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Department of Molecular and Cellular Biology and Biochemistry, Brown University, Providence, Rhode Island 02912, USA
1Correspondence: Department of Molecular and Cell Biology and Biochemistry, Brown University, 69 Brown St., Providence, RI 02912, USA. E-mail: rhet{at}brown.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: GTP binding protein • vesicle dynamics • membrane fusion • cytokinesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, eukaryotic cells, consisting of
a variety of membrane-bound organelles, must partition each organelle
type into daughter cells for viability. In particular, the nuclear
envelope breaks down shortly after chromosome condensation during
mitosis and, like the membranes of the Golgi and endoplasmic reticulum
(ER), are thought to fragment into small vesicles (2
, 3)
.
Once these organelle membranes have been partitioned between daughter
cells, the nuclear envelope, ER, and Golgi must be reconstituted
through a series of homotypic membrane fusion events to recreate
functional organelles. What regulates the membrane fusion events
required for cell division?
Recently we provided evidence that sea urchin syntaxin (t-SNARE) is
essential for cell division progression, presumably by regulating the
fusion of cortical vesicles seen in early blastomeres (4)
.
This result is consistent with observations that syntaxin family
members are required for cell division in Arabidopsis
(5)
and for cellularization in Drosophila
(6)
. However, observations that recombinant v- and t-SNARE
proteins reconstituted into separate lipid bilayer vesicles
spontaneously assemble into SNARE complexes and fuse (7)
suggest that these integral membrane proteins function as the core
machinery driving membrane fusion. Whereas SNAREs appear to provide the
motive force required for membrane fusion and possibly membrane fusion
specificity, these proteins are unlikely to regulate the timing of the
fusion event. The timing of vesicle fusion may instead be regulated by
proteins sensitive to calcium fluxes (e.g., synaptotagmin or rabphilin)
and/or by rabs, small GTP binding proteins of the ras
superfamily. Rab proteins have been implicated in regulating membrane
fusion events in almost every step of the secretory pathway
(8)
. They act as molecular switches, cycling between
active and inactive GDP-bound states and are targeted to, and appear to
function in, specific membrane compartments. For example, rab1 and rab2
family members appear to function in vesicle traffic from the
endoplasmic reticulum to the Golgi apparatus (9)
; rab6 has
been found to localize to intra-Golgi membranes (10)
and
rab5 appears to control early endosome fusions in vitro
(11)
, whereas rab3 family members have been shown
essential for the final steps of regulated exocytosis (12
, 13)
. The precise function of rabs is ambiguous; they are modeled
to control either vesicle docking by controlling SNARE complex
formation (14)
, modulating SNARE complex stability
(15)
, or controlling the timing of the fusion event
(16)
.
We previously reported that the sea urchin rab3 homologue associates
with cortical granules in the unfertilized egg (17)
and
functions in their exocytosis at a step after vesicle docking using
rab3 effector domain peptide microinjections (18)
. Here we
found rab3 on vesicles enriched at the cortex of dividing cells and
thus hypothesized that rab3 could be functioning to regulate membrane
fusion events necessary for cell division. To test this hypothesis, we
injected affinity-purified antibodies to the rab3 effector domain and
effector domain peptides to either inactivate or compete for rab3
function. We found that microinjection of either reagent has inhibitory
effects specifically on cell division, whereas heat-inactivated
antibodies or scrambled control effector domain peptides have no
effect. These data suggest that rab3 might function to regulate
membrane fusion events that are required for cell division.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Effector domain peptides were custom synthesized by
W. M. Keck Foundation Biotechnology Resource Center (New Haven,
Conn.) and Quality Controlled Biochemicals (Hopkinton, Mass.):
VSTVGISFKVKTVFRQ; the scrambled effector domain, VFVSVTKQVSGRFTIK (Keck
Foundation, New Haven, Conn.); and hypervariable domain,
CDKMSETIDTDQTLRPSTT (Quality Controlled Biochemicals), all resuspended
in deionized water at 4.95 mM.
