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First published online 24 January 2007
doi: 10.1242/dev.02784
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Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
* Author for correspondence (e-mail: schupbac{at}princeton.edu)
Accepted 14 December 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fzy, Cort, Cks, APC, Drosophila, Cell cycle, Meiosis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). One example is in the regulation of anaphase - the
coordinated series of events that results in the segregation of chromosomes to
produce two daughter nuclei. In mitotically dividing cells, anaphase
progression crucially depends on the inactivation of the mitotic kinase Cdk1
(also known as Cdc2) and on the subsequent release of sister chromatid
cohesion through the destruction of cohesin complexes. These events are
controlled by an E3 ubiquitin ligase - the anaphase-promoting complex (APC) -
in association with an adaptor protein, Fzy, and this complex targets mitotic
cyclins and securin for destruction (reviewed in
Peters, 2002). The role of the
APC in meiosis appears to be more complex than in mitotic cells. For example,
the APC only partially inhibits Cdk1 activity between meiotic divisions
(Gross et al., 2000) and
sister chromatid cohesion persists at centromeres through anaphase I
(Katis et al., 2004;
Kitajima et al., 2004). It is
not yet clear how the activity of the APC is modified in these specialized
cell divisions. In most eukaryotes, the meiotic cell cycle is followed by another atypical cell cycle - the cleavage divisions of early embryogenesis. In Drosophila, these cleavage cycles occur as a series of synchronized, rapid nuclear divisions and are referred to as syncytial divisions. The female meiotic cell cycle is not only closely linked to the syncytial mitotic cell cycle in time, but it also occurs within a shared cytoplasm - that of the egg. Therefore, these two distinct cell cycles share a common pool of cell-cycle regulators, and may share common strategies for spatially and temporally regulating cell-cycle progression within a syncytium.
One way in which the syncytial cell cycle is modified appears to be in the
limited destruction of mitotic cyclins in each cell cycle, apparently by
restricting their destruction to the area of the mitotic nuclei. Although
there is evidence that cyclin destruction is spatially regulated in somatic
cells (Kallio et al., 1998;
Rieder et al., 1997), this
strategy appears to be of particular importance in the syncytial embryo of
Drosophila as a means to conserve mitotic cyclins for the duration of
the rapid syncytial divisions. Several lines of evidence suggest that at least
one cyclin, cyclin B, undergoes limited local destruction on mitotic spindles
in the syncytial embryo (Edgar et al.,
1994; Huang and Raff,
1999; Raff et al.,
2002; Su et al.,
1998). It is not yet known what mediates this local cyclin B
destruction, and it is also not known whether this is unique to the syncytial
mitotic cell cycle or if it occurs in the preceding meiotic divisions.
Drosophila represents an excellent model system for understanding
how the canonical cell-cycle machinery is developmentally modified, and how
novel cell-cycle regulators are used to control meiosis and syncytial
divisions. cortex (cort) encodes a Cdc20/Cdh1 (Cdh1 is also
known as Fzr and Rap)-related protein, which appears to be required
specifically in female meiosis (Chu et al.,
2001; Lieberfarb et al.,
1996; Page and Orr-Weaver,
1996) and functions with a germline-specific Cks gene,
Cks30A, to mediate the destruction of cyclin A
(Swan et al., 2005;
Swan and Schupbach, 2005).
Here, we show that the canonical APC adaptor Fzy functions together with Cort
to target mitotic cyclins for destruction, and to drive anaphase in both
meiosis I and meiosis II. Female meiosis, like the subsequent syncytial
mitotic cell cycles, appears to involve the local destruction of cyclin B, and
we find that both Cort and Fzy are required for this process.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) have similar meiotic phenotypes
(Page and Orr-Weaver, 1996)
and were analyzed as transheterozygotes. cortQW55 has a
conserved Y303C change and cortRH65 encodes a truncated
protein lacking the seventh WD repeat (Chu
et al., 2001). Cks30A corresponds to the remnants gene,
and Cks30AKO was generated by site-directed mutagenesis
and is a molecular null, lacking a start codon
(Swan et al., 2005).
fzy6 and fzy7
(Dawson et al., 1995) are
temperature-sensitive lethal alleles that are female sterile at 22°C.
These were analyzed as transheterozygotes. Double mutant fzy; cort
chromosomes were generated by recombining cortQW55 with
fzy6 and recombining cortRH65 with
fzy7. All experiments with fzy mutants and
fzy; cort double mutants were performed on eggs from females kept at
29°C for 3-5 days. UAS-HA-cort was made by PCR amplification of
genomic cort (including introns), followed by cloning into pUASp with
a 2x hemaglutin (HA) tag at the N-terminus. Expression of UAS-HA-Cort
using the nosGal4-VP16 driver resulted in the rescue of the female
sterility of cort mutants (data not shown). UAS-Fzy and UAS-Cdh1 were
obtained from Christian Lehner (Sigrist
and Lehner, 1997). UAS-CyclinB-TPM-GFP was obtained from Jordan
Raff (Raff et al., 2002). Wing
expression of UAS-HA-Cort, UAS-Fzy and UAS-Cdh1 was driven by ptcGal4 and
enGal4. With these drivers, UAS-Fzy and UAS-Cdh1 expression results in pupal
lethality (data not shown). To observe the wing phenotype in enGal4-UAS-Fzy,
flies were raised at 18°C to reduce expression levels of this protein and
permit survival to adult.
