Seminars in Cell and Developmental Biology xxx (xxxx) xxx

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Seminars in Cell and Developmental Biology

journal homepage: www.elsevier.com/locate/semcdb

Review

Symmetry-breaking of animal cytokinesis

Kenji Sugioka a, b, *
a
Life Sciences Institute, The University of British Columbia, Vancouver, BC V6T1Z3, Canada
b
Department of Zoology, The University of British Columbia, Vancouver, BC V6T1Z3, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: Cytokinesis is a mechanism that separates dividing cells via constriction of a supramolecular structure, the
Cytokinesis contractile ring. In animal cells, three modes of symmetry-breaking of cytokinesis result in unilateral cytokinesis,
Cell polarity asymmetric cell division, and oriented cell division. Each mode of cytokinesis plays a significant role in tissue
Cell cortex
patterning and morphogenesis by the mechanisms that control the orientation and position of the contractile ring
Cytoskeleton
relative to the body axis. Despite its significance, the mechanisms involved in the symmetry-breaking of cyto­
Asymmetric cell division
Contractile ring kinesis remain unclear in many cell types. Classical embryologists have identified that the geometric relationship
between the mitotic spindle and cell cortex induces cytokinesis asymmetry; however, emerging evidence suggests
that a concerted flow of compressional cell-cortex materials (cortical flow) is a spindle-independent driving force
in spatial cytokinesis control. This review provides an overview of both classical and emerging mechanisms of
cytokinesis asymmetry and their roles in animal development.

1. Introduction cycle-dependent regulation of the chromosome passenger complex

Cytokinesis is the final step of cell division that physically separates
dividing cells through the function of supramolecular machinery called
the contractile ring. Failure of cytokinesis results in duplication of the
entire genome and centrosomes that eventually facilitates the develop­
ment of aneuploidy (reviewed in [1]). Although the causal relationship
between cytokinesis failure and spontaneous tumorigenesis in humans
remains unclear, it is well-established that genome-doubling and
centrosome-amplification events caused by cytokinesis failure can
induce tumors in model organisms [1–4]. Despite the critical require­
ment for faithful cell division, cytokinesis is not stereotypical during
animal development. Stereotypical cytokinesis describes a mode of
cytokinesis that is often observed in cell biology textbooks, wherein the
contractile ring formed at the cell equator constricts in a concentric
manner (Figs. 1A and 2A). In this review, this mode of cytokinesis is
termed “symmetric cytokinesis” for convenience.
In symmetric cytokinesis, a contractile ring is formed at the equa­
torial region of the cell cortex (Fig. 1A). Central to the contractile ring
formation is the cortical activation of small GTPase RhoA (Fig. 1B) [5].
Ect2 RhoGEF (Guanine nucleotide Exchange Factors) regulates dissoci­
ation of GDP from inactive RhoA-GDP and accelerates the formation of
active RhoA-GTP. However, Ect2 activity is limited due to auto­
inhibition [6–9]. Timely activation of Ect2 requires the cell
(CPC) and centralspindlin. The CPC is composed of Aurora B kinase,
INCENP, Borealin, and Survivin while centralspindlin consists of
kinesin-6/MKLP1 and MgcRacGAP/CYK-4 [5,10]. Until the anaphase of
cell division, M-Cdk inhibits the CPC and centralspindlin (Fig. 1Ai) [11,
12]. 14–3–3 protein also inhibits centralspindlin oligomerization
through interacting with kinesin-6 [13,14]. Upon degradation of Cyclin
B at anaphase, the CPC recruits centralspindlin to the spindle midzone
and activates its oligomerization through Aurora B-mediated disruption
of kinsin-6–14–3–3 interaction (Fig. 1Aii) [13,14]. Furthermore, cell
cycle-dependent dephosphorylation of Ect2 and Polo-like kin­
ase-dependent phosphorylation of CYK-4 promote CYK-4-Ect2 interac­
tion that in turn activates Ect2 (Figure 1 Aiii, and B) [15–18]. Forced
inhibition of M-Cdk prematurely induces cytokinesis without chromo­
some segregation [19,20]. Upon Ect2 activation, Ect2-RhoA interaction
stimulates RhoA that in turn promotes Ect2 activity through an allosteric
activation mechanism [9]. While centralspindlin plays critical roles in
RhoA activation, there are centralspindlin-independent pathways. The
CPC may activate contractile ring assembly in a
centralspindlin-independent manner, while the molecular mechanisms
remain unclear [21–24]. Other centralspindlin-independent factors in
Caenorhabditis elegans (C. elegans) include the worm-specific NOP-1,
which activates RhoA in parallel to CYK-4 [25].
The mitotic spindle plays a critical role in specifying the site of

* Correspondence address: 2407–2350 Health Sciences Mall, Vancouver V6T1Z3, Canada.

