Coupling Changes in Cell Shape to Chromosome Segregation

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Coupling changes in cell shape to chromosome segregation

Nitya Ramkumar and Buzz Baum Abstract | Animal cells undergo dramatic changes in shape, mechanics and polarity as they progress through the different stages of cell division. These changes begin at mitotic entry, with cell–substrate adhesion remodelling, assembly of a cortical actomyosin network and osmotic swelling, which together enable cells to adopt a near spherical form even when growing in a crowded tissue environment. These shape changes, which probably aid spindle assembly and positioning, are then reversed at mitotic exit to restore the interphase cell morphology. Here, we discuss the dynamics, regulation and function of these processes, and how cell shape changes and sister chromatid segregation are coupled to ensure that the daughter cells generated through division receive their fair inheritance.

Astral microtubules During cell division, the entire set of cellular compo different systems remains to be determined, the spheri A subpopulation of dynamic nents must be segregated before being partitioned into cal shape is likely to provide a suitable environment for microtubules, which originate two daughter cells. This includes cellular constituents the spindle to assemble, while also delimiting the space from the centrosomes but do that are present in relatively large numbers, such as in which astral microtubules have to search to capture not attach to the 9 chromosomes. They are ribosomes, which are partitioned by stochastic pro mitotic chromosomes . In addition, by simplifying cell present only during mitosis. cesses at division; cellular components that are pres geometry, mitotic rounding might contribute to the ent in smaller numbers, such as mitochondria and accurate partitioning of cellular contents into the two the Golgi, which tend to be fragmented and actively daughter cells. In a similar manner, the apical move transported to opposing cell poles to facilitate their ment of cell mass accompanying mitotic rounding in fair inheritance1; and structures that must be segre cells dividing in tall epithelia, such as neuroepithe gated with high precision, because their function is lia10, facilitates the preservation of apical polarity cues dependent on exact copy number, such as centrioles and cell–cell adhesion during a symmetrical division. and chromosomes. Therefore, although the basic goal Rounding also helps to set the stage for the dramatic of division is the same across all kingdoms of life2,3, it is redistribution of cell mass that is associated with especially challenging for eukaryotic cells, in which anaphase elongation and cytokinesis, which is impor segregation of genetic material must be coordinated tant for the restoration of normal cell packing in a in space and time with organelle segregation and cyto tissue that has been subject to mechanical stretching11. kinesis. The problem is further compounded for cells To aid division, changes in cell shape during mitotic in multicellular tissues such as epithelia, in which entry and exit are coupled to the cell cycle clock by the cells are highly polarized in shape and structure and activity of the master mitotic kinase cyclin-dependent physically connected to one another. In the context kinase 1 (CDK1)–cyclin B. Cellular architecture begins of a tissue, cell divisions must also be resistant to the to be remodelled during prophase, as CDK1–cyclin B potentially adverse influence of extrinsic forces, while levels rise, causing cells to adopt a typical, spherical responding to cues that determine the axis of cell mitotic cell shape12, while the centrosomes separate division to facilitate the maintenance of polarity and and begin to nucleate microtubules to capture and tissue relaxation4,5. align chromosomes. These changes in cell shape are In animal cells, all of these processes are facilitated reversed following the drop in CDK1–cyclin B levels MRC Laboratory of Molecular Cell Biology, University by ‘mitotic rounding’. This is the process by which cells at anaphase. Then, as sister chromatids separate, cells College London, Gower Street, adopt a relatively simple, symmetric and spherical leaving mitosis elongate to make space for the extend London WC1E 6BT, UK. shape as they enter mitosis. Although this is not a uni ing spindle, while simultaneously assembling the con 6,7 Correspondence to B.B. versal phenomenon , most eukaryotic cells that lack tractile ring machinery to generate two daughter cells. [email protected] a rigid cell wall8, and therefore have a flexible form, The two processes must be highly coordinated in space doi:10.1038/nrm.2016.75 begin to ‘round up’ in this way as they enter mitosis. and time to ensure the equal inheritance of genetic Published online 29 June 2016 Although the precise function of mitotic rounding in material in the daughter cells.

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In this Review, we discuss the dynamics of cell shape the phosphorylation of the proteome16–18. As a result, cell changes and their contribution to mitosis in cell cul shape and structure are rapidly altered within minutes ture and in the context of an epithelial tissue. Finally, of mitotic entry12. we explore the importance of crosstalk between the cell cortex and the mitotic spindle for precise cell division. Adhesion remodelling during mitotic entry. One of the factors contributing to this rapid change in cell shape is Entry into mitosis loss of attachment to the underlying substrate. This loss For many animal cells, entry into mitosis begins with a of integrin-based adhesions is sufficient to cause cells in dramatic series of changes in cell shape, movement and culture to adopt a near-spherical shape — the minimum polarity10,13,14 (FIG. 1a,b). Although these changes have energy conformation for a cell with high cortical tension not been investigated in much detail, they are among (as the sphere has the least surface area for a given vol the first signs of a cell approaching mitosis. The changes ume). At a molecular level, this rapid change in adhesion are triggered by rising levels of CDK1–cyclin B15, along is probably triggered by activation of CDK1–cyclin B19–22. with rising activities of a cohort of mitotic kinases (most Although CDK1–cyclin B directly phosphorylates sev notably Aurora A, Aurora B and Polo-like kinase 1 eral adhesion complex components23, including integ (PLK1)). This sets up a positive feedback loop that rins24, focal adhesion kinase and paxillin25, the major culminates in full kinase activation and a global shift in molecular event responsible for the loss of adhesions in

a NEB Anaphase ARP2/3 complex activity Isotrophic cortical network Contractile ring Actomyosin network