Antibody staining
To observe early meiotic events in wild type, mature eggs were activated to
undergo meiosis in vitro, as previously described
(Page and Orr-Weaver, 1997).
To observe later meiotic events in wild type, eggs from 0 to 20 minute egg-lay
collections were used. To detect cyclin B, eggs from 0-2 hour collections or
from activated oocytes were fixed in 100% methanol, rehydrated gradually,
blocked in PBST, 1% BSA and incubated with rabbit anti-cyclin B antiserum
(from Jordan Raff) at 1/500. Rat anti-
-Tubulin (Cappel) was used at
1/500 and DNA was labeled either with mouse anti-Histones (Chemicon) at 1/1000
or OliGreen (Molecular Probes) at 1/500. Rat anti-Subito (anti-Sub) antibody
(Jang et al., 2005) was used
at 1/3000. FISH was performed on 0-2-hour-old eggs or dissected oocytes using
a probe to a repeated 359 bp repeat sequence unique to the centromeric region
of the X-chromosome (Dernburg,
2000). For immunostaining of wing discs, third instar larvae were
collected from crosses of UAS-HA-Cort, UAS-Cdh1 or UAS-Fzy to ptcGal4. Discs
were fixed for 30 minutes in 3.7% formaldehyde/PBST, extracted for 1 hour in
PBST +0.3% Triton X-100 and labeled with rabbit anti-cyclin B, B3 (from
Christian Lehner, University of Bayreuth, Germany) at 1/500 or with rabbit
anti-cyclin A (from David Glover, Cambridge University, England) at 1/500.
Discs were also labeled with rat anti-HA antiserum (Roche) at 1/500 and mouse
anti-ß-gal antiserum (Promega) at 1/500.
Western analysis
Extracts were prepared in 2x sample buffer from wild-type and mutant
eggs collected over a 2-hour period. Wild-type eggs were derived from
unfertilized females (crossed to XO males). Western blotting was performed by
standard techniques. Antibodies were mouse anti-cyclin A and mouse anti-cyclin
B (both from Developmental Studies Hybridoma Bank), rabbit anti-cyclin B3
(Sigrist et al., 1995), rabbit
anti-Pim (Stratmann and Lehner,
1996) and rabbit anti-PSTAIR (Santa Cruz).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Fzr2
appears to be exclusively transcribed in the male germline
(Jacobs et al., 2002), whereas
Cdh1 is transcribed in the female germline
(Sigrist and Lehner, 1997),
but the protein is not detectable in early embryos, either by western blot
analysis or by in vivo functional assays
(Jacobs et al., 2002;
Raff et al., 2002). To
determine the role of APC complexes in female meiosis, we focused on the
canonical Cdc20 (fzy), and a female-specific
Cdc20/Cdh1 homologue, cort, both of which are
highly expressed in the female germline
(Chu et al., 2001;
Dawson et al., 1995). We
re-examined the meiotic phenotypes of cort and fzy mutants
separately and in double-mutant combinations by observing spindles and DNA,
and by following chromosome segregation using FISH against an X-chromosome
probe. Temperature-sensitive fzy mutants were analyzed at 29°C
and, to control for temperature effects, wild-type and cort mutants
were therefore examined at both room temperature and at 29°C. In female
Drosophila, meiosis arrests in metaphase of the first meiotic
division until ovulation (for a review see
McKim et al., 2002). At this
stage, the egg contains a single spindle near the anterior cortex; this
spindle contains two X-chromosome signals representing the two pairs of sister
chromatids (Fig. 1A,A').
Upon ovulation, meiosis resumes. In metaphase of meiosis II, two tandemly
arranged spindles form around the products of the first meiotic division. Both
metaphase spindles contain a single sister chromatid pair
(Fig. 1B,B'). In anaphase
II, sister chromatids separate, resulting in four meiotic products, each with
a single X-chromosome (Fig.
1C,C'). Meiosis is completed very rapidly after ovulation
and, at 22°C, only 1% (n=220) of eggs from a 0-2-hour-old
collection were still in meiosis. The remainder of eggs contained arrested
meiotic products (polar bodies). Similarly, in eggs from females kept at
29°C, only 4% (n=113) were in meiosis. In addition, 3% of eggs
contained aberrant spindles near the cortex, suggesting low-level disruption
of meiosis at this temperature. As previously described, eggs from
cort-mutant females (hereafter referred to as cort eggs)
contain two spindles near the anterior cortex of the egg, indicative of an
arrest in meiosis II (Chu et al.,
2001; Lieberfarb et al.,
1996; Page and Orr-Weaver,
1996) (Fig. 1D).
