RESEARCH ARTICLE 2005
Development 140, 2005-2014 (2013) doi:10.1242/dev.088310
© 2013. Published by The Company of Biologists Ltd
PAR-2, LGL-1 and the CDC-42 GAP CHIN-1 act in distinct
pathways to maintain polarity in the C. elegans embryo
Alexander Beatty, Diane G. Morton and Kenneth Kemphues*
SUMMARY
In the one-cell C. elegans embryo, polarity is maintained by mutual antagonism between the anterior cortical proteins PAR-3, PKC3, PAR-6 and CDC-42, and the posterior cortical proteins PAR-2 and LGL-1 on the posterior cortex. The mechanisms by which these
proteins interact to maintain polarity are incompletely understood. In this study, we investigate the interplay among PAR-2, LGL-1,
myosin, the anterior PAR proteins and CDC-42. We find that PAR-2 and LGL-1 affect cortical myosin accumulation by different
mechanisms. LGL-1 does not directly antagonize the accumulation of cortical myosin and instead plays a role in regulating PAR-6 levels.
By contrast, PAR-2 likely has separate roles in regulating cortical myosin accumulation and preventing the expansion of the anterior
cortical domain. We also provide evidence that asymmetry of active CDC-42 can be maintained independently of LGL-1 and PAR-2 by
a redundant pathway that includes the CDC-42 GAP CHIN-1. Finally, we show that, in addition to its primary role in regulating the
size of the anterior cortical domain via its binding to PAR-6, CDC-42 has a secondary role in regulating cortical myosin that is not
dependent on PAR-6.
INTRODUCTION
Cell polarity is crucial for embryonic development. In the one-cell
C. elegans embryo, anterior-posterior polarity occurs in two
temporally distinct phases referred to as establishment and
maintenance (Cuenca et al., 2003). Polarity establishment is
mediated by an asymmetric contraction of the cortical meshwork
that instructs the segregation of two antagonistic sets of conserved
polarity regulators known as the partitioning-defective (PAR)
proteins (Nance and Zallen, 2011). A cue associated with the sperm
centrosome triggers a non-muscle myosin II-dependent asymmetric
contraction of the actomyosin cytoskeleton and an associated
anteriorly directed cortical flow (Cheeks et al., 2004; Mayer et al.,
2010; Munro et al., 2004). This flow requires the small GTPase
RhoA (RHO-1) (Motegi and Sugimoto, 2006; Schonegg and
Hyman, 2006) and functions to concentrate PAR-3, PAR-6 and the
atypical protein kinase C PKC-3 into an anterior cortical domain,
likely via advective transport (Goehring et al., 2011b). As the
anterior PAR proteins become enriched in the anterior, the posterior
proteins, including PAR-1, PAR-2, and LGL-1, become enriched in
a reciprocal domain (Nance and Zallen, 2011).
Following the attenuation of cortical flow, polarity maintenance
is necessary to perpetuate the cortical asymmetries generated during
establishment. Although a number of the key proteins involved in
polarity maintenance have been identified, the molecular
mechanisms by which the proteins contribute to polarity
maintenance, as well as the level of interaction between the
components, are not well understood.
The Rho GTPase CDC-42 is an important regulator of polarity
maintenance (Aceto et al., 2006; Kumfer et al., 2010; Motegi and
Sugimoto, 2006; Schonegg and Hyman, 2006). During
maintenance, CDC-42 is enriched on the anterior cortex (Aceto et
Department of Molecular Biology and Genetics, Cornell University, 433
Biotechnology Building, Ithaca, NY 14853, USA.
*Author for correspondence ([email protected])
Accepted 26 February 2013
al., 2006; Motegi and Sugimoto, 2006; Schonegg and Hyman,
2006), and its active form binds PAR-6 (Aceto et al., 2006; Gotta et
al., 2001). In cdc-42(RNAi) embryos, PAR-6 and PKC-3 become
asymmetrically enriched on the anterior cortex at a reduced level
during establishment, and are lost from the cortex around the time
of nuclear envelope breakdown. PAR-3 remains cortical but often
extends into the posterior and can overlap with PAR-2 (Gotta et al.,
2001; Kay and Hunter, 2001). Embryos expressing a PAR-6 mutant
unable to bind CDC-42 exhibit defects similar to cdc-42(RNAi)
embryos, suggesting that CDC-42 functions in polarity primarily
via its physical interaction with PAR-6 (Aceto et al., 2006).
Reduction of CDC-42 function also results in alterations in nonmuscle myosin II (NMY-2) localization. In embryos depleted for
CDC-42, cortical myosin is largely lost during the transition to the
maintenance phase when myosin foci are normally reorganized and
replaced by finer fibers (Kumfer et al., 2010; Motegi and Sugimoto,
2006; Schonegg and Hyman, 2006). Because CDC-42 is required
for the maintenance of cortical PAR-6/PKC-3 as well as cortical
NMY-2, CDC-42 appears to provide a functional link between the
anterior PAR proteins and the actomyosin cytoskeleton during
polarity maintenance.
In addition to signaling through CDC-42, polarity maintenance is
also mediated by mutual exclusion between the anterior and
posterior PAR proteins. During the maintenance phase, the sizes of
reciprocal PAR domains are stable, but the individual PAR proteins
exchange between the cortex and the cytoplasm (Cheeks et al.,
2004; Goehring et al., 2011a; Nakayama et al., 2009), and cortical
PAR proteins can diffuse freely across the anterior-posterior domain
boundary (Goehring et al., 2011a). Mathematical modeling suggests
that mutual inhibition between the anterior and posterior PAR
proteins occurs via a reaction-diffusion system that does not require
the actomyosin cytoskeleton (Dawes and Munro, 2011; Goehring et
al., 2011a; Goehring et al., 2011b).
On the posterior cortex of the one-cell embryo, the putative E3
ubiquitin ligase PAR-2 is required to prevent the anterior cortical
domain from expanding during the maintenance phase (Boyd et al.,
1996; Cuenca et al., 2003; Hao et al., 2006). Recently, LGL-1, the
DEVELOPMENT
KEY WORDS: Embryo, PAR genes, Polarity
Development 140 (9)
2006 RESEARCH ARTICLE
MATERIALS AND METHODS
Nematode strains
Nematodes were grown using standard conditions (Brenner, 1974) and N2
(Bristol) was wild type. Mutations used in this analysis include par-2(it5)
(Kemphues et al., 1988), par-2(lw32) (Cheng et al., 1995), par-2(ok1723),
par-6(zu222) (Watts et al., 1996), unc-119(ed4) (Maduro and Pilgrim,
1995), lgl-1(tm2616), lgl-1(it31) (Beatty et al., 2010), cgef-1(gk261)
(Kumfer et al., 2010) and mrck-1(or586) (Gally et al., 2009). par-2(ok1723)
was out-crossed to N2 five times. We also used the transgenes zuIs45
[Pnmy-2::nmy-2::gfp+unc-119(+)] (Nance et al., 2003), ojIs40 [Ppie1::mgfp::wsp-1(G-protein binding domain) + unc-119(+)] (Kumfer et al.,
2010), itIs0256 [Plgl-1::lgl-1::gfp, unc-119(+)], itIs279 [Plgl-1::lgl1::mChr + unc-119(+)] (Beatty et al., 2010) and itIs167 [Ppie-1::gfp::par6 + unc-119(+)] (Li et al., 2010).