CDK1–cyclin B activity

Interphase Prophase Prometa Metaphase Anaphase Telophase

b Actin Chromatin Centrosome Furrow formation Microtubule Cell spreading

Substrate Adhesion Contractile Decreased actomyosin ring cell-substrate Cell division adhesion and movement

Cell rounding Polar relaxation

Assembly of cortical Cell elongation actomyosin network Figure 1 | Cell shape changes during mitosis. As animal cells become committed to mitosis, their internal architecture and physical connections to the environment are rapidly remodelled, causing cells to assumeNature a Reviewstypical, spherical | Molecular mitotic Cell cellBiology shape. These structural changes are reversed during mitotic exit as cells divide and revert to their initial interphase morphology. Structural changes are coupled to the cell cycle clock, and many are directed by changes in protein phosphorylation driven by the rise and fall of cyclin-dependent kinase 1 (CDK1)–cyclin B activity. a | As the levels of CDK1– cyclin B activity rise (blue line), the cell begins the assembly of its cortical actomyosin network (red line); both of these events are accelerated by loss of the nuclear–cytoplasmic compartment boundary. The cortical actomyosin network that is assembled upon entry into mitosis is largely independent of the activity of the actin nucleator ARP2/3 complex (purple line), which is inhibited at the onset of prophase. At anaphase, this mitotic actomyosin network is remodelled to generate a contractile actomyosin ring at the cell centre. The ARP2/3 complex is then reactivated as cells re-enter interphase. b | At the onset of mitosis, cells stop moving and reduce their adhesions to the substrate with rising levels of CDK1–cyclin B activity. Centrosomes separate, and cells begin to assemble a cortical actomyosin network and increase in height to round up. Rounding is expedited by nuclear envelope breakdown (NEB). Chromosomes (blue) then align at the metaphase plate. Once all the chromosomes are attached to the spindle and the spindle assembly checkpoint has been satisfied, the activation of the anaphase-promoting complex (APC) triggers the cleavage of cohesin, allowing sister chromatids to move to the opposite poles, and the degradation of cyclin B, leading to a collapse in CDK1 activity. This is accompanied by the relaxation of the cortical actomyosin network at cell poles and by the concentration of actomyosin at the cell centre to form a contractile ring. As cells divide, chromatids begin to de-condense, and the nuclear envelope and lamina reform.

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extracellular matrix. Importantly, for cells in culture with Plasma pERM pSLIK membrane a isotropic interphase shape, it has also been suggested DIA that these retraction fibres provide cells with a physi DIA cal memory of the pattern of interphase cell–substrate adhesions, which then guides mitotic spindle orienta Actin tion27 (BOX 1) and the re‑establishment of these adhesions Myosin when cells re‑spread at mitotic exit28,29. Similar structures miniﬁlament have been observed in mitotic cells within an epithe lium, which retain long basal retraction fibres as they Actin nucleation DIA RhoA ROK Myosin activity round up during mitosis. These fibres maintain physical ECT2 contact between cells and their underlying basal extra cellular matrix and seem to aid the restoration of apical basal polarity upon mitotic exit30,31. Importantly, epi adherens Actin thelial cells also retain their E‑cadherin-based Myosin junctions and tight junctions during the process of division pERM to preserve tissue integrity32,33. Chromosome Centrosome Assembling the mitotic cortical actin network. The adhesion remodelling that takes place as cells enter Microtubule mitosis is accompanied by profound changes in the Plasma membrane organization of cortical actin cytoskeleton (FIG. 2), which culminate in the assembly of a dense cortical actin net work characteristic of mitotic cells34. The loss of basal actin cables (also known as stress fibres) is likely to be a direct consequence of the disassembly of cell–substrate adhesions13. There is also a shift in the dominance of Substrate Focal adhesion different cortical actin nucleators at the onset of mitosis, Figure 2 | Cortical actomyosin network reorganization during mitotic rounding. from the ARP2/3 complex to formins14,35,36. For cells enter Nature Reviews | Molecular Cell Biology In isolated cells in culture, several factors contribute to the changes in the actomyosin ing mitosis in culture, this is apparent from the loss of network that aid in mitotic cell rounding. The activity of the actin nucleator ARP2/3 lamellipodia, which are actin-based membrane pro complex is reduced, whereas that of negative regulators of actin turnover is increased. trusions that are generated by branched actin filament Increasing levels of cyclin-dependent kinase 1 (CDK1)–cyclin B activity lead to nucleation downstream of Rac, the SCAR/WAVE com progressive loss of integrin-based cell–substrate adhesion. The rise in nuclear CDK1– plex37–39 and the ARP2/3 complex. Although the extent to cyclin B also leads to the export of ECT2 from the nucleus. This activates RhoA and its which residual ARP2/3 activity contributes to the gener targets, Diaphanous (DIA), Rho-associated protein kinase (ROK) and myosin (blue), 14,35,36 leading to the formation of a contractile actomyosin network. Following its mitotic ation of mitotic structures is currently unclear , this activation, the active phosphorylated SLIK (pSLIK) kinase (sterlie 20 kinase), now loss of lamellipodial actin is accompanied by the activ phosphorylates and activates ERM (Ezrin, Radixin, Moesin) proteins (Moesin in flies). ation of the formin-based actin nucleator Diaphanous Phosphorylated ERM (pERM; yellow) proteins tether this actomyosin network to the (DIA)14, which drives the assembly of a cortical net

