Aurora B‐dependent polarization of the cortical actomyosin network during mitotic exit
2021; Springer Nature; Volume: 22; Issue: 10 Linguagem: Inglês
10.15252/embr.202152387
ISSN1469-3178
AutoresNitya Ramkumar, Jigna V. Patel, Jannis Anstatt, Buzz Baum,
Tópico(s)Cellular Mechanics and Interactions
ResumoArticle24 August 2021Open Access Transparent process Aurora B-dependent polarization of the cortical actomyosin network during mitotic exit Nitya Ramkumar Corresponding Author Nitya Ramkumar [email protected] orcid.org/0000-0002-4086-4562 MRC LMCB, UCL, London, UK Search for more papers by this author Jigna V Patel Jigna V Patel MRC LMCB, UCL, London, UK Search for more papers by this author Jannis Anstatt Jannis Anstatt MRC LMCB, UCL, London, UK Search for more papers by this author Buzz Baum Corresponding Author Buzz Baum [email protected] orcid.org/0000-0002-9201-6186 MRC LMCB, UCL, London, UK Search for more papers by this author Nitya Ramkumar Corresponding Author Nitya Ramkumar [email protected] orcid.org/0000-0002-4086-4562 MRC LMCB, UCL, London, UK Search for more papers by this author Jigna V Patel Jigna V Patel MRC LMCB, UCL, London, UK Search for more papers by this author Jannis Anstatt Jannis Anstatt MRC LMCB, UCL, London, UK Search for more papers by this author Buzz Baum Corresponding Author Buzz Baum [email protected] orcid.org/0000-0002-9201-6186 MRC LMCB, UCL, London, UK Search for more papers by this author Author Information Nitya Ramkumar *,1,2, Jigna V Patel1, Jannis Anstatt1 and Buzz Baum *,1,3 1MRC LMCB, UCL, London, UK 2Present address: Duke University, Durham, NC, USA 3Present address: MRC-LMB, Cambridge, UK *Corresponding author. Tel: +1 919 684 5775; E-mail: [email protected] *Corresponding author. Tel: +44 0 1223 267000; E-mail: [email protected] EMBO Reports (2021)22:e52387https://doi.org/10.15252/embr.202152387 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The isotropic metaphase actin cortex progressively polarizes as the anaphase spindle elongates during mitotic exit. This involves the loss of actomyosin cortex from opposing cell poles and the accumulation of an actomyosin belt at the cell centre. Although these spatially distinct cortical remodelling events are coordinated in time, here we show that they are independent of each other. Thus, actomyosin is lost from opposing poles in anaphase cells that lack an actomyosin ring owing to centralspindlin depletion. In examining potential regulators of this process, we identify a role for Aurora B kinase in actin clearance at cell poles. Upon combining Aurora B inhibition with centralspindlin depletion, cells exiting mitosis fail to change shape and remain completely spherical. Additionally, we demonstrate a requirement for Aurora B in the clearance of cortical actin close to anaphase chromatin in cells exiting mitosis with a bipolar spindle and in monopolar cells forced to divide while flat. Altogether, these data suggest a novel role for Aurora B activity in facilitating DNA-mediated polar relaxation at anaphase, polarization of the actomyosin cortex, and cell division. Synopsis Aurora B facilitates DNA-mediated polar relaxation at anaphase and polarization of the actomyosin cortex to ensure a robust coupling of anaphase spindle elongation and cytokinesis. The clearance of actin from cell poles at anaphase does not depend on actomyosin ring assembly or on the centralspindlin complex. Aurora B activity is required for clearance of cortical actin network during anaphase and for actin clearance in the vicinity of chromosomes in artificially flattened cells. The role of Aurora B in cortical relaxation is independent of its role in regulating centralspindlin activity. Introduction Cell division involves the segregation of genetic material and cellular components into two daughter cells. Cytokinesis is the final step in this process, whereby an actomyosin ring constricts to cleave the cell into two. This process begins with anaphase cell elongation, as spherical cells exiting mitosis adopt an ellipsoid shape. Concomitantly, as the DNA begins to separate and reach the poles, there is a break in the symmetry of the uniform spherical cortical actomyosin network as the cortical actomyosin network