Department of Cellular Biology, University of Georgia, Athens, GA 30602-2607, USA
Author for correspondence (e-mail:
ekipreos{at}cb.uga.edu)
Accepted 30 April 2004
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SUMMARY |
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Key words: PAR-2, CUL2, Cullin, Polarity, Meiosis, Metaphase II, C. elegans
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Introduction |
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In many metazoa, the anteroposterior (AP) body axis is defined at a very
early stage, when patterning molecules are asymmetrically distributed in the
embryo (Pellettieri and Seydoux,
2002). In the nematode C. elegans, AP polarity is
initiated in the zygote immediately after meiosis by the sperm
pronucleus/centrosome complex (SPCC)
(Goldstein and Hird, 1996
). In
response to the SPCC, the PDZ-domain polarity protein PAR-6 becomes excluded
from the posterior cortex. PAR-6 functions in a complex with the PDZ-domain
protein PAR-3 and the atypical protein kinase C PKC-3
(Tabuse et al., 1998
;
Hung and Kemphues, 1999
;
Joberty et al., 2000
). The
exclusion of PAR-6 from the posterior allows the RING finger protein PAR-2 to
accumulate on the posterior cortex (Watts
et al., 1996
; Boyd et al.,
1996
; Hung and Kemphues,
1999
; Cuenca et al.,
2003
). This redistribution of PAR-2 and PAR-6 is required for
subsequent AP asymmetries, including the polarized localization of maternal
proteins to specify different cell fates and the asymmetric orientation of the
mitotic spindle to generate unequal cell divisions
(Schneider and Bowerman, 2003
;
Pellettieri and Seydoux,
2002
).
Ubiquitin-mediated protein degradation is an essential aspect of many
dynamic cellular processes, including cell cycle progression, signal
transduction and transcription (Pickart,
2001). The covalent attachment of poly-ubiquitin chains to
proteins can signal degradation by the 26S proteasome
(Pickart, 2001
). The addition
of ubiquitin to proteins is highly regulated and requires the action of an
ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and an
ubiquitin ligase (E3). The substrate specificity of ubiquitination derives
from the recognition of the substrate by the ubiquitin ligase. A major class
of E3s is the cullin/RING finger ubiquitin ligases
(Tyers and Jorgensen, 2000
).
The cullin gene family comprises five major groupings in metazoa, CUL-1
through CUL-5 (Tyers and Jorgensen,
2000
). In mammals, CUL2 functions in an E3 complex that contains
the core components elongin C, elongin B and the RING-H2 finger protein
RBX1/ROC1 (Kim and Kaelin,
2003
).
Here, we show that CUL-2, as well as orthologs of elongin C and RBX1, is required for the initiation of meiotic anaphase II but not for anaphase I. The meiotic delay in cul-2 mutants causes a reversal of AP polarity. By titrating the length of the delay, we demonstrate that there is a tight linkage between meiotic timing and the placement of the AP axis. We also show that CUL-2 has an additional role to prevent the ectopic localization or spreading of PAR-2 on the cortex.
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Materials and methods |
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RNAi
RNAi was performed either by injection of dsRNA into hermaphrodites or by
feeding with bacteria expressing dsRNA, as described
(Feng et al., 1999;
Timmons et al., 2001
). To
produce a partial loss of CUL-2 function, embryos were observed between 4 and
12 hours post-injection of cul-2 dsRNA into adult hermaphrodites. The
cul-2; rec-8 double RNAi experiment followed the protocol of Davis et
al. (Davis et al., 2002
),
except that after injection of rec-8 dsRNA into adult hermaphrodites,
L4-stage progeny were placed on cul-2 RNAi bacteria feeding plates
for 24 hours prior to observation. Inactivation of cul-2 in double
RNAi experiments was assessed by observation of the multinuclei and
cytoplasmic extensions that occur in cul-2 mutant embryos
(Feng et al., 1999
). The
effectiveness of cyb-1 RNAi was confirmed by the elimination of
CYB-1::GFP signal.
