1 Ludwig Institute for Cancer Research, Department of Cellular and Molecular
Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA
92093, USA
2 Scionics Computer Innovation, Pfotenhauerstrasse 110, Dresden 01307,
Germany
* Authors for correspondence (e-mail: amaddox{at}ucsd.edu; koegema{at}ucsd.edu)
Accepted 18 March 2005
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SUMMARY |
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Key words: Cytokinesis, Cellularization, Polarity, Cortical flows, Contractility
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Introduction |
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A characteristic sequence feature of all anillin family members is a
C-terminal pleckstrin homology (PH) domain. The PH domains of human anillin
and the S. pombe anillin-related protein Mid2 are required for
targeting to the contractile ring (Berlin
et al., 2003; Oegema et al.,
2000
; Tasto et al.,
2003
). The PH domain may mediate interactions with plasma membrane
lipids and/or with cortical proteins such as the septins
(Kinoshita et al., 2002
;
Oegema et al., 2000
;
Field et al., 2005
). Septins
are conserved guanine nucleotide-binding proteins that are thought to be
components of a membrane-associated cytoskeletal filament system
(Kinoshita, 2003
). Isolated
septin filaments associate specifically with actin bundled by human anillin,
but not by other actin-binding proteins
(Kinoshita et al., 2002
). The
interaction between anillin and the septins defined in vitro is supported by
similar cellularization and cytokinesis defects observed in septin and anillin
mutant Drosophila embryos (Adam et
al., 2000
; Neufeld and Rubin,
1994
; Field et al.,
2005
). Similarly, S. pombe cells deleted for Mid2 or null
for septin function exhibit an identical non-lethal delay in septation
(Berlin et al., 2003
;
Longtine et al., 1996
;
Tasto et al., 2003
).
Anillins have primarily been implicated in cortical remodeling during
cytokinesis and cellularization. In Drosophila and human cells in
which anillin is inhibited by RNAi (Echard
et al., 2004; Eggert et al.,
2004
; Kiger et al.,
2003
; Rogers et al.,
2003
; Somma et al.,
2002
; Straight et al.,
2005
) or mutation (Field et
al., 2005
), contractile rings form and ingress, but cytokinesis
fails to complete. Cytokinesis failure is accompanied by cortical blebbing
surrounding the bridge that connects the two daughter cells, suggesting that
anillin acts late in cytokinesis to facilitate completion
(Echard et al., 2004
;
Somma et al., 2002
;
Straight et al., 2005
).
Although neither of the two anillin-related proteins in S. pombe is
essential, both contribute to cytokinesis. Mid1 is the first protein recruited
to the medial ring and is required for its proper placement
(Bahler et al., 1998
;
Sohrmann et al., 1996
;
Wu et al., 2003
). Mid2 acts
later to stabilize septin localization and facilitate cell separation
(Berlin et al., 2003
;
Tasto et al., 2003
). In
addition to its role in cytokinesis, Drosophila anillin also has a
crucial role during cellularization of the syncytial embryo
(Field et al., 2005
).
Cellularization occurs when the
6000 nuclei created during the first 13
synchronous nuclear divisions are simultaneously partitioned by membranes
deposited behind furrows that ingress inwards from the embryo surface
(Mazumdar and Mazumdar, 2002
;
Miller and Kiehart, 1995
).
Embryos of the nematode C. elegans exhibit stereotypical cortical
contractile events, including polar body extrusion, cortical ruffling,
pseudocleavage and cytokinesis (Wood,
1988). In addition, oogenesis in C. elegans is a gradual
cellularization event that involves extensive cortical remodeling in a
syncytial gonad that bears some resemblance to the cellularizing
Drosophila embryo (Hubbard and
Greenstein, 2000
). We describe the functional characterization of
three C. elegans anillin homologs, ANI-1, ANI-2 and ANI-3. We show
that ANI-2 functions specifically in the syncytial gonad, where it is required
to maintain gonad structure and promote proper oogenesis. By contrast, ANI-1
modulates cortical contractility in the early embryo and also functions later
in development. ANI-1 is required to concentrate myosin II into cortical
patches to generate productive ingressions during ruffling and pseudocleavage,
which normally occur concurrent with the establishment of polarity in the
one-cell stage embryo (Cowan and Hyman,
2004
). ANI-1 is also required to target the septins, but not
myosin II, to contractile rings during polar body formation and cytokinesis.
