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First published online 30 August 2006
doi: 10.1242/dev.02544
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Department of Molecular Biology and Genetics, 101 Biotechnology Building, Cornell University, Ithaca, NY 14853, USA.
* Author for correspondence (e-mail: kjk1{at}cornell.edu)
Accepted 20 July 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Embryo, Polarity, Protein stability, CDC-42, PAR proteins
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
PAR proteins are conserved widely in animals and play roles in such diverse
cellular functions as asymmetric cell division, apicobasal polarity in
epithelial cells, oriented cell migration, neuronal axis specification and
tight junction formation (Nishimura et
al., 2004; Ohno,
2001; Wiggin et al.,
2005).
Considerable progress has been made in understanding events leading up to
the first asymmetric division of C. elegans. After fertilization, the
egg completes the meiotic divisions and produces egg coverings. Upon
completion of meiosis, the zygotic surface begins to ruffle, the result of
local cortical contractions. At the same time, the anterior PAR proteins
(PAR-3, PAR-6 and PKC-3) appear uniformly at the cell periphery. Initially,
the contractions are uniformly distributed over the surface, but shortly after
the sperm pronucleus decondenses, the sperm centrosome signals to the cortex,
leading to a local cessation of cortical ruffling and initiation of a cortical
flow away from the posterior (Cheeks et
al., 2004; Cowan and Hyman,
2004; Munro et al.,
2004). The anterior PAR proteins move with the flow toward the
anterior, clearing them from the posterior, and PAR-1 and PAR-2 proteins are
recruited from the cytoplasm to the smooth cortex in the posterior. Flow
depends upon the activities of the PAR proteins
(Cheeks et al., 2004;
Munro et al., 2004). Mutations
in the anterior PAR proteins allow an initial smoothing of the posterior
cortex, but either block or greatly attenuate the flow into the anterior
(Cheeks et al., 2004;
Kirby et al., 1990;
Munro et al., 2004). After the
establishment phase, PAR-2 protein mediates the maintenance of asymmetry
(Cuenca et al., 2003), in part
by inhibiting a posterior-directed flow of cortical cytoplasm
(Munro et al., 2004), and in
part by antagonizing its own removal from the cortex by PKC-3 phosphorylation
(Hao et al., 2006).
The establishment and maintenance of stable polar PAR domains is based on
the principle of mutual exclusion. In the absence of the anterior PAR
proteins, PAR-1 and PAR-2 are not restricted to the posterior, and, in the
absence of PAR-2, the anterior proteins, as already described, are not stably
restricted to the anterior (Boyd et al.,
1996; Cuenca et al.,
2003; Etemad-Moghadam et al.,
1995; Tabuse et al.,
1998; Watts et al.,
1996). The mutual exclusion of anterior and posterior PAR proteins
depends upon the 14-3-3 family member PAR-5
(Morton et al., 2002).
Cortical localization of the anterior PAR proteins is dependent upon the
activities of all three proteins: PAR-3, PAR-6 and PKC-3
(Hung and Kemphues, 1999;
Tabuse et al., 1998;
Watts et al., 1996). In
par-3 mutants, neither PAR-6 nor PKC-3 protein is detectable at the
cell cortex. Similarly, in par-6 mutants, PKC-3 is not detectable at
the cortex, and in pkc-3(RNAi) embryos PAR-6 is absent. However, in
par-6 mutant and pkc-3(RNAi) embryos, PAR-3 accumulates
weakly and somewhat asymmetrically at the cortex early in the cell cycle, but
is undetectable after metaphase of mitosis. Cortical localization of the
anterior PAR proteins also depends on a conserved interaction of PAR-6 with
CDC-42, a Rho-family GTPase (Gotta et al.,
2001; Kay and Hunter,
2001) (D. Aceto, M.B. and K.K., unpublished).
To find other proteins with possible roles in establishing polarity in
C. elegans, we identified homologs of a set of human proteins that
co-purified with PAR proteins (Brajenovic
et al., 2004) and tested them by RNA interference (RNAi). RNAi
knockdown of one of them, W08F4.8, a homolog of human CDC37, caused a PAR-like
phenotype. Concurrent with our analysis, a genome-wide RNAi screen identified
this same gene as one of a small number of genes whose knockdown results in
PAR-like early cleavages (Sonnichsen et
al., 2005). In addition, Gunsalus and colleagues reported the
distribution of GFP::CDC-37 in the early embryo
(Gunsalus et al., 2005).
CDC37 was initially identified by a temperature-sensitive mutation
defective in the cell cycle at Start in G1 in Saccharomyces
cerevisiae (Reed, 1980).
