Update
TRENDS in Cell Biology
115
Vol.14 No.3 March 2004
Pathways for membrane trafficking during cytokinesis
Laila I. Strickland and David R. Burgess
Boston College, Chestnut Hill, MA, USA
The molecular mechanisms underlying targeted deposition of new membrane at the advancing furrow of a
dividing cell have long been intriguing to cell biologists.
Three recent studies have made use of Drosophila cellularization to explore current questions in this field.
These findings indicate that both the secretory pathway
and endosomal recycling contribute membrane to the
advancing furrow. Furthermore, new work reveals that
vesicles derived from the Rab11 recycling endosome
(RE) promote actin remodeling at the furrow.
Cytokinesis, or the cytoplasmic division of a single cell into
two, requires the assembly and activation of an actomyosin
ring, and membrane insertion at the site of cleavage.
Addition of new membrane satisfies the geometric requirement for an increase in surface area in order for a cell to
divide. New membrane could be added to the surface
globally as the cell prepares to divide, or it could be
targeted to a specific region for insertion. In spherical sea
urchin embryos, it has been observed that, rather than
global deposition, membrane is delivered specifically to the
cleavage furrow [1]. Similar observations have also been
made in cleaving Xenopus eggs [2]. Targeting of vesicles to
this location might facilitate organization of the cell
surface, resulting in the composition of membrane at the
furrow being distinct from that of the rest of the cell [3].
Localization of structural and signaling molecules
required for the execution of cytokinesis might be coupled
to membrane deposition at the furrow. For example,
vesicles destined for the furrow might contain important
transmembrane proteins and lipids whose enrichment at
the cleavage site contributes to contractility. Indeed,
specialized interaction between the plasma membrane
and the underlying cytoskeleton at the cleavage site is
required to coordinate the contractile and exocytic events
necessary for the execution of cytokinesis [4].
Drosophila embryogenesis provides a unique system for
studying membrane trafficking. The events of early
Drosophila development have been reviewed recently
[5]. In Drosophila, the first thirteen mitotic cycles occur
in a syncytium. At the tenth cycle, the nuclei migrate to the
actin-rich cortex, where they induce so-called pseudofurrows around each mitotic apparatus at metaphase of
subsequent cycles. The metaphase furrows ingress only
partially between the many mitotic apparatuses as each
segregates its associated chromosomes during cycles 10–13.
Cellularization occurs during the fourteenth cycle and
involves formation of furrow canals and full cytokinetic
contraction between each nucleus (Figure 1). Completion
Corresponding author: David R. Burgess ([email protected]).
www.sciencedirect.com
of this process results in a polarized epithelial sheet
surrounding the embryo.
The molecular and mechanical elements required for
cellularization of the Drosophila embryo are often homologous to those that drive cytokinesis in other systems
(Figure 2). Specifically, mutations that result in cellularization defects in Drosophila commonly interfere with
cytokinesis when examined in other cell types. Membrane
trafficking and vesicle fusion is widespread during
cytokinesis in animal [6] and plant cells [7]. Accordingly,
Nucleus
Actin cortex
Recycling pathway
(Rab11/Nuf)
(EV)
Secretory
pathway
(Strabismus)
RE
(TV)
?
XX
X
X
X
X
Post-REVs X
X
F-actin
Nucleus
Golgi
TRENDS in Cell Biology
Figure 1. Membrane trafficking during Drosophila embryo cellularization. Components of the secretory pathway and the membrane recycling pathway are
required for membrane deposition during cellularization of the Drosophila
embryo. Transport vesicles (TVs) might be trafficked directly to the furrow by a
mechanism dependent on Strabismus and other Golgi proteins or might be sorted
through the recycling endosome (RE). Endocytic vesicles are sorted through the
RE and redirected to the furrow by Nuf and Rab11. At the furrow, post-RE vesicles
promote recruitment of actin to the contractile ring.