Microscopy
Time-lapse movies were made of embryos in utero. Young adult hermaphrodites
were anesthetized either with 0.1% tricaine, 0.01% tetramisole (Sigma) in M9
solution or 10 mM levamisole (Sigma) in M9 solution. To observe the initiation
of PAR-2 cortical localization, zygotes were cut from the uterus into EBGM
medium (Shelton and Bowerman,
1996) on 12x12 mm coverslips. The coverslip was mounted on
4% agarose pads with a thin layer of petroleum jelly applied between the
coverslip and the pad to eliminate pressure on the zygotes. Automatic
time-lapse imaging was performed with a Zeiss Axioplan microscope equipped
with a Hamamatsu ORCA-ER CCD camera, LUDL hardware controller, automated
filter wheels and shutters, and an Apple G4 computer running Openlab software
(Improvision). Movies were made with pulsed 100 msec exposures for DIC and
epifluorescence every 40-80 seconds; epifluorescent illumination was from a
100 W HBO mercury lamp that was filtered to 25% of normal levels. PAR-2::GFP
cortical patches were only scored when they were of higher epifluorescent
intensity than the interior cytoplasm, except in
Table 2 where they were noted
as being transient and weak. For the timing of meiosis I, the following number
of embryos were observed: wild type, n=5; cul-2(RNAi), n=5;
cul-2(ek1), n=5; elc-1(RNAi), n=3; rbx-1(RNAi),
n=4; elb-1(RNAi), n=6; zyg-11(RNAi), n=9;
zyg-11(mn40), n=3; and zyg-11(mn40); cul-2(ek1), n=5. For
the timing of meiosis II, the following number of embryos were observed: wild
type, n=5; cul-2(RNAi), n=5; cul-2(ek1), n=5;
elc-1(RNAi), n=5; rbx-1(RNAi), n=4; elb-1(RNAi),
n=9; zyg-11(RNAi), n=9; zyg-11(mn40), n=4; and
zyg-11(mn40); cul-2(ek1), n=5. Quantitation of CYB-1::GFP levels was
performed by measuring the fluorescent signal with OpenLab software and
subtracting background fluorescence.
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Results |
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cul-2 and zyg-11 mutants have a similar meiosis II arrest
The failure of cul-2 mutant embryos to initiate anaphase II was
reminiscent of the zyg-11 mutant phenotype. In a study by Kemphues et
al. (Kemphues et al., 1986),
embryos homozygous for the hypomorphic allele zyg-11(bn2) were found
to progress through meiosis I with normal timing, whereas the time for meiosis
II was extended to 26 minutes. We analyzed embryos homozygous for the null
allele zyg-11(mn40), as well as zyg-11(RNAi) embryos, and
found a longer meiosis II delay of 50.2±4.0 minutes (n=5) and
43.6±3.1 minutes (n=9), respectively, comparable to the delay
observed in cul-2 mutants (Fig.
1A,B). To test whether simultaneous inactivation of both genes
increased the severity of the meiotic arrest, as might occur if the two genes
affected meiotic progression through independent pathways, we created a double
heterozygous mutant strain (ET144) using null alleles for each gene. Double
homozygous zyg-11(mn40); cul-2(ek1) embryos from this strain remained
in meiosis II for 53±4.5 minutes (n=5), equivalent to the
timings seen upon individual inactivation of each gene
(Fig. 1B). This result is
consistent with the two genes functioning in the same pathway to regulate
anaphase II.
In addition to defective meiosis II, zyg-11 and cul-2
mutant embryos share other phenotypes, including the presence of multinuclei
and cytoplasmic extensions during mitotic division
(Feng et al., 1999;
Kemphues et al., 1986
). One
difference between the two mutants is the presence of G1-phase arrested germ
cells in cul-2 mutants (Feng et
al., 1999
), which are not observed in zyg-11 mutants. The
decreased number of germ cells in cul-2 mutants leads to a lower
numbers of eggs. We sought to test whether combining the cul-2 mutant
with the zyg-11 mutant would enhance the cul-2 egg
production defect. zyg-11(mn40); cul-2(ek1) double homozygotes
produced a low number of eggs (35.2±4.7; n=18), which was
comparable to the egg numbers in cul-2(ek1) homozygotes
(Feng et al., 1999
) or
hermaphrodites that are homozygous for cul-2(ek1) and heterozygous
for zyg-11(mn40) (29.3±5.5; n=19). Conversely,
animals homozygous for zyg-11(mn40) but heterozygous for
cul-2(ek1) had egg numbers (333±3.2; n=10) similar to
wild type, indicating that the loss of zyg-11 cannot induce a
germline arrest phenotype in cul-2(ek1) heterozygotes. These results
suggest that ZYG-11 is unlikely to function with CUL-2 in promoting germline
proliferation.