softens at the poles and stiffens and constricts at the cell centre. The timely execution of this process requires extensive crosstalk between the spindle and the overlying cortical actomyosin network and is essential for the accurate segregation of sister chromatids at division (Ramkumar & Baum, 2016). Multiple pathways have been shown to be involved in this crosstalk and in the regulation of cytokinesis in both time and space. Although these events differ between cell types and across evolution, pioneering work from Ray Rappaport provided a crucial framework for understanding this process (Rappaport, 1996). He demonstrated that overlapping antiparallel microtubule from opposing spindle poles position the cleavage furrow—helping to ensure that the actomyosin ring bisects the spindle. As this contractile actomyosin ring is specified, anaphase cells elongate along their long cell axis. This change in shape has often been attributed to the effects of the gradient in cortical contractility induced via actomyosin ring formation (Pollard, 2010; Turlier et al, 2014). However, it has long been appreciated that polar relaxation itself may play a critical part in this process (Wolpert, 1960). In fact, the softening of cell poles is a likely pre-requisite for the division of animal cells which have undergone prior cortical stiffening during the process of mitotic rounding (Taubenberger et al, 2020). In addition, polar re-spreading has been shown to be sufficient to drive cell division in some cells that lack contractile components of the cleavage furrow (Kanada et al, 2005; Dix et al, 2018). While the pathways involved in this process of polar relaxation are not yet clear, recent work has suggested a role for DNA-dependent cues in polar relaxation (Kiyomitsu & Cheeseman, 2012; Rodrigues et al, 2015). These data point towards a role for the advancing chromosomes in softening cell poles in early anaphase as an aid to division. Two pathways have been suggested to regulate polar relaxation. A chromatin-based Ran-GTP gradient (Kiyomitsu & Cheeseman, 2012) has been proposed to act via importin to limit the accumulation of Anillin at cell poles. In addition, a kinetochore-associated pool of Sds22/PP1 has been proposed to dephosphorylate cortical ERM proteins (Rodrigues et al, 2015). Since ERM proteins link actin cytoskeleton to the membrane, localized ERM dephosphorylation at cell poles was proposed to de-couple the actomyosin network from the plasma membrane to reduce the mechanical rigidity of the actomyosin network at the poles (Kunda et al, 2012). The activation of Arp2/3 at the polar cortex could assist this process by driving polar cell re-spreading in some systems (Kanada et al, 2005; Dix et al, 2018). Due to the dynamic nature of the changes in cell shape and cytoskeletal organization during mitotic exit, it is important that these spatially distinct processes—polar relaxation, contractile ring specification and furrowing, be coordinated. While it is not understood precisely how this is achieved, Aurora B, the core member of the chromosome passenger complex (CPC), likely plays a role in this process. It has been implicated in multiple events at mitotic exit (Carmena et al, 2015; Afonso et al, 2016; Liu & Pellman, 2020), including in cell and nuclear division (Carmena et al, 2015; Petersen & Hagan, 2003; Afonso et al, 2014; Roubinet et al, 2021), and in spindle assembly checkpoint function (Krenn & Musacchio, 2015). Midzone localized Aurora B has been shown to regulate the oligomerization and accumulation of centralspindlin proteins, which guide the construction of the contractile ring during cytokinesis (Basant et al, 2015; Adriaans et al, 2019), and to assist in the final steps of abscission (Guse et al, 2005; Ahonen et al, 2009; Carmena, 2012). In addition, the kinase appears to function at a distance, since a gradient of Aurora B phosphorylation is thought to regulate the separation of DNA during anaphase and to guide the progressive formation of the nuclear envelope soon after (Fuller et al, 2008; Afonso et al, 2014; Liu & Pellman, 2020). The ability of Aurora B to function in so many distinct processes is likely due to its dynamic localization. Thus, at the metaphase-anaphase