E-mail address: sugioka@zoology.ubc.ca.

https://doi.org/10.1016/j.semcdb.2021.12.008
Received 30 July 2021; Received in revised form 5 December 2021; Accepted 16 December 2021
Available online 23 December 2021
1084-9521/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Please cite this article as: Kenji Sugioka, Seminars in Cell and Developmental Biology, https://doi.org/10.1016/j.semcdb.2021.12.008
K. Sugioka
contractile ring formation, as predicted by Rappaport’s classic experi­
ments [26]. First, astral microtubules promote contractile ring forma­
tion (astral stimulation model; Fig. 1B). In sea urchin embryos, Ect2 and
centralspindlin localize from the spindle midzone to the plus-end of
astral microtubules [27]. Additionally, a CYK-4 homolog in Drosophila
shows microtubule plus-end localization in a manner dependent on the
microtubule plus-end binding protein EB1 [28]. Second, the mitotic
spindle positions the spindle midzone at the cell equator, where cen­
tralspindlin enriches and promotes bundling of anti-parallel interpolar
microtubules (central spindle stimulation model; Figure 1Aiii and B)
[29]. Finally, astral microtubules can also inhibit contractile ring as­
sembly in the polar region, with the resulting relaxation of the polar cell
cortex referred to as astral relaxation or polar relaxation (Fig. 1C) [30].
In sea urchin embryos, selective disassembly of astral microtubules and
laser ablation of aster result in the extension of active RhoA zone to the
area devoid of surface astral microtubules [31]. Furthermore, the
mitotic spindle that positions in proximity to the cell cortex can suppress
cortical myosin enrichment in C. elegans [32]. Different mechanisms of
polar relaxation have been proposed (Fig. 1C). In C. elegans, active
Aurora A at the astral microtubules and dynein-dependent removal of
myosin II from the cell cortex drive polar relaxation [33,34]. In HeLa
cells, astral microtubule-dependent inhibition of diaphanous formin
through the microtubule plus-tip binding protein CLIP170 reduces polar
actin assembly [35]. Interaction between astral microtubules and cell
cortex is mediated by anillin in both worms and HeLa cells [36,37].
Interestingly, an astral microtubule-independent pathway exists in
human HeLa cells, where chromatin-derived RanGTP,
kinetochore-localized protein phosphatase 1 (PP1) and PP1 regulatory
subunit Sds22 inhibit polar F-actin assembly, while the relationship
between RanGTP and PP1 remains unclear [38,39].
The relationship between the three microtubule-dependent con­
tractile ring formation pathways have been studied extensively. Astral
microtubules and central spindle specify contractile ring position
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Seminars in Cell and Developmental Biology xxx (xxxx) xxx
independently [40,41]. Indeed, cells without astral microtubules pre­
dominantly rely on spindle midzone-dependent contractile ring forma­
tion [31]. In C. elegans, astral microtubules at the equator stimulate
contractile ring assembly in early anaphase while those in late anaphase
restrict contractile ring assembly at the equator through inhibitory
mechanisms [41–43].
After establishment of the active RhoA zone at the equatorial cortex,
RhoA activates myosin II motor protein through Rho kinase and the
actin nucleation/polymerizing factor formin to assemble the contractile
ring (Fig. 1B) [44,45]. Importantly, optogenetic recruitment of RhoGEF
to the plasma membrane is sufficient to induce cleavage furrow inde­
pendent of the spindle position [46]. Thus, theoretically
spindle-independent cytokinesis is possible as long as there are factors
that spatio-temporally control the active RhoA zone. Readers interested
in detailed information on stereotypical cytokinesis are advised to read
recent reviews [5,6].
In various developmental contexts, contractile ring formation and
function are asymmetrically controlled due to spatially localized factors.
Symmetry breaking of cytokinesis is involved in epithelial cell division,
asymmetric cell division, and oriented cell division and plays significant
roles in animal morphogenesis. Based on the types of asymmetry, we can
categorize symmetry breaking of cytokinesis into three different modes
(Fig. 2). First, unilateral cytokinesis involves eccentric contractile ring
closure (Fig. 2A). Note that some studies refer unilateral cytokinesis as
“asymmetric cytokinesis,” but it is conceptually distinct from asym­
metric cell division. Second, asymmetric cell division involves polarized
contractile ring positioning, resulting in the generation of daughter cells
of different sizes (Fig. 2B). Finally, oriented cell division involves tilting
of the contractile ring as the ring constricts (Fig. 2C). We refer to this
type of cytokinesis as oriented cytokinesis. The symmetry-breaking of
cytokinesis is not a new concept, as it was reported by 19th-century
biologists through studies of early embryogenesis (summarized in
[47]). Early studies have demonstrated that some symmetry-breaking
Fig. 1. Mitotic Spindle-dependent Regulation
of Cytokinesis. (A) Stereotypical cell division at
metaphase (i), anaphase (ii, iii), and cytokinesis
(iv). The chromosome passenger complex (CPC)
and centralspindlin relocalize from the kineto­
chore to the spindle midzone and cell cortex to
activate contractile ring formation. (B) The
mechanisms of RhoA activation at the cell
equator. RhoA activator Ect2-RhoGEF is acti­
vated through interaction with centralspindlin
component CYK-4. Astral microtubule- and
central spindle-dependent activation of RhoA
via Ect2 results in the formation of active RhoA
zone at the equatorial cell cortex. RhoA-GTP
then activates myosin II (through Rho kinase)
and formin to initiate contractile ring assembly.