116
Update
TRENDS in Cell Biology
(a)
(d)
Fly embryo
Frog egg
(b)
Vol.14 No.3 March 2004
Worm embryo
(e) Fission yeast
(c)
Sea urchin egg
(f)
Plant cell
TRENDS in Cell Biology
Figure 2. Trafficking of membrane to the cleavage furrow is an important event in many types of cells. Shown is a sample of cell types in which membrane trafficking during
cytokinesis has been studied. Fruit fly embryos (a) require mobilization of internal membranes to the surface to complete cytokinesis. Secretion is essential in nematode
worm embryos (b), which divide asymmetrically owing to asymmetric positioning of the mitotic apparatus. Targeted secretion specifically at the cleavage furrow has also
been observed in sea urchin eggs (c). Deposition of new membrane in cleaving frog eggs (d) is clearly visible by the appearance of light pigment on the cell surface at the
site of furrow ingression. Fission (d) and budding yeast both require vesicle delivery to the division site to complete cytokinesis. In plant cells (f), cytokinesis occurs as
Golgi-derived vesicles accumulate and fuse at the phragmoplast in the cell center. Vesicle fusion produces a compartment of cell-wall precursors that eventually merges
with the mother cell membrane to produce two daughters. In all of these cell types, vesicle trafficking relies on interactions between proteins on the surface of the vesicles
themselves with various elements of the cytoskeleton. The surface proteins of each vesicle are characteristic of the membrane from which the vesicle originates. The membrane sources that supply vesicles to the cleavage furrow are of great interest to the broad field of cytokinesis.
many of the genes involved in membrane dynamics are
highly conserved. For example, the syntaxins represent a
large family of proteins that perform homologous functions
in vesicle fusion in many cell types. Cellularization fails in
Drosophila embryos mutant for syntaxin [8]. Syntaxin has
also been shown to be necessary for cell division in budding
yeast [9], sea urchin eggs [10], plants [11], nematodes [12]
and mammalian cells [13]. These studies highlight the
importance of membrane trafficking and vesicle fusion in
cytokinesis. While the general requirements for membrane trafficking during cytokinesis are well established
[14], several current papers reviewed here raise unanticipated possibilities as to the breadth of membrane
trafficking in cytokinesis. Drosophila cellularization
lends itself well to molecular dissection of these processes
because of its amenability to genetic analysis, microinjection and microscopy. Furthermore, cellularization provides an opportunity to observe thousands of simultaneous
division events in a single embryo. New work discussed
here has utilized this system to address questions
regarding trafficking of membrane to the furrow and
provides an exciting link between membrane addition and
actin dynamics.
A role for the secretory pathway in membrane
deposition
One model by which vesicles could be targeted to the
cleavage furrow proposes that vesicles derived from the
secretory pathway are directed to the site of furrow
ingression (Figure 1). Evidence that the secretory pathway
provides an important source of membrane during cell
division is the finding that the inhibitor of anterograde
trafficking brefeldin A (BFA) disrupts cytokinesis in
Caenorhabditis elegans embryos [15] and Drosophila
cellularization [16]. However, BFA does not prevent
membrane addition or interfere with cytokinesis during
www.sciencedirect.com
the cleavage of early sea urchin embryos [1], suggesting
that different embryos might stockpile differing amounts
of ready-to-exocytose membrane during oogenesis.
Additional support for a role for the secretory pathway
in membrane deposition comes from studies demonstrating that various Golgi proteins are required for cytokinesis
[16]. New findings by Lee et al. show that an integral
membrane protein called Strabismus (Stbm) localizes
primarily to the Golgi and is required for membrane
deposition during cell division [17]. In wild-type embryos,
the tumor-suppressor and adaptor protein Discs-Large
(Dlg) localizes to the plasma membrane along the newly
formed cell – cell interface that results from cellularization
[18]. Another protein, Discs-Lost (Dlt), is specifically
deposited in the region of the advancing furrow [19]. The
authors identified an interaction between Stbm and Dlg
using a yeast two-hybrid screen and showed that, in
embryos mutant for Stbm, both Dlg and Dlt fail to be
deposited at their respective locations on the cell surface.
Intriguingly, the very process of membrane formation was
shown to be impaired in stbm mutants, by visualization of
the membrane marker concanavalin A. These findings
demonstrate that the Golgi and secretory pathway provide
a necessary contribution of new membrane during
cellularization. It remains to be determined whether
transport vesicles from the Golgi are delivered directly
to the plasma membrane or are sorted through the
recycling endosome (Figure 1).
A role for endocytic recycling in membrane deposition
A second model proposes that vesicles destined for the
cleavage furrow are derived from endocytosis and sorted
through the recycling endosome (RE; Figure 1). Endocytosis has been observed to occur specifically at the furrow
in the embryos of zebrafish [20], as well as cleaving
Xenopus eggs [2]. Intriguingly, clathrin has been shown to
Update
TRENDS in Cell Biology
be required for appropriate localization of myosin II to the
contractile ring and is therefore required for cytokinesis in
Dictyostelium cells [21]. This work suggests that trafficking of endocytic vesicles to the cleavage site is important
for cytokinesis and that these vesicles might contribute to
the appropriate assembly and activation of the contractile
ring, as well as provide a source of new membrane.
