Department of Biology, University of Washington, Seattle, WA 98195-1800, USA
Author for correspondence (e-mail:
jji{at}partners.org)
Accepted 11 February 2005
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SUMMARY |
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Key words: Cdk1-CycB, three rows, Separase, Anaphase, Drosophila
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Introduction |
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Because Separase is the enzyme that disrupts the Cohesin complex, the
reliability of chromatid separation depends on the precise control of Separase
activity. One widely accepted model to explain how Separase activity is
regulated is that Separase activity is inactive when bonded to Securin but is
activated when Securin is degraded by the 26S proteasome. This process
requires poly-ubiquitination of Securin by a highly conserved ubiquitin
protein ligase APC/C (anaphase-promoting complex or cyclosome)
(Murray, 2004;
Zachariae and Nasmyth, 1999
).
This model is based on many genetic and biochemical studies using yeasts,
Xenopus egg extracts and mammalian cell lines
(Haering and Nasmyth, 2003
;
Harper et al., 2002
). However,
several observations do not support the idea that Separase activity is only
regulated by the inhibition of Securin. For example, the budding yeast Securin
(Pds1) null mutation causes chromatid separation defects without cell cycle
arrest (Yamamoto et al., 1996
;
Shirayama et al., 1999
).
Non-degradable Drosophila CycA causes metaphase arrest without
inhibiting Securin (Pim) degradation
(Leismann and Lehner, 2003
),
thus Securin destruction is not sufficient to induce anaphase in
Drosophila. Furthermore, three types of mitotic cyclins in
Drosophila are also degraded in succession: CycA is degraded at
metaphase, CycB during anaphase and CycB3 during telophase
(Huang and Raff, 1999
;
Jacobs et al., 1998
;
Lehner and O'Farrell, 1989
;
Parry and O'Farrell, 2001
;
Sigrist et al., 1995
).
Non-degradable cyclins result in blockage of cell-cycle progression at stages
when the cyclins are normally degraded
(Parry and O'Farrell, 2001
;
Sigrist et al., 1995
;
Su et al., 1998
). Therefore,
in addition to Separase activity regulated by Securin, the order of
degradation of mitotic cyclins must also control proper anaphase initiation
(Follette and O'Farrell, 1997
;
Parry and O'Farrell,
2001
).
There are observations indicating that Separase activity is inhibited by
Cdk1-CycB1 modification on Separase. For example, in Xenopus, a
slightly above the physiological level of non-degradable CycB1 causes
metaphase arrest because Cdk1-CycB1 either directly or indirectly
phosphorylates Separase, thereby inhibiting Separase activity
(Stemmann et al., 2001).
Similar observations have been made with Australian rat kangaroo PtK1 cells,
and it was estimated that a 1.5- to 2-fold excess of CycB1 inhibits sister
chromatid separation even when Securin degradation occurs
(Hagting et al., 2002
). Thus,
it is likely that Separase activity is inhibited by both phosphorylation and
binding to Securin in vertebrate cells
(Hagting et al., 2002
;
Stemmann et al., 2001
).
However, it is not known whether Cdk1-CycA or Cdk1-CycB in Drosophila
can modify Separase activity in a similar way.
In Drosophila, a novel protein Thr (encoded by three
rows) is involved in regulating Separase (Sse) activity and Cohesin
cleavage. thr mutant embryos have reduced rows of epidermal
denticles, presumably caused by cell division defects
(Nüsslein-Volhard et al.,
1984). Thr protein forms a trimeric protein complex with Sse and
Securin (Pim, encoded by pimples)
(Leismann et al., 2000
;
Herzig et al., 2002
). This
three-protein complex is present but inactive during interphase and metaphase
(Leismann et al., 2000
;
Jäger et al., 2001
). Sse
activation occurs when Pim is degraded shortly before anaphase begins. The
Thr-Sse heterodimer is now active and presumably cleaves Drad21/Scc1, a
subunit of the Cohesin complex in Drosophila
(Vass et al., 2003
;
Warren et al., 2000
). After
chromatids separate, Thr is cleaved by Sse, which presumably inhibits Sse
activity (Herzig et al.,
2002
).