CUL-2 is not required for the removal of REC-8 from sister chromatids
A failure to segregate chromosomes during meiosis could result from defects
in either chromosome separation or chromosome movement. In yeast, cohesin
degradation is sufficient for the initiation of anaphase chromosome
segregation (Uhlmann et al.,
2000). In C. elegans, RNAi depletion of the cohesion
REC-8 produces chromosome separation at diakinesis
(Pasierbek et al., 2001
).
To determine whether REC-8 is removed from chromosomes in
cul-2(RNAi) zygotes, we studied REC-8 localization using
immunofluorescence with anti-REC-8 antibody
(Pasierbek et al., 2001). We
found that the anti-REC-8 staining pattern in cul-2(RNAi) zygotes was
similar to that in wild type. For both wild-type and cul-2(RNAi)
zygotes, REC-8 is located along the axes of sister chromatids during metaphase
I, and is located at the junction of the sister chromatids during metaphase II
(Fig. 2A)
(Pasierbek et al., 2001
). In
wild type, REC-8 is completely lost from the separating wild-type chromatids
during anaphase II (Fig. 2A).
In cul-2(RNAi) zygotes, although chromosomes remain in a metaphase II
pentagonal array for an extended period, the majority of metaphase chromosomes
do not have anti-REC-8 staining (70%, n=26;
Fig. 2A). When
cul-2(RNAi) zygotes are in the later stages of the extended meiosis
II, the meiotic spindle often wanders away from the anterior pole of the egg.
None of these late stage meiosis II zygotes were observed to have anti-REC8
staining (data not shown).
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A further indication that a defect in chromosome cohesion was not
responsible for the CUL-2 meiotic defects was obtained from experiments in
which zygotes were depleted of REC-8. Inactivation of REC-8 produces a loss of
chromatid cohesion from the meiotic diakinesis stage onwards
(Pasierbek et al., 2001). In
both rec-8(RNAi) and cul-2; rec-8 double RNAi
zygotes, chromosomes segregate during anaphase I
(Davis et al., 2002
). However,
in both zygotes we observed that chromosomes failed to segregate during
meiosis II. Nevertheless, rec-8(RNAi) zygotes had normal meiosis II
timing (16.1±0.7 minutes, n=9)
(Fig. 2B). By contrast,
cul-2; rec-8 double RNAi zygotes had a meiosis II delay
comparable to that observed in cul-2(RNAi) zygotes (55.2±6.5
minutes, n=4), indicating that the cul-2 meiotic delay was
not rescued by removing REC-8 (Fig.
2B).
An interesting observation was that in the majority of rec-8(RNAi)
zygotes (7/8), a monopoleward movement of chromosomes toward the cortex was
observed at the time when the second polar body would be extruded in wild type
(Fig. 2B). The mechanistic
basis of this monopoleward chromosome movement is not known, but it is similar
to the chromosome behavior reported in air-2(RNAi) zygotes, which
have a failure to remove REC-8 from meiotic chromosomes
(Rogers et al., 2002).
Interestingly, cul-2; rec-8 double RNAi zygotes did not
exhibit monopoleward chromosome movement (n=9), indicating that this
aspect of meiotic chromosome movement was also defective
(Fig, 2B).
Cyclin B1 is not degraded during meiosis in cul-2(RNAi) zygotes
The cyclin B/CDK1 complex is essential for entry and completion of mitosis
and meiosis (Nebreda and Ferby,
2000). A failure to degrade cyclin B1 blocks the metaphase to
anaphase transition in mitotically-dividing mammalian cells and is required
for meiotic homolog disjunction in mice
(Stemmann et al., 2001
;
Hagting et al., 2002
;
Chang et al., 2003
;
Herbert et al., 2003
). To
analyze whether the degradation of cyclin B1 (CYB-1), was normal in
cul-2(RNAi) embryos, we used the pie-1 promoter to express
CYB-1::GFP in the germline. In wild type, CYB-1::GFP fluorescence was detected
in mature oocytes, but rapidly disappeared during meiosis I and II following
fertilization (Fig. 3A,B). By
contrast, in cul-2(RNAi) embryos, CYB-1::GFP signal decreased only
modestly during meiosis and remained at elevated levels in mitotic-stage
embryos (Fig. 3A,B). Elevated
levels of CYB-1 were also detected in cul-2(RNAi) embryos with an
anti-CYB-1 antibody kindly provided by Sander van den Heuvel (MGH Cancer
Center, USA) (data not shown). A similar defect in CYB-1::GFP degradation was
observed in zyg-11(RNAi) embryos
(Fig. 3A).
