Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA
Author for correspondence (e-mail:
lsrose{at}ucdavis.edu)
Accepted 11 August 2003
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SUMMARY |
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Key words: Asymmetric division, Polarity, Spindle orientation, C. elegans, Nuclear rotation
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Introduction |
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The early development of Caenorhabditis elegans is characterized
by asymmetric divisions that produce diverse cell fates
(Rose and Kemphues, 1998b;
Lyczak, 2002). In the one-cell embryo (P0), the spindle is oriented
on the polarized anterior/posterior (AP) axis. First cleavage is unequal and
generates a larger anterior cell called AB and a smaller posterior cell called
P1. P1 divides unequally with its spindle oriented on
the AP axis to produce a larger EMS and a smaller P2 cell, both of
which divide asymmetrically. Divisions of the P lineage are intrinsically
programmed (Goldstein et al.,
1993
; Goldstein,
1995
). By contrast, the asymmetric division of EMS along the AP
axis absolutely requires contact with its sister P2
(Goldstein, 1995
). However,
the AP orientation of spindles in both the P and EMS cells results from a
90° rotation of the nuclear-centrosome complex during prophase, which does
not occur in AB.
The intrinsic polarity in P lineage cells is established through the
asymmetric distributions of several PAR proteins, which are conserved in many
organisms (Ohno, 2001). In the
one-cell (P0), a complex of PAR-3, PAR-6 and PKC-3 (atypical
protein kinase-3) are present on the anterior cortex, while the PAR-2 and
PAR-1 proteins are present on the posterior cortex. At the two-cell stage, the
PAR-3 complex and PAR-2/PAR-1 become asymmetrically localized in anterior and
posterior domains again in P1. The PAR-3 complex is also present
uniformly at the cortex of AB. The asymmetric distributions of the PAR
proteins result in the polarized distribution of downstream cell fate
determinants (Rose and Kemphues,
1998b
; Lyczak, 2002). This intrinsic PAR-3/PAR-2 asymmetry is also
essential for nuclear rotation and for the asymmetric spindle elongation that
results in unequal cleavage in P lineage cells
(Tsou et al., 2003
;
Cheng et al., 1995
). The
PAR-dependent mechanism causes nuclear rotation to occur centrally in the
P0 and P1 cells, when the effects of cell shape
asymmetry are removed (Tsou et al.,
2002
; Tsou et al.,
2003
). The uniform distribution of PAR-3 in AB is also required to
prevent ectopic nuclear rotation directed towards the cell cortex that is
caused by the geometry of the cell shape
(Tsou et al., 2003
). The
precise mechanism by which the PARs coordinate polarity with spindle
orientation remains to be elucidated, but several key players have been
identified.
Heterotrimeric G proteins are required for several aspects of spindle
positioning (Zwaal et al.,
1996; Gotta and Ahringer,
2001
). Using RNA interference experiments, it was shown that
asymmetric spindle elongation in the one-cell embryo is dependent on two
partially redundant G
proteins encoded by goa-1 and
gpa-16 (Gotta and Ahringer,
2001
). It was proposed that Gß
, encoded by
gpb-1 and gpc-2, are important in regulating migration of
the centrosomes around the nucleus because oblique migration paths were seen
in mutant embryos. These observations, coupled with G
;
Gß double mutant analysis led to the interpretation that G
and Gß
control distinct microtubule-dependent processes that are
required for proper spindle positioning in C. elegans embryos
(Gotta and Ahringer, 2001
).
However, depletion of Gß
also resulted in late nuclear rotation,
while G
(RNAi) embryos showed a complete failure of
nuclear rotation at the two-cell stage, indicating potential involvement in
common processes as well.
Although canonical heterotrimeric G protein signaling pathways are
primarily activated via cell-surface receptors, recent work in
Drosophila and rat has revealed receptor-independent mechanisms for
activation of G-protein signaling
(Schaefer et al., 2001;
Takesono et al., 1999
). In
particular, in Drosophila neuroblasts the GoLoco domain protein,
PINS, is localized asymmetrically and is required for proper spindle
orientation. PINS binds to the GDP form of G
(GDP-G
) and can
cause Gß
to be released from G
. C. elegans
homologs of PINS, called GPR-1 and GPR-2 (GPR-1/2), are required for proper
spindle positioning in P lineage cells
(Gonczy et al., 2000
). This
observation suggests that intrinsically activated G-protein signaling may be a
conserved pathway for spindle positioning among species. Recent work suggests
that G
and GPR-1/2 act together in the generation of forces needed for
anaphase spindle elongation in C. elegans
(Gonczy et al., 2000
;
Dechant and Glotzer, 2003
;
Srinivasan et al., 2003
;
Colombo et al., 2003
;
Gotta et al., 2003
). In
addition, two of three recent studies found that GPR-1/2 are enriched at the
posterior pole of the embryo in response to PAR-3
(Colombo et al., 2003
;
Gotta et al., 2003
). These
observations, together with previous work
(Grill et al., 2001
), leads to
the model that the asymmetric enrichment of GPR-1/2 results in higher cortical
forces at the posterior that cause asymmetric anaphase spindle elongation
(Colombo et al., 2003
;
Gotta et al., 2003
).