RNA interference
RNAi was performed by feeding (Timmons et al., 2001). Adults were
allowed to lay embryos on RNAi feeding plates, and the progeny were
grown to adulthood prior to dissection (60-72 hours). All RNAi feedings
were carried out at 25°C with the exception of experiments involving par2(lw32) and par-2(it5), which were carried out at 20°C and 16°C,
respectively. In experiments in which PAR-6 was depleted and NMY2::GFP was examined, we controlled for the effectiveness of PAR-6
knockdown by quantifying embryonic lethality prior to dissection and by
monitoring loss of asymmetry in NMY-2::GFP during polarity maintenance.
Any experiments with less than 100% lethality or residual NMY-2::GFP
asymmetry were excluded from the data sets.
Imaging
Differential interference contrast (DIC) and wide-field fluorescence images
of live embryos were captured using a Leica DM RA2 microscope with a
63× Leica HCX PL APO oil immersion lens, a Hamamatsu ORCA-ER
digital camera and Openlab software (Improvision/PerkinElmer, Waltham
MA). Blastomere cross-sectional areas were measured using Openlab.
Confocal images were obtained with a PerkinElmer UltraVIEW LCI
confocal scanner with a Nikon Eclipse TE2000-U microscope using
UltraVIEW Imaging Suite v5.5. The sections were stacked and processed
using ImageJ and Adobe Photoshop CS4.
Immunohistochemistry
PAR-6 immunostaining was carried out according to Hung and Kemphues
(Hung and Kemphues, 1999).
Measuring cortical NMY-2 levels
The fraction of the cortex with NMY-2::GFP signal was measured using
ImageJ. Twelve confocal sections from the top of the cortex to the
midsection and spaced at 1.0 μm were stacked. Threshold levels were
selected such that most of the NMY-2::GFP fibers in each of the stacks were
recognized as particles by ImageJ. Then the area of the particles relative to
the total area of the embryo cross-section was calculated using ImageJ, and
the ratio was referred to as the ‘fraction of the cortex with signal’. The same
threshold was applied to all the data sets being analyzed, and the means
were compared. As an additional method for quantifying cortical NMY2::GFP signal, the average signal intensity across the area of two to three
stacked cortical sections was measured using ImageJ, and these values were
compared after subtracting the background. Means were compared using
Welch’s t-test. The threshold for statistical significance corresponded to a Pvalue of 0.05. Both methods yielded similar results.
Measuring PAR-6 levels
Cortical and cytoplasmic PAR-6 levels were measured using ImageJ. For each
embryo, three confocal sections (0.5 μm spacing) centered on the midsection
were stacked. Signal for the entire embryo was assumed to be proportional to
the signal measured for these stacked cross-sections. To determine the average
cortical PAR-6 signal, nine one-pixel wide lines of roughly equal length were
traced along the cortical domain (three near the anterior pole and six laterally),
and measured the average signal intensity for each. The average signal
intensities were averaged after subtracting the background signal to yield an
average cortical signal for the embryo. The average cytoplasmic signal was
determined similarly using three lines per embryo. The area of the embryo
cross-section occupied by the cortical domain was determined by selecting a
signal threshold so that the cortical PAR-6::GFP signal, and not the
cytoplasmic signal, was recognized as a particle by ImageJ, and by measuring
the corresponding area (see supplementary material Fig. S1). The cortical
signal was determined by multiplying the area of the cortical domain by the
average cortical signal. To validate this approach, we also calculated the
average cortical signal by multiplying the mean signal intensity for the cortical
domain by the area of the cortical domain for some of the data sets (see
supplementary material Table S1). The cytoplasmic signal was determined
by multiplying the total area of the cross-section minus the area of the anterior
cortical domain by the average cytoplasmic signal. Three independent sets of
5 to 10 embryos each were examined for each genotype, except for par2(RNAi) and lgl-1(tm2616); cgef-1(RNAi), in which two sets were used.
Means for individual experiments were compared using Student’s t-test.
Results for independent trials were combined using Fisher’s method.
Measuring cortical enrichment of GFP::GBDwsp-1
Cortical enrichment of GFP::GBDwsp-1 was measured as above for PAR6. The anterior and posterior signals were determined by drawing three lines
on the anterior or posterior cortex, respectively, and measuring the average
signal intensity for each. These values were averaged and the background
subtracted to yield the cortical signal. The cytoplasmic signal was
determined similarly.
RESULTS
LGL-1 and PAR-2 affect myosin distribution by
different mechanisms
During polarity maintenance in wild-type embryos, myosin is
asymmetrically localized to the anterior cortex (Fig. 1A) (Munro et
al., 2004). In par-2(RNAi) embryos, LGL-1 negatively regulates the
posterior cortical accumulation of myosin during polarity
DEVELOPMENT
homolog of the Drosophila tumor suppressor protein Lethal Giant
Larvae, was found to localize asymmetrically to the posterior cortex
and function redundantly with PAR-2 to maintain polarity (Beatty
et al., 2010; Hoege et al., 2010). LGL-1 is not required for polarity
maintenance, but lgl-1; par-2 double mutants have a stronger
phenotype than par-2 alone. In par-2 mutants, the anterior PARs
expand into the posterior but retain a graded distribution there. In
lgl-1;par-2 double mutants, the anterior PAR proteins become even
more symmetric, although reduced levels of anterior PARs are still
apparent in the extreme posterior (Beatty et al., 2010). Although it
is clear that PAR-2 and LGL-1 act in polarity maintenance, the
molecular mechanisms by which the proteins function are not well
understood. Both PAR-2 and LGL-1 inhibit the cortical
accumulation of NMY-2 on the posterior cortex (Beatty et al., 2010;
Munro et al., 2004), consistent with the hypothesis that pathways
regulated by the two proteins converge on NMY-2. An alternative
hypothesis is that the effects of LGL-1 on NMY-2 are indirect and
mediated by the anterior PAR proteins. Hoege and co-workers have
suggested that LGL-1 contributes to polarity maintenance via a
mutual elimination mechanism (Hoege et al., 2010). This model
proposes that PAR-6 and PKC-3 physically interact with LGL-1,
likely near the interface of the anterior and posterior cortical
domains. The interaction leads to phosphorylation of LGL-1 by
PKC-3, which results in the cortical removal of the entire complex.