plasma membrane through binding transmembrane proteins and the phospholipid PIP2. work of actin filaments. Importantly, once activated, The cells retain actin-rich retraction fibres, which both direct spindle orientation (BOX 1) through physical association with Rho‑family GTPases, and help in cell re‑spreading following division. DIA generates non-branched actin filaments. These provide an ideal substrate for non-muscle myosin II mini filaments40, which walk along antiparallel actin filaments many cell types seems to be inactivation of the RAP1 in a stepwise fashion to generate cortical tension. GTPase12. This is supported by the fact that many cul In human and fly cells, assembly of this DIA- tured cells expressing a constitutively activated form of dependent cortical actin mesh seems to be regulated RAP1 retain adhesions and remain flat as they enter by ECT2 (called Pbl in Drosophila melanogaster)13,14 mitosis, despite extensive CDK1–cyclin B-mediated — a potent Rho‑family GTPase activating protein41. phosphorylation of adhesion proteins12. This experiment ECT2 is released from the nucleus as levels of nuclear 13 Retraction fibres demonstrates the importance of adhesion remodelling CDK1–cyclin B increase at the onset of mitosis . Once Actin-rich fibres retained by for normal mitotic cell rounding. in the cytoplasm, ECT2 activates Rho‑family GTPases cells as they round up as they Although the loss of cell–substrate adhesions during at the plasma membrane (FIG. 2). These include RhoA42,43 enter mitosis. These hold the entry into mitosis is rapid, most animal cells maintain which, when bound to GTP, activates DIA and ROK cells in place and are thought to guide spindle orientation to physical connections with their environment through (Rho-associated protein kinase), which in turn activ 44 determine the division axis. out mitosis (FIG. 2). For animal cells in culture, the ates non-muscle myosin II . In addition, it has been actin-based structures that remain are called retraction suggested that ECT2 associates with the polarity pro Adherens junctions fibres26 (FIG. 2). Although these lack myosin II and do teins atypical protein kinase C (aPKC), partitioning Protein complexes consisting not provide robust cell–substrate adhesion (enabling defective 6 (PAR6) and CDC42, leading to the activ of cadherins and catenin, (REFS 14,45) which are involved in cell–cell mitotic cells to be dislodged by a physical blow to the ation of the Rho‑family GTPase CDC42 . junctions in epithelial and cell culture flask), it has been proposed that they act In this way, ECT2 may direct a shift in polarized actin endothelial cells. like tent cables5 — keeping rounded cells tethered to the organization, enabling epithelial cells to assemble an