Interestingly, polar body extrusion fails in ANI-1-depleted embryos, but
ingression of the cleavage furrow during cytokinesis appears to occur
normally. Our results highlight both the conserved functions of the anillin
protein family, and the adaptations that enable it to meet the specific
requirements of the C. elegans life cycle.
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Materials and methods |
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RNA-mediated interference
dsRNA was prepared as described (Oegema
et al., 2001). DNA templates were prepared using primers (see
Table S2 in the supplementary material) to amplify regions of N2 genomic DNA,
N2 cDNA or specific cDNAs, as indicated. L4-stage hermaphrodites were injected
with dsRNA and incubated at 20°C for 45-48 hours. Soaking RNAi was
performed as described (Maeda et al.,
2001
), except worms were incubated in a drop of RNA solution on
parafilm in a humid chamber for 24 hours at 20°C. Worms were then
recovered to a seeded NGM (nematode growth medium) plate and incubated for 48
hours at 20°C.
Embryonic lethality and Brood size tests
RNAi-treated worms were placed on individual plates 48 hours after
injection or 72 hours after soaking was initiated. After 24 hours at 20°C,
the worms were removed from each plate and embryos and young larvae were
grouped together and counted (average=brood size). Embryo viability was
evaluated 24 hours or more later; unhatched eggs were counted and calculated
as a percentage of total laid for that plate (embryonic lethality).
Microscopy
Immunofluorescence images were acquired and processed as described
(Cheeseman et al., 2004). For
live imaging, newly fertilized embryos were mounted as described
(Oegema et al., 2001
). DIC
imaging was performed as described (Gonczy
et al., 1999
). For embryos expressing NMY-2:GFP, three
z-sections were acquired at 1 µm intervals near the embryo surface
using a spinning disc confocal equipped with a 60x, 1.40 NA Nikon
PlanApo objective and 2 x2 binning. Images were collected at 10 second
intervals and the three images for each time point were projected for
presentation.
Immunofluorescence and immunoblotting
Immunofluorescence was performed as described previously
(Desai et al., 2003;
Oegema et al., 2001
).
Polyclonal antibodies against ANI-1 (residues 460-768), ANI-2 (residues
890-1015), NMY-2 (residues 945-1368), UNC-59 (C-terminal peptide:
(C)SGTMKKRMGGLGLFNRN), UNC-61 [C-terminal peptide: (C)TEERMKLMTKVSKKLRK] were
generated as described previously (Desai et
al., 2003
). For fixation of gonads, adult worms in 5% sucrose, 100
mM NaCl were nicked with a scalpel to extrude the gonads. Western blotting of
extracts prepared from embryos and adult hermaphrodites was performed using
standard protocols and blotting of RNAi-depleted and control worms was
performed as described (Hannak et al.,
2001
). To control for protein loading, blots were re-probed with
antibodies to
-tubulin (DM1-
; Abcam).
Dextran injections
Dextran (70 kDa) labeled with tetramethylrhodamine (Molecular Probes) was
reconstituted to 1 mg/ml in injection buffer (1 mM potassium citrate, 6.7 mM
KPO4, pH 7.5, 0.67% PEG) and injected into the gonad rachis of
adult worms. After 5-15 minutes, worms were mounted whole in M9 buffer (22 mM
KH2PO4, 19 mM NH4Cl, 48 mM
Na2HPO4, 9 mM NaCl) on multiwell glass slides. DIC and
wide-field fluorescent images were acquired using a 20 x 0.75 NA Nikon
PlanApo objective, and 2 x2 binning.