The LET-99 protein also plays a crucial role in spindle positioning
(Rose and Kemphues, 1998a;
Tsou et al., 2002
). LET-99 is
required for nuclear rotation and asymmetric anaphase spindle movements in
P0 and P1, and LET-99 is enriched in an asymmetrically
positioned band in P lineage cells in response to PAR polarity cues
(Rose and Kemphues, 1998a
;
Tsou et al., 2002
).
Furthermore, the mislocalization of LET-99 correlates with failures in nuclear
rotation in par-3 and par-2 mutant P0 and
P1 cells, as well as alterations in anaphase spindle movements in
par-3 embryos (Tsou et al.,
2002
; Tsou et al.,
2003
). These observations have led to the model that the cortical
LET-99 band is an intermediate that transmits PAR cues into the asymmetric
forces needed for nuclear rotation and anaphase spindle movement
(Tsou et al., 2002
;
Tsou et al., 2003
).
Interestingly, the LET-99 protein contains a DEP domain, which is found in
many other molecules involved in G-protein signaling. Thus, LET-99 could
provide an asymmetric cue to the G protein signaling pathway.
Asymmetric cell division that occurs in the EMS cell is driven by extrinsic
signals from the P2 cell that both polarizes EMS and orients the
spindle (Goldstein, 1995). The
conserved Wnt/wingless signaling pathway functions in P2/EMS
signaling, in a partially redundant manner with the MES-1/SRC-1 tyrosine
kinase pathway (Schlesinger et al.,
1999
; Bei et al.,
2002
). Nuclear rotation in EMS cells is directed toward the
posterior cell contact with P2
(Schlesinger et al., 1999
),
which is different from the free central nuclear rotation driven by the
PAR-dependent mechanism in P0 and P1 cells
(Tsou et al., 2002
;
Tsou et al., 2003
). In
addition, just after rotation in EMS cells, the posterior spindle pole is
closely associated with the cortex at the EMS/P2 boundary
(Berkowitz and Strome, 2000
),
which is not observed for the posterior spindle pole in P0 and
P1 cells. These differences in nuclear rotation and spindle
movements driven by PAR and Wnt/MES-1/SRC-1 signaling suggest that the spatial
control of forces that act on the spindle may be different in these cell
types. It is not known, however, whether any of the proteins used in
positioning spindles in the P lineage also function in EMS.
Whether intrinsically or extrinsically programmed, the coupling between
polarity and spindle orientation is essential for asymmetric division. Two
characteristics of force generation that must be regulated and coordinated to
properly position the spindle are the magnitude of the force and the asymmetry
of forces. In this report, we provide evidence for a model that the
G/GPR-1/2 signaling pathway upregulates the magnitude of force
generation in P-lineage cells to drive nuclear and spindle movements, and that
LET-99 provides an asymmetric cue and acts antagonistically to
G
/GPR-1/2 signaling. Furthermore, our results indicate that aspects of
the Gß phenotype are due to gain of G
/GPR-1/2 activity, rather
than reflecting a separable role for Gß in spindle positioning. Finally,
we show that G
/GPR-1/2 signaling and LET-99 are both involved in the
asymmetric cell division of EMS cells. Thus, the different polarizing cues
used in intrinsic and extrinsically controlled asymmetric divisions use common
downstream signaling components.
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Materials and methods |
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RNA interference
Antisense and sense RNAs were transcribed in vitro from linearized
full-length cDNA templates (Ambion MEGAscript). Double-stranded RNAs (dsRNA)
were annealed as described by Fire et al.
(Fire et al., 1998). Young
adult worms were soaked in dsRNA solution (1.5 mg/ml) for 8-10 hours at room
temperature. The progeny of soaked worms were analyzed between 16 and 32 hours
post-soaking.
Microscopy and analysis of living embryos
Embryos were mounted to avoid flattening the embryo, and examined under DIC
optics using time-lapse video microscopy
(Rose and Kemphues, 1998a).
Nuclear and spindle positions were measured from video images as described in
Table 1. Hyperactive centrosome
movements were quantified by measuring the angular velocity of the
nuclear-centrosome complex, which was then converted to a linear velocity
using the radius of the complex. Spherical cells were generated as described
previously (Tsou et al.,
2002
). All filming was at room temperature of 23-25°C.
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For in situ immunolocalization, worms were cut in egg buffer on poly-lysine
coated slides, freeze-fractured and fixed with methanol
(Miller and Shakes, 1995). For
staining of let-99ts embryos, embryos were temperature shifted as
described in the text, and those undergoing a normal P1 division were fixed
during prophase of the EMS cell cycle. Antibody incubation was carried out at
4°C overnight for both anti-LET-99 and anti-GPR-2 (1:50 in PBS) and at
room temperature for 1-2 hours with FITC-conjugated goat anti-rabbit or
Rhodamine-conjugated goat anti-rat (1:200 in PBS). Primary and secondary
antibodies were pre-absorbed with acetone powders of GST-expressing bacteria
and wild-type worms respectively. Embryos were staged by DAPI
(4',6-diamidino-2-phenylindole dihydrochloride) staining of the nuclei.