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Box 1 | Spindle orientation guided by cortical cues in cellular biochemistry and cyto-architecture. This is augmented by osmotic swelling59 and a sudden influx Components of the molecular machinery that guide the orientation of the mitotic of water, leading to a significant (>10%) increase in cell spindle in response to cues in the cortex were first identified in genetic screens. These volume that is independent of the actin cytoskeleton60,61 include a small set of highly conserved proteins, G protein subunit G , LGN (also known αi and a dramatic increase in cell height59,62,63. In combin as GPSM2 or Pins) and nuclear mitotic apparatus protein 1 (NuMA; also known as Mud). Following nuclear envelope breakdown, these proteins localize to the cell cortex, ation, changes in cell architecture, actin remodelling, where they recruit the minus-end-directed motor dynein–dynactin146. As dynein walks cell–substrate adhesion and osmotic swelling, cause along astral microtubules, towards their minus ends and the spindle pole, it generates cells to assume a spherical shape by the time they have the forces required to reposition the spindle147. assembled a metaphase plate (FIGS 1,3a). Although many actin regulators (for example, afadin, myosin X, LIM kinase) have been implicated in spindle positioning, it is not clear whether individual actin regulators relay Changes in mechanical properties. Although spherical specific information to guide polarization of the cortex or whether they function mitotic cells resemble non-adherent interphase cells, collectively in construction of the mitotic cortical actin network. The latter is important, their mechanical properties are profoundly differ as the loss of cortical actin leads to the accumulation of cortical polarity cues together ent13,59,63–65. In fact, entry into mitosis is accompanied with dynein at centrosomes148. In this case, it is clear that actin is not needed for dynein by a rapid, nearly tenfold decrease in cell compliance to generate pulling forces but to provide a physical platform on the plasma membrane upon which polarity cues can be stably bound and read. This situation is further (equivalent to an increase in cell stiffness). This is complicated by the fact that spindle assembly depends on mitotic cell dependent on both osmotic swelling, which can be + + 59,60 geometry — which is another actin-dependent process. inhibited by the Na /H antiporter poison (EIPA) , and the cortical actomyosin network13,52. These forces are sufficient to deform neighbouring cells within a crowded epithelial tissue11,66 (FIG. 3b), can drive the api isotropic cortical actomyosin network as they round up cal movement of cell mass in a columnar epithelium63 to divide14. Interestingly, the same set of proteins drives and are strong enough to induce morphogenetic tissue both the assembly and the polarization of a cortical buckling in the developing fly and lumen growth in contractile actomyosin network before division of the zebrafish epithelial tissues66,67. Thus, the stable, rounded Caenorhabditis elegans zygote46. shape that is characteristic of metaphase cells reflects a Tight junctions (FIG. 3) Cell–cell junctions, consisting Many other changes in the dynamics and organiza balance of forces : the mitotic cortical actomyosin of occludins, claudins and tion of actin filaments contribute to the assembly and network, which is crosslinked to the plasma membrane associated proteins, which remodelling of the mitotic cortical actomyosin network. by active ERM proteins52, functions like the elastic skin bring membranes of adjacent For example, phosphorylation-induced changes in the of a balloon to counter the internal pressure established cells into close proximity to activities of proteins that negatively regulate actin fila by the activity of osmotically active ion pumps59,60. form a permeability barrier. They are positioned apical to ment formation, including LIM kinase, cofilin and WD the adherens junctions in repeat-containing protein 1 (WDR1), may contribute Mitotic exit vertebrates and (as septate to an increase in the actin filament turnover rate upon The duration of mitosis depends both on mitotic timers junctions) basal to adherens entry into mitosis35,47–50. Furthermore, the activation of and on satisfaction of the spindle assembly checkpoint68–71. junctions in most invertebrate epithelia. actin filament-binding proteins, such as ERM (Ezrin, When a minimal time has elapsed and the last pair of Radixin, Moesin) proteins, brought about by the SLIK chromosomes has been aligned at the metaphase plate ARP2/3 complex kinase51,52 leads to the physical crosslinking of the actin (reviewed in REF. 72), the anaphase-promoting complex A protein complex consisting filaments to proteins embedded in the plasma mem (APC; reviewed in REF. 73) is activated. This induces of seven polypeptides brane52,53 (FIG. 2). These changes in actin organization cleavage of cohesin, the protein complex that holds sis (including actin-related protein 2 (ARP2) and ARP3), are probably aided by a shift from the phospholipid ter chromatids together, enabling the movement of sister 54,55 which regulates the nucleation PIP3 towards PIP2 in the plasma membrane , which chromatids to opposing poles of the elongating anaphase of branched actin filament has been shown to activate ERM binding56. Together, spindle74 (FIG. 4a). At the same time, APC-mediated pro networks from the filament these changes in actin filament nucleation, turnover tein degradation reverses many of the regulatory changes minus end. and dynamics lead to the assembly of a thin but dense that accompany entry into mitosis, triggering the rapid Formins network of actin filaments and non-muscle myosin II degradation of cyclin B and the loss of mitotic kinase Proteins defined by the motors underneath the plasma membrane34. activity (reviewed in REF. 75). This drives cells out of presence of a formin Although this reorganization of the cytoskeleton mitosis — a process that culminates in cell division homology 2 (FH2) domain, begins in prophase, cytoskeletal remodelling acceler (reviewed in REF. 76). which nucleate actin filaments from growing (plus) ends to ates following the loss of the nuclear–cytoplasmic com generate parallel or antiparallel partment barrier (a process known as nuclear envelope Assembly and positioning of the actomyosin ring. filaments that are a good breakdown (NEB))57. One might speculate that this is Although many of the cell biological changes induced substrate for non-muscle due to the release of proteins previously sequestered in upon entry into mitosis are reversed during mitotic myosin II. the nucleus. In addition to residual nuclear ECT2, this exit (for example, reassembly of the nuclear compart Non-muscle myosin II includes RanGTP, which catalyses the release of pro ment; reviewed in REF. 77) the same is not true of actin An ATP-dependent motor teins from importins to alter microtubule dynamics and remodelling. In fact, the mitotic cortical actin network protein that forms large bipolar cortical actomyosin58. Simultaneously, the mixing of must not be disassembled at mitotic exit. This is because ‘minifilaments’. In non-muscle cytoplasm and nucleoplasm induced by the loss of the the machinery used to generate the actomyosin network cells, these both crosslink actin filaments and walk along nuclear compartment is likely to induce a sudden drop that establishes the characteristic spherical shape of the actin filaments to drive in the concentration of proteins localized to only one of metaphase cortex also controls the assembly of the acto- network contraction. the two compartments — inducing profound changes myosin contractile ring that is used to drive cytokinesis.

