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First published online 4 October 2006
doi: 10.1242/dev.02615
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1 Department of Biology, Ursinus College, Collegeville, PA 19426, USA.
2 Institute of Molecular Biology, University of Oregon, Eugene, OR 97403,
USA.
* Author for correspondence (e-mail: rlyczak{at}ursinus.edu)
Accepted 6 September 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: C. elegans, Anteroposterior axis, Meiotic exit, PAM-1, CYB-3, Aminopeptidase
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Maturation
cues entry into metaphase I (Greenstein,
2005), but fertilization is required for extrusion of the first
polar body and the second meiotic division
(McNally and McNally, 2005).
During meiosis, the sperm chromosomes remain condensed near the future
posterior pole. Immediately following extrusion of the second polar body, the
embryo exits meiosis, the sperm and egg chromosomes decondense, and pronuclei
appear as the embryo enters the first mitotic interphase
(Albertson and Thomson, 1993;
Schneider and Bowerman,
2003).
Near the end of meiosis II or during the first interphase, the
anteroposterior (AP) axis is established. During axis formation, the sperm
pronucleus/centrosome complex (SPCC) is closely apposed to the cell membrane
at the cortex of the zygote (Rappleye et
al., 2002), and its position defines the posterior pole
(Albertson, 1984;
Goldstein and Hird, 1996;
Schneider and Bowerman, 2003).
Evidence points to the centrosome as the key SPCC component that orchestrates
axis polarization (Cowan and Hyman,
2004b; Hamill et al.,
2002; O'Connell et al.,
2000; Sadler and Shakes,
2000; Sonneville and
Gönczy, 2004). The centrosome, by an unknown mechanism,
appears to destabilize the cortical actomyosin network in the posterior
(Munro et al., 2004). This
destabilization results in a flow of cortical F-actin and nonmuscle myosin to
the anterior pole, along with the PDZ-domain polarity proteins PAR-3 and PAR-6
(Cuenca et al., 2003;
Munro et al., 2004). Following
this initial polarization, the ring-finger protein PAR-2 becomes enriched at
the posterior cortex, where it stabilizes polarity
(Cuenca et al., 2003;
Munro et al., 2004). These
reciprocal domains are then maintained due to interactions between the PAR
proteins themselves (Cuenca et al.,
2003). The PAR proteins are required for all but the initial AP
asymmetries, which occur in response to the centrosome, including posterior
displacement of the first mitotic spindle and the polarized distribution of
the germline P granules and developmental determinants
(Cowan and Hyman, 2004a;
Lyczak et al., 2002;
Schneider and Bowerman,
2003).
One process that is important for axis formation is cell cycle progression,
as polarity establishment occurs immediately after meiotic exit. Moreover,
many mutants with meiotic defects also have polarity defects, although these
are in some cases separable (Liu et al.,
2004; Rappleye et al.,
2002; Shakes et al.,
2003; Sonneville and
Gönczy, 2004). For example, the scaffolding protein CUL-2 and
its putative adaptor ZYG-11 are components of a cullin-based E3 ligase
required for progression through meiosis and AP axis formation, most likely
through degradation of different targets
(Liu et al., 2004;
Sonneville and Gönczy,
2004). Nevertheless, proper progression through meiosis may be
important for axis formation. Mutants with a partial loss of the
anaphase-promoting complex (APC) sometimes completely bypass meiosis II, and
when this bypass occurs these mutants exhibit AP axis defects
(Shakes et al., 2003). These
results suggest that either cell cycle progression is important for AP
polarity establishment, or that the machineries governing cell cycle
progression and cell polarity share some components.
If common machinery links meiotic completion and axis establishment, it may
be proteolytic. All the proteins mentioned above are components of either the
APC or SCF E3 ubiquitin ligase complexes, proposed to target specific
substrates for degradation during the cell cycle
(Bowerman and Kurz, 2006;
Koepp et al., 1999).
Furthermore, CUL-2-based ECS E3 ligase(s) have been shown to regulate cell
polarity by degrading developmental determinants in the anterior or posterior
cytoplasm during and after the first asymmetric division of the one-cell
zygote (DeRenzo et al., 2003).
Acting downstream of these E3 ubiquitin ligases is the 26S proteasome.