(C) The mechanisms of polar relaxation. Astral
microtubules interact with the cell cortex via
anillin. Dynein-dependent myosin II removal
from the cell cortex, and CLIP-170-dependent
inhibition of formin rely on astral microtu­
bules. Although Aurora A is required for polar
relaxation in C. elegans, it is unclear whether it
regulates dynein-dependent myosin II trans­
port. Ran-GTP and protein phosphatase PP1-
Sds22 on the chromosome can also inhibit
polar F-actin assembly in an astral microtubule-
independent manner. Note that these mecha­
nisms are relatively divergent among organisms
and tissues (see text).
K. Sugioka
events can be explained by the biased positioning of the mitotic spindle,
because the spindle can specify the furrow position, as described above.
However, emerging evidence suggests that spindle-independent mech­
anisms also break cytokinesis symmetry. Interestingly, these new studies
proposed that the cortical flow, a concerted movement of cell cortex
materials, plays significant roles in the spindle-independent mecha­
nisms. This review focuses on recent advances in knowledge concerning
symmetry breaking of cytokinesis.
2. Unilateral cytokinesis
It has been known since the 19th century, that cleavage of the
ctenophore zygote is strongly asymmetric (Fig. 3A) [48]. The tiny
mitotic spindle in this embryo is eccentric, leading to the formation of a
cleavage furrow at the cell cortex near the spindle. The opposite side of
the cell cortex shows no furrows or indentations. The cleavage furrow,
which is formed between the nuclei, cuts across the zygote to complete
cytokinesis. Yatsu termed this process and the leading edge of the furrow
“unilateral cleavage” and “cleavage head,” respectively [49]. Unilateral
cytokinesis is observed in single cells, such as the zygote of many ani­
mals [48,50,51], as well as in multicellular tissues, such as epithelia of
wide animals, including humans [52–56]. In this section, we discuss the
mechanisms and roles of unilateral cytokinesis.
2.1. Spindle geometry-dependent regulation of unilateral cytokinesis
Unilateral cleavage is induced by eccentric spindle positioning. Here,
eccentric positioning specifically refers to the transverse displacement of
the spindle relative to the division axis (Fig. 3A). Yatsu performed pio­
neering embryo dissection experiments using ctenophore zygotes that
undergo unilateral cytokinesis. After dissection of the zygote just below
the cleavage head, the cell half containing the mitotic spindle completed
cytokinesis, whereas the other half devoid of a spindle did not form
cleavage [49]. Rappaport confirmed these findings and showed that
artificially-induced centering of the mitotic spindle resulted in
Fig. 2. Symmetry Breaking of Cytokinesis. (A) Stereotypical cytokinesis. (B) Unilateral cytokinesis. The contractile ring closure is eccentric. (C) Symmetry breaking
of cytokinesis during asymmetric cell division. The contractile ring positioning is polarized along the division axis. (D) Symmetry-breaking of cytokinesis during
oriented cell division. The contractile ring undergoes tilting as the ring constricts. The green arrows summarize representative cortical flow patterns observed.
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Seminars in Cell and Developmental Biology xxx (xxxx) xxx
symmetric cytokinesis [57]. Rappaport further showed that normally
symmetrically-dividing sand dollar zygotes undergo unilateral cytoki­
nesis due to spindle displacement upon embryo flattening [57]. Similar
manipulation in sea urchin embryos resulted in RhoA activation and
unilateral cleavage at the cell cortex closest to the displaced spindle
[58]. Thus, it is widely accepted that a transverse shift in spindle posi­
tioning causes unilateral cytokinesis.
What causes transverse spindle shift in vivo? A key to answering this
question is the spindle centering-force that prevents transverse spindle
shift. A pioneering in vitro reconstitution experiment showed that an
artificial microtubule-organizing center (a bead emanating microtu­
bules; MTOC) positions in the center of the microfabricated chambers
without motor proteins [59]. On the other hand, an artificial MTOC with
short microtubules positioned eccentrically, suggesting that the
spindle-centering force is likely generated by the
microtubule-polymerization-dependent pushing of the chamber wall
[59]. A recent analysis using magnetic tweezers revealed the
spindle-centering force in vivo. The centrosome in the mitotic spindle of
the C. elegans zygote showed higher resistance against the transverse
shift when more astral microtubules are emanated from it [60] (Fig. 3B).
