Seminars in Cell & Developmental Biology xxx (2014) xxx–xxx
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Seminars in Cell & Developmental Biology
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haping up to divide: Coordinating actin and microtubule cytoskeletalemodelling during mitosis
scar M. Lancaster ∗,1, Buzz Baum ∗
RC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK
r t i c l e i n f o
rticle history:vailable online xxx
eywords:
a b s t r a c t
Cell division requires the wholesale reorganization of cell architecture. At the same time as the micro-tubule network is remodelled to generate a bipolar spindle, animal cells entering mitosis replace theirinterphase actin cytoskeleton with a contractile mitotic actomyosin cortex that is tightly coupled to theplasma membrane – driving mitotic cell rounding. Here, we consider how these two processes are coor-
dinated to couple chromosome segregation and cell division. In doing so we explore the relative roles ofcell shape and the actin cortex in spindle morphogenesis, orientation and positioning.
To achieve accurate segregation of genetic information duringukaryotic cell division requires dramatic reorganization of cellu-ar structures in a highly ordered and coordinated fashion. Most
a new population of shorter, more dynamic microtubules is nucle-ated from centrosomes [1]. Following nuclear envelope breakdownthe plus ends of centrosome-nucleated microtubules establish con-tacts with chromosomes at kinetochores [2]. Then, working inconcert with microtubules nucleated by chromatin and augmin
Please cite this article in press as: Lancaster OM, Baum B. Shapingremodelling during mitosis. Semin Cell Dev Biol (2014), http://dx.doi.
mportantly, interphase microtubules need to be remodelled toenerate a mitotic spindle; the macromolecular machine responsi-le for chromosome segregation. Cytoskeletal remodelling begins
n prophase, when interphase microtubules are disassembled and
together with microtubule motors and other accessory proteins,centrosome-nucleated microtubules establish a bipolar spindle.Kinetochores play key roles in this process. They serve as the majormechanical anchors between microtubules and chromosomes andcouple microtubule-based spindle forces to chromosome move-ment [3]; they also act as signalling platforms for the spindleassembly checkpoint, which monitors the physical state of chro-
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mosome attachment to microtubules [4] to ensure that everychromosomal pair is properly attached to the spindle before theinitiation of chromosome segregation.
Fig. 1. Roles for the actin cortex in spindle morphogenesis. (A) Metazoan cellschange shape in mitosis and become round. (B) Cell rounding is required for efficientchromosome capture and spindle pole stability. The actin cortex is only requiredwhen cells are required to round against a deformable constraint (see [19]). (C) Theactin cortex and cell rounding are required for planar spindle orientation in epitheliain vivo [93], and the actin cortex may buffer mitotic cell shape against tissue forces
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It is possible to recapitulate the events of spindle assem-ly in vitro, in cytoplasmic extracts from mitotic cells in thebsence of cell membranes and kinetochores. Mitotic Xenopusgg extracts will self-assemble bipolar spindles around beadsoated with plasmid DNA [5]. This both provides a useful sys-em with which to study the mechanisms of spindle assemblynd makes it clear that spatial cues from the cell and its sur-oundings are not essential for spindle formation. This observationas inspired a number of theoretical and biochemical studieshose aim is to study the minimal set of components required for
ipolar spindle self-organisation. It also begs the question: whathen is the relationship between the spindle and the rest of theell?
In most eukaryotic systems, the events of chromosome segrega-ion and cell division are tightly coupled. This coordination is mostritical at mitotic exit, when overlapping microtubules at the cen-re of the elongating anaphase spindle recruit accessory proteinso form a “centralspindlin complex”. This centralspindlin complexecruits a RhoGEF called Ect2, which activates RhoA GTPase to trig-er the assembly of an actomyosin network just beneath the plasmaembrane across the centre of the spindle [6]. The contraction
f this cortical actomyosin network, together with relaxation atpposing cell poles [7–9], then drives cytokinesis. Once membraneas been delivered to the cleavage site and the actin cortex haseen disassembled [10], abscission follows [11]. The result: twoaughter cells each of which carries a single, complete copy of thearental cell’s duplicated genome.
