1 Swiss Institute for Experimental Cancer Research (ISREC), Swiss Federal
Institute of Technology (EPFL), CH-1066 Lausanne, Switzerland
2 Department of Pharmacology, Lineberger Comprehensive Cancer Center and
Neuroscience Center, The University of North Carolina, Chapel Hill, NC
27599-7365, USA
* Author for correspondence (e-mail: pierre.gonczy{at}isrec.unil.ch)
Accepted 16 August 2005
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SUMMARY |
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Key words: C. elegans embryos, Spindle positioning, G protein
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Introduction |
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The one-cell stage C. elegans embryo has emerged as an attractive
model to analyze the mechanisms of cell-intrinsic spindle positioning during
unequal cell division (for a review, see
Schneider and Bowerman, 2003).
In the wild type, in response to anteroposterior (AP) polarity cues, the
spindle is displaced towards the posterior by the end of anaphase, resulting
in unequal cleavage into a larger anterior and a smaller posterior blastomere.
Asymmetric spindle displacement results from unbalanced cortical force
generators acting on astral microtubules and pulling on spindle poles
(Grill et al., 2001
;
Grill et al., 2003
). Because
more force generators are active on the posterior cortex, there is a larger
net force pulling on the posterior spindle pole
(Grill et al., 2003
).
Although the molecular nature of cortical force generators is not known,
their activity relies on two subunits of the heterotrimeric
G-proteins: GOA-1 and GPA-16 (see Fig. S1 in the supplementary material).
These components act in a partially redundant manner, as pulling forces are
decreased in only a modest manner in embryos lacking either GOA-1 or GPA-16
(Afshar et al., 2004
), still
allowing asymmetric spindle elongation and unequal cleavage
(Gotta and Ahringer, 2001
). By
contrast, simultaneous inactivation of GOA-1 and GPA-16 results in an extreme
decrease of pulling forces (Colombo et al.,
2003
), yielding symmetric spindle elongation and equal first
cleavage (Gotta and Ahringer,
2001
). Conversely, inactivation of Gß
results in
excess pulling forces (Afshar et al.,
2004
). Furthermore, the triple inactivation of GOA-1, GPA-16 and
Gß
also yields an equal first cleavage, as in
goa-1/gpa-16(RNAi) embryos
(Gotta and Ahringer, 2001
;
Tsou et al., 2003
), indicating
that Gß
dampens G
-dependent force generation in one-cell
stage embryos.
A phenotype analogous to that of goa-1/gpa-16(RNAi) is
observed after inactivation of GPR-1/2
(Colombo et al., 2003;
Gotta et al., 2003
;
Srinivasan et al., 2003
), a
GoLoco protein which acts as a guanine nucleotide dissociation inhibitor (GDI)
for GOA-1 (Afshar et al., 2004
;
Gotta et al., 2003
) or of
LIN-5, a coiled-coil protein that physically interacts with GPR-1/2
(Lorson et al., 2000
;
Srinivasan et al., 2003
).
GPR-1/2 and LIN-5 are present at the cortex of one-cell stage embryos
(Colombo et al., 2003
;
Gotta et al., 2003
;
Srinivasan et al., 2003
).
During mitosis, cortical GPR-1/2 distribution is asymmetric, with a slight
enrichment at the posterior (Colombo et
al., 2003
; Gotta et al.,
2003
; Tsou et al.,
2003
). This raises the possibility that GPR-1/2 is responsible for
the larger net pulling force exerted on the posterior spindle pole. RIC-8,
which acts as a guanine nucleotide exchange factor (GEF) for GOA-1
(Afshar et al., 2004
;
Hess et al., 2004
), is also
required for pulling force generation
(Afshar et al., 2004
).
Co-immunoprecipitation experiments and biochemical analyses indicate that
RIC-8 is required for the interaction between GPR-1/2 and GOA-1, raising the
possibility that RIC-8 acts before GPR-1/2 in the GOA-1 activation cycle
(Afshar et al., 2004
).
Moreover, inactivation of the Gß subunit GPB-1 alleviates the requirement
for RIC-8 during spindle positioning, suggesting that RIC-8 promotes
generation of GOA-1 free from Gß
, thus making it available for
binding to GPR-1/2 (Afshar et al.,
2004
) (for a review, see
McCudden et al., 2005
).