The investigation of the Pim-Thr-Sse complex have provided us with a
molecular description of the changes in this complex necessary to induce
chromatid movements in anaphase (Herzig et
al., 2002; Jäger et al.,
2001
; Jäger et al.,
2004
; Leismann et al.,
2000
). However, it is not known whether Cdk1-CycB interacts with
Pim-Thr-Sse in a dose-dependent manner to act as a timer in regulating the
onset of anaphase. Based on the observation that increasing maternal Cdk1-CycB
activity causes abnormal nuclear distribution and morphology at cycle 14
interphase, we performed a loss-of-function genetic screen for modifiers of
Cdk1-CycB (Ji et al., 2002
).
Here, we report genetic interactions between Cdk1-CycB and components of the
Pim-Thr-Sse complex, and document the dosage effects of these factors in
anaphase initiation and early embryonic development in Drosophila.
Both CycB and Cdk1 are overexpressed in many human malignant cancers, such as
colorectal, breast, liver and lung cancers (for examples, see
Ito et al., 2000
;
Sarafan-Vasseur et al., 2002
;
Soria et al., 2000
).
Therefore, our results suggest that higher Cdk1-CycB activity in cancer cells
may contribute to generating aneuploidy by directly affecting Separase
activity and thereby the onset of anaphase.
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Materials and methods |
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Genetic crosses and the nomenclature of genotypes have been described
previously (Ji et al., 2002).
Briefly, `mutation/six cycB' refers to embryos from females that were
heterozygous for a specific mutant gene or deficiency, and also carried six
copies of the cycB+ gene. We used the description
`thr1/pim1/six cycB' for flies with the
genotype +; thr1, pim+,
cycB+/thr+, pim1, cycB+;
2P[w+ cycB+]; and
`thr1/sse13m/six cycB' for the genotype +;
thr1, cycB+/thr+, cycB+,
2P[w+ cycB+]; sse13m/sse+,
2P[w+ cycB+]. For time-lapse two-photon microscopy,
embryos with the maternal genotype +; cycB+/cycB+;
histone-GFP/+ were referred to as `two cycB' embryos or
controls; +; cycB+/cycB+, 2P[w+
cycB+]; histone-GFP/+ as `four cycB' embryos; +;
thr1, cycB+/thr+, cycB+,
2P[w+ cycB+]; histone-GFP/+ as `thr/four
cycB' embryos; +; pim1, cycB+/pim+,
cycB+, 2P[w+ cycB+]; histone-GFP/+ as
`pim/four cycB' embryos; and +;
cycB+/cycB+, 2P[w+ cycB+];
sse13m/sse+, histone-GFP as `sse/four
cycB' embryos. The phenotypes of the four cycB and six
cycB embryos are not different at cycle 10 and cycle 14
(Table 1). Compared with
two cycB (wild type) embryos, we observed a similar increase of
Cdk1-CycB kinase activity in four cycB and six cycB embryos
(Ji et al., 2002
;
Stiffler et al., 1999
).
|
Synthesis and injection of double-stranded RNA of thr
We prepared dsRNA as described
(Kennerdell and Carthew,
1998). Both PCR strand fragments of the thr gene from
positions 888 to 1442 were used as templates for simultaneous in vitro
transcription using Ambion MEGAscript T7 kit. The complementary RNA products
were annealed during the transcription reactions to form dsRNAs. About 250-370
pL of 5 µM dsRNA dissolved in injection buffer (5 mM KCl, 0.1 mM
NaH2PO4, pH 7.5) was injected into the posterior part of
histone-GFP embryos, as well as the buffer-only injection control. We also
used RNAi to downregulate endogenous bicoid as a positive control and
obtained the bicoid phenotype as reported
(Kennerdell et al., 2002
).
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Results |
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To exclude possible non-lethal dominant maternal effects that were independent of increased Cdk1-CycB activity, we analyzed the enhancer alleles in a two cycB background (enhancer/two cycB). We found that thr heterozygous embryos are not different from wild-type embryos in their hatching rate (data not shown) or in their blastoderm phenotype at cycle 14 (Table 1). Similar observations were made with the three enhancing deficiencies identified in cytogenetic region 54-55 (Table 1).
In addition to thr, we tested four other maternal effect mutations
in the cytogenetic map region 54/55: early (eay, at
cytogenetic region 54F6-55B12), subito (sub, at 54E7),
staufen (stau, at 55B5-7) and halted (hal,
at 55A1) (Schüpbach and Wieschaus,
1989). None of them enhanced the six cycB blastoderm
phenotype at cycle 14 (data not shown).