In this way, LGL-1 could contribute to polarity maintenance by
facilitating the cortical removal of PAR-6 and PKC-3 that otherwise
would diffuse into the posterior cortical domain.
In this study, we investigate the interplay among LGL-1, PAR-2,
cortical myosin and the anterior PAR protein PAR-6, and uncover
a third redundant polarity maintenance pathway that includes the
CDC-42 GTPase-activating protein CHIN-1.
Three pathways maintain C. elegans polarity
RESEARCH ARTICLE 2007
Fig. 1. The effect of LGL-1 on myosin accumulation in par-2 is
epistatic to par-6(RNAi). (A) Confocal projections of cortical NMY-2::GFP
prior to symmetry breaking (left), at pseudocleavage (center) and at
nuclear envelope breakdown (NEB) of the first mitotic division (right).
(B) The left column shows confocal projections of cortical NMY-2::GFP in
embryos at NEB. The column on the right depicts the threshold mask (red
overlay) applied to the images on the left to quantify the fraction of the
cortex with NMY-2::GFP signal. Anterior is towards the left in all figures.
(C) The average fraction of the cortex with NMY-2::GFP signal±s.d. based
on the threshold mask exemplified in B. The values for the average
fraction of the cortex with signal are provided in Table 1 in the ‘Area
method’ column. Asterisks indicate significant differences relative to par6(RNAi). P<0.002.
maintenance (Beatty et al., 2010). However, it is unclear whether
LGL-1 affects asymmetric cortical myosin accumulation
independently of the anterior PAR proteins, or whether the asymmetry
in cortical myosin in the absence of PAR-2 is a secondary
consequence of the removal of the anterior PAR proteins from the
posterior cortex by the action of LGL-1 (Hoege et al., 2010). To
distinguish between these two potential mechanisms, we measured
cortical NMY-2::GFP levels in the presence or absence of LGL-1 in
wild-type and par-2 mutant embryos using two different methods (see
Materials and methods). Although we can readily detect an increase
in the cortical area occupied by myosin in par-2;lgl-1 embryos
compared with par-2 embryos (Beatty et al., 2010) we could detect
no significant difference in total cortical myosin levels between lgl-
PAR-6 levels in the early embryo are increased in
lgl-1(RNAi)
If LGL-1 functions solely by removing PAR-6 from the cortex in the
one-cell embryo (Hoege et al., 2010), we hypothesized that cortical
levels of PAR-6 should be increased and cytoplasmic levels should be
reciprocally decreased after depleting LGL-1. To test this, we
compared the relative amount of cortical and cytoplasmic PAR6::GFP in control and lgl-1(RNAi) during polarity maintenance at the
time of nuclear envelope breakdown (see Materials and methods). In
contrast to our predictions, we found that both cortical and
cytoplasmic PAR-6::GFP levels were reproducibly higher after
depleting LGL-1 (Fig. 2). The cortical and cytoplasmic amounts of
PAR-6::GFP were increased by 15.1±4.4% (P=0.009) and 26.0±6.1%
(P=0.0001), respectively, in lgl-1(RNAi) embryos compared with the
controls (n=24 for each genotype). Despite this increase in PAR-6
levels, the area of the PAR-6 cortical domain with respect to the total
area of the embryo cross-section was similar in lgl-1(RNAi) and
control embryos; the measured change in the PAR-6 domain in lgl1(RNAi) embryos with respect to wild type was −4.2±2.9% (Fig. 2;
P=0.61). Thus, LGL-1 normally acts to reduce levels of PAR-6
protein, but our experiment does not address whether LGL-1 might
also remove PAR-6 from the cortex (see Discussion).
We performed a similar experiment comparing PAR-6 levels with
and without depleting PAR-2. In par-2(RNAi) embryos, consistent
with expectations, the area of the cortical PAR-6::GFP domain was
expanded by 24.2±4.8% (P=2×10−5); however, neither the cortical
nor cytoplasmic PAR-6 levels changed compared with the control
(Fig. 3; P=0.36, 0.22, respectively; n=18). Consistent with the
previously described role for PAR-2 in polarity maintenance
(Cuenca et al., 2003; Hao et al., 2006), we conclude that PAR-2
negatively regulates the size of the cortical PAR-6 domain, but not
the steady state level of PAR-6. By contrast, in the presence of PAR2, LGL-1 has no effect on anterior domain size, but instead regulates
the overall amount of PAR-6.
Depletion of CHIN-1 blocks the ability of LGL-1
overexpression to rescue par-2
Overexpression of LGL-1 is sufficient to maintain cortical
asymmetry in par-2 mutants (Beatty et al., 2010; Hoege et al.,
DEVELOPMENT
1 and wild type or between par-2 and par-2;lgl-1 (Table 1). This
suggested that LGL-1 affected the distribution of cortical myosin
without affecting the overall accumulation of myosin or that the
difference was too small to reliably detect. To test the dependence of
the observed LGL-1-mediated change in myosin distribution, and
possibly levels, on PAR-6, we examined myosin distribution and
measured the cortical myosin levels in par-6(RNAi), par-6(RNAi);
par-2(lw32) and par-6(RNAi); par-2(lw32); lgl-1(tm2616) embryos
during polarity maintenance (see Materials and methods). Consistent
with a previous report of cortical NMY-2 distribution and levels in
par-2; par-3 double mutants (Munro et al., 2004), myosin asymmetry
is lost when the anterior PAR proteins are eliminated from the cortex
and the cortical levels of NMY-2 in par-6(RNAi); par-2(lw32)
embryos were substantially higher than in par-6(RNAi) embryos
(Fig. 1B,C; Table 1). Furthermore, we found no significant difference
in the distribution or amount of cortical myosin between par-6(RNAi);
par-2(lw32) and par-6(RNAi); par-2(lw32); lgl-1(tm2616)
(Fig. 1B,C; Table 1). Therefore, we conclude that PAR-2 negatively
regulates the cortical accumulation of NMY-2 on the posterior cortex
in a manner that is independent of the asymmetry generated by the
anterior PAR proteins, whereas LGL-1 reduces cortical myosin in the
posterior via its effect on the anterior PAR proteins.