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Importins This includes ECT2, RhoA, DIA, actin, non-muscle formation, which divides the cell into two daughter cells 78 Protein complexes that myosin II and Anillin , all of which seem to be further (FIGS 1,4). The cortical remodelling that is triggered at transport proteins containing activated by the drop in CDK1–cyclin B activity at mitotic exit to facilitate these shape changes cannot nuclear localization sequences mitotic exit79,80. In this regard, mitotic rounding can be be subject to autonomous regulation; that is, an acto (NLS) into the nucleus. The complex consists of importin‑α seen as a prelude to division. Although the activity of this myosin ring cannot be simply assembled and constricted and importin‑β. set of actin regulators is required for the assembly of the at a fixed time and place following the activation of the actomyosin network that is necessary for both round APC. If each daughter cell is to inherit a single copy of Spindle assembly ing and cytokinesis, the assembly of an actomyosin ring the nuclear genome, cells must ensure that the acto check point probably involves additional factors including the loss myosin ring bisects the plane of the elongating anaphase The checkpoint that delays 52,81 anaphase onset until of actin-binding proteins from cell poles and the spindle. Given the speed at which animal cells change 82–84 chromosomes are properly recruitment of septins and BAR-domain proteins to their shape (elongation and cleavage furrow formation) attached to the the site of actomyosin ring formation, where they aid during mitotic exit, this requires active crosstalk between metaphase spindle. membrane deformation85,86. the spindle and the cortex (see REF. 87 for information Structural changes at mitotic exit begin with elong about the duration of these signals). This contrasts with ation of the spherical mitotic cells, which now adopt a the situation during mitotic rounding, when changes more elliptical shape. This is followed by cleavage furrow in cell shape and actin organization seem to be regu lated relatively independently of microtubules and microtubule-based structures13 (an exception being a a Plasma membrane potential role for the cortex in centrosome separation at 9,88,89 Chromosome mitotic entry ). Similarly, spindle assembly seems to Microtubule be largely independent of the presence of actin filaments Centrosome both in vivo9 and in vitro90. This difference implies that mitotic exit must be accompanied by profound changes Retraction ﬁbre in spindle–cortex communication — as has long been appreciated (we suggest reading Rappaport’s book87 for an in-depth review of the early literature). Insights into the mechanism came from Rappaport, who carried out a series of seminal experiments showing how over lapping antiparallel astral microtubules can direct the local formation of an ectopic furrow during mitotic Substrate Actin exit87. Although ectopic furrows do not form in analo Myosin 91 pERM gous experiments in all systems , in most animal cells tested, the overlapping microtubules that form between b the poles of the anaphase spindle92 direct the formation and position of the actomyosin furrow to ensure that the division plane bisects the spindle. At a molecular level, this is achieved through recruit Adherens centralspindlin complex junction ment of the , a set of proteins that orchestrate the ensuing molecular events92. In brief, overlapping antiparallel microtubules of the anaphase spindle recruit, and are further organized by, a set of kinesin motors (reviewed in detail in REF. 76). Following the APC-induced fall in CDK1–cyclin B activity, these in turn recruit a scaffolding protein, RacGAP1 (also known as MgcRacGAP) together with the RhoGEF ECT2 to the spindle midzone. This shift in ECT2 localization leads to the repolarization of the cortical actomyosin network (and associated proteins such as Anillin93), through the local activation of RhoA94,95. RacGAP1 facilitates ECT2 in the activation of RhoA96, possibly by inactivating Figure 3 | Forces experienced by the cells duringNature mitotic Reviews rounding. | Molecular An Cellisolated Biology cell RacGTP at the midzone97, and therefore helps to estab dividing in culture (a) and a cell dividing in an epithelium (b) experience a plethora of lish a gradient of Rac and ARP2/3 activity — low at forces (indicated by arrows). At metaphase, these are balanced to give cells their the midzone and high at the poles — aiding anaphase characteristic stable rounded shape. Cells retain retraction fibres, which tether them in elongation. In some cells, the repolarization of cortical place. Osmotic swelling causes an increase in the internal pressure (purple arrows), actomyosin may be further augmented by the establish which is counteracted by a contractile actomyosin network (red arrows). The actomyosin 98 network is tethered to the cortex by active (phosphorylated) ERM (Ezrin, Radixin, Moesin) ment of a PIP2/PIP3 gradient in anaphase (PIP2 high at proteins (pERM; yellow). Cortically localized dynein motors then pull on astral the furrow, facilitating Rho activation; and PIP3 high microtubules to align the spindle and to determine the plane of cell division. In at the poles, facilitating Rac activation). Finally, this pro comparison to cells in culture, cells in an epithelium (b) have additional complexities, as cess is promoted by the local action of the mitotic kinases they are surrounded by neighbouring cells that constantly push back on the dividing cell PLK1 and Aurora B94,99 (a component of the chromosomal (black arrows). In addition, they retain their adherens junctions during cell division. passenger complex (CPC)), which persist at the midzone.