Intriguingly, a puromycin-sensitive aminopeptidase (PSA) appears to colocalize
with the 26S proteosome and to participate in proteolytic events to regulate
the cell cycle in mammalian cells (Constam
et al., 1995). PSA family members are metalloproteases that
hydolyze N-terminal amino acids and are members of the M1 metalloprotease
family, characterized by the HEXXH(X)18E metal coordination site
and an upstream GAMEN motif (Laustsen et
al., 2001). These peptidases are widely conserved and include a
human family member (Taylor,
1993). Caenorhabditis elegans has one PSA homolog, PAM-1,
a cytoplasmic aminopeptidase localized to neurons and intestinal cells in
larvae and adults (Brooks et al.,
2003). While PSA has been implicated in cell cycle regulation in
mammals, its requirements remain largely unstudied. Here we show that C.
elegans PAM-1 is contributed by both the sperm and the egg, and is
required for timely exit from meiosis and AP axis specification. These two
processes appear to be independently regulated by PAM-1. We propose
that PAM-1 is part of proteolytic machinery in the early embryo used to
trigger meiotic completion and trigger axis formation through regulated
protein degradation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). N2 Bristol was
used for wild-type and CB4856 for SNP mapping. The following alleles and
balancers were used: LG I dpy-5(e61); LG II rol-6(e187); LG
III unc-32(e189); LG IV unc-5(e53), bli-6(sc16), unc-8(e49),
pam-1(or282ts), pam-1(or403ts), pam-1(or347ts), pam-1(or547ts),
pam-1(or370ts), unc-24(e138), dpy-20(e1282), him-8(e1489), DnT1 (LG IV/V
translocation balancer); LGV him-5(e1409), dpy-11(e224), fog-2(q71),
unc-51(e1189); X lon-2(e678).
GFP lines: JH227 pie-1::GPF axEx73[pJH3.92;pRF4]
(Reese et al., 2000); WH204
pie-1::GFP::ß-TUBULIN
(Strome et al., 2001); and
AZ212 pie-1::GFP::H2B (Praitis et
al., 2001) were used to construct strains EU929
pam-1(or282ts)/DnT1 IV; +/DnT1 V; pie-1::GFP
axEx73[pJH3.92;pRF4], EU881 pam-1(or282ts) IV;
pie-1::GFP::ß-TUBULIN, and EU947 pam-1(or282ts)/DnT1
IV; +/DnT1 V; pie-1::GFP::H2B. The CYB-3::GFP strain was
provided by P. Gönczy (Sonneville and
Gönczy, 2004).
Homozygous temperature-sensitive strains were propagated at the permissive temperature of 15°C. L4 larvae were shifted to 25°C overnight before analysis of the mutant embryos.
Isolation and characterization of pam-1 alleles
Five pam-1 alleles (or282ts, or347ts, or370ts, or403ts,
or547ts) were identified in a screen for temperature-sensitive embryonic
lethal mutants (Encalada et al.,
2000). This locus was originally named scu-1 (sperm cue
abnormal) but subsequently changed to pam-1 after gene identity was
determined. Complementation tests were performed for each allele with
or282ts. All alleles showed weak temperature sensitivity for
embryonic lethality when homozygous hermaphrodites were shifted to 25°C as
L4s. pam-1(or282ts) was 85.3% embryonic lethal at 15°C
(n=495), and 97.0% embryonic lethal at 25°C (n=396).
Similarly, pam-1(or403ts) was 88.7% embryonic lethal at 15°C
(n=1029), and 98.5% embryonic lethal at 25°C (n=470).
All phenotypic characterization was performed with these two alleles.
To test for a zygotic requirement, hermaphrodites of strain pam-1(or282ts) unc-24(e138)/+ or pam-1(or403)/dpy-13(e184) unc-24(e138) were allowed to make embryos at 25°C: nearly all embryos hatched (705/716; 425/428, respectively). To test for paternal requirements, fog-2 females from strain CB4108 were mated at 25°C with EU983 pam-1(or403); him-5 mutant males, which had been raised at 25°C from the L1 stage. To test for maternal requirements, pam-1(or403); unc-51 fog-2 females were crossed with N2 males at 25°C. From each cross, females were removed after mating for embryo analysis. As a control, CB4108 fog-2 females were crossed with N2 males.
For deficiency analysis, pam-1(or282ts) males were mated into hermaphrodites from the following strains: RW1324 fem-1(e1991) unc-24(e138) unc-22(s12)/stDf7 IV and RW1333 fem-1(e1991) unc-24(e138) unc-22(s12)/stDf8 IV at the permissive temperature. F1 animals were shifted to 25°C and embryos from pam-1(or282ts)/stDf7 (n=3) or pam-1(or282ts)/stDf8 (n=3) worms were imaged using DIC microscopy.