There are two models describing generation of the spindle-centering
force. The first model involves the microtubule
polymerization-dependent pushing of the plasma membrane as
described above (Fig. 3B). The second model is based on the observation
of cytoplasmic dynein-dependent transport of organelles such as endo­
some, lysosome, and yolk granules in C. elegans zygote [61]. The cor­
relation between microtubule minus-end-directed organelle movements
and centrosome positioning indicates that the resistive force during
organelle transport generates spindle-pulling forces (Fig. 3B). It is pro­
posed that resistive force is the drag force generated when the organelles
move inside the viscous cytoplasm. In this model, more pulling forces
are generated along the longer microtubules due to the increased
number of cytoplasmic dynein molecules per fiber. In both models, the
number and length of the astral microtubules scale with
spindle-centering forces. Thus, it is likely that the spindle with fewer and
K. Sugioka
shorter astral microtubules more likely undergoes transverse shift due to
the lower spindle centering-force. These embryos are likely large in size:
the comparative embryo analysis shows that extremely large embryos
tend to have small mitotic spindles in proportion to cell size, whereas the
sizes of embryo and spindle normally scale [62].
Other factors can also induce transverse spindle shift. The non-
uniform distribution of cytoplasmic materials in cells can bias spindle
positioning in both Xenopus and zebrafish. In these embryos, yolk is
enriched in the vegetal side and is proposed to inhibit the growth of
astral microtubules [63,64]. Additionally, the infection of HeLa cells
with Chlamydia trachomatis results in biased positioning of the spindle
and unilateral cytokinesis due to the physical presence of Chlamydia
inclusion at the cell equator [65].
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Fig. 3. Spindle Geometry-dependent Unilateral
Cytokinesis. (A) Unilateral cytokinesis of the
ctenophore zygote. The cleavage head is formed
between small nuclei (two circles near the cell
outlines). Spindle transverse shift induces uni­
lateral cytokinesis (right). (B) The spindle-
centering force prevents transverse spindle
shift. Microtubule pushing against the cell cor­
tex might account for the spindle-centering
force. The cytoplasmic dynein-dependent
organelle transport toward the centrosome
might also generate pulling forces that prevent
the transverse shift. A centrosome with more
astral microtubules is more resistant against the
magnetic tweezer-dependent shift of centro­
some position. Green spheres are magnetic
beads.
(left; adapted from Ziegler [48]).
2.2. Spindle geometry-independent regulation of unilateral cytokinesis
Cells can also undergo unilateral cytokinesis in a spindle-positioning-
independent manner. These types of unilateral cytokinesis have been
intensely studied in C. elegans zygotes and animal epithelia. Studies in
both systems provide unique insights into the mechanism of unilateral
cytokinesis, since cell-intrinsic and -extrinsic mechanisms control
C. elegans zygote and epithelia unilateral cytokinesis, respectively.
The C. elegans zygote undergoes unilateral cytokinesis without a
transverse shift in spindle positioning [51]. Furthermore, the loss of
spindle midzone in spd-1 mutants does not impair unilateral cytokinesis
[66]. Maddox et al., showed that contractile ring components, such as
myosin, actin, anillin, and septin, are enriched in the cleavage head,
thereby establishing structural asymmetry in the contractile ring
Fig. 4. Spindle Geometry-independent Regula­
tion of Unilateral Cytokinesis. (A) Unilateral
cytokinesis in the C. elegans zygote. The active
gel model considers the cell cortex as a
compressive gel. The myosin-dependent
compression of unit gel at the active Rho A
zone induces cortical flow towards cell equator
that in turn enriches myosin at the cleavage
head by transporting cell cortex materials (left).
Cortical flow also aligns actin filaments (mid­
dle). Schematic diagram of unilateral contrac­
tile ring constriction (right). The involvement
of membrane curvature and the mechanism of
cortical flow asymmetry remain unclear. (B)
Unilateral cytokinesis in Drosophila embryonic
epithelium. Basal-to-apical cleavage results in
the apical positioning of the ring (left). The
cleavage becomes symmetric upon the loss of
adherens junctions (middle). Mechanical resis­
tance prevents the contractile ring constriction
(right). (C) Apical lumen formation in the 3D
culture of epithelial cells. The first cytokinesis is
symmetric while the consecutive divisions
involve unilateral cytokinesis, leading to the
maintenance of apical lumen site. The defects in
unilateral cytokinesis result in multiple lumen
formation in some tissues.