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Anaphase-promoting Until recently, it was thought that this same set of Polar relaxation. Physical models of cytokinesis suggest complex cues emanating from the spindle positions the furrow in that the essential factor required for a successful divi (APC). An E3 ubiquitin ligase the same way across all animal cell divisions. However, sion is the establishment of a gradient of cortical con that targets specific cell cycle spindle–cortex crosstalk is flexible. Thus, asymmetries tractility across the cell76, whereby the cortex is more proteins for degradation by 100–102 the 26S proteasome upon in the cortex can bias the site of furrow formation . contractile at the centre and relatively compliant at cell satisfaction of the spindle In addition, pre-existing cortical asymmetries can mod poles. Thus, the assembly and constriction of a central assembly checkpoint, to trigger ify the rate of anaphase elongation101 to alter the size of actomyosin ring may not be sufficient to drive cyto the separation of sister daughter cells. Although the instances identified so far kinesis in cells with a rigid mitotic cortex52,104. In line chromatids and the transition are not as extreme as that in budding yeast, in which the with this, division involves both the assembly of an to anaphase. spindle is moved so that it comes to lie across the bud actomyosin ring, induced by overlapping microtubules neck103, it is clear from these few instances that the actin- that are positioned between spindle poles (FIG. 4b,c), based cortex can contribute to the positioning of the and concomitant softening of the cortical actomyosin division plane. network at cell poles105,106 — a process we term polar

a Centralspindlin complex b CPC Actin Plasma membrane pMyosin pERM Sister chromatid Anillin Kinetochore Microtubule Centrosome

PP1–SDS22

Retraction ﬁbre

Substrate PP1–SDS22 PP1–SDS22

Aurora B Aurora B c d Actin

Centralspindlin complex CPC Centrosome PP1 ERM Microtubule (inactive) Sister chromatid

Myosin Microtubule

Plasma membrane Plasma membrane Anillin pERM RhoA Actin

Figure 4 | Polar relaxation and coupling of chromosome segregation to with PP1 dominating at the poles Natureand the Reviews CPC dominating | Molecular at theCell midzone. Biology cytokinesis. a | As sister chromatids separate and cyclin-dependent kinase 1 This, together with the local RanGTP gradient generated by the association (CDK1)–cyclin B activity declines at the onset of anaphase, the of the Ran GEF RCC1 (regulator of chromosome condensation 1) with centralspindlin complex and the chromosomal passenger complex (CPC; mitotic chromatin, probably facilitates the local dephosphorylation of a complex containing active Aurora B) move to the spindle midzone. non-muscle myosin II and ERM (Ezrin, Radixin, Moesin) proteins and induces Centralspindlin recruits ECT2 to the spindle midzone. By contrast, the the loss of Anillin at cell poles, establishing a gradient of actomyosin phosphatase PP1 (in complex with SDS22), remains on the kinetochores. As a across the cell that helps to drive cell division. c | High magnification view of result, the activity domain of the phosphatase and kinase begin to separate the spindle midzone showing the localization of CPC and centralspindilin in space. b | As the sister chromatids begin to approach the poles, the cell complex to the overlapping microtubules, where they induce the local elongates. Anaphase cell elongation is regulated by a combination of forces formation of a contractile actomyosin ring. d | High magnification view of the and signals, many of which emanate from the anaphase spindle. Following cell poles during anaphase. Kinetochore-localized PP1–SDS22 chromosome separation, the activity gradients of the CPC and PP1 separate approaches cell poles at mid-anaphase leading to local dephosphorylation to oppose one another, leading to an opposing kinase–phosphatase gradient of active ERM proteins at the cortex, polar relaxation and local blebbing.