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Results |
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ANI-1 and ANI-2 have distinct roles in embryo production and viability
The divergence in the protein sequences of the three C. elegans
anillin homologs suggests that they perform different functions. To determine
whether the C. elegans anillins are required for embryo production or
viability, we depleted each individually by RNAi using either injection or
soaking. We then measured the number of embryos laid by the treated worms
(brood size) and their viability. Consistent with the results of a large-scale
screen (Kamath et al., 2003),
RNAi of ANI-3 by either method had no effect on either brood size or embryonic
viability (Fig. 1B,C). RNAi of
ANI-3 also did not enhance any of the phenotypes resulting from depletion of
ANI-1 or ANI-2 (discussed in detail below). Therefore, we did not characterize
ANI-3 further.
Consistent with the results of previous screens
(Gonczy et al., 2000;
Kamath et al., 2003
;
Piano et al., 2000
;
Simmer et al., 2003
), the
number of embryos laid by ani-1(RNAi) worms was similar to controls
(Fig. 1B), but highly penetrant
embryonic lethality was observed (Fig.
1C). Quantitative western blotting using affinity-purified
antibodies revealed that ANI-1 levels were reduced to
3% of control by
injection RNAi (Fig. 1D).
Soaking also resulted in increased embryonic lethality but with lower
penetrance. Consequently, for all subsequent experiments, injection of dsRNA
was used to deplete ANI-1.
|
The different consequences of depleting ANI-1 or ANI-2 on embryo production and viability suggest that these related proteins have distinct functions. Indeed, western blotting of extracts prepared from whole worms or isolated embryos revealed that ANI-1 is selectively enriched in embryos relative to adult hermaphrodites, whereas ANI-2 is highly enriched in adults relative to embryos (Fig. 1E). Immunofluorescence analysis of fixed embryos further underscored this difference. ANI-1 was detected on the cortex and localized prominently to the cytokinetic furrow of telophase embryos, whereas antibodies to ANI-2 did not recognize specific structures in embryos (Fig. 1F). From these results, we conclude that ANI-1 is enriched in embryos and is required for their viability. By contrast, ANI-2, which contributes to both embryo production and viability, is primarily present in adult worms.
Polar body extrusion, ruffling and pseudocleavage fail in ani-1(RNAi) embryos, but cytokinesis occurs normally
The penetrant embryonic lethality resulting from depletion of ANI-1
suggested a role in embryonic development. To characterize the function of
ANI-1, we analyzed cortical dynamics between fertilization and the first
embryonic cytokinesis using DIC microscopy
(Fig. 2; see Movies 1 and 2 in
the supplementary material). In control wild-type embryos, immediately
following fertilization the oocyte-derived nucleus undergoes two rounds of
meiotic division, each of which ends with a small asymmetric cytokinesis-like
event that extrudes a polar body. During the meiotic divisions, membrane
ruffling occurs over the entire embryo surface. As embryonic polarity is
established, ruffling becomes limited to the embryo anterior
(Fig. 2A)
(Cowan and Hyman, 2004). The
oocyte pronucleus subsequently migrates towards the sperm pronucleus and the
cortical ruffles resolve to form a transient invagination called the
pseudocleavage furrow (Fig. 2B) that regresses as the pronuclei meet. Following nuclear envelope breakdown,
spindle assembly and chromosome segregation, a cytokinetic furrow ingresses
between the separated chromosome masses to generate the two daughter cells
(Fig. 2E).
In ANI-1-depleted embryos, defects were observed in all of the early contractile events. Polar body extrusion was either abnormal, leading to the formation of a large bleb containing yolk granules and cytoplasm (Fig. 2A', arrowhead), or failed completely, allowing the polar body chromatin to reenter the embryo (Fig. 2B',E', arrow). Cortical ruffles and the pseudocleavage furrow also failed to form following ANI-1 depletion (Fig. 2A',B'). However, in surprising contrast to the complete failure of early contractile events, the positioning and ingression of the cytokinetic furrow appeared normal (Fig. 2D'). Cytokinesis also completed successfully and the cell-cell boundary remained intact beyond the four-cell stage in all ANI-1-depleted embryos (n=20). Simultaneous depletion of either ANI-2 or ANI-3 with ANI-1 did not exacerbate any of the defects observed in the ANI-1-depleted embryos (data not shown), suggesting that they do not function redundantly with ANI-1 in the early embryo. Thus, ANI-1 is required for early contractile events, including polar body formation, ruffling and pseudocleavage, but appears to be dispensable for cytokinesis.