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The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase promotes
cyclin B degradation during mitosis in yeast and metazoa
(Peters, 2002). APC/C also
promotes cyclin B degradation during meiosis in Xenopus and mouse
(Peter et al., 2001
,
Nixon et al., 2002
). In C.
elegans, loss of APC/C produces a one-cell embryonic arrest at metaphase
of meiosis I (Furuta et al.,
2000
; Davis et al.,
2002
). Levels of CYB-1::GFP were stabilized in
apc-11(RNAi) embryos (Fig.
3B), suggesting that both CUL-2 and APC/C are required for CYB-1
degradation.
The meiotic spindle maintains normal morphology for an extended period in cul-2(RNAi) zygotes
We studied the morphology and dynamics of the meiotic spindle in
cul-2(RNAi) zygotes by both anti--tubulin antibody staining
and live ß-tubulin::GFP movies. In meiosis I, the meiotic spindles of
cul-2(RNAi) and wild-type embryos were indistinguishable (data not
shown). Meiosis II spindles in cul-2(RNAi) embryos maintained a
normal barrel-shaped structure for an extended period of time
(Fig. 4A). Eventually, the
cul-2(RNAi) meiotic spindle shrunk and lost its barrel structure. On
average, the cul-2(RNAi) meiosis II spindle lasted three times as
long as in wild type (36.2±2.5 versus 12.2±0.7 minutes,
n=6 for each) (Fig.
4A). Meiotic spindle morphology characteristic of anaphase II
(Fig. 4B) was never observed in
cul-2(RNAi) embryos.
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In wild type, maternal pronuclei migrate from the anterior to meet the sperm pronucleus in the posterior (Fig. 5E). This aspect of polarity is also reversed in cul-2(RNAi) embryos. In the majority of cul-2(RNAi) embryos (8/10), the sperm pronucleus migrates further than the maternal pronucleus (Fig. 5E). Pronuclei often meet in the anterior of the embryo rather than in the posterior, and the anterior daughter cell is smaller than the posterior cell in over half of cul-2(RNAi) embryos (16/31; Fig. 5B). Similar polarity defects were observed in elc-1 and rbx-1 RNAi embryos (Fig. 5B; data not shown).
To study whether the polarity reversal correlated with the extended meiosis
II delay in cul-2(RNAi) zygotes, we varied the penetrance of
cul-2 RNAi depletion by altering the length of time adult
hermaphrodites were exposed to cul-2 dsRNA prior to fertilization.
The partial cul-2 RNAi treatment produced meiosis II timing ranging
from that of wild type to delays similar to that of cul-2(ek1) null
mutants (Table 2). In wild
type, meiosis II lasted 16.2±1.3 minutes and PAR-2::GFP accumulated on
the posterior cortex approximately 2 minutes after the completion of meiosis
II (18.4±0.9 minutes after meiosis I;
Table 2). In two of five
wild-type zygotes, transient, weak, anterior cortical PAR-2::GFP was observed
during meiosis II, similar to previous reports
(Boyd et al., 1996;
Cuenca et al., 2003
)
(Table 2). Transient, weak,
anterior PAR-2 localizations were also observed in two cul-2(RNAi)
embryos that had meiosis II timing similar to wild type
(Table 2). cul-2(RNAi)
zygotes with meiosis II timing of
28 minutes developed intense, stable
PAR-2::GFP patches on anterior or lateral cortexes
(Table 2). An extended meiotic
delay coupled with an anterior PAR-2::GFP patch was associated with a reversal
in the longitudinal placement of the mitotic spindle, so that the first
division produced a posterior cell larger than the anterior cell
(Table 2).