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relaxation (FIG. 4b,d). Depending on the relative timing In support of this idea, the approach of kinetochores of this local polar loss of phosphorylated ERM (pERM) carrying PP1 and SDS22 (REF. 106) to cell poles is accom proteins, actin and non-muscle myosin II, cell elong panied by PP1–SDS22‑dependent dephosphorylation ation can be a smooth and gradual process, or it can be of ERM proteins51,52,81 and the loss of polar actomyo accompanied by furious rounds of blebbing — as physi sin118, before the accumulation of actomyosin at the cell cal connections between the plasma membrane and the midzone. As PP1 promotes the large-scale reversal of underlying cortical actin network are repeatedly lost mitotic phosphorylation at mitotic exit119, potentially and re-established105. Moreover, failure to induce timely aiding inactivation of the spindle checkpoint120, the rise relaxation of the polar cortex can lead to catastrophic in PP1 activity at anaphase effectively couples cortical failure of cell division105. mechanics (that lead to the shape changes) to the change Three signals have been identified that may contrib in mitotic state. In addition, as the kinases that oppose ute to relaxation of the cortex at opposing cell poles PP1 activity (notably Aurora B and Polo kinase) remain (FIG. 4). First, polar relaxation is likely to occur as an at the centre of the spindle during anaphase, where they indirect consequence of the movement of the acto localize to regions of microtubule overlap (reviewed in myosin network from cell poles towards the central REFS 121,122), the anaphase spindle may set up a phos region of the furrow at anaphase105. Anaphase cell phorylation gradient across the cell — with high levels elongation has been shown to depend on ROK and non- of kinase-mediated phosphorylation near the spindle muscle myosin II activity in fly cells69,107, both of which midzone and high levels of PP1–SDS22 mediated are also required for furrow formation, indicating an dephosphorylation towards the cell poles. In principle, association between the two processes. In addition, polar such a phosphorylation gradient could bridge the physi relaxation may be induced by signals that emanate from cal gaps between the spindle, the cytoplasm and the cor the anaphase spindle and are independent of overlapping tex, thereby helping to coordinate events across the cell microtubules and centralspindlin. In previous decades, and promoting polarization of the cortical actomyosin this type of activity was often attributed to the ability of before cytokinesis. the growing, plus ends of astral microtubules to induce cortical softening at cell poles108. Although the complex Completion of division ity of microtubule-based signals are yet to be resolved At this stage, the contraction of the centrally positioned (for example, furrow components accumulate at the tips actomyosin ring transforms the cell into a dumbbell Actomyosin contractile ring of microtubules in the absence of microtubule overlap shape. Although this ring usually closes in a symmetrical A contractile structure in cells exiting mitosis with a monopolar spindle 109,110), fashion in cells in culture (FIG. 5a), the ring moves apically composed of actin and myosin it is now clear that the anaphase spindle106,111–113 also as it constricts in many epithelia83,84,123 (FIG. 5b). In some filaments that deforms the plasma membrane to drive signals to the cortex via microtubule-independent sig fly epithelia, this basal-to-apical movement of the ring cytokinesis. nals. In support of this, polar relaxation still occurs may be a consequence of the lack of actomyosin at the near anaphase chromatin in cells that lack spindle poles apical surface of mitotic cells14. In addition, it has been Centralspindilin complex and/or microtubules106. suggested that this polarized geometry is a simple con A protein complex that Recent work suggests that one or more of the signals sequence of tissue mechanics, as the contractile forces localizes to overlapping antiparallel microtubules in may be chromatin-based. In one study in human cells in that neighbouring cells exert on mitotic cells through anaphase to recruit proteins culture, this was attributed to the action of RCC1 (regu apically positioned cell–cell junctions resist ring closure that drive local actomyosin ring lator of chromosome condensation 1), a Ran GEF112. in fly epithelia83,84,124. The opposing forces of ring clo formation, so that closure of It was postulated that the association of RCC1 with sure and tissue tension have other consequences. In fly the ring bisects the spindle. mitotic chromatin establishes a local RanGTP gradient epithelia, they physically uncouple the actomyosin ring Chromosomal passenger in anaphase that is high at cell poles and low at the cell and the associated plasma membrane from the existing complex centre. RanGTP then functions via importin‑β to influ junction (FIG. 5b), leading, eventually, to the formation (CPC). A protein complex ence the activity and/or localization of a wide range of of a straight, new planar interface between the two consisting of kinase Aurora B actin regulators that carry an NLS (nuclear localization daughter cells123. The formation of this new interface and its regulatory subunits 111,114 115 borealin, INCENP and survivin, signal) . These include Anillin , which is lost from may be aided by the reactivation of the ARP2/3 com 123 which relocalize from the polar cortex following the approach of chromo plex at the cortex close to the midbody , through the kinetochores to overlapping somes112, leading to weakening of the mitotic cortical delivery of membrane from vesicular stores125,126 and/or antiparallel microtubules at the actomyosin network (as is the case during meiosis)116. through the generation of pushing forces on opposing midzones of cells in anaphase, 123 to regulate actomyosin ring This RanGTP pathway, it has been argued, aids spindle membranes . At the same time, these forces may help formation and, later, positioning by negatively regulating local recruitment of trigger abscission, as a loss of tension across the division abscission. nuclear mitotic apparatus protein (NuMA), LGN111 and site has been shown to be a prerequisite for the physical Anilllin112 when anaphase chromatin in an off-centre separation of animal cells in culture127. Midbody spindle approaches one cell pole more closely than the The completion of the division process also Also called a Flemming body, the midbody is a structure that other, to ensure equal partitioning of cell mass during requires disassembly of the actomyosin ring and the connects the two daughter symmetric divisions. microtubule-rich midbody. This is associated with cells towards the end of In a separate study it was proposed that a kinetochore- the binding of RacGAP1 to the membrane, the recruit cytokinesis. It controls localized phosphatase117, PP1, functions together with a ment of FIP3 (also known as NEMO) in place of abscission, a process that (REF. 128) 129 leads to the physical regulatory subunit, SDS22, to promote the loss of cor ECT2 , ECT2 degradation , and the loss of (REF. 130) 98,131 separation of the two tical actomyosin network from the poles of cells dur PIP2 , Rho and actin from the midzone . daughter cells. ing mid-anaphase, helping to trigger polar relaxation. Following disassembly of the microtubule-based

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a Actin Anillin Myosin RhoA pERM ESCRTIII Plasma membrane Microtubule Centrosome Chromatin