Whereas GOA-1 is known to be present at the cortex of early embryos
(Afshar et al., 2004;
Gotta and Ahringer, 2001
;
Miller and Rand, 2000
), the
subcellular distribution of GPA-16 has not been investigated. Moreover,
although yeast two-hybrid assays indicate that GPA-16 can physically interact
with RIC-8 (Afshar et al.,
2004
) and GPR-1/2 (Li et al.,
2004
), the latter interaction was not detected in two other
studies (Colombo et al., 2003
;
Gotta et al., 2003
), and
whether such interactions occur in C. elegans embryos is not known.
Furthermore, the consequence of putative interactions of GPA-16 with RIC-8 and
GPR-1/2 has not been investigated. In addition, the nature of the partial
redundancy between GPA-16 and GOA-1 has not been addressed. For example,
GPA-16 and GOA-1 could each be essential for activation of separate pathways
that trigger distinct effectors, which together ensure force generation.
Alternatively, GPA-16 and GOA-1 could each contribute to partial activation of
the same pathway.
We investigated these issues in this study. We report that GPA-16 is
present predominantly at the cell cortex and that it interacts with RIC-8 and
GPR-1/2, both in vitro and in vivo. We show that GPA-16 and GOA-1 become
entirely redundant after GPB-1 inactivation, suggesting that the two G
proteins can activate the same pathway. Furthermore, we establish that GPR-1/2
acts as a GDI for GPA-16, whereas RIC-8 does not act as a GEF for GPA-16.
Importantly, we find that RIC-8 is required for GPA-16 cortical localization
and that this novel requirement is distinct from its known role in enabling
interaction between G
proteins and GPR-1/2.
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Materials and methods |
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Bacterial RNAi feeding strains were as described
(Afshar et al., 2004;
Colombo et al., 2003
). The
conditions for RNAi by feeding were as follows, starting with L3/L4 larvae:
gpa-16, goa-1, gpb-1 and gpr-1/2: 48-60 hours at
20°C; ric-8: 40-44 hours at 20°C. RNAi-mediated inactivation
of ric-8 in ric-8(md1909) mutant animals was performed as
described (Afshar et al.,
2004
). For experiments where two genes were inactivated with RNAi,
appropriate controls were performed in parallel to ensure the efficiency of
each RNAi condition.
Microscopy and spindle severing
Time-lapse DIC microscopy was performed capturing 1 image every 5 seconds
(Gönczy et al., 1999).
Spindle severing experiments and measurement of peak velocities were performed
essentially as before (Grill et al.,
2001
), using a Leica LMD microscope equipped with a pulsed N2
laser (
=337 nM).
Antibody production and use
For generating GPA-16 antibodies, the full-length gpa-16 cDNA
(Colombo et al., 2003) was
cloned into pGEX-6P-2. GST-GPA-16 was expressed, purified from inclusion
bodies, run on an SDS-PAGE gel and injected into a rabbit (Eurogentec).
Antibodies were strip-purified against GST-GPA-16, eluting with 0.1 M glycine
(pH 2.5). Affinity-purified antibodies were dialyzed against PBS and kept at
-20°C in 50% glycerol.
For revealing RIC-8 following immunoprecipitation with GPA-16 antibodies
(Fig. 2A,
Fig. 5E), we generated RIC-8
antibodies by first cloning the full length ric-8 cDNA into a pGEX
derivative. Bacterially expressed GST-RIC-8 was purified from inclusion
bodies, purified on an SDS-PAGE gel and injected into a rabbit (Eurogentec).
Antibodies were column affinity purified against His6-RIC-8 bound
to a HiTrap NHS Hp column (Pharmacia), dialyzed and stored as above. These
antibodies (see Fig. S4 in the supplementary material) were used following
immunoprecipitation with GPA-16 antibodies in place of previously described
antibodies (Miller et al.,
2000) (which were used in the case of
Fig. 2B) because they are more
sensitive.
Fixation and staining of embryos for indirect immunofluorescence was as
described (Afshar et al.,
2004). The following primary antibodies were used: 1:200 mouse
anti-
tubulin (DM1A, Sigma), 1:300 rabbit anti-GPA-16 (this study),
1:300 rabbit anti-GOA-1 (Afshar et al.,
2004
) and 1:150 rabbit anti-GPR-1/2
(Colombo et al., 2003
).
Secondary antibodies were 1:500 goat anti-mouse conjugated to Alexa-488
(Molecular Probes) and 1:1000 goat anti-rabbit conjugated to Cy3. Slides were
counterstained with
1 µg/ml Hoechst 33258 (Sigma) to detect DNA.
Approximately 1 µm optical slices were collected on an LSM510 Zeiss
confocal microscope and processed in Adobe Photoshop. Quantification of
GPR-1/2 signal was performed on images collected on a Zeiss Axioplan 2 with at
12-bit Diagnostic Instrument Spot RT Camera controlled by Metamorph software
(Universal Imaging). Average cortical intensities were determined in a
5
µm-long region between the EMS and ABp blastomeres, after subtraction of
the average cytoplasmic signal from a neighboring region.
Generation of embryonic extracts, immunoprecipitation (using 3 µg
of GPA-16 antibodies without addition of guanine nucleotides, unless specified
otherwise) and western blot analysis were performed as described
(Afshar et al., 2004
). All
primary antibodies for western blot analysis were used at 1:1000, except the
new RIC-8 antibodies, which were used at 1:2000. For secondary antibodies, we
used 1:10,000 HRP-conjugated goat anti-rabbit (Amersham), except when
revealing GPR-1/2, where HRP-conjugated Protein A (Amersham) was used at
1:2000 in place of secondary antibodies because the heavy chain of
immunoglobulins is similar in size to GPR-1/2.
Protein purification
Purification of GOA-1 (amino acids 28-351) and His6-RIC-8 was as
described (Afshar et al.,
2004). The RIC-8 open reading frame (ORF) was cloned into
pGEX4TEV2 (Kimple et al.,
2004
) and GST-RIC-8 purified by chromatographic methods as
previously described (Willard and
Siderovski, 2004
). We have previously observed aggregation of
RIC-8 during purification (Afshar et al.,
2004
); accordingly chromatography buffers were supplemented with
400 mM NaCl and 5-10% (v/v) glycerol. GST-GPR-1/2 (amino acids 374-476) was
cloned into pGEX4TEV2 and purified using the methods described above.
All baculoviral and insect cell culture reagents were obtained from
Invitrogen. DNA encoding GPA-16 (amino acids 5-357) was cloned into
pFastBacHTb and recombinant bacmid DNA was generated using the Bac-to-Bac
method. Insect cell transfection and viral amplification were performed by the
Tissue Culture Core Laboratory (University of Colorado Cancer Center). Protein
was expressed by infecting Hi5 cells (1.0x106 cells/ml) grown
in Express Five SFM (containing 16.5 mM L-glutamine) with baculovirus at a
multiplicity of infection of 1.0. After incubation at 27°C for 2 days,
cells were pelleted by centrifugation at 3000 g, and
resuspended in buffer N [50 mM Tris/HCl (pH 7.5), 100 mM NaCl, 5% (v/v)
glycerol, 10 mM imidazole, 50 µM GDP, 20 mM NaF, 30 µM AlCl3,
5 mM MgCl2, 0.5% (w/v) sodium cholate and 1 mM 2-mercaptoethanol].
His6-GPA-16 was then purified using Ni2+-affinity
chromatographic methods as described
(Willard and Siderovski,
2004). Protein-containing fractions were pooled and subjected to
HiTrapQ (Amersham) anion exchange chromatography. The eluent was 20 mM
Tris/HCl (pH 8.0), 10 mM NaCl, 5% (v/v) glycerol, 10 µM GDP and 1 mM DTT;
protein was eluted with a linear gradient of 0-300 mM NaCl over 20 column
volumes. Protein fractions were analyzed for the presence GPA-16 by
SDS-PAGE/Coomassie Blue staining, immunoblot with
-His6
antibodies (Covance) and GTP
S binding. His6-GPA-16
containing fractions were pooled and concentrated using a Vivaspin 30 kDa
cutoff centrifugal filter (Sartorius).
Biochemical assays
[35S]-GTPS binding assays were performed as described
(Afshar et al., 2004
). Rates of
GTP
S binding were calculated using a one-site exponential model (Prism
v4.0; GraphPad Software). Protein-protein interactions were measured by
surface plasmon resonance (SPR) spectroscopy as described
(Kimple et al., 2004
). Eluent
buffer [20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 0.005% (v/v)
NP-40] was supplemented with GDP (50 µM), GTP
S (50 µM), or
GDP·AlF4- (50 µM GDP, 20 mM NaF, 30 µM
AlCl3). GOA-1 and GPA-16 were diluted to 2 µM in the GDP,
GTP
S, and GDP·AlF4- buffers, and incubated
for 30 minutes at 25°C to ensure full nucleotide loading.