Similarly, at 29°C, 90% (n=78) of cort eggs contained
two meiotic spindles. Both of the spindles contained a single X-chromosome
signal (Fig. 1D'),
indicating an arrest in metaphase, prior to sister chromatid separation.
Cks30A, like cort, is required for the proper completion
of meiosis II, consistent with a model in which Cks promotes the activation of
APCCort (Swan et al.,
2005). However, whereas cort mutants invariably arrest in
the second meiosis, in Cks30 mutants, most oocytes eventually
complete meiosis, although they are delayed in doing so
(Swan et al., 2005). In
0-2-hour-old collections of Cks30AKO eggs, 26% were in
meiosis II (n=46). In 58% of these, both spindles had a single
X-chromosome signal and were therefore in metaphase of meiosis II
(Fig. 1E,E'), while the
remaining 42% had two X-chromosomes per spindle and were therefore in anaphase
of meiosis II (Fig.
1F,F'). Therefore, loss of Cks30A results in a
meiotic phenotype similar to, but weaker than, cort, suggesting that
Cks30A activity enhances but is not essential for the function of the
APCCort.
In Drosophila, as in most eukaryotes, Fzy is the crucial APC
adaptor in mitosis, and is essential for anaphase progression in most cell
types (Dawson et al., 1993;
Dawson et al., 1995;
Sigrist et al., 1995). It is
not yet known if Fzy is also required for anaphase progression in the meiotic
divisions. To address this question, we analyzed female meiosis in eggs
produced by fzy females. fzy, unlike cort or
Cks30A, is essential for viability, and germline clones of a null
allele did not produce eggs (data not shown). However, temperature-sensitive
allele combinations raised at a permissive temperature are viable and have
been used to study the role of fzy in early embryogenesis
(Dawson et al., 1995).
fzy6/fzy7 mutants raised at the
permissive temperature of 22°C are female-sterile and embryos arrest in
the first mitosis (Dawson et al.,
1993). Meiosis appeared to be unaffected in these eggs (data not
shown). To achieve a stronger phenotype, we shifted
fzy6/fzy7 females to the restrictive
temperature of 29°C. In addition to the mitotic arrest, eggs from
fzy6/fzy7 females kept at 29°C
(hereafter referred to as fzy eggs) displayed defects in meiosis. 74%
(n=78) of fzy eggs contained two spindles near the cortex
(Fig. 1G), indicative of a
delay or arrest in meiosis II. In most cases, both spindles contained two
X-chromosome signals (Fig.
1G'), indicating that sister chromatid separation had
occurred and that they were therefore in anaphase of meiosis II. Often, as
shown in Fig. 1G', the
two X-chromosomes were not properly aligned along the spindle axis, probably
as a result of prolonged arrest. In rare cases, we detected more than two
X-chromosome signals per spindle (data not shown), suggesting that DNA
replication can occur during the aberrant meiosis in fzy eggs. We did
not observe meiotic spindles with only a single X-chromosome, indicating that
meiosis did not detectably delay or arrest in metaphase of meiosis II in these
eggs. Eggs often contained, near the two major spindles, one or more smaller
spindles with associated chromatin (arrow,
Fig. 1G), possibly resulting
from chromosome loss at the first meiotic division. In total, 13% of embryos
contained one or more spindles at the anterior cortex in addition to a polar
body, suggesting a partial completion of meiosis, whereas 6% of embryos
contained only polar bodies at the anterior cortex, and therefore appear to
have completed meiosis.
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)],
suggesting the possibility that cort and fzy play partially
redundant roles in meiosis I. To test this possibility, we analyzed the
phenotype of a fzy; cort double mutant raised at 29°C. In total,
74% (n=57) of fzy; cort double-mutant eggs contained two
spindles, each with a single X-chromosome signal
(Fig. 1H,H'), indicating
that they arrested in metaphase of the second meiotic division. The remaining
26% of the eggs contained only a single spindle containing two X-chromosome
signals (Fig. 1I,I'),
indicating an arrest in meiosis I. We conclude that the two APC adaptors Cort
and Fzy are necessary for anaphase progression in both meiotic divisions,
performing partially redundant roles in meiosis I and non-redundant roles in
meiosis II.