Ectopic lateral PAR-2 localization is associated with loss of CUL-2
We were intrigued by the observation in cul-2(RNAi) zygotes of
PAR-2 patches (8/67 embryos), and exclusions of PAR-3 (3/33) and PAR-6 (2/10),
on the lateral cortex that were often distant from both the meiotic spindle
and the SPCC (Fig. 5A;
Table 1). As microtubule (MT)
organizing foci have been implicated in PAR-2 localization
(O'Connell et al., 2000;
Wallenfang and Seydoux, 2000
),
we sought to determine whether the lateral PAR-2 arose from prior transient
association of the meiotic spindle or SPCC with the lateral cortex. To address
this, we followed the initiation of cortical PAR-2 in living embryos
expressing either a combination of PAR-2::GFP and ß-tubulin::GFP (to
visualize the meiotic spindle), or PAR-2::GFP and histone H2B::GFP (to
visualize oocyte and sperm DNA). In the two strains, PAR-2 patches appeared to
initiate distant from the meiotic spindle or distant from both oocyte and
sperm DNA (5/20 and 3/10 embryos, respectively)
(Fig. 6A). With the PAR-2::GFP
strains, we observed a higher percentage of lateral accumulation of PAR-2 upon
cul-2 RNAi than was observed for cul-2(RNAi) embryos probed
with anti-PAR-2 antibodies (Table
1). In particular, we observed a substantial percentage of embryos
in which PAR-2::GFP spread extensively on the cortex to form a circular band
around the embryo (Fig. 6B;
Table 1). One third of the
circular PAR-2 patterns were observed distant from the meiotic spindle
(Fig. 6B;
Table 1). These observations of
PAR-2 accumulation distant from the meiotic spindle suggest that in
cul-2(RNAi) embryos, the initiation or spreading of cortical PAR-2 is
under less stringent control.
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Interestingly, the percentage of lateral and circular PAR-2 was significantly lower in zyg-11(RNAi) embryos, with no examples of PAR-2 patches distant from the meiotic spindle observed (Table 1). Although this may reflect a reduced effectiveness of the RNAi procedure in depleting ZYG-11 relative to CUL-2, it nevertheless suggests that the lateral accumulation of PAR-2 does not arise directly from the meiotic delay, as RNAi of both genes gave comparable meiotic lengths but only cul-2(RNAi) embryos had lateral patches distant from the meiotic spindle.
To further test whether the accumulation of PAR-2 distant from the meiotic
spindle was a secondary consequence of the meiotic delay or reflected the loss
of a separable CUL-2 function, we analyzed PAR-2 localization in an
emb-30 mutant background. emb-30 encodes the APC/C component
APC-4 (Furuta et al., 2000).
At the restrictive temperature of 24°C, emb-30(tn377ts)
embryos arrest at the metaphase I stage of meiosis
(Furuta et al., 2000
). As
described above, CUL-2 is not required for progression through meiosis I;
emb-30(tn377); cul-2(RNAi) embryos have the same metaphase I-arrest
phenotype and spindle morphology as emb-30(tn377) embryos
(Fig. 6E; data not shown).
Therefore, any differences in PAR-2 localization between the two strains
should reflect the loss of CUL-2 functions that are independent of its role in
meiotic progression. As expected from previous work on emb-30 by
Wallenfang and Seydoux (Wallenfang and
Seydoux, 2000
), we observed that a high percentage of both
emb-30(tn377) and emb-30(tn377); cul-2(RNAi) embryos had
anterior PAR-2 (89%, n=54, and 83%, n=47, respectively). In
emb-30(tn377) zygotes, only a small minority of embryos had lateral
or posterior PAR-2 patches (3.7%, 2/54)
(Fig. 6E), as was also observed
by Wallenfang and Seydoux (Wallenfang and
Seydoux, 2000
). In emb-30(tn377); cul-2(RNAi) zygotes,
however, the percentage of embryos with lateral or posterior PAR-2 patches was
significantly higher (23%, 11/47; P<0.01); most of these embryos
also had anterior PAR-2 patches (Fig.
6E). Similarly, 27% of emb-30(tn377); cul-2(RNAi) zygotes
(n=56) exhibited an exclusion of PAR-6 on lateral or posterior
cortexes compared with 4% of emb-30(tn377) zygotes (n=28)
(significance of P<0.05) (Fig.
6E). There was no difference in the MT distributions observed in
emb-30(tn377) and emb-30(tn377); cul-2(RNAi) embryos, both
of which had disorganized spindles at comparable percentages (43% and 33%,
respectively). These results provide evidence that CUL-2 has a separable
function to prevent the ectopic initiation or spreading of cortical PAR-2.