Nucleotide-locked G
proteins (2 µM) were injected (KINJECT, 30
µl, 300 s dissociation time, 5 µl/minute) over an anti-GST
antibody-conjugated CM5 sensor chip, loaded with GST (1500 RU), GST-RIC-8 (800
RU) and GST-GPR-1 (amino acids 374-476) (1400 RU). Background binding to the
GST surface was subtracted from all sensorgrams (BIAevaluation v3.0;
Biacore).
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Results |
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GPA-16 interacts with RIC-8 and GPR-1/2 in vivo
We investigated whether GPA-16 is present in a complex with RIC-8 and
GPR-1/2 in C. elegans embryos. As shown in
Fig. 2A (lane 7), we found that
GPA-16 antibodies co-immunoprecipitate both RIC-8 and GPR-1/2 from wild-type
embryonic extracts. Furthermore, we found that the interaction between GPA-16
and RIC-8 is severely compromised in embryonic extracts derived from
ric-8(md1909) or ric-8(md303) mutant animals
(Fig. 2A, lane 8 and data not
shown). These observations establish that GPA-16 normally associates with both
RIC-8 and GPR-1/2 in vivo.
We next addressed the order in which GPA-16 interacts with RIC-8 and GPR-1/2 by conducting co-immunoprecipitation experiments in embryonic extracts depleted of RIC-8 or GPR-1/2. These experiments established that the interaction between GPA-16 and RIC-8 is not altered in gpr-1/2(RNAi) embryonic extracts (Fig. 2A, lane 11, compare with lane 7). By contrast, the interaction between GPA-16 and GPR-1/2 is essentially abolished in ric-8(md1909) or ric-8(md303) embryonic extracts (Fig. 2A, lane 8, compare with lane 7 and data not shown). We conclude that RIC-8 is required for efficient assembly of a complex containing GPA-16 and GPR-1/2.
These findings prompted us to test whether RIC-8 may activate GPA-16 by
supporting stable levels of Gß-free GPA-16. If this were the case,
then inactivation of Gß
might enable GPA-16 to interact with
GPR-1/2 in the absence of RIC-8. Accordingly, we found that the interaction
between GPA-16 and GPR-1/2 is partially restored in ric-8(md1909)
gpb-1(RNAi) embryos (Fig.
2A, lane 10, compare with lane 8). Therefore, as for GOA-1
(Afshar et al., 2004
),
inactivation of Gß
alleviates the need for RIC-8 to permit
association of GPR-1/2 with GPA-16.
GOA-1 and GPA-16 become completely redundant during asymmetric cell division when GPB-1 is inactivated
As our findings suggest that generation of both GOA-1 and GPA-16 free from
Gß is important for asymmetric spindle positioning, we
investigated the nature of the partial redundancy between the two G
proteins. We reasoned that if GPA-16 and GOA-1 are essential for distinct
pathways, an excess of GPA-16 liberated from Gß
should not
compensate for loss of GOA-1. Similarly, an excess of GOA-1 liberated from
Gß
should not compensate for loss of GPA-16.
To test whether this is the case, we investigated the extent of pulling
forces using laser microbeam-mediated spindle severing experiments, analyzing
the resulting spindle pole movements with time-lapse differential interference
contrast (DIC) microscopy (Grill et al.,
2001). In these experiments, we used goa-1(sa734), a
deletion allele (Robatzek and Thomas,
2000
), gpa-16(it143), a strong reduction of function
allele that results in a G202D substitution in the switch II region of the
GTPase domain (Bergmann et al.,
2003
), and RNAi-mediated inactivation of goa-1 or
gpa-16. We inactivated gpb-1 using RNAi, because
gpb-1 homozygous mutant animals die as larvae
(Zwaal et al., 1996
).
|
An examination of GPR-1/2 cortical distribution in early two-cell stage
embryo corroborated the outcome of the spindle severing experiments
(Fig. 4; see also Figs S2 and
S3 in the supplementary material for a quantitative assessment in four-cell
stage embryos). Cortical GPR-1/2 is essentially absent in
goa-1/gpa-16(RNAi) embryos (Fig.