Subsequent analysis revealed that Cdc37p acts by maintaining the
cyclin-dependent kinase Cdc28p in a state that is competent to associate with
G1 and mitotic cyclins (Gerber et al.,
1995). Vertebrate Cdc37 homologs have been identified in a number
of studies as 
50 kDa proteins that associate with Hsp90 and a variety of
other proteins, particularly kinases
(Hunter and Poon, 1997). The
prevailing view is that Cdc37 functions as a cochaperone of Hsp90, interacting
with protein kinases to regulate their activity, either by enabling them to
fold correctly for proper activation or by preventing them from being degraded
(Hunter and Poon, 1997). Among
the many kinases regulated by Cdc37 and Hsp90 are Raf1
(Grammatikakis et al., 1999;
Perdew et al., 1997;
Silverstein et al., 1998;
Stancato et al., 1993), Cdk4
(Dai et al., 1996;
Stepanova et al., 1996), Akt
(Basso et al., 2002) and Lkb1,
a homolog of C. elegans PAR-4
(Boudeau et al., 2003;
Nony et al., 2003). However,
evidence is accumulating that Cdc37 can act independently of Hsp90 and has a
wider range of client proteins than has previously been appreciated
(MacLean and Picard,
2003).
In this paper, we report that reduction of CDC-37 in the C. elegans early embryo alters the dynamic interactions among the PAR proteins, leading to Par-like embryonic phenotypes. Our analysis of the CDC-37 phenotype has provided insight into the mechanisms of anterior PAR protein accumulation at the cortex.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The Bristol N2
strain was used as wild type. Strains used for this analysis were KK0818,
par-6(zu222) unc-101(m1)/hIn1 [unc-54(h1040)]I; KK0653,
par-3(it71) unc-32(e0189)/qC1; JH1448, axEx1125 [pKR2.04,
Ppie-1::GFP::MEX-5, pRF4, N2 genomic]
(Cuenca et al., 2003); SS629,
[pie-1::gfp::pgl-1, unc-119(+)]
(Cheeks et al., 2004); KK0881,
itIs160 [Ppie-1::GFP::PAR-6 unc-119(+)]; unc-119(ed3); JJ1473,
zuIs45 [Pnmy-2::NMY-2::GFP unc-119(+)]; unc-119(ed3)
(Nance et al., 2003); KK0911,
itIs161 [Ppar-3::PAR-3 S863A::GFP unc-119(+)]; unc-119(ed3)] (B. Li
and K.K., unpublished). The PAR-3::GFP protein in KK0911 carries an alanine
substitution in the PKC-3 phosphorylation site of PAR-3 (Serine 863). PAR-3
S863A::GFP rescues par-3(it71) and gives a stronger cortical signal
than does wild-type PAR-3::GFP.
RNA interference
For some experiments dsRNA was injected into the gonad arms of the worms
(Fire et al., 1998). In this
case, dsRNA was made in vitro from L4440 plasmids
(Timmons and Fire, 1998)
containing the gene of interest, using the RiboMAX kit (Promega). In other
experiments, the L4440 plasmids containing the gene of interest were induced
with IPTG in HT115(DE3) bacteria to produce dsRNA, and the bacteria were fed
directly to worms to produce the RNAi effect
(Timmons et al., 2001). For
most RNAi studies, treated worms were incubated at 25°C for at least 18
hours before either antibody staining or time-lapse microscopy. For
cdc-42(RNAi) studies, injected worms were incubated at 20°C for 8
hours, then 25°C for at least 16 hours before analysis. The efficiency of
each RNAi experiment was determined by assessing the number of dead embryos
laid by a subset of treated worms. Because of variable effectiveness of RNAi
in C. elegans, comparisons between the results of different
experiments are only reliable where the penetrance of each experiment is
high.
Imaging
Observations of live embryos with either DIC or fluorescence microscopy
were made on a Leica DM RA2 microscope with a 63x or 100x Leica
HCX PL APO oil emersion lens and Hamamatsu ORCA-ER digital camera. Digital
images were captured using Openlab software (Improvision). All embryos were
imaged at 22°C immersed in egg buffer (118 mM NaCl, 40 mM KCl, 3.4 mM
CaCl2, 3.4 mM MgCl2, 5 mM Hepes, pH 7.4) on agar pads.
The center of the embryo was chosen as the focal plane for the live
observations. For cortical images, we focused at the cortex of partially
flattened fixed embryos. We adjusted each image in PhotoShop, to remove
background signal, by bringing the posterior cortical signal to zero. To count
puncta, we sampled two similar-sized, non-overlapping windows from the center
of the anterior cortex by laying an acetate sheet sequentially over the
grayscale images of each fluorochrome and marking the positive puncta. We took
the average of two samples for each embryo. Egg length was measured using
Openlab software. Two-cell blastomere sizes were compared by measuring the
length along the long axis of the embryo of AB and P1 in control and
cdc-37(RNAi) embryos. Confocal images were collected on the Leica TCS
SP2 system with the Leica DMRE-7 microscope and an HCX PL APO 63x oil
immersion lens. Images were processed using the Leica Confocal SP2 software
program and Adobe PhotoShop.
