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Biochemical Society Transactions (2005) Volume 33, part 6
Membrane traffic in cytokinesis
J. Matheson, X. Yu, A.B. Fielding and G.W. Gould1
Henry Wellcome Laboratory of Cell Biology, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Davidson Building,
University of Glasgow, Glasgow G12 8QQ, U.K.
Abstract
A crucial facet of mammalian cell division is the separation of two daughter cells by a process known as
cytokinesis. An early event in cytokinesis is the formation of an actomyosis contractile ring, which functions
like a purse string in the constriction of the forming furrow between the cells. Far less well characterized are
the membrane-trafficking steps which deliver new membrane to the cell surface during the plasma membrane expansion known to accompany furrow formation. It is now clearly established that the plasma
membrane at the cleavage furrow of mammalian cells has a distinct lipid and protein composition from
the rest of the plasma membrane. This may reflect a requirement for both increased surface area during
furrowing and for the co-ordinated delivery of intracellular signalling or membrane re-modelling activities
to the correct spatial coordinates during cleavage. In this review, we discuss recent work within the area of
membrane traffic and cytokinesis.
Introduction
A crucial facet of mammalian cell division is the separation
of two daughter cells by a process known as cytokinesis. An
early event in cytokinesis is the formation of an actomyosin
contractile ring, which functions like a purse string in the constriction of the forming furrow between the two cells [1]. This
furrow constricts, leading to the formation of a thin cytoplasmic bridge between the cells (the mid-body), which
is ultimately cleaved in the terminal step of cytokinesis –
abscission. It is now clear that membrane dynamics is required
at, at least, two stages of cytokinesis. First, membrane delivery to the surface of the cell is required during cleavage
furrow ingression in order to provide the increased surface
area necessary to form two new daughter cells. Secondly, once
the cleavage furrow has completed its ingression, the cells
remain connected by a narrow intracellular bridge. Membrane dynamics must take place in order to finally separate
the mother cell into two separate daughter cells and to seal the
two newly formed cells. In this brief review, we will highlight
recent advances in our understanding of membrane traffic
during cytokinesis.
Flies, worms and furrows
It is clear from studies in several different organisms that
membrane trafficking is required for successful cytokinesis
(for recent reviews, see [2–5]). Perhaps the most striking
example of this is in plant cells. These completely lack an
actomyosin ring and cytokinesis proceeds by the delivery of
membrane vesicles along microtubules to the centre of the cell
Key words: ADP-ribosylation factor 6 (Arf6), cytokinesis, endosome, exocyst, membrane traffic,
Rab11.
Abbreviations used: Arf, ADP-ribosylation factor; FIP, family of Rab11-interacting proteins; GFP,
green fluorescent protein; RNAi, RNA interference; SNARE, soluble N-ethylmaleimide-sensitive
fusion protein attachment protein receptor.
1
To whom correspondence should be addressed (email [email protected]).
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where they accumulate and fuse, forming the ‘phragmoplast’.
Continued addition of vesicles eventually leads to the fusion
of the phragmoplast with the mother cell membrane, hence
dividing the cell into two [3].
In animal cells, an actomyosin ring physically constricts
the cells until the two daughter cells remain connected by a
narrow intracellular bridge (the mid-body). This constriction
requires significant additional membrane to accommodate the
increased surface area of the two daughter cells. Studies in
Xenopus have indicated clearly that the additional membrane
has a different lipid and protein composition from the original
membrane, arguing that the membrane is not derived from
expansion of the pre-existing surface membrane, but instead
forms through insertion of membrane from internal stores
[3,5,6]. It is thought that the unique composition of the
furrow plasma membrane may underscore its ability to be deformed during ingression, as well as possibly generating the
signals that regulate progression of cytokinesis [6–8]. Thus, in
addition to the delivery of the membrane to compensate for
the expanding plasma membrane surface, membrane traffic
during cytokinesis could also mediate the delivery of proteins
that control the ingression of the cleavage furrow as well as
cell–cell abscission. However, the source of this new membrane and the mechanism(s) by which this traffic is controlled
remained, until recently, fairly obscure.