Images were obtained on a Leica Confocal. Single-section confocal images taken
at a mid-embryo focal plane were analyzed using IP Images software
(Scanalytic). To quantify levels of GPR-1/2 staining, the line tool was used
to mark the entire cortex, and the average pixel value of the marked region
was measured. The unit of relative intensity in
Fig. 4 is expressed as a ratio
of cortical staining to cytoplasmic staining as in
(Tsou et al., 2002
).
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Results |
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In addition to the phenotypes described previously
(Gotta and Ahringer, 2001;
Zwaal et al., 1996
), we
observed that gpb-1(RNAi) embryos (also referred to as
Gß mutants) exhibit hyperactive nuclear and spindle movements
(rocking) in all cells from early prophase to metaphase. Instead of the
centering and smooth rotational movement of the nucleus seen in wild-type
embryos, the nuclear-centrosome complex in gpb-1(RNAi) embryos rocked
vigorously and did not center completely
(Fig. 1A,
Table 1). However, the
centrosomes aligned along the AP axis by metaphase
(Fig. 1A). The speed of nuclear
rocking during prophase (0.55±0.09 µm/second, n=8) was six
times faster than the speed of the nuclear rotation seen in wild-type embryos
(0.09 µm/second) (Tsou, 2002). During anaphase in gpb-1(RNAi)
embryos, the oscillations of the spindle poles were asymmetric and resemble
those seen in wild type (Fig.
1A), suggesting that asymmetric forces are present
(Grill et al., 2001
). At the
two-cell stage in gpb-1(RNAi) embryos, the nuclei exhibited rocking
during prophase (not shown), and P1 nuclear rotation often occurred late,
during nuclear envelope breakdown (Gotta
and Ahringer, 2001
; Zwaal et
al., 1996
). These results together suggest that the net forces
acting on nuclei and spindles in Gß mutant cells are hyperactive
but still act asymmetrically. We therefore propose that GPB-1 is required for
controlling the magnitude of the net forces acting on centrosomes, but is not
required for generating asymmetric forces in early C. elegans
embryos.
|
Although nuclear rotation was observed in some one-cell G
mutants embryos (6/18 embryos), in many embryos the two centrosomes were
prematurely positioned on the AP axis before pronuclear meeting (12/18
embryos). To further examine nuclear rotation in G
mutants, we
examined embryos in which the eggshell was removed by chitinase digestion. Our
recent studies showed that although wild-type embryos have a PAR
polarity-dependent mechanism for nuclear rotation that is cell-shape
independent (Hyman and White,
1987
; Tsou et al.,
2003
), ectopic rotation in certain polarity and spindle
orientation mutants can be driven by the oval shape of the embryo
(Tsou et al., 2002
;
Tsou et al., 2003
). Thus,
removal of the eggshell to produce a spherical embryo is essential to
determine if the intrinsic polarity-dependent mechanism of nuclear rotation
remains functional. In spherical G
(RNAi) embryos in
which the centrosomes were normally positioned at pronuclear meeting, nuclear
rotation failed to occur (n=3;
Fig. 1B). This result indicates
that G
not only is required for asymmetric anaphase spindle positioning
as described previously, but is also essential for intrinsically controlled
nuclear rotation in the one-cell embryo.
Overall, the less active nuclear and spindle movements of G
mutant embryos suggest that the net forces acting on nuclei and spindles are
much smaller than in wild-type and Gß mutant cells. It is not
clear whether G
is directly required for the asymmetry of forces, or if
there is simply insufficient force to respond to asymmetric cues in the
absence of G
. Taken together, these results suggest that G
and
Gß depletion cause opposite effects in the one-cell embryo: depletion of
Gß causes hyperactive but polarized nuclear and spindle movements, while
depletion of G
causes less active and non-polarized nuclear and spindle
movements.
The hyperactive spindle movements of gpb-1(RNAi) embryos are
due to excess G activity
The opposite phenotypes of G and Gß mutant embryos described
above can be explained by three hypotheses: (1) G
and Gß
have distinct downstream effectors and function independently to affect
nuclear and spindle movements; (2) Gß
is the major regulator while
the less-active nuclear and spindle movements seen in G
mutant embryos
are due to gain of Gß
activity; and (3) G
is the major
regulator while the hyperactive nuclear and spindle movements seen in the
Gß
mutant embryos are gain of G
phenotypes. Simultaneous
depletion of both G
and Gß should distinguish among these
possibilities. Previous triple RNAi analyses were interpreted as support for
the first hypothesis, however those analyses did not examine all of the
phenotypes reported here. Therefore, we re-examined G
;
Gß double mutants using RNA interference of gpa-16 and
gpb-1 function in a goa-1 mutant background.
The phenotype of goa-1(n1134); gpa-16(RNAi); gpb-1(RNAi) embryos
(n=13) was indistinguishable from that of G single
mutant embryos. Significantly, the centration defects and hyperactive nuclear
movements during prophase and metaphase that are indicative of Gß
depletion were completely suppressed (Fig.