4B, compare with
4A)
(Colombo et al., 2003;
Gotta et al., 2003
;
Tsou et al., 2003
), as well as
following triple inactivation of goa-1 gpa-16 and gpb-1
(data not shown). By contrast, we found that cortical GPR-1/2 appears merely
diminished compared with wild type in goa-1(RNAi) or
gpa-16(RNAi) embryos (Fig.
4C,D). Similar results were obtained with goa-1(sa734)
and gpa-16(it143) (data not shown). Therefore, GPA-16 and GOA-1 each
contribute to GPR-1/2 cortical targeting. As anticipated from the fact that
gpb-1(RNAi) embryos in which either goa-1 or gpa-16
is also inactivated exhibit an indistinguishable phenotype from
gpb-1(RNAi) embryos, we found that cortical GPR-1/2 is similar to
wild type in such doubly inactivated embryos (compare
Fig. 4F with 4C and 4A, as well
as Fig. 4G with 4D and 4A).
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GPR-1/2 is a GDI for GPA-16, but RIC-8 is not a GEF for GPA-16
Next, we set out to determine whether RIC-8 and GPR-1/2 exhibit the same
biochemical activity towards GPA-16 as they do toward GOA-1. To this end, we
first conducted surface plasmon resonance (SPR) binding assays to investigate
the nucleotide dependency of the interaction between GPA-16 and GPR-1/2, as
well as that between GPA-16 and RIC-8. Recombinant GPA-16 was injected over
SPR surfaces after pre-incubation with either GDP, the non-hydrolyzable GTP
analogue GTPS, or GDP·AlF4- to mimic the
transition state of GTP hydrolysis. We found that a GST fusion protein
encompassing the GoLoco motif of GPR-1/2 binds exclusively to
GPA-16·GDP (Fig. 5A). We
found also that a surface of immobilized GST-RIC-8 binds robustly to
GPA-16·GDP, but to a much lesser extent to GTP
S-bound or
AlF4--activated GPA-16
(Fig. 5B).
|
We reasoned that if RIC-8 also does not act as a GEF towards GPA-16 in
vivo, it could remain associated with GPA-16 even after spontaneous nucleotide
exchange has occurred, especially in light of the slight binding of RIC-8 to
GPA-16·GTPS and
GPA-16·GDP·AlF4- observed in vitro (see
Fig. 5B). Compatible with this
view, we found that GPA-16 antibodies co-immunoprecipitate RIC-8 equally well
in the presence of excess GDP or GTP
S
(Fig. 5G). By contrast, GOA-1
antibodies co-immunoprecipitate RIC-8 preferentially in the presence of excess
GDP (Fig. 5H) (see also
Afshar et al., 2004
). As
expected, we found also that GPA-16 antibodies co-immunoprecipitate GPR-1/2
only in the presence of excess GDP (Fig.
5E), as is the case with GOA-1 antibodies
(Afshar et al., 2004
).
Overall, our findings indicate that whereas GPR-1/2 acts as a GDI towards both GOA-1 and GPA-16, RIC-8 exhibits GEF activity towards GOA-1, but not GPA-16.
RIC-8 ensures normal cortical localization and protein levels of GPA-16
Despite RIC-8 not acting as a GEF towards GPA-16, RIC-8 is required for
GPA-16 function during asymmetric cell division, as RIC-8 inactivation results
in a phenotype analogous to inactivating both GOA-1 and GPA-16
(Afshar et al., 2004). To begin
investigating how RIC-8 exerts its requirement towards GPA-16, we examined the
distribution of GPA-16 in ric-8 mutant embryos. Strikingly, we found
that GPA-16 distribution at the cortex is extremely diminished when RIC-8
function is compromised (Fig.