Injections
Eggs and/or embryos were placed in a Kiehart chamber
(20)
in artificial sea water (ASW; ref 19
)
and injected with deionized water or peptides resuspended in water at
the various concentrations indicated or affinity-purified polyclonal
antibodies against rab3 in phosphate-buffered saline (PBS). An oil
droplet of dimethylpolysiloxane (Sigma, St. Louis, Mo.) was coinjected
into cells as a marker. Injection volumes never exceeded 5% of the
cell volume.
Antibody generation and purification for immunolocalization in
vivo
Antibody generation and purification were performed as described
previously (18)
. To generate antibodies to the effector
region of rab3, a partial rab3 protein was engineered using the
nucleotide sequence representing amino acids 1–153 [DNKW... QLGL]
of the rab3 cDNA clone (17)
ligated into a pGEX-3C vector
for fusion with glutathione S-transferase (GST) and transformed into
BL21(DE3) cells for overexpression with 0.1 mM IPTG induction as
described below. The resultant overexpressed protein was isolated from
cell lysates by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Protein was then electroeluted and dialyzed
extensively against deionized water and lyophilized. The rab3 fusion
protein immunogen was resuspended in Freund’s adjuvant, which was
injected subcutaneously into New Zealand White rabbits every 3 wk for 3
months. One week after the last boost, plasma was collected from the
central ear artery (21)
. Resultant immunoreactivity to GST
showed no detectable cross-reactivity in eggs or zygotes when used in
immunoblot or immunolocalization assays.
To affinity purify antibodies, protein A-purified rab3 effector domain
sera, isolated as described (21)
and previously conjugated
to the Cy3 fluorochrome using the Fluorolink CyDye labeling kit
(Amersham Life Science, Arlington Heights, Ill.), was incubated with
nitrocellulose blotted with affinity-purified rab3-GST fusion protein
(see below), the exact same protein used for antibody generation, in
PBS for 30 min. The blots were then washed with PBS and the Cy3-labeled
antibodies were eluted from the nitrocellulose with 100 mM glycine (pH
2.5), dialyzed extensively against PBS, and concentrated to 2 mg/ml
using Ultrafree-4 centrifugal filters with a 10 kDa cutoff (Millipore,
Bedford, Mass.). Affinity-purified antibodies labeled with Cy3 were
tested by immunolocalization in thick sections of eggs (see below) and
shown to react specifically with rab3 sequences (rab3 expressed protein
and effector peptides but not scrambled peptides or nonrelevant
proteins).
Rab3-GST fusion protein was isolated as follows. Rab3-GST fusion protein-expressing BL21(DE3) cells were induced at 23°C with 0.1 mM IPTG for 3 h. Cells were then pelleted by centrifugation at 4000 rpm for 10 min, resuspended in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4), lysed with high pressure using a French press, and solubilized with 1% Triton X-100 for 30 min. Cellular debris was then pelleted at 10,000 g at 4°C for 20 min. The resulting supernatant was passed over a glutathione-agarose column (Sigma); the column was washed with 10 column volumes of PBS. Rab3-GST fusion protein was specifically eluted with PBS containing 10 mM reduced glutathione (Sigma) and the purity of column elutant rab3-GST protein was verified by SDS-PAGE and immunoblot analysis using either rab3 effector domain or anti-GST (Sigma) antibodies. Affinity-purified rab3-GST protein was blotted to nitrocellulose in PBS and blocked with preimmune sera for 10 min. The blot was then washed with PBS and used for antibody isolation.
Immunolocalization assays in situ
Immunofluorescence localization was performed in whole mounts
and on embryo sections that were fixed and processed as described
previously (22)
. The polyclonal antibodies against the
rab3 carboxyl-terminal hypervariable domain (17)
were
diluted 1:50 (~20 µg/ml) and the polyclonal antibodies against the
rab3 effector domain were diluted 1:200 (~5 µg/ml). The secondary
antibodies, Cy3-conjugated affinity-purified goat anti-rabbit IgG
(Kirkegaard & Perry Labs, Gaithersburg, Md.), were diluted 1:20–100
(~1–5 µg/ml). Signals were recorded by epifluorescence with a
Zeiss Axioplan or by confocal microscopy with a Zeiss LSM 410.