1A, Table 1).
During anaphase, neither spindle pole exhibited oscillations and division was
symmetric. In addition, these embryos showed the nuclear mispositioning defect
and multiple nuclei seen in G
embryos
(Fig. 1A). Control RNAi
experiments done in parallel using RNAi of gpb-1 in wild-type worms
produced the characteristic gpb-1 phenotype, and RNAi of
gpb-1 in goa-1(n1134) worms produced a phenotype
intermediate between that of goa-1 and gpb-1 mutant embryos
(not shown). These results indicate that RNAi of gpb-1 was effective
and therefore we conclude that G
loss of function is epistatic to
Gß loss of function. The absence of the nuclear rocking and centration
defects in the G
;Gß double mutants is consistent
with the hypothesis that the hyperactive movements of nuclei in
gpb-1(RNAi) embryos are due to excess G
activity, rather than
the loss of a gpb-1 specific function.
Our results are in contrast to previous studies in which
G;Gß triple RNAi embryos were generated that
exhibited intermediate or additive phenotypes compared with G
and Gß single RNAi mutants
(Gotta and Ahringer, 2001
;
Srinivasan et al., 2003
). Our
use of a strong loss-of-function mutant and only double RNAi may have resulted
in a stronger phenotype because the efficacy of RNAi drops for some genes when
carried out using three or more RNAs
(Gonczy et al., 2000
). In
addition, G
and Gß
naturally sequester each other, and
reducing one subunit will release and increase the free form of the other
subunit. Thus, if G
double RNAi does not completely deplete the
protein, any remaining G
will probably be sequestered by excess
Gß
, resulting in a strong G
phenotype. This G
could
then be released and become active when Gß is removed by RNAi, resulting
in a `synthetic' intermediate or weaker phenotype.
RNA interference of the receptor independent G-protein regulators
GPR-1/2 produces a similar phenotype to that of G(RNAi) and
suppresses the Gß(RNAi) phenotype
Recent work has shown that two C. elegans homologs of receptor
independent activators of G protein signaling, called GPR-1 and GPR-2
(GPR-1/2), are involved in spindle positioning. gpr-1/2(RNAi) embryos
have a phenotype very similar to that of G (RNAi)
(Gonczy et al., 2000
;
Srinivasan et al., 2003
;
Colombo et al., 2003
;
Gotta et al., 2003
), and thus
GPR-1/2 is thought to be required positively for G
signaling. Based on
studies of GPR homologs in other systems
(Schaefer et al., 2001
), loss
of GPR should result in more inactive G
Gß
trimeric
complexes and thus cause a loss of function for both G
and Gß. The
strong similarity in phenotype between G
(RNAi) and
gpr-1/2(RNAi) embryos is thus consistent with our interpretation that
the Gß phenotype is due to excess G
, rather than to a loss of
Gß specific effector function. Furthermore, if this hypothesis is
correct, then gpr-1/2 should mimic G
in all double mutant
combinations.
To provide a baseline for double mutant analysis, we first examined the
gpr-1/2(RNAi) phenotype using RNA interference of gpr-2.
Because gpr-2 is 96% identical to gpr-1 at the nucleotide
level, RNAi is expected to inhibit the function of both genes. Antibody
staining with anti-GPR-2 antibodies showed that RNA interference did deplete
GPR-1/2 protein (see below). In gpr-1/2(RNAi) one-cell embryos, less
active nuclear and spindle movements like those seen in G mutants were
observed (Fig. 2). During
prophase, no rocking of the nucleus was observed (n=13). During
anaphase, no oscillations of the spindle poles were observed in any embryos,
but in many embryos (62%) the spindle still elongated asymmetrically toward
the posterior pole. This asymmetric spindle elongation in
gpr-1/2(RNAi) embryos was reduced compared with wild type, but did
result in slightly unequal cleavage (compare
Fig. 2 with
Fig. 1,
Table 1). Nuclear
mispositioning after cell division was also observed in gpr-1/2(RNAi)
cells (Fig. 2), as in G
mutants.
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Asymmetric localization of GPR-1/2 at the cortex depends on PAR-3 and
LET-99
GPR-1/2 were recently shown to be asymmetrically localized at the cell
cortex in response to PAR proteins (Colombo
et al., 2003; Gotta et al.,
2003
). Because LET-99 is also asymmetrically localized in a
PAR-dependent manner, we sought to determine the relationship between PAR-3,
LET-99 and GPR-1/2 localization. We first confirmed the asymmetric
localization of GPR using affinity-purified antibodies against a full-length
GPR-2 fusion protein, which are also expected to recognize the 97% identical
GPR-1 protein. In early wild-type embryos, GPR-1/2 were localized both on the
asters and the cell cortex (Fig.
3A-I). These staining patterns were absent in
gpr-1/2(RNAi) embryos, suggesting that they are specific
(Fig. 3J); by contrast, nuclear
staining observed in all cells was present in gpr-1/2(RNAi) embryos,
suggesting that this staining is not GPR-specific
(Fig. 3J). The cortical
localization of GPR-1/2 changed with the cell cycle. In one-cell embryos,
GPR-1/2 were uniformly present at a low level on the cortex from early
prophase to metaphase. The level of cortical localization of GPR-1/2 increased
and became weakly enriched at the posterior pole of most embryos during
anaphase (78%, n=42; Fig.
