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First published online 9 August 2006
doi: 10.1242/dev.02527
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Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
* Author for correspondence (e-mail: schonegg{at}mpi-cbg.de)
Accepted 7 July 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cell polarity, Rho GTPase, PAR, Myosin, Contractility, C. elegans
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Etemad-Moghadam et al., 1995;
Hung and Kemphues, 1999;
Tabuse et al., 1998;
Watts et al., 1996). The
posterior domain is defined by PAR-1 and PAR-2
(Boyd et al., 1996;
Cuenca et al., 2003;
Guo and Kemphues, 1995). The
establishment of the anterior and posterior PAR domains is a dynamic process.
Following fertilization, the anterior and posterior PAR proteins co-localize
throughout the entire cortex (Boyd et al.,
1996; Cuenca et al.,
2003; Etemad-Moghadam et al.,
1995; Hung and Kemphues,
1999; Munro et al.,
2004). During meiosis II, PAR-2 leaves the cortex, while the
anterior PAR proteins remain localized over the whole cortex. The apposition
of the sperm-derived centrosome at the posterior cortex triggers regression of
the anterior PAR proteins to the anterior cortex of the embryo. PAR-2 returns
exclusively to the posterior cortex, the region devoid of the anterior PAR
proteins (Cowan and Hyman,
2004b; Cuenca et al.,
2003; Munro et al.,
2004). Throughout the paper we will refer to this PAR-2
localization cycle as the `meiotic PAR-2 cycle'.
The initiation of polarity in C. elegans induces dramatic
cytoskeletal rearrangements that lead to a morphological polarization, which
was termed `contractile polarity' (Cowan
and Hyman, 2004a). At the end of meiosis, small transient cortical
ruffles can be seen over the entire cortex. Later, the ruffling ceases in the
area where the centrosome becomes juxtaposed with the posterior cortex
(Cheeks et al., 2004;
Cowan and Hyman, 2004b;
Cuenca et al., 2003;
Munro et al., 2004). This
smooth area gradually expands towards the anterior until it is about 50% of
the egg-length. A constriction called the pseudocleavage furrow separates the
smooth posterior domain from the anterior domain, which remains contractile
(Hirsh et al., 1976;
Strome, 1986). Fixed sample
studies revealed that actin becomes asymmetrically localized in the embryo
(Strome, 1986;
Strome and Hill, 1988), and
suggested that the establishment of contractile polarity is associated with
the segregation of the acto-myosin cytoskeleton. More recent studies imaging
the non-muscle myosin II heavy chain (NMY-2) fused to GFP revealed that,
during contractile polarity establishment, a uniform contractile acto-myosin
meshwork disassembles in close vicinity to the posterior nucleus/centrosome
complex and segregates towards the anterior pole
(Munro et al., 2004). The
signal-inhibiting local contractility appears to come from the centrosome
(Cowan and Hyman, 2004b;
Munro et al., 2004).
Cell polarization depends on communicating a symmetry-breaking event to
induce a reorganization of the actin-myosin cytoskeleton, leading to polarized
cellular domains and an asymmetric distribution of cytoskeletal functions. The
Rho family GTPases Cdc42 and RhoA play important roles in signaling to the
downstream cellular machinery that controls actin cytoskeleton organization
and, therewith, cell polarity. The activity of GTPases is controlled by
regulatory proteins: guanine nucleotide exchange factors (GEFs) activate
GTPases by catalyzing the exchange of GDP for GTP
(Schmidt and Hall, 2002),
whereas GTPase activating proteins (GAPs) inactivate GTPases by stimulating
the intrinsic GTPase activity (Bernards,
2003). Cdc42 was identified in Saccharomyces cerevisae
and shown to be involved in bud site selection
(Drubin, 1991;
Johnson, 1999). Further
analysis in different systems showed that Cdc42 is required for numerous
aspects of polarity establishment. For example, in migrating cells, Cdc42 is
implicated in the orientation and maintenance of polarized morphology,
whereas, in epithelial cells, Cdc42 is implicated in the formation of tight
junctions, which separate the apical and the basolateral membranes. Cdc42
plays a further role in the polarized vesicular trafficking required for
polarized protein distribution (reviewed by
Etienne-Manneville, 2004).