Brefeldin A treatment
Eggs were fertilized with sperm in ASW and after 10 min were
transferred to ASW containing brefeldin A (Calbiochem, La Jolla,
Calif.) at the indicated concentrations (stock solution was 4 mg/ml in
methanol) or ASW containing identical concentrations of methanol as
that of the brefeldin A-treated embryos as a control. Methanol in the
ASW of the experimental and control samples was given 30 min at room
temperature to evaporate before embryo transfer.
Membrane topology and endocytosis
DiOC6 (3)
(Molecular Probes,
Eugene, Oreg.) was resuspended in methanol at 1 mg/ml, then transferred
to Hollywood safflower oil (Big Daddy Wesley’s, Beaufort, N.C.) by
mixing 500 µl of the methanol/DiOC6
(3)
solution with the 500 µl safflower oil.
DiOC6 (3)
resuspended in safflower
oil was then used for microinjection into cells for membrane labeling.
The volume of oil containing DiOC6
(3)
did not exceed 5% of the cell volume. FM1–43
(Molecular Probes) was resuspended in methanol at 1 mg/ml, then diluted
in ASW to give a working concentration of 1 µM. To evaluate
endocytosis, experimentally manipulated embryos were transferred to the
FM1–43 in ASW and visualized after a 15–45 min incubation at room
temperature using confocal microscopy with a Zeiss LSM 410.
Endocytosis quantitation
To quantitate FM1–43 endocytosis, 5–10 confocal sections of
each embryo were observed with the Zeiss LSM 410 laser scanning
microscope and analyzed using Adobe PhotoShop (Adobe Systems Inc.,
Mountain View, Calif.). For each confocal section, the average FM1–43
brightness and area of each cell were determined by using the histogram
function. Vesicles were counted in each section, and this number was
then divided by the average brightness and area to obtain a
standardized value. Standardized values from 5–10 confocal sections
were averaged to obtain the FM1–43 endocytosis value for each cell.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Rab3 homologs are
modeled to function in the final steps of the secretory pathway; since
a single rab3 homologue has been identified in the sea urchin that is
present throughout embryogenesis (S. D. Conner and G. M.
Wessel, unpublished results), we asked where rab3 localizes in
vivo in the developing embryo. Microinjection of
fluorochrome-labeled, Fab fragment antibodies (~200 nM)
raised against the effector domain reveals rab3 immunolabeling
concentrated on vesicles that are enriched at the cortex of one- and
two-cell embryos (Fig. 1B, E
). The injection of increased levels of labeled antibody
(~900 nM) also show a reticular pattern (Fig. 1G
,
brightfield; H, immunolocalization) whereas injection of nonrelevant
fluorochrome-tagged antibodies at the same concentration show no
detectable localization within the cell (data not shown). It is
possible that rab3 effector domain antibodies could be binding to other
rab family members in the egg. Thus, we performed immunolocalizations
in fixed cells with antibodies against a region distinct between rab
family members, the hypervariable domain, and found that labeling
patterns are consistent with those observed in vivo (Fig. 1I
, the middle embryo has not divided whereas the other two
have completed cell division). Since we saw consistent labeling
patterns between antibodies raised against two different regions of
rab3 and found no cross-reactivity with antibodies against mammalian
rab3 (a highly conserved family member) by either immunoblot or
immunolocalization (data not shown), we conclude that the rab3 labeling
patterns observed using the effector domain antibodies are specific for
rab3. The reticular pattern may reflect rab3 vesicle origins, whereas
the distinct vesicles at the cortex may be involved in transport of
cell surface or extracellular proteins. Do rab3-positive vesicles play
a role in cell division?
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Rab3 antibodies block cell division
To test the function of rab3 in cell division, we used a
microinjection approach using rab3 affinity-purified monovalent Fab
antibody fragments against the rab3 effector domain. This domain was
selected because rab function is thought to be mediated through this
domain by interaction with other proteins, possibly rabphilin
(23)
or Mss4 (24)
. We found that injection of
~500 nM rab3 antibody Fab fragments into a single cell of a two-cell
embryo blocks cell division (Fig. 2D
, E
, F
; progression of an embryo is shown in each row). Whereas
cells sometimes progress through another round of cell division after
antibody injection before being inhibited, once inhibition occurs both
cytokinesis and karyokinesis are halted (Fig. 2F
). Injection
of ~700 nM heat inactivated affinity-purified Fab fragments, however,
has no effect on cell division (Fig. 2G
, H
, I
), nor are any
effects observed when single blastomeres are injected with nonrelevant
Fab fragment antibodies (anti-rabbit IgG molecules at 580 nM, Fig. 2A
, B
, C
). These observations suggest that it is the specific
inactivation of rab3 that results in a cessation of cell division and
not the injection or protein mass.