Positional cloning
Linkage group and 3-factor mapping
(Brenner, 1974) were used to
position the or282ts allele to +3.39 on chromosome IV. Single
nucleotide polymorphism (SNP) mapping
(Wicks et al., 2001) was then
done using SNPs identified from The University of Washington Genetics and
Genome Sequencing Center. Unc-nonPam recombinants were found from strains
heterozygous for CB4856 DNA and either unc-8(e49) pam-1(or282) or
pam-1(or282) unc-24(e138). PCR-amplified DNA was isolated from
recombinants using a kit from Gentra Systems and scored for the SNPs to narrow
the region to between cosmids R05G6 and C06A6.
The entire coding region of candidate genes in the region were sequenced directly from PCR products produced from purified DNA for each allele. For each sequencing reaction, four independent PCR reactions were combined and purified using the QUIAquick PCR purification kit (Quiagen) and sequenced at the Nucleic Acid/Protein Research Core Facility at the Children's Hospital of Philadelphia. Sequences and chromatograms were aligned and compared to the published sequence using DNASIS MAX software (Hitachi). All potential sequences were confirmed via a secondary PCR and sequencing reaction. The pam-1 gene was amplified in two separate reactions using primers f49e8.3a: CAAAATTGACGAGAGGGG with f49e8.3b: GTGATCCAGGAGTCACG and f49e8.3c: GCCAAAGATCAGTCCACC with f49e8.3d: AAGCAAGATGATGCCACG.
Mutations found in all five alleles are detailed below. Nucleotide numbers are based on position in the confirmed sequence for F49E8.3a. In or370, a G to A substitution was found at nucleotide 1022 and resulted in an A278T change. In or347, a C to T substitution was at nucleotide 1023 and resulted in A278W. In or403 a G to A change was observed at nucleotide 1849 and resulted in W538X. In or547 a C to T substitution at nucleotide 2027 was observed, which resulted in Q598X. In or282 a deletion from nucleotide 283 to 873 was found in the genomic DNA. This deletion resulted in an amino acid sequence change following K56 to NNFSX.
Microscopy and immunoflorescence
For time-lapse imaging, embryos were placed on a 3% agarose cushion under a
coverslip. DIC images were captured every 5 seconds using a Dage MT1 VE1000
camera and Scion Image software, or Nikon Eclipse E600 and SPOT Basic
software. Cortical flows were time-lapsed as previously described
(Severson et al., 2002).
PIE-1::GFP and TUBULIN::GFP images were acquired using epifluorescence and a
Micromax EBF512 cooled CCD camera (Roper Scientific) under the control of
Metamorph imaging software (Universal Imaging). HISTONE::GFP images were
captured similarly except on a spinning disk confocal (Perkin Elmer) or a
Nikon C1 Confocal Imaging System. For time-lapse GFP imaging, a Z-series of
5-15 frames was acquired at 1.5 µm intervals every 20-60 seconds and in
some experiments one center-plane DIC image was captured. Imaging of meiosis
was performed in utero with worms anesthetized in 0.1% tricane and 0.01%
tetramisole (McCarter et al.,
1999). Measurements of pronuclear meeting position and centrosome
movements were made using Object-Image software.
The following primary antibodies and dilutions were used: rabbit anti-PAR-2
(1:5), rabbit anti-PAR-3 (1:20), both a gift from K. Kemphues, rabbit
anti-PGL-1 (1:10,000), a gift from S. Strome, mouse anti-tubulin (1:250)
(Sigma clone DM 1A), and mouse anti-actin (1:200) (ICN clone C4).
Immunostaining was done using a methanol fix protocol
(Severson et al., 2002). The
DNA was stained with 0.2 µm TOTO3 (Molecular Probes) or DAPI in Antifade
mounting media (Invitrogen). Embryos were imaged on a BioRad MRC 1024 laser
scanning confocal microscope or a Nikon C1 confocal microscope system.
RNA interference
Feeding RNAi was used to inactivate cyb-3 as described
(Kamath et al., 2003).
Bacteria were grown on plates containing 1 mmol/l IPTG and 75 µg/µl
carbenacillin and placed at 37°C overnight. L4 worms were picked to the
plates and grown at 25°C for 24 hours before analysis.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To identify the molecular lesion in pam-1 mutants, the gene was
mapped to a small interval (see Materials and methods). We sequenced candidate
genes in the region and found, in all five mutant alleles, mutations in the
puromycin-sensitive aminopeptidase gene called pam-1
(Fig. 1). This gene is closely
related to mouse and human PSA genes (Fig.