Immunohistochemistry
Immunostaining for PAR-2, PAR-3, PAR-6, PKC-3 and GFP was performed using a
standard methanol fixation (Guo and
Kemphues, 1995). Antibodies used were: anti-PAR-2 rabbit
polyclonal (Boyd et al., 1996),
1:3; anti-PAR-3 mouse monoclonal (Nance et
al., 2003), 1:75; anti-PAR-6 rabbit polyclonal
(Hung and Kemphues, 1999),
1:15; anti-PKC-3 rat polyclonal (Hung and
Kemphues, 1999), 1:10; and anti-GFP goat polyclonal (Rockland
Immunochemicals), 1:3000. Slides were incubated with primary antibodies for
12-18 hours at 4°C. Slides were then washed three times in PBS, 0.5% Tween
20. All secondary antibodies were from Jackson Immunochemicals: goat
anti-rabbit FITC, 1:200; goat anti-mouse Cy3, 1:250; donkey anti-rat Cy3,
1:400; donkey anti-goat FITC, 1:100. Embryos were treated with secondary
antibodies for 3 hours at 37°C. Slides were washed as before and then
covered with VectaShield (Vector Laboratories).
GFP::CDC-37 transgenic animals
We created a vector for expressing GFP::CDC-37 by inserting cdc-37
cDNA (W08F4.8a, 1113 nucleotides, amplified from the Gibco Proquest Library)
into the pJunc vector, a derivative of pMW1.03, constructed by Aaron Schetter
(Gunsalus et al., 2005), which
contains a 5.7 kb genomic fragment of unc-119 (pPDMM016)
(Maduro and Pilgrim, 1995) and
a par-2 coding sequence fused to GFP and driven by the pie-1
promoter (Wallenfang and Seydoux,
2000). The par-2 gene was removed from the vector by
Spe1 cleavage and cdc-37 cDNA was inserted into the same
site. Low-copy integrated lines expressing GFP::CDC-37 in the germline were
identified among unc-119(ed3) worms following microparticle
bombardment (Praitis et al.,
2001).
Testing for rescue of cdc-37(RNAi) by GFP::CDC-37
To test for rescue, worms carrying the GFP::CDC-37 transgene were injected
with dsRNA specific to the first 160 nucleotides of the endogenous
cdc-37 3'UTR. dsRNA specific to the 3'UTR of a gene is an
effective RNAi trigger (Parrish et al.,
2000). Progeny of the injected worms were assayed for lethality 48
hours after injection. Eighty-five to 100% of embryos from 20 wild-type worms
were dead compared with 32-50% of embryos from 28 GFP::CDC-37-expressing
worms.
Western blots
Populations of control or RNAi worms were collected from feeding plates 20
hours after feeding began; adults were bleached to obtain early embryos; and
embryos were lysed in SDS sample buffer. Samples were analyzed by Bradford
Assay (Bradford, 1976) to
determine total protein present in each sample. Equal amounts of total protein
were loaded onto an 8% SDS PAGE gel. The proteins were then transferred to
nitrocellulose membrane for western blotting
(Burnette, 1981) and stained
with the MemCode Reversible Membrane Staining Kit (Pierce) to verify equal
loading of samples. All primary antibodies were diluted 1:1000 in 1xTBST
with 0.5% milk and incubated overnight at 4°C. Appropriate HRP-conjugated
secondary antibodies (Jackson ImmunoResearch) were diluted 1:5000 in
1xTBST with milk, incubated for one hour at 22°C, then washed with
TBST three times for a total of 30 minutes. Bands were visualized using a
chemiluminescence detection reagent (Amersham Biosciences).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). On
the basis of this result, we sought to test Cdc37 homologs for roles in
polarity establishment in C. elegans. Of the three potential Cdc37
homologs identified by sequence similarity, only one, W08F4.8, was predicted
to encode a protein similar in overall size and domain structure to the H.
sapiens CDC37 protein (see Fig. S1 in the supplementary material).
W08F4.8 protein was found to interact with the Hsp90 family member DAF-21 in a
yeast two-hybrid assay (Li et al.,
2004). In spite of sequence similarity to CDC37 and conservation of HSP-90 binding, we found that W08F4.8 cDNA expressed under the control of the yeast GAL1/10 promoter was unable to complement a temperature-sensitive cdc37 yeast mutation. Even though C. elegans W08F4.8 failed this test for orthology with yeast CDC37, it is the C. elegans gene most similar in sequence to yeast, fruit fly and mammalian Cdc37 proteins. Therefore we refer to the gene as cdc-37.
To test whether CDC-37 has a role in embryonic polarity in C.
elegans, we depleted the message from early embryos by using RNAi.