K. Sugioka
organization [51]. Knockdown of anillin, septin, or formin results in
symmetric contractile ring assembly and closure [51,67]. Interestingly,
it is still unclear whether anillin is essential for unilateral cleavage, given
that further knockdown of the worm-specific RhoA activator NOP-1
rescues the anillin RNAi phenotype [25]. How is the cell cortex asym­
metry established? Two major models are currently available. Menon
et al. proposed that the cortical flow plays a critical role in unilateral
cytokinesis [68]. Cortical flow is a concerted flow of the compressible
cell cortex that controls various biological processes, including cell po­
larity, cell migration, and cytokinesis [69–73]. Theoretical analysis
showed that asymmetric cortical flow towards the ring from the pole
resulted in a transverse shift in contractile ring position [68]. On the
other hand, Dorn et al. suggested that the initial membrane curvature
aligns circumferential actin filaments in the contractile ring, thereby
facilitating the ring constriction [66] (Fig. 4A, middle). A positive
feedback loop between filament alignment, membrane curvature, and
ring constriction might amplify the initial spontaneous asymmetry in
filament alignment, resulting in unilateral cleavage. Insights from recent
studies might integrate these two models. Khaliullin et al. showed that
cortical flow from the pole toward the ring is indeed asymmetric, and
that the area with higher cortical flow toward the ring coincides with the
site of cleavage head formation [74]. They proposed that
myosin-dependent compression of the cell cortex at the active RhoA
zone and membrane expansion at the polar region induce cortical flow
toward the equator from the polar region. The cortical flow then recruits
more myosin to the active RhoA zone to form a positive feedback loop
(Fig. 4A, left and right). Cortical flow also aligns the circumferential
actin filament around the contractile ring through compression and
filament-guided filament assembly (Fig. 4A, middle) [71,75]. Thus,
asymmetric cortical flow appears to be the critical regulator of unilateral
cytokinesis (Fig. 4A; right). However, the mechanisms underlying
symmetry-breaking of cortical flow remain unclear.
Epithelial cells undergo unilateral cytokinesis in wide organisms
including humans [52–55,57]. Larvae and embryos of Drosophila mela­
nogaster, Xenopus laevis, and C. elegans were used as models to study the
mechanism of unilateral cytokinesis in this tissue. Despite the signifi­
cance of spindle off-centering in unilateral cytokinesis, the geometrical
relationship between the mitotic spindle and cell cortex has been
quantitatively analyzed in a few studies [76,77]. Apparently, mitotic
spindle positioning is not extremely off-centered but geometrically
closer to the apical cell cortex in some cell types [76–79]. In many
epithelial cells, the cleavage head is formed at the basal side and ad­
vances toward the apical side. However, there are exceptions: the
cleavage head is formed at the apical side and advances toward the basal
side in the blastula stage Xenopus embryo and in rat kidney tubular cells
[55,80,81]. While these cell types are interesting and important
considering the fact that most studies on epithelial unilateral cytokinesis
have been done using basal-to-apical cleavage, this review focuses on
basal-to-apical unilateral cytokinesis due to the limited knowledge on
the mechanisms underlying apical-to-basal cleavage.
Although the C. elegans zygote forms a structurally asymmetric
contractile ring (Fig. 4A), those in Drosophila embryonic epithelial cells,
follicular epithelia, and pupal wing epithelia do not show clear enrich­
ment of contractile ring components to the basal side [79,82,83]
(Fig. 4B). Thus, these epithelial cells form structurally symmetric con­
tractile ring. By contrast, myosin and septin are enriched in the cleavage
head of Drosophila pupal dorsal thorax epithelia as in the C. elegans
zygote [83,84]. Consistent with this difference, contractile ring
constriction is symmetric and asymmetric in the cells with structurally
symmetric and asymmetric contractile rings, respectively [82,83]. Un­
like the C. elegans zygote, unilateral cleavage in embryonic epithelial
cells and adult dorsal thorax epithelia does not require anillin and septin
while they are required for the normal rate of constriction [82,84]. How
is unilateral cytokinesis regulated in epithelia with the structurally
symmetric contractile ring? In Drosophila embryos, knockdown of
adherens junction components results in symmetric cytokinesis,
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Seminars in Cell and Developmental Biology xxx (xxxx) xxx
suggesting that the apical anchorage of the contractile ring causes uni­
lateral cytokinesis (Fig. 4B) [82]. The anchorage-dependent unilateral
cytokinesis has also been reported in permeabilized fission yeast cells
[69]. On the other hand, the follicle epithelium shows randomly ori­
ented cytokinesis after knockdown of adherens junctions or Rho kinase
[85,86]. Furthermore, some normal epithelia undergo apical-to-basal
unilateral cleavage as described above. Thus, adherens
junction-dependent anchorage of the contractile ring appears to not be
the only mechanism of epithelial unilateral cytokinesis. Because tension
of the surrounding cells suppresses cleavage, anisotropic tension in the
tissue might drive unilateral cytokinesis in other cases (Fig. 4B, right)
[82,84].