Cell division however is not the only morphological changehat accompanies mitotic progression. Almost all metazoan cellsecome round and spherical as they enter mitosis (Fig. 1A). Thisrocess requires a profound change in cell organization, and hap-ens in a few minutes right at the onset of mitosis. Significantly,his is the case in nearly all eukaryotes that lack a cell wall, fromictyostelium to humans [12], and even accompanies cell division inome protozoa like Leishmania [13]. For this reason, mitotic round-ng is a familiar sight to cell biologists working with cells in culture,o developmental biologists [14,15,8], and to pathologists look-ng at tissue samples from cancer patients [16]. The process of
itotic rounding has been described in most detail for animal cellsn culture. Here, typically over a period of 5–20 min, animal cellsetract their margins to reduce their spread area and increase ineight, taking on a near-spherical shape [17–19]. For a long whilehe events of mitotic cell rounding were relatively understudied.owever, a picture of the molecular mechanisms underlying therocess has begun to emerge over the last few years. As with cytoki-esis, the actin cytoskeleton is a key player in the story. Manytudies have used the availability of small molecule inhibitors thatct near instantaneously and reversibly to perturb actin cytoskele-al dynamics and actomyosin-dependent force generation in livingells [20], revealing complex cross-talk between the actin cortexnd the developing mitotic spindle. Although much remains to beiscovered, our aim here is to look at the relationship between thepindle and the dividing cell in which it resides: to explore the rolesf cell shape and the actin cortex in the morphogenesis, positioningnd orientation of mitotic spindles.
. Mitotic rounding and spindle formation
For cells growing in culture, rounding begins during the earliesttages of mitotic prophase [21,18] with a progressive actomyosin-riven retraction of the cell margin [21,22,18]. This is followed by
Please cite this article in press as: Lancaster OM, Baum B. Shapingremodelling during mitosis. Semin Cell Dev Biol (2014), http://dx.doi.
smotic swelling that begins at the onset of nuclear envelope break-own [17], by the loss of actin stress fibres [23,18] and by a coor-inated remodelling of focal adhesions [24]. While these processesork together to ensure rounding, the loss of adhesions is essential
in a manner analogous to that shown in (B).
and cells over-expressing a GTP-locked version of the adhesionregulator Rap1 fail to round up [24,19]. Strikingly, however, mostanimal cells entering mitosis in culture do not completely detachfrom the substratum as they round. Instead, the cell margin retractsto leave behind thin tubular strands of cytoplasm called retractionfibres that tether mitotic cells to the growth surface [25,23]. Theseare rich in actin filaments [23] and contain activated ERM proteins
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[26], which couple actin filaments to proteins embedded in theplasma membrane [27,28]. Although these fibres do not containhigh levels of activated Myosin II, they support tensile forces gener-ated by the retraction of the interphase cell margin at mitosis onset
23], and perform an important function in guiding later events initosis [29]. As cells round up, an actomyosin cortex is assembled
30–32], which is crosslinked to the overlying plasma membraney activated ERM proteins [33,27]. The forces generated throughhe action of this cortical pool of Myosin II help to give mitotic cellsheir mechanically stiff rounded form [27,17], and are large enougho drive epithelial buckling in a developing embryo [34].
Although the actin and microtubule cytoskeletons are coordi-ately remodelled at the onset of mitosis as the result of changes inhe activities of mitotic kinases, the two filament systems appearo be largely independent at this stage in mitosis. In tissue cul-ure cells, mitotic rounding does not depend on spindle assembly18]. Conversely, spindle assembly in mitotic cytoplasmic extractsn vitro is inhibited rather than aided by the presence of actin fila-
ents [5]. Despite this, several cell biological studies have pointedo a requirement for actin during spindle formation (reviewed in35]), based on defects in spindle morphogenesis observed in cellsith a compromised actin cytoskeleton [30,36,31,33,27,14,37], and
n reports describing the presence of actin filaments within thepindle itself [38–41]. While the impact of the actin cytoskeletonn mitosis is clear, the wide-ranging effects of perturbing the actinytoskeleton on cell shape, signalling, endocytosis and adhesion,omplicates the interpretation of this type of experiment. In anttempt to disentangle some of these variables, we recently car-ied out a study to determine the relative contributions made byhe actin cortex and mitotic cell shape to spindle assembly [19].sing a series of complementary genetic and physical methods toontrol mitotic cell geometry, the importance of mitotic roundingor spindle assembly was made clear (Fig. 1B); mitotic roundingrovides an appropriate space in which centrosomally nucleatedicrotubules can efficiently capture chromosomes. Further, bipo-
ar spindle collapse was a frequent occurrence in cells confined toeights of less than 7 �m (Fig. 1B; [42,19]). Again, this tendencyf flattened cells to form multipolar spindles could be attributedo simple geometrical constraints in the system. Cell confinement,ike the active compression of mitotic cells [43], leads to an increasen the width and length of spindles, which because of limits in theength of mitotic microtubules tends to destabilize spindle poleseading to the formation of multipolar spindles [44]. Consistent
ith this idea, lengthening mitotic microtubules rescued pole split-ing in HeLa cells entering mitosis under conditions of confinement19].