Development 140 (9)
2008 RESEARCH ARTICLE
Table 1. Comparison of cortical myosin levels
Genotype
n
Area method
Signal intensity method
Wild type
lgl-1(tm2616)
par-2(RNAi)
par-2(RNAi); lgl-1(tm2616)
par-6(RNAi)
par-6(RNAi); par-2(lw32)
par-6(RNAi); par-2(lw32); lgl-1(tm2616)
7
8
10
10
15
8
10
0.27±0.09
0.24±0.07 (P=0.70)
0.25±0.10
0.20±0.07 (P=0.24)
0.21±0.08
0.49±0.11 (P=2.8×10–5)
0.43±0.14 (P=0.42)
42.0±4.8
41.0±5.3 (P=0.69)
41.5±5.7
37.9±6.4 (P=0.20)
42.1±5.6
54.9±5.2 (P=3.7×10–5)
52.5±7.1 (P=0.21)
The values provided by the area method represent the average fraction of the embryo with NMY-2::GFP signal. P-values shown compare wild type with lgl-1(tm2616) in
row 2, par-2(RNAi) to par-2(RNAi); lgl-1(tm2616) in row 4, par-6(RNAi) with par-6(RNAi); par-2(lw32) in row 7 and par-6(RNAi); par-2(lw32) with par-6(RNAi); par-2(lw32);
lgl-1(tm2616) in row 8.
Fig. 2. Depletion of LGL-1 increases the levels of PAR-6::GFP in the
one-cell embryo. (A,B) Confocal midsection of PAR-6::GFP in
representative (A) control and (B) lgl-1(RNAi) embryos at NEB.
(C) The measured percentage change in the cortical and cytoplasmic
levels of PAR-6::GFP±s.e.m. in the one-cell embryos after LGL-1 depletion,
relative to the negative controls.
53.9±1.5% (n=11) of the total embryo area in lgl-1::gfp; par2(lw32) as compared to 51.1±2.4% (n=17) in lgl-1::gfp; par2(lw32); chin1-1(RNAi) (P=0.0004). We examined the subcellular
localization of LGL-1::GFP in one-cell lgl-1::gfp; par-2(lw32);
chin-1(RNAi) embryos. In these embryos, LGL-1::GFP became
asymmetrically localized to the posterior cortex during polarity
establishment, but the cortical domain was not maintained (n=8/8,
see Movies 1 and 2 in the supplementary material), suggesting
CHIN-1 is required to maintain the posterior cortical domain in lgl1::gfp; par-2(lw32).
Consistent with a role for CHIN-1 in maintenance, we found that
depleting CHIN-1 enhanced the lethality and early polarity defects
of par-2(it5ts) at the permissive temperature of 16°C, similar to lgl1 (Beatty et al., 2010). Embryos from par-2(it5); chin-1(RNAi) were
82.3±15.6% (n=1428) embryonic lethal compared with chin1(RNAi) and par-2(it5) worms at 16°C, which yielded 1.6±1.2%
(n=377) and 11.4±3.8% (n=1071) dead embryos, respectively
(Fig. 4).
One possible explanation for these results is that chin-1 and lgl1 are components of a common genetic pathway, and LGL-1 acts
through CHIN-1. An alternative explanation is that chin-1 and lgl1 are components of parallel genetic pathways, both of which
contribute to polarity maintenance. To distinguish between these
hypotheses, we depleted CHIN-1 in the presumed null mutation lgl1(tm2616). If CHIN-1 acts downstream of LGL-1 in a linear
pathway, we expected the embryonic lethality of lgl-1(tm2616);
chin-1(RNAi) to be no greater than the sum of the embryonic
lethality from lgl-1(tm2616) (1.7±0.3%; n=3787) and chin-1(RNAi)
(0.8±0.1%; n=4100). By contrast, if CHIN-1 and LGL-1 function in
parallel, we expected to see synthetic lethality when both proteins
were compromised. lgl-1(tm2616); chin-1(RNAi) yielded 5.0±2.0%
lethality [n=3420; P<0.0001, χ2 test assuming an expected lethality
of 2.5% for lgl-1(tm2616); chin-1(RNAi)]. Although the magnitude
of the difference is not large, leaving open the possibility that it is
due to variation in RNAi effectiveness, the results are consistent
with the hypothesis that chin-1 and lgl-1 are components of distinct
genetic pathways.
Loss of CGEF-1 function rescues par-2(lw32)
A putative CDC-42 GEF, CGEF-1, appears to antagonize CHIN-1
(Kumfer et al., 2010). Because CHIN-1 depletion enhanced the
phenotype of par-2(it5), we hypothesized that depletion or mutation
of CGEF-1 may be sufficient to suppress par-2. Consistent with this
hypothesis, embryos from par-2(lw32); cgef-1(RNAi) were
65.0±20.2% viable (n=618), whereas par-2(lw32) worms fed on
control bacteria yielded no viable progeny (Fig. 5A; n=653). Similar
results were observed using par-2(it5) at the restrictive temperature;
viability of embryos from par-2(it5) and par-2(it5); cgef-1(RNAi)
DEVELOPMENT
2010). In an attempt to understand how LGL-1 influences polarity
maintenance, we screened a small set of known cytoskeletal
regulators by RNAi depletion to identify any that prevented LGL1::GFP overexpression from rescuing par-2(lw32), but were not
required in wild type (Beatty et al., 2010). One protein that
emerged from the screen was the putative CDC-42 GAP, CHIN1, which has been shown to inhibit the accumulation of NMY-2
fibers on the posterior cortex during polarity maintenance (Kumfer
et al., 2010). Embryos from chin-1(RNAi) were mostly viable
(0.6±0.9% lethality, n=872), as were embryos from lgl-1::gfp;
par-2(lw32) (10.3±8.0% lethality, n=436). However, lgl-1::gfp;
par-2(lw32); chin-1(RNAi) embryos were 83.9±10.7% lethal
(Fig. 4A; n=1286). We obtained a similar result using a different
lgl-1 transgene and different par-2 allele. Embryos from par2(ok1723); lgl-1::mCherry were 48.5±22.6% lethal (n=912),
while embryos from par-2(ok1723); lgl-1::mCherry; chin1(RNAi) were 98.1±1.4% lethal (n=1312). Thus, depletion of
CHIN-1 severely impairs the ability of LGL-1 overexpression to
rescue par-2.
We determined that the increased lethality in the CHIN-1
knockdown was due to a loss of polarity maintenance. Early
embryos from lgl-1::gfp; par-2(lw32); chin-1(RNAi) exhibited a
characteristic Par-2 phenotype. In contrast to chin-1(RNAi) and lgl1::gfp; par-2(lw32) embryos, lgl-1::gfp; par-2(lw32); chin-1(RNAi)
embryos exhibited similarly sized blastomeres and transverse
spindle orientations in the P1 cell, like par-2(lw32) embryos
(Fig. 4B) (Kumfer et al., 2010). The AB blastomere accounted for
Three pathways maintain C. elegans polarity
RESEARCH ARTICLE 2009
Fig. 3. PAR-2, CHIN-1 and CGEF-1 regulate PAR-6 in early embryo.