In addition to its role in anaphase, Cks30A is required earlier in meiosis,
for the assembly or maintenance of the first meiotic spindle
(Pearson et al., 2005;
Swan et al., 2005). To
determine whether spindle assembly or metaphase I arrest is affected in
cort or fzy mutants, we analyzed chromosome alignment in
unactivated oocytes using the X-chromosome FISH probe. In metaphase I in wild
type, the autosomes are aligned at the spindle equator while the X-chromosomes
are typically precociously segregated to either pole
(McKim et al., 2002). We found
that chromosomes were properly aligned in both cort and fzy
mutants, as well as in fzy; cort double mutants (see Fig. S1 in the
supplementary material). Therefore, with the caveat that we are not able to
study null alleles of cort and fzy, we conclude that the
first requirement for cort and fzy in meiosis is in anaphase
of meiosis I.
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). The importance
of cyclin destruction in the two meiotic divisions is less clear. To determine
whether cyclin destruction is necessary for female meiosis in
Drosophila, we examined meiotic progression in eggs from females
expressing a destruction-box (D-box) mutated form of cyclin B - cyclin
B-TPM-GFP (Raff et al., 2002).
When expressed in the female germline, cyclin B-TPM-GFP results in mitotic
arrest at a variable stage of the syncytial mitotic cycle in the majority of
embryos, indicating that cyclin B destruction is necessary for anaphase
progression in these cell cycles (Raff et
al., 2002). To determine whether a failure to destroy cyclin B
also disrupts meiosis, we expressed cyclin B-TMP-GFP with the strong germline
driver nosGal4VP16 at 29°C (to induce higher expression). Under these
conditions, almost all embryos arrested in the first mitotic division (data
not shown). In addition to this mitotic arrest, only 38% (n=51)
appeared to complete female meiosis, as judged by the presence of polar bodies
and the absence of spindles at the dorsal anterior of the egg. A total of 50%
of eggs contained multiple small spindles in the dorsal anterior, possibly as
a result of meiotic spindle breakdown and/or chromosome mis-segregation
(Fig. 1J,J'). The
remaining 14% of eggs appeared to arrest in meiosis. A small proportion of the
eggs (4%) had two spindles with either one or two X-chromosomes, indicative of
an arrest in either metaphase or anaphase of meiosis II
(Fig. 1K,K'). In
addition, 10% of the eggs contained a single spindle at the dorsal anterior,
typically with two X-chromosome signals, indicative of a meiosis I arrest
(Fig. 1L,L'). Therefore,
cyclin B destruction is necessary for the proper completion of female meiosis
in Drosophila.
Cort and Fzy are required for the destruction of mitotic cyclins in the egg
The above results suggest the possibility that the meiotic arrest in
cort and fzy eggs could be caused by a failure to destroy
mitotic cyclins. In Drosophila, it is not known whether the
APCFzy has any role in cyclin destruction during meiosis. On the
other hand, the APCCort has been implicated with Cks30A in
cyclin A destruction in the female germline
(Swan et al., 2005). To
determine the respective roles of cort and fzy in cyclin
destruction in female meiosis, we compared cyclin levels in egg extracts from
cort, fzy and Cks30A single mutants, and from fzy;
cort double mutants. All of these mutants arrest at or before entry into
the first mitotic cell cycle and we therefore used unfertilized, and therefore
non-cycling, wild-type eggs for control extracts. As previously reported,
Cks30A and cort eggs contain high levels of cyclin A protein
(Swan et al., 2005)
(Fig. 2). Cyclin A levels were
not elevated in egg extracts from fzy mutants raised at 22°C
(data not shown). However, eggs from fzy females kept at 29°C
showed a clear elevation in cyclin A levels, and fzy; cort double
mutants had an even-greater elevation in cyclin A levels
(Fig. 2). Therefore,
fzy and cort are both required for cyclin A destruction in
the Drosophila egg. Cyclin B and cyclin B3 levels were also elevated
in fzy and cort single mutants, and more so in fzy;
cort double mutants (Fig.
2), indicating that Cort and Fzy cooperate in the destruction of
all three mitotic cyclins. Comparing the relative effects of cort and
fzy mutants on the different cyclins suggests that Cort is more
important for cyclin A and cyclin B3 destruction, whereas Fzy is more
important for cyclin B destruction. Therefore, the two APC adaptors may have
different target preferences
In Xenopus and mice, Cks2 is necessary for the activation of the
APCFzy complex by associating with Cdk1 and promoting its
phosphorylation of the APC subunits Cdc27 and Cdc16
(Patra and Dunphy, 1998;
Spruck et al., 2003). In
Drosophila, Cks30A interacts with Cdk1 in the germline and is
required for cyclin A destruction (Swan et
al., 2005). Cks30A eggs also have elevated cyclin B3
levels, and both cyclin A and cyclin B3 were at levels higher than in
cort or fzy single mutants, and were approaching levels
observed in fzy; cort double mutants
(Fig. 2). This could be
explained if Cks30A activity is required for the function of both
APCFzy and APCCort complexes. Cyclin B, by contrast, is
not strongly affected in Cks30A mutants
(Fig. 2), indicating that
Cks30A plays a lesser role in promoting the activity of APCFzy and
APCCort in cyclin B destruction.