Wild-type worms injected with dsRNA specific to cdc-37 show
increasing levels of embryo lethality: 30% at 0-17 hours versus >80% 18-24
hours post-injection. Embryos collected at this time show an approximately 80%
reduction in steady-state GFP::CDC-37 levels (see Fig. S2 in the supplementary
material). All embryos used for phenotypical analysis were dissected during
this latter interval. Injected worms become sterile approximately 24 hours
after injection. Sterile animals have sperm in the spermatheca as determined
by DAPI staining, and developing oocytes are present; however, oocytes
accumulate behind the spermatheca, suggesting a failure in egg maturation or
ovulation (McCarter et al.,
1999). Approximately 18% of the worms that escape embryonic
lethality show a protruding vulva phenotype. Other less frequently observed
post-embryonic phenotypes include larval arrest and tail rupture.
The affected embryos from cdc-37(RNAi) worms exhibit defects consistent with a loss of embryonic polarity (Fig. 1). The extent of cortical ruffling in cdc-37(RNAi) embryos varies from almost normal to barely detectable (Fig. 1F,G). The position of the pseudocleavage furrow in cdc-37(RNAi) embryos does not differ from that of controls (n=9 wild type and 11 cdc-37(RNAi); P=0.24). However, the pseudocleavage invaginations in cdc-37(RNAi) embryos are shallow (37.9±13.2 µm versus 47.4±5.1 µm in wild type, P=0.012), tend to be unilateral (Fig. 2G; 6/11 embryos), and are short lived (113±22 seconds, n=11, versus 191±56 seconds in wild type, n=11; P=0.0003). In cdc-37(RNAi) embryos, the two pronuclei meet more centrally (Fig. 1C,H; 56±8% egg length versus 70±3% egg length in wild type; n=14; P<0.0001) and the first cleavage is more equal (Fig. 1I; AB, the anterior two-cell stage blastomore, occupies 52±4% of total egg length, n=39, versus 58±2% in wild type, n=17; P<0.0001). The second cleavage is more synchronous after cdc-37(RNAi) (Fig. 1J) and is slightly slower than wild type. In cdc-37(RNAi) embryos, the period between nuclear envelope breakdown (NEB) in the zygote, P0, to NEB in its daughter cells AB and P1 was 1070±82 seconds, whereas the time between NEB in P0 and AB in wild type was 859±49 seconds (P<0.0001), and the time between NEB in P0 and P1 was 966±58.5 (P=0.0018; n=9, wild type; n=11, cdc-37(RNAi)). In contrast to the reproducible orthogonal spindle orientations of wild-type two cell embryos (n=11), cdc-37(RNAi) embryos display a variety of spindle orientations, with 85% exhibiting phenotypes different from wild type (Fig. 1J; n=39): 56% of the two-cell embryos divide with both spindles transverse to the long axis.
We also examined molecular markers of embryonic polarity, GFP::MEX-5
(Cuenca et al., 2003;
Schubert et al., 2000) and P
granules using GFP::PGL-1 (Cheeks et al.,
2004; Strome and Wood,
1982; Strome and Wood,
1983). We found that asymmetry of both markers is dependent upon
CDC-37 (Fig. 2; for GFP::MEX-5,
asymmetry was normal in 8/8 wild-type versus 1/10 cdc-37(RNAi)
embryos; for GFP::PGL-1, asymmetry was normal in 12/12 wildtype
versus 6/16 cdc-37(RNAi) embryos).
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; Kirby et al.,
1990; Morton et al.,
2002; Tabuse et al.,
1998; Watts et al.,
1996). We tested whether depletion of CDC-37 alters the
distributions of the PAR proteins. Five PAR proteins have a highly
reproducible asymmetry in the developing embryo. PAR-3, PAR-6 and the atypical
protein kinase C PKC-3 colocalize to the anterior cortex of the one-cell
embryo (Etemad-Moghadam et al.,
1995; Hung and Kemphues,
1999; Tabuse et al.,
1998), and PAR-1 and PAR-2 colocalize to the posterior cortex of
the embryo (Boyd et al., 1996;
Guo and Kemphues, 1995). We
found that the distribution of both sets of proteins is dramatically altered
by depletion of CDC-37.
In contrast to control embryos (Fig.
3A-C, n=32; Fig.
4, column 3, n=13) PAR-1, PAR-2, PAR-6 and PKC-3 are
evenly distributed around the entire cortex of one-cell cdc-37(RNAi)
embryos throughout the cell cycle beginning just after the completion of
meiosis (Fig. 3D-I;
Fig. 4, column 4;
n=12; data not shown for PAR-1). The abundance of PAR-1 and PAR-2 at
the cortex appears to be reduced relative to control embryos. The relative
abundance of PAR-6 and PKC-3 does not appear to be affected, although PKC-3
levels are lower by western blot assays (see below). GFP::PAR-6 in control
embryos enters the nucleus at nuclear envelope breakdown in 10 out of 10
embryos (not shown) (Cuenca et al.,
2003). This fails to occur in eight out of nine
cdc-37(RNAi) embryos, and these embryos consistently exhibit a
mitosis-specific peri-centrosomal accumulation of GFP::PAR-6 that is never
seen in control embryos (Fig.