1). Two of the alleles contained missense mutations. The A278T
mutation in pam-1(or370) and the A278V mutation in
pam-1(or347) both altered the GAMEN motif that is conserved in all M1
aminopeptidases and has been shown in other systems to be important for full
enzyme activity (Laustsen et al.,
2001). Two of the alleles encoded nonsense mutations. Both the
W538X mutation in pam-1(or403) and the Q598X mutation in
pam-1(or547) are predicted to truncate the protein downstream of the
active site (Fig. 1). A 590 bp
deletion was found in pam-1(or282), resulting in a change following
K56 to NNFSX and thus a truncated protein product. All five alleles are
recessive and pam-1(or282) is most likely a null, as this deletion
results in a frameshift and stop codon before the active site. Additionally,
embryos produced by hemizygous mothers (with one mutant copy of pam-1
in trans to a chromosomal deficiency that removes pam-1) were
indistinguishable from those produced by homozygous pam-1(or282)
mothers (data not shown).
pam-1 embryos exhibit a meiotic exit defect
The first defects observed in pam-1 mutant embryos occurred during
meiosis. The timing of meiosis was compared in wild-type and pam-1
embryos in utero using a HISTONE::GFP line (H2B::GFP) to fluorescently label
chromosomes in live embryos (Table
1). We detected no significant difference in the timing of
completion of either meiosis I or II in pam-1(or282) mutants,
compared to wild-type embryos. For these experiments we defined the end of
meiosis as the time when polar body extrusion was evident. However, we did
observe meiosis II defects in 33% (4/12) of pam-1(or282) embryos. In
these embryos, a DNA bridge connected the separating anaphase chromosomes
(Fig. 2B).
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Meiotic exit defects are rescued by loss of cyclin B3
Previous studies have shown that inactivation of B-type cyclins is
necessary for exit from meiosis in several organisms. For example, reduction
of the cyclin encoded by cyb-3 in C. elegans has been shown
to rescue meiotic exit delays in zyg-11 mutants
(Sonneville and Gönczy,
2004). We therefore asked if cyb-3 was required for the
meiotic exit delay in pam-1 mutants, using RNAi to deplete CYB-3. As
previously shown (Sonneville and
Gönczy, 2004), we found that cyb-3(RNAi) embryos
exhibit a prolonged meiosis II, but a timely exit from meiosis
(Table 1). In pam-1(or282);
cyb-3(RNAi) embryos that completed meiosis II and extruded a second polar
body, we observed a marked decrease in the time of meiotic exit, similar to
wild type (Table 1). Thus
depletion of CYB-3 rescues the meiotic exit defect in pam-1 mutants,
suggesting that PAM-1 may promote CYB-3 degradation to permit exit from
meiosis.
Interestingly, in some pam-1(or282);cyb-3(RNAi) embryos, a second polar body was never extruded. Embryos entered into metaphase II, but never progressed into a clear anaphase stage before chromosome decondensation became evident. In these embryos, the timing from meiosis II to appearance of the pronuclei appeared to be lengthened even over cyb-3(RNAi) alone (Table 1), suggesting a possible role for PAM-1 in meiosis II.
pam-1 embryos lack early signs of AP polarity
Because polarity defects are associated with meiotic progression defects in
other C. elegans mutants, we next examined AP axis formation in
pam-1 mutant and wild-type embryos using DIC optics. As the SPCC
contacts the posterior cortex and polarizes the AP axis in wild type, cortical
changes are apparent in which the posterior cortex smoothens and the anterior
cortex ruffles while cortical flows occur
(Fig. 3A). The oocyte
pronucleus then migrates to meet the sperm pronucleus in the posterior
(Fig. 3B). Together the
pronuclei and associated centrosomes move to the center of the embryo and set
up the first mitotic spindle, which displaces posteriorly during anaphase,
resulting in an asymmetric division (Fig.
3C,D). The two daughters differ in size, cell-cycle timing and
spindle orientation (Fig. 3E)
(Hyman and White, 1987;
Sulston et al., 1983).
pam-1 mutant embryos failed to exhibit the earliest signs of AP axis polarization. In most embryos the SPCC failed to make a tight association with the cortex (54/60) and was often near the center of the embryo when it first became visible (46/60; Fig. 3F). In addition, SPCC-cued polarity was not apparent. The cortex did not smoothen at either pole, membrane ruffling persisted around the entire periphery, and a pseudocleavage furrow failed to form (54/60; Fig. 3F). Furthermore, cortical flows were absent (n=6; data not shown). Thus it seems that the cortex did not polarize in pam-1 mutant embryos.
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pam-1 is required for localization of polarity determinants
Because most pam-1 mutant embryos lacked early signs of polarity,
we next used indirect immunofluorescent labeling of fixed embryos to examine
the localization of the cortical polarity regulators called the PAR proteins.