|
ANI-1 targets independently of the septins and myosin II during mitosis and meiosis
To determine whether ANI-1 and the septins are interdependent for their
localization, we analyzed septin-depleted embryos using worms homozygous for
an unc-61 mutant allele (e228)
(Nguyen et al., 2000), or
embryos depleted of UNC-59 and UNC-61 by double RNAi. Both ANI-1 and NMY-2
targeted to contractile rings during cytokinesis
(Fig. 3A) and polar body
formation (Fig. 3D) in septin
loss-of-function embryos (only unc-61 embryos are shown). The
targeting of NMY-2 is consistent with the fact that that cytokinesis and polar
body formation are successful in both types of septin loss-of-function embryos
(Nguyen et al., 2000
).
Cumulatively, our targeting analysis places ANI-1 upstream of the septins in
contractile ring assembly.
|
ANI-1 organizes myosin II and the septins into cortical patches to promote ruffling
In contrast to cytokinesis, ruffling and pseudocleavage fail in
ANI-1-depleted embryos (Fig.
2). To characterize these defects further, we examined the
localization of NMY-2 and the septins in embryos fixed at times between the
completion of meiosis II and pronuclear migration, when ruffling and
pseudocleavage normally occur. During this period in control embryos, ANI-1,
the septins and NMY-2 all concentrate in cortical patches
(Fig. 4A,C). Although membrane
invaginations are not preserved by the fixation used here, timelapse imaging
of embryos expressing NMY-2:GFP (see Fig.
5B), GFP:UNC-59 or GFP:ANI-1 (data not shown) indicates that these
patches correspond to the base of ingressing ruffles.
In ANI-1-depleted embryos, distinct cortical patches containing NMY-2 and
the septins fail to form. Instead, both remain homogeneous at the cortex
(Fig. 4A,C; results for UNC-61
not shown). A similar defect in the organization of the cortical cytoskeleton
was observed in living ANI-1-depleted embryos expressing NMY-2:GFP (see
Fig. 5B) or GFP:UNC-59 (data
not shown). Interestingly, distinct patches of ANI-1 and the septins formed in
embryos in which NMY-2 was depleted below the level of detection by
immunofluorescence (Fig. 4A).
NMY-2 depleted embryos failed to ruffle, further indicating that neither NMY-2
protein nor contractility is required for the organization of ANI-1 and the
septins (and presumably other components of the cortical cytoskeleton)
(Munro et al., 2004) into an
inhomogeneous network. We conclude that ANI-1 promotes ruffling via a crucial
role in organizing the cortical cytoskeleton to form inhomogeneities that
become contractile in the presence of NMY-2 (see
Fig. 9).
|
To analyze the dynamics of cortical NMY-2, we acquired timelapse sequences
of control and ANI-1-depleted embryos expressing NMY-2:GFP. In control
embryos, NMY-2:GFP was organized into distinct cortical patches that moved
towards and concentrated in the embryo anterior
(Fig. 5B, top right)
(Munro et al., 2004). By
contrast, in ani-1(RNAi) embryos, NMY-2:GFP was homogeneously
distributed at the cortex. Although it failed to organize into patches,
cortical NMY-2:GFP formed a diffuse anterior cap as polarity was established
in ani-1(RNAi) embryos. We observed similar homogeneous cortical
staining and anterior enrichment of endogenous NMY-2 in fixed
ani-1(RNAi) embryos (data not shown). Kymographs revealed that the
small discontinuities of NMY-2:GFP fluorescence visible in ANI-depleted
embryos moved towards the anterior (Fig.
5B, bottom right). We conclude that although its organization is
disrupted, NMY-2 localizes to the cortex and flows to the anterior in
ANI-1-depleted embryos. These results also suggest that organization of NMY-2
into patches and the resulting plasma membrane ingressions are not required
for movement of cortical myosin to the anterior, or to generate polarity (see
Fig. 9).
|
ANI-2 localizes to the surface of the rachis in the adult gonad
ANI-2 contributes to embryonic viability but was not, somewhat
paradoxically, detected in embryos by immunofluorescence or western blotting
(Fig. 1E,F). ANI-2-depleted
embryos also did not exhibit contractile defects detectable by DIC microscopy
(see Fig. S4 and Movie 3 in the supplementary material). One possible
explanation for these observations is that ANI-2 functions in the gonad to
facilitate the formation of oocytes capable of developing into viable embryos.
To test this idea, we examined the localization of ANI-2, NMY-2 and DNA in
fixed gonads (Fig. 6). The
C. elegans hermaphrodite gonad is a syncytium (see schematic in
Fig. 6A). Mitotic divisions
occur near the distal tip, but the majority of the gonad is lined with nuclei
progressing through meiotic prophase in a common cytoplasmic environment. The
meiotic nuclei are closely apposed to the gonad surface. Partitions extending
from the surface compartmentalize each nucleus in a membrane-bound chamber, or
pseudo-cell. Round `windows' connect each pseudo-cell to the rachis, the
central cytoplasmic core of the gonad (Fig.
6A, arrows). At the turn of the gonad, the pseudo-cells increase
dramatically in size. Full-sized oocytes populate the proximal gonad, and are
fertilized as they pass through the spermatheca.
In control gonads, ANI-2 localized strikingly and exclusively to the surface of the rachis (Fig. 6; green in schematics). ANI-2 framed the windows between the pseudo-cells and the rachis in the distal and middle gonad, and also localized to the surface of the rachis between the windows (Fig. 6A,B). At the turn of the gonad, where developing oocytes increase in size, the `windows' to the rachis enlarge (Fig. 6A, arrows; Fig. 6C). In the proximal gonad, the ANI-2-stained rachis gradually decreases in diameter, and runs alongside some of the full-sized oocytes in the oviduct. All but approximately the four oocytes that are nearest to the spermatheca appear to be connected to the rachis (Fig. 6A, arrows).
Other cortical cytoskeletal proteins, NMY-2
(Fig. 6), the septin UNC-59
(see Fig. S5 in the supplementary material) and ANI-1 (data not shown), also
localized to cortical surfaces in the gonad, but their distributions were
distinct from that of ANI-2. The other cortical markers were less specific for
the rachis surface and also localized prominently to the lateral surfaces of
the developing oocytes. ANI-2 is one of the few proteins known to specifically
demarcate the boundaries of the rachis
(Vogel and Hedgecock, 2001;
Thompson et al., 2002
), a
localization consistent with its playing an important role in oogenesis and
embryo production.
ANI-2 is required for the structural organization of the gonad
To determine how ANI-2 contributes to gonad structure and function, we
analyzed ani-2(RNAi) worms. Gonads from ANI-2 depleted worms were
grossly disorganized. The region of the gonad that ordinarily contains
regularly spaced nuclei (Fig.
6) was dramatically shortened: nuclei were randomly positioned and
commonly filled the rachis in ani-2(RNAi) worms
(Fig. 7C). At the bend in the
gonad, where oocytes should be midsized and continuous with the rachis, they
instead appeared small and tightly packed, eliminating the rachis
(Fig. 7A,B, arrowheads).
Oocytes in the oviduct were smaller and more variable in size than in control
gonads (Fig. 7A,B, arrows).
Staining of the septin UNC-59 (see Fig. S5 in the supplementary material) and
NMY-2 (Fig. 7) suggested that
the partitions between nuclei were either fragmented and disordered or absent
in ani-2(RNAi) gonads (compare
Fig. 6C with
Fig. 7C). Both NMY-2
(Fig. 7) and UNC-59 (see Fig.
S5 in the supplementary material) still localized to the remaining partitions
between developing oocytes, suggesting that their cortical localization does
not require ANI-2. However, targeting dependencies are difficult to assess
because of the disorganization of the residual structures. Interestingly, in
the gonads of unc-61(e228) worms in which septins fail to localize to
cortical structures (Nguyen et al.,
2000), ANI-2 targeted normally to the rachis surface (see Fig. S5
in the supplementary material). Thus, consistent with its localization, ANI-2
plays an important role in gonad structure and oocyte formation.
|
It seemed likely that the small oocytes in the ani-2(RNAi) worms would generate small embryos following fertilization. Indeed, embryos from ani-2(RNAi) worms were significantly smaller (average length 40±8.0 µm; n=85) than controls (average length 50±2.6 µm; n=85; Fig. 8B,C). In addition, embryos from ANI-2-depleted worms were highly variable in size (Fig. 8B,C). Depletion of ANI-1 or ANI-3 did not affect embryo size and the effects of ANI-2 depletion were not exacerbated by simultaneous depletion of ANI-1 or ANI-3 (data not shown), indicating that these other anillin family proteins do not function redundantly with ANI-2 in the gonad. Preliminary studies, in which individual embryos were isolated and their viability assessed, indicate that the partial loss of embryo viability following ANI-2 depletion (Fig. 1C) is due to increased lethality of embryos that are smaller or larger than normal (data not shown). Interestingly, the surviving progeny from ANI-2 depleted worms matured normally and had no adult phenotypes other than significantly reduced brood sizes, demonstrating compromised gonad function [control: 223±16 (s.d.); n=4, ani-2(RNAi) progeny: 70±67 (s.d.); n=21, P=0.00018; two-tailed t-test]. Cumulatively, these results suggest that ANI-2 functions specifically in the gonad to maintain the structure of the rachis.
|
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Discussion |
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Functional interactions between anillin family proteins and septins
In vivo and in vitro studies of vertebrate anillins suggest that the PH
domain, together with a small adjacent portion of the AH domain, interacts
with the septins (Kinoshita et al.,
2002; Oegema et al.,
2000
). The existence of a physical interaction suggests that
anillins and septins influence each other's localization or stability. We show
that ANI-1 is required for enrichment of the septins in contractile rings
during both meiosis and mitosis, whereas the septins are not required to
target ANI-1 to contractile rings. A role for anillin in septin recruitment is
consistent with analysis of anillin mutants in Drosophila, where the
phenotypic severity of maternal effect mutations correlates with the degree of
disruption of septin recruitment to contractile structures
(Field et al., 2005
). In
S. pombe, the anillin related protein Mid2 is not required to recruit
septins to the medial ring but does affect the rate at which they turn over
(Berlin et al., 2003
;
Tasto et al., 2003
). Like
ANI-1, ANI-2 also has C-terminal AH and PH domains but the relationship
between ANI-2 and the septins is less clear. ANI-2 localizes to the rachis
surface in unc-61(e228) septin mutant worms (see Fig. S5 in the
supplementary material), indicating that the septins are not required for
ANI-2 targeting. The septins also target to residual cortical structures in
the gonads of ani-2(RNAi) worms (see Fig. S5 in the supplementary
material), but the extent of septin recruitment is difficult to assess because
of the severe disruption of gonad structure.
The phenotypes we observed when ani-1(RNAi) was initiated later in
embryogenesis (bypassing the polar body extrusion defect; see Fig. S3 in the
supplementary material) are similar to those reported for septin mutant worms
(Nguyen et al., 2000). Defects
in somatic cytokinesis and cell migration have been reported for septin mutant
worms and further studies are necessary to test whether these defects occur in
adult ani-1(RNAi) animals (Nguyen
et al., 2000
; Finger et al.,
2003
). Genetic analyses in Drosophila and S.
pombe provide additional support for a close functional relationship
between anillin family proteins and the septins. Mutational inactivation of
either results in similar phenotypes in both systems
(Adam et al., 2000
;
Berlin et al., 2003
;
Neufeld and Rubin, 1994
;
Tasto et al., 2003
;
Field et al., 2005
).
|
A conserved role for anillin family proteins in syncytial structures
Like the early Drosophila embryo, the C. elegans gonad is
a syncytium in which nuclei are cellularized. But unlike the
Drosophila embryo, where cellularization of all nuclei occurs
simultaneously, in the C. elegans gonad, meiotic nuclei are
cellularized one by one as they move gradually from the distal mitotic zone to
the gonad proximal end. In the cellularizing Drosophila embryo,
anillin promotes the ingression of membrane furrows to create new cell surface
between adjacent nuclei and is also required for structural integrity of the
newly established partitions (Field et
al., 2005). The finding that ANI-2 is important for structural
organization of the C. elegans gonad during oogenesis highlights a
conserved role for anillin family proteins in syncytial structures.
ANI-1 organizes the cortical cytoskeleton to form an inhomogeneous contractile network prior to mitosis
One striking aspect of the ANI-1 depletion phenotype is the complete
inhibition of cortical ruffling and pseudocleavage during interphase/prophase.
During this cell cycle phase, anillins in other metazoans are sequestered in
the nucleus. By contrast, ANI-1 is not detected in the nucleus, has no
predicted nuclear localization sequences (the same is true for ANI-2), and is
present in patches at the base of ingressing ruffles on the cell cortex,
together with septins and myosin II. In ANI-1-depleted embryos, both NMY-2 and
the septins appear homogenous at the cortex (Figs
4 and
5). In striking contrast, an
inhomogeneous network of patches containing AN1-1 and the septins was still
present in NMY-2-depleted embryos (Fig.
4), indicating that neither NMY-2 nor productive contractility is
required for cortical patch formation. Cumulatively, these results indicate
that ANI-1 has a more important role than NMY-2 in organizing the cortical
cytoskeleton. Based on the predicted conservation of its F-actin and myosin
II-binding domains, we propose a model for the role of ANI-1 in patch
formation (Fig. 9). In this
model, cortical ANI-1 binds F-actin and bound F-actin recruits additional
ANI-1. These two steps might constitute a positive feedback loop that clusters
ANI-1, F-actin and the septins to form an inhomogeneous network of cortical
patches. NMY-2 also concentrates in the patches (possibly via binding to
ANI-1), and provides the contractile force for cortical ingressions. In
ani-1(RNAi) embryos, the patches fail to form and ruffling does not
occur.
|
Role of anillin family proteins in contractile ring structure and function
Consistent with the failure of cytokinesis completion following anillin
disruption in other metazoans, polar body formation is abnormal or fails in
ANI-1-depleted C. elegans embryos. As myosin II-containing rings form
at the site of meiotic cytokinesis in the depleted embryos, it is not known
whether this failure results from defects in contractile ring ingression or
completion. Determining this will require live imaging of polar body
formation, which has so far proven difficult because of their small size and
mobility on the embryo surface and the fragility of the embryo egg shell at
this stage.
Surprisingly, cytokinesis appears normal in ani-1(RNAi) embryos.
Although we cannot rule out the possibility that a small amount of residual
ANI-1 is sufficient for successful cytokinesis, we think this is unlikely.
Quantitative western blotting revealed that ANI-1 is reduced to 3% of
wild-type levels and little or no residual ANI-1 was detected on contractile
structures by immunofluorescence. The robust effects of ANI-1 depletion on
polar body formation and cortical ruffling also argue against the success of
cytokinesis being a consequence of residual ANI-1.
In other systems, anillins are dispensable for the majority of furrow
ingression (Echard et al.,
2004; Somma et al.,
2002
; Straight et al.,
2005
). Thus, one possible explanation for our observation of
successful cytokinesis following ANI-1 depletion is the dramatically different
geometry of cleavage in C. elegans embryos. When Drosophila
and human cultured cells undergo cytokinesis, they become dumbbell shaped, and
an extended bridge is the only connection between the incipient daughter
cells. By contrast, in C. elegans embryos a new cell-cell boundary
forms as the cleavage furrow ingresses. The extensive contacts between the
daughter cells may stabilize the small intracellular bridge sufficiently to
allow completion of cytokinesis even in the absence of cortical
organizing/stabilizing proteins such as anillin.
Given the essential role for ANI-1 in organizing cortical contractility
during ruffling and meiotic cytokinesis, why does the mitotic contractile ring
form and ingress normally in ani-1(RNAi) embryos? Our results suggest
two possibilities: (1) an unrelated protein substitutes for ANI-1 in
organizing the contractile ring; or (2) the role of ANI-1 in contractile ring
function is compensated for by a redundant organizing activity that acts
specifically during mitotic cytokinesis. The conservation of anillins and
their role in targeting the septins to the contractile ring
(Field et al., 2005) lead us
to favor the second possibility, but further work is necessary to determine if
this is indeed the case.
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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/132/12/2837/DC1
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