1 Department of Systems Biology, Harvard Medical School, Boston MA 02115,
USA
2 Five Prime Therapeutics, South San Francisco CA 94080, USA
3 Howard Hughes Medical Institute, Developmental Genetics Program, Skirball
Institute and Department of Cell Biology, New York University School of
Medicine, New York, NY 10016, USA
4 Department of Molecular, Cell and Developmental Biology, Sinsheimer
Laboratory, University of Santa Cruz, Santa Cruz, CA 95064, USA
Author for correspondence (e-mail:
cfield{at}hms.harvard.edu)
Accepted 1 April 2005
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SUMMARY |
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Key words: Cellularization, cytokinesis, Anillin, septin, PH domain, Drosophila
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Introduction |
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Cellularization is more complex than conventional cytokinesis. It requires
an estimated 25-fold increase in surface area during a single cell cycle
(Lecuit and Wieschaus, 2000),
and is thus useful for exploring the coupling between actomyosin contraction
and insertion of new plasma membrane. It also requires ingression in two
different planes. Initially, furrows ingress perpendicularly to the embryo
surface. The tips of cellularization furrows are called furrow canals, and
they interconnect as an almost hexagonal network surrounding each nucleus.
Ingression of the plasma membrane occurs in at least two stages that differ in
rate (initially slow, then fast) and mechanism, with certain mutations
selectively affecting one stage (Schejter
and Wieschaus, 1993
). Later, once the furrow canals pass the
nuclei, ingression also occurs parallel to the embryo surface. The furrow
canals broaden and become almost triangular-shaped in cross-section, and the
network transforms into an almost hexagonal array of contractile rings. These
constrict around the base of each nucleus, individualizing the cells.
Constriction is incomplete. The newly formed cells remain connected to the
yolk mass by a thin neck of cytoplasm or `stalk' as the embryo initiates
gastrulation movements (Rickoll,
1976
).
Given their rich contractile biology, Drosophila embryos have been
useful for investigating the mechanism of furrowing. As expected, embryonic
furrows contain the contractile proteins F-actin
(Warn and Robert-Nicoud, 1990)
and cytoplasmic Myosin II (Young et al.,
1991
; Royou et al.,
2004
). They also contain septins and Anillin, conserved furrow
components whose function is less clear. Septins were discovered as CDC
mutants in budding yeast and were implicated in animal cytokinesis by analysis
of mutations in the Drosophila septin Peanut (reviewed by
Field and Kellogg, 1999
;
Trimble, 1999
;
Mitchison and Field, 2002
).
Peanut was later shown to be involved in cellularization
(Adam et al., 2000
).
Biochemical investigation in Drosophila embryos showed that septins
bind GTP and assemble into heteromeric complexes and filaments
(Field et al., 1996
). Their
molecular function is unknown, although septins have been implicated in
vesicle trafficking (Beites et al.,
1999
).
Anillin was originally isolated from Drosophila embryos by
affinity chromatography on F-actin (Field
and Alberts, 1995) and homologs were later found in vertebrates
(Oegema et al., 2000
;
Straight et al., 2005
) and
C. elegans (see Maddox et al.,
2005
). Anillin is required for cytokinesis in Drosophila
and vertebrate tissue culture cells
(Oegema et al., 2000
;
Somma et al., 2002
;
Kiger et al., 2003
;
Rogers et al., 2003
;
Echard et al., 2004
;
Straight et al., 2005
), but
its function during furrowing remains unclear. Mid1 and Mid2, two proteins
with more limited homology to Anillin, play central roles in cytokinesis in
S. pombe (Berlin et al.,
2003
; Paoletti and Chang,
2000
; Tasto et al.,
2003
).
Anillin is a multi-domain protein that physically interacts with several
other cleavage furrow components in vitro. Its N terminus contains a region
that binds and bundles F-actin (Field and
Alberts, 1995; Oegema et al.,
2000
), and a second region that binds phosphorylated cytoplasmic
Myosin II (Straight et al.,
2005
). Its C-terminus comprises a predicted PH domain, an
100
amino acid module often implicated in binding to membranes via inositol lipids
(Lemmon, 2004
;
Lemmon et al., 2002
). The
C-terminal region of vertebrate Anillin was implicated in septin binding by
expression of truncated protein and biochemical assays
(Oegema et al., 2000
;
Kinoshita et al., 2002
), but
the physiological relevance of that proposed interaction was not tested. To
explore the function of Anillin, in particular its potential role in coupling
cytoskeletal and membrane dynamics, we analyzed the effects of a series of
mutations in Anillin on the diverse, cell cycle regulated furrows in the early
Drosophila embryo.