As expected, in wild-type one-cell embryos, PAR-2 and PAR-3 localized to the
cortex in reciprocal domains, with PAR-2 in the posterior and PAR-3 in the
anterior (Table 2;
Fig. 4A,D)
(Rose and Kemphues, 1998). In
one-cell pam-1 mutant embryos we observed defects in the localization
of both proteins (Table 2;
Fig. 4B,C,E). PAR-2 was not
detected at the cortex in 44.5% of pam-1 mutant embryos
(Table 2;
Fig. 4B). In 60% of embryos
with detectable PAR-2 at the cortex, it was mislocalized to a small lateral
patch, which did not correlate with meiotic spindle position or polar body
location (Table 2;
Fig. 4C). In about 56% of
pam-1 mutant embryos, PAR-2 was detected in puncta near the
centrosomes and their asters (Fig.
4B). While this peri-centrosome staining occurred most frequently
in embryos lacking cortical PAR-2, it was also observed in embryos in which
cortical PAR-2 was mislocalized or properly localized. Only 20% of one-cell
pam-1 mutant embryos showed normal cortical PAR-2 staining restricted
to the posterior pole. As a control, cortical actin localization appeared
normal in these embryos, despite the loss of PAR-2 at the cortex
(n=10; data not shown). In contrast to the loss of PAR-2 at the
cortex, PAR-3 was distributed throughout the cortex in most pam-1
mutant embryos (Table 2;
Fig. 4E). Thus in most
pam-1 mutant embryos, the initial polarization that normally
restricts PAR-3 to the anterior pole appears to be defective.
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;
Strome and Wood, 1982).
Similarly, in fixed, wild-type embryos (n=19), we observed posterior
localization in one-cell embryos and localization to the P1 cell at
the two-cell stage (Table 3;
Fig. 4F,F'). By contrast,
P granules were not localized to one pole in 67% of fixed pam-1
mutant one-cell embryos and were also mislocalized in later stage embryos
(Table 3;
Fig. 4G,G').
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). By contrast, PIE-1::GFP was distributed throughout the
cytoplasm in 41% of one-cell pam-1 embryos (7/17;
Fig. 4I). Of the embryos with
mislocalized PIE-1::GFP, some divided asymmetrically (3/7) and others divided
symmetrically (4/7) during the first mitosis. By contrast, the majority of
embryos that properly segregated PIE-1::GFP went on to divide asymmetrically
(data not shown; 7/10). By the late two-cell stage, most asymmetric embryos
exhibited normal PIE-1::GFP localization (12/13), and all symmetric embryos
showed diffuse mislocalized PIE-1::GFP (16/16;
Fig. 4I). We conclude that
PAM-1 is necessary to promote proper segregation of developmental determinants
along the AP axis during the first mitotic division.
pam-1 polarity defects are separable from meiotic exit defects
As meiotic exit and axis polarization occur in rapid succession in the
one-cell embryo, we asked if the polarity defects in pam-1 mutants
might be a secondary consequence of the meiotic exit defect. As described
above, CYB-3/Cyclin B depletion rescued the meiotic exit defect in
pam-1 mutants. We next asked whether the AP polarity defects were
also rescued in CYB-3-depleted pam-1 mutant embryos. pam-1;
cyb-3(RNAI) embryos were observed through the first cell division for
signs of polarity, such as pseudocleavage and asymmetric spindle positioning.
While all cyb-3(RNAi) embryos observed showed normal pseudocleavage
and a posteriorly displaced spindle (n=7), many pam-1;
cyb-3(RNAi) embryos lacked pseudocleavage (n=16/23) and had
spindles positioned toward the anterior (n=5/20) or center
(n=6/20) (Fig. 5A-D).
As only some pam-1; cyb-3(RNAi) embryos exit meiosis promptly, due to
meiosis II defects in others (see above), we examined whether embryos with
rescued meiotic exit were additionally rescued for polarity. We saw no such
correlation; pam-1; cyb-3(RNAi) embryos that promptly exited meiosis
showed no signs of polarity 55% of the time (n=9). As an additional
marker, we examined P granule localization in cyb-3(RNAi) and in
pam-1(or403); cyb-3(RNAi) embryos
(Table 3;
Fig. 5). All
cyb-3(RNAi) embryos examined showed normal posterior localization of
the P granules during the first mitosis
(Fig. 5E,G;
Table 3) (see also
Sonneville and Gönczy,
2004). By contrast, even though pam-1(or282); cyb-3(RNAi)
embryos often exhibited meiotic exit timings similar to wild type
(Table 1), P granule
localization was still severely defective in many embryos
(Fig. 5F-I;
Table 3). As a control to
determine if CYB-3 levels could be eliminated effectively by RNAi treatment,
we exposed CYB-3::GFP-expressing worms
(Sonneville and Gönczy,
2004) to cyb-3 RNAi. While all untreated worms
(n=6), displayed strong oocyte GFP expression, we detected no GFP in
all treated worms (n=8), suggesting that incomplete elimination of
CYB-3 does not account for the persistent polarity defects (data not shown).