Identification of the gene pimples and the gene separase as suppressors of the six cycB phenotype
Since thr is an enhancer of the six cycB phenotype, we
tested whether Cdk1-CycB genetically interacts with other proteins related to
Thr, such as Pim and Sse (Herzig et al.,
2002; Jäger et al.,
2001
). We tested four alleles of pim and found that three
of them (pim1, pim3 and
pim4) suppressed the six cycB phenotype at cycle
14 (Table 1). With the
exception of pim2, reducing one copy of the other three
pim alleles normalized the cycle 14 blastoderm phenotype
(Table 1).
The pim2 allele was generated by X-ray and has a small
inframe deletion that leads to a protein missing amino acids 110-114
(Jäger et al., 2001). It
was proposed that the small deletion in Pim specifically abolishes the binding
to Sse but does not result in destabilization or complete misfolding of the
mutant Pim2 protein (Jäger
et al., 2001
). The other three pim alleles
(pim1, pim3 and pim4) are
nucleotide substitutions that lead to defective mRNA splicing
(pim1) or premature translation stop
(pim3 and pim4)
(Stratmann and Lehner, 1996
).
Homozygous embryos of all four pim alleles show the same zygotic
phenotype: failure of sister chromatid separation in the centromeric region at
cycle 15 and cycle 16 (Leismann et al.,
2000
; Stratmann and Lehner,
1996
).
We investigated why the pim2 allele behaved differently in the presence of elevated CycB when compared with the other three amorphic pim alleles. We found that the hatching rates of both pim2/two cycB and pim2/six cycB embryos were significantly lower than two cycB or six cycB embryos, but the frequencies of normal cycle 14 blastoderm embryos were not different from six cycB embryos (Table 1). These results and further analyses with this allele at cycle 10 and cycle 14 (see below) suggest that the pim2 allele is semi-dominant (see Discussion).
Previously, we observed that Df(2L)J77 and the neomorphic allele
cdk1D57 (at cytogenetic map position 31D11), but not the
null allele of cdk1, suppressed the six cycB phenotype
(Fig. 1B), thus we concluded
that another suppressor gene was present within Df(2L)J77 whose
product genetically interacted with Cdk1-CycB
(Ji et al., 2002). The gene
pim maps to cytogenetic region 31D10 and is uncovered by
Df(2L)J77 (31C; 31E7), suggesting that Df(2L)J77 indeed
uncovers at least two suppressor genes
(Fig. 1B).
To further test the genetic interaction between Cdk1-CycB and proteins of
the Pim-Thr-Sse complex, we tested the separase (at 61E1) null allele
sse13m and the deficiency Df(3L)SseA
(Fig. 1C) (Jäger et al., 2001). The
allele sse13m has a deletion of four bases leading to a
frame shift and premature translation stop, thus the mutant Sse13m
protein lacks the invariant cysteine that is involved in catalysis
(Jäger et al., 2001
). As
shown in Table 1, both
Df(3L)SseA and sse13m have the same suppressive
effect on the six cycB phenotype at cycle 14, supporting the idea
that sse13m is an amorphic allele
(Jäger et al., 2001
).
Dose-dependent interaction between Cdk1-CycB and Pim-Thr-Sse
To genetically confirm that Cdk1-CycB interacts with the Pim-Thr-Sse
complex in regulating a common process, we combined the enhancer (thr
mutation) with suppressor mutations in double heterozygous combinations. We
generated mothers that had six copies of the cycB+ gene
and were heterozygous for both Thr and Pim (referred as
thr1/pim1/six cycB), or both Thr and Sse
(referred as thr1/sse13m/six cycB). We found
that compared with the thr/six cycB embryos, double heterozygous
embryos (thr1/pim1/six cycB and
thr1/sse13m/six cycB) were normalized at cycle
14 (Table 1) and had increased
hatching rates (data not shown). Thus, reducing either Pim or Sse partially
suppressed the enhancing effects caused by lower Thr in the six cycB
background, probably by restoring stoichiometry among the Pim-Thr-Sse
heterotrimer complex. Taken together, our genetic analyses demonstrate a
dosage-dependent interaction between Cdk1-CycB and the Pim-Thr-Sse
complex.