1E,F, compare with
1C). Moreover, western blot
analysis revealed a severe reduction in GPA-16 protein levels in
ric-8 mutant embryonic extracts
(Fig. 2B, lanes 5-7, compare
with lane 1).
|
Compromising the function of GOA-1 and RIC-8 is more detrimental than compromising that of GPA-16 and RIC-8
The above observations suggest that compromising RIC-8 function may be more
detrimental to embryos depleted of GOA-1 than to embryos depleted of GPA-16,
because GPA-16 levels are already severely compromised in the absence of
RIC-8. To test this prediction, we performed time-lapse DIC microscopy of
one-cell stage embryos. We compromised goa-1 and gpa-16 by
RNAi, rather than through the use of mutants for the following reasons. First,
using time-lapse DIC microscopy and spindle severing, we found that the
spindle positioning phenotypes in goa-1(RNAi) and the deletion allele
goa-1(sa734) are indistinguishable (see Table S1 in the supplementary
material). Moreover, ric-8(md1909) goa-1(RNAi) embryos already have a
penetrant spindle positioning phenotype. Second, null allele of
gpa-16 are not available (Bergmann
et al., 2003), and we found using spindle severing that the
impairment of force generation in gpa-16(RNAi) embryos is as severe
as that in gpa-16(it143) embryos (see Table S1 in the supplementary
material).
In the wild type, asymmetric spindle positioning is accompanied by
transverse oscillations of the posterior spindle pole that reflect the extent
of pulling forces and results in unequal cleavage
(Fig. 6A; see Movie 8 in the
supplementary material). In goa-1(RNAi) or gpa-16(RNAi)
embryos, transverse oscillations are dampened, but asymmetric spindle
positioning is nevertheless achieved, resulting in unequal cleavage
(Fig. 6B,C; see Movies 9 and 10
in the supplementary material) (Miller and
Rand, 2000). The same is true in ric-8(md1909) mutant
embryos and ric-8(md1909) gpa-16(RNAi) embryos
(Fig. 6D,E; see Movies 11 and
12 in the supplementary material). By contrast, in ric-8(md1909)
goa-1(RNAi) embryos, the spindle remains centrally located and sometimes
even drifts towards the anterior, resulting in equal cleavage
(Fig. 6F; see Movie 13 in the
supplementary material). An analogous behavior is observed in
goa-1/gpa-16(RNAi) embryos
(Fig. 6G,H; see Movie 14 in the
supplementary material) (Gotta and
Ahringer, 2001
).
|
Although we cannot formally exclude that these results reflect partial inactivation of gpa-16, we view this as unlikely because GPA-16 protein levels are significantly diminished in gpa-16(RNAi) embryonic extracts (Fig. 2B, lane 2) and because the gpa-16(RNAi) phenotype is as severe as that of the strong reduction of function allele gpa-16(it143) (see Table S1 in the supplementary material). Therefore, compromising simultaneously GOA-1 and RIC-8 appears more detrimental to asymmetric spindle positioning than compromising simultaneously GPA-16 and RIC-8, as expected from the fact that GPA-16 cortical localization and protein levels are already severely diminished in the absence of RIC-8.
The requirement of RIC-8 for normal GPA-16 cortical localization and protein levels is distinct from that enabling interaction between GPA-16 and GPR-1/2
Reduced GPA-16 cortical localization and protein levels in ric-8
mutant embryos could be due to the lack of interaction between GPA-16 and
GPR-1/2 or instead uncover a novel requirement for RIC-8. If the former was
the case, then mutations that impair the interaction between GPA-16 and
GPR-1/2 should necessarily result in reduced GPA-16 cortical localization and
protein levels. Contrary to this prediction, we found that GPA-16 distribution
and levels are normal in gpa-16(it143) mutant embryos
(Fig. 1G and
Fig. 2B, lane 3, compare with
lane 1) (Bergmann et al.,
2003), despite the interaction between GPA-16 and both RIC-8 and
GPR-1/2 being severely diminished (Fig.
2A, lane 12, compare with lane 7). In addition, we found that
GPA-16 cortical localization and protein levels are compromised in
gpa-16(it143) ric-8(RNAi) (Fig.
1H; data not shown), indicating that the mutation in
gpa-16(it143) does not render GPA-16 insensitive to RIC-8. Overall,
these findings suggest that the requirement of RIC-8 for ensuring normal
GPA-16 cortical localization and protein levels is novel and distinct from its
known role in enabling interaction between G
proteins and GPR-1/2.
|
An examination of GPA-16 distribution in gpb-1(RNAi) embryos leads us to favor the latter scenario. We found that GPA-16 protein levels are severely diminished in such embryos (Fig. 2A, lane 3, compare with lane 1), to an extent comparable with that observed in ric-8(md1909) mutant embryos (Fig. 2A, compare lanes 2 and 3). Importantly, in addition, we found that GPA-16 is present at the cortex of gpb-1(RNAi) embryos (Fig. 1I). Although we cannot exclude that GPA-16 in gpb-1(RNAi) embryos is targeted to the cortex in a manner that does not occur in the wild type, these observations render it unlikely that lack of GPA-16 cortical localization in ric-8(md1909) mutant embryos is due merely to diminished protein levels. Therefore, we propose that RIC-8 is required for cortical localization of GPA-16 during asymmetric division of C. elegans embryos.