4). Finally, whereas PAR-6 accumulates in cortical puncta in
wild-type embryos (Hung and Kemphues,
1999), in cdc-37(RNAi) embryos it has a much smoother
cortical distribution (data not shown).
Surprisingly, PAR-3 behaves differently from PAR-6 and PKC-3 in
cdc-37(RNAi) embryos. In control embryos, PAR-3 is evenly distributed
along the entire cortex early in the cell cycle and becomes restricted to the
anterior of the cell along with PAR-6 and PKC-3
(Etemad-Moghadam et al., 1995)
(Fig. 3B;
Fig. 4; n=82). In
cdc-37(RNAi) embryos, PAR-3, like PKC-3 and PAR-6, has a significant
overlap with PAR-2 (Fig. 3D-F;
n=57). However, in these embryos PAR-3 forms large cortical
aggregates, and is more patchily distributed than are PAR-6 and PKC-3
(Fig. 3E,F;
Fig. 4). Furthermore, unlike
PAR-6 and PKC-3, which remain uniformly distributed around the cortex in
cdc-37(RNAi) embryos, PAR-3 clears from the posterior cortex during
pronuclear migration. In control embryos, PAR-3 clears from the posterior
cortical region to 49%±5% of the total egg length (n=10); in
cdc-37(RNAi) embryos, the maximal clearing reaches only
40%±11% (n=19) of the egg length. Finally, whereas PAR-6 and
PKC-3 remain cortical throughout the cell cycle in cdc-37(RNAi)
embryos, PAR-3 consistently disappears from the cortex at metaphase
(Fig. 4, column 2, fourth
image). PAR-3 returns to the cortex late in the cell cycle and is distributed
to both daughter cells in a patchy and variable distribution, disappearing
again at the next metaphase and coming back to the cortex late in the second
cell cycle. This pattern of PAR-3 dynamics is similar to that observed in
par-6 mutant and pkc-3(RNAi) embryos
(Tabuse et al., 1998;
Watts et al., 1996).
These results were unexpected because in the cdc-37(+) background,
cortical localization of PAR-6 and PKC-3 is dependent upon PAR-3
(Hung and Kemphues, 1999;
Tabuse et al., 1998).
Therefore, we hypothesized that CDC-37 activity contributes to the normal
dynamics of these proteins at the cortex, but when it is reduced or absent
PAR-6 and PKC-3 can accumulate at the cortex independently of PAR-3.
To test this hypothesis, we compared the distribution of PAR-6 and PKC-3 in
par-3(it71) embryos with or without RNAi-mediated depletion of CDC-37
(Fig. 5). As previously
reported (Hung and Kemphues,
1999; Tabuse et al.,
1998) in cdc-37(+); par-3(it71) embryos, PAR-6
and PKC-3 are not detectable at the cortex at any stage in the cell cycle
(Fig. 5A,C; n=50).
However, in cdc-37(RNAi); par-3(it71) embryos, PAR-6 and
PKC-3 accumulate at the cortex but are not asymmetrically localized
(Fig. 5B,D; n=42).
Thus, cortical accumulation of PAR-6 and PKC-3 is dependent upon PAR-3 only in
the presence of normal levels of CDC-37.
To determine whether CDC-37 acts preferentially through PAR-6 or PKC-3, we
depleted CDC-37 in par-6(zu222) worms
(Fig. 5G), and doubly depleted
PKC-3 and CDC-37 in wild-type worms (Fig.
5H), and assayed the remaining anterior complex proteins by
immunofluorescence microscopy. As previously shown by Hung and Kemphues, in
par-6(zu222) worms, PKC-3 is not detectable at the cortex
(Hung and Kemphues, 1999)
(Fig. 5G; n=21). PKC-3
is also absent from the cortex in cdc-37(RNAi); par-6(zu222) embryos
at all stages of the first cell cycle (Fig.
5H; n=25). As shown by Tabuse and colleagues, in
pkc-3(RNAi) embryos, PAR-6 is not detectable at the cortex
(Tabuse et al., 1998)
(Fig. 5E; n=102).
However, after double RNAi of PKC-3 and CDC-37, PAR-6 is present symmetrically
at the cortex of one-cell embryos at all stages of the first cell cycle
(Fig. 5F; n=170).
Thus, in a cdc-37(RNAi) background, PAR-6 is required for PKC-3
localization to the cortex, but PKC-3 is not required for PAR-6 cortical
localization. We interpret these results to mean that CDC-37 is required to
prevent PAR-6 from associating with the cortex, and that PAR-3 normally acts
to block the action of CDC-37.