Thus, Cdc42 is a component of many cell polarization pathways. RhoA is also
essential for many types of cell polarity, as polarized cell shape and cell
migration depend largely on the acto-myosin cytoskeleton. RhoA is required for
the assembly of actin filaments and myosin II into contractile filaments that
provide the mechanical force for cortical contractions, motility and
cytokinesis (reviewed by
Etienne-Manneville and Hall,
2002; Glotzer,
2005). Thus, both RhoA and Cdc42 are essential for many types of
cell polarity. However, it remains unclear how their functions are coordinated
in cell polarity.
CDC-42 and RHO-1 play essential roles in the C. elegans one-cell
embryo. Previous studies have shown that after depletion of CDC-42, PAR-2 was
found uniformly at the cortex and PAR-6 was either anteriorly enriched, as in
wild type, or scattered throughout the entire cortex at the two-cell stage
(Gotta et al., 2001; Kay and
Hunter et al., 2001). As CDC-42 was shown to interact with PAR-6 in many
systems, including C. elegans
(Gotta et al., 2001;
Hutterer et al., 2004;
Joberty et al., 2000;
Johansson et al., 2000;
Lin et al., 2000;
Qiu et al., 2000), it seemed
likely that CDC-42 would act through PAR-6 to regulate polarity. These studies
demonstrated the involvement of CDC-42 in C. elegans polarity,
although which specific process in polarity establishment is affected by
CDC-42 remains unclear.
RHO-1 was shown to function in cytokinesis
(Jantsch-Plunger et al.,
2000), presumably by regulating acto-myosin activity. It has been
demonstrated that polarity establishment in C. elegans embryos
requires the acto-myosin cytoskeleton. Embryos treated with
actin-depolymerizing drugs or depleted of myosin II subunits, or the actin
nucleators profilin or formin, do not establish polarity: PAR-2 is unable to
localize correctly to the cortex and PAR-3/PAR-6 remain uniformly distributed
around the entire cortex (Cuenca et al.,
2003; Guo and and Kemphues,
1996; Severson and Bowerman,
2003; Shelton et al.,
1999) (S.S. and A.A.H., unpublished). Thus, in C. elegans
embryos, RHO-1 may be involved in polarity establishment, possibly through
regulation of the acto-myosin cytoskeleton, although this remains to be
tested.
Here, we investigate the roles of CDC-42 and RHO-1 in polarity establishment in C. elegans embryos and examine the interaction between these two signaling pathways in cell polarity. Our data suggest that RHO-1 and CDC-42 have separable functions in polarity establishment. We show that RHO-1 activity is required for acto-myosin contractility and organization of the NMY-2 meshwork, which, in turn, is essential for localizing CDC-42 to the anterior half of the embryo. CDC-42, in turn, is required to stabilize the acto-myosin network and for localizing PAR-6 in the anterior. In addition, CDC-42 removes PAR-2 from the cortex during meiosis. We have found that during polarity establishment the roles of RHO-1 and CDC-42 are interdependent, and appear to be coordinated, in part, through the acto-myosin contractile network in C. elegans one-cell embryos.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The following
strains were used: N2 (wild type), JH1380 (GFP-PAR-2), TH25 (GFP-PAR-6),
JJ1473 (NMY-2-GFP), TH71 (NMY-2-GFP;GFP-PAR-2), TH72 (YFP-CDC-42), KK571
[par-3(it71)]. The TH71 strain was constructed by crossing JH1380 to
JJ1473 and progeny were selected that expressed both GFPs. TH72 was crossed to
KK571 to analyze YFP-CDC-42 localization in a par-3(it71) background.
The cdc-42 coding sequence (R07G3.1) was identified using the
WormBase web site (release WS155;
http://www.wormbase.org)
and was amplified by PCR from wild-type strain N2 genomic DNA using the
primers 5'-ggccactagtggaATGCAGACGATCAAGTGCGTC-3' and
5'-ggcccccgggCTAGAGAATATTGCACTTCTTCT-3', containing
SpeI or XmaI sites (bold). The N-terminal YFP-fusion was
generated in pTH-YFP(N) (a modified version of the pAZ132 plasmid, a gift from
Andrei Pozniakovsky, Max Planck Institute of Cell Biology and Genetics,
Dresden), expressing YFP under the control of the pie-1 promoter.