Space is therefore a key factor in spindle assembly. By contrast,n an isolated rounded mitotic HeLa cell, the near-complete loss ofhe actin cortex had no significant impact on the kinetics of spin-le formation (Fig. 1B; [19,45]). Conversely when forced to roundp against an externally imposed restraint, in this case a poly-crylamide gel designed to mimic the mechanical environment ofells growing in a crowded tissue environment (Fig. 1B), HeLa cellsacking a rigid actin cortex suffered mitotic catastrophe. Thus, actin-ased cortical tension is critically important under confinement toranslate osmotic swelling into an effective rounding force [17].aken together these data argue that the actin cytoskeleton con-ributes to spindle morphogenesis primarily through its protectiveffect on mitotic cell shape.
. Centrosome separation and spindle positioning
Although the role of actin in spindle assembly functions throughitotic cell shape, there is evidence in both cell culture and devel-
ping animals pointing to a more direct role for the mitotic actin
Please cite this article in press as: Lancaster OM, Baum B. Shapingremodelling during mitosis. Semin Cell Dev Biol (2014), http://dx.doi.
ortex in establishing spindle bipolarity through centrosome sepa-ation and in coordinating positioning of spindles within cells. Sincerosstalk between the anaphase spindle and cortex governs theosition of the cleavage furrow at cytokinesis, it follows that spindle
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position plays a critical role in determining the size of daughter cellsand in determining the polarity and fate of cells dividing withinpolarized developing tissues [46].
3.1. Actin and centrosome separation
During spindle assembly, duplicated centrosomes move toopposite sides of mitotic chromosomes (Fig. 1A). This is an impor-tant prerequisite for bipolar spindle formation, spindle positioningand for accurate chromosome segregation, and appears to rely onseveral functionally redundant mechanisms. Centrosome separa-tion can occur at different stages of mitosis [47]: either centrosomesseparate during prophase, such that spindle bipolarity is alreadyachieved by the time of nuclear envelope breakdown, or laterduring prometaphase. During prophase, Eg5 kinesin is thought tohelp drive centrosome separation by sliding antiparallel micro-tubules apart [48,49]; a process that is aided by the accumulationof Dynein at the nuclear envelope [50]. In prometaphase, theseprocesses are augmented by pushing forces derived from kineto-chore microtubules [51] [52] (for a detailed review of mechanismsof centrosome separation see [53]).
Given the functional redundancy in the system, most studieshave relied on live imaging to determine whether the loss of actinfilaments results in kinetic changes in the process of centrosomeseparation in cells entering mitosis. Using RNAi and inhibitors todepolymerize actin or to inhibit myosin II motor activity in a varietyof cultured cells, the cortical actomyosin cytoskeleton was sug-gested to play a novel role in the process of centrosome separationduring prometaphase [54]. By following the movement of fluores-cently labelled beads on the surface of cells, the authors went onto show that cortical actin and myosin II contraction on one sideof the cell leads to cortical expansion on the opposing side, gener-ating cortical flows that, via contacts between astral microtubulesand the cell cortex, move centrosomes apart. However, the pre-cise timing and the molecular nature of the contact between astralmicrotubules and actin cortex remain unclear. Delays in centro-some separation during prophase have also been observed whenactin filaments are depolymerised in HeLa cells and other celllines [48,55,56,19]. However, perhaps because of functional redun-dancy in the system, loss of the actin cortex does not compromisespindle formation in HeLa cells with an intact spindle checkpoint,and does not lead to a significant delay in the overall timing ofmitotic progression [19] (although centrosome separation duringprometaphase is associated with a small increase in chromosomesegregation errors during anaphase [57,58]). In addition, actin hasalso been implicated in prophase centrosome separation duringDrosophila syncytial development, where an actin network over-lying the nucleus appears to contribute to centrosome separation[59]. In this case, however, a small molecule inhibitor of myosinII activity did not effect centrosome separation; implying that theeffect is not due to actomyosin cortical flows like those describedby Rosenblatt and colleagues [54].