(A) Confocal midsections of PAR-6::GFP embryos of the specified
genotype. (B) The percentage change in the cortical and cytoplasmic
levels of PAR-6::GFP±s.e.m. in par-2(RNAi), chin-1(RNAi) or cgef-1(RNAi)
relative to the respective negative controls. Asterisks indicate significant
differences. P<0.007.
CHIN-1 and CGEF-1 can regulate CDC-42 in par2(lw32); lgl-1(tm2616)
To determine the functional relationship between CHIN-1/CGEF-1,
PAR-2 and LGL-1 with respect to CDC-42 regulation, we used a
Fig. 4. Knockdown of CHIN-1 blocks the ability of LGL-1
overexpression to rescue par-2. (A) The percentage of embryos that
failed to complete embryogenesis for each of the labeled genotypes.
Data are mean±s.e.m. (B) DIC images of two-cell embryos during
interphase (left column) and prior to the second mitotic division (right
column). The black lines indicate the orientation of the mitotic spindles.
biosensor, GFP::GBDwsp-1, that reports high concentrations of
available active GTP-bound CDC-42 (Kumfer et al., 2010). During
polarity maintenance at the time of nuclear envelope breakdown,
GFP::GBDwsp-1 is asymmetrically enriched on the anterior cortex
of the embryo (Fig. 6A) (Kumfer et al., 2010). In Fig. 6B we show
measurements of the signal at the cortex relative to the cytoplasm
in the anterior and the posterior. Control embryos had cortical to
cytoplasmic enrichments of 60.1±0.9% and 5.8±0.9% at the anterior
and posterior pole, respectively (n=10). Consistent with published
results (Kumfer et al., 2010), both chin-1(RNAi) and cgef-1(RNAi)
altered the distribution of the probe during polarity maintenance
(Fig. 6A). In chin-1(RNAi) embryos, GFP::GBDwsp-1 was
cortically enriched in the anterior (70.9±8.7%), like controls, but
DEVELOPMENT
were 11.2±2.2% (n=1049) and 42.9±29.0% (n=462), respectively
(Fig. 5A). Furthermore, embryos from par-2(lw32); cgef-1(gk261)
were 76.4±4.5% viable (Fig. 5; n=505). gk261 is a likely null allele
(Kumfer et al., 2010). We conclude that loss of cgef-1 function
suppresses the embryonic lethality associated with par-2 mutations.
In an effort to learn the relationship between CGEF-1 function
and LGL-1, we also tested whether CGEF-1 depletion could
suppress par-2; lgl-1. We made a series of par-2; lgl-1 double
mutants by generating all possible pairwise combinations of two
likely null alleles (lw32 and tm2616) and two hypomorphic alleles
(it5 and it31) of par-2 and lgl-1. We treated each of the par-2; lgl1 double mutants with cgef-1(RNAi) and quantified embryonic
lethality (supplementary material Table S2). We observed that cgef1(RNAi) can suppress par-2; lgl-1 when a hypomorphic allele of
lgl-1 is coupled with a null allele of par-2, but not when a
hypomorphic allele of par-2 is coupled with a null allele of lgl-1.
Thus, lgl-1 function is required for loss of cgef-1 function to rescue
embryonic viability in par-2. Although this result is consistent with
a role for regulating active CDC-42 in polarity maintenance, it does
not distinguish whether LGL-1 acts in the same pathway as CDC42 or in a parallel pathway.
2010 RESEARCH ARTICLE
Development 140 (9)
Fig. 5. Loss of CGEF-1 function suppresses par-2(lw32). (A) The percentage of embryonic lethality for the indicated genotypes (±s.e.m.). (B) DIC
images comparing par-2(lw32) and par-2(lw32); cgef-1(gk261) at the two-cell stage (left column), and during the second mitotic division (right column).
The black lines indicate the orientation of the mitotic spindles.
Mis-regulation of CDC-42 results in increased
amounts of PAR-6 in the early embryo
Next, we asked what role active CDC-42 plays in controlling the
amount and cortical distribution of PAR-6 in the early embryo by
comparing the cortical and cytoplasmic amounts of PAR-6::GFP in
control, chin-1(RNAi) and cgef-1(RNAi) embryos at nuclear
envelope breakdown. As in lgl-1(RNAi) embryos, both the cortical
and cytoplasmic PAR-6::GFP amounts were increased in chin1(RNAi) embryos (Fig. 3; 26.9±16.1% increase in cortical amount,
P=0.0005; 19.3±10.6% increase in cytoplasmic amount, P=0.006;
n=21). Additionally, the anterior cortical domain extended
14.8±11.6% further into the posterior after depleting CHIN-1
(Fig. 3A; P=0.0002).
In embryos depleted of CGEF-1, the area of the PAR-6 cortical
domain was 16.5±3.2% smaller than in the controls (P=0.0006,
n=27) and was sometimes positioned laterally (Fig. 3A; n=5/27).
Cortical PAR-6 amounts were similar to controls (−1.2±10.0%;
P=0.18); but, surprisingly, the cytoplasmic amounts were
34.8±16.6% greater in cgef-1(RNAi) embryos compared with the
controls (Fig. 3; P=6×10−5; n=27); indeed, the total PAR-6 (sum of
the cortical and cytoplasmic signal) was increased after knocking
down CGEF-1 (P=0.0001).
Thus, elevating the cortical levels of active CDC-42 during
polarity maintenance results in an expansion of the PAR-6 cortical
domain, as well as an increase in the amount of cortical PAR-6. By
contrast, reducing active CDC-42 at the cortex leads to a reduction
in both the size of the PAR-6 domain and amount of cortical PAR6. However, in both situations, total PAR-6 amounts are elevated.
CHIN-1 and CGEF-1 regulate anterior cortical
domain size and PAR-6 levels in lgl-1(tm2616)
If LGL-1 and CHIN-1 are components of parallel pathways that both
independently contribute to polarity maintenance, we predicted that
depletion of CHIN-1 in lgl-1(tm2616) would result in an expansion
of the anterior cortical domain and an increase in the total amount of
PAR-6 in the one-cell embryo. For two independent par-6::gfp; lgl1(tm2616) lines, we found that the area of the cortical PAR-6::GFP
domain was increased in chin-1(RNAi) embryos with respect to the
controls; the anterior cortical domain was expanded by 25.9±11.3%
(P=6×10−5; n=24) in one of the lines and 23.7±6.1% (P<1×10−8;
n=26) in the other (Fig. 7A,B). Additionally, depletion of CHIN-1
resulted in an increase in cortical PAR-6::GFP in both lines
(25.3±21.0%; P=0.03; n=24; 35.1±15.5%; P=0.003; n=26) and a
statistically significant increase in cytoplasmic PAR-6 signal in one of
the two lines (Fig. 7; 9.7±7.0%; P=0.02; n=24). The other line showed
a similar, but not statistically significant, change in the cytoplasmic
signal following CHIN-1 depletion (8.6±1.3%; P=0.10; n=26).