New work by Pellisier et al. has sought to examine this
hypothesis in Drosophila embryos [22]. If endocytic
vesicles are important during cellularization, then disruption of endocytosis before cellularization would be
expected to have consequences for this process. In their
current paper, Pelissier et al. have examined the phenotypes of embryos that were defective for endocytosis, either
by microinjection of a dominant-negative Rab5 construct
or examination of a temperature-sensitive mutant for
shibire. Rab5 is a small GTPase involved in endocytosis,
and shibire is the Drosophila homolog of dynamin, also
important for endocytosis. Neither of these embryos was
able to undergo cellularization, supporting the hypothesis
that endocytic vesicles might be an important route for
membrane delivery to the furrow. However, in the case of
the shibire mutant, dynamin has also been shown to
mediate vesicle budding from other compartments, including the trans-Golgi network and the RE [23]. Using the
membrane protein neurotactin as a marker for trafficking
through the secretory pathway, the authors observed that,
in shibire mutants, neurotactin accumulated in a nonGolgi compartment, apical to the nucleus. Based on
colocalization with Rab11, the pericentriolar RE was
identified as the site of accumulation.
Rab11 is a small GTPase and resident of the RE whose
activity is required for the budding of vesicles from this
compartment [24]. The authors reasoned that, if vesicles
derived either from endocytosis or the secretory pathway
are being redirected to the cleavage furrow through such a
compartment, then Rab11 activity should be essential for
cellularization. This was shown to be the case by
microinjection of a dominant-negative Rab11 construct
before cellularization. These embryos displayed defects in
membrane addition and furrow morphology very similar to
Nuclear Fallout (Nuf) mutants [25], suggesting a role in a
common pathway for these two proteins.
Rab11, Nuf and a link between the RE and actin
remodeling
Several lines of evidence implicate vesicle trafficking in the
cytoskeletal rearrangements that culminate in cytokinesis, including the previously mentioned work demonstrating a role for clathrin in myosin II localization to the
contractile ring [21]. Additionally, dynamin has been
shown to effect changes in actin organization at the
leading edge of migrating cells [26] and is required for
cytokinesis in C. elegans embryos [27]. Exciting new
findings by Riggs et al. support a role for vesicle trafficking
in actin remodeling at the furrow and execution of
cellularization [28]. The authors explore a link between
the membrane addition and actin remodeling defects at
cellularization in embryos mutant for either Rab11 or Nuf.
This study implicates trafficking of vesicles through the
RE in membrane addition and recruitment of actin to the
www.sciencedirect.com
Vol.14 No.3 March 2004
117
furrow, providing intriguing new evidence that these
processes might be coupled.
Nuf is homologous to the mammalian protein arfophilin-2, which is a member of the Arf family of small
GTPases, and a known binding partner of Rab11 [29].
Notably, Arf family members have been shown to be
involved in trafficking of recycled vesicles back to the
plasma membrane [30] and in actin assembly [31].
Previously, Nuf mutants had been observed to be
defective both in recruitment of actin to the contractile
ring [25] and in delivery of membrane to the site of
furrow ingression [32].
In their investigation, Riggs and colleagues used a GFPtagged version of Nuf to examine its localization during
Drosophila embryogenesis. They found that Nuf concentrates in the centrosomal region during prophase in a
pattern reminiscent of the recycling endosome. Immunofluorescence techniques were applied to confirm the
colocalization of Nuf with Rab11 at the RE. Pull-down
assays demonstrated that the observed colocalization
reflects a physical association between the two proteins.
To determine whether this interaction also had functional
implications, the authors used genetic techniques to
deplete embryos of either Nuf or Rab11. Their results
showed that the physical association between Nuf and
Rab11 is a requirement for the correct localization of both
proteins to the RE. Comparison of both mutant phenotypes
revealed similar actin-remodeling and membrane-delivery
defects.
The authors propose that Rab11 and Nuf stimulate the
delivery of RE-derived vesicles to the furrow and that
either the vesicles themselves are associated with small
actin filaments that incorporate into the contractile ring
upon fusion with the plasma membrane or the vesicles
carry another protein that stimulates actin remodeling
upon arrival at the cortex. The findings of Riggs et al. that
membrane trafficking and actin remodeling are linked
processes are very intriguing, and future work will
undoubtedly elucidate the nature of this coordination.
Concluding remarks
While the recent publications reviewed here argue that
both the Golgi and the recycling endosome are important
compartments for directing new membrane to the cleavage
furrow, key questions remain. Recycling of endocytic
membrane might be a way of moving excess microvillar
’apical’ membrane to the advancing furrow; however, other
sources might also be involved. An endocytosis-based
system of membrane delivery would allow for reorganization of the cell surface, facilitating the concentration of
specific membrane proteins and lipids required for cell
division to the furrow. However, the recycling of endocytic
membrane alone does not account for the increase in
surface area that accompanies cell division. Other sources
of membrane must also be involved. Indeed, previous work
has demonstrated the importance of the Golgi, but it has
yet to be determined whether Golgi-derived transport
vesicles fuse directly with the furrow or are directed first
through the recycling endosome.
Much debate remains over the status of the secretory
pathway during mitosis. It is not clear, for example,
118
Update
TRENDS in Cell Biology
whether the Golgi fragments into vesicle clusters during
mitosis [33] or whether it is resorbed by the ER [34].
Cellularization of Drosophila embryos occurs during
interphase, rather than during the final stages of mitosis.
Interestingly, the embryonic Drosophila Golgi is fragmented into vesicles, similar to what has been observed during
mitosis in dividing sea urchin eggs [35]. While transport
vesicles are necessary for cell division in many systems, it
is difficult to draw conclusions about the nature of their
contribution in the context of the secretory pathway as the
organelles that comprise the secretory pathway are
themselves in an altered state during mitosis and cell
division. Future investigation into the intracellular source
of furrow-directed vesicles, the membrane composition at
the furrow and the interplay between membrane and the
cytoskeleton will greatly enhance our current understanding of cell division.
Acknowledgements
We are grateful to Brad Shuster for his contribution to the design of
Figure 2. Supported in part by NIH GM58231.
References
1 Shuster, C.B. and Burgess, D.R. (2002) Targeted new membrane
addition in the cleavage furrow is a late, separate event in cytokinesis.
Proc. Natl. Acad. Sci. U. S. A. 99, 3633 – 3638
2 Danilchik, M.V. et al. (2003) Furrow microtubules and localized
exocytosis in cleaving Xenopus laevis embryos. J. Cell Sci. 116,
273 – 283
3 Bluemink, J.G. and de Laat, S.W. (1973) New membrane formation
during cytokinesis in normal and cytochalasin B-treated eggs of
Xenopus laevis. I. Electron microscope observations. J. Cell Biol. 59,
89 – 108
4 Emoto, K. and Umeda, M. (2000) An essential role for a membrane lipid
in cytokinesis. Regulation of contractile ring disassembly by redistribution of phosphatidylethanolamine. J. Cell Biol. 149, 1215– 1224