The first clue regarding the role of membrane traffic originated from experiments using the genetic tractability of Drosophila. Drosophila’s early embryogenesis involves 13 nuclear
divisions in a syncytium. The first nine occur in the interior
of the embryo, but by interphase of cycle 10 the nuclei reach
the cortex and undergo four more synchronous divisions as
an even monolayer underneath the plasma membrane (the
syncytial blastoderm stage) [26]. Cellularization occurs
during the interphase of cycle 14 when the plasma membrane
invaginates the 6000 or more cortical nuclei to form individual
cells. These highly ordered events are highly dependent upon
Cell Architecture: from Structure to Function
cytoskeletal organization, and at cycle 14 the actin cytoskeleton is associated with the invaginating membranes [27].
Evidence suggests that insertion of the membrane at the apex
of cleavage furrow is crucial for the successful completion of
cellularization, in which approx. 6000 nuclei are cellularized
in approx. 45 min.
Nuclear fallout (Nuf ) is an essential maternal-effect gene,
whose product is required for this cellularization [28].
Our laboratory and others recently identified two human
proteins with sequence identity to Nuf, termed Rab11–FIP3
(where FIP stands for a family of Rab11-interacting proteins) and Rab11–FIP4 (see below) [9–12]. Interestingly,
these proteins were identified by virtue of their interaction
with the GTPases Rab11 and Arf (ADP-ribosylation factor),
prompting the examination of the role of Rab11 and recycling
endosomes in cellularization. Riggs et al. [13] showed that
Nuf and Rab11 co-localize and physically associate with each
other at the recycling endosome, that each of them are dependent on the other for this localization and that there are similar
defects in both membrane delivery to and actin remodelling at the cleavage furrow when either of them is genetically
deleted. As Rab11 is a small GTPase that is resident in
the recycling endosome and is required for the budding of
vesicles from this compartment [14], these authors concluded that trafficking through the Rab11 compartment
is required for cellularization. Riggs et al. [13] proposed
two potential models that explain their findings. Both of
these suggest that Rab11 and Nuf are required for the
delivery of recycling endosome-derived membrane to the cleavage furrow. The first model proposes that actin filaments
are associated with the membrane vesicles being delivered to
the furrow. The second suggests that rather than actin being
included in the vesicles, it is actually an actin-remodelling
factor that is co-delivered with the membrane that then has
its effects on the actin cytoskeleton once delivered to the
cleavage furrow. In either case, this work suggests that membrane delivery to and actin remodelling at the cleavage furrow
are linked processes and that the compartment providing the
driving force for both of these processes is the recycling
endosome. Further in support of this, Pelissier et al. [15]
have shown that functional endocytosis is required prior to
cellularization, implicating recycled endosomes as a source of
membrane required for cellularization. Further to this, they
showed that knocking down Rab11 activity before cellularization results in defects in membrane addition at the cleavage
furrow. These results clearly underscored the importance
of membrane traffic in the formation of a cellularization
furrow.
Mammalian cells also require Rab11
for cytokinesis
Rab11 is a small GTPase that plays a key role in regulating
the trafficking of plasma membrane receptors through endosomes. The cycling between GTP- and GDP-bound forms
of Rab proteins regulates the recruitment of various effectors
to membranes that regulate the targeting and fusion of trans-
Figure 1 Rab11 is required for cytokinesis
HeLa cells were infected with an adenovirus expressing a dominant
negative mutant of Rab11 [Rab11-S25N (Ser25 → Asn)]. Note that cells
positive for expression of this protein (green, left panel) often exhibit
a binucleate phenotype (see tubulin stain in red, right panel). Quantification of these data revealed that approx. 15% of cells expressing this
mutant exhibited a cytokinesis defect. Similarly, knockdown of Rab11
using siRNA (small interfering RNA) induced a cytokinesis defect, with
some 25% of cells exhibiting a binucleate phenotype.
port vesicles [16]. Recently, we and others have identified
a novel FIP [17–21], all members of which share a highly
conserved, 20-amino-acid motif at the C-terminus of the
protein, known as the RBD (Rab11-binding domain) [19,22].