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Discussion |
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Distinct regulation of GPA-16 and GOA-1 during asymmetric cell division
In this study, we establish that the interaction of GPA-16 with GPR-1/2
requires RIC-8, and that this requirement is alleviated when Gß is
inactivated. Moreover, we find that GPR-1/2 acts as a GDI towards GPA-16.
Whereas these findings mirror those made with GOA-1
(Afshar et al., 2004
), we show
also that GPA-16 differs in two important ways from GOA-1 with respect to its
relationships with RIC-8. First, RIC-8 does not exhibit GEF activity towards
GPA-16, in contrast to its effect on GOA-1. Consistent with our findings,
yeast two hybrid experiments indicate that rat Ric8 isoforms interact with
both wild type and mutant GTP-ase deficient, G
q and
G
o (Tall et al.,
2003
). Interestingly, rat Ric8A also exhibits differential GEF
activity towards distinct G
proteins in vitro
(Tall et al., 2003
), and our
work with GOA-1 and GPA-16 provides the first evidence that such differential
activity occurs in a physiological setting.
A second important difference is that RIC-8 is required for normal cortical
localization of GPA-16, but not GOA-1. We found also that RIC-8 is needed for
efficient cortical localization of GPB-1, but this most likely reflects the
requirement of RIC-8 for GPA-16 cortical localization, because a diminution of
GPB-1 is observed in gpa-16(RNAi) embryos (data not shown).
Importantly, we found in addition that GPA-16 cortical localization is not
altered in gpa-16(it143) mutant embryos, despite the interaction of
GPA-16 with RIC-8 and GPR-1/2 being essentially abolished. This indicates that
the requirement of RIC-8 for GPA-16 cortical localization is distinct from its
known role in ensuring interaction between G proteins and GPR-1/2.
It will be interesting to investigate the mechanism by which the novel
requirement of RIC-8 for GPA-16 cortical localization is exerted. One
possibility is suggested by the fact that myristoylation and palmitoylation of
G subunits is important for their cortical localization (reviewed by
Wedegaertner, 1998
). In view
of this, RIC-8 may be needed for lipid modification of GPA-16. Alternatively,
RIC-8 could help fold GPA-16 to make it competent for cortical localization.
Yet an alternative possibility is suggested by the fact that G
subunits
in vertebrate cells can redistribute from the cortex to the cytosol in
response to agonist stimulation (Allen et
al., 2005
; Wedegaertner and
Bourne, 1994
). In this scenario, RIC-8 may be a negative regulator
of GPA-16 removal from the cortex. Regardless of the underlying mechanism, our
findings uncover a novel function for RIC-8, that of ensuring cortical
localization of a G
protein. By extension, our results raise the
possibility that RIC-8 family members modulate G-protein signaling in other
organisms in an analogous manner.
Activation mechanism of GPA-16 during asymmetric cell division
Our previous work suggested a model for the activation mechanism of GOA-1
in which RIC-8 GEF activity first generates GOA-1·GTP, after which the
intrinsic GTPase activity of GOA-1 converts GOA-1 to the GDP-bound form
capable of binding GPR-1/2 (Afshar et al.,
2004). Our present findings suggest a simpler model for GPA-16, in
which nucleotide exchange does not occur prior to interaction of GPA-16 with
GPR-1/2 (Fig. 7). As Ric-8A is
inactive on heterotrimer-complexed G
·GDP
(Tall et al., 2003
), it is
likely that RIC-8 acts on Gß
-independent GPA-16·GDP.