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Rab3 effector peptides inhibit cytokinesis in a concentration
dependent manner
As an alternative approach to test rab3 function, rab3 effector
domain peptides made to compete for rab3 function were used to test the
role of rab3 during cell division. In other systems, the effector
domain directly interacts with a downstream protein, rabphilin-3A
(23)
, and has been successfully used to block rab3
function in cortical granule exocytosis in the sea urchin egg
(18)
, although the identity of the sea urchin rab3
effector proteins is currently unknown. After the first cell division,
we injected a single cell of the two-cell stage embryo with 19 µM
effector peptide and found that 36% of embryos showed significant
delays in cell division (Table 1
). However, these embryos recover and develop normally to the larval
stage (Fig. 3F
). Note that if a cell is injected with peptide at the
beginning of cytokinesis, the actin contractile ring continues to
contract; however, cytokinesis is aborted until the next round of
division (Fig. 3G
, H
, I
). When exposed to 48 µM effector
peptide (one-fifth the concentration required to block vesicle fusion
in the sea urchin egg; ref 18
), embryos begin to show
greater delays in cell division (Fig. 3J
, K
, L
) with greater
frequency (70%, Table 1
). When cells are injected with 136 µM
peptide, 67% of the embryos stop dividing. In some of these cases
karyokinesis is completed, resulting in a cell with two nuclei (Fig. 3O
, arrowheads mark nuclei); however, additional rounds of
karyokinesis are not observed. Peptide concentrations found to inhibit
cell division here are less than those previously reported to block the
exocytosis of cortical granules in the egg at fertilization (218 µM;
ref 18
) and similar to that used to perturb rab function
in mast cells (125 µM; ref 25
). To eliminate the
possibility of artifacts due to peptide preparation, effector domain
peptides from two independent sources (see Materials and Methods) were
tested and both gave similar results. The numbers in Table 1
represent
the cumulative totals from the use of both synthesized peptides.
Injection of rab3 effector peptides at 136 µM whose sequence has been
scrambled has no effect on cell division or development (Fig. 3A
, B
, C
), arguing that neither the injection, the peptide
mass, nor the peptide charge affects the ability of the embryo to
undergo cell division.
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Does effector peptide injection disrupt the distribution or number of rab3-positive vesicles? Currently we are unable to follow single rab3-positive vesicles or visualize these vesicles independent of rab3. But when we examined immunolocalization patterns of rab3-positive vesicles in effector peptide-injected cells and uninjected controls, we found significant variability. Some effector peptide-injected cells possess a greater number of rab3-positive vesicles than controls whereas others have fewer (data not shown). We do not know whether this reflects a slightly different stage in the cell cycle, variability in cell size vs. vesicle number, or rab3 accessibility. Moreover, interpretation of these results is especially difficult since it is unclear whether these rab3-positive vesicles are a homogeneous population or whether rab3 associates with a variety of vesicle types of which only a subset are essential for cell division. Thus, it will be important to identify vesicle content proteins so as to distinguish between these possibilities and better define the role of rab3 vesicles in cell division.
Rab3 immunolocalizations, both in vivo and in fixed
sections, indicate a reticular pattern in addition to vesicles at the
cell cortex. Thus, we hypothesized that cessation in cell division that
results from rab3 effector peptide and/or antibody injections might
result from a general paralysis of the secretory pathway. However,
embryos are unaffected in the first few cell divisions when they are
treated with brefeldin A (Fig. 4
; 4
), a compound that specifically disrupts the secretory
pathway by preventing anterograde vesicle transport from the
endoplasmic reticulum to the Golgi apparatus while leaving the
retrograde pathway unaffected (26)
. Newly fertilized eggs
divide as normal when treated with 10 µM brefeldin A (Fig. 4A, B
), a concentration known to disassemble the Golgi. No effects on
cell division are observed even at concentrations of 100 µM brefeldin
A (data not shown), 10-fold greater than that required to block the
secretion of the hatching enzyme (Fig. 4C
). During
embryogenesis, sea urchins are surrounded by the fertilization envelope
until the blastula stage, when the embryo secretes the hatching enzyme
that digests the fertilization envelope and releases the ciliated
embryo (27)
). These results strongly argue that antibody-
and/or effector peptide-induced cell division blocks are not the result
of inhibiting the general secretory pathway. Instead, we conclude that
the cell division defects we observed derive from effects on the
post-Golgi, rab3-positive vesicles that are enriched at the cell
cortex.