Next, we depleted CGEF-1 in lgl-1(tm2616) expressing PAR6::GFP and observed a 35.6±4.4% reduction in the area of the PAR6 cortical domain (Fig. 7A,C; P=1×10−7; n=15) along with a
21.7±16.1% reduction in cortical PAR-6::GFP relative to the controls
(Fig. 7; P=0.02; n=15). In contrast, the cytoplasmic and total amount
of PAR-6 were increased in lgl-1(tm2616); cgef-1(RNAi) (Fig. 7;
P=0.0004 and 0.001, respectively). The cytoplasmic PAR-6::GFP
DEVELOPMENT
posterior cortical enrichment increased roughly sixfold relative to
the control (35.6±7.6%; n=5). By contrast, cgef-1(RNAi) resulted
in a marked reduction of cortical GFP::GBDwsp-1 (6.7±6.6%
enrichment on the anterior cortex, −4.0±15.3% on the posterior
cortex; n=5).
To determine whether PAR-2 and LGL-1 are required for CHIN1 or CGEF-1 function, or both, we examined and quantified the
localization of GFP::GBDwsp-1 in par-2(lw32); lgl-1(tm2616) with
and without RNAi-mediated depletion of CHIN-1 or CGEF-1. In
contrast to expectations from the hypothesis that levels of active
CDC-42 are regulated exclusively by either PAR-2 or LGL-1, or
both, GFP::GBDwsp-1 was distributed asymmetrically in par2(lw32); lgl-1(tm2616) (Fig. 6C). We observed a 38.5±4.0% cortical
enrichment of GFP::GBDwsp-1 in the anterior, and no enrichment
in the posterior (−2.2±5.3%, n=15). Furthermore, the cortical levels
of the biosensor were increased in the posterior in par-2(lw32); lgl1(tm2616) embryos depleted of CHIN-1 (Fig. 6C; 43.2±14.0%
anterior enrichment and 34.3±21.3% posterior enrichment; n=5).
By contrast, when CGEF-1 was knocked down in par-2(lw32); lgl1(tm2616), GFP::GBDwsp-1 levels were reduced throughout the
cortex (Fig. 6C, 10.3±4.3% anterior enrichment, −3.5±0.4%
posterior enrichment, n=10). We conclude that active CDC-42
asymmetry is maintained, at least in part, by a mechanism that is
independent of both PAR-2 and LGL-1. In addition, CHIN-1 and
CGEF-1 can function in the absence PAR-2 and LGL-1, consistent
with the hypothesis that these proteins are likely components of a
genetic pathway that does not include PAR-2 or LGL-1.
Three pathways maintain C. elegans polarity
RESEARCH ARTICLE 2011
cortical NMY-2::GFP was reduced in cgef-1(gk261); par-6(zu222)
embryos compared with par-6(zu222) embryos (Fig. 8; n=8 for each
genotype). Results were similar using par-6(RNAi). Thus, unlike
LGL-1, CGEF-1 regulates cortical myosin accumulation in a PAR6-independent manner.
Suppression of par-2 by cgef-1 is not mediated by
MRCK-1
Kumfer and colleagues reported that the loss-of-function phenotype
of the gene mrck-1/tag-59 is similar to cgef-1(gk261) with respect
to cortical NMY-2 accumulation, suggesting that CDC-42 functions
through MRCK-1 to influence cortical myosin association (Kumfer
et al., 2010). If the observed suppression of par-2 by cgef-1 is a
result of a reduction in cortical NMY-2 during polarity maintenance,
then loss or depletion of mrck-1 should also suppress the embryonic
lethality associated with loss or depletion of par-2. Embryos from
par-2(lw32) were 98.8±1.6% lethal (n=562), whereas par-2(lw32);
mrck-1(RNAi) worms yielded no viable embryos (n=512). We
obtained similar results when we depleted PAR-2 by RNAi in wild
type, cgef-1(gk261) and mrck-1(ok586): lethality of 93.9±0.4%
(n=1609), 12.6±9.6% (n=1045) and 99±0.1% (n=1242),
respectively. Thus, loss or depletion of MRCK-1 is not sufficient to
suppress loss or depletion of par-2.
Although loss of cgef-1 affects cortical myosin accumulation
independently of PAR-6, it is possible that loss of cortical myosin
has an effect on the anterior PARs. If so, our expectation would be
that the PAR-6 domain size in mrck-1 mutants, which lack cortical
myosin accumulation at maintenance, would be larger than in wild
type. Instead we found that the average PAR-6 domain size in
pooled mrck-1(ok856) one-cell embryos in late prophase, metaphase
and anaphase (56.5±4.7% egg length) was slightly smaller than that
of wild type (59.5±5.5% egg length) and the difference was not
statistically significant (n=30 for each genotype, P>0.05, two-tailed
t-test).
amount was elevated 31.6±7.4% and the total was 27.2±8.1% greater
than the controls (n=15). Furthermore, 40% (6/15) of lgl-1(tm2616);
cgef-1(RNAi) had laterally skewed PAR-6 domains (Fig. 7C). Thus,
CHIN-1 and CGEF-1 function to regulate the size of the PAR-6
cortical domain and PAR-6 amounts independent of LGL-1.
Mutation of CGEF-1 reduces cortical myosin levels
independent of the anterior PAR proteins
Consistent with the data published by Kumfer et al. (Kumfer et al.,
2010), we noted a substantial decrease in cortical NMY-2::GFP in
cgef-1(gk261) compared with wild type at nuclear envelope
breakdown (Fig. 8; n=21 for each genotype). The residual cortical
myosin puncta appeared sparser than in wild type, but were still
modestly enriched in the anterior. To determine whether the
decrease in cortical myosin fibers was dependent on the anterior
PAR proteins, we compared cortical NMY-2::GFP distribution in
par-6(zu222) and cgef-1(gk261); par-6(zu222), and found that
PAR-2 does not require the anterior PAR proteins
to affect myosin accumulation in the posterior
During polarity maintenance in the one-cell embryo, PAR-2 is
required to prevent the expansion of the anterior cortical domain
(Cuenca et al., 2003; Goehring et al., 2011b; Hao et al., 2006) and
also prevents the formation of NMY-2 filaments on the posterior
cortex (Beatty et al., 2010; Munro et al., 2004). How these two
functions relate to one another is not clear. However, our results rule
out one possibility – that the accumulation of posterior myosin is a
secondary consequence of the effect of PAR-2 on the anterior PAR
proteins. Consistent with an earlier report examining par-3; par2(RNAi) (Munro et al., 2004), we found that par-2; par-6(RNAi)
embryos have increased cortical myosin during polarity
maintenance compared with par-6(RNAi). This observation is
consistent with the previously proposed hypothesis that PAR-2
influences the size of the anterior domain indirectly by preventing
the formation of myosin filaments in the posterior (Munro et al.,
2004). Also consistent with this possibility, we observed that PAR-
DEVELOPMENT
Fig. 6. CHIN-1 and CGEF-1 appear to regulate the distribution of
active CDC-42 in par-2(lw32); lgl-1(tm2616). (A,C) Confocal midsection
of GFP::GBDwsp-1 at NEB in a (A) wild-type embryo or a (C) par-2(lw32);
lgl-1(tm2616) embryo treated with empty vector, chin-1(RNAi) or cgef1(RNAi). (B,D) The percentage cortical enrichment of GFP::GBDwsp-1 on
the anterior and posterior cortex±s.e.m. in (B) wild type and (D) par2(lw32); lgl-1(tm2616) following the indicated RNAi treatment.