transition, Aurora B undergoes a sudden change in localization from centromeres to the developing spindle midzone and the equatorial cortex (Van Der Horst & Lens, 2014). It is clear from these studies that, despite system specific differences in its function, Aurora B helps to choreograph many of the dramatic changes in cell organization that accompany mitotic exit. In this paper we identify an additional role for Aurora B in polar relaxation in human cells leaving mitosis. Strikingly, this appears to be independent of Aurora B's role in midzone stabilization and centralspindlin activity, and appears to be based on a requirement of Aurora B for chromatin-based cortical remodelling during anaphase. These data lead us to propose that Aurora B coordinates the softening of the cortical cytoskeleton at cell poles close to advancing chromatin and the enhancement of midzone actomyosin ring formation and contraction to ensure a robust coupling of anaphase spindle elongation and cytokinesis. Results and Discussion Centralspindlin proteins are key players in regulating cytokinesis (White & Glotzer, 2012). In human cells, a heterotetrametric complex consisting of two molecules of the GTPase activating protein, RACGAP1, and two molecules of the kinesin, MKLP1, are recruited to the overlapping microtubules of the spindle midzone where they stabilize the midzone and recruit ECT2, a conserved GEF that activates RhoA to drive the local accumulation of contractile actomyosin necessary for cytokinesis (Su et al, 2011; Basant & Glotzer, 2018). To assess the relative timing of contractile ring assembly and polar actin clearance during mitotic exit, we performed live cell imaging of cells depleted for RACGAP1 using previously validated siRNAs and a HeLa cell line stably expressing LifeAct GFP and H2B-mCherry. Cells begin to change shape at anaphase onset. As they begin exiting mitosis, control cells elongate along the spindle axis and constrict in width along a perpendicular axis while the cleavage furrow is still being specified in early anaphase (Fig 1A). These shape changes are further enhanced following the assembly and constriction of the cleavage furrow (Fig EV1A and B). At early stages of anaphase, these changes in cell shape are accompanied by a modest but significant clearance of actin filaments from cell poles (Fig 1A–E), as seen by average actin intensity levels dipping below 1 (normalized to metaphase levels). We observed a similar clearance of LifeAct from the poles of RACGAP1 siRNA-treated Hela cells exiting mitosis (Fig 1A–E), which exhibit moderate cell elongation and midzone flattening at early anaphase (Fig EV1A and C). This was the case even though RACGAP1 depleted anaphase cells show no sign of assembling an actomyosin ring (Figs 1A, EV1A–C, and 4A and B), eventually leading to division failure (Fig EV1A and C). Figure 1. Actin clearance and dynamic re-localization of Aurora B in early anaphase upon centralspindlin depletion Stills from time-lapse sequence of representative Hela cells expressing LifeAct GFP and H2B-mCherry exiting mitosis under different conditions, i.e. Control siRNA and knockdown of the centralspindlin protein RACGAP1. Scale bar = 10 µm. High magnification view of boxed regions in (A) pseudo-coloured to show actin depletion in anaphase. Scale bar = 1 µm. Schematic representation for quantifying actin intensity along the cortex as in (D). Segmented line was drawn along the cortex from one equatorial region to the other. Actin intensity across the line is plotted in (D). Quantification of actin intensity profile of representative siControl and siRACGAP1-treated cells shown in (A). While actin levels were uniform across the cortex at metaphase (black line), actin is depleted from the polar regions (Blue line—segments 2–8) of both siControl and siRACGAP1-treated cells in early anaphase. Quantification of average actin intensity at the poles of cells at 6 min post-anaphase onset under different conditions as in (A). Despite reduced elongation, actin is cleared from the poles in both Control cells and following RACGAP1 depletion. Data represented as mean ± SD. siControl-n = 44, 0.98 ± 0.051, siRACGAP1-n = 48, 0.99 ± 0.041. Unpaired Welch's t-test comparing siControl