Transgenic worms were created by high-pressure ballistic bombardment (BioRad)
of DP38 unc-119(ed3) homozygotes, as described previously
(Praitis et al., 2001). The YFP-CDC-42 transgene was tested for functionality by using double-stranded RNA against the 3'UTR of cdc-42 to deplete endogenous CDC-42 in wild-type N2 and YFP-CDC-42 worms. Injected worms were then assayed for embryonic hatching. The YFP-CDC-42 fusion uses the pie-1 3'UTR, and therefore was not targeted by the cdc-42 3'UTR double-stranded RNA. To test whether RNAi against the 3'UTR of cdc-42 gives the same phenotype as cdc-42(RNAi), cdc-42 3'UTR RNA was injected into GFP-PAR-2. We found that PAR-2 was uniformly localized, as in cdc-42(RNAi) embryos (n=7, data not shown). To determine embryonic hatching, injected worms were placed on individual plates for 56 hours at 25°C and allowed to lay eggs for 5 hours at 25°C. These embryos were checked for hatching 48 hours later. The progeny of N2 worms injected with cdc-42 3'UTR RNA showed 0% embryonic hatching (21 worms, 193 embryos), whereas the progeny of injected YFP-CDC-42 worms (19 worms, 178 embryos) showed 96.2% hatching, indicating that the YFP-CDC-42 is functional.
RNA-mediated interference
RNAi experiments were performed as described
(Oegema et al., 2001). Primers
used to amplify regions from N2 genomic DNA are listed in
Table 1. Worms were incubated
depending on the individual double-stranded RNA for 10-26 hours at 25°C
after injection. Cdc-42(RNAi);rho-1(RNAi) was performed by
co-injection of both RNAs, combined with feeding of cdc-42(RNAi)
(Timmons and Fire, 1998).
Cdc-42(RNAi);spd-2(RNAi) was performed by co-injection of both RNAs,
combined with feeding of cdc-42(RNAi) and spd-2(RNAi). Worms
were placed on feeding plates after injection and maintained at 25°C for
22-48 hours.
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; Kay and Hunter,
2001). The discrepancy between the different results is probably
due to a difference in RNAi penetrance, as Gotta et al.
(Gotta et al., 2001) used
different RNAi conditions. After RNAi of CDC-42 for 26 hours at 25°C, we
also found some PAR-6 on the anterior cortex, but at very reduced levels (data
not shown).
Time-lapse microscopy
Worms were shifted to 25°C before recording. Embryos were dissected and
mounted in a solution containing 0.1 M NaCl and 4% sucrose, with and without
2% agarose. GFP, YFP and differential interference contrast (DIC) recordings
were acquired at 10-15 second intervals (exposure time 400 mseconds, 2x2
binning) with a Hamamatsu Orca ER 12 bit digital camera mounted on a spinning
disk confocal microscope (Zeiss Axioplan using a 63x 1.4 NA
PlanApochromat objective and Yokogawa disk head). Illumination was via a 488
nm Argon ion laser (Melles Griot). Movies acquired for
Fig. 2 were done on a
wide-field microscope (Zeiss Axioplan II using a 63x 1.4 NA
PlanApochromat objective equipped with a Hamamatsu Orca ER 12 bit digital
camera). Image processing was done with MetaView Software (Universal Imaging
Corporation).
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).
For the PAR-2 immunostaining, the GFP-PAR-2 strain (JH1380) was used. A sheep
polyclonal antibody to GFP (1:1000; a gift from Francis Barr,
Max-Planck-Institute of Biochemistry, Martinsried, Germany) was used to
visualize PAR-2. DM1
(1:300, Sigma) and SPD-2, 1:5000,
(Pelletier et al., 2004) were
used to detect microtubules and centrosomes. PAR-6 was stained with a
C-terminal peptide (amino acid 291-308) anti-PAR-6 antibody. The antibodies
were visualized with TR- and Cy5-conjugated antibodies (Jackson
Immunochemicals), and with a donkey anti-sheep antibody coupled to Alexa 488
(Molecular Probes). Imaging was performed on a DeltaVision microscope and
stacks were deconvolved as described
(Oegema et al., 2001). SPD-2
and DNA images are projections of z-sections representing the entire
cell. PAR-2, PAR-6 and Tubulin images are projections of four to 10
z-sections.