We also examined the distribution of PAR-3 in these doubly depleted embryos. We found, as expected, that PAR-3 becomes partially restricted to the anterior cortex in par-6 mutant embryos, pkc-3(RNAi) embryos, cdc-37(RNAi) embryos, pkc-3(RNAi); cdc-37(RNAi) and par-6(zu222); cdc-37(RNAi) embryos (data not shown). However, we noted that removal of PAR-6 or depletion of PKC-3 in cdc-37(RNAi) embryos led to a slight increase in the levels of cortical PAR-3 (data not shown).
Cortical localization of PAR-6 in cdc-37(RNAi) embryos depends upon CDC-42
As described above, cdc-37(RNAi) allows constitutive cortical
accumulation of PAR-6. If this reflects a role for CDC-37 in the normal
mechanism for PAR-6 cortical accumulation and asymmetry, we would expect this
cortical accumulation to be dependent upon the Rho-GTPase CDC-42. Binding to
CDC-42 is required for the proper accumulation and maintenance of PAR-6 at the
cortex of the one-cell embryo (Gotta et
al., 2001; Kay and Hunter,
2001) (D. Aceto, M.B. and K.K., unpublished). In
cdc-37(RNAi) embryos, GFP::PAR-6 is evenly distributed around the
entire cortex (Fig. 4, column
4). By contrast, in cdc-42(RNAi) embryos, a reduced amount of
GFP::PAR-6 accumulates at the cortex early in the first cell cycle and becomes
partially restricted to the anterior, but is undetectable after metaphase
(Fig. 6A,B; D. Aceto, M.B. and
K.K., unpublished). We found that depleting both CDC-42 and CDC-37 by RNAi
results in a phenotype that differs from either single RNAi. In
cdc-42(RNAi); cdc-37(RNAi) embryos, GFP::PAR-6 does not accumulate at
the cortex (7/11 embryos; Fig.
6C,D). Thus, CDC-42 is required for the constitutive cortical
accumulation of PAR-6 in cdc-37(RNAi) embryos, and CDC-37 is required
for the residual PAR-6 in cdc-42(RNAi) embryos.
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), and thus is consistent with the hypothesis that most or
even all cortical PAR-6 in cdc-42(RNAi) embryos is associated with
PAR-3. The proportion of puncta that contained only PAR-3 did not show a
statistically significant difference between control and cdc-42(RNAi)
embryos (see Table S1 in the supplementary material). These results suggest
that there are two sites for PAR-6 cortical association, one dependent on
CDC-42, and the other independent of CDC-42 and colocalizing with PAR-3.
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). Just
after the completion of meiosis in wild-type embryos, NMY-2::GFP is found in
distinct foci and filaments throughout the entire cortex. In response to the
polarization signal, the cortical cytoskeleton, including the NMY-2::GFP foci,
flows into the anterior (Munro et al.,
2004). To determine the effect of knockdown of CDC-37 on NMY-2
localization, we examined cdc-37(RNAi) and control embryos expressing
NMY-2::GFP. We found that, in cdc-37(RNAi) embryos, NMY-2::GFP forms
foci and filaments like wild-type controls do. The foci flow into the
anterior, clearing approximately 40% of the posterior cortex in contrast to
the 50% clearing observed in control embryos. Based on descriptions by Munro
and colleagues (Munro et al.,
2004), this behaviour is similar to that seen when anterior PAR
proteins are reduced or eliminated. As the two pronuclei migrate to the center
of the embryos, the large NMY-2 foci in the anterior are replaced by fine
fibers, as occurs in controls; however, in contrast to controls, asymmetry is
lost. Cortical myosin drops to a level normally seen in the posterior (see
Fig. S3 in the supplementary material).
CDC-37 protein is uniformly distributed in the cytoplasm and accumulates weakly on the spindle at mitosis
Previous work had shown that GFP::CDC-37 exhibited a distribution typical
of free cytosolic proteins (Gunsalus et
al., 2005). We independently constructed a GFP::CDC-37
translational fusion expressed under the control of the pie-1
promoter and generated transgenic lines by biolistic bombardment
(Praitis et al., 2001). We
recovered nine independent lines showing similar GFP expression in the embryo.
The Ppie-1::gfp::cdc-37 transgene suppresses the lethality of
cdc-37(RNAi), indicating that the transgene is functional (see
Materials and methods). As previously reported, from meiosis until NEB,
GFP::CDC-37 is evenly distributed throughout the cytoplasm in a pattern
consistent with free cytosolic proteins
(Fig. 8A-C). In our strains, at
NEB, GFP::CDC-37 remains predominantly cytoplasmic but becomes transiently
enriched around the chromosomes (Fig.
8D). As mitosis progresses, CDC-37 becomes slightly enriched on
the spindle (Fig. 8E). Upon
nuclear envelope reformation, CDC-37 is excluded from the nucleus
(Fig. 8F).