and siRACGAP1 shows no significant difference between them, P = 0.5199. One sample t-test comparing siControl and siRACGAP1 to theoretical mean 1 (implying no change in actin compared with metaphase levels) shows significance for both treatments, with *P = 0.0195 for siControl and *P = 0.0392 for siRACGAP1. The depletion seen for both treatments, as means are lower than 1 (red dashed line), suggests significant clearance of actin from cell poles. Stills from time-lapse sequence of representative Hela cells exiting mitosis, expressing mNeonGreen Aurora B and Cell Mask to label the plasma membrane, showing the dynamic re-localization of Aurora B from DNA to the overlapping microtubules and cleavage furrow in control siRNA cells (arrows top). Following RACGAP1 silencing, Aurora B still re-localizes from the DNA to the microtubules and furrow in the midzone in early anaphase (arrows bottom), but the microtubule localization is lost at later stages (asterisk bottom). Scale bar = 10 µm. Maximum projection of 2 z slices of Hela-Cdk1as cells stained with anti-RACGAP1 antibody when treated with DMSO or 2 µM ZM447439 (ZM) at metaphase and early anaphase, showing that RACGAP1 relocates to the spindle midzone in early anaphase even upon Aurora B inhibition. Scale bar = 10 µm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Mitotic exit progression upon centralspindlin depletion Stills from time-lapse sequence of representative Hela cells expressing LifeAct GFP and H2B-mCherry exiting mitosis upon Control siRNA treatment or following siRNA-mediated silencing of the centralspindlin protein RACGAP1 (extension from Fig 1A). Maximum projection of 10 z slices around the middle of the cells shown in Fig 1A. While control-treated cells form an actomyosin contractile ring that completes ingression by 10 min, upon RACGAP1 depletion cells fail to furrow and remain binucleate. Scale bar = 10 µm. Quantification of cell shape changes—Cell elongation and compression (cell width) in siControl (B) and siRACGAP1-treated cells (C). siControl-treated cells begin elongation and midzone flattening at anaphase onset while the cleavage furrow is still being specified. Following furrow specification, around 6–8 min after anaphase onset, the rate of shape changes is enhanced. Average track for 10 representative cells, data are presented as mean ± SD. In siRACGAP1-treated cells, moderate shape changes are initiated at anaphase onset, seen by increase in cell length and compression of cell. However, unlike in control cells, these cells fail to specify a furrow and therefore these changes are not enhanced and cells begin re-spreading. Plot shows average track for 10 representative cells. Data are presented as mean ± SD. Quantification of Aurora B levels in the spindle midzone in siControl and siRACGAP1-treated cells as in Fig 1. Levels were normalized with respect to metaphase levels for each cell. There was a moderate reduction in Aurora B levels at the midzone upon RACGAP1 silencing. Data are represented as mean ± SD, siControl—0.94 ± 0.023, siRACGAP1—0.91 ± 0.033. Unpaired t-test comparing siControl and siRACGAP1, *P = 0.0172. Download figure Download PowerPoint These observations show that polar actin clearance is not an indirect consequence of midzone actomyosin ring formation. Instead, the data support the existence of a second pathway that breaks cortical symmetry at the transition from spherical metaphase to elongated anaphase cells (Kiyomitsu & Cheeseman, 2012; Rodrigues et al, 2015). This led us to search for additional regulators that might cooperate with centralspindlin proteins in the polarization of the mitotic cortical actomyosin network. For this analysis, we focussed on Aurora B kinase, as it is a conserved regulator of cytokinesis that functions as part of the chromosome passenger complex (Carmena et al, 2015; Afonso et al, 2016). To follow Aurora B dynamics, we endogenously tagged Aurora B with mNeonGreen in HeLa cells using CRISPR/Cas9 and imaged these cells as they progressed through mitosis, using red CellMask as a marker of the plasma membrane. As observed previously, Aurora B localizes to centromeres at metaphase in control HeLa cells. Following satisfaction of spindle assembly checkpoint (SAC), Aurora B then re-localizes to overlapping microtubules within the spindle midzone and to the cleavage furrow (Fig 1F), where it remains to aid abscission (Steigemann et al, 2009). In line with previous reports showing that Aurora B re-localization is unaffected upon knockdown of MKLP1, another centralspindlin protein (Gruneberg et al, 2004), this dynamic pattern of Aurora B re-localization appeared largely unaltered following the silencing of RACGAP1 using RNAi. Although there was a slight reduction in levels of Aurora B::mNeonGreen at the midzone upon RACGAP1silencing (Fig EV1D), the kinase re-localized from the DNA to midzone microtubules and cleavage furrow in early anaphase with similar timing in both RACGAP1 RNAi and control cells (Fig 1F). Further, Aurora B failed to maintain its later telophase midzone localization in the absence of RACGAP1. The converse also proved true. Aurora B inhibition did not affect the localization of RACGAP1, as assessed by immunofluorescence, in cells exiting mitosis that had been fixed following treatment with 2 µM of ZM447439 (ZM), a small molecule inhibitor of the Aurora B kinase (Ditchfield et al, 2003). Thus, in both control cells and in cells inhibited for Aurora B activity, RACGAP1 was seen localizing to the mitotic spindle in metaphase and to overlapping microtubules within the midzone of the spindle in early anaphase (Fig 1G). Although the midzone localization of RACGAP1 was maintained upon Aurora B inhibition, there was a moderate reduction in its accumulation at the midzone, as seen by increase in cytoplasmic levels. These data suggest that the dynamic re-localization of Aurora B and RACGAP1 are largely independent of one another in early anaphase HeLa cells. To better disentangle the different roles played by Aurora B, it was important to study Aurora B localization and the impact of Aurora B inhibition during the very early part of anaphase in cells that lacked a functional midzone. To do so, we forced cells to exit mitosis with a monopolar spindle, i.e. in the absence of overlapping microtubules by treating cells arrested in prometaphase using STLC, an Eg5 inhibitor (Skoufias et al, 2006) with a Cdk1 inhibitor (RO3306) (Canman et al, 2003; Hu et al, 2008). During this "monopolar cytokinesis", Aurora B was observed re-localizing from centromeres to the cell cortex most distant from anaphase chromatin as the DNA moved poleward (Fig 2A), as previously reported (Canman et al, 2003). This was coincident with the break in cortical actomyosin symmetry. Thus, phosphorylated ERM proteins (pERM-Fig 2A), which link actin filaments to the cell membrane (Bretscher et al, 2002) and have been associated with mitotic cell shape control (Kunda et al, 2008, 2012; Rodrigues et al, 2015), were lost from the cortex close to the DNA within 5 min of Cdk1 inhibitor addition. The resulting pERM gradient (Fig 2A) strengthened over time so that within ∼15 min of Cdk1 inhibitor addition most of the cells had a well-defined cortical gradient of phosphorylated ERM localization (Fig 2A–D). Figure 2. Aurora B is required for polarization of the actomyosin network in the absence of a midzone Maximum projection of 2 z slices of Hela cells immunostained for Aurora B (top) and actin-membrane cross-linker pERM (bottom), showing the change in protein localization upon Cdk1 inhibition with RO3306 (exit onset). As DNA moves to one side, Aurora B re-localizes from DNA to cortical region of the side opposite the DNA. In addition to Aurora B, the membrane-actin cross-linker pERM also polarizes in the direction opposite to DNA localization during the course of mitotic exit. Scale bar = 10 µm. Schematic representation of a cell, showing the quantification of actin and pERM along the cortex during mitotic exit. Regions 5 and 6 are cortex close to the DNA, while regions 1 and 10 are the furthest away. Maximum projection of 2 z slices of Hela cells immunostained for pERM following 10 min of forced mitotic exit in presence of DMSO and 2 µM ZM447439. Cells fail to polarize their pERM along the perimeter following Aurora B inhibition, as quantified in (D). Scale bar = 10 µm. Quantification of pERM intensity across the perimeter of the cells as shown in (B) where 5 and 