2.3. Roles of unilateral cytokinesis
The role of unilateral cytokinesis in zygotes has been studied in
C. elegans. First, symmetrically-closing-contractile ring tends to fail
cytokinesis upon reduction of RhoA activity [51]. Second, uniaxial
compression of embryos promotes unilateral cleavage apparently
through rotational cortical flow activation [87]. Third, embryo
compression causes cytokinesis failure in the absence of unilateral
cytokinesis regulators anillin and septin [87]. These results collectively
suggest that unilateral cleavage contributes to the cytokinesis robustness
against naturally occurring RhoA activity fluctuations and embryo
compression.
In some epithelia, unilateral cytokinesis maintains epithelial polarity
through the polarized positioning of the cytokinetic ring remnant called
the midbody. In Drosophila pupal wing epithelial cells, unilateral cyto­
kinesis positions midbody to the apical surface and regulates normal
cell-cell contact formation through midbody-guided actin assembly
[83]. Unilateral cytokinesis also regulates lumen formation [88]. In 3D
cell cultures of Madin-Darby canine kidney (MDCK) cells, LLC-PK1
kidney cells, human intestinal Caco-2 cells, and the rat hepatocyte cell
line Can10, the first mitosis is symmetric cytokinesis, during which the
midbody specifies the apical lumen at the center of the dividing cell
(Fig. 4C) [89–92]. Successive cell division involves basal-to-apical uni­
lateral cytokinesis and midbody positioning toward the
previously-formed apical lumen. Abnormal midbody positioning is
associated with the formation of multiple lumens, suggesting a link
between unilateral cytokinesis and lumen formation [89,92,93]. How­
ever, it is important to note that unilateral cytokinesis and midbody
positioning after ring closure can be independently regulated in some
tissues [78,94,95]. Thus, tissue-specific live-imaging is required to
determine the specific role of unilateral cytokinesis in epithelial
polarity.
3. Symmetry-breaking of cytokinesis during asymmetric cell
division
Asymmetric cell division is one of the major strategies for generating
cellular diversity during development (reviewed in [96–98]). When
contractile ring positioning is biased along the division axis, cytokinesis
generates daughter cells of different sizes. In this section,
we review spindle-dependent and -independent regulation of asym­
metric contractile ring positioning.
3.1. Spindle geometry-dependent regulation of asymmetric cell division
Asymmetric cell division requires a horizontal shift in spindle posi­
tion, whereas unilateral cytokinesis requires a transverse shift (Fig. 5A).
Although the sea urchin zygote normally undergoes symmetric cytoki­
nesis, magnetic tweezer-dependent horizontal spindle shift can induce
asymmetric contractile ring positioning [100].
In Drosophila neuroblasts and C. elegans zygotes that undergo
asymmetric cell division, horizontal spindle shift is induced by the
polarized distribution of microtubule-associated proteins (MAPs) and
K. Sugioka
Fig. 5. Spindle Geometry-dependent Asymmetric Cytokinesis. (A) Asymmetric
cytokinesis of the sea snail Crepidula plana. The horizontal shift of the spindle
results in asymmetric contractile ring positioning (right). (B) The spindle-
pulling force induces a horizontal spindle shift. Cortically-tethered dynein
pulls depolymerizing microtubules. Polymerizing microtubules generate push­
ing forces to counteract the pulling forces. The balance of pulling and pushing
forces are modulated by cell polarity and microtubule associated protein
(MAPs).
(A) (left; adapted from Conklin [99]).
motor proteins at the cell cortex (the polarization mechanisms are
reviewed in [96,98,101]). The most conserved regulator is a complex of
dynein, NuMA, LGN, and Gα (the α subunit of the heterotrimeric G
proteins) (Fig. 5B). Gα localizes to the plasma membrane, presumably
through lipid modification [102]. NuMA and LGN form a
hetero-hexameric complex and interact with the GDP-bound form of Gα
[103–106].
NuMA:LGN then recruits the microtubule minus-end-directed motor
protein dynein to the cell cortex [107,108]. The minus-end-directed
motor activity of cortically-tethered dynein pulls microtubules toward
the cell cortex, leading to spindle displacement [106,109–112].
Optogenetically-induced cortical localization of NuMA is sufficient to
recruit dynein to the cell cortex and induce a horizontal shift of the
mitotic spindle in C. elegans and HeLa cells [108,113].
The precise positioning of mitotic spindle is regulated by additional
factors. Spindle-centering forces counteract the dynein-dependent
pulling force, preventing a premature horizontal spindle shift (Fig. 5B)
[60,114]. Additionally, polarized localization of MAPs or asymmetric
centrosome activities can control the horizontal spindle shift by modu­
lating the number of astral microtubules and microtubule-residence
time at the cell cortex, given that both parameters influence spindle
pulling and centering forces (Fig. 5B) [115–119].
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Seminars in Cell and Developmental Biology xxx (xxxx) xxx
3.2. Spindle Geometry-independent Regulation of Asymmetric Cell
Division
In some cases, spindle-independent mechanisms control asymmetric
cytokinesis. In asymmetric cell division of Drosophila neuroblasts that
divide along the apical-basal tissue axis, the contractile ring positions
relative to the basal side, leading to generation of a larger neuroblast and
a smaller ganglion mother cell (Fig. 6). Asymmetric cleavage occurs
even after the depolymerization of microtubules by colcemid, suggest­
ing that the spindle-independent cue specifies the cleavage site [120].
Several lines of evidence suggest that the cortical flow from apical to
basal cortex leads to the clearance of myosin from the apical cortex,
resulting in asymmetric polar relaxation and expansion of the apical cell
cortex (Fig. 6) [121–123]. In neuroblasts without colcemid treatment,
asymmetric positioning and organization of the spindle also contribute
to cortical flow and asymmetric cytokinesis [122]. The cortical
flow-dependent mechanism might be evolutionarily conserved, given
that similar myosin relocalization and cortex relaxation are observed in
C. elegans asymmetric neuroblast division, although spindle-dependency
has not been directly evaluated in this system [124].
4. Oriented cytokinesis
Oriented cell division contributes to animal morphogenesis through
asymmetric positioning of daughter cells. Cortically-tethered dynein,
cellular geometry, and other factors can control oriented division by
influencing the forces acting on astral microtubules [97,125]. However,
cytokinesis also regulates oriented division likely through oriented
cytokinesis, a spindle-independent contractile ring tilting relative to
body axes. In this section, we review its mechanism and role.
In many groups of animals with bilateral symmetry, organs and tis­
sues are arranged differently with respect to the axis of symmetry.
However, in rare individuals, total inversion of organ handedness is
observed (e.g., situs inversus in 0.01% of humans). This so-called chiral
morphogenesis has attracted the attention of early developmental bi­
ologists. Pioneering observations of oriented cytokinesis have been un­
dertaken in an attempt to understand snail shell chirality [127]. Some
snail species, such as Lymnaea stagnalis and Physa acuta, typically show
dextral and sinistral shell chirality, respectively [127,128]. Snail shell
chirality is specified during 4-cell-stage cytokinesis (Fig. 7A, left). All
blastomeres synchronously enter mitosis and undergo cytokinesis
accompanied by division-axis skew, resulting in formation of a chiral
blastomere arrangement. The counter-clockwise and clockwise rotations
of the division axes dictate dextral and sinistral shell types, respectively
[127,129]. Similar oriented cytokinesis has been observed during
cytokinesis in C. elegans 4-cell-stage embryos (Fig. 7A, right) [130].
Until anaphase onset, the division axes of ABa and ABp cells orient along
the left-right body axis. During cytokinesis, division-axis rotates in a
clock-wise manner when seen from the dorsal side. The resulted chiral
blastomere positioning specifies the chiral arrangement of organs, such
as the intestine and gonads, in adult worms. In both C. elegans and snails,
manipulation of division axes by a glass capillary results in the reversal
of chirality [130,131].
What does trigger oriented cytokinesis? Meshcheryakov and
Beloussov tracked cell-surface movement during oriented cytokinesis of
snails using carbon particles [129]. They found that the cell surfaces of
the left and right halves of the dividing cell exhibited opposite move­
ments (Fig. 7A, black arrows). Naganathan et al. showed a similar
counter-rotating cell-surface movement during oriented cytokinesis in
C. elegans and termed this as chiral cortical flow [72]. Using RNAi, they
demonstrated association between the extent of RhoA activity, chiral
cortical flow, and division-axis skew. How is chiral cortical flow
generated? It is speculated that the molecular chirality of actin might
drive chiral cortical flow based on the fact that myosin twists actin fil­
aments in vitro [72,132]. A recent study showed that diaphanous
formin/CYK-1 is required for chiral cortical flow in C. elegans zygotes
K. Sugioka
[133]. Interestingly, diaphanous is also required for chiral morpho­
genesis in snails and frogs [128,134]. These studies indicate that
evolutionarily conserved chiral cortical flow and oriented cytokinesis
regulate chiral morphogenesis. It is important to note that
cytokinesis-independent mechanisms also drive chiral morphogenesis in
C. elegans [135], Drosophila [136–138], and mammals (reviewed in
[139,140]).
C. elegans dorsal-ventral axis formation in 4-cell stage embryos is also
regulated by oriented cytokinesis. In a 2-cell stage embryo, anterior
blastomere AB divides in parallel to the cell contact with posterior
blastomere P1. The contractile ring is oriented perpendicular to the
contact plane, even after depolymerization of the microtubules [141].
Moreover, attachment of adhesive polystyrene beads to isolated AB cells
was sufficient to orient the contractile ring (Fig. 7B). Contact with cells
or beads resulted in myosin inactivation and retardation of cortical flow
near the contact site, leading to anisotropic cortical flow. The aniso­
tropic cortical flow was proposed to rotate the dividing cell, as its loss
leads to an abnormal AB division axis [141]. During cytokinesis of AB
cells, dividing cells also undergo rotation inside the eggshell, which
contributes to the establishment of the dorsal-ventral axis. The pertur­
bation of the chiral cortical flow prevents AB cell rotation [142].
Importantly, AB cell rotation did not occur in the absence of the vitelline
membrane [143]. Thus, it is likely that the interaction between the
7
Seminars in Cell and Developmental Biology xxx (xxxx) xxx
Fig. 6. Spindle Geometry-independent Regula­
tion of Asymmetric Cytokinesis. Asymmetric
cytokinesis of Drosophila neuroblasts in the
absence of microtubules. A mutant of the spin­
dle assembly checkpoint component was used
to bypass cell cycle arrest due to the microtu­
bule depolymerization. Apical-to-basal cortical
flow results in myosin reduction and enrich­
ment in the apical and basal cortices, respec­
tively. Removal of myosin from the cortex
promotes cell extension presumably in a
manner similar to polar relaxation.
Fig. 7. Spindle Geometry-independent Regula­
tion of Oriented Cytokinesis. (A) Oriented
cytokinesis during chiral morphogenesis. Snail
4- to 8-cell (third) cleavages undergo oriented
cytokinesis, as well as asymmetric cytokinesis.
The peripheral larger cell and the inner smaller
cell are siblings. Counter-clockwise division-
axis skew specifies the formation of the dextral
shell. C. elegans 4- to 6-cell (fourth) cleavages
undergo oriented cytokinesis. Clockwise
division-axis skew specifies the chiral arrange­
ment of the intestine (green) and gonad (light
blue). See text for the detail. (B) Oriented
cytokinesis induced by adhesion. Isolated
C. elegans or mouse embryos undergo oriented
cytokinesis relative to the adhesive bead or cell.
The contact area experiences reduced myosin
regulatory light-chain phosphorylation, indi­
cating low myosin activity. Cortical flow and
division-axis skew are identified by black and
white/black arrows, respectively. Dotted ar­
rows in the diagram indicate unclear relation­
ships.
(A) (Adapted from Clessin et al. (1886) [126]).
chiral rotating AB cell and the vitelline membrane surrounding the
embryo induces further AB division-axis rotation. Note that
cytokinesis-independent mechanisms also contribute to the
dorsal-ventral axis establishment [144]. It is unclear whether oriented
cytokinesis also plays important roles in mammals, although mouse
2-cell stage blastomeres undergo oriented cytokinesis in contact with
adhesive beads [141].
5. Conclusion
Symmetry-breaking of cytokinesis plays important roles in animal
morphogenesis by contributing to the maintenance of epithelial polar­
ity, asymmetric cell division, chiral morphogenesis, and oriented cell
division. In a single cell, unilateral cytokinesis might be required for
cytokinesis robustness. Emerging evidence suggests that unilateral
cytokinesis, cytokinesis during asymmetric cell division, and oriented
cytokinesis all involve uniquely-patterned cortical flow. Thus, it will be
critical to expand our understanding of the mechanisms underlying
symmetry-breaking of cortical flow in future studies.
Recent studies identified genes required for the normal cortical flow
patterning [69,72,73,87,133,141,145,146]; however, the alteration of
spindle geometry might confound data interpretation, given that spindle
geometry influences the regulator of cortical flow RhoA. Thus, it is
K. Sugioka
important to quantitatively report the geometrical relationship between
the spindle and the cell cortex to understand the direct effects of newly
identified regulators on the cortical flow. Ideally, the role of cortical
flow regulators should be tested in the absence of microtubules in order
to clearly separate the contribution of spindle geometry. Although
analysis of cytokinesis in the absence of microtubules is challenging due
to cell cycle arrest, the combined use of spindle assembly checkpoint
mutants and microtubule drugs should enable such analyses, as in the
case of Drosophila neuroblasts [120]. C. elegans embryo is also a useful
model because of its weak spindle assembly checkpoint, which only
delays the cell cycle upon microtubule depolymerization [147].
Finally, the mechanical understanding of the relationship between
cortical flow, cytokinesis, and morphogenesis is limited. In addition to
theoretical and genetic analyses, it is imperative to measure the forces
generated by cortical flow in order to better understand their direct
effects on symmetry-breaking of cytokinesis and animal morphogenesis.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We would like to thank reviewers for valuable feedback on the
manuscript. The author apologizes for not citing some relevant papers
due to space limitation. This work was supported by the Canadian In­
stitutes of Health Research, Canada (Project Grant; PJT-169145) and the
Michael Smith Foundation for Health Research, Canada (Scholar Award;
SCH-2020-0406) to K.S.
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