We conclude that the polarity defects in pam-1 mutants occur even in
the absence of meiotic exit defects.
Aberrant centrosome positioning in pam-1 mutants
The abnormally late appearance of pronuclei and the central appearance of
the pronuclei in pam-1 mutant embryos suggested that the SPCC that
normally specifies the posterior pole might not be mature or associated with
the cortex at the time needed for proper axis specification. We therefore
monitored centrosome maturation and position in wild-type and
pam-1(or282) mutant embryos, using transgenic ß-TUBULIN::GFP
lines (Fig. 6A-C). When
centrosomes first became visible in wild type, they were together and in close
association with both the sperm pronucleus and the posterior cortex
(n=6; Fig. 6A,B,C,
part a). In the first few minutes after centrosomes could be detected, they
separated from one another and increased in size as they began to nucleate
more microtubules (Fig. 6C,
parts b,c). During this time, wild-type centrosomes remained in the posterior
25% of the embryo (Fig. 6A-C).
By contrast, pam-1 mutant embryos exhibited multiple defects in
centrosome positioning. In all embryos examined, the centrosomes first
appeared normally near the presumptive posterior cortex, but then rapidly
moved toward the center of the embryo (n=13;
Fig. 6A,B,C, parts d-f). As a
result, the time centrosomes spent near the cortex in pam-1 mutant
embryos was dramatically decreased (Fig.
6A). In addition, in most embryos, this centrosome movement
occurred prior to the appearance of the sperm pronucleus, suggesting that
centrosome movement occurred during the delayed meiotic exit stage
(n=10/13). This aberrant centrosome movement was also observed in
pam-1; cyb-3(RNAi) embryos (n=6; data not shown). It is
possible that this change in centrosome localization in pam-1 mutants
prevents polarization of the AP axis, through a failure of the centrosomes to
influence cortical actomyosin and trigger axis formation.
We next used a combination of DIC and TUBULIN::GFP time-lapse videomicroscopy to track both centrosome-associated microtubules and prouclei at the same time in live embryos. While all pam-1 embryos showed rapid centrosome movement from the cortex, we observed an additional defect in 32% of pam-1 mutant embryos (10/31). In these embryos, centrosomes nucleated robust microtubule asters before the appearance of pronuclei. Centrosomes enlarged until they were visible using DIC microscopy and resembled mitotic centrosomes (Fig. 6D, part d). In most cases the enlarged centrosomes diminished in size as the pronuclei became visible (Fig. 6D, part e), but subsequently nucleated more microtubules once again before and during the first mitotic division (n=7/10; Fig. 6D, part f). There was no correlation of this premature microtubule nucleation with the subsequent polarity of the division.
Oocyte- or sperm-supplied PAM-1 is sufficient for development
As pam-1 mutant embryos are defective in events triggered by
fertilization, we asked if there might be any paternal contribution of PAM-1
from sperm. We crossed pam-1(or403) feminized hermaphrodites and
wild-type males, or wild-type feminized hermaphrodites and
pam-1(or403) males, recorded subsequent hatching rates, and observed
early embryonic cell divisions using time-lapse videomicroscopy
(Table 4). When pam-1
feminized hermaphrodites were mated with wild-type males, about 32% of the
embryos hatched, a significant increase over pam-1 mutant
hermaphrodite self-progeny hatching rates (1.5%). Furthermore, 25% of embryos
observed by DIC optics exhibited early development that was indistinguishable
from wild type (3/12), suggesting that paternal PAM-1 is sufficient in some
embryos for normal development. The remaining embryos showed phenotypes like
those produced by pam-1 mutant hermaphrodites (n=9/12; data
not shown). Thus, embryos produced from mutant mothers and wild-type males
either appeared normal, or exhibited fully expressed pam-1 mutant
phenotypes. When feminized pam-1(+) hermaphrodites were mated to
pam-1(or403) mutant males, nearly all embryos hatched and exhibited
normal early embryonic cell divisions (12/13; data not shown), indicating that
maternally supplied PAM-1 is sufficient for embryogenesis. Thus while the
paternal contribution can partially rescue pam-1 mutant oocytes, it
is not necessary for wild-type oocytes to develop normally after
fertilization.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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pam-1 encodes a puromycin-sensitive aminopeptidase
PAM-1 is the only C. elegans member of the puromycin-sensitive
aminopeptidase (PSA) family. This protein is highly conserved, being 36-37%
identical to human PSA, with key domains showing higher levels of homology
(Brooks et al., 2003). PSA is a
member of the M1 class of metalloproteases, which cleave N-terminal amino
acids from a variety of substrates
(Taylor, 1993). These cleavage
events have been implicated in inactivation of neuropeptides in mammalian
systems (Hui et al., 1995) and
processing of MHC class I peptides (Saric
et al., 2001; Stoltze et al.,
2000). However, few in vivo substrates are known. This family of
proteases include a zinc-binding motif preceded by the GAMEN sequence, which
is necessary for protease specificity and enhanced activity
(Laustsen et al., 2001). The
recessive mutations identified in our alleles affect conserved residues or
truncate the protein to reduce or eliminate function. Two mutations affect the
GAMEN alanine residue, highlighting its importance.