The above results indicate that Cort, like other Fzy/Cdh1-family proteins, functions in the targeting of mitotic cyclins for destruction. To further test the ability of Cort to target cyclins for destruction, we expressed HA-tagged Cort in a stripe of cells in the wing imaginal disc using the Gal4-UAS system and then looked at cyclin levels by immunolocalization. The expression of HA-Cort resulted in a corresponding decrease in cyclin A, cyclin B and cyclin B3 (Fig. 3A-C), consistent with these cyclins being targeted for destruction by Cort. A similar effect was observed upon the overexpression of Fzy or Cdh1 (Fig. 3D and data not shown). Therefore, Cort is able to target all of the mitotic cyclins for destruction, consistent with a proposed role as an APC adaptor.
The reduction of cyclin levels would be expected to inhibit mitosis in the
wing imaginal disc. Each cell in the wing secretes a single bristle, and
mitotic failure results in fewer, but larger, cells; consequently, there are
fewer wing hairs (Weigmann et al.,
1997). Indeed, the expression of Fzy or HA-Cort in the posterior
compartment of the wing disc, using the enGal4 driver, led to fewer but larger
cells, as judged by an increase in the spacing between the wing hairs
(Fig. 3E,F). To test the
possibility that Cks30A is required for the activation of the
APCCort, we used enGal4 to express HA-Cort in Drosophila
that also lacked zygotic expression of Cks30A. In the Cks30A
background, the wing-hair-spacing phenotype was suppressed
(Fig. 3G). It was largely
restored if Flag-Cks30A is coexpressed with HA-Cort in the
Cks30A-mutant background (Fig.
3H), whereas the expression of Flag-Cks30A alone had no effect
(Fig. 3I). Therefore, Cks30A is
required for Cort activity.
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;
Huang and Raff, 1999;
Raff et al., 2002;
Su et al., 1998). It is not
known how this local destruction is mediated, or whether localized cyclin B
destruction is unique to the syncytial mitotic cycles or whether it also
occurs in the preceding meiotic divisions. To determine if cyclin B is subject
to localized destruction in female meiosis, we first determined the
localization of cyclin B in wild-type meiosis. In Drosophila females,
meiosis is arrested in metaphase of the first meiotic division until ovulation
and cyclin B accumulates at high levels on the metaphase I spindle
(Fig. 4A)
(Pearson et al., 2005). This
cyclin B accumulation is nonuniform and appears to be focused at the meiotic
spindle mid-zone - the region of the meiotic spindle where non-kinetochore
microtubules from either pole overlap. The meiotic spindle mid-zone (or
meiotic metaphase central spindle) appears to play a specialized role in
establishing spindle bipolarity and in recruiting chromosomal passenger
proteins to the meiotic spindle (Jang et
al., 2005). To confirm that cyclin B associates with the spindle
mid-zone, we double-labeled oocytes for both cyclin B and the spindle mid-zone
component Subito (Sub) (Jang et al.,
2005). Cyclin B and Sub appeared to colocalize precisely
(Fig. 4B), confirming that
cyclin B specifically associates with the spindle mid-zone in metaphase of
meiosis I. In anaphase of meiosis I, the spindle mid-zone extends as the
spindle elongates, and chromosomes segregate to either pole
(Jang et al., 2005). Cyclin B
persisted on the spindle mid-zone throughout anaphase I
(Fig. 4C,D). Upon assembly of
the second meiotic spindle, cyclin B appeared to redistribute to the spindle
mid-zone of the newly formed meiosis II spindles
(Fig. 4E). The protein
persisted at the spindle mid-zone after the onset of anaphase
(Fig. 4F), but was no longer
detected later in anaphase (Fig.
4G). Therefore, cyclin B is associated with the meiotic spindle
mid-zone throughout meiosis, and dissociates from the spindle late in anaphase
II. This pattern of accumulation suggests that cyclin B, presumably in complex
with Cdk1, plays a unique role at the meiotic mid-zone in meiosis I and
meiosis II, and that it is targeted for destruction at this site in anaphase
II.
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The difference in site of cyclin B accumulation on the meiotic spindle between cort and fzy could be a result of Cort and Fzy having distinct sites of activity. In this model, Cort would mediate cyclin B destruction at the spindle mid-zone while Fzy targeted cyclin B along the length of the spindle. One consequence of this model would be that fzy; cort double mutants might have a cyclin B accumulation that is the sum of that of the two single mutants. Alternatively, Cort and Fzy may mediate cyclin B destruction at different stages of meiosis. In this model, Cort would mediate cyclin B destruction in metaphase when cyclin B is primarily at the mid-zone, and Fzy would function in anaphase along the entire spindle. This model fits with the time of arrest of cort and fzy in metaphase and anaphase, respectively (Fig. 1), and it predicts that fzy; cort double mutants would arrest in metaphase, with cyclin B localized at the mid-zone. We find that fzy; cort double mutants do indeed accumulate cyclin B largely at the spindle mid-zone and not along the length of the spindle (Fig. 5E), and are therefore identical to cort single mutants. Therefore, the different site of accumulation of cyclin B in cort and fzy may reflect different temporal requirements for the APCCort and APCFzy in meiosis.