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Since rab3 associates with endocytic vesicles after fertilization
(18)
, we suspected that the effector peptide might be
blocking the endocytic pathway and indirectly inhibit cells from
dividing. To test this hypothesis, we asked whether effector
peptide-injected cells were still capable of endocytosing a
membrane-impermeant lipophilic dye (FM1–43) that fluoresces when
associated with membranes previously shown useful in studying
endocytosis in the sea urchin embryo (28)
. Single cells of
a two-cell embryo were injected with 136 µM effector peptide and
allowed to develop for ~1 h (Fig. 5A
). Injected embryos were then transferred to artificial sea
water containing FM1–43 to assay for endocytosis. We found that
peptide-injected cells accumulate endocytic vesicles in their cytoplasm
to levels comparable to uninjected cells (Fig. 5B, C
). This
indicates that the endocytic pathway is active in injected blastomeres
despite a cessation of cell division.
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In an embryo dividing every 45 min-1 h, we suspected that if rab3
functions in general vesicle fusion events, peptide injections, which
compete for rab3 function, could lead to major changes in membrane
topology of the cell and hence secondarily block the ability of a cell
to divide. To test this possibility, we used the lipophilic dye,
DiOC6 (3)
, which labels any
contacting membrane (29)
. Fertilized eggs were injected
with DiOC6 (3)
and allowed to
develop as normal. Then a single cell of a two-cell embryo was injected
with 136 µM effector peptide, the highest concentration used in this
study, to ask if changes in gross membrane morphology or distribution
could be observed. Although effector peptide-injected cells are
inhibited in their cell division, we found no detectable difference in
membrane labeling patterns as compared to uninjected cells (Fig. 6D
, E
, F
). These observations suggest that rab3 effector
peptide injections do not cause global membrane trafficking problems,
inhibit general metabolic processes, or detectably alter membrane
distribution or topology by preventing or causing spurious membrane
fusion events. Instead, we believe that rab3-positive vesicles are not
only important in the cargo they may carry for exocytosis in this or
other cell types, but also for the regulation of membrane dynamics
critical for cell division.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and here we found that disruption of rab3 with
antibodies or competition for its function with effector peptides
blocks cell division. Recent observations suggest that syntaxin family
members are required for cytokinesis in Arabidopsis
(5)
and cellularization in Drosophila at the
syncytial blastoderm stage (6)
. Moreover, we recently
found rab3 colocalization with syntaxin on vesicles enriched at the
cortex of dividing cells and that syntaxin inactivation either with
Botulinum C1 or affinity-purified antibodies against
syntaxin also block cell division (4)
. It is possible that
by disrupting rab3 function, SNARE complex formation and/or stability
is inhibited, thus blocking membrane fusion events in the same manner
as blocking syntaxin function. This is consistent with the
demonstration that Ypt1p, a yeast rab, transiently interacts with a
t-SNARE to displace a negative regulator, Sly1p, allowing the
subsequent formation of the SNARE complex (30)
. These
results, in addition to those presented here, suggest that SNARE and
rab3 protein family members play a role in mediating membrane dynamics
important for cell division.
Rab3 family members function in the final steps of the secretory
pathway (13)
; in the sea urchin egg, the single rab3
homologue associates with and appears to function in the exocytosis of
specialized secretory vesicles, cortical granules whose contents give
rise to the fertilization envelope at fertilization (18)
.