DISCUSSION
Our analysis of relationships among PAR-2, LGL-1, cortical myosin
accumulation and the anterior PAR proteins in polarity maintenance
in the C. elegans zygote provides evidence that PAR-2 and LGL-1
influence myosin distribution via different mechanisms and that
LGL-1 acts to reduce the overall level of PAR-6 in the embryo. It
also reveals a third redundant pathway for polarity maintenance
defined by the action of CHIN-1.
2012 RESEARCH ARTICLE
Development 140 (9)
Fig. 7. LGL-1 is not required for CHIN-1 and
CGEF-1 to influence PAR-6 distribution and
amount. (A-C) Confocal midsection of PAR6::GFP in representative (A) lgl-1(tm2616), (B) chin1(RNAi); lgl-1(tm2616) or (C) cgef-1(RNAi); lgl1(tm2616) embryos at NEB. (D) The measured
percentage change±s.e.m. in the cortical and
cytoplasmic amounts of PAR-6::GFP in chin1(RNAi); lgl-1(tm2616) and cgef-1(RNAi); lgl1(tm2616) relative to lgl-1(tm2616).
Fig. 8. Cortical myosin is greatly reduced in cgef-1(gk261); par6(zu222) at maintenance. Confocal projections of cortical NMY-2::GFP in
one-cell embryos of the specified genotype at NEB.
(Beatty et al., 2010), we conclude that MRCK-1 does have a role in
polarity maintenance.
LGL-1 contributes to regulating the overall
amount of PAR-6 in the one-cell embryo
LGL-1 functions redundantly with PAR-2 to maintain polarity in
the early embryo, and negatively regulates cortical myosin
accumulation in the absence of PAR-2 (Beatty et al., 2010; Hoege
et al., 2010). In contrast to our results with PAR-2, we found that
the effect of LGL-1 on cortical myosin in embryos lacking PAR2 was dependent on the anterior PAR proteins, precluding a direct
role for LGL-1 in regulating cortical myosin, but consistent with
a mutual elimination mechanism proposed by Hoege and
colleagues (Hoege et al., 2010). However, our measurements of
PAR-6 levels are not consistent with the mutual elimination
model. If LGL-1 acts solely by removing the anterior PAR
proteins at the boundary between anterior and posterior cortical
domains, we expected that removing LGL-1 would lead to an
increase in PAR-6 at the cortex and a reciprocal decrease in
cytoplasmic PAR-6. Instead we observed an increase in both
cortical and cytoplasmic levels of PAR-6 in the embryo,
suggesting LGL-1 is involved in regulating PAR-6 turnover.
Because this regulation could affect cortical and cytoplasmic PAR6 differentially, our experiment does not test the mutual
elimination model. Notably, whereas loss of LGL-1 results in
anterior domain expansion in the absence of PAR-2 (Beatty et al.,
2010), lgl-1(RNAi) in the presence of PAR-2 does not, despite
increasing both cortical and cytoplasmic PAR-6 levels. Thus,
limiting cytoplasmic pools of PAR proteins is likely not the only
constraint on domain expansion (Goehring et al., 2011b).
Taken together, our results suggest that LGL-1 negatively
regulates the overall amount of PAR-6 in the early embryo and thus
may buffer against expansion of the anterior cortical domain
(Dawes and Munro, 2011). Although the molecular mechanism by
which LGL-1 acts remains elusive, we suggest that polarity
maintenance in the one-cell C. elegans embryo may represent
another instance where LGL functions as a molecular buffer, but in
a method that is distinct from that reported for the peripheral
nervous system of Drosophila (Wirtz-Peitz et al., 2008).
DEVELOPMENT
2 regulated the size of the anterior domain without significantly
affecting the amount of PAR-6 in the one-cell embryo. A role for
PAR-2 in regulating PAR-6 through myosin is in apparent
contradiction to a report that the actomyosin network is not required
for polarity maintenance (Goehring et al., 2011a).
We propose, however, that PAR-2 has separate roles in regulating
cortical myosin accumulation and anterior cortical domain size. We
found that knockdown of MRCK-1, a likely effector of active CDC42, fails to suppress par-2. Kumfer and colleagues (Kumfer et al.,
2010) reported, and we confirmed, that a loss-of-function mutation
in mrck-1 resulted in a robust decrease in cortical myosin during
maintenance. If PAR-2 contributes to maintenance only by
preventing aberrant cortical flow, we would predict that
compromising the actomyosin network during maintenance would
suppress par-2 mutants. Because this was not the case, we conclude
that PAR-2 affects the size of the anterior PAR domain by another
mechanism. However, because depletion of MRCK-1 partially
blocks the ability of LGL-1 to compensate for the loss of PAR-2
CHIN-1 and CGEF-1 are involved in controlling
anterior domain size and PAR-6 levels
CHIN-1 and CGEF-1 regulate cortical myosin localization during
polarity maintenance, presumably by modulating the activity of
CDC-42 (Kumfer et al., 2010). Additionally, these CDC-42
regulatory proteins appear to contribute to controlling the size of
the PAR-6 cortical domain and the overall levels of PAR-6 in the
early embryo. In agreement with their antagonistic roles in
regulating CDC-42, we found that cgef-1(RNAi) caused a reduction
in the PAR-6 cortical domain, whereas chin-1(RNAi) resulted in an
expansion of the PAR-6 domain. However, depletion of either
protein led to an increase in the overall amount of PAR-6 in the early
embryo, suggesting there must be a regulatory mechanism curtailing
anterior domain growth in cgef-1(RNAi) embryos, despite an excess
of cytoplasmic PAR-6 relative to wild type. One likely explanation
for this observation is that depletion of CGEF-1, through its effect
on levels of activated CDC-42, reduces the ability of PAR-6 to
localize to the cortex. It has previously been reported that PAR-6
must interact with CDC-42 in order to be robustly maintained at the
cortex (Aceto et al., 2006). Modulating the affinity of the cortex for
PAR-6 may provide an additional layer of regulation to a reaction
diffusion mechanism of polarity maintenance. The reason for the
increase in the overall amount of PAR-6 in both chin-1(RNAi) and
cgef-1(RNAi) embryos is unclear, but may be evidence of a feedback
loop between CDC-42 and the anterior PAR proteins.