Contractility tracking
The ruffle kymographs were performed as described
(Cowan and Hyman, 2004b).
Briefly, the ruffles were tracked starting around the time of the beginning of
pronuclear appearance for an interval of 1000 seconds. The position of
cortical ruffles was manually tracked and projected onto a calculated ellipse.
One half of the ellipse was straightened to generate the x-axis, the
anteroposterior axis. This procedure was done for each time point and laid
down sequentially along the y-axis (time). Lines connect ruffles
within nearest neighbor groups.
Tracking of PAR-2 and PAR-6 domain extent
The extent of the GFP-PAR-2 domain was manually tracked after the domain
reached its maximal size. The extent of the GFP-PAR-6 domain was tracked after
pseudocleavage regression. The domain size was calculated as a fraction of the
respective embryo circumference. Manual tracking was performed using a
custom-written macro (Stephan Grill) for NIH-Image (NIH). Further analysis was
done with Mathematica 4.1 (Wolfram Research).
Kymograph analysis
Kymographs were done with Metamorph Software (Universal Imaging
Corporation) from cortical YFP-CDC-42 time-lapse recordings (7-12 minutes
total). Kymographs were made from a straight line along the long axis of the
embryo.
Measurement of the position of the posterior boundary of YFP-CDC-42
Position was measured with Metamorph Software (Universal Imaging
Corporation) as a distance along the long axis from the anterior pole. The
distance was standardized to total embryo length (100%). 0% indicates the
anterior (ANT) pole.
Tracking of NMY-2 foci
GFP-NMY-2 foci were manually tracked with Metamorph Software (Universal
Imaging Corporation).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; O'Connell et al.,
2000). Previous work has shown that in CDC-42-depleted embryos,
PAR-2 was uniformly distributed throughout the cortex
(Gotta et al., 2001;
Kay and Hunter, 2001);
however, the reason for this uniform distribution remained unclear. To
investigate whether the uniform PAR-2 localization in cdc-42(RNAi)
embryos is dependent on the centrosomal signal, CDC-42 was depleted together
with SPD-2, a centrosomal protein essential for polarity establishment
(Cowan and Hyman, 2004b;
O'Connell et al., 2000). In
spd-2(RNAi);cdc-42(RNAi) embryos, GFP-PAR-2 localized uniformly at
the cortex (Fig. 2), showing
that the uniform PAR-2 distribution in cdc-42(RNAi) embryos is
independent of the centrosome-dependent polarity signal. These data suggest
that the aberrant PAR-2 distribution could be caused by an earlier defect in
the PAR-2 localization mechanism. We next examined the meiotic cycle of
GFP-PAR-2 in cdc-42(RNAi) embryos. We found that PAR-2 did not leave
the cortex during meiosis II, but instead remained uniformly distributed
throughout the cortex during the entire cell cycle (n=10,
Fig. 1; data not shown). This
data suggests that the defect in PAR-2 localization in cdc-42(RNAi)
embryos results from a failure to remove PAR-2 from the cortex during the
meiotic cycle.
CDC-42 is required to localize PAR-6 to the cortex
The uniform distribution of PAR-2 in cdc-42(RNAi) embryos is
similar to the PAR-2 distribution seen in par-6 and par-3
mutant embryos, as the localization of the anterior and posterior PAR proteins
is interdependent (Etemad-Moghadam et al.,
1995; Hung and Kemphues,
1999; Tabuse et al.,
1998; Watts et al.,
1996). PAR-6 and CDC-42 physically interact in C. elegans
and other systems (Gotta et al.,
2001; Hutterer et al.,
2004; Joberty et al.,
2000; Johansson et al.,
2000; Lin et al.,
2000; Qiu et al.,
2000), and studies in C. elegans have suggested that
CDC-42 is required to maintain PAR-6 in the anterior half
(Gotta et al., 2001;
Kay and Hunter, 2001). We
re-examined the requirement of CDC-42 for PAR-6 localization
(Fig. 3A). Because complete
depletion of CDC-42 leads to sterility (data not shown), we determined the
maximum depletion that would still yield embryos, and fixed and stained for
PAR-6. Polarity formation can be divided into two phases: an establishment
phase in which symmetry is broken and a PAR-6 domain is formed independently
of PAR-2; and a maintenance phase, in which the maintenance of the PAR-6
domain in the anterior requires PAR-2
(Cuenca et al., 2003). We found
that in four out of four embryos examined during polarity establishment, PAR-6
was absent from the cortex. In four out of six embryos examined during the
polarity maintenance phase, we also could not detect PAR-6 at the cortex (in
two out of these six embryos, PAR-6 was weakly present in the anterior). This
indicated that CDC-42 is required for localization of PAR-6 to the cortex
during all stages of the mitotic cell cycle.