|
;
Osada et al., 2001a;
Osada et al., 2001b).
Additionally, in Drosophila, males with only one wild-type copy of
dPSA show reduced fertility (Schulz et
al., 2001). In Arabidopsis, the single PSA homolog is
essential for meiosis (Sanchez-Moran et
al., 2004). Less is known about C. elegans PSA, although
a previous study showed that PAM-1 is expressed in intestinal cells and in
neurons of the head, and in the male tail
(Brooks et al., 2003). However,
this study did not address maternal, paternal or early embryonic expression,
due to germline silencing of the transgene reporter
(Brooks et al., 2003). The
aminopeptidase activity of C. elegans PAM-1 is similar to other PSA
molecules, except that it is less sensitive to puromycin
(Brooks et al., 2003). When
RNAi was used to reduce pam-1 function in adult hermaphrodites, 30%
embryonic lethality resulted, although early embryos were not examined
(Brooks et al., 2003). We
conclude that RNAi does not diminish pam-1 function as effectively as
the mutations we identified, and our studies reveal new roles for this
protease in the regulation of meiotic exit and the establishment of the AP
body axis.
A role for PAM-1 in meiotic exit
The most penetrant early defect in pam-1 mutant embryos was a
delayed exit from meiosis II. During meiotic exit in wild-type embryos, the
sperm and oocyte chromosomes rapidly decondensed and nuclear envelopes formed,
followed by entry into S phase before the first mitosis
(Edgar and McGhee, 1988;
Vidwans and Su, 2001). In
pam-1 mutant embryos, chromosome decondensation and formation of the
pronuclei were delayed after extrusion of the second polar body. Surprisingly,
in some pam-1 mutant embryos, centrosome maturation and microtubule
nucleation were not delayed, and thus the centrosomes matured and nucleated
large microtubule asters before prouclear formation. Mitotic exit was not
delayed in pam-1 mutants (data not shown), suggesting that the
mechanisms governing meiotic and mitotic exit in the early embryo are at least
partially distinct. However, PAM-1 might act redundantly with other factors to
regulate mitotic exit.
|
; Deshaies,
1999). Specifically, the APC and cullin-based SCF and ECS E3
ligase complexes are required for progression through meiosis or mitosis in
many species. For example, studies in fission yeast have implicated mfr1 as a
meiotic-specific activator of the APC necessary to ensure meiotic exit and
sporulation following meiosis (Blanco et
al., 2001). Similarly, in C. elegans, the APC regulates
the metaphase to anaphase transition of meiosis I
(Golden et al., 2000), and
hypomorphic alleles of APC components sometimes exhibit meiotic exit
abnormalities (Shakes et al.,
2003). Also in C. elegans, inactivation of the ubiquitin
E3 ligase scaffold, a cullin called CUL-2, or a potential adaptor component
for this E3 ligase called ZYG-11, delays the metaphase to anaphase transition
during meiosis II, resulting in elevated levels of B-type cyclins and meiotic
exit defects (Liu et al.,
2004; Sonneville and
Gönczy, 2004). Timely meiotic exit is restored in
cul-2 and zyg-11 mutants when RNAi is used to deplete the
B-type cyclins, implicating this E3 ligase in targeting B-type cyclins for
degradation and meiotic exit (Liu et al.,
2004; Sonneville and
Gönczy, 2004).
Our results indicate that the aminopeptidase PAM-1 also acts upstream of
CYB-3 to regulate meiotic exit. However, PAM-1 may act more specifically than
the known ubiquitin E3 ligases to regulate meiotic exit. Most pam-1
embryos advance through meiosis normally, with the first defect appearing
during meiotic exit. This is in contrast to many ubiquitin E3 ligase mutants,
which display more severe meiotic defects. How PAM-1 and ubiquitin-mediated
proteolysis interact to influence cell cycle progression and other processes,
and whether PAM-1 directly degrades B-type cyclins, are important topics for
further study. However, the potential colocalization of PSA, the mammalian
PAM-1 homolog, to the 26S proteasome suggests a role downstream of the E3
ligases, perhaps in conjunction with the proteasome
(Constam et al., 1995). Studies
of a mouse PSA have also implicated these aminopeptidases in mitotic exit
(Constam et al., 1995). Thus
regulation of cell cycle exit may be a widely conserved role for
puromycin-sensitive aminopeptidases.