Although roles for actin in centrosome separation have beendemonstrated, there is mechanistic redundancy in centrosomeseparation pathways [53], so it is not essential for this process.Moreover, it is now clear that mitotic progression in many sys-tems does not absolutely require centrosomes, since chromosomesegregation and cell division continue in flies lacking centrosomes[60] and in mammalian cells following the ablation of centrosomes[61].
3.2. Actin and spindle positioning
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Centrosome separation and establishment and maintenance ofspindle bipolarity in turn permits controlled positioning of thespindle within the cell. Since spindle position helps to guide the
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No astral microtubules No actinA
y
x
z
x
microtubules actinGαi/LGN/NuMA/DyneinDNA
Mitosis
B Female meiosis No microtubules No actin
Myosin II Rab11a/MyosinVb vesiclesArp2/3 Arp2/3-dependent cytoplasmic streaming
micropatterned substrate
Fig. 2. Roles for the actin cortex in spindle positioning. (A) Cells growing in culture align their mitotic spindles parallel to the surface on which they are growing and relative tothe position of their retraction fibres. Spindle positioning depends on astral microtubules which read cortical positioning cues from G�i-LGN-NuMA/Dynein. Correct spindlep Dyneo
piolirirllein(
3
lhuoIiebt
ositioning also depends on actin and cortical localization of G�i-LGN-NuMA andocytes relies on actin dependent mechanisms but not microtubules.
ositioning of the cleavage furrow [62], accurate spindle position-ng in cells plays a crucial role in determining both the size and fatef daughter cells. This is particularly apparent when cell divisioneads to asymmetries in daughter cell size and fate. Studies look-ng at developmentally regulated asymmetric cell divisions haveevealed a set of conserved proteins that control spindle positioningn both asymmetric (see [63,64] for detailed reviews), and symmet-ical divisions [65]. These suggest a model in which the asymmetricocalization of the G�i-LGN-NuMA signalling module guides theocal recruitment and activation of cortical Dynein. Dynein thenxerts a pulling force on astral microtubules to guide the spindlento position. As a result, spindles lacking these signalling compo-ents or astral microtubules fail to align their spindles correctlyFig. 2A; [29,65–68]).
.2.1. Actin in meiotic spindle orientation and positioningInterestingly, female meiotic spindles in many systems
ack centrosomes and astral microtubules despite undergoingighly asymmetric divisions. These divisions function to removenwanted sets of chromosomes (in the form of polar bodies) with-ut the oocyte suffering an accompanying loss of cytoplasm [69].n large mammalian oocytes this process of spindle positioning
Please cite this article in press as: Lancaster OM, Baum B. Shapingremodelling during mitosis. Semin Cell Dev Biol (2014), http://dx.doi.
s highly dependent on cytoplasmic actin filaments (Fig. 2B). Forxample, meiosis I spindles form near the centre of mouse oocytesefore migrating along their long axis towards the closest cell cor-ex in a process that requires dynamic actin filaments [70], actin
in is stabilized by the actin cortex [80]. (B) Meiotic spindle positioning in mouse
filament nucleators Formin-2, Spire-1, Spire-2 and Arp2/3 [71–73],Myosin II [74] and Myosin Vb [75].
Several recent studies have proposed mechanisms to accountfor the role of actin in the displacement of the meiotic spindle tothe cortex. Myosin II is located at the poles of meiosis I spindles[74] where it has been proposed to couple spindle poles to thedynamic cytoplasmic actin network. This biased pulling force actingacross a cytoplasmic actin mesh that is nucleated by Rab11a vesi-cles [75], working together with changes in cortical rigidity [76] andaccompanying cytoplasmic flows [77], likely drives meiotic spindlemigration. These roles for cytoplasmic actin in the repositioning ofmeiotic spindles in oocytes prior to division at the cell cortex, arelikely explained by the need to link the cortex and the spindle inenormous cells that lack centrosomes (Fig. 2; [78]). Roles for cyto-plasmic actin in oocytes are not restricted to spindle positioning;in starfish oocytes a cytoplasmic actin mesh is required to gathermeiotic chromosomes towards the nascent spindle [79].