Abscission

Substrate

Daughter cells Chromatin Centrosome Microtubule Plasma membrane

Substrate Adhesion

Cell junction b Centrosome Microtubule Chromatin

↑ ARP2/3

Figure 5 | Structural changes at mitotic exit. a | During mitotic exit, the actomyosin network used to drive mitotic Nature Reviews | Molecular Cell Biology rounding is remodelled to form an actomyosin ring that is used to drive cytokinesis. As cells exit from mitosis, the nuclear lamina begins to reform from the poles, thus allowing time for lagging chromosomes to become incorporated into the nascent nucleus. ESCRTIII-mediated abscission leads to the formation of two daughter cells, which form new cell– substrate adhesions and re-spread following abscission. b | In cells dividing in an epithelium, the actomyosin ring moves from basal to apical as it contracts. Additionally, these cells need to form a new cell–cell interface. This may be aided by reactivation of ARP2/3 complex, which pushes adjacent cell membranes together. Finally abscission mediated by ESCRTIII leads to the formation of two daughter cells.

midbody, which is regulated by centrosomes132, mitotic changes in the cell. Further, proteins containing an NLS kinases and Spastin133,134, ESCRTIII is recruited to the — such as Anillin, RacGAP1 and ECT2 — that drive thin membrane tube that connects daughter cells, actin-dependent changes in mitotic cell shape can then where it triggers abscission as cells enter G1 (reviewed return to the nucleus to re‑establish interphase cell in REF. 134). An abscission checkpoint has been pro organization. Simultaneously, an increase in the activ posed to function as an additional layer of control to ity of RAP1 is thought to promote the assembly of cell– coordinate division with chromosome segregation, as substrate adhesions12. This involves Aurora B‑mediated lagging chromosomes act through Aurora B to delay phosphorylation of targets including FHOD1 ESCRTIII-mediated abscission135,136. (FH1/FH2 domain-containing protein 1; a formin) and Finally, as cells return to an interphase state, the the microtubule-binding protein EB3 (also known as nuclear–cytoplasmic compartment boundary is re‑ MAPRE3)139,140, which promote cell re‑spreading. established through the reassembly of nuclear pores and ESCRTIII-mediated remodelling of the nuclear Conclusions and perspectives envelope137. In addition to its other effects138, this Cell division necessitates a rapid, near-complete restoration of the nuclear compartment boundary is remodelling of cell shape, structure and polarity, which likely to insulate the cortex from the direct influence of partitions the entire suite of cellular components into chromatin-based signals to prevent further structural two daughter cells. This is an incredibly complex

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physical process, especially for animal cells dividing Although some of the events of cell division are now in the context of a multicellular tissue, and it must be relatively well understood, much remains to be discov precisely regulated. During entry into mitosis, events ered. The precise mechanisms underlying spindle–cortex such as adhesion remodelling, assembly of a cortical crosstalk remains an interesting and unsolved problem. actomyosin network, microtubule remodelling and In addition, little is known about the mechanisms that are spindle morphogenesis are coordinated by entrain used to ensure that mitosis and cell division are mechani ment to the same clock — that is, increasing levels of cally robust62 and that the various organelles are precisely mitotic kinase activity — as well as by the dramatic segregated during division142–144, or about the mech biochemical changes that accompany the sudden loss anisms that are used to bias these processes to achieve of the compartment boundary that separates the cyto the same precision during asymmetric divisions. Finally, plasm and nucleus at the transition from prophase to very little is known about the evolution of eukaryotic cell prometaphase. Thus, for most purposes, the micro division, which appears to have its origins in Archaea2,145. tubule and actin-based structures that are essential for As progression through mitosis is accompanied by rapid, chromosome segregation and cell division, respectively, sequential and wholesale changes in cell structure, the are constructed in parallel. Nevertheless, a successful study of cell division provides researchers with an ideal division requires spatial coordination of the cortex and window into the dynamic regulation of cell architecture. the spindle, necessitating crosstalk between these two The bulk of these events are driven by changes in the systems. This communication is essential to ensure that activities of a small set of kinases and phosphatases, the metaphase spindle is properly oriented with respect making it relatively easy to identify the switches and to interphase and mitotic cell shape, cell–cell junctions molecules that underpin these changes. Moreover, this and tissue tension; and that the cytokinesis ring is posi type of research is being aided by the advent of live tioned so that it bisects the anaphase spindle. Finally, super-resolution microscopy, which allows changes the return to interphase organization also requires in cell architecture to be imaged at an unprecedented both temporal (cyclin degradation, the loss of mitotic level, together with CRISPR–Cas9‑mediated genome kinase activity and the increasing dominance of mitotic editing and optogenetics, which provide new tools to phosphatases) and spatial (gradients of activity and re‑ test hypotheses and reveal the underlying molecular establishment of compartment boundaries) control. As mechanisms. Therefore, although the mass of cell divi an example of this, the nuclear envelope seals from the sion literature (only some of which we have been able to poles to the centre138, enabling lagging chromosomes summarize here) can seem overwhelming and the field to be gathered up into daughter nuclei to prevent the saturated, there are rich untapped seams waiting to be formation of micronuclei and chromothripsis141. mined and the perfect tools with which to do it.

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