3C). In some embryos with enriched posterior staining, GPR-1/2
also appeared to be slightly enriched at the anterior pole of the embryo,
compared with lateral regions (Fig.
3D and Fig. 4B).
GPR-1/2 asymmetry became more pronounced during cytokinesis and interphase of
the two-cell stage. In the P1 blastomere during interphase
(Fig. 3E,F), GPR-1/2 were
highly enriched around the posterior pole of the cell (100%, n=53),
and were present at low levels uniformly around the cortex of AB. As the cell
cycle progressed, GPR-1/2 asymmetry in P1 disappeared; GPR-1/2 were
uniformly localized around the cortex through out prophase, metaphase and
early anaphase (Fig. 3G,H).
During late anaphase and telophase, GPR-1/2 were once again enriched at the
posterior part of the P1 cell
(Fig. 3I). These results
indicate that GPR-1/2 is asymmetrically localized in the P lineage.
|
Recent studies showed that asymmetric enrichment of GPR-1/2 in the P
lineage depends on the PAR-3 polarity protein, and it was observed that
GPR-1/2 levels are high in both the AB and P1 cells just after division in
par-3 embryos (Colombo et al.,
2003; Gotta et al.,
2003
). Our observations confirm this result, but we also note that
the higher levels appear late in the first cell cycle. In prophase and
metaphase of one-cell par-3 embryos, cortical GPR-1/2 staining
appeared similar to wild type (compare Fig.
4E with 4A). During late anaphase and telophase in one-cell
par-3 embryos, the posterior enrichment of GPR-1/2 was not observed
(n=11; Fig. 4F) and
instead the entire cortex showed higher staining intensity for GPR. During
interphase of the next cell cycle, strong GPR1/2 staining was observed in both
daughter cells (n=13; Fig.
4G), instead of being restricted to the posterior pole of P1
(Fig. 4C). In some embryos
GPR-1/2 levels appeared higher at the poles of both AB and P1
(Fig. 4G). In four-cell
embryos, during interphase, many cells showed a cap of GRP-2 enrichment at the
cell periphery away from cell contact regions (n=17;
Fig. 4H,I).
Next, we asked if the PAR-dependent asymmetric enrichment of GPR-1/2 is
mediated through the let-99 gene, as LET-99 functions in spindle
positioning and is asymmetrically localized in response to PAR-3
(Tsou et al., 2002). In
let-99 embryos, where the PAR proteins are distributed normally
(Rose and Kemphues, 1998a
),
the posterior enrichment of GPR-1/2 was no longer observed. Instead GPR-1/2
were uniformly distributed around the entire cortex of P0
(n=19; Fig. 4J-M).
Interestingly, unlike in par-3 embryos, the fluorescence intensity of
the cortical GPR-1/2 during prophase appeared higher than in wild-type
embryos. This change in GPR staining during prophase appeared similar to that
seen in Gß mutant embryos
(Fig. 4P). In early interphase
of the two-cell stage, no polar enrichment was seen in P1
(n=17; Fig. 4N), and
the staining intensity of the AB cell cortex appeared similar to that of the
P1 cortex. These data indicate that the asymmetry of GPR-1/2
localization requires both PAR-3 and LET-99. Conversely, in
gpr-1/2(RNAi) embryos, LET-99 is asymmetrically enriched at the
cortex of P lineage cells as in wild-type embryos
(Fig. 4O; n=14).
Together these results support the hypothesis that LET-99 acts upstream of, or
at the level of G
/GPR-1/2 signaling.
LET-99 functions antagonistically to G/GPR-1/2 signaling
pathway in the P lineage
We have shown that LET-99 is required for asymmetric GPR-1/2 localization.
However, the cortical localizations of LET-99 and GPR-1/2 in cells do not
overlap but instead are somewhat reciprocal at anaphase. In addition, the
hyperactive and dynein-dependent nuclear and spindle oscillations exhibited by
let-99 mutants (Tsou et al.,
2002) are similar to those shown here for Gß mutant embryos,
which appear to be due to excess G
/GPR-1/2 activity
(Fig. 4K). Together, these
observations suggest that let-99 functions antagonistically to
G
/GPR-1/2 signaling.