3.2.2. Actin in mitotic spindle orientation and positioningWhat then might be the role for actin filaments in spindle posi-
tioning during mitosis in proliferating animal cells? Cells growingin culture typically divide parallel to the substrate, providing a sim-
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ple experimental system in which to study the role of the actincytoskeleton in mitotic spindle orientation (Fig. 2A). Moreover,with the development of micropatterning and microfabricationtechniques, it has become clear that isolated animal cells can
example via the circulating Arp2/3-dependent pool of cytoplasmicactin structures observed in many cells in culture [88]. In addition,
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recisely orient their spindle in the plane of the dish in responseo defined biochemical and physical cues from the environment.ignificantly, spindle orientation and positioning in these cell cul-ure systems requires an intact actin cortex [26,45,31,80], togetherith a number of actin regulators, including ERM family pro-
eins [33,27], LIMK [81] and Cdc42 [82] and regulators of corticalignalling Abl [83] and PtdIns(3,4,5)P3 [84]. Components of focaldhesions such as �1-integrin [45] and MISP [85] also appear to beequired.
In exploring the connection between the substrate and the spin-le in cells on micropatterned substrates, Théry and colleaguesound that the distribution of adhesions in interphase cells pre-isely predicts the future orientation of the mitotic spindle (Fig. 2A;26,29]). This focused attention on the distribution of retractionbres formed during mitotic rounding, which physically inter-onnect the substrate to the rounded mitotic actin cortex, anded the authors to suggest that retraction fibres represent a formf cellular memory by which cells remember their interphasehape. Using laser ablation to selectively cut retraction fibres inells plated on micropatterned substrata, Fink and colleagues [86]ater confirmed their role in guiding spindle orientation. Moreover,y applying external forces to the substrate, Fink and colleaguesere able to show that mitotic cells continually monitor the force
mparted on the cell body by retraction fibres enabling them toeorient their spindle in response to a change in their mechanicalnvironment.
How might the mitotic spindle read external forces, adhesionsnd retraction fibres? While a precise mechanistic understandings lacking, it is clear that the actin cortex plays a key role. Spin-les align with the longest axis of mitotic cells [87], and whilstost mitotic cells are uniformly round in mitosis, it is possible
hat forces exerted by retraction fibres lead to small anisotropiesn cell shape that are read by spindles. Alternatively, as suggestedy the work of Fink and colleagues [86], forces exerted by retrac-ion fibres lead to biased organization of sub-cortical Arp2/3 basedctin structures in mitotic cells [88], which may then be coupled tostral microtubules and influence spindle positioning. While this isn attractive idea, there has yet been a good case for a role for therp2/3 complex functioning in mitosis. Since actin-based retrac-
ion fibres are directly embedded in the actomyosin cortex of theell body, a further possibility is that forces exerted by retractionbres on the cortex and/or resulting changes in local cortical orga-ization co-opt G�i-LGN-NuMA and, therefore, Dynein. If this werehe case, astral microtubules emanating from the spindle would beble to probe the inner face of the cortex, using the local accumu-ation of G�i-LGN-NuMA and cortical Dynein as a proxy for corticalorces induced through the application of external forces or by
itotic rounding itself. That said, myristolation of G�i likely aidshe localization of LGN-NuMA at the plasma membrane [89] and a
echanistic link between cortical actin and cortical localization of�i-LGN-NuMA has not been clear. Interestingly however, a recenttudy from Zheng and colleagues [80] revealed a critical role for thectin cortex in countering the action of Dynein, which in the pres-nce of astral microtubules tends to strip G�i-LGN-NuMA from theortex (Fig. 2A). Using this as the basis of a model, it is easy to imag-ne how differences in cortical actin organization, for example thoseriven by retraction fibres, could translate into different effectiveortical Dynein-dependent pulling forces. Converting this into sta-le central positioning of a spindle may in turn rely on feedbackrom the bipolar spindle itself, as recently suggested by Kiyomitsund Cheeseman [65]. Thus, while the role of actin in spindle orien-ation is not yet understood, it is possible that it provides mitoticells with a mechanically stable platform upon which informationbout a cell’s micro-environment, adhesion, shape and force can
Please cite this article in press as: Lancaster OM, Baum B. Shapingremodelling during mitosis. Semin Cell Dev Biol (2014), http://dx.doi.
e integrated and read by the microtubule motors that position thepindle.