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Materials and methods |
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In rescue experiments, full-length anillin cDNA was inserted into the Germ9
P-element transformation vector (Serano et
al., 1994) and injected into 1-hour-old Drosophila
embryos.
Eggs were collected from homozygous anillinRV/RV females carrying the wild-type transgene, and the percentage that hatched was scored. For complementation tests (see Table S1 in the supplementary material), scraps/anillin alleles were maintained over a balancer chromosome containing Cy. In all tests, the ratio of homozygous scraps adults to the total number of viable adults (anillin homozygotes plus anillin/Cy heterozygotes) was scored. If the anillin mutation does not affect adult viability, the expected ratio would be 1/3 (33%) (see Table S1, column 2 in the supplementary material). When examining maternal/zygotic combinations, anillinmaternal/Cy flies were crossed with anillinzygotic/Cy and adult viability scored as above. The strong anillin maternal alleles could not rescue the lesions caused by the Heitzler zygotic alleles or deficiencies (Schupach and Wieschaus, 1989), indicating that the anillin gene probably has the same function both maternally and zygotically. For the maternal genes, combinations of alleles were mated, adult survival scored and then egg hatchability was examined.
Sequencing of scraps alleles
Sequencing was carried out at the Biopolymers Facility at Harvard Medical
School (HMS)
(http://genome.med.harvard.edu)
and the HHMI sequencing facility at Rockefeller University. At HMS, the entire
anillin genomic region for alleles HP and RS was sequenced. In addition, the
3' end (corresponding to the C-terminal 266 amino acids) was sequenced
in the genotypes PQ/RS, HP/Cy, RS/Cy, RV/Cy and RV/RV. To generate sequencing
templates, a series of overlapping PCR products of 1-1.5 kb in length were
produced using Turbo Pfu polymerase. At the HHMI facility, sequencing
templates encompassing the full-length gene were generated by PCR of the
genomic region of anillinB26-35,
anillinC82-45 and
anillinPE/anillin8 using a Long-Range
High Fidelity PCR kit (Roche). In addition to the wild-type anillin
sequence in FlyBase, sequences were compared with a full-length
anillin genomic sequence from the stock P3427, generated during the
cloning of the blownfuse gene
(Doberstein et al., 1997).
Immunofluorescence
Three different fixations were used for embryos. The fixative for F-actin
was 18.5% formaldehyde in PBS/heptane (1:1). Embryos were gently swirled for
20 minutes and then hand devitellinized with a tungsten needle. For Peanut,
the fixative was cold methanol/heptane (1:1) for 30 seconds, followed by
methanol popping. For Myosin II and Neurotactin a combination of heat
treatment and methanol fixation was performed
(Peifer et al., 1994).
Imaginal discs were dissected from 3rd instar larvae into PBS and immediately
fixed in 10% formaldehyde in PBS with 5 mM EDTA, swirling slowly for 20
minutes. Topro Dye, T-3605 (Molecular Probes) was used to stain DNA in
imaginal discs. Anti-Anillin (Field and
Alberts, 1995
), anti-Peanut
(Field et al., 1996
) and
anti-Myosin II (Foe et al.,
2000
) were all used at 1 µg/ml. Anti-Neurotactin (Developmental
Studies Hybridoma Bank, University of Iowa) was used at 1/200. F-actin was
stained with Rhodamine-phalloidin (Sigma).
Imaging
For immunofluorescence, samples were imaged on a Nikon TE2000 inverted
microscope with a PerkinElmer Spinning Disk confocal (Nikon Imaging Facility
at Harvard Medical School). For DIC imaging of cellularization, the chorion
was removed by hand, embryos covered with Halocarbon oil, Series 700
(Halocarbon Products, Hackensack, NJ) and filmed (the center of the dorsal
embryo surface) at 22°C using a Nikon TE3000 inverted microscope with a 60
x water immersion lens. Kymographs were generated using Metamorph
software (Universal Imaging). For each genotype, four to eight embryos were
filmed and ingression rates measured. Of these, two or three embryos/genotype
were kymographed.
Western blots
Blots were carried out using standard conditions. Zero- to 2-hour-old
embryos were homogenized in 2 x PAGE sample buffer, boiled for 2 minutes
and then diluted 1:1 with 8 M urea. Actin is probed with a monoclonal antibody
from ICN.
Transmission electron microscopy
Staged embryos were dechorinated for 1.5-2.0 minutes in 50% bleach, fixed,
thin sectioned (65-70 nm sections) and stained with uranyl acetate and
lead citrate using a previously described method
(Rickoll, 1976
). Fresh
acrolein was used in each fixation and each fixation included one or more
wild-type embryos as controls. Sections were examined on a JOEL 12000. For the
majority of embryos, thick sections (
1 µm) were also generated and
stained for light microscopy with 1% Toluidine Blue. These were examined to
determine developmental stage and confirm that the phenotypes observed were
representative.
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Results |
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To confirm that Anillin is the product of the scraps gene, we
performed rescue experiments by injecting anillin cDNA. All the embryos from
scrapsRV/RV mothers that received the transgene were
rescued to hatching, and 90% of these developed into fertile adults (data
not shown). We conclude that Anillin is essential for embryonic viability and
is the product of the scraps gene. We propose renaming the gene
anillin and maintaining the same allelic superscripts, and will
follow this convention for the remainder of the manuscript.
|
To identify the location of the genetic lesions, the entire genomic region containing anillin was sequenced in five maternal alleles. Two additional maternal alleles were sequenced at their C terminus only (see Table S2 in the supplementary material). Mutations were found in anillin exons in every case. The three strong maternal alleles contained mis-sense mutations resulting in changes in conserved amino acids at the N-terminal region of the PH domain (Fig. 1B,C). An additional amino acid change (V1055S) was present at the junction between the PH domain and the rest of the protein in all five of the Schupach/Wieschaus alleles examined (both weak and strong). The possible contribution to the phenotype of this additional mutation was not separately evaluated. Western blotting revealed that the quantity of Anillin protein and its migration on SDS-PAGE was similar in extracts of wild type and all of the maternal alleles tested. (Fig. 1D). We conclude that the amino acid substitutions compromise Anillin function, but not its expression or stability.
|
When cellularization begins in nuclear cycle 14, F-actin is enriched in
cortical `caps' that form above each of the nuclei, while Anillin localizes to
the region between and around the base of the F-actin caps, where furrow
canals will subsequently assemble. Anillin in `intercap' regions forms a
hexagonal network in a plane parallel to the embryo surface
(Field and Alberts, 1995). In
all anillin-derived embryos examined, F-actin and Anillin localized
correctly to their respective cortical domains at this early stage (not
shown).
The first significant defects observed were in cellularization. In wild-type embryos, early furrow canals are enriched in Anillin and F-actin (Fig. 2A, arrowheads). In late cellularization/gastrulation, the contractile rings at the base of the newly formed cells are strongly enriched in Anillin which is colocalized with F-actin (Fig. 2C,c,c'). The contractile rings sit on top of membranous stalks connecting the newly formed cell to the yolk mass (see Fig. S1 in the supplementary material). In embryos from anillinPQ/RS mothers, early furrow canals lacked the characteristic `tear-drop' shape, and the level of Anillin was reduced (Fig. 2B, arrowhead). As these embryos progressed through cellularization, Anillin staining at the cellularization front was further reduced, and the contractile rings failed to form. When gastrulation initiated, little Anillin remained at the cellularization front (Fig. 2D,d) and F-actin staining revealed a broken, hexagonal mesh in place of the normal lattice of rings (Fig. 2D,d'). In summary, in anillinPQ/RS-derived embryos there is a progressive loss of Anillin from ingressing furrow canals and the transition from hexagonal network into contractile rings fails. The newly formed cells are open at their bases.
Myosin II localization is disrupted at the cellularization front and cortical nuclei are disorganized
We next probed the effect of anillin maternal effect mutations on
targeting of Myosin II during cellularization. Early, during slow phase,
Myosin II, like Anillin, is strongly enriched in wild-type furrow canals;
however, the two signals are not perfectly co-incident and our
anillin alleles did not exhibit much effect on Myosin II localization
(not shown). Later, when contractile rings form (fast phase), Myosin II and
Anillin precisely colocalize in the contracting rings
(Fig. 3a,a',a''). In
anillinPQ/RS-derived embryos, where contractile rings fail
to form, the Myosin II localization was reduced at the cellularization front
(Fig. 3b,b',b''). Myosin II colocalized with mutant Anillin in bar-shaped structures within the
disorganized F-actin network (Fig.
3b,b',b''). A similar disruption of Myosin II
localization was previously observed in scraps/anillin mutants
(Thomas and Wieschaus,
2004).
|
Anillin is required for Peanut localization at the cellularization front
Septin localization was more strongly perturbed by anillin
mutations. We used antibodies to Peanut, a component of a three-septin complex
in embryos (Adam et al., 2000;
Fares et al., 1995
;
Field et al., 1996
). Peanut is
present and precisely colocalized with Anillin at the cellularization front
throughout the entire cellularization process in wild-type embryos
(Fig. 4A,C). Peanut alone also
localizes to the new plasma membranes between nuclei
(Fig. 4C, arrowhead). In
embryos derived from anillinPQ/RS
(Fig. 4B,D) and
anillinHP/RS (not shown) Peanut localization is strongly
perturbed. It fails to colocalize with mutant Anillin in the cellularization
front, and it deposits into abnormal puncta at the apical cortex
(Fig. 4B, arrowhead) and the
plasma membrane (Fig. 4D,
arrowhead). By contrast, in embryos derived from weak maternal alleles
(anillinPE/PE and anillinRV/RV) or
from weak maternal alleles over a zygotic allele
(anillinB26-35 and
anillinC82-45/anillin8), Peanut
localization was normal during slow phase, and the evolution to rings occurred
normally in fast phase. However, the rings were less robust and defects in the
meshwork were observed (see Fig. S2 in the supplementary material). Because
strong anillin alleles perturbed the localization of septins to a
greater extent than that of F-actin, Myosin II or even Anillin itself, we
conclude that these anillin mutations, which alter the PH domain,
interfere directly with the targeting of septins to their correct cell
locations.
Anillin is required for furrow canals to ingress at normal rates
Despite defects in furrow canal structure and septin targeting, the
cellularization front still ingresses in anillin mutant embryos. To
measure ingression kinetics, we examined wild-type and three different mutant
genotypes by DIC time-lapse video microscopy. In wild-type, a pronounced
cellularization front was evident (Fig.
5A, black arrows). This front was less distinct in all mutant
genotypes examined, though its ingression could still be tracked in time-lapse
sequences (Fig. 5B, black
arrows). Ingression kinetics were measured using kymographs, a method that
converts a movie of furrow canal ingression into a graph, with cellularization
front depth on the y-axis and time on the x-axis. This
method is more sensitive and accurate than tracking the front manually,
especially in mutant embryos where it is indistinct in still images. In
wild-type embryos (Fig. 5C,D),
kymography revealed three stages of ingression identified by two inflection
points (white arrows). Each stage lasted 22 minutes. The first
corresponds to organization of furrow canals with little ingression
('initiation phase'). The second and third correspond to slow and fast phases
defined previously. The transition between slow and phase phases occurred
10 µm from the embryo surface. This analysis is similar to that of
Lecuit et al. (Lecuit et al., 2000) with our initiation phase approximately
corresponding to their phases 1 and 2, our slow phase to their phase 3, and
our fast phase to their phase 4.
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|
Anillin is required for pole cell formation
TEM also revealed effects of anillin mutations on pole cell
separation, a specialized type of cytokinesis that defines the presumptive
germ cells (Foe et al., 1993).
Analysis by TEM indicated that wild-type embryos late in cycle 14 possess
well-separated, almost spherical pole cells, containing characteristically
round nuclei at their posterior pole. Pole cells occupy a layer outside the
cellularizing blastoderm and are also interspersed between incipient
blastoderm cells (Fig. 8A). In
anillinHP/RS-derived embryos (a strong allele
combination), pole cells were properly formed and were enclosed by intact
spherical plasma membranes as in wild type
(Fig. 8B,b). This normal
morphology was remarkable given the abnormally positioned nuclei and
vesiculated lateral plasma membranes (Fig.
8b') of incipient blastoderm cells in the same embryo. By
contrast, anillinPE/PE-derived embryos completely lacked
separated pole cells by TEM. Numerous pole cell nuclei were present at the
posterior pole, but no plasma membranes separated them from the rest of the
blastoderm (Fig. 8C) and no
membranes were observed between nuclei
(Fig. 8c). In keeping with the
weaker allele strength, there is less vesiculation of the lateral membrane in
this genotype (Fig.
8c').
|
|
To test for a zygotic requirement for Anillin in cytokinesis, we examined cells in imaginal discs isolated from mutant 3rd instar larvae (genotype anillin7/anillinPQ) probing for F-actin and DNA. Embryos of this genotype survive to pupation but die before eclosion. All discs dissected were much smaller than wild type and difficult to identify. The rudimentary discs contained many large bi-nucleate cells (Fig. 9D), implying failure of cytokinesis. We conclude Anillin is required for normal cytokinesis in Drosophila embryos, and that strong maternal alleles block recruitment of Peanut (but not of Anillin or Actin) to cleavage furrows.
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Discussion |
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PH domains in many proteins have been implicated in lipid binding, and the
amino acid changes in our strong anillin alleles fall in the region
of this domain known to interact with lipids
(Lemmon, 2004;
Rameh et al., 1997
). However,
the PH domain of Anillin lacks the positively charged amino acids that mediate
specific binding to phosphoinositides, and GFP fusions to the C terminus of
human Anillin transiently expressed in mammalian cells did not localize to the
plasma membrane, but instead assembled into small septin-containing foci
(Oegema et al., 2000
). It
therefore seems possible that the PH domain of Anillin mediates a
protein-protein interaction with septins and not a protein-lipid interaction.
However, Drosophila Anillin can target to the cellularization front
in the absence of Peanut (Adam et al.,
2000
) and the C. elegans ortholog ANI-1 can target to the
furrow normally in the absence of the septins
(Maddox et al., 2005
). Many PH
domains do not bind inositol lipids with high affinity and require
oligomerization or additional motifs within the same protein to impart
membrane localization (Lemmon et al.,
2002
). It is therefore possible that the PH domain of Anillin
mediates association with membranes by mechanisms other than septin
binding.
The role of Anillin role in membrane stabilization
Morphologically, the most dramatic phenotype of strong anillin
alleles was the appearance of sheets of vesicles (that appear as lines in thin
sections) between nuclei during cellularization, in place of the intact,
apposed plasma membranes deposited behind the cellularization front in
wild-type embryos. We interpret the presence of some intact membranes in
mutant embryos fixed early in cellularization
(Fig. 7), as evidence that
lateral plasma membranes are initially deposited in anillin mutant
embryos, but they are unstable and subsequently vesiculate. Other membranes in
the mutant embryos, including pole cell and apical plasma membranes, are
unaffected, arguing that vesiculation is not a fixation artifact, and that the
new lateral plasma membranes have a specific requirement for Anillin and
septins for stability. These membranes are special in at least three ways that
might account for their fragility: they assemble very rapidly, by highly
dynamic exo- and endocytosis (Lecuit and
Wieschaus, 2000; Pelissier et
al., 2003
); they are probably under tension from the ingressing
furrow canals; and they are closely apposed to each other. Anillin might
regulate vesicle trafficking dynamics; for example, decreasing exocytosis
could lead to a build up of tension and membrane fragmentation. Septins have
been argued to regulate exocytosis in mammalian cells but, in this case,
inactivation of septins leads to increased, rather than decreased, exocytosis
(Beites et al., 1999
).
Alternatively, Anillin might directly regulate physical stability of
membranes. Plasma membranes are physically stabilized in most situations by
attachment to a cortical actin cytoskeleton, and loss of Anillin might
destabilize them by weakening the cortex or its attachment to the membrane.
Destabilization of membranes under tension might lead to the fusion of closely
apposed membranes and the lines of vesicles we observe are reminiscent of some
stages of programmed cell fusion, e.g. myoblast fusion
(Doberstein et al., 1997
).
Anillin itself does not localize to the cortex of the apposed plasma
membranes, but it might function to recruit and leave behind other proteins
required for stability under tension. Septins are normally present and
localized ectopically in anillin mutants; although targeting of
F-actin is less affected, its organization in mutants is unknown. Loss of
septins or F-actin bundling could result in a more fragile, or more weakly
attached, cortical cytoskeleton, causing the membranes to fragment as tension
builds up during cellularization. A role for septins in stabilizing membranes
could be tested by TEM of peanut mutant embryos, which are known to
exhibit defects in nuclear positioning similar to those we observe in
anillin mutants (Adam et al.,
2000
). Roles for Anillin in regulating membrane trafficking
compared with physical stability might be distinguished by live imaging of
membrane markers to measure exo- and endocytosis, and by imaging thermal
fluctuations of the new plasma membranes to estimate their stiffness, as a
function of anillin genotype.
An interesting aspect of the vesiculation phenotype is the tendency of
plasma membrane-derived vesicles to remain localized in sheets behind the
cellularization front, rather than diffusing away. We suspect they may be
adhering to the baskets of microtubules that surround each nucleus
(Foe et al., 1993), that were
unaffected in anillin mutant embryos. Remarkably, the physical
organization of vesicles is sufficient to allow gastrulation movements (not
shown), even though cellularization has failed completely in terms of
generating cells bounded by plasma membranes.
A role for Anillin in organizing contractile structures
Although defects in septin recruitment mirrored our allelic series, septin
recruitment is clearly not the only function of Anillin. We observed similar
defects in the timing and rate of cellularization front ingression in all
alleles examined, including weak alleles that had no obvious defects in septin
recruitment. We also observed defects in F-actin and Myosin II
localization/organization that are consistent with previously identified
biochemical interactions (Field and
Alberts, 1995; Straight et
al., 2005
). As our alleles did not alter the N terminus of
Anillin, where it interacts with F-actin and Myosin II, these defects probably
result from reduced localization of functional Anillin. A role for Anillin in
scaffolding contractile structures has been demonstrated in C.
elegans, where the Anillin homolog ANI-1 is required to organize foci
containing Myosin II that pull on the plasma membrane during polarity
establishment (Maddox et al.,
2005
). Focusing of actomyosin contraction by Anillin is also
suggested by excessive membrane blebbing and mislocalization of myosin II seen
during cytokinesis after knocking down Anillin by RNAi
(Echard et al., 2004
;
Somma et al., 2002
;
Straight et al., 2005
).
A more structural role for Anillin may be important late in cytokinesis and
cellularization. Myosin II, and most F-actin, typically leave furrows before
cytokinesis is complete, and it is important that something prevents the
furrow from opening back up when it is no longer actively contractile. Anillin
and septins may assemble into a structure under the plasma membrane to
stabilize the neck of cytoplasm late in cytokinesis. One allele,
anillinPE/PE, exhibited a severe effect on pole cell
formation because of re-opening of the neck of cytoplasm connecting the pole
cell to the yolk mass. Interestingly, this allele exhibited milder defects in
cellularization, was viable over zygotic mutations and thus was termed `weak'.
The mutation maps to a region in the middle of Anillin not implicated in any
protein interactions. In other systems, removal of Anillin also leads to
defects late in cytokinesis, with furrows reopening
(Echard et al., 2004;
Somma et al., 2002
;
Rogers et al., 2003
;
Straight et al., 2005
).
Anillin and septins are strongly enriched as rings or short tubes in stable
intracellular bridges, including male ring canals during spermatogenesis
(Hime et al., 1996
;
Robinson and Cooley, 1996
),
and the stalks that connect cells to the yolk mass after cellularization (see
Fig. S1 in the supplementary material, Fig.
2 and Fig. 4). They
also remain in mid-bodies after the contractile proteins leave during
conventional cytokinesis (Field and
Alberts, 1995
). Cumulatively, these data suggests that ring- or
tube-shaped Anillin-septin assemblies [rings are the preferred assembly state
of mammalian septins (Kinoshita et al.,
2002
)] may stabilize intracellular bridges to facilitate the
completion of normal cytokinesis, and to allow communication between sister
cells following incomplete cytokinesis.
Given its interaction with multiple conserved furrow proteins, and its functional involvement in both contractility and membrane stability, further study of Anillin is likely to reveal detailed aspects of how the cytoskeleton and membrane systems work together during cytokinesis, and related furrowing processes in embryos.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/12/2849/DC1
* Present address: Swiss Parliamentary Services, Federal Parliament Building,
3003 Bern, Switzerland
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