Polarity defects in pam-1 embryos
In addition to exhibiting defects in meiotic exit, pam-1 embryos
fail to polarize the anteroposterior axis. In most pam-1 mutant
embryos, all the earliest AP asymmetries are absent, including cytoplasmic
flows, pseudocleavage and asymmetric localization of the PAR proteins. In
addition, cytoplasmic determinants such as the P granules and PIE-1 fail to
localize, and many embryos divide symmetrically. Thus PAM-1 is important for
an early step in axis polarization.
|
;
Rappleye et al., 2002;
Sonneville and Gönczy,
2004), our results provide an additional link between AP axis
formation and proteolytic regulation.
PAM-1 may regulate polarity through centrosome association with the cortex
Around the time of meiotic exit, the sperm-donated centrosome duplicates,
matures and nucleates microtubules
(O'Connell, 2000). Both proper
maturation of the centrosome (Hamill et
al., 2002; O'Connell et al.,
2000) and a close SPCC association with the posterior cortex
(Cowan and Hyman, 2004b;
Rappleye et al., 2002) are
required for axis formation. Centrosomes mature and microtubule asters form in
pam-1 mutants. However, both the sperm chromosomes and the
centrosomes move away from the cortex prematurely, during the meiotic exit
delay. Thus the lack of axis polarization in pam-1 mutants may result
from the absence of close centrosome association with the cortex. Current
models suggest that the SPCC centrosomes downregulate cortical microfilament
contractility, resulting in cortical flows and a movement of PAR-3 away from
the SPCC, to form an anterior pole opposite the site of the SPCC
(Cuenca et al., 2003;
Jenkins et al., 2006;
Munro et al., 2004). As the
centrosome moves quickly away from the cortex in pam-1 mutants, it
may fail to induce the cortical changes. This hypothesis is consistent with
the lack of posterior PAR-2 localization, and the presence of PAR-3 throughout
the entire cortex, in most pam-1 mutant embryos.
Paternal contribution of PAM-1
The pam-1 gene product is contributed to the zygote by both the
sperm and the egg. Egg contribution is necessary in nearly all embryos for
viability and is also fully sufficient for normal development. By contrast,
the sperm contribution of PAM-1 is not necessary for embryo viability but is
sufficient in many cases. It is unlikely that the rescue of pam-1
mutant oocytes by wild-type sperm is due to zygotic expression of
pam-1, as the rescued processes occur shortly after fertilization,
before the zygotic genome is activated
(Seydoux and Fire, 1994). Thus
PAM-1 is partially contributed as a paternal gene product, although this
contribution appears nonessential. It is interesting to note that, although
cues from the sperm are known to promote resumption of meiosis and axis
polarization, only one true paternal-effect mutation, spe-11, has
been identified to date in C. elegans
(Browning and Strome, 1996).
Despite this, many mutants that affect early development of the embryo,
including pam-1, show both maternal and paternal contributions of the
gene product (Gönczy et al.,
1999; Lee et al.,
2001; O'Connell et al.,
1998). Perhaps the addition of multiple sperm-contributed proteins
at fertilization helps to produce a threshold concentration of factors
necessary for the progression into embryogenesis.
Possible mechanisms for PAM-1 action
While in vivo substrates of puromycin-sensitive aminopeptidases are poorly
understood, these enzymes have been implicated in a variety of processes,
including neuropeptide inactivation (Hui
et al., 1995), MHC class I peptide processing
(Saric et al., 2001;
Stoltze et al., 2000) and cell
cycle regulation in conjunction with the proteasome
(Constam et al., 1995). The
role of PAM-1 role in the early embryo may be complex, causing the activation
of some peptides through N-terminal trimming and the degradation of other
proteins, perhaps in conjunction with the proteasome. One intriguing
possibility is that PAM-1 may trigger peptides for degradation through the
N-end rule, with removal of N-terminal residues by PAM-1 exposing amino acids
that target the protein for ubiquitin-mediated proteolysis
(Varshavsky, 1996).
Our study provides new insights into the PAM-1 aminopeptidase and its roles in early development. Because PAM-1 is a member of the highly conserved PSA gene family, its role in cell cycle progression and cell polarity may provide important new insights into these processes in other systems.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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