Interestingly, the C-termini of FIP3 and FIP4 (also known as
arfophilin1 and arfophilin2) have identity with Nuf.
We investigated the importance of endocytic membrane traffic during cytokinesis in mammalian cells and characterized the regulatory interactions that control membrane
targeting to the cleavage furrow [23]. First, we demonstrated
that Rab11-containing recycling endosomes accumulate near
the cleavage furrow and that Rab11 is required for successful
completion of cytokinesis in mammalian cells (Figure 1) [23].
Secondly, using a combination of dominant negative mutants
and RNAi (RNA interference), we found that Rab11 is a key
component involved in the delivery of endosomes to the
cleavage furrow; these endosomes are characterized by
the presence of FIP3 and FIP4. Consistent with this, we found
that both FIP3 and FIP4 accumulate on endosomes in the
furrow and in the mid-body, and that Rab11, in complex
with the FIP, is required for localization [23]. Moreover,
we found that the FIP3–Rab11 complex was required for
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Biochemical Society Transactions (2005) Volume 33, part 6
Figure 2 FIP3–Rab11 interaction is required for cytokinesis
HeLa cells were transfected with an FIP3 mutant unable to bind Rab11
(FIP3-I737E). Expression of this mutant protein resulted in a significant
fraction of cells exhibiting a binuclear phenotype (shown) and an inability
to recruit FIP3 to membranes. See [23] for details. Reprinted from
Molecular Biology of the Cell (Mol. Biol. Cell 2005 16: 849–860; published
online before print as 10.1091/mbc.E04-10-0927) with the permission
of the American Society for Cell Biology.
packed, antiparallel microtubules derived from the mid-zone
microtubules that have been squeezed into a dense bundle.
Where these microtubues overlap is the electron dense central
‘Flemming body’. Surrounding the Flemming body is a littledescribed ring-like structure that contains, amongst other
proteins, MKLP1 (mitotic kinesin-like protein 1) and which
here will be referred to as the ‘mid-body ring’. It is known
that many proteins reside in the mid-body and that these
play a number of roles in the final stages of cytokinesis. For
example, Skop et al.’s [24] recent functional proteomic
approach to identify proteins in the mid-body found 172 proteins present in the mammalian mid-body, of which
100 homologous proteins showed cytokinesis defects when
they were knocked down by RNAi in Caenorhabditis elegans
[24]. The proteins present at the mid-body carry out a variety
of functions, including mid-body formation and actin ring
disassembly [24]. However, it is also clear that membrane
events underpin abscission.
Membrane events leading to abscission
completion of cytokinesis, as a mutant FIP3 that does not
bind Rab11 exhibited a late cytokinesis defect (Figure 2).
Significantly, while FIP3 recruitment to endosomes is Rab11dependent, we find that the targeting of FIP3 to the midbody during late cytokinesis is independent of Rab11. Finally,
using GFP (green fluorescent protein)–FIP3, we show that
the localization of FIP3 is subject to spatial and temporal
regulation, and FIP3 is localized to the centrosome during
early anaphase before rapidly moving to the furrow at the
onset of cytokinesis. After abscission, FIP3 then returns to
the centrosome [23].
Together, these results suggest that accumulation of FIP3 in
the mid-body and its interaction with Rab11-containing endosomes allows the docking and subsequent fusion of endocytic vesicles with the apex of the cleavage furrow. We
propose that the dynamic Rab11–FIP3 interaction controls
the delivery, targeting and perhaps fusion of vesicles derived
from recycling endosomes with the furrow. The redistribution of FIP3 during the mitosis couples recycling endosome-derived membrane vesicle traffic to the furrow with
the cell cycle, thus regulating furrowing and ultimately
abscission.