6 represent regions closest to DNA, and 1 and 10 furthest. In DMSO-treated cells (n = 28 cells) pERM levels increase away from the DNA. By contrast, pERM fails to polarize in cells treated with ZM447439 (n = 26 cells) during forced mitotic exit. Data are presented as mean ± SD. Stills of representative Hela cells expressing LifeAct GFP and H2B-mCherry at 10 min following forced mitotic exit with different treatments. Control siRNA, RACGAP1 siRNA and DMSO-treated cells show clearance of actin from the cortex close to the DNA, while ZM447439-treated cells fail to clear actin, as quantified in (F). Note, the failure in actin accumulation following RACGAP1 depletion, asterisk. Scale bar = 10 µm. Quantification of actin intensity across the perimeter of the cells, measured based on distance from DNA, with 5 and 6 being closest to DNA and 1 and 10 furthest away, as shown in schematic (B). Graph shows decrease in actin intensity closer to the DNA in siControl (n = 26), siRACGAP1 (n = 37) and Control DMSO-treated cells (n = 38 cells), seen by average levels lower than 1 (dotted red line), while this local decrease is not seen in ZM447439-treated cells (n = 42 cells). Data are presented as mean ± SD. Download figure Download PowerPoint This cortical ERM phosphorylation gradient provided an excellent read-out of cortical polarization (Fig 2A–C). While pERM accumulated on the cortex furthest from the DNA in control HeLa cells undergoing a monopolar exit, cortical pERM remained isotropic in cells treated with ZM447439, an Aurora B inhibitor (Fig 2C and D). Since Aurora B inhibition also impacts spindle elongation (Hu et al, 2008), it was important to measure pERM levels as a function of distance from the DNA in these experiments (Fig 2B). When taking this into account by studying cortical polarization in cells where the DNA succeeded in moving close to one side, pERM still failed to polarize in the absence of Aurora B activity (Fig 2C and D). Furthermore, this function of Aurora B appears specific, since cortical polarization was also compromised in cells exposed to another Aurora B specific inhibitor, AZD1152 (Yang et al, 2007) and following RNAi silencing of INCENP, another member of the chromosomal passenger complex (Fig EV2-EV5), but was unaffected by the addition of an Aurora A inhibitor (Fig EV2A and B). Interestingly, cortical polarization was affected in a similar way by depletion of MKLP2 (Fig EV2C and D), a kinesin required for Aurora B re-localization during anaphase (Serena et al, 2015). This suggests a role for spatial control of Aurora B activity in polarizing the cortex at mitotic exit. Click here to expand this figure. Figure EV2. Midzone microtubule-independent polarization of the cortical actomyosin network by Aurora B during mitotic exit Maximum projection of 2 z slices of Hela cells immunostained for pERM following 10 min of forced mitotic exit in presence of DMSO, 1 µM AZD1152 and 1 µM Aurora A inhibitor I. Cells fail to polarize cortical pERM when treated with another Aurora B inhibitor AZD1152, but are not affected by Aurora-A inhibition, suggesting that this function is specific to Aurora B during mitotic exit. Scale bar = 10 µm. Quantification of pERM intensity across the perimeter of cells as shown in Fig 2B, where 5 and 6 represent regions closest to DNA, and 1 and 10 furthest. DMSO (n = 28 cells) or Aur-A inhibitor-treated cells (n = 8 cells) display an increase in pERM levels as the distance from the DNA increases, whereas cells treated with AZD1152 (n = 19 cells) during exit fail to polarize cortical pERM. Data are represented as mean ± SD. Maximum projection of 2 z slices of Hela cells immunostained for pERM following 10 min of forced mitotic exit after siControl, siINCENP or siKLP2 treatment. While Control siRNA-treated cells polarize their cortex, depletion of INCENP or MKLP2 leads to a failure in polarization. Note, the clearance of pERM close to DNA in control conditions, which does not occur following INCENP or MKLP2 depletion. Scale bar = 10 µm. Quantification of pERM intensity across the perimeter of the cells shown in (C), where 5 and 6 represent regions closest to DNA, and 1 and 10 furthest. Control siRNA-treated cells (n = 21 cells) show polarization of pERM. By contrast, this polarization is not seen following siRNA-mediated silencing of INCENP (n = 19 cells) or MKLP2 (n = 19 cells). Data are represented as mean ± SD. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Aurora B activity is required for DNA-dependent clearance of actin Maximum projection of 2 z slices of the basal most region of Hela cells expressing LifeAct GFP and H2B-mCherry forced to undergo flat monopolar cytokinesis. This reveals the ability of DNA to clear basal actomyosin at 15 min post-exit onset, which is lost following Aurora B inhibition with ZM447439 treatment, as quantified in (B). Dotted mask shows position of DNA in the actin channel. Scale bar = 10 µm. Quantification of the fraction of cells in which cortical actin is cleared from underneath the DNA within 15 min of exit onset. 82% of DMSO cells (n = 35 cells) clear actin from underneath the DNA (51% completely and 31% partially), whereas only 70% of the cells manage to do so following Aurora B inhibition (20% completely, 50% partially) (n = 46 cells). Chi-square test comparing distribution of cleared vs partial and no clearance shows significant difference between DMSO and ZM treatments, P = 0.0026. Quantification of cell shape changes—Cell elongation and midzone flattening and constriction (cell width) in siControl and ZM-treated cells. siControl-treated cells begin elongation and midzone flattening in early anaphase. This is further enhanced by furrow constriction. In ZM-treated cells, the shape changes initiated at early anaphase are slower than those seen in control cells. However, these shape changes are enhanced following furrow specification and constriction, and eventually become comparable to those seen in control cells. Average track for 10 representative cells, control cells track from Fig EV1. Data are represented as mean ± SD. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Cooperative effect of Aurora B and centralspindlin on cell shape changes during anaphase Maximum projection of 2 z slices of representative Hela-Cdk1as cells immunostained for PLK1 which accumulates on spindle midzone with different treatments. Midzone is specified at early anaphase in siRACGAP1-treated cells, but unlike siControl treated cells, it fails to be stabilized at later stages. In contrast, ZM-treated cells have a diffuse midzone region during anaphase. In the double treatment condition there is no recognizable midzone region. Scale bar = 10 µm. Stills from time-lapse of representative Hela cells expressing LifeAct GFP and H2B-mCherry exiting mitosis. Cells were treated with either siECT2 or siECT2 plus ZM. ECT2 silencing leads to increased cytoplasmic actin and reduced anaphase cell elongation. This effect is further enhanced upon ZM inhibition, which completely abrogates any shape changes. Scale bar = 10 µm. Quantification of cell shape changes during mitotic exit of cells following ECT2 depletion, with or without Aurora B inhibition with ZM, as in (B). While Hela cells with ECT2 depletion undergo some initial shape changes, this is completely abrogated following Aurora B inhibition. Unpaired Welch's t-test comparing aspect ratio of siECT2- and siECT2+ZM-treated cells shows significant difference between them, P = 0.0002. Data are represented as mean ± SD. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Centralspindlin-independent function of Aurora B during mitotic exit in RPE1 cells A. Stills from time-lapse sequence of RPE1 cells expressing LifeAct GFP, proceeding through anaphase in Control DMSO treated cells and in cells treated with the Aurora B inhibitor, 2 µM ZM. Scale bar = 10 µm. B. Quantification of cell shape changes during mitotic exit of cells treated as in (A). Cells are able to furrow upon Aurora B inhibition, but the furrow eventually regresses, leading to the formation of binucleate cells. Note that there is a significant delay in the progression of cell shape changes upon ZM treatment, an indication of a defect in polar relaxation. Data are represented as mean ± SD. C. Stills from time-lapse sequence of RPE1 cells expressing LifeAct GFP, treated with siRNAs targetting ECT2 and with DMSO or with ZM, quantified
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