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); in
addition the phenotypes of CDC-42 and PAR-6 depletion are similar.
Alternatively, the aberrant localization of PAR-2 may prevent PAR-6 from
localizing to the cortex. We attempted to distinguish between the two
possibilities by performing double RNAi of cdc-42 and par-2
(Fig. 3A). We found that PAR-6
can localize to the cortex after the reduction of both proteins, although at
reduced levels relative to wild-type embryos (n=16/16). These data
suggest that the aberrant PAR-2 localization may prevent PAR-6 from localizing
to the cortex in cdc-42(RNAi) embryos, similar to recent work showing
that mutant PAR-2 expressed ectopically in embryonic somatic blastomeres can
displace PAR-3 from the cortex (Hao et
al., 2006). However, because we are not working under
loss-of-function conditions for RNAi of CDC-42 (stronger RNAi conditions
produce sterile worms), we cannot rule out the possibility that residual
CDC-42 activity could directly recruit PAR-6 to the cortex after the removal
of PAR-2. In support of this idea, the residual PAR-6 in the double RNAi
embryos still localizes to the anterior cortex. Therefore, we conclude that a
CDC-42-dependent activity is required to remove PAR-2 from the cortex and a
CDC-42-dependent activity is necessary to localize PAR-6 to the cortex.
CDC-42 localizes to the anterior cortex
Because CDC-42 and PAR-6 form a complex
(Gotta et al., 2001), we
hypothesized that the requirement of CDC-42 for PAR-6 localization may be
reciprocal. To examine whether the anterior PAR proteins are required for
CDC-42 localization, we generated a YFP-labeled CDC-42. The YFP-CDC-42
transgene rescues the loss of endogenous CDC-42 (see Materials and methods),
suggesting that the fusion protein complements the function of endogenous
CDC-42. YFP-CDC-42 formed dynamic structures at the cortex
(Fig. 4; see Movie 2 in the
supplementary material) that segregated to the anterior cortex during polarity
establishment (n=8), recapitulating the behavior of anterior PAR
proteins (Cuenca et al., 2003;
Munro et al., 2004). Cortical
YFP-CDC-42 disappeared around the time of pronuclear rotation (data not
shown). To test whether the anterior PAR proteins are required for CDC-42
localization, we examined YFP-CDC-42 dynamics in the par-3(it71)
loss-of-function mutant and in par-6(RNAi) embryos
(Fig. 4; Movie 3 in the
supplementary material). We made kymographs from time-lapse recordings in
control, mutant and RNAi embryos. In par-3(it71) (n=8) and
par-6(RNAi) (n=7) embryos, CDC-42 segregated to the
anterior, although segregation was slower than in control embryos
(Fig. 4B). Importantly,
YFP-CDC-42 eventually localized in the anterior half as it did in control
embryos.
Acto-myosin contractility is required to form an anterior cortical domain of CDC-42
CDC-42 segregation to the embryo anterior occurred coincident with the
segregation of contractility. Therefore, we speculated that CDC-42 segregation
might by regulated by the acto-myosin cytoskeleton. To test this idea, we
followed the dynamics of CDC-42 distribution in embryos depleted of myosin II
(NMY-2). We found that in nmy-2(RNAi) embryos, YFP-CDC-42 localized
to the cortex, but did not segregate into an anterior domain
(Fig. 4, n=5). Thus,
similar to the establishment of PAR polarity, the asymmetric distribution of
CDC-42 requires acto-myosin activity.