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PKC-3 and PAR-4 protein levels, however, were affected by cdc-37(RNAi). PKC-3 levels were reduced in two out of three experiments by more than 80% (Fig. 9A). The effect on PAR-4 was less pronounced. When wild-type embryonic proteins were separated by gel electrophoresis and probed with a PAR-4 antibody, two predominant bands were observed: a faster migrating band representing about 90% of the antibody-reactive protein and a slower band (Fig. 9B). The slower migrating band is a phosphorylated form of the protein, as this band is eliminated by treatment of the sample with phosphatase (data not shown). Several independent samples of cdc-37(RNAi) embryos have shown a consistent reduction of the slower migrating PAR-4 band.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Depletion of CDC-37 causes Par-like phenotypes
cdc-37(RNAi) embryos exhibit polarity-defective phenotypes,
including altered cortical dynamics, equal first cleavage and failure to
localize MEX-5 and P granules. These phenotypes apparently arise through the
effect of CDC-37 on the distribution of the PAR proteins. It is probable that
the embryonic phenotypes we describe do not represent the null condition, as
at the time we sample embryos some CDC-37 protein remains.
Some cdc-37(RNAi) phenotypes can be explained by depletion of PKC-3
In wild-type embryos, stable cortical localization of the anterior PAR
proteins, PAR-3, PAR-6 and PKC-3, requires the proper function of all three
proteins (Hung and Kemphues,
1999; Tabuse et al.,
1998; Watts et al.,
1996). PAR-6 and PKC-3 are absolutely dependent upon PAR-3
(Hung and Kemphues, 1999;
Tabuse et al., 1998), whereas
PAR-3 exhibits only a partial dependence on the other two proteins. The
reduced PAR-3 asymmetry, its patchy distribution and its disappearance at
metaphase in cdc-37(RNAi) embryos is identical to what is observed in
par-6(zu222) and pkc-3(RNAi) embryos
(Tabuse et al., 1998;
Watts et al., 1996). Indeed,
PAR-3 distribution and dynamics are identical in cdc-37(RNAi)
embryos, par-6(zu222); cdc-37(RNAi) embryos and pkc-3(RNAi);
cdc-37(RNAi) embryos. A simple explanation for the effect on PAR-3
distribution and dynamics comes from the observation that PKC-3 levels are
consistently reduced after cdc-37(RNAi) (see below). Reduced levels
of PKC-3 can also account for the failure of PAR-2 to become restricted to the
posterior, as exclusion of PAR-2 from the anterior requires the
phosphorylation of PAR-2 by PKC-3 (Hao et
al., 2006). Similar arguments may also hold for PAR-1
(Hao et al., 2006). NMY-2::GFP
behaviour in cdc-37(RNAi) embryos can also be explained by reduced
levels of PKC-3.
PAR-6 and PKC-3 cortical accumulation is independent of PAR-3 in cdc-37(RNAi) embryos
A reduction of PKC-3 cannot explain the entire cdc-37(RNAi)
phenotype, however, as pkc-3(RNAi) and cdc-37(RNAi)
phenotypes differ. In contrast to the failure of PAR-6 to accumulate
cortically in pkc-3(RNAi) embryos
(Tabuse et al., 1998), PAR-6
and the residual PKC-3 remain at the cortex throughout the cell cycle in
cdc-37(RNAi) embryos, and do so even in the absence of PAR-3. Our
epistasis analysis reveals that PAR-3-independent cortical localization of
PKC-3 requires PAR-6, but that PAR-6 does not require PKC-3. Thus, reduction
of CDC-37 renders PAR-6 and PKC-3 independent of PAR-3 for cortical
localization, and renders PAR-6 independent of PKC-3 for its cortical
localization.
We interpret these results to mean that CDC-37 activity normally influences the dynamics of the anterior complex proteins. We propose that CDC-37 and one or more of its client protein(s) function in wild-type cells to block the binding of PAR-6 to an unknown cortically associated protein. We further propose that binding to this cortical protein is part of the normal mechanism by which PAR-6 and PKC-3 localize to the anterior cortex. In wild-type cells, PAR-3 acts locally to antagonize the action of the CDC-37 client and allows PAR-6 to bind only in the anterior. In wild-type cells, this binding also requires PKC-3, perhaps because PAR-3 exerts its blocking effect via PKC-3.