To test this hypothesis, we examined G(RNAi);
let-99(or81) and gpr-1/2(RNAi); let-99(or81) double mutant
embryos during the first cell cycle. In both G
(RNAi);
let-99 and gpr-1/2(RNAi); let-99 double mutant embryos, the
phenotype resembled that of G
(RNAi) embryos alone. In
particular, the let-99 centration defects and nuclear and metaphase
rocking phenotypes were completely suppressed
(Fig. 5;
Table 1), consistent with the
idea that the hyperactive nuclear movements observed in let-99
embryos are due to an excess of G
/GPR-1/2 signaling. During anaphase in
G
(RNAi); let-99 and gpr-1/2(RNAi); let-99
double mutant embryos, the spindle poles did not exhibit oscillations, and
spindle elongation and first cleavage were symmetric as seen in
G
(RNAi) embryos
(Fig. 5). Two-cell double
mutant embryos also showed G
phenotypes, such as mispositioned nuclei
(Fig. 5). Interestingly, the
slightly asymmetric spindle elongation movements observed during anaphase in
gpr-1/2(RNAi) embryos were not observed in
gpr-1/2(RNAi);let-99 double mutant embryos (compare
Fig. 2 with
Fig. 5,
Table 1). The remaining
asymmetry in gpr-1/2(RNAi) embryos suggests that although force
generation is greatly reduced in these embryos, the spindle is still
responding to an asymmetric cue. The loss of asymmetry in
gpr-1/2(RNAi);let-99 embryos thus suggests that LET-99 is part of
that asymmetric cue.
|
GPR-1/2 and LET-99 are asymmetrically localized in opposite patterns
at the EMS/P2 boundary in response to MES1/SRC-1 signaling
To see if G/GPR-1/2 and LET-99 are also involved in the asymmetric
division of EMS cells, we first determined the localization of both proteins.
Strikingly, in EMS cells from prophase to prometaphase, when nuclear rotation
normally occurs, GPR-1/2 were asymmetrically enriched at the EMS/P2
boundary (Fig. 6B,C,G,I;
n=33). As we previously reported, there is no cortical LET-99 band in
the EMS cell. However, we observed a cell cycle dependent change in LET-99
localization at cell contacts. In contrast to the enrichment of GPR-1/2 at
prophase, LET-99 was greatly reduced at the EMS/P2 boundary
compared with other cell boundaries during the same stage
(Fig. 6E,F,H,I; n=25),
even though LET-99 was initially present at this boundary during interphase
(Fig. 6D). To test whether
GPR-1/2 and LET-99 asymmetries depend on either of the signaling pathways
known to function in EMS spindle orientation, we examined their localization
in mutant embryos defective in either the MES-1/SRC-1 or the Wnt signaling
pathways. In mom-5 mutant embryos (MOM-5 is the Frizzled receptor in
the Wnt pathway), GPR-1/2 and LET-99 asymmetries were still observed at the
EMS /P2 boundary (Fig.
6J, n=9 and 11 embryos respectively), just as in wild
type. However, in mes-1 mutant embryos, GPR-1/2 were no longer
enriched at the EMS/P2 boundary
(Fig. 6J, n=14).
Similar results were recently reported for GPR-1/2 asymmetry by others
(Srinivasan et al., 2003
).
Interestingly, LET-99 was now present at the EMS/P2 boundary during
prophase/prometaphase in mes-1 mutant embryos
(Fig. 6J, n=20).
Similar results were obtained in mom-5; mes-1 double mutants (data
not shown). Thus, the asymmetric patterns of both LET-99 and GPR-1/2 at the
EMS/P2 cell boundary are MES-1/SRC-1 signaling dependent, which
suggests that LET-99 and G
signaling act downstream of MES-1/SRC-1 to
promote spindle orientation in EMS.
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![]() |
Discussion |
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The G proteins and GPR-1/2 are localized at the cortex and the microtubule
asters. The adapter protein LIN-5 forms a complex with G and GPR-1/2,
and is required for the overall cortical and astral localization of the
G
/GPR-1/2 complex (Srinivasan et
al., 2003
). G
signaling at either the cortex or the asters
could influence microtubules and their interactions with the cortex, and thus
regulate force generation. Several observations support the hypothesis that
the cortex is the active site of G
signaling. Previous work showed that
G
is absent from the asters in Gß mutant embryos but is
still present at the cortex (Gotta and
Ahringer, 2001
). The observation that the phenotype of
Gß mutants is opposite to that of G
mutants thus
suggests that the asters are not the sites of G
signaling for spindle
positioning. In addition, the cortical localization of GPR-1/2 depends on
G
but not Gß (Colombo et al.,
2003
) (this report). Indeed, there appears to be more GPR on the
cortex during prophase in Gß mutants, and loss of G
/GPR
activity suppressed the Gß phenotype. These correlations suggest that the
primary site of G
/GPR-1/2 activity for spindle positioning is at the
cortex.
The conclusion that G/GPR-1/2 are the key regulators of spindle
positioning in C. elegans is different from what has been reported in
Drosophila neuroblast cells
(Schaefer et al., 2001
). When
GTP-G
was overexpressed in neuroblasts, no spindle orientation
phenotype was observed. However, when GDP-G
was overexpressed, random
spindle orientations were seen, similar to those observed in Gß13F
mutants (which lack both G
and Gß13F)
(Schaefer et al., 2001
). It
was predicted that GDP-G
would sequester the free Gß
, and
thus it was concluded that Gß
is the key signaling molecule that
regulates polarity and spindle orientation in neuroblast cells
(Schaefer et al., 2001
). The
difference between Drosophila and C. elegans could reflect
differential usage of conserved molecules. An alternative interpretation is
that overexpression of GDP-G
could result in a gain-of-function spindle
orientation phenotype, as we have shown here for loss of Gß. That is,
loss or gain of G
activity could both produce defects in spindle
orientation, which may not be distinguishable without live imaging of the
spindle movements.