The level of Thr, Pim, Sse and CycB affects anaphase onset
In four cycB and six cycB embryos, we observed a higher
Cdk1-CycB activity and a delayed onset of anaphase
(Ji et al., 2002;
Ji et al., 2004
). Because
components of the Pim-Thr-Sse complex are involved in regulating sister
chromatid separation, we asked whether reducing the levels of each of these
proteins had any effect on the onset of anaphase. We analyzed the duration of
cell-cycle phases by using a two-photon laser-scanning microscope. Reducing
Thr in embryos with more maternal CycB (thr1/four cycB)
led to significantly longer prophase-metaphase duration
(Fig. 2). By contrast, reducing
either Pim or Sse in embryos with more maternal CycB significantly shortened
the prophase-metaphase time, normalizing the timing of anaphase onset.
Furthermore, varying the dosage of Thr, Pim or Sse in embryos that have higher
levels of CycB had no effect on interphase (data not shown) and
anaphase-telophase duration (Fig.
2), suggesting that the levels of these proteins specifically
define when anaphase begins.
|
To test whether Thr levels affected anaphase onset without increasing Cdk1-CycB activity, we injected dsRNA of thr into two cycB embryos labeled with histone-GFP. We found that compared with embryos injected only with buffer, embryos injected with thr dsRNA at cycle 6 have a significantly delayed onset of anaphase at cycle 12 and cycle 13 (26% and 41% longer prophase-metaphase, respectively), but not before cycle 12. This observation indicates that RNAi process takes about 60 minutes to downregulate endogenous thr mRNA, and that reducing Thr alone can postpone the onset of anaphase in two cycB embryos as well.
Cycle 10 phenotype of thr/six cycB and pim/six cycB embryos
The earliest mitoses in the Drosophila embryo have been well
studied previously. The first four cycles occur in the interior and slightly
towards the anterior end of the embryo. Between cycles 4-7, nuclei move along
the anteroposterior axis of the embryo in a process known as `axial expansion'
(Baker et al., 1993). Later,
during cycles 8-10, nuclei are pushed to the cortex by the expanding
microtubule network, a process known as `cortical migration'
(Baker et al., 1993
). Because
nuclei migrate to the cortex almost simultaneously in two cycB
embryos (Foe and Alberts,
1983
), embryos fixed at cycle 10 have somatic nuclei evenly
distributed at the cortex in 97% of the embryos
(Fig. 3A,
Table 2).
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|
To test whether Thr, Pim or Sse affects nuclear cortical migration, we analyzed the cycle 10 phenotype in fixed pim/six cycB and sse/six cycB embryos. We observed that reducing Pim with the amorphic pim alleles pim1, pim3 and pim4 in the six cycB genetic background suppressed the six cycB phenotype at cycle 10 and cycle 14; the pim2 allele had no suppressing effect (Tables 1, 2). Reducing Sse with either the amorphic allele sse13m or the deficiency Df(3L)SseA normalized the cycle 10 phenotype (Fig. 3C, Table 2). Therefore, both pim and Sse alleles suppressed the six cycB phenotype at both cycle 10 and cycle 14.
The cycle 10 phenotype in thr/six cycB embryos (Fig. 3D) was noticeably worse than in six cycB embryos (Fig. 3B, Table 2). In addition to the defects of nuclear cortical migration (Table 2), we frequently observed asynchronous mitoses (Fig. 3D'), macro/micro-nuclei and chromosomal bridges before cycle 10 (Fig. 3D'), suggesting that the enhancing effect of lower maternal Thr occurs prior to cycle 10.
Effects of lower levels of Pim or Sse on nuclear movement
The observations made with fixed materials led us to focus on when and how
cortical migration is normalized in pim/six cycB and Sse/six
cycB embryos. For this, we analyzed in vivo timelapse recordings of
embryos labeled with histone-GFP and focused on cycles 9 and 10. We defined
velocity and pattern of nuclear migration in two cycB, four cycB, pim/four
cycB and sse/four cycB embryos. In all four genotypes, nuclear
cortical migration was initiated at telophase of cycle 9 and ends 1.5 min
into early interphase of cycle 10. However, the velocity and pattern of
nuclear migration were different. During cortical migration, nuclei moved
slower in four cycB embryos (6.2±3.3 µm/min, 28
measurements from 8 embryos) than in wild-type embryos (8.8±3.2
µm/min, 26 measurements from 9 embryos), confirming our previous
observation using DIC microscopy (Stiffler
et al., 1999
). Compared with both the four cycB and
two cycB embryos, significantly faster nuclear movements were
observed in both pim1/four cycB embryos (11.1±3.2
µm/min, 30 measurements from 9 embryos) and sse13m/four
cycB embryos (12.5±3.6 µm/min, 42 measurements from 10
embryos).
