped microptterns without (b) nd with horizontl stretch (c+d). Chromosome pltes were visulized by Hoechst stining of DNA nd imged every 3 minutes. Initil represents chromosome plte orienttion in lte prometphse once chromosome plte cn be clerly detected nd t lest 1 minutes before nphse onset. Finl represents chromosome plte orienttion t nphse onset. Cells were stretched directly fter imging Initil orienttion. Grphics showing unstretched nd stretched cells dividing on ovl ring shped microptterns (left column, first nd second row) re lso shown in Fig 3. (d) Stretching of RPE1 cells dividing on ovl ring shped microptterns in presence of 2 nm nocodzole (NZ). Low nocodzole concentrtion llowed depolymeriztion of strl microtubules without ffecting spindle microtubules, s cells were not blocked in metphse. Imges show mitotic spindles (tubulin), ctin cortex (phlloidin) nd DNA (DAPI) in fixed control (left) nd nocodzole treted cells (right). White rrows highlight strl microtubules. Micro-ptterns, ornge; chromosome pltes, red; membrne, green. Scle br, 1 μm Mcmilln Publishers Limited. All rights reserved.

15 SUPPLEMENTARY INFORMATION Z- projection Equtoril plne Lifect-mCherry GFP-Utr-CH Lifect-mCherry GFP-Utr-CH 3.9X Figure S5 Comprison of Lifect nd Utr-CH probes to visulize filmentous ctin during mitosis. HeL cell dividing on br shped micropttern nd co-expressing Lifect-mCherry nd GFP Utr CH. Z-projections (left column) consist of imges tken t 1 μm intervl. Convincingly both ctin probes visulize polrized ctin structures found in the equtoril plne (right column). Note tht rings ppering on the Z projection re due to the lrge spcing of the successive confocl plnes. Actin filments inside retrction fibers, which hve been shown to be very stble 14, cn be clerly visulized using Lifect-mCherry. However GFP Utr-CH ppers to stin only ctin filments close to the cell body. Scle br is 1 μm Mcmilln Publishers Limited. All rights reserved.

16 SUPPLEMENTARY INFORMATION RPE1 Lifect-mCherry Figure S6 Subcorticl ctin structures in RPE1 cells. Timelpse sequence of dividing RPE1 cell expressing Lifect-mCherry. White rrows point out subcorticl ctin structures. Scle br, 1µm. Time is in min ( ) sec ( ) Mcmilln Publishers Limited. All rights reserved.