LET-99 serves as an asymmetric cue that functions antagonistically to
the G/GPR-1/2 signaling pathway
Forces must also be polarized in response to PAR polarity cues in order to
achieve proper spindle positioning. The localization of GPR-1/2 has led to the
model that the enrichment of GPR-1/2 at the posterior provides higher pulling
forces on the posterior spindle pole, thus mediating anaphase spindle
positioning (Srinivasan et al.,
2003; Colombo et al.,
2003
; Gotta et al.,
2003
). This model does not address a role for GPR in nuclear
rotation, however. Posterior enrichment of GPR-1/2 was seen in only some
embryos during nuclear rotation (Colombo et
al., 2003
). Such asymmetry at this time is actually predicted to
be counter-productive, as it would potentially hold the nucleus at the
posterior and prevent centration and rotation.
We previously proposed that the asymmetric enrichment of LET-99 in a
cortical band provides the asymmetric cue to polarize forces during both
rotation and anaphase (Tsou et al.,
2002). Loss of LET-99 results in an absence of nuclear rotation
and an absence of the normal asymmetric spindle pole movements during anaphase
(Rose and Kemphues, 1998a
;
Tsou et al., 2002
). Based on
the hyperactive movements of nuclei and metaphase spindles, we proposed that
the ultimate effect of LET-99 activity is a downregulation of cortical forces
that act on centrosomes. Because LET-99 is enriched in a cortical band that
encircles P lineage cells, downregulation of cortical forces in this region
during prophase would result in higher net anterior and posterior forces that
would produce a rotational movement of the nuclear-centrosome complex
(Fig. 8, left). After rotation,
the posterior centrosome/spindle pole lies partially underneath the LET-99
band. Downregulation of cortical forces in the LET-99 band region at this
stage would affect lateral astral microtubule interactions, producing higher
net forces directed towards the posterior and thus asymmetric anaphase spindle
elongation (Tsou et al.,
2002
). The results reported here on the genetic interactions
between LET-99 and G
/GPR signaling are consistent with this model. Loss
of LET-99 causes gain of G
/GPR-1/2-like phenotypes, hyperactive nuclear
and spindle movements. These hyperactive movements are completely suppressed
in G
(RNAi); let-99 or gpr-1/2(RNAi); let-99
mutant embryos, suggesting that LET-99 opposes G
/GPR-1/2 signaling. The
antagonistic role of let-99 to G
/GPR-1/2 signaling is further
supported by the observation that partially reducing let-99 activity
suppresses the lethality caused by loss of gpa-16 activity alone.
Finally, the weak asymmetry of spindle positioning that we observed in
gpr-1/2(RNAi) embryos was no longer observed in gpr-1/2(RNAi);
let-99 double mutant embryos. These results suggest that let-99
not only functions oppositely to G
/GPR-1/2 signaling, but also indeed
provides an asymmetric cue. Based on these results and the pattern of cortical
LET-99 localization, we propose that LET-99 antagonizes G
/GPR-1/2
signaling, thus downregulating cortical forces asymmetrically during both
rotation and anaphase spindle elongation.
|
The models for GPR-1/2 and LET-99 function during anaphase are not mutually
exclusive. Indeed, LET-99 could be acting solely through localization of
GPR-1/2. However, both LET-99 and G are also required for
polarity-dependent nuclear rotation in the one cell, when asymmetry of GPR-1/2
is not evident, which suggests that the main role of LET-99 may not be
localization of GPR-1/2. Rather, we propose that LET-99 antagonizes
G
/GPR-1/2 signaling in addition to, or as part of, its effect on
GPR-1/2 localization. One speculative model that fits the current data is that
LET-99 directly or indirectly inhibits the association of G
and
GPR-1/2. This would downregulate G
signaling in the region of the
LET-99 band, causing nuclear rotation during prophase. As the cell cycle
progresses, the dissociated GPR-1/2 would then be free to reassociate with the
posterior cortex where neither PAR-3 nor LET-99 is present at high levels.
During anaphase, the inhibition of G
signaling by LET-99, the
enrichment of GPR-1/2 posteriorly, or both could function in asymmetric
anaphase spindle elongation. Biochemical experiments and identification of
LET-99-interacting proteins will be required to elucidate the molecular
mechanism of the interactions of LET-99 with G-protein signaling.
We also found that Gß function antagonized G/GPR signaling.
However, it appears that LET-99 acts separately from Gß. Significantly,
Gß mutant embryos exhibit late nuclear rotation and asymmetric
oscillations of spindle poles during anaphase, neither of which occurs in
let-99 embryos (this report)
(Tsou et al., 2002
). The
asymmetric localization of GPR-1/2 was also observed in Gß
mutants but not in let-99 mutants. These observations strongly
suggest that the cues for polarizing forces that act on centrosomes still
exist in Gß mutants but not in let-99 mutant embryos,
and that polarized force is essential for asymmetric spindle oscillations
during anaphase. Although a loss of polarized forces is consistent with the
absence of wild-type anaphase spindle pole oscillations in let-99
embryos, it is unclear why random oscillations similar to prophase nuclear
rocking are not seen at anaphase. It is possible that changes in microtubule
to cortex interactions during the cell cycle could explain this phenotypic
effect. In anaphase, more astral microtubules appear to reach the cortex and
these microtubules are more cold stable than prophase microtubules (L. R.