RHO-1 is required for organization of the cortical myosin II network
During polarity establishment, acto-myosin contractility has to be
temporally and spatially regulated such that contractile polarity is
coordinated with other events in cell polarization. RhoA is a conserved
regulator of acto-myosin contractility. Therefore, we investigated whether
C. elegans RHO-1 and the putative RhoGEF ECT-2 regulate contractility
during polarization. In all our experiments (see below), RNAi of
ect-2 phenocopied the defects observed in rho-1(RNAi)
embryos, but did not yield defects characteristic of CDC-42 depletion. This
suggests that ECT-2 acts primarily on RHO-1 and not on CDC-42 during polarity
establishment.
Depletion of RHO-1 or ECT-2 abolished actin-dependent processes such as
cortex ruffling and pseudocleavage furrow formation. Thus, contractile
polarity was not established (Fig.
5). The embryos also failed to extrude the polar bodies (data not
shown) and cytokinesis failed, as has been previously shown
(Jantsch-Plunger et al.,
2000). These results suggest that depletion of RHO-1/ECT-2
disrupts the dynamics of the acto-myosin cytoskeleton.
|
) to
monitor myosin organization and dynamics in control and RNAi embryos
(Fig. 6A). In control embryos,
NMY-2-GFP first forms a dynamic network throughout the entire cortex
consisting of clustered foci interconnected by small filaments. At the onset
of polarity, concomitant with the apposition of the centrosome at the
posterior cortex, this network begins to disassemble in the vicinity of the
nucleus/centrosome complex, and the remaining network segregates towards the
anterior half (Munro et al.,
2004) (see Movie 4 in the supplementary material). Reducing the
function of either RHO-1 (n=10, data not shown) or ECT-2
(n=21) by RNAi altered the NMY-2-GFP organization
(Fig. 6A; Movie 5 in the
supplementary material). Specifically, the early network of interconnected
foci clusters did not form. Instead, small foci were uniformly distributed
throughout the cortex, reminiscent of the small foci that appear after
polarity establishment in control embryos
(Fig. 6A, t=750
seconds, 914 seconds). Despite these defects in myosin organization, we
noticed that in ect-2(RNAi) (Fig.
6A, t=742 seconds, t=918 seconds) and
rho-1(RNAi) (data not shown) embryos the small myosin foci
collectively segregated into an anterior cap in a concerted direction at
similar speeds (average velocity 0.17 µm/second, n=10 foci in one
embryo; Fig. 6C). However, this
cap was not stable (data not shown). In some ect-2(RNAi) embryos (six
out of 21), the segregation occurred off the long embryo axis, as shown in
Fig. 6A. This might reflect a
failure in posteriorization (reviewed by
Cowan and Hyman 2004a), but as
little is known about the molecular details of posteriorization, we did not
analyze this further. In four out of 21 ect-2(RNAi) embryos, NMY-2
segregation did not take place at any time. Similar results were found for
embryos depleted of RHO-1 (data not shown). The cdc-42(RNAi) embryos did not display any obvious structural alterations during the initial assembly of the NMY-2-GFP network (n=9; Fig. 6A, see also Movie 6 in the supplementary material). The contractile network formed and retracted towards the anterior to form a cap as in control embryos. However, the NMY-2-GFP cap was unstable. While the pseudocleavage furrow was regressing, small bright foci appeared and moved back towards the posterior (Fig. 6A, t=763 seconds, t=913 seconds), implicating CDC-42 in stabilizing the acto-myosin network in the anterior half. Ruffle kymographs of cdc-42(RNAi) embryos revealed that the establishment of contractile polarity occurred, but the ruffles were more pronounced and less dynamic (Fig. 5).
RHO-1 is required to form an anterior cortical domain of CDC-42
We have demonstrated above that the anterior cortical localization of
CDC-42 depends on acto-myosin activity and that RHO-1 regulates the
organization of the acto-myosin network. The segregation of the acto-myosin
network, however, could occur independently of RHO-1 activity. We therefore
investigated whether CDC-42 segregation could occur in RHO-1- and
ECT-2-depleted embryos.
In ect-2(RNAi) (n=6) and rho-1(RNAi) (n=5) embryos, YFP-CDC-42 remained localized over the whole cortex and did not segregate into an anterior domain (Fig. 4; data not shown; see Movie 7 in the supplementary material). From this, we conclude that RHO-1 activity is essential for CDC-42 segregation to the anterior, but not its localization to the cortex. To test whether PAR-6 localization also requires RHO-1 activity, we made time-lapse movies of GFP-PAR-6 in rho-1(RNAi) and ect-2(RNAi) embryos. In all rho-1(RNAi) (n=6) and ect-2(RNAi) (n=11) one-cell embryos studied, GFP-PAR-6 remained localized throughout the cortex during the whole cell cycle and failed to segregate into an anterior domain (Fig. 3B; see Movies 8 and 9 in the supplementary material; data not shown). Thus, RHO-1 activity is also essential for the establishment of an anterior PAR-6 domain. Because CDC-42 distribution appears to dictate cortical PAR-6 localization, it is possible that the symmetric distribution of PAR-6 in rho-1(RNAi)/ect-2(RNAi) embryos reflects the defect in CDC-42 segregation. Interestingly, embryos depleted of RHO-1 (n=9/10, data not shown) or ECT-2 (Fig. 6A, n=17/21) sometimes segregated myosin to the anterior, whereas in all embryos studied under the same RNAi conditions, PAR-6 localization remained uniform [ect-2(RNAi), n=63/63; rho-1(RNAi), n=6/6]. RHO-1 activity may therefore couple the anterior movement of myosin II with the anterior segregation of CDC-42 and PAR-6. In support of this idea, co-depletion of RHO-1 and CDC-42 resulted into an additive phenotype (see Fig. S1 in the supplementary material), from which we conclude that RHO-1 and CDC-42 function in separate pathways to localize the PAR proteins.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One interesting aspect of the rho-1(RNAi) phenotype is that the
PAR-2 domain was often expanded, and, in extreme cases, uniformly distributed
along the cortex (see Fig. S2 in the supplementary material). By contrast,
PAR-6 was always uniformly localized (Fig.
3B). Depletion of PAR-5 or proteins implicated in the regulation
or formation of the cytoskeleton showed overlapping anterior and posterior PAR
domains (Cuenca et al., 2003;
Guo and Kemphues, 1996;
Hill and Strome, 1990;
Severson et al., 2002;
Severson and Bowerman, 2003;
Shelton et al., 1999).
However, an aberrant spreading of PAR-2 along the cortex leading, in some
cases, to almost uniform PAR-2 distribution has not been observed previously.
This implicates RHO-1 activity in the regulation of PAR-2 domain size and
suggests that RHO-1-dependent acto-myosin contractility may also help to
define the boundaries between anterior and posterior cortical domains in the
embryo. One model of cortical polarity establishment suggests that the
cortical acto-myosin network is under tension. A local break in the meshwork
causes the meshwork to collapse away from the break point
(Hird and White, 1993),
leaving the voided region of the cortex available for PAR-2 localization.
RHO-1 activity might modulate the contractile forces within the network,
resulting in an alteration of the boundary between the cytoskeleton network
and the PAR-2 domain.
|
). Recent studies in MDCK II epithelial cells have also shown
that PAR-3 modulates actin dynamics by regulating the Rac activity through the
interaction with a Rac-specific GEF, Tiam1
(Chen and Macara, 2005). So
far, no evidence exists that Rac activity or a C. elegans homolog of
Tiam1 is required for polarity establishment in one-cell embryos. However, it
is possible that CDC-42 might regulate acto-myosin dynamics as part of a
PAR-3/PAR-6/aPKC/CDC-42 complex. This idea is supported by studies that have
shown that ECT-2 interacts with PAR-6 in a two hybrid assay
(Liu et al., 2004), and that
PAR-3 inactivates cofilin by LIM kinase 2 (LIMK2), a downstream effector of
the RHO-1 and CDC-42, which alters contractility in MDCK II cells
(Chen and Macara, 2006).
Our data indicate that a key function of CDC-42 during polarity
establishment is to facilitate the localization of PAR-6 to the cortex. In the
absence of CDC-42, PAR-6 is unable to localize to the cortex and PAR-2 is
uniformly distributed along the cortex
(Fig. 1,
Fig. 3A). This differs from
previously published data, which suggested that CDC-42 was required to
maintain PAR-6 in an anterior domain. However, in our study we obtained no
evidence of PAR-6 localization to the anterior cortex after RNAi of CDC-42,
either during the establishment or during the maintenance phase of polarity
(Cuenca et al., 2003). The most
likely reason for this difference is that we are working under stronger RNAi
conditions (see Materials and methods).
How might CDC-42 act to localize PAR-6? In other systems, CDC-42 binds to
PAR-6 and activates its PDZ domain, enabling it to bind other partners. Thus,
one likely possibility is that CDC-42 localizes to the cortex, where it in
turn recruits PAR-6, triggering the assembly of the anterior PAR complex. In
cdc-42(RNAi) embryos, PAR-2 stays localized uniformly over the cortex
as the embryo enters mitosis. This is similar to the phenotype of
par-6(RNAi), providing additional support for the idea that CDC-42
operates in concert with the anterior PAR complex. We show that this aberrant
PAR-2 localization in cdc-42(RNAi) embryos excludes PAR-6 from
localizing to the cortex (Fig.
3A). In support of this idea, recent work has shown that ectopic
mutant PAR-2 excludes PAR-3 from the apical cortex of embryonic somatic
blastomeres (Hao et al.,
2006). Therefore, our data, and the findings of Hao et al.
(Hao et al., 2006), would
support a model in which ectopic PAR-2 localization, in cdc-42(RNAi)
embryos, is sufficient to displace PAR-6 from the cortex. However, it is
unclear whether these two experimental situations rely on the same molecular
machinery. Hao et al. (Hao et al.,
2006) examined the ability of PAR-2 to displace PAR-3 from the
cortex in cells that exhibit epithelial (apical-basal) polarity, and it has
not been investigated whether localization of the anterior PAR complex in
somatic blastomeres has the same molecular requirements, such as for CDC-42,
as in one-cell embryos. Additionally, one important caveat in our experiments
is that we are not working under full loss-of-function conditions for
cdc-42(RNAi) embryos (see Materials and methods) and, thus, we cannot
rule out the possibility that residual CDC-42 in
cdc-42(RNAi);par-2(RNAi) embryos can localize PAR-6 after removal of
PAR-2. Interestingly, PAR-6 is enriched in the anterior cortex in
cdc-42(RNAi);par-2(RNAi) embryos. Because PAR-2 has been depleted,
one might expect that PAR-6 would spread back to the posterior during the
maintenance phase. This anterior enrichment of PAR-6 in
cdc-42(RNAi);par-2(RNAi) embryos could suggest some partially
counterbalancing antagonistic activities, as proposed by Gotta et al.
(Gotta et al., 2001).
In wild-type embryos, PAR-2 does not prevent the localization of PAR-6 at the cortex during meiosis. PAR protein distribution changes from co-localization at meiosis to mutually exclusive domains at mitosis, which suggests that the distribution of PAR proteins is controlled differently during meiosis and mitosis. Thus, it is possible that the mechanism of PAR-6 localization to the cortex differs between the meiotic and mitotic cell cycle, and that cortical PAR-6 localization is CDC-42 independent during meiosis but CDC-42 dependent during mitosis. The cortical PAR-6 localization we observed in par-2(RNAi);cdc-42(RNAi) embryos would reflect the meiotic localization pathway. Ultimately, our experiments cannot distinguish between the varieties of models at this time. We conclude that CDC-42 has two activities: to remove PAR-2 from the cortex at the end of meiosis, and to localize PAR-6 throughout the cell cycle.
Taken together, our data suggest the following model: prior to polarity establishment, active RHO-1, perhaps regulated by ECT-2, organizes the acto-myosin into a contractile network. Both actin polymerization and NMY-2 activity contribute to the structure of the network and could be regulated by RHO-1. RHO-1-independent localization and/or activation of CDC-42 at the cortex triggers assembly of the anterior PAR complex. Upon perception of the centrosomal polarization signal, the myosin network, together with CDC-42 and the anterior PAR complex, segregates to the embryo anterior. This segregation is dependent on RHO-1 activity. At the same time, PAR-2 responds to the altered cortical structure resulting from the anterior segregation of myosin and establishes a posterior cortical domain.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/18/3507/DC1
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ACKNOWLEDGMENTS |
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