Abscission: the final frontier of cytokinesis
Once the actomyosin ring has contracted and the cleavage
furrow has fully ingressed, the two daughter cells remain
connected by a narrow intracellular bridge, known as the midbody. The final stages of cytokinesis result in the resolution of
this bridge into two separate, sealed daughter cells (reviewed
in [4]). The mid-body is the term given to the narrow band
of cytoplasm that links the two daughter cells after cleavage
furrow ingression. One of its main components is tightly
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Prior to abscission, the cells are connected by the midbody and the actomyosin ring has disassembled and cannot
therefore constrict the cell further. It must be membrane
events that ultimately divide one cell into two and ensure
that these two new cells are sealed. It is not yet clear exactly
how this event occurs, although two major models can be
envisaged. First, division may occur at the centre of the midbody in a process similar to plant cell division, i.e. membrane
vesicles accumulate and fuse with each other and eventually
the plasma membrane, thus dividing the cell into two. The
second model is one of endocytosis at the mid-body, with
endocytosed membrane leading to the division of the cell
into two [24]. Recent data from our laboratory hints at the
former as a key mechanism in mammalian cells.
The vesicle delivery and fusion model seems to be feasible
as microtubules are already in place, terminating in the centre
of the mid-body. Here they overlap, providing an ideal situation for vesicles travelling from opposite sides of the midbody to meet and fuse. Components of the membrane fusion
machinery localize to the centre of the mid-body and play
important roles in cytokinesis. For example, Low et al. [25]
have shown that two members of the SNARE (soluble
N-ethylmaleimide-sensitive fusion protein attachment protein receptor) membrane fusion machinery, syntaxin 2 and
endobrevin/VAMP 8 (vesicle-associated membrane protein 8), localize to the mid-body and that when mutant
forms of these proteins are expressed, it results in binucleate
cells. By using time-lapse microscopy, they show that this
failure in cytokinesis occurs specifically at the final abscission
stage [25]. These data also confirm that membrane events for
abscission are distinct from earlier cleavage furrow ingression
events. Also, Gromley et al. have been studying a novel,
centrosomal protein, centriolin, which they have shown to
be required for the final stage of cytokinesis [26,27]. More
recently, they have reported that this protein binds to both
sec15, a member of the exocyst complex (a complex that has
Cell Architecture: from Structure to Function
been shown to be essential for cytokinesis; for a review, see
[28–30]), and to the SNARE protein snapin [26,27]. Knocking
these two proteins down by RNAi produces abscission
defects. These and other SNARE and exocyst components
co-localize in the ‘mid-body ring’ structure. They conclude
from all of these observations that membrane is delivered to
the ‘mid-body ring’ structure at the centre of the mid-body
where the membrane fusion and exocytic proteins identified
above carry out their functions, leading to abscission of the
mid-body.
The other model is one of endocytosis at the midbody, with endocytosed membrane somehow leading to the
division of the cell into two. Endocytosis has certainly been
shown to occur earlier in cytokinesis, during cleavage furrow
ingression, in zebrafish [31]. It has also been shown to be
essential for successful cytokinesis in C. elegans [32]. However, there are some hints that suggest that it may be important
at a later stage of cytokinesis in mammalian cells. For example,
dynamin, which is a key endocytic protein, has been shown
to localize to the central spindle and later to the mid-body in
both C. elegans and mammalian cells. In addition, depletion
of dynamin in C. elegans has been shown to cause both early
and late, possibly abscission, cytokinesis defects [32]. It has
also been shown that the small GTPase Arf6 plays a role
in mammalian cytokinesis [33]. Wild-type Arf6 was seen to
accumulate at the cleavage furrow during cytokinesis, whilst a
constitutively active mutant of Arf6 localizes to the mid-body
late during cytokinesis, and when expressed at high levels
causes various defects in cytokinesis [33]. More recently, they
have suggested that the function that Arf6 is carrying out in
the late stages of cytokinesis may be endocytosis [4].
We recently addressed these kinds of models. Recall that
FIP3 and FIP4 were originally identified in a two-hybrid
screen interacting with Arf GTPases. We have found that both
FIP3 and FIP4 interact strongly and in a nucleotide-dependent manner with Arf6, and that this interaction is crucial
for the recruitment of FIP3 and FIP4 into the mid-body
(A.B. Fielding, E. Schonteich, J. Matheson, G. Wilson, X.