We also observed that the paths of nuclear movement were different. In
two cycB embryos, nuclei migrated perpendicular to the cortex in
straight paths (Fig. 4A)
(Foe and Alberts, 1983). In
four cycB embryos, nuclei moved to the cortex like `drunken
soldiers': they moved in meandering lines towards the cortex
(Fig. 4B). By contrast, we
observed that nuclei migrated in curved paths and at an angle to the cortex in
both pim1/four cycB
(Fig. 4C) and
sse13m/four cycB (Fig.
4D) embryos. The same novel nuclear migration pattern was also
observed with pim3/four cycB and Df(3L)SseA/four
cycB embryos (data not shown). These observations indicate that faster
nuclear movement and a novel cortical migration pattern contribute to the
normal cycle 10 phenotype in both pim1/four cycB and
sse13m/four cycB embryos.
|
To test whether normalized microtubule function underlies the normalized cycle 10 phenotype in pim/six cycB and Sse/six cycB embryos, we analyzed microtubule morphology during early interphase of cycle 9. At this stage, we observed an extensive microtubule network within two cycB embryos (Fig. 5A). Increasing maternal CycB (six cycB embryos) reduced the microtubule network (Fig. 5B, also compare Fig. 5A' with 5B'). However, in pim1/six cycB and sse13m/six cycB embryos the microtubule morphology was restored (Fig. 5C, compare Fig. 5C' with Fig. 5B'). Similar differences in microtubule configurations were seen at cycles 7 and 8. Thus the normalized microtubule network could account for the faster nuclear movement during cortical migration.
|
Overall, we conclude that reducing either pim or Sse suppressed the six/cycB phenotype at both cycle 10 and cycle 14 by normalizing the onset of anaphase, restoring microtubule morphology in interphase and by inducing faster nuclear movement during cortical migration. By contrast, reducing thr enhances the six/cycB phenotype at both cycle 10 and 14 by further delaying the initiation of anaphase.
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Discussion |
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Indeed, further reducing maternal thr by one copy in embryos with higher Cdk1-CycB activity led to an even greater delay of anaphase onset (Fig. 2), resulting in more frequent and severe nuclear defects. We propose that a greater delay of anaphase onset is the result of fewer Thr-Sse dimers, thereby causing an increase in the time taken to cleave Cohesin. This idea is based on the observation that the majority of the thr/six cycB embryos had many macro/micro-nuclei, and had disrupted synchrony and chromosomal bridges both before and after cycle 10 (Fig. 3D'), which indicates that these defects result from abnormal chromatid separation. This scenario would explain why thr becomes haplo-insufficient in the presence of higher Cdk1-CycB activity (six cycB background, Table 1 and Fig. 2), but not in the wild-type (two cycB) background (Table 1).
Suppression of the six cycB phenotype by reducing pim levels
Sse and Cdk1-CycB activities have opposite effects on the onset of
anaphase: higher Sse activity leads to earlier anaphase onset whereas higher
Cdk1-CycB delays it. If this is so, reducing Pim, the inhibitor of Sse
(Leismann et al., 2000;
Jäger et al., 2001
),
would lead to slightly earlier activation of Sse than in six cycB
embryos, and thus correct the timing of anaphase initiation
(Fig. 2).
Alternatively, both Pim and CycB need to be degraded to initiate anaphase
(Peters, 2002;
Pickart, 2001
), thus reducing
pim in a six cycB genetic background might suppress the
six cycB phenotype if Pim and CycB compete for destruction by the
ubiquitin/proteasome system. Both CycB and Securin contain a similar
N-terminal sequence motif, known as the `destruction box'
(Peters, 2002
). The idea that
CycB and Securin compete for degradation is supported by the observation that
the N-terminal fragments of CycB and Securin compete with the full-length
protein for the destruction machinery in yeast
(Funabiki et al., 1996
).
According to this scenario, Pim degradation would be delayed in six
cycB embryos because more CycB needs to be degraded. Reducing Pim, as in
pim/six cycB embryos would relieve the inhibition of Pim on Sse, thus
suppressing the six cycB phenotype.