However, rab3 is also present throughout the development of the sea
urchin embryo, and here we show that it associates with vesicles
enriched at the cortex of dividing cells. Although rabs are generally
ubiquitous in their expression in mammalian cells, they are usually
found only within discrete and limited secretory compartments. Yet a
rab6 family member associates with the entire Golgi stack in liver
cells (31)
and in post-Golgi vesicles in retinal cells
(32)
and Torpedo marmorata electrocytes
(10)
. This distribution pattern has led investigators to
speculate that rab6 may function in several steps of the secretory
pathway (33)
. Similarly, sea urchin rab3 could also be
functioning in various steps of the secretory pathway, and by
disrupting membrane flow through the various secretory compartments,
cell division is halted. We do not favor this idea, however, since
treatment of cleavage stage embryos with brefeldin A, a drug that
blocks the secretory pathway by preventing anterograde vesicular
membrane flow from the endoplasmic reticulum to the Golgi, has no
effect on the ability of the embryo to undergo the first few cell
divisions (4)
.
Instead, rab3 may regulate the fusion of maternally derived, post-Golgi
cortical vesicles with the plasma membrane, and these vesicles may
possess proteins that are essential for cell division. What do these
vesicles contain and why are they important? Even though no vesicle
contents have yet been identified, we speculate that they may contain
proteins that need to be specifically targeted within the cell to
activate cell division machinery. It is possible that the aggregation
of actin into a contractile ring or the localization of motor proteins
requires targeting factors provided by these vesicles. Also, since
cells must increase their membrane surface area roughly 25% for cell
division (1)
, a simple explanation could be that vesicles
enriched with rab3 are important for contributing essential membrane
surface area.
Alternatively, rab3 could control homotypic membrane fusion events like
those required for the reconstitution of the Golgi, endoplasmic
reticulum, or nuclear envelope after their breakdown during the cell
cycle. This latter idea agrees with recent data that have implicated a
rab GTPase in regulating homotypic fusion of mammalian ER membranes
in vitro (34)
. Balch and colleagues
(34)
found that microsome membrane fusion was specifically
inhibited by treatment with a guanine nucleotide dissociation
inhibitor, a protein that extracts GDP-bound rabs from membranes. Thus,
it is possible that the observed block in cell division results from
the cell stalling at a cell cycle checkpoint that monitors the status
of intracellular membranes, ensuring that the cell does not progress
through the cell cycle before proper organelle reconstitution.
What are the rab3 effectors in this rapidly dividing sea urchin embryo?
Several proteins have been found in other cell types to directly
interact with rabs in their GTP-bound state and could potentially
stabilize active rabs: 1) rabphilin, a protein containing a conserved
protein kinase C calcium binding motif (35)
, 2) the
cytosolic protein, rabaptin-5, found essential for early endosome
membrane trafficking (36)
, and 3) RIM, a zinc finger
protein, thought to regulate synaptic vesicle fusion in a
rab3-dependent fashion (37)
. In addition, Mss4, a putative
guanine nucleotide exchange factor, stimulates GDP release from and the
association of GTP with various rab family members (24
, 38)
, thereby cycling the protein from an inactive to active
form. Most recently, a molecular motor, rabkinesin-6, has been
identified that specifically interacts with a GTP-bound rab6 family
member (39)
, suggesting that rabs may use a
microtubule-based cytoskeleton to direct vesicle traffic. It is
possible that by perturbing rab3 interaction with one of these proteins
and/or close family member homologs by antibody or effector peptide
injection, rab3 could be locked in an (in)active state or misdirect
vesicles and lead to membrane fusion abnormalities and cessation of the
cell division.
Whereas rab and SNARE proteins have been clearly demonstrated to
function in vesicular trafficking in a variety of systems (33
, 40)
, we provide evidence here that rab3 is required for cell
division. However, the next important question is, which membrane
fusion events are important for cell division? Though membrane fusion
events leading to the reformation of fragmented membrane-bound
organelles is of obvious import, it is possible that key membrane
fusion events are required that target membrane proteins or vesicle
contents to cell locations that are spatially or temporally required
for cell division.
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ACKNOWLEDGMENTS |
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Received for publication September 2, 1999.
Revision received November 5, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  C. B. Shuster and D. R. Burgess Targeted new membrane addition in the cleavage furrow is a late, separate event in cytokinesis PNAS, March 19, 2002; 99(6): 3633 - 3638. [Abstract] [Full Text] [PDF]
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