DeBella and L.S.R., unpublished results). With a large number of
cortical-microtubule contacts during anaphase, the stochastic loss or gain of
a few contacts would have little effect on the balance of forces. By contrast,
with fewer microtubule contacts during prophase, a similar stochastic effect
could lead to a dramatic imbalance of forces and hence cause nuclear rocking.
Alternatively, we cannot rule out the possibility that LET-99 functions
differently during prophase and anaphase to regulate forces.
G proteins and LET-99 function in spindle positioning in both
intrinsically and extrinsically determined asymmetric divisions
We propose that the fundamental roles of G signaling and LET-99 are
the same in both the P lineage and the EMS cell: G
upregulates force
generation from the cortex on centrosomes, and LET-99 downregulates force
generation. Furthermore, we propose that the differences in the spatial and
temporal regulation of LET-99 and GPR-1/2 localization provide an explanation
for the different types of movements that lead to asymmetric spindle placement
and orientation in these two cell types
(Fig. 8). In P lineage cells,
an intrinsic PAR-dependent mechanism causes `free nuclear rotation' that
occurs in the center of the cell when the extrinsic effects of cell shape are
removed (Tsou et al., 2003
).
Distinct asymmetric elongation movements then position the spindle towards the
posterior during anaphase. In these cells, GPR-1/2 is uniformly localized in
most cells at the time of rotation, but LET-99 is enriched in a band and
antagonizes G
/GPR-1/2 activity as proposed earlier
(Fig. 8, left). In contrast to
free central rotation, previous studies have shown that nuclear rotation in
the EMS cell occurs directly toward the EMS/P2 boundary in both
intact embryos and in isolated blastomeres
(Schlesinger et al., 1999
). We
showed that in EMS cells LET-99 is not present as a band, but rather is absent
from the EMS/P2 cell contact region, where GPR-1/2 localization is
enriched. Both patterns are present at the time of nuclear rotation, and the
opposite localization of these two proteins at the EMS/P2 boundary
is consistent with our model that LET-99 antagonizes G
/GPR-1/2
signaling. The absence of LET-99 would be necessary in order for the enriched
GPR-1/2 at the cell boundary to have highest activity. High G
/GPR-1/2
activity could then cause nuclear rotation directed towards the
EMS/P2 boundary, simultaneously positioning the spindle
asymmetrically on the AP axis (Fig.
8, right). Interestingly, we also found that LET-99 is required
for the asymmetry of GPR-1/2 at the EMS/P2 boundary. Although we
cannot rule out a role for LET-99 in the ability of the P2 cell to
signal to EMS and thus produce an enrichment of GPR-1/2, LET-99 could function
in the EMS cell in a manner analogous to that proposed for the P lineage. That
is, the presence of LET-99 at the other cell contacts could inhibit
G
/GPR-1/2 signaling there, further enhancing the asymmetry of
G
/GPR-1/2 signaling. The inhibition could also result in more free
GPR-1/2, which would then associate with the EMS/P2 boundary in
response to MES-1 signaling.
In let-99 mutants, nuclear rotation in the EMS failed completely
while in mes-1 mutants, nuclear rotation failed in only 10% of EMS
cells (Bei et al., 2002).
Similarly, the gpa-16 EMS rotation phenotype is stronger than that of
mes-1 mutants. This is not surprising, because mes-1 mutants
cause a loss of asymmetry of LET-99 and GPR-1/2, not a total loss of protein.
Similarly, mutations in par-3 result in symmetric GPR-1/2 and LET-99
localization during prophase in the P lineage, but par-3, let-99 and
gpr-1/2 mutants have different nuclear rotation phenotypes.
Nonetheless, the finding that let-99 and G
mutant EMS
cells have stronger defects in rotation suggest that these proteins play a
basic role in the interaction between microtubules and the cortex in multiple
cells. Consistent with this view, both G
and let-99 mutants
have defects in nuclear and centrosome positioning in the AB lineage
(M.-F.B.T. and L.S.R., unpublished) (Gotta
and Ahringer, 2001
; Rose and
Kemphues, 1998a
). In addition, the activity of LET-99 and GPR-1/2,
rather than their localization, could also be modulated in the EMS cell in
response to Wnt signaling, which acts redundantly with MES-1 to promote EMS
spindle orientation.
In summary, our results provide evidence that in C. elegans as in
Drosophila, G-protein signaling is used for spindle positioning in
asymmetric divisions that are both intrinsically and extrinsically determined
(Knoblich, 2001). Further work
to elucidate the molecular mechanisms by which G protein signaling and LET-99
regulate forces on microtubules during asymmetric division will provide
insight into both types of divisions.
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ACKNOWLEDGMENTS |
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Footnotes |
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