Both scenarios could explain why reducing Pim in embryos with higher Cdk1-CycB normalizes anaphase onset (Fig. 2). However, additional assumptions are necessary for the second hypothesis. For example, it is not known whether Pim degradation is affected by its binding with Thr and/or Sse, or by levels of Thr and/or Sse. Interestingly, there are indications that degradation of Securin may be affected by its binding with Separase in human cells (H. Zou, personal communication).
How do we explain the dominant effect of the pim2 allele
(Table 1 and
Table 2)? Since Pim2
can still bind to Thr even though it does not bind to Sse
(Jäger et al., 2001),
Pim2 may inhibit Thr by titrating it into an ineffective
Pim2-Thr complex that cannot recruit Sse
(Jäger et al., 2001
).
Accordingly, Pim2 would inactivate both Pim and Thr, thus it might
have a phenotype similar to that seen with other pim alleles when
they were combined with a thr mutation
(Table 1).
Suppression of the six cycB phenotype by reducing Sse levels
Lehner and colleagues have proposed that after the active Thr-Sse
heterodimer cleaves the Cohesin subunit, Thr itself is cleaved by Sse, which
presumably inactivates Sse at the end of anaphase
(Herzig et al., 2002). Because
of this negative feedback, Thr-Sse heterodimer activity is likely to be
limited to a short time after anaphase begins. It is not known whether Thr is
cleaved by the same Sse molecule that it binds or by another Thr-Sse dimer. A
similar negative-feedback mechanism in Separase regulation was found in
Xenopus and human cells, where Separase undergoes auto-cleavage.
However, the cleaved fragments are still active and remain associated, thus
the function of the auto-cleavage in regulating anaphase onset is not resolved
(Waizenegger et al., 2002
;
Zou et al., 2002
).
If our hypothesis that levels of Thr and Pim affect the onset of anaphase
by modifying Sse activity is correct, we expect Sse to be an enhancer.
However, both the amorphic allele sse13m and the
deficiency Df(3L)SseA are suppressors
(Table 1). This presents a
challenge. We propose two scenarios to explain this unexpected result. First,
if cleavage of Thr by Thr-Sse inactivates Sse
(Herzig et al., 2002), we
speculate that both Thr-Sse heterodimers and Sse monomers have protease
activity to cleave Thr bound to Sse. If so, compared with six cycB
embryos, reducing Sse in sse/six cycB embryos would reduce the
concentration of Thr-Sse, and thus the cleaveage of Thr and the inactivation
of Sse would take longer. The delay in Sse inactivation could have similar
effects as increasing Thr-Sse levels (i.e. Separase activity) does, helping to
overcome the inhibitory effect of a higher Cdk1-CycB activity on sister
chromatid separation. Our explanation of the effect of Separase activity on
the onset of anaphase is consistent with observations that depletion of a
Cohesin subunit Drad21/Scc1 in Drosophila cultured cells and embryos
by RNAi leads to premature chromatid separation and abnormal spindle
morphology (Vass et al.,
2003
), suggesting that the onset of anaphase is defined by the
cleavage efficiency of Drad21/Scc1.
Alternatively, the suppressive effect of Sse could be caused by
Sse possessing functions other than the ability to cleave the Cohesin subunit.
This possibility is supported by the following observations in budding yeast.
(1) Besides cleaving Securin, Separase can also cleave the kinetochore and the
spindle associated protein Slk19 at the onset of anaphase. Cleaved Slk19
localizes to the spindle midzone and is required to maintain spindle stability
in anaphase, preventing elongated spindle from breaking down prematurely
(Sullivan et al., 2001). (2)
Separase may also promote phosphorylation of Net1, the inhibitor of
phosphatase Cdc14, thereby causing the release of Cdc14 from the nucleolus, a
key step in mitotic exit (Sullivan and
Uhlmann, 2003
). It is still an open question whether Separase has
additional substrates (Pellman and
Christman, 2001
). Although it is not known whether similar
mechanisms also occur in Drosophila, it is possible that the
suppressive effect we observed by reducing Sse may be caused by affecting the
exit of mitosis through other Sse targets.
Effects of Sse and Pim on cytoskeleton stability and nuclear migration pattern
We observed that reducing either Pim or Sse restores the microtubule
morphology in interphase, but not in metaphase
(Fig. 5,
Table 2). In these embryos,
nuclei show a faster and novel pattern in cortical migration, but this still
leads to a normal nuclear distribution at cycle 10
(Fig. 4 and
Table 2). Although it is not
clear whether levels of Separase, Securin or APC/C modulate microtubule
stability, it has been observed that Separase, Securin and components of the
APC/C complex co-localize with spindle microtubules. For examples, in budding
yeast, phosphorylated Pds1 (Securin) binds with Esp1 (Separase) and the
complex is targeted to the spindle apparatus
(Agarwal and Cohen-Fix, 2002).
In Drosophila, both Sse and Pim co-localize with spindle microtubules
(Herzig et al., 2002
).
Furthermore, components of APC/C, such as CDC16 and CDC27,
co-immunoprecipitate with microtubules in Drosophila embryos
(Huang and Raff, 1999
).
Finally, Securin co-localizes with mitotic spindles in HeLa cells
(Hagting et al., 2002
).
Based on these observations, several hypotheses may explain the dosage effects of Pim and Sse on microtubule morphology at different cell-cycle phases. The most compelling one is that if CycB and Pim compete for poly-ubiquitination by APC/C on microtubules, reducing Pim may lead to faster CycB degradation, resulting in the restoration of microtubule morphology in interphase compared with in six cycB embryos. By contrast, because there is no degradation of either Pim or CycB in metaphase, the effect of degradation competition between Pim and CycB is absent, thus explaining why astral microtubule morphology is not restored in pim/six cycB embryos. As mentioned earlier, if Sse levels affect Pim degradation, reducing either Pim or Sse could have similar effects on CycB degradation. Thus we speculate that the interplay between the different kinetics of Cdk1-CycB activity and Separase activity over the cell cycle may contribute to the different effects of Sse/Pim dosage on microtubule stability.
To understand why reducing either Pim or Sse led to faster nuclear
movements and a different nuclear migration pattern, the mechanics involved in
the process of cortical migration need to be considered. Two major
cytoskeletal networks are reorganized during this process: microtubules are
stabilized in late telophase and early interphase, which pushes nuclei to the
cortex (Baker et al., 1993);
and the microfilament network is denser in the cortex than in the interior
(von Dassow and Schubiger,
1994
). Thus, the velocity and pattern of nuclear movement will be
defined both by the pushing force generated by microtubules and by the
resistance generated by the microfilament matrix.
In embryos with more Cdk1-CycB, microtubules become less stable
(Ji et al., 2002). This may
generate a weaker force to push nuclei to the cortex, resulting in the slower
and less direct nuclear movement that we observed. When we further reduce
either Pim or Sse, microtubule morphology is restored in early interphase
(Fig. 5). This may contribute
to the observation of faster nuclear cortical migration than in the six
cycB embryos. However, why do nuclei in Sse/four cycB or
pim/four cycB embryos move even faster than in two cycB
embryos? This observation is puzzling to us. The simple explanation would be
that the microtubule network is more robust in Sse/four cycB or
pim/four cycB embryos than in two cycB embryos. Previously,
we suggested a model in which microtubule and microfilament networks
antagonistically interact with each other, and suggested that Cdk1-CycB
activity negatively affects this interaction in early Drosophila
embryos (Ji et al., 2002
).
Accordingly, a more robust microtubule network would result in a weaker
microfilament network, presumably reducing the resistance for nuclear movement
because of the less dense microfilament matrix in the extended cortex. The
novel pathway of nuclear movement may reflect the disrupted balance between
microtubule and microfilament networks because of the over-corrected
microtubules in interphase. Consistent with this scenario, we also observed
dramatic global cytoplasmic movements in pim1/four cycB
and sse13m/four cycB embryos
(Fig. 5C) during the nuclear
cortical migration. Thus, an increased microtubule network and the less dense
microfilament matrix might account for accelerated nuclear movements.
Our genetic screen has identified modifiers of the six cycB
phenotype (Ji et al., 2002).
The studies have documented an interplay between Cdk1-CycB, microtubules and
microfilaments. Here, we report three new modifiers that affect the six
cycB phenotype. One of them, thr, is an enhancer. Interestingly,
when the enhancer thr is combined with the suppressor quail
(which encodes a villin-like protein), we find that the six cycB
phenotype is restored (J.C. and G.S., unpublished). This indicates that, at
least at the genetic level, the amount of Cdk1-CycB modulates many parameters
of gene products regulating nuclear behavior and cