Biochem. J. (2011) 437, 13–24 (Printed in Great Britain)
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doi:10.1042/BJ20110153
REVIEW ARTICLE
Vesicle trafficking and membrane remodelling in cytokinesis
Hélia NETO, Louise L. COLLINS and Gwyn W. GOULD1
Henry Wellcome Laboratory of Cell Biology, Davidson Building, Institute for Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of
Glasgow, Glasgow G12 8QQ, Scotland, U.K.
All cells complete cell division by the process of cytokinesis.
At the end of mitosis, eukaryotic cells accurately mark the site
of division between the replicated genetic material and assemble
a contractile ring comprised of myosin II, actin filaments and
other proteins, which is attached to the plasma membrane. The
myosin–actin interaction drives constriction of the contractile
ring, forming a cleavage furrow (the so-called ‘purse-string’
model of cytokinesis). After furrowing is completed, the cells
remain attached by a thin cytoplasmic bridge, filled with two antiparallel arrays of microtubules with their plus-ends interdigitating
in the midbody region. The cell then assembles the abscission
machinery required for cleavage of the intercellular bridge, and
so forms two genetically identical daughter cells. We now know
much of the molecular detail of cytokinesis, including a list of
potential genes/proteins involved, analysis of the function of some
of these proteins, and the temporal order of their arrival at the
cleavage site. Such studies reveal that membrane trafficking and/or
remodelling appears to play crucial roles in both furrowing and
abscission. In the present review, we assess studies of vesicular
trafficking during cytokinesis, discuss the role of the lipid
components of the plasma membrane and endosomes and their
role in cytokinesis, and describe some novel molecules implicated
in cytokinesis. The present review covers experiments performed
mainly on tissue culture cells. We will end by considering how
this mechanistic insight may be related to cytokinesis in other
systems, and how other forms of cytokinesis may utilize similar
aspects of the same machinery.
INTRODUCTION
that Rab11 is required for cellularization in Drosophila embryos
[6,7]. Subsequent studies revealed that Rab11-containing vesicles
accumulate in the midbody, and that depletion of Rab11 or
expression of a dominant-negative mutant perturbs abscission in
mammalian cells [8].
Such observations support an important role for membrane
traffic in cytokinesis, but it is unclear whether this reflects
movement into the furrow/intercellular bridge from both daughter
cells, an asymmetric delivery of membrane from only one of
the two daughters, or a combination of both. Furthermore, other
important questions surrounding aspects of these trafficking
steps remain unanswered: (i) do the vesicles trafficked to the
midbody coalesce into a cell-plate-like structure, or do they
remain distinct?; (ii) what controls the accumulation of vesicles
in the intercellular bridge?; (iii) how are the normal cellular
trafficking pathways modulated during cytokinesis?; and (iv) what
is the function of the vesicles which accumulate in the intercellular
bridge? Further studies have begun to address these questions.
Membrane traffic is required for cytokinesis
Mammalian cells exhibit pronounced shape changes during
mitosis: cells become rounded in early mitosis, before compacting
in metaphase and furrowing in telophase [1]. Such changes
are underscored by alterations in membrane trafficking. Over
the past decade, clear evidence for a role of membrane
traffic in mammalian cytokinesis has been described, including
the accumulation of membrane vesicles in the furrow and
intercellular bridge, the localization of key trafficking proteins
{e.g SNAREs [SNAP (soluble N-ethylmaleimide-sensitive fusion
protein-attachment protein) receptors], dynamin and clathrin}
to the cleavage furrow and intercellular bridge, and by several
studies reporting a functional role for these proteins in abscission
[2–5]. Plant cells cannot divide by an actomyosin-based ring
because of the rigid cell wall. Instead, plant cells construct a new
membrane at the division plane by directing post-Golgi traffic
to this site where the vesicles fuse into a transient compartment,
the cell plate, which expands towards the cell edge then fuses
with the PM (plasma membrane). Similarly, mammalian cells
deliver secretory vesicles to the site of abscission, but other
membrane compartments also traffic into the intercellular bridge,
in particular recycling endosomes (Figure 1). A role for recycling
endosomes in cytokinesis was first suggested from observations
Key words: cholesterol, cytokinesis, cytoskeleton, membrane
trafficking, phospholipid, soluble N-ethylmaleimide-sensitive
fusion protein-attachment protein receptor (SNARE).
TRAFFIC FROM ONE CELL, OR TWO?
Using a GFP (green fluorescent protein) marker engineered to
localize to secretory vesicles (lum-GFP), Gromley et al. [9]
studied the dynamics of secretory vesicles during cytokinesis.
Early in cytokinesis, lum-GFP vesicles do not accumulate in
Abbreviations used: Bru, Brunelleschi ; BRUCE, baculovirus inhibitor of apoptosis repeat-containing ubiquitin-conjugating enzyme; Cdk1, cyclindependent kinase 1; Cep55, centrosome protein 55; EMMA, exocytic mid-zone membrane accumulation; ER, endoplasmic reticulum; ESCRT, endosomal
sorting complex required for transport; F-actin, filamentous actin; fwd , Four Wheel Drive ; FYVE-CENT, FYVE domain-containing centrosomal protein;
GAP, GTPase-activating protein; GEF, guanine-nucleotide-exchange factor; GFP, green fluorescent protein; JIP, JNK (c-Jun N-terminal kinase)-interacting
protein; PE, phosphatidylethanolamine; PH, pleckstrin homology; Plk, Polo-like kinase; PM, plasma membrane; PX, Phox homology; SNAP, soluble N ethylmaleimide-sensitive fusion protein-attachment protein; SNARE, SNAP receptor; TRAPP, transport protein particle; t-SNARE, target SNARE; VAMP8,
vesicle-associated membrane protein 8; v-SNARE, vesicle SNARE; VSVG, vesicular somatitis virus glycoprotein; WAVE, Wiskott–Aldrich syndrome protein
verprolin homologous; YFP, yellow fluorescent protein.
1
To whom correspondence should be addressed (email [email protected])
c The Authors Journal compilation c 2011 Biochemical Society
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Figure 1
H. Neto, L. L. Collins and G. W. Gould
Membrane traffic in cytokinesis
Top panel: during metaphase/anaphase, endocytosis occurs mainly from the poles of the cell,
where endosomal Rab proteins such as Rab5 are localized to the side of the daughter nucleus
facing away from the furrow. Although endocytosis occurs normally, exocytosis (recycling)
of membrane back to the cell surface is inhibited. Rab11-positive vesicles are located more
proximal to the furrow, and traffic into the midbody in late telophase along curvi-linear paths.
Secretory traffic (shown as light grey-shaded vesicles on the right-hand daughter cell for
clarity) into the furrow is also observed. EMMAs are localized to the midbody in telophase.
The midbody ring is shown in red. Lower panel: a cross-section through the midbody in late
telophase. Rab11/FIP3-positive vesicles are recruited to the midbody: (i) by interaction with the
centralspindlin component Cyk4 (after Ect2 is released); or (ii) via a Rab11- and Arf6-dependent
interaction with the Exocyst (which in turn is recruited to the midbody ring (red) by centriolin
(not shown for clarity). See the text for further details. (iii) Secretory and/or endosomal vesicles
accumulate in the midbody prior to abscission and may represent a ‘platform’ on to which the
abscission machinery is assembled (see the text).
the intercellular bridge, but as the midbody thins later in
cytokinesis, lum-GFP-containing vesicles accumulate in this
region. Strikingly, in all cells analysed, Gromley et al. [9] reported
that lum-GFP vesicles accumulated on only one side of the
midbody, suggesting that they were delivered from only one of
the daughter cells. As cytokinesis progressed, the GFP signal
decreased, presumably reflecting the fusion of these vesicles with
the furrow PM. Finally, in all cells examined, the intercellular
bridge cleaved on the side that received the accumulation of
vesicles, albeit some time after the disappearance of the GFP
signal. These observations led to the suggestion that the compound
fusion of these asymmetrically delivered vesicles with each other
and the PM could mark the site of abscission (Figure 1) [9].
Using three-dimensional confocal time-lapse imaging, Goss
and Toomre [10] reached somewhat different conclusions. They
examined the distribution of a temperature-sensitive mutant
of VSVG–YFP (vesicular somatitis virus glycoprotein–yellow
fluorescent protein). At 39.5 ◦ C, this protein accumulates in
the ER (endoplasmic reticulum); this accumulation can then
be released as a synchronous pulse by shifting cells to 32 ◦ C.
One crucial finding of this study was that 1 h after release of
VSVG–YFP from the ER, 63 % of cells undergoing cytokinesis
exhibited bright punctae at the cleavage furrow. These structures
were given the name of EMMAs (exocytic mid-zone membrane
accumulations) (Figure 1), and do not co-localize with Golgi
or trans-Golgi network markers, suggesting that EMMAs are
c The Authors Journal compilation c 2011 Biochemical Society
constituted from post-Golgi cargo [10]. Interestingly, EMMAs
exhibited a markedly heterogeneous distribution in mitotic cells,
with one-third of cells examined exhibiting no EMMAs, onethird with EMMAs only on one side of the midbody, and
one-third on both sides of the midbody. Since this analysis
was performed in fixed cell populations, Goss and Toomre [10]
postulated that this distribution reflected the cells transitioning
between these structures at different stages of cytokinesis. Realtime analysis revealed that both daughter cells traffic both VSVG–
YFP-positive and YFP–glycosylphosphatidylinositol-anchored
protein-positive vesicles into the furrow, and both daughter
cells symmetrically accumulate these post-Golgi vesicles in
the furrow (Figure 1). However, the authors observed a
transition to asymmetrical accumulation, suggesting that cells
can vary the trafficking and/or accumulation of these vesicles
in different phases of cytokinesis [10]. Further support for
asymmetric vesicle delivery was provided from studies of a
large multi-functional protein, BRUCE (baculovirus inhibitor of
apoptosis repeat-containing ubiquitin-conjugating enzyme; see
below) [11]. Expression of a mutant form of BRUCE revealed
that the midbodies of these cells accumulated ‘balloon-like’
accumulations of secretory vesicles in close proximity to the
intercellular bridge, but only on one side. Similarly, BRUCE
depletion resulted in the asymmetric accumulation of GFP–Rab11
on one side of the midbody [11].
IS THERE A CELL-PLATE-LIKE STRUCTURE IN MAMMALIAN CELLS?
A key question arising from the observation of EMMAs and
the accumulation of secretory vesicles in the intercellular bridge
is whether these structures are akin to a cell-plate; in other
words do these accumulations of vesicles homotypically fuse with
each other to generate a larger compartment? Using fluorescence
recovery after photo-bleaching, Goss and Toomre [10] observed
that VSVG–YFP vesicles remain as distinct entities after reaching
the midbody. Similar conclusions were reached by Gromley
et al. [9], arguing against homotypic fusion into a cell-plate-like
organelle.
The SNAREs required for abscission {Sx2, VAMP8 (vesicleassociated membrane protein 8); [12]} have also been implicated
in homotypic fusion [13]. Hence, the possibility remains that
homotypic fusion may occur in EMMAs, but the nature of the
VSVG–YFP cargo (an integral membrane protein) or a property
of the vesicles may place spatial restrictions on the ability of this
protein to diffuse into the limiting membrane of any fused vesicles.
The presence of lum-GFP-containing vesicles in the midbody of
heterogeneous size [11] could imply that some of these vesicles
have fused with each other. High-resolution imaging will be
required to definitively answer this issue.
Regardless of this point, there is agreement that there is fusion
of both lum-GFP vesicles and VSVG–YFP vesicles with the
PM in the furrow, and that this fusion precedes the completion
of abscission. This may be important mechanistically, as the
intercellular bridge is long lived, often persisting for several
hours. Although it is generally viewed as being a stable structure,
the bridge undergoes gradual thinning, with a notably thinned
area often visible at one side of the midbody (reviewed in
[14]). This thinning of the bridge may be an important prerequisite to the abscission event, and the notion that the fusion
of vesicles with the PM in the bridge may underpin this is gaining
support [14]. Thus a present model suggests that the fusion of
vesicles with the PM of the intercellular bridge (and perhaps their
homotypic fusion) results in the gradual thinning of the midbody
until the final abscission step, which is mediated by components
Membrane remodelling in cytokinesis
of the ESCRT (endosomal sorting complex required for
transport) complex (discussed further below; for a recent review,
see [15]).
WHAT CONTROLS THE ACCUMULATION OF VESICLES IN THE
INTERCELLULAR BRIDGE?
The observation that vesicles derived from recycling endosomes
and post-Golgi secretory vesicles accumulate in the intercellular
bridge begs the question of how this is controlled. A first clue
regarding this came with the identification of the centrosomal
protein centriolin, which was found to accumulate on the
midbody ring in late telophase [16]. Centriolin was subsequently
found to interact with proteins involved in vesicle docking,
specifically components of the Exocyst complex [17], and the
SNARE regulatory protein SNAPIN [9]. Depletion of centriolin
impairs the localization of Exocyst components to the midbody,
and depletion of Exocyst components results in an enhanced
accumulation of lum-GFP-containing vesicles in the midbody [9].
Such data would apparently argue against a role for the Exocyst
in tethering these vesicles, but rather implicates the Exocyst in
their subsequent fusion.
The localization of the Exocyst complex to the midbody also
requires Septin 9. Septins are a family of GTP-binding proteins
that regulate aspects of membrane trafficking and have previously
been implicated in cell division. Depletion of Septin 9 results in
a 5-fold increase in the number of cells remaining joined by a
midbody [18]. HeLa cells depleted of Septin 2, 11 or 17 reveal
abnormal furrow ingression. By contrast, cells depleted of Septin
9 furrow normally but abscission fails in 70 % of the cells, with
midbodies remaining intact for many hours [19]. In an effort to
determine the mechanism responsible for this, Estey et al. [19]
examined the localization of known midbody proteins upon Septin
9 depletion. Although molecules such as Cep55 (centrosome
protein 55), Plk1 (Polo-like kinase 1) and VAMP8 were correctly
localized, the characteristic enrichment of the Exocyst component
Sec8 in midbodies was impaired. This result is in agreement with
studies in yeast, where septins act to localize Exocyst components
[20]. Collectively, such data point to an important role for Septin
9 (possibly acting in concert with centriolin) in the localization of
the Exocyst to the midbody.
The mechanism responsible for the tethering of recycling
endosomal vesicles in the midbody is incompletely understood,
but some studies are beginning to provide us with clues (Figure 1).
The recycling endosome-associated protein FIP3 was originally
identified as a Rab11 and Arf6 GTPase-binding protein that is
required for abscission [21]. Both Rab11 and Arf6 are known
to be involved in endosomal trafficking into the midbody [22],
and it has been shown that FIP3-associated endosomes associate
with centrosomes until late telophase, whereupon they rapidly
move into the midbody and accumulate there late in telophase
[8,21]. Rab11 and Arf6 are known to interact with components
of the Exocyst complex [23], and depletion of Exo70 was found
to decrease the localization of FIP3-containing endosomes into
the midbody [24]. This is consistent with a model in which the
tethering of Rab11/Arf6/FIP3-positive vesicles in the intercellular
bridge involves the Exocyst (Figure 1). However, previous
work has revealed that FIP3 can also bind the centralspindlin
component Cyk-4 and offered a different view of how these
vesicles could be tethered [25]. The centralspindlin complex
[comprised of Cyk-4/MgcRacGAP (GTPase-activating protein)
and MLKP1] regulates the formation of the central spindle and
controls actomyosin ring constriction [26]. Cyk-4 recruits the
Rho GEF (guanine-nucleotide-exchange factor) ECT2 to the
15
centralspindlin complex; ECT2 activates RhoA and so regulates
contractile ring function [26]. It has now been shown that in
late telophase, FIP3 interacts directly with Cyk-4 and it does
so at the same site on Cyk-4 that binds ECT2 [25]. This led
Prekeris and co-workers to propose a model whereby in early
telophase (during furrow ingression), centralspindlin-localized
Cyk-4 interacts with ECT2 and so controls RhoA activity during
contractile ring constriction [25]. Later in cytokinesis, there is
a delocalization of ECT2 from centralspindlin (by an as-yet
uncharacterized mechanism), resulting in Cyk-4 now interacting
with FIP3 localized on recycling endosomal vesicles trafficking
into the furrow (Figure 1). Consistent with this model, Prekeris
and co-workers showed that ECT2 and FIP3 are sequentially
recruited to the midbody as cells progress through cytokinesis
[25]. These data make a compelling case for the tethering of
recycling endosomal vesicles in the midbody in late telophase
involving the centralspindlin complex.
So how can one integrate these observations into a model?
A clue may come from the observation that FIP3 vesicle
dynamics in telophase is complex [8,25]. Early in anaphase,
FIP3 is associated with vesicles around the centrosome. As
anaphase proceeds, traffic of FIP3-positive vesicles to and from
the cleavage furrow is observed, with the majority of FIP3containing organelles re-localizing to the furrow side of the
nucleus. In late telophase, FIP3-containing vesicles accumulate at
the midbody, and are notably more ‘static’ [25], suggesting that
different tethering/targeting machinery operates at different stages
of the cell cycle. Alternatively, multiple mechanisms of tethering
may operate simultaneously, acting as a ‘belt-and-braces’ to insure
that appropriate accumulation takes place. Consistent with this,
Prekeris and co-workers noted that ECT2 knockdown alone is
insufficient to fully drive FIP3 accumulation in the midbody in
anaphase [25], and is also supported by data showing that the
simultaneous depletion of Rab11 and Arf6 results in a more
pronounced cytokinesis defect than either alone [27].
Further studies have identified other vesicle-tethering
complexes, including the GARP (Golgi-associated retrograde
protein) complex, and TRAPP I (transport protein particle I)
and TRAPP II complexes [28], which may also function in
cytokinesis. Mutation of the Drosophila homologue of the TRAPP
II-specific gene TRS120 [Bru (Brunelleschi)] results in the failure
of furrow ingression in male meiotic cells. The localization of
Rab11 to the cleavage furrow requires Bru, and Bru and Rab11
genes exhibit synthetic lethality [29]. Bru also exhibits a genetic
interaction with a PtdIns 4-kinase [fwd (Four Wheel Drive); see
below], prompting the suggesting that this tethering complex
acts in concert with phosphoinositides to regulate membrane
delivery to the cleavage furrow [29]. These data clearly implicate
membrane-tethering complexes, in addition to the Exocyst, as key
players in cytokinesis, and further studies regarding their specific
function are clearly warranted.
The identification of a new player in abscission dynamics may
also have an impact on vesicle tethering in the midbody. BRUCE
is a large (528 kDa) protein which is necessary for abscission
[11]. This protein localizes to the spindle midzone and midbody
in cytokinesis, where it adopts a ring-like structure adjacent
to the mitotic kinase Aurora A. BRUCE interacts with Rab8
and Rab11 and also with components of the Exocyst complex
[11]. Expression of a mutant form of BRUCE (or its depletion)
modulated membrane traffic into the midbody (see above), which
led Pohl and Jentsch [11] to argue that BRUCE targets vesicles
to the midbody via an N-terminal domain to bind the Exocyst
and endosomal components, and a C-terminal-targeting domain
for midbody localization. Although it remains unclear whether
BRUCE is a bona fide tethering factor, it is clear that this
c The Authors Journal compilation c 2011 Biochemical Society
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H. Neto, L. L. Collins and G. W. Gould
protein plays a vital role in the accumulation of vesicles in the
midbody.
HOW ARE NORMAL CELLULAR TRAFFICKING PATHWAYS
MODULATED DURING CYTOKINESIS?
During mitosis, cells exhibit dramatic shape changes [1]. Detailed
quantitative analysis of cell volume changes in the cell cycle reveal
that as a cell transitions from the characteristic spread morphology
in interphase through prophase to metaphase, its volume
decreases by approximately 30 % (‘pre-mitotic condensation’)
[1]. Furrowing during cytokinesis results in the volume of the
two daughter cells becoming equivalent to that of the mother cell
before pre-mitotic condensation.
These changes in cell volume/surface area are underpinned
by dramatic, cell-cycle-dependent changes in membrane traffic
to and from the PM. Clathrin-dependent endocytosis continues
throughout the cell cycle, but in anaphase there is a dramatic
decrease in the recycling of internalized material back to the
PM, resulting in the accumulation of internalized material
in endosomal compartments (Figure 1) [30]. In anaphase, this
endocytosis occurs predominantly from the poles of the cells. As
mitosis progresses, recycling of the internalized material back to
the PM resumes, with traffic now directed towards the furrow
[30]. This is consistent with the dynamics of FIP3 distribution
(recycling endosomes) described above: in early telophase there is
an accumulation of FIP3-positive structures on the furrow side of
the nucleus, which then traffic into the intercellular bridge [25].
Similar changes in transferrin receptor trafficking were also
reported [30,31]. These latter studies also revealed that the
recovery in cell surface area during late stages of mitosis does
not require a functional Golgi complex, suggesting that de novo
synthesis of lipids and proteins is not required, but rather is
underpinned by the redistribution of existing stores, consistent
with the suggestion that the delivery of recycling endosomal
vesicles plays a key role in this step. [30] Additionally, these
changes in endocytic recycling are essential for cytokinesis, as
blockade of clathrin-dependent endocytosis resulted in significant
cytokinesis defects [31]. Studies in Drosophila spermatocytes
revealed similar changes in membrane dynamics, but further
showed that there appear to be two distinct phases of membrane
addition to the growing furrow [32,33]. The first is a slow increase in surface area during anaphase, followed by a dramatic
increase during cytokinesis. Interestingly, this latter increase
requires the GTPase Arf6, which was observed to localize to Rab11 and Rab-4 positive structures in the midzone region, further
supporting a role for recycling endosomes in cytokinesis [32].
Changes in cell shape during the cell cycle are co-ordinated
by changes to the actin cytoskeleton and the microtubule
network. Similar networks participate in the regulation of integrindependent cell adhesion [34]. When viewed in the context of
a study showing that adherent cells held in suspension fail
cytokinesis [35], such observations prompt the question of
whether integrin recycling plays a role in cytokinesis. Pellinen et
al. [36] showed that the Rab21-dependent trafficking of integrins
to and from the cleavage furrow is required for the completion of
cytokinesis, where the integrins anchor the ingressing furrow to
the matrix. Importantly this group also identified a chromosomal
deletion and loss of Rab21 expression in human cancer which
resulted in the appearance of multi-nucleated cells, strongly
supporting the idea that Rab21-dependent integrin traffic into the
furrow is a crucial facet of cytokinesis [36]. It is well established
that integrins act to regulate other facets of the mitotic machinery,
including spindle positioning, orientation and bipolarity. For
c The Authors Journal compilation c 2011 Biochemical Society
example, integrins regulate the production and localization of
PtdIns(3,4,5)P3 (see below), which acts as a cortical signal to
direct spindle orientation (for a review, see [37]). It is therefore
likely that endosomal recycling of integrins play roles in the
cell cycle beyond cytokinesis. What is clear, however, is that
endosomal recycling to direct traffic into the furrow and midbody
(and perhaps other regions of the cell) is carefully co-ordinated
during the cell cycle.
So how is cell-cycle-dependent modulation of endocytic traffic
achieved? At present, very little is known regarding how cells can
turn endocytic recycling on and off. In mitosis, phosphorylation
of Golgi structural proteins by Cdk1 (cyclin-dependent kinase 1),
Plks and mitogen-activated protein kinases are known to underlie
structural changes in the Golgi ribbon [38,39]. Similarly, the Arf
exchange factor GBF1 (Golgi-specific brefeldin A-resistant GEF
1) is phosphorylated by Cdk1-cyclin B in mitosis, resulting in
its dissociation from Golgi membranes [40]. Such observations
suggest that phosphorylation/dephosphorylation of key target
proteins in mitosis probably underlies these changes in membrane
trafficking characteristics.
Previous studies offer a potential clue to this mechanism. Traffic
of endosomal vesicles to the intercellular bridge is a major mechanism contributing to the completion of cytokinesis, and Arf6
is key to that process. Arf6 localizes to both the PM and
endosomal vesicles and regulates both actin remodelling and the
targeting of vesicles to the intercellular bridge [22,41]. Of
relevance to this latter function, Arf6 interacts with the JNK (c-Jun
N-terminal kinase)-interacting proteins JIP3 and JIP4 [42]. JIP3
and JIP4 bind the microtubule-associated proteins kinesin-1 and
dynactin/dynein, suggesting that Arf6 may control trafficking via
modulation of these motor protein interactions [42]. Montagnac
et al. [42] showed that endosomal traffic into and out of the
intercellular bridge occurs constitutively, with traffic occurring
in both directions without apparent vesicle fusion. Based upon
elegant biochemical analysis, Montagnac et al. [42] proposed that
FIP3/Rab11/Arf6-positive vesicles move within the intercellular
bridge towards the plus ends of microtubules, via an interaction
between GDP-Arf6 and kinesin-1. Upon activation of Arf6 at the
midbody, GTP-Arf6 interacts with the JIP4–dynein–dynanctin
complex and moves towards the microtubule minus ends and so
carries vesicles out of the bridge [42]. Arf6/JIP4 would then act
as a ‘motor switch’ controlling a continuous vesicle polarized
trafficking to and from the midbody. Presumably, the availability
of docking machinery within the midbody (e.g. Cyk-4 and
Exocyst, see above) would then dictate whether a vesicle moves
back into the cell body, or is ‘captured’ [42]. A recent study using
cryo-electron tomography revealed that, just before abscission, the
two daughter cells are connected by a thin cytoplasmic bridge,
dense with tightly packed microtubules. The midbody contains
both a core of continuous microtubules between the two daughter
cells, and anti-parallel polar microtubules whose plus ends overlap
at the midbody ring [43]. This dense, overlapping region possibly
acts as a barrier to vesicle trafficking between the daughter cells,
and may facilitate the switching of motors on vesicles delivered
into and out of the bridge.
WHAT IS THE FUNCTION OF THE VESICLES WHICH ACCUMULATE
IN THE INTERCELLULAR BRIDGE?
A prevailing view of the midbody is that it provides a platform for
the assembly of the machinery required for abscission. Proteomic
analysis of midbodies identified over a hundred proteins, many
of which were subsequently shown to be essential for abscission
[2]. Hence one view of membrane traffic in this context may
Membrane remodelling in cytokinesis
be to ensure delivery of key components to the midbody
in the correct temporal sequence. An alternative role for the
accumulated vesicles in the midbody prior to abscission is to
deliver membrane area, such that upon fusion with each other
and the PM there is a thinning of the intercellular bridge (see
above).
A further model proposes that the assembly of a pre-abscission
‘platform’ of densely packed endosomal/secretory vesicles within
the midbody acts both as a diffusion barrier prior to abscission
and facilitates the formation of the abscission machinery [44].
A clue as to the mechanism in which this may operate comes
from studies of protein recruitment to the PM. The presence of
phosphoserine (10–20 % of PM lipids) and phosphoinositides
endows the PM with a net negative charge important for the
targeting of proteins containing polycationic motifs [45]. Studies
from Grinstein and co-workers revealed that receptor activation
alters the PM inner surface potential during phagocytosis. This
results in the release of molecules such as K-Ras, Rac1 and cSrc from the membrane [46], and thus modulation of surface
charge may re-direct proteins normally resident on PMs on to
endosomal compartments [47]. We have speculated that similar
alterations in surface charge (arising from alterations in lipid
composition; see below) on endosomes localized in distinct
cellular regions could facilitate distinct ‘endosome functionality’,
serving to recruit the abscission machinery on to accumulated
vesicles in the midbody (Figure 1) [44]. This notion of the coordinated recruitment of factors that regulate abscission is similar
to that proposed for the recruitment of coat components to specific
membrane domains during transmembrane protein sorting. For
example, the adaptor protein AP-1A coat assembly triggers the
concomitant recruitment of many other proteins, including Rac1,
the Scar/WAVE (Wiskott–Aldrich syndrome protein verprolin
homologous) complex, Rab11 and Rab14 in a fashion dependent
upon the lipid composition of the membrane [48]. Baust et al. [48]
concluded that “the combinatorial use of low-affinity binding sites
present on the same membrane domain accounts not only for a
selective coat assembly but also for the co-ordinated assembly
of selected machineries required for actin polymerization and
subsequent membrane fusion”. The identification of a range of
protein domains that interact with anionic phospholipids {such as
the FYVE, PH (pleckstrin homology) and PX (Phox homology)
domains that interact with different phosphoinositides [49]}
or domains which recognise more general physical properties
of membranes such as charge or shape (e.g. annexins, BAR and
C2 [50]) offers support for the concept of coincidence detection
playing an important role in the assembly of an abscission
machine on the surface of vesicles in the intercellular bridge.
Finally, a recent report has identified a new membrane-targeting
domain, kinase-associated-1 domain, that appears to modulate
the association of key signalling molecules with the PM [51].
We hypothesize that similar machinery operates on the surface of
vesicles accumulated in the midbody to regulate cytokinesis, and
in the following section, we offer some ideas of how the membrane
itself may help co-ordinate aspects of trafficking in cytokinesis.
THE ROLE OF LIPIDS IN REGULATING CYTOKINESIS
In most membranes, phospholipids are distributed asymmetrically
between the two leaflets of the bilayer, forming distinct PM
sub-domains enriched in specific species of phospholipids.
For instance, in eukaryotic cells, phosphatidylserine and PE
(phosphatidylethanolamine) are present predominately in the
inner leaflet of the PM, whereas phosphatidylcholine, glycolipids
and sphingomyelin reside in the outer leaflet [52]. It is becoming
17
increasingly clear that localized domains of different lipid species
can result in the congruent localization of (for example) signalling
domains. With this in mind, it is worth re-visiting the role of lipid
species on cytokinesis.
Phospholipids
It has been long-established that, during cytokinesis, PE
accumulates in the outer leaflet of the PM in the cleavage furrow
[53,54]. The movement of the charged PE head-group through
the hydrophobic core of the bilayer is energetically expensive,
and a family of type 4 P-type ATPases (‘flippases’) has been
implicated in mediating the transport of phospholipids between
inner and outer leaflets of the PM [55]. Perhaps the localized
activity of flippases may mediate the transient enrichment of PE at
the cell surface in the furrow. Consistent with this, a phospholipid
scramblase has been reported to localize symmetrically over the
PM of metaphase cells, but to become asymmetrically localized
to the furrow in telophase [56].
This exposure of PE at the surface is important for cytokinesis,
since inhibition of the retro-translocation of PE to the inner leaflet
by a PE-binding peptide blocks abscission (Figure 2) [53,54].
HeLa cells treated with a PE-binding peptide initiate furrow
formation normally, and furrow with normal kinetics, but fail to
complete abscission and remain connected for many hours after
initiation of cytokinesis [54,57]. Examination of a mutant CHO
(Chinese-hamster ovary) cell line defective in PE biosynthesis
grown in PE-depleted medium revealed that the contractile ring
formed and contracted normally, but did not disassemble, resulting
in cell division arresting in late telophase. This suggests that
PE transposition is intimately coupled to the disassembly of
the contractile ring (Figure 2) [53,58]. Consistent with this,
Emoto et al. [53,59] discovered that the immobilization of PE
in the exofacial leaflet inhibited the disassembly of contractile
ring components such as myosin II and radixin. In bacteria,
cell division involves the formation of a septal ring (the FtsZ
ring). This ring invaginates the cell wall and results in cell
cleavage. In a PE-deficient bacterial strain, FtsZ proteins and other
components of the cell division apparatus are correctly recruited
to the division site, but fail to constrict [60]. Such observations
offer the tantalizing view that PE is required for the cytoskeletal
rearrangements required for cytokinesis in prokaryotes as well as
eukaryotes.
So how might coupling between contractile ring dynamics and
PE levels be achieved? PE adopts an inverted-cone (hexagonal
II-structures) in model membranes. Such structures have been
shown to modulate the behaviour of integral membrane proteins
[61,62] and the activity of signalling molecules [56]. Perhaps
the role of PE in cytokinesis is to form a unique PM domain
within the furrow to control key machinery for cytokinesis. A
central component of the contractile ring assembly pathway is
the RhoA GTPase [26]. Similar to most small GTPases, RhoA is
regulated by GEFs and GAPs. In the case of RhoA, the critical
GEF appears to be the centralspindlin component ECT2, and
the GAP is Cyk-4 (see above). RhoA-GTP activates pathways
which culminate in actin polymerization and myosin II activation,
so driving contractile ring constriction, and active RhoA has
been localized to the ingressing furrow membrane [26,63–65].
In late cytokinesis, down-regulation of RhoA activity in the
midbody is needed for contractile ring disassembly. In cells
treated with the PE-binding peptide, RhoA remained localized
in the furrow membrane late in telophase and accumulated in
the intercellular bridge, suggesting that the PE-binding peptide
prevents RhoA inactivation [57,59]. Such observations offer
c The Authors Journal compilation c 2011 Biochemical Society
18
Figure 2
H. Neto, L. L. Collins and G. W. Gould
Lipid distribution in the furrow
The asymmetric accumulation of cholesterol/sphingolipids into raft domains may influence the ability of exocytic vesicles to fuse with the PM. (i) One possible mechanism for this is that the
localization of t-SNAREs to these raft domains may result in their preferentially adopting a ‘closed’ conformation (in which the SNARE domain required for fusion is inaccessible to the incoming
v-SNARE). The syntaxin (t-SNARE) is shown in blue, with the N-terminal Habc domain folded back on to the SNARE domain. Note that PtdIns(4,5)P 2 may also inhibit syntaxin protein function (see
the text). (ii) We speculate that within the furrow or midbody, the absence of raft domains in certain areas may facilitate the t-SNARE adopting an ‘open’ conformation (where the Habc domain is
moved away from the SNARE domain) and thus may represent areas of the furrow/midbody in which fusion may be enhanced. (iii) PE has been shown to be localized to the exofacial leaflet of the
furrow. This PE ‘flipping’ is required for the down-regulation of RhoA activity in the furrow, and may represent a crucial control point for the disassembly of the actomyosin ring (shown in red). (iv)
Cholesterol and/or PtdIns(4,5)P 2 domains may also act as foci for the assembly of signalling domains, possibly including Ras and phospholipase C-γ . See the text for details.
the intriguing hypothesis that PE microdomains may modulate
the interaction of RhoA and the contractile ring, and so affect
contractile ring dynamics.
Cholesterol, PM rafts and cytokinesis
Sterols, such as cholesterol, have been implicated in cytokinesis
in many organisms, including yeast, zebrafish and mammalian
cells [66–69]. The localization of sterols is cell-cycle-dependent:
cholesterol accumulates at the site of cytokinesis in mammalian
cells and Schizosaccharomyces pombe [66,67], and depletion
of cholesterol perturbs cytokinesis in many systems [66–69].
The role of cholesterol enrichment at the equatorial ring is
unresolved. The sterol-rich membrane domain of the midline
has been proposed to be involved in actomyosin ring positioning
and maintenance, as prolonged treatment with the cholesterolsequestering agent filipin disrupts the localization of the
actomyosin ring marker Cdc4–GFP [66]. This was mirrored by
overexpression of C-4 sterol methyl oxidase, an enzyme in the
sterol biosynthetic pathway [66]. Additionally, Bgs4–GFP, an
integral membrane protein which co-localizes with the sterol ring,
is mis-localized on overtreatment of filipin [66]. Placement of the
division machinery and sterol enrichment at the site of division
may be linked, since a mutant of S. pombe with a mis-positioned
actomyosin ring and septa has a similarly misplaced sterol ring
[66]. Further mutants of S. pombe blocked in G2 /M-phase show
that the actomyosin ring forms and nuclear division occurs before
sterol ring formation in a medial band, but this band persists as the
ring constricts and the septum forms [66]. Membrane traffic may
be involved in formation of a cholesterol-rich band at the equator
since, in S. pombe, a functional secretory pathway is required as
c The Authors Journal compilation c 2011 Biochemical Society
brefeldin A-treated cells show limited filipin localization to the
midline [66]. This notion is supported by studies of Supervillin, a
protein that binds tightly to cholesterol-rich lipid rafts. Supervillin
interacts with F-actin (filamentous actin) and myosin-II in the
furrow, and with the kinesins such as KIF14 in the midbody, and
acts with Arf6-associated vesicle recycling at the cell periphery,
thus co-ordinating trafficking and signalling with motor functions
in these raft domains [70,71].
An alternative view of the role of cholesterol in membrane
function has received impetus from the development of the lipid
raft hypothesis, which posits that the preferential association of
sterols (such as cholesterol) and sphingolipids endows PMs with
the ability to form laterally segregated structures (rafts) that can
act as platforms for the selective association of specific molecular
machines (e.g. cell signalling platforms) (Figure 2) [72]. Although
there remains some debate regarding the size and dynamics of
these structures, the prevailing view of lipid rafts as organizing
platforms is generally accepted [72]. Several observations have
suggested that PM raft-like domains are formed in the furrows of
dividing cells, raising the intriguing possibility that raft domains
play a fundamental role in cytokinesis, perhaps acting as foci
for the assembly of signalling systems. Using sea urchin eggs,
Burgess and co-workers demonstrated that the ganglioside GM1, a
marker for cholesterol-rich membrane domains, exhibits a striking
re-distribution during mitosis [67]. As eggs elongate in anaphase,
GM1 moves towards the site of the future furrow. As the furrow
ingresses, GM1 concentrates in the furrow by moving away from
the poles (compare with the movement of endosomes discussed
above); a similar re-distribution of cholesterol into the furrow
was also observed. By contrast, analysis of the fluidity of the PM
revealed that the furrow membrane was more liquid ordered than
the rest of the PM. The raft domains in the furrow were enriched
Membrane remodelling in cytokinesis
in signalling molecules such as Src and phospholipase C-γ in
the raft domains, supporting the notion that these domains act as
foci for the formation of signalling units which control furrowing
(Figure 2) [67]. Although we agree with this thesis, the possibility
exists that a further role for cholesterol-rich domains may be to
modulate SNARE protein function in the furrow.
SNAREs, CYTOKINESIS AND LIPID RAFTS
As discussed above, membrane traffic plays a central role in
cytokinesis, with the cell exhibiting a carefully orchestrated
set of trafficking events during mitosis, the fusion of vesicles
at the tips of the growing furrow during cellularization in
Drosophila providing a particularly striking example [73].
Membrane trafficking events in all eukaryotic cells, from yeast
to humans, are controlled by the formation of specific SNARE
complexes [74]. Members of the t-SNARE (target SNARE) family
of proteins mark specific organelles. Formation of a SNARE
complex between t-SNAREs and their cognate v-SNARE (vesicle
SNARE) on the appropriate donor membrane is sufficient to
drive bilayer fusion [75,76], thus controlling SNARE complex
formation enables the cell to regulate membrane traffic in space
and time. Studies of the distribution of t-SNAREs in other systems
offer an interesting perspective on the potential role that PM
domains may play in cytokinesis.
Exocytic t-SNAREs (comprising of one member of the syntaxin
family, and one member of the SNAP25/SNAP23 family [74]) are
at least partially clustered within cholesterol/sphingolipid-rich
raft domains which are thought to mark the site of exocytosis,
and disruption of these rafts by cholesterol depletion modulates
exocytosis, suggesting that lipid rafts may act to restrict or localize
t-SNAREs into functional domains and so may contribute to
the spatial control of membrane traffic (Figure 2) [77–80]. By
generating a range of mutants of the t-SNARE proteins SNAP23
or SNAP25 with different affinities for lipid rafts, Salaün et al.
[79,80] observed that mutants of SNAP25 with an increased
affinity for lipid rafts displayed a reduced ability to support
exocytosis, and that SNAP23 mutants with decreased affinities
for lipid rafts supported increased exocytosis. Such studies argue
that the localization of t-SNAREs to rafts may decrease SNAREdependent fusion. These studies have been further supported by
the demonstration that two different t-SNARE conformations
exist that are separated both spatially and functionally into
distinct clusters in which one of either conformation predominates
[81]. Intriguingly, cholesterol depletion results in an increase in
the proportion of t-SNAREs adopting a conformation which is
functionally competent for fusion [81], prompting the suggestion
that cells could regulate the position of exocytosis sites by
controlling the lipid microenvironment within which the tSNAREs reside (Figure 2). This is further supported by a
recent study of lysosomal SNARE proteins in lysosomal storage
diseases which are characterized by the abnormal accumulation
of cholesterol in endosomal/lysosomal compartments [82]. Under
these conditions, there is a pronounced reduction in the ability
of lysosomes to fuse with either endocytic or autophagocytic
compartments. Fraldi et al. [82] showed that the SNARE proteins
involved in these fusion steps are abnormally sequestered into
cholesterol-rich regions of lysosomal membranes, accounting for
their diminished fusion capacity. With such studies in mind, the
apparent accumulation of cholesterol and raft domains within
the furrow of cells during cytokinesis could perhaps reflect a
mechanism by which exocytic fusion is confined, perhaps to the
growing tip of the furrow, or regions adjacent to the furrow region,
so driving invagination (Figure 2). Clearly such a model is highly
19
speculative, but super-resolution imaging approaches offer the
ability to test these hypotheses directly.
Both genetic and biochemical studies have suggested that lipid
species may themselves directly modulate SNARE-dependent
fusion (reviewed in [83]). It is now generally accepted that
SNARE-mediated fusion occurs through a hemi-fused state, in
which the outer leaflets fuse first followed by the inner leaflets
[84,85]. This imposes geometrical constraints on the two leaflets,
since the formation of the hemi-fused state will require the outer
leaflet to bend with high negative curvature, and progression to
the fused state requires the inner membrane to adopt a positive
curvature. Such models suggest that the presence of lipid species
with geometries that favour these highly curved states could
influence fusion directly [85–87]. PtdIns(4,5)P2 is localized to the
inner leaflet of the PM and is an inverted cone-shaped lipid [88].
As such, it would be expected that this species would antagonize
the high negative curvature which is required for the formation
of the hemi-fused intermediate (Figure 2). Consistent with this,
James et al. [89] showed that incorporation of PtdIns(4,5)P2 into
proteoliposomes containing SNARE proteins significantly slowed
the rate of SNARE-dependent fusion. Similarly, incorporation
of phosphatidic acid (which favours the formation of negative
membrane curvature) enhances SNARE-dependent fusion in vitro
[90,91]. Such studies support the hypothesis that the asymmetric
distribution of phospholipids and membrane microdomains in the
furrow provides an organizing platform for the control of furrowing/abscission (Figure 2). Consistent with this notion, the rate of
fusion of secretory vesicles with the PM in the furrow has been
observed to be significantly higher than that in the cell body [10].
PHOSPHOINOSITIDES: PtdIns(4,5)P 2
Genetic studies implicate phosphoinositide metabolism as a
crucial facet of cytokinesis. For example, a mutation in the
Drosophila gene fwd, which encodes PtdIns 4-kinase, resulted
in defective cytokinesis during male meiosis [33,92]. In S.
pombe, both PtdIns4P 5-kinase and its product PtdIns(4,5)P2
are concentrated in the medial ring during cytokinesis, and both
PtdIns 4-kinase and PtdIns4P 5-kinase are required for completion
of cytokinesis [88]. Studies using reporter constructs comprised
of different PH domains to inform on the localization of different
phosphoinositides have established that there is an accumulation
of PtdIns(4,5)P2 in the cleavage furrow of mammalian cells
(Figure 3) [88]. In a contemporaneous study, PtdIns(4,5)P2 was
also observed in the cleavage furrow of Drosophila spermatocytes
[93]. Both these studies established that PtdIns(4,5)P2 in the
cleavage furrow is important for the completion of cytokinesis.
Although expression of the PH domain reporters at low levels
(for image analysis) did not impair cytokinesis, expression at
higher levels resulted in cytokinesis failure. A range of approaches
led both groups to concur that interference with PtdIns(4,5)P2
production in the furrow interfered with the adhesion of the PM
to the contractile ring, and that a given level of PtdIns(4,5)P2
production is required in the furrow to sustain ingression and
maintain an active link with the underlying actin cytoskeleton
(Figure 3) [88,93]. This suggestion is supported by the observation
that (at least in vitro) PtdIns(4,5)P2 binds to septins and ERM
(ezrin/radixin/moesin)-family proteins that are known to link the
actin cortex to the PM (reviewed in [94]).
PHOSPHOINOSITIDES: PtdIns4P
Previous studies have added a further level of understanding to the
role of fwd and PtdIns4P dynamics in cytokinesis, and begun to
c The Authors Journal compilation c 2011 Biochemical Society
20
Figure 3
H. Neto, L. L. Collins and G. W. Gould
Phosphoinositides in cytokinesis
Top panel: PtdIns(4,5)P 2 accumulates in the furrow and may control actin dynamics and the constriction of the actomyosin ring. PtdIns4P is localized to Rab11-positive vesicles trafficking to
the furrow. PtdIns3P localizes to vesicles observed in the furrow/midbody in late telophase. Lower panel: Vps34/Beculin generates PtdIns3P on vesicles in the midbody. PtdIns3P then recruits
FYVE-CENT via the FYVE domain, which trafficks into the midbody as a ternary complex with KIF13A (a kinesin superfamily member which uses microtubules to deliver the complex) and TTC19.
TTC19 interacts with CHMP4B, a component of the ESCRT complex proposed to mediate the final abscission step. See the text for details.
integrate these pathways with those of the endosomal trafficking
system. Fwd is required for the synthesis of PtdIns4P on Golgi
membranes and for the formation of PtdIns4P-containing vesicles
that move to the midzone during cytokinesis in fly spermatocytes
[95]; importantly, these vesicles were shown to be Rab11-positive
(Figure 3). PtdIns4P has been suggested to play an important role
in the recruitment of both Rab11 and actin-regulatory proteins
(Rac1, Scar/WAVE) during AP-1-dependent protein sorting at
the Golgi [92]. Since (i) membrane vesicular transport is known
to deliver actin to the midzone in Drosophila embryos [7,73],
and (ii) the Rab11-effector Nuf promotes the polymerization
of actin in the furrow [96], Brill and co-workers suggested
that PtdIns4P-containing organelles may serve to concentrate or
recruit factors that maintain F-actin in the contractile ring [92].
In support of this hypothesis, they note that mutations in fwd,
nuf and rab11 are all associated with a failure to maintain actin
organization during cytokinesis [92]. How these studies of fwd
in spermatocyte cytokinesis relates to cytokinesis in other cell
types is not clear, as fwd is dispensable for normal development
and female fertility. It is likely that (an)other gene product(s)
is/are capable of compensating for fwd mutation outside of the
male germline. PtdIns4P localizes to the cell plate during plant
cytokinesis [97], further emphasizing commonality between plant
and mammalian cytokinetic mechanisms.
PHOSPHOINOSITIDES: PtdIns3P
A role of PtdIns3P in cytokinesis was first suggested from studies
in Ustilago maydis and Arabidopsis. In U. maydis, there is an
c The Authors Journal compilation c 2011 Biochemical Society
asymmetrical accumulation of vesicles enriched in PtdIns3P on
the daughter side of the primary septum [98], and in plants,
PtdIns3P-enriched vesicles accumulate around the cell plate
[97]. Until recently, the role of PtdIns3P in mammalian cell
cytokinesis was largely unexplored. Using a GFP-tagged tandem
FYVE-domain reporter to localize to PtdIns3P, Stenmark and coworkers observed PtdIns3P-positive vesicles in the intercellular
bridge which co-stained with endocytosed transferrin and thus
represent recycling endosomal-derived vesicles (Figure 3) [99].
Knockdown of VPS34 (the main phosphoinositide 3-kinase)
or its regulator, Beclin-1, delayed abscission and perturbed
cytokinesis. Both VPS34 and Beclin-1 localize to the intercellular
bridge, suggesting that local production of PtdIns3P is intimately
involved in abscission [100]. These studies complement work
in plant cells, where disruption of AtVps34 results in a severe
growth defect [101], and in Trypanosoma brucei, where RNAi
(RNA interference) depletion of TbVps34 results in a cytokinetic
block [102], supporting the notion that evolutionarily conserved
accumulation of PtdIns3P at the site of cytokinesis is important
for abscission.
To delve into the mechanism underpinning this, Stenmark and
co-workers performed an siRNA (small interfering RNA) screen
in which the effect of depletion of approximately 80 proteins
containing either FYVE or PX domains on cytokinesis was
examined [99]. Depletion of one such protein, termed FYVECENT (FYVE domain-containing centrosomal protein), was
found to result in severe cytokinesis defects, similar to those
observed upon knockdown of VPS34 or Beclin-1. FYVE-CENT
is centrosomally localized in interphase, but localizes to the
Membrane remodelling in cytokinesis
midbody during cytokinesis. The midbody association requires
an intact FYVE domain (i.e. is PtdIns3P-dependent), whereas the
centrosomal localization does not [99]. Further insight into the
biology of FYVE-CENT came from an examination of its binding
partners: FYVE-CENT interacts with KIF13A (a kinesin superfamily member) and TTC19 (a protein which contains four
tetratricopeptide repeats, identified as protein–protein interaction
domains in cell-cycle proteins). Similar to FYVE-CENT, these
proteins localize to the centrosome in interphase, but move into the
midbody during late telophase and are required for completion of
abscission. The proteins exist in a ternary complex, and working
models suggest that the kinesin superfamily member KIF13A
transports the FYVE-CENT–TTC19 complex into the midbody
along microtubules from the centrosomal location (Figure 3) [99].
So what may be the function of this delivery of FYVE-CENT–
TTC19 to the midbody? A tantalizing clue was provided by the
same study which identified TTC19 as a binding partner for
CHMP4B [99]. The ESCRT machinery has been implicated as
playing a key role in abscission [15,103]. In particular, ESCRTIII complex proteins have been proposed to mediate abscission via
interactions with centrosomal proteins and membrane/membrane
vesicles in the midbody. CHMP4B is a member of the ESCRTIII complex [104] that localizes to the midbody in cytokinesis,
suggesting perhaps that a crucial role for PtdIns3P is to contribute
to the proper assembly of ESCRT-III machinery in the midbody in
late telophase via delivery of TTC19 (Figure 3) [99]. This exciting
model awaits further experimental verification, but certainly
supports the view that membrane remodelling represents a crucial
facet of cytokinesis; in this case by defining the assembly of a
molecular machine (the ESCRT-III complex) in the correct spatial
and temporal co-ordinates.
THE ESCRT COMPLEX AND CYTOKINESIS
A central facet of the completion of abscission is the ability of
the dividing daughter cells to cleave the final narrow ‘neck’
of membrane in the intercellular bridge. Studies have focussed
on the ESCRT complex in this event, largely because of the
remarkable in vitro studies from the Hurley laboratory (see e.g.
[105]) which have revealed that ESCRT proteins have the ability
to cleave such membrane necks in vitro, and from studies in which
various components of the ESCRT complex were localized to the
midbody and/or shown to be required for cytokinesis (for reviews,
see [15,103,105]). As both the structure and function of ESCRT
complexes, and their role in cytokinesis, have previously been
reviewed, we will not delve into this area here. However, it is
clear that cytokinesis requires ESCRT-III components, but not
ESCRT-0 or ESCRT-II. One model proposes that the centrosomal
protein Cep55 is recruited to the membrane neck, followed by
ESCRT-I and ALIX (vps31 in yeast), which then recruits ESCRTIII to drive the final abscission [103,106,107]. In this context,
ESCRT-III is thought to recruit the microtubule-severing enzyme
spastin, thus facilitating cleavage of the central spindle at the
proposed site of abscission [108,109]. Remarkably, some of this
machinery (notably ESCRT-III and vps4-like proteins) function
in cell division in archaea, strongly suggesting that this vital facet
of the abscission machinery is evolutionaily conserved [110,111].
Several crucial questions remain to be addressed. These include
determining the temporal order of arrival of the ESCRT machinery
at the site of abscission, and defining how the lipid environment
of the membrane neck is modulated to control the interaction
with the ESCRT machinery (or vice versa). Super-resolution
imaging will no doubt soon provide answers to some of these
issues.
21
SUMMARY AND PERSPECTIVES
How membrane traffic and/or membrane remodelling are
integrated and controlled during cytokinesis represents an
important challenge for cell biologists. As discussed above,
changes in the dynamics and nature of membrane trafficking
pathways and the composition of the PM and the accumulated
vesicles in the midbody all offer potentially important mechanistic
control points: our challenge now is to develop a clearer picture
of how these systems operate collectively. The application of
ultra-fast high-definition imaging approaches, coupled to genetic
manipulation, offers one route to address some of these issues.
The work described above was largely performed on
model cell lines grown in culture, reflecting the ease of
genetic/pharmacological manipulation and imaging that such
systems offer. Cultured cells use a contractile ring to divide (the
‘purse-string’ mode of cytokinesis, sometimes called cytokinesis
A). However, it is clear that under certain circumstances and in
some cell types, cytokinesis proceeds apparently normally without
a contractile ring. For example, a mutant form of Dictyostelium
discoideum lacking myosin-II uses traction forces to drive the
separation of the two daughter cells when grown on adherent
surfaces [112,113]. Similarly, some mammalian cells exhibit a
form of cytokinesis which is adhesion-dependent but contractile
ring-independent (often referred to as ‘cytokinesis B’) [114]. It is
presently unclear whether these two forms of cytokinesis represent
back-up mechanisms/redundancies, or whether these different
forms of cytokinesis are utilized differentially, e.g. in tissues or
when a cell is surrounded by a matrix. The studies described
above revealing a role for integrin trafficking in cytokinesis could
be interpreted to imply that adhesion forces may operate in
‘cytokinesis A’ as well as in ‘cytokinesis B’, consistent with
the evolutionary conservation of key facets of cytokinesis (e.g.
Rab11-dependent endosomal vesicle trafficking in mammals,
flies and worms; ESCRT function from archaea to mammals,
etc.). A further challenge for the field will be to determine
whether the molecular machinery identified in tissue culture
cells (e.g. endosomal trafficking into the intercellular bridge,
the accumulation of cholesterol in the furrow, etc.) also operate
during cytokinesis in tissues, or in other more specialized forms
of cytokinesis such as the division of larger cells (e.g. in early
embryonic division). The next few years will undoubtedly provide
us with new insight into these questions.
ACKNOWLEDGEMENTS
The authors thank Nia J. Bryant for helpful comments on the manuscript and Figures. We
apologize to those authors whose work was not cited for space constraints.
FUNDING
Work in the Gould laboratory is supported by Cancer Research U.K., the Association for
International Cancer Research, Diabetes U.K. and The Wellcome Trust.
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