Our observations suggested that the lateral PAR-2 patches could occur distant from the meiotic spindle or sperm DNA. To rigorously test whether PAR-2 can localize to the cortex in the absence of the meiotic spindle in cul-2(RNAi) embryos, we disrupted MTs by depleting the ß-tubulin gene tbb-2, which had the effect of eliminating the meiotic spindle and visible MTs during meiosis. The elimination of MTs made distinguishing the two meiotic stages problematic because of the loss of chromosome and spindle dynamics. Therefore, all meiotic embryos were analyzed rather than just those in meiosis II, the stage in which ectopic PAR-2 localization generally occurs. We observed that in tbb-2; cul-2 double RNAi embryos, the overall percentage of cortical PAR-2 was markedly reduced, particularly in the anterior where PAR-2 is normally associated with the meiotic spindle (Table 1). However, discrete PAR-2 patches were still observed in 11.2% of meiotic embryos, most of which was localized to the lateral cortex (Fig. 6D; Table 1). Wild-type embryos subjected to tbb-2 RNAi had no meiotic PAR-2 staining (Table 1). These results indicate that PAR-2 localization can occur in cul-2(RNAi) embryos in the absence of the meiotic spindle. However, the reduction in the percentage of lateral PAR-2 localization in tbb-2; cul-2 RNAi embryos relative to cul-2(RNAi) embryos suggests that the presence of MTs potentiates the ectopic PAR-2 localization.
Interestingly, depletion of tbb-2 alone produced a high percentage
of post-meiotic embryos with condensed mitotic prometaphase chromosomes and
cortical PAR-2 localized in the same half of the embryo. In 13 out of 15 of
these embryos, -tubulin staining was observed in one or two small
circles of approximately 0.3 µm diameter near the condensed chromosomes,
presumably the centrosomes. For tbb-2, cul-2 RNAi embryos with
mitotic prometaphase chromosomes, 8 out of 12 had one or two similar small
-tubulin circles, with PAR-2 either localized on the cortical half of
the embryo with the chromosomes and
-tubulin circles (9/12) or on
lateral cortexes distant from
-tubulin circles (3/12). The observation
of
-tubulin at the centrosome suggests that even though visible MT
filaments were eliminated after tbb-2 RNAi, a residual capacity for
tubulin to be organized into the centrosome still exists and potentially this
structure is capable of localizing PAR-2. Therefore, although our results
demonstrate that the loss of CUL-2 during meiosis can localize cortical PAR-2
in the absence of the meiotic spindle, we cannot conclude that the PAR-2
localization is independent of all MT activity.
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Discussion |
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Three lines of evidence indicate that the meiosis II defects in
cul-2 mutants do not arise from a failure of chromosome separation.
First, the cohesin REC-8, which is required to hold sister chromatids together
(Pasierbek et al., 2001),
dissociates from metaphase II chromosomes in cul-2 mutants. Second, a
small percentage of cul-2 mutant zygotes are observed in which sister
chromatids separate during meiosis II but still fail to move to the spindle
poles. And third, in cul-2(RNAi); rec-8(RNAi) zygotes, all chromatids
separate from each other, yet exit from meiosis II is still delayed.
Mitotic anaphase chromosome movement has been extensively analyzed. Both MT
spindle dynamics and MT motors are required for mitotic anaphase poleward
force generation (Wittmann et al.,
2001). By contrast, meiotic anaphase has not been extensively
studied, and it is unclear to what extent similar mechanisms will function to
move chromosomes during meiosis. Interestingly, the morphology and dynamics of
the C. elegans meiotic spindle are quite different from the mitotic
spindle. The mitotic spindle is composed of long polar MTs and relatively
short midzone MTs. Ablation of the mitotic midzone MTs increases the velocity
and distance of chromosome separation, suggesting that they are not required
to `push' chromosomes apart but instead function to limit the speed and/or
extent of chromosome movement (Grill et
al., 2001
). By contrast, during meiotic anaphase, the MTs near the
meiotic spindle poles appear to depolymerize, whereas the amount of midzone
MTs between the separating chromosomes increases dramatically
(Albertson and Thomson, 1993
)
(Fig. 4B). During late
anaphase, all MTs appear to be located between the separating chromosomes
(Fig. 4B). The reorganization
of MTs to the midzone that occurs during meiotic anaphase suggests that
meiotic chromosomes are `pushed' apart by MT polymerization between the
chromosomes rather than being `pulled' to the spindle poles as occurs during
mitosis. In cul-2 mutants, the meiotic spindle maintains its normal
size and shape for an extended period of time during metaphase II, indicating
a defect in the initiation of MT depolymerization at the spindle poles and
polymerization between sister chromatids. CUL-2 therefore appears to function
upstream of the dramatic MT rearrangements that occur during meiotic chromatid
segregation.
CUL-2 and ZYG-11 are required for the degradation of Cyclin B1 during meiosis
Using a CYB-1::GFP reporter, we observed that maternally-provided cyclin B1
is degraded upon fertilization. In both cul-2(RNAi) and
zyg-11(RNAi) embryos, CYB-1::GFP is stabilized during both meiosis I
and II. This is not a secondary consequence of the meiosis II arrest, as the
failure to degrade CYB-1::GFP precedes the arrest. Surprisingly, depletion of
CYB-1 by RNAi in cul-2(ek1) mutants produced a synthetic phenotype of
loss of the pentagonal chromosome array in meiosis II, indicating that
cul-2 mutant zygotes have additional meiotic defect(s) that are
exacerbated by loss of CYB-1. The depletion of CYB-1 partially rescued the
meiosis II delay of cul-2(ek1) embryos, suggesting that the failure
to degrade CYB-1 may be contributing to the meiotic delay.
The APC/C ubiquitin ligase promotes cyclin B degradation during mitosis in
yeast and metazoa (Peters,
2002). APC/C has also been shown to promote meiotic cyclin B
degradation in budding yeast, fission yeast, Xenopus and mouse
(Cooper et al., 2000
;
Blanco et al., 2001
;
Peter et al., 2001
;
Nixon et al., 2002
). In C.
elegans, inactivation of the APC component apc-11 also
stabilized cyclin B1 levels. Further research will be required to assess the
roles of CUL-2 and APC/C in regulating cyclin B1 levels.
The spindle dynamics of anaphase I and II appear to be similar to each
other in wild type (Albertson and Thomson,
1993). Therefore it is surprising that in cul-2(ek1)
embryos, anaphase I is normal but anaphase II is severely delayed or
abolished. This is not due to the perdurance of cul-2 maternal
product through meiosis I because these embryos arose from cul-2(ek1)
homozygous hermaphrodite parents; furthermore, cul-2(ek1) embryos
completely lack anti-CUL-2 staining (Feng
et al., 1999
). The initiation of the metaphase to anaphase
transition in the two meiotic stages therefore appears to be differentially
regulated.
CUL-2 ubiquitin ligase complex components share meiotic and polarity functions
In mammals, elongin C has been shown to interact with CUL2 and CUL5, but
not with other cullins (Pause et al.,
1999; Kamura et al.,
2001
). CUL2 and CUL5 share homology in the N-terminal region,
which is required for elongin C binding
(Pause et al., 1999
;
Pintard et al., 2003b
).
Inactivation of CUL-5 in C. elegans does not produce visible
phenotypes (Kamath et al.,
2003
), suggesting that it is not involved in the meiotic or
polarity defects described in this work. RNAi depletion of elc-1
phenocopies all visible cul-2 mutant phenotypes, including the G1
arrest of germ cells (Hui Feng and E.T.K., unpublished), as well as the
degradation of embryonic CCCH proteins
(DeRenzo et al., 2003
),
suggesting that ELC-1 participates in all known CUL-2 functions. RBX-1
depletion in C. elegans phenocopies multiple cullin loss-of-function
phenotypes (Sasagawa et al.,
2003
) (data not shown), consistent with its interaction with
multiple cullins in mammals (Ohta et al.,
1999
). The observation of meiotic and polarity defects upon
inactivation of homologs of both core CUL-2 complex components suggests that
CUL-2 functions in the context of a conserved ubiquitin ligase complex.
A substrate recognition component (SRC) is required for the core CUL-2
complex to bind substrates. A number of mammalian CUL2 SRCs have been
identified that function in distinct CUL2 E3 complexes, including the von
Hippel-Lindau tumor suppressor protein (VHL)
(Kim and Kaelin, 2003;
Kamizono et al., 2001
;
Brower et al., 2002
).
Inactivation of the C. elegans VHL ortholog produces no embryonic
phenotypes (Epstein et al.,
2001
), indicating that SRCs other than VHL are required for the
CUL-2 meiotic and embryonic functions.
Currently, the only genes in C. elegans known to specifically
affect the meiosis II metaphase-to-anaphase transition are CUL-2 complex
components and zyg-11. Combining homozygous null alleles of both
cul-2 and zyg-11 in the same embryo did not enhance the
meiotic phenotype, which is consistent with both genes functioning in the same
pathway. Both genes also share other embryonic phenotypes, including multiple
nuclei, cytoplasmic extensions, and embryonic arrest with approximately 24
cells (Kemphues et al., 1986;
Feng et al., 1999
) (data not
shown). ZYG-11 is a leucine-rich repeat protein and this protein-interaction
motif is found in a number of SRCs for CUL-1-based SCF E3 complexes
(Tyers and Jorgensen, 2000
).
It is therefore possible that ZYG-11 functions as an SRC in one of the
CUL-2-based E3 complexes to promote meiosis; although our data suggests that
ZYG-11 does not function with CUL-2 to promote germ cell proliferation. As the
localization of PAR-2 independently of MT-foci was not observed in
zyg-11(RNAi) embryos, currently there is no evidence that ZYG-11
functions to restrain PAR-2 localization in regions distant from MT-organizing
centers.
Meiotic timing and AP polarity
During the extended meiosis II in cul-2 mutants, the majority of
embryos accumulate stable PAR-2 on the anterior cortex near the meiotic
spindle. It has been shown that in APC/C mutant zygotes arrested in meiosis I,
the meiotic spindle can initiate the localization of PAR-2 onto the anterior
cortex (Wallenfang and Seydoux,
2000). The disorganized `frayed' appearance of the meiotic spindle
observed in older meiosis I-arrested embryos was proposed to initiate the
PAR-2 reversal (Wallenfang and Seydoux,
2000
). By contrast, our study found that stable anterior PAR-2
localizations occur with meiosis II spindles of normal morphology and with
timing similar to the appearance of PAR-2 on the posterior cortex in wild
type. In addition, we observed that P granules segregate toward the
mislocalized PAR-2, that the SPCC migrates further than the meiotic
pronucleus, and that the cell division plane is often asymmetrically
positioned to produce a reversal in daughter cell size. None of these
downstream polarity changes are observed in meiosis I-arrested APC/C mutants
(Wallenfang and Seydoux,
2000
), suggesting either that the meiosis II spindle is
fundamentally different than the meiosis I spindle in effecting polarity or
that these changes can only manifest upon entry into interphase.
Transient and weak PAR-2 patches occasionally arise on the anterior cortex
during meiosis II in wild-type zygotes, which suggests that the intact meiosis
II spindle has an intrinsic ability to direct PAR-2 localization
(Table 2)
(Boyd et al., 1996;
Cuenca et al., 2003
). We
observed that the longer cul-2(RNAi) zygotes remained in meiosis, the
more stabilized the anterior PAR-2 became and the less likely that a posterior
PAR-2 patch would form. With only a modest meiosis II delay, a full reversal
of PAR localization was observed, with PAR-2 exclusively on the anterior and
the asymmetric cleavage of the zygote reversed
(Table 2). The failure of PAR-2
to localize to the posterior in these cul-2(RNAi) embryos may derive
in part from the aberrant migration of the SPCC to the anterior, which begins
during meiosis (Fig. 5E). In
cul-2(RNAi) embryos, sperm asters generally form during meiosis but
are very small until meiosis is completed, presumably because of the effect of
the MT-severing katanin MEI-1, which is degraded after meiosis
(Pintard et al., 2003a
).
Therefore, the failure of the SPCC to form full mitotic asters while in the
posterior may contribute to the lack of PAR-2 in that region. In total, our
results suggest that only a short window of meiotic timing is compatible with
the proper placement of the AP axis.
CUL-2 limits the inappropriate localization of PAR-2
The establishment of AP polarity in the C. elegans zygote has been
proposed to be a MT-directed process
(O'Connell et al., 2000;
Wallenfang and Seydoux, 2000
).
In cul-2(RNAi) embryos, PAR-2 was observed to initiate cortical
localization distant from the meiotic spindle or sperm DNA. Upon elimination
of the meiotic spindle by RNAi depletion of the ß-tubulin TBB-2, lateral
cortical PAR-2 was still observed in cul-2(RNAi) embryos at
significant although somewhat reduced levels. This suggests that the aberrant
PAR-2 localization is not dependent on the presence of a MT organizing center,
although MTs may potentiate the localization. The ectopic PAR-2 localization
in cul-2 mutants does not appear to arise as a secondary consequence
of the meiosis II delay, as the depletion of CUL-2 produces a significant
increase in ectopic PAR-2 localization even in an emb-30 mutant
background in which embryos are arrested in metaphase I. Furthermore, although
ZYG-11 is also required for meiotic progression, the localization of PAR-2
distant from the meiotic spindle was not observed in zyg-11(RNAi)
embryos. We propose that CUL-2 has a separate function to restrain ectopic
PAR-2 association with the cortex during meiosis. In combination with the
recently described role for CUL-2 in degrading germ cell determinants in
anterior cells of the early embryo
(DeRenzo et al., 2003
), our
results indicate that CUL-2 promotes the initiation of AP polarity through
multiple mechanisms.
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ACKNOWLEDGMENTS |
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Footnotes |
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