Yu, G.R.X. Hickson, S. Srivastava, S.A. Baldwin, R. Prekeris
and G.W. Gould unpublished work). Such observations are
interesting since the regulated delivery of membrane vesicles
to the furrow/mid-body during cytokinesis is most probably
accompanied by alterations in actin dynamics [1,34–36],
and Arf6 has previously been reported to control actin
dynamics in mammalian cells [37–39]. In support of such a
contention, it is interesting to note that there is a well-established dynamic interplay between the mitotic spindle and
the actomyosin cortex; FIP3 and FIP4 may provide a focus
for this interaction, regulating membrane delivery and actin
dynamics through the same molecule. This is a particularly
interesting suggestion, since both FIP3 and FIP4 are frequently found, together with Arf6, on spindle microtubules.
However, we believe that a further potential explanation
for the Arf6 interaction with FIP3 and FIP4 may be to facilitate docking of vesicles at the furrow and mid-body prior to
fusion. This hypothesis is based upon the fact that Exo70p, a
component of the mammalian exocyst complex, was recently
reported to interact with Arf6. The exocyst is involved
in budding events in yeast [40,41], is thought to control
membrane vesicle docking during exocytosis [29,42,43] and
may also play a key role in cytokinesis, since components of
the exocyst have been identified in mid-bodies [27]. Moreover, Arf6 localizes to the furrow and mid-body and is required for abscission. Hence, we propose the following model
for membrane trafficking during cytokinesis: Rab11 recruits
FIP3 or FIP4 to recycling endosome-derived vesicles for
traffic along microtubules into the cleavage furrow or midbody. Perturbation of the function of either Rab11 or FIP3
results in defective abscission [23]. We propose that an
interaction of FIP3 or FIP4 with active Arf6 at the cleavage
furrow or in the mid-body serves to tether these vesicles in
this region, via interaction with Exo70p, prior to membrane
fusion. In support of this model, we have found that (i) Arf6
binds both FIP3 and FIP4 in a GTP-dependent manner,
(ii) Arf6 is localized to the furrow and mid-body and
(iii) Arf6-GTP recruits FIP3/FIP4 to the mid-body of
dividing cells. Furthermore, we have shown that Exo70p is
localized to the furrow of dividing cells and that depletion
of Exo70p resulted in a profound cytokinesis defect (A.B.
Fielding, E. Schonteich, J. Matheson, R. Lucas, X. Yu,
G.R.X. Hickson, R. Prekeris and G.W. Gould, unpublished
work). These results clearly implicate Exo70p as an important
component of the cytokinesis machinery. Strikingly, we show
that antibodies specific for Exo70p co-immunoprecipitate
FIP3 and FIP4 from CHO (Chinese-hamster ovary) cells.
Consistent with the model described above, Rab11 was
also observed in these immunoprecipitates (A.B. Fielding,
E. Schonteich, J. Matheson, R. Lucas, X. Yu, G.R.X. Hickson,
R. Prekeris and G.W. Gould, unpublished work). The model
we propose suggests that the interaction of FIP3 or FIP4 with
active Arf6 at the cleavage furrow/mid-body serves to regulate a docking event involving Exo70p. It is tempting to speculate that this docking event could involve the tethering of
vesicles around the mid-body ring prior to a compound
fusion event that results in separation of the cells. However,
further work is required to definitively show this.
In summary, during cytokinesis vesicles derived from
recycling endosomes and identified by the presence of Rab11–
FIP3 and Rab11–FIP4 protein complexes traffic to the furrow
and mid-body along microtubules. The recruitment of these
complexes to the mid-body is controlled by active Arf6 and
the ability of FIP3/FIP4 to bind Arf6-GTP, perhaps in a
ternary complex with Rab11. In the mid-body, the interaction of FIP3/FIP4 with Arf6 may facilitate tethering via
Exo70p, perhaps prior to homotypic compound membrane
fusion, thus implicating FIP3 and FIP4 in the abscission
event.
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Received 10 June 2005