Analysis by western blot showed that Cks30A has little effect on overall cyclin B levels (Fig. 3). However, the immunostaining of eggs from Cks30A revealed that cyclin B was enriched on meiotic spindles (Fig. 5F). Therefore, Cks30A is also required for the destruction of cyclin B on spindles in female meiosis, consistent with a role in the activation of the APCCort and APCFzy complexes.
In the syncytial embryonic cell cycles, cyclin B associates with the
mitotic spindle at metaphase (Huang and
Raff, 1999) (Fig.
5G), and its destruction on spindles may play a role in anaphase
progression. Given that the APCCort and APCFzy are both
required for the destruction of cyclin B on the meiotic spindle, it seems
likely that either or both APC complexes would also be involved in local
cyclin B destruction on mitotic spindles. cort mutants arrested prior
to the assembly of a mitotic spindle and, therefore, the role of Cort in
localized cyclin B destruction in mitosis could not be determined.
Fzy and Cks30A, however, enter into, and arrest in, the
first mitosis. In both of these mutants, the mitotic arrest is associated with
a failure to locally destroy cyclin B (Fig.
5H and data not shown), arguing that Cks30A and Fzy are necessary
for the local destruction of cyclin B in syncytial mitosis, as well as in
meiosis.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cort is a functional Fzy/Cdh1 homologue
The cort gene encodes a diverged member of the Fzy/Cdh1 family
(Chu et al., 2001). Fzy/Cdh1
homologues interact with the APC and with specific sequences (D-box, KEN box
or A-box) found on cyclins and on other APC targets. As such, Fzy/Cdh1
proteins act as specificity factors to target proteins for ubiquitination and
eventual destruction. Cort protein, like all Fzy/Cdh1-family proteins,
contains seven WD domains in the C-terminal-half of the protein, implicated in
substrate recognition (Pfleger et al.,
2001). We also found that Cort has an N-terminal C-box (amino
acids 482, 483) and a C-terminal IR tail (amino acids 54-60), both implicated
in binding to the APC (Passmore et al.,
2003; Schwab et al.,
2001; Vodermaier et al.,
2003). In addition to containing these conserved functional
domains, Cort displays a conserved ability to mediate cyclin destruction.
cort mutations result in the overaccumulation of cyclin A, cyclin B
and cyclin B3 in the egg (Swan et al.,
2005) (Fig. 2),
whereas the ectopic expression of Cort in the wing disc leads to a reduction
in the levels of these mitotic cyclins
(Fig. 3). Taken together, these
results indicate that Cortex encodes a functional member of the Fzy/Cdh1
family.
Fzy and Cort cooperate to promote cyclin destruction and meiotic progression
Although the Drosophila genome has four genes that encode Fzy/Cdh1
proteins, only two of these proteins, Fzy and Cort, are expressed at
detectable levels in the female germline
(Raff et al., 2002;
Jacobs et al., 2002;
Chu et al., 2001). We have
studied the role of these two APC adaptors both individually and in double
mutants, and have found that they function together to promote anaphase in
both the first and second meiotic divisions of female meiosis. In most cell
types in Drosophila and other eukaryotes, a single APC complex,
APCFzy, is responsible for cyclin destruction and anaphase
progression. It is therefore surprising that, in the female germline of
Drosophila, two APC adaptors are necessary for meiotic progression.
In the case of meiosis I, Cort and Fzy appear to play largely redundant roles,
as only removing both genes results in a significant block in meiosis I. The
two APC complexes may also be functionally redundant with respect to global
cyclin levels. Mutations in either fzy or cort result in an
increase in the levels of cyclin A, cyclin B and cyclin B3, whereas mutation
in both genes results in even-further increases in cyclin levels.