PAR-6 requires CDC-42 to bind to the cortex in cdc-37(RNAi) embryos
It is also possible that the PAR-3-independent, cortical-binding site for
PAR-6 in cdc-37(RNAi) embryos is spurious and has no normal role in
anterior PAR protein function. Although we cannot rule this out, two
observations make this unlikely. First, in keeping with previous
immunolocalization results (Hung and
Kemphues, 1999), we found that in wild-type embryos a substantial
fraction of cortical PAR-6 does not colocalize in puncta with PAR-3. Second,
the accumulation of PAR-6 at the cortex in cdc-37(RNAi) embryos is
dependent upon CDC-42, just as it is in cdc-37(+) embryos. We also
found that, in cdc-42(RNAi) embryos, all or most of the residual
PAR-6-containing puncta also contain PAR-3. A further complexity of our
results is that double depletion of CDC-37 and CDC-42 results in a complete
absence of GFP::PAR-6 at the cortex, a phenotype that differs from that
resulting from depletion of CDC-42 alone. This indicates that CDC-37 is
required for the residual GFP::PAR-6 puncta in cdc-42(RNAi) embryos,
suggesting that these puncta are not the result of partial depletion of
CDC-42, but rather represent a different class of PAR-6-binding site.
|
). In these studies, the PAR-6 PDZ domain was found to be able
to bind to a carboxy-terminal-type synthetic ligand in a CDC-42-regulated
manner. PAR-6 was also found to bind weakly to the N-terminal PDZ domain of
Bazooka (PAR-3) in a CDC-42-independent manner. Furthermore, the peptide
ligand did not compete with PAR-3 for binding
(Peterson et al., 2004). We
also infer from our results and previously published results the existence of
a class of PAR-3-containing puncta that do not contain PAR-6, and that in the
early part of the cell cycle are independent of CDC-42, CDC-37, PKC-3 and
PAR-6 for their cortical localization.
Our results contribute to a growing body of evidence that supports a
dynamic regulation of anterior PAR protein distribution, as opposed to the
simple concept of an obligate complex suggested by early biochemical and
co-localization results. PAR-6 can bind to a variety of partners including
Lethal Giant Larva (Betschinger et al.,
2003; Plant et al.,
2003; Yamanaka et al.,
2003), Crumbs (Kempkens et
al., 2006; Lemmers et al.,
2004) and Pals1 (Hurd et al.,
2003; Wang et al.,
2004). Distinct distributions for Bazooka (PAR-3) versus PAR-6 and
aPKC have been described in Drosophila epithelial cells and
photoreceptors (Harris and Peifer,
2005; Nam and Choi,
2003), and Par3-independent cortical recruitment of Par6 and aPKC
occurs in mammalian astrocytes
(Etienne-Manneville and Hall,
2001). It will be interesting to learn to what extent the
mechanisms for regulating these complexes are evolutionarily conserved.
cdc-37(RNAi) causes the depletion of the steadystate levels of PKC-3 and of phosphorylated PAR-4
CDC-37 is known to function as a protein chaperone in many systems,
regulating the stability and function of many proteins, especially kinases
(MacLean and Picard, 2003). We
found that the steady-state level of PKC-3 is affected by depletion of CDC-37.
Cdc37 was identified among proteins complexed with aPKC
(Brajenovic et al., 2004), and
thus may directly regulate its stability and, perhaps, its activity.
Biochemical support for our genetic evidence that another kinase (or
kinases) might also be a client of CDC-37 comes from the observation that
cdc-37(RNAi) consistently reduced the level of a phosphorylated form
of PAR-4. The effect on this minor PAR-4 isoform, however, cannot account for
all of the polarity phenotypes of the cdc-37(RNAi) embryos, because
these phenotypes differ substantially from those of par-4
loss-of-function mutants (Watts et al.,
2000). We considered that PKC-3 might be functioning to
phosphorylate PAR-4, but the phosphorylated form of PAR-4 was unaffected by
pkc-3(RNAi) (M.B. and K.K., unpublished). We also considered the
possibility that the unique cdc-37(RNAi) phenotype might be revealing
a redundant function among the three known polarity kinases PAR-1, PAR-4 and
PKC-3. However, doubly or triply depleted embryos (e.g. par-4(it57);
par-1(RNAi); pkc-3(RNAi)) did not replicate the cdc-37(RNAi)
phenotype (M.B. and K.K., unpublished). We also ruled out the possibility that
the phenotype was a gain-of-function defect due to an imbalance between PAR-4
isoforms, by showing that cdc-37(RNAi); par-4(it57) embryos had early
polarity defects identical to those of cdc-37(RNAi) alone (M.B. and
K.K., unpublished).
We have shown that C. elegans CDC-37 has a role in the establishment of polarity in the one-cell embryo. We also present evidence that we interpret as indicating the existence of two modes of binding for PAR-6 at the cortex: CDC-42-dependent binding at a site that is normally blocked by the action of a CDC-37 client; and CDC-42-independent binding at a site that co-localizes with PAR-3 and that is also dependent upon a CDC-37 client. We propose that, in wild-type embryos, CDC-37-mediated inhibition of the CDC-42-dependent binding site and PAR-3-mediated release of this inhibition provide a key mechanism for the anterior accumulation of PAR-6. Identifying the ligand or ligands for cortical binding, and the relevant client or clients of CDC-37 that regulate this binding will advance our understanding of the mechanism of action of the anterior PAR proteins.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/19/3745/DC1
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ACKNOWLEDGMENTS |
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