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3.2.3. Spindle orientation in an epithelial contextFinally, what might be the role of a cell’s local environment
and actin cytoskeleton in governing mitotic spindle orientationin vivo (Fig. 1C)? In the context of a mechanically active epithe-lium, spindle orientation plays a vital role in ensuring tissue growthand integrity, and in the control of tissue-scale forces [15]. Spindlealignment in the plane of epithelial tissues promotes tissue growthand spreading, whereas perpendicular spindle alignment leads totissue thickening and differentiation [46]. As for planar spindle ori-entation in tissue culture cells, many of the same actin regulatorsand adhesion proteins play similar roles in the control of spindleorientation in epithelia in vitro [90], and in the context of tissues inliving animals [14,83,91–93]. Recent studies have highlighted rolesfor actomyosin in this process. Thus, a balance of astral microtubuleand Myosin 10 basally directed forces and apically directed actinand Myosin II flow accurately positions mitotic spindles within thecorrect plane in epithelial cells in the early Xenopus embryo [94]. Inpseudo-stratified epithelia in Drosophila, mitotic cell apical migra-tion and planar spindle alignment rely on balanced actomyosincontractility and mitotic cell rounding [95,93,96]. Moreover, indeveloping fly tissues, spindle alignment influences tissue growthby responding to local and tissue-scale forces that influence cellshape [97–99]. While it remains unclear whether the mitotic actincytoskeleton communicates information about tissue force and/orshape to the spindle in a manner analogous to that described fortissue culture cells, it is clear that this role for the actin cytoskeletonin mitotic rounding [34] and spindle orientation will be a factor inguiding the morphogenesis of tissues during development, homeo-stasis, regeneration and disease.
4. Conclusion
The actin filament based cortex lies at the interface between ananimal cell and its environment. It sculpts the membrane to controlcell shape and acts as a physical boundary through which interac-tions between a cell, its neighbours and its material environmentare mediated. Thus, in the context of mitosis, the rigid actin cortexassembled during mitotic rounding buffers the delicate process ofchromosome segregation from the potentially disturbing influenceof external forces, while at the same time enabling cells to senseand respond to force anisotropies. While this level of sophisticationin a cell in a culture dish may seem surprising, these dual functionsare likely to be critical for cells growing in complex polar tissueenvironments, where forces are continually shifting, and wherethe orientation of the cell division plane has important implica-tions for the preservation of cell polarity and tissue topology. Forcells in culture, actin rich retraction fibres generated during cellrounding act as key intermediaries in this dialogue; by acting as amemory of interphase cell shape, they enable cells to read changesin the mechanics of the matrix to which they are attached and guideorientation of the mitotic spindle. How this physical image of theworld is imprinted in the cortex remains a mystery, but it seemslikely that it is read out as differences in the localization of corticalmarks that bind Dynein, which binds astral microtubules to reposi-tion the spindle. And how these models translate into the influenceof cell–cell contacts and forces in tissues on spindle orientationin vivo remains an active area of investigation.
Given recent findings documenting roles for cytoplasmic actinin chromosome segregation and spindle positioning in oocytes,it will be important in the future to determine whether or notnon-cortical actin plays a similar role in mitotic animal cells, for
up to divide: Coordinating actin and microtubule cytoskeletalorg/10.1016/j.semcdb.2014.02.015
while strong evidence remains to be found to support actin func-tioning within the mitotic spindle itself, it is clear that cytoplasmic
ctin must be tightly regulated in mitosis, since the deregulation ofctin polymerization during mitosis can profoundly disrupt chro-osome movements [36,37]. In conclusion, mitosis is accompanied
y changes in cortical actin organization and dynamics – similar inagnitude to the wholesale changes in the microtubule cytoskele-
on required for spindle formation. Since these alter cell shape,rganelle organization [100], vesicle trafficking [101], as well asentrosome separation, spindle positioning and cell division, it islear that this is likely to be the beginning of the story of the role ofctin in mitosis.
cknowledgements
We would like to thank Andrea Dimitracopoulos, Helenatthews, Nunu McHedlishvili, Yanlan Mao and Maël Le Berre for
ritical reading of and suggestions on the manuscript. Buzz Baums funded by a Cancer Research UK Senior Research Fellowship.
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