Because RIC-8 does not exhibit GEF activity towards GPA-16, we propose that
nucleotide exchange does not occur on GPA-16 prior to interaction with GPR-1/2
and the associated protein LIN-5. The GPA-16·GDP-GPR-1/2-LIN-5 complex
may then promote generation of pulling forces along astral microtubules. Such
a complex may still be associated with RIC-8, because some GPR-1/2 is present
following co-immunoprecipitation with RIC-8 antibodies from wild-type
embryonic extracts (Afshar et al.,
2004
). Spontaneous nucleotide exchange ensues, after which the
intrinsic GTPase activity of GPA-16, most probably accelerated by RGS-7
(Hess et al., 2004
),
terminates the activation cycle.
|
Mechanism of partial redundancy: GOA-1 and GPA-16 counter the effect of Gß to ensure accurate asymmetric spindle positioning
Our study contributes to understanding the partial redundancy between GOA-1
and GPA-16. We find that simultaneous inactivation of the Gß subunit
GPB-1 and of either GOA-1 or GPA-16 results in the same phenotype as that
observed after GPB-1 inactivation alone. Therefore, GOA-1 and GPA-16 become
entirely redundant for asymmetric spindle positioning following Gß
inactivation. These findings illustrate the importance of the balance between
G
proteins and Gß
during receptor-independent activation of
heterotrimeric G proteins. This is reminiscent of the interplay between
G
and Gß
proteins in modulating receptor-dependent
heterotrimeric G protein signaling (for a review, see
Gilman, 1987
).
Our findings suggest that in wild-type C. elegans embryos, the
presence of both G subunits provides substantial G
molecules
free from Gß
, which can then associate in a RIC-8-dependent manner
with GPR-1/2-LIN-5 to generate appropriate pulling forces. When either GPA-16
or GOA-1 is inactivated, less G
is available for interaction with
GPR-1/2-LIN-5, resulting in lower pulling forces. When GPB-1 is inactivated in
addition, no Gß
-dependent dampening occurs and either GOA-1 or
GPA-16 can alone sustain full generation of pulling forces. Interestingly,
this is the case in embryos simultaneously compromised for GPB-1 and GOA-1
(see Fig. 3; Table S1), despite
GPA-16 protein levels being diminished in the absence of GPB-1. However, we
found that the bulk of residual GPA-16 protein in gpb-1(RNAi) embryos
is present at the cortex (see Fig.
1I), which appears sufficient to recruit GPR-1/2-LIN-5 and
generate pulling forces in the absence of GOA-1.
G requirement during spindle positioning: beyond C. elegans
Whereas two G proteins act in concert in one-cell stage C.
elegans embryos, a single G
that belongs to the
G
i class is known to be required for proper spindle
orientation in Drosophila neuroblasts and sensory organ precursor
cells (Schaefer et al., 2001
).
Although a second G
that belongs to the G
o class is
expressed in these cells, it is not essential for spindle orientation
(Yu et al., 2003
). However,
overexpression of either G
o or G
i yields
identical spindle positioning defects, possibly owing to depletion of free
Gß
(Yu et al.,
2003
). Therefore, reminiscent of the situation in C.
elegans, the balance between G
proteins and Gß
is also
crucial in Drosophila. However, the mechanisms of activation may
differ between the two species, as overexpression of G
o or
G
i in Drosophila results in a phenotype independent
of the GoLoco protein PINS (Yu et al.,
2003
). By contrast, Gß
inactivation in C.
elegans, which presumably results in excess free G
proteins,
results in a GPR-1/2-dependent phenotype
(Tsou et al., 2003
).
The requirement for G proteins and their regulators in spindle
positioning extends to vertebrate cells. The GoLoco motif and RGS
domain-containing protein RGS14, which exerts both GDI and GAP activities
towards G
i/o subunits, is crucial for spindle assembly in
the mouse zygote (Martin-McCaffrey et al.,
2005
). LGN, a mammalian GoLoco protein more closely related to
GPR-1/2 and PINS, is recruited to the cell cortex through association with a
G
i (Du and Macara,
2004
). LGN also associates with the coiled-coil protein NuMA, thus
targeting it to the cell cortex, where it may function as a spindle
positioning effector. It has been suggested that GPR-1/2 and PINS may serve an
analogous function, perhaps targeting LIN-5 and Inscuteable, respectively
(Du and Macara, 2004
;
Willard et al., 2004
). If this
view were correct, our work would indicate that both GPA-16 and GOA-1 are
needed to provide sufficient binding sites for GPR-1/2 to ensure efficient
LIN-5 cortical recruitment and generation of pulling forces.
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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/132/20/4449/DC1
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