5 Mazumdar, A. and Mazumdar, M. (2002) How one becomes many:
blastoderm cellularization in Drosophila melanogaster. Bioessays 24,
1012 – 1022
6 Finger, F.P. and White, J.G. (2002) Fusion and fission: membrane
trafficking in animal cytokinesis. Cell 108, 727– 730
7 Verma, D.P.S. and Gu, X. (1996) Vesicle dynamics during cell-plate
formation in plants. Trends Plant Sci. 1, 145– 149
8 Burgess, R.W. et al. (1997) The synaptic protein syntaxin1 is required
for cellularization of Drosophila embryos. J. Cell Biol. 138, 861– 875
9 Jantti, J. et al. (2002) Characterization of temperature-sensitive
mutations in the yeast syntaxin 1 homologues Sso1p and Sso2p, and
evidence of a distinct function for Sso1p in sporulation. J. Cell Sci. 115,
409 – 420
10 Conner, S.D. and Wessel, G.M. (1999) Syntaxin is required for cell
division. Mol. Biol. Cell 10, 2735– 2743
11 Muller, I. et al. (2003) Syntaxin specificity of cytokinesis in
Arabidopsis. Nat. Cell Biol. 5, 531 – 534
12 Jantsch-Plunger, V. and Glotzer, M. (1999) Depletion of syntaxins in
the early Caenorhabditis elegans embryo reveals a role for membrane
fusion events in cytokinesis. Curr. Biol. 9, 738 – 745
13 Low, S.H. et al. (2003) Syntaxin 2 and endobrevin are required for the
terminal step of cytokinesis in mammalian cells. Dev. Cell 4, 753– 759
www.sciencedirect.com
Vol.14 No.3 March 2004
14 Xu, H. et al. (2002) Membrane trafficking in cytokinesis. Semin. Cell
Dev. Biol. 13, 77 – 82
15 Skop, A.R. et al. (2001) Completion of cytokinesis in C. elegans requires
a brefeldin A-sensitive membrane accumulation at the cleavage furrow
apex. Curr. Biol. 11, 735 – 746
16 Sisson, J.C. et al. (2000) Lava lamp, a novel peripheral Golgi protein, is
required for Drosophila melanogaster cellularization. J. Cell Biol. 151,
905– 918
17 Lee, O.K. et al. (2003) Discs-large and Strabismus are functionally
linked to plasma membrane formation. Nat. Cell Biol. 5, 987 – 993
18 Bhat, M.A. et al. (1999) Discs lost, a novel multi-PDZ domain protein,
establishes and maintains epithelial polarity. Cell 96, 833 – 845
19 Bilder, D. (2001) PDZ proteins and polarity: functions from the fly.
Trends Genet. 17, 511 – 519
20 Feng, B. et al. (2002) Furrow-specific endocytosis during cytokinesis of
zebrafish blastomeres. Exp. Cell Res. 279, 14 – 20
21 Gerald, N.J. et al. (2001) Cytokinesis failure in clathrin-minus cells is
caused by cleavage furrow instability. Cell Motil. Cytoskeleton 48,
213– 223
22 Pelissier, A. et al. (2003) Trafficking through Rab11 endosomes is
required for cellularization during Drosophila embryogenesis. Curr.
Biol. 13, 1848 – 1857
23 van Dam, E.M. and Stoorvogel, W. (2002) Dynamin-dependent
transferrin receptor recycling by endosome-derived clathrin-coated
vesicles. Mol. Biol. Cell 13, 169 – 182
24 Ullrich, O. et al. (1996) Rab11 regulates recycling through the
pericentriolar recycling endosome. J. Cell Biol. 135, 913 – 924
25 Rothwell, W.F. et al. (1998) Nuclear-fallout, a Drosophila protein that
cycles from the cytoplasm to the centrosomes, regulates cortical
microfilament organization. Development 125, 1295– 1303
26 Krueger, E.W. et al. (2003) A dynamin-cortactin-Arp2/3 complex
mediates actin reorganization in growth factor-stimulated cells. Mol.
Biol. Cell 14, 1085– 1096
27 Thompson, H.M. et al. (2002) The large GTPase dynamin associates
with the spindle midzone and is required for cytokinesis. Curr. Biol. 12,
2111 – 2117
28 Riggs, B. et al. (2003) Actin cytoskeleton remodeling during early
Drosophila furrow formation requires recycling endosomal components Nuclear-fallout and Rab11. J. Cell Biol. 163, 143– 154
29 Hickson, G.R. et al. (2003) Arfophilins are dual Arf/Rab 11 binding
proteins that regulate recycling endosome distribution and are related
to Drosophila nuclear fallout. Mol. Biol. Cell 14, 2908 – 2920
30 D’Souza-Schorey, C. et al. (1998) ARF6 targets recycling vesicles to the
plasma membrane: insights from an ultrastructural investigation.
J. Cell Biol. 140, 603 – 616
31 Schafer, D.A. et al. (2000) Actin assembly at membranes controlled by
ARF6. Traffic 1, 892 – 903
32 Rothwell, W.F. et al. (1999) The Drosophila centrosomal protein Nuf is
required for recruiting Dah, a membrane associated protein, to
furrows in the early embryo. J. Cell Sci. 112, 2885 – 2893
33 Jokitalo, E. et al. (2001) Golgi clusters and vesicles mediate mitotic
inheritance independently of the endoplasmic reticulum. J. Cell Biol.
154, 317 – 330
34 Zaal, K.J. et al. (1999) Golgi membranes are absorbed into and
reemerge from the ER during mitosis. Cell 99, 589 – 601
35 Terasaki, M. (2000) Dynamics of the endoplasmic reticulum and Golgi
apparatus during early sea urchin development. Mol. Biol. Cell 11,
897– 914
0962-8924/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tcb.2004.01.006