We found that reducing the levels of active CDC-42 via cgef-1
mutation during polarity maintenance results in a substantial
decrease in cortical myosin, even in par-6 mutants, suggesting that
CDC-42 has two separable functions in polarity maintenance. In
contrast to the role of CDC-42 in binding to PAR-6, the role of
CDC-42 in cortical myosin regulation via MRCK-1 during polarity
maintenance appears to be dispensable, consistent with the
observation that the actomyosin cytoskeleton is not required for the
maintenance of distinct cortical domains (Dawes and Munro, 2011;
Goehring et al., 2011a; Goehring et al., 2011b).
A third pathway for polarity maintenance
Our results lead us to propose that there are at least three pathways
contributing to polarity maintenance in the early C. elegans embryo:
a primary pathway that requires PAR-2, a second redundant
pathway that requires LGL-1 and a third independent pathway that
requires CHIN-1, the putative CDC-42 GAP . This third pathway is
either consistent with or supported by the following evidence: (1)
chin-1(RNAi) blocks the ability of LGL-1 overexpression to rescue
par-2 loss-of-function mutations; (2) depletion of CHIN-1 in an lgl1-null mutant resulted in modest but statistically significant
synthetic lethality; (3) embryos lacking functional PAR-2 and LGL1 exhibited persistent asymmetry of active CDC-42; (4) the
distribution of active CDC-42 in the double par-2; lgl-1 mutant is
responsive to knockdown of CHIN-1 or CGEF-1; (5) loss of LGL1 and depletion of CHIN-1 have an additive effect on the level of
PAR-6::GFP and synergistic effect on the size of the PAR-6::GFP
domain; (6) LGL-1 affects myosin levels through the anterior PAR
proteins whereas CGEF-1 does not.
Putting it all together
In Fig. 9 we present a summary of the proposed relationships among
LGL-1, PAR-2, myosin and regulation of CDC-42 during polarity
maintenance. Negative regulatory activities of LGL-1, PAR-2 and
CHIN-1 independently contribute to polarity maintenance, but each
acts differently. LGL-1 acts by regulating PAR-6 cortical
accumulation and overall levels. PAR-2 appears to have two
RESEARCH ARTICLE 2013
Fig. 9. Summary of proposed relationships during maintenance.
Proteins localized to the anterior or posterior cortex are indicated in green
and red, respectively. The thickness of the arrows reflects relative
importance of the interaction. The distribution of MRCK-1 is not known,
but it presumably acts in the anterior. The relevant CDC-42 GEFs appear
to act throughout the embryo. The dotted line representing aPARmediated inhibition of CHIN-1 is based on the observation that CHIN-1 is
delocalized upon depletion of PAR-6 (Kumfer et al., 2010).
independent roles, restricting the anterior PAR domain and
preventing myosin accumulation in the posterior. Because
eliminating myosin accumulation during maintenance by mutating
MRCK-1 fails to suppress the absence of PAR-2, the main activity
of PAR-2 must not require its blocking cortical myosin
accumulation; unfortunately, the nature of that activity remains
elusive. The role of CHIN-1, presumably acting through CDC-42,
is minor compared with that of PAR-2.
A key finding is that CDC-42 has a dual role: a major role in
regulating the size of the anterior cortical domain via its binding
to PAR-6 and a minor role in regulating cortical myosin during
maintenance. The basis for this conclusion is that cgef-1 can
suppress par-2 mutation or depletion but lowering the level of
MRCK-1, a downstream effector of CDC-42, fails to suppress
par-2 depletion; indeed, it appears to enhance it. It is well
established that lowering the steady state level of PAR-6 can
suppress par-2 mutations (Hyenne et al., 2008; Watts et al., 1996).
Lowering active CDC-42 levels by mutation of cgef-1 will affect
both cortical PAR-6 accumulation (Aceto et al., 2006) and
MRCK-1 activity (Kumfer et al., 2010). We propose that the two
effects exert unequal influence on polarity maintenance and that
suppression of PAR-2 by CGEF-1 occurs because reduced active
CDC-42 will restore the balance between the antagonistic forces
of the anterior and posterior domains. Lowering MRCK-1 levels
lowers the levels of cortical myosin in the anterior (Kumfer et al.,
2010) but appears to have no effect on the size of the PAR-6
domain, consistent with the observation that the actomyosin
cytoskeleton is not required for the maintenance of distinct cortical
domains (Dawes and Munro, 2011; Goehring et al., 2011a;
Goehring et al., 2011b). As mentioned above, however, the ability
of mrck-1(RNAi) to partially block LGL-1 overexpression rescue
of par-2 argues that MRCK-1 normally contributes to maintaining
the anterior cortical domain, although this contribution is nonessential in a normal genetic background.
An unexpected finding of our analysis is that the embryo has a
mechanism to maintain an asymmetric distribution of active CDC42 that is independent of PAR-2 and LGL-1. Whether this
asymmetric CDC-42 activity is dependent on the centrosome-
DEVELOPMENT
Three pathways maintain C. elegans polarity
mediated polarity signals remains to be tested. The relationship
between CDC-42 activity and steady state PAR-6 levels is also
unclear. Increasing posterior CDC-42 activity by depleting CHIN1 results in expansion of the PAR-6 domain and increased amounts
of cytoplasmic PAR-6. Although this could be explained if cortical
PAR-6 was protected from turnover, this explanation would predict
that lowering levels of active CDC-42 by depleting CGEF-1 should
lower steady-state amounts of cytoplasmic PAR-6. Because the
latter is not the case, we clearly have more to learn.
Overall, our results reveal an additional level of redundancy that
ensures faithful polarization of the early embryo, clarify the role of
CDC-42, and add to a growing body of evidence indicating that
numerous regulatory processes function in concert to consistently
establish and maintain cell polarity.
Acknowledgements
We thank Ed Munro for helpful suggestions on the manuscript, Rich
McCloskey for valuable discussions and the Caenorhabditis elegans Genetics
Center for strains.
Funding
This work was supported by grants from the National Institutes of Health
[HD27689 and GM79112]. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.088310/-/DC1
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