Although Cort and Fzy have overlapping roles in promoting anaphase I, both
are essential for meiosis II. This could simply reflect a greater quantitative
requirement for APC activity in meiosis II. Alternatively, the two APC
complexes could have distinct roles in the second meiotic division. Consistent
with this latter possibility, mutations in either cort or
fzy both result in arrest at different stages of meiosis II:
cort mutants arrest with the sister chromatids associated, and
therefore in metaphase, whereas fzy mutants almost invariably arrest
with separated sister chromatids, and are therefore in anaphase. cort
and fzy also result in different patterns of cyclin B stabilization
on the arrested spindles, suggesting roles in metaphase and anaphase,
respectively. Therefore, Cort may function to initiate sister chromatid
separation at the onset of anaphase II and Fzy may primarily function later,
in anaphase II. Alternatively, the later arrest observed in fzy could
simply reflect the fact that the fzy alleles that we have used are
not nulls, and it is possible that a complete loss of Fzy activity would also
result in a metaphase arrest, as seen in cort. However, comparing the
meiosis II phenotypes of fzy with Cks30A-null mutants
suggests that the later arrest in fzy is not simply due to residual
activity. Cks30A-null mutants have a weaker meiotic arrest than
fzy, as they complete meiosis at high frequency
(Swan et al., 2005), but they
display a higher frequency of metaphase arrest or delay. The fact that
fzy does not similarly cause a delay in metaphase of meiosis II
suggests that it is only required at anaphase. Therefore, it is possible that
Fzy is crucial at anaphase, whereas Cort is necessary for the metaphase to
anaphase transition.
The different temporal requirements for Cort and Fzy prior to and after
sister chromatid separation, respectively, could be related to their apparent
differences in substrate specificity. Western analysis
(Fig. 2) reveals that Cort is
more important for the destruction of cyclin A and cyclin B3, whereas Fzy
appears to play a greater role in cyclin B destruction in the egg. In mitotic
cells, cyclin destruction occurs sequentially. Cyclin A is destroyed first, in
prometaphase, and this is a prerequisite for sister chromatid separation.
Cyclin B destruction occurs at anaphase onset and is necessary for later
anaphase events, subsequent to sister chromatid separation
(Sigrist et al., 1995).
Therefore, it is possible that Cort promotes the early stages of meiotic
anaphase by targeting cyclin A for destruction, whereas Fzy is more crucial
later, through its targeting of cyclin B for destruction.
Role of the APC in meiosis
The meiotic cell cycle differs in many respects from the standard mitotic
cycle. Whereas APC-mediated destruction of mitotic regulators appears to be
required for anaphase progression in most or all mitotic cells, the role of
the APC and cyclin destruction in meiosis is not as well-understood. Our
analysis of the two APC adaptors Cort and Fzy has permitted an evaluation of
the role of the APC complex in female meiosis in Drosophila. We found
that the APC is required for anaphase progression in both meiotic divisions.
Correlating with its requirement for the completion of meiosis, the APC is
required for the destruction of mitotic cyclins. At least one of these
cyclins, cyclin B, is a crucial substrate in meiosis, because the expression
of a stabilized form of cyclin B disrupts this process
(Fig. 1). Therefore, APC
activity and cyclin destruction are required for anaphase progression in both
meiotic divisions, in addition to in mitosis. APC activity has been implicated
in both meiotic divisions in C. elegans
(Furuta et al., 2000;
Golden et al., 2000) and in
the mouse (Salah and Nasmyth,
2000; Terret et al.,
2003), and in the second, but not the first, meiotic division in
Xenopus (Peter et al.,
2001; Taieb et al.,
2001). In yeast, two APC complexes, the mitotic APCFzy
and a meiosis-specific complex (APCAma1 in S. cerevisiae
and APCMfr1 in S. pombe) function together to mediate
protein destruction in meiosis (Asakawa et
al., 2001; Blanco et al.,
2001; Izawa et al.,
2005; Salah and Nasmyth,
2000). It now appears that Drosophila also uses two APC
complexes in female meiosis, and this may turn out to be a common strategy in
other eukaryotes.
The role of Cks30A in activating the APC
Cks30A belongs to a highly conserved family of proteins that bind to and
stimulate the activity of the mitotic kinase Cdk1. In Xenopus, the
Cks30A homologue Xep9 stimulates the Cdk-dependent phosphorylation of APC
subunits, and thereby promotes the activation of the APCFzy complex
(Patra and Dunphy, 1998). Our
results suggest that Cks30A may have a similar role in stimulating both the
APCFzy and APCCort in female meiosis in
Drosophila. First, Cks30A, as are cort and
fzy, is required for the completion of meiosis II and, like
fzy, it is required for the completion of the first mitotic division
of embryogenesis (this study, Fig.
1) (Lieberfarb et al.,
1996; Page and Orr-Weaver,
1996; Swan et al.,
2005). Second, Cks30A, as are Cort and Fzy, is necessary for
global cyclin destruction in the Drosophila egg and for local cyclin
B destruction on the meiotic spindle (Figs
2,
5). Global levels of cyclin A
and cyclin B3 are elevated to a greater extent in Cks30A mutants than
in single mutants for cort or fzy, consistent with the idea
of Cks30A activating both Cort and Fzy. Third, we have shown that Cks30A is
necessary for the activity of ectopically expressed Cort in the adult wing
(Fig. 3). Cks30A may also play
a role in activating APCFzy in mitotic cells. We have found that
the temperature-sensitive fzy6 allele is lethal at all
temperatures in a Cks30A-null background (A.S. and T.S.,
unpublished), suggesting that the Cks30A-dependent activation of
APCFzy becomes essential when Fzy activity is compromised.
Although Cks30A appears to promote the activity of the APCCort
and the APCFzy, these complexes seems to retain some activity in
the absence of Cks30A. Whereas cort and fzy cause an arrest
in meiosis II, Cks30A-null mutants are typically delayed only in
meiosis II (Swan et al.,
2005). Also, although cyclin A and cyclin B3 levels are elevated
more in Cks30A eggs than in either fzy or cort, their levels
are still not as high as in fzy; cort double mutants, indicating that
Fzy and Cort can destroy cyclin A and cyclin B3 to some degree in the absence
of Cks30A. Cyclin B destruction is even less dependent on Cks30A, because
cyclin B levels are affected less in Cks30A mutants than in either
cort or fzy single mutants. Therefore, Cks30A may be more
crucial for the activity of APCCort and APCFzy complexes
on cyclin A and cyclin B3, and less crucial for their activity on cyclin B.
The relatively weaker meiotic arrest in Cks30A mutants compared to
fzy; cort double mutants may also indicate that the APC has other
meiotic targets that can be destroyed in the absence of Cks30A.
Localized cyclin destruction in Drosophila meiosis
Cyclin B undergoes local oscillations in its association with mitotic
spindles in syncytial embryos, appearing transiently along the full length of
the mitotic spindle in early metaphase and gradually disappearing from the
spindle starting at the centrosomes and ending at the kinetochores
(Huang and Raff, 1999). The
timing of this loss of cyclin B from the spindle, at the onset of anaphase,
corresponds with the timing of cyclin B destruction in other cell types,
suggesting the possibility that cyclin B is locally destroyed on the spindle
in anaphase. We now show that cyclin B is subject to similar local
oscillations in the female meiotic cycles
(Fig. 4), and that cyclin B
destruction is necessary for the completion of female meiosis
(Fig. 1J-L). Importantly, we
demonstrate that the local loss of cyclin B from the spindle in meiosis is
dependent on the APC adaptors Cort and Fzy, and that the local loss of cyclin
B from the spindle in mitosis depends on Fzy
(Fig. 5). These results
strongly suggest that the local loss of cyclin B from the spindle in anaphase
of meiosis II and anaphase of mitosis is actually due to its local
destruction.
The pattern of accumulation and loss of cyclin B from the spindle in
meiosis differs in some respects compared to syncytial mitotic cycles. First,
in metaphase of mitosis, cyclin B initially accumulates throughout the spindle
microtubules (Huang and Raff,
1999), whereas, in metaphase of the meiotic divisions, cyclin B
first appears exclusively at the spindle mid-zone. This difference may reflect
the fact that the meiotic spindle does not contain centrosomes and cyclin B
may, therefore, not load onto spindles from centrosomes and progress along the
spindles to the kinetochores, as has been proposed for mitosis
(Huang and Raff, 1999).
Second, the timing of cyclin B destruction appears to be different between the
meiotic and mitotic cycles. Most strikingly, there is no loss of cyclin B from
the spindle in anaphase of meiosis I, implying that local cyclin B destruction
is not necessary for the completion of the first meiotic division. In
addition, the loss of cyclin B from the spindle following meiosis II only
occurs late in anaphase rather than at the onset of anaphase, as occurs in the
syncytial mitotic cycles. We do not yet know how cyclin B destruction is
prevented in anaphase I and early in anaphase of meiosis II. One possibility
is that the spindleassembly checkpoint is locally active during these stages.
This checkpoint is required for the proper completion of female meiosis in
Drosophila (Fischer et al.,
2004; Gilliland et al.,
2005), and it will be interesting to see if this requirement
reflects a role in inhibiting either APCFzy or APCCort
activity.
The specific accumulation of cyclin B at the spindle mid-zone in meiosis
may reflect the unique properties of the meiotic spindle. The mid-zone
microtubules or central spindle microtubules are a subset of spindle
microtubules that do not end in kinetochores, but instead overlap at the
mid-zone with microtubules from the other pole. In dividing cells, the central
spindle is crucial for cytokinesis, but, in female meiosis, it appears to have
a role in spindle assembly (Jang et al.,
2005). Along with cyclin B, the chromosomal passenger proteins
Aurora B and Incenp are recruited to the spindle mid-zone. It will be of great
interest to determine what these proteins do at the mid-zone and how cyclin B
destruction at this site may be important for anaphase in meiosis. It will
also be important to determine how the APCCort targets cyclin B at
the spindle mid-zone. We have not been able to detect any specific
localization of GFP or HA-tagged Cortex in meiosis or in the syncytial embryo
(A.S. and T.S., unpublished), but it is possible that its activity is
spatially regulated.
In conclusion, our results support a model in which two APC complexes, APCFzy and APCCort, cooperate to mediate the destruction of meiotic cyclins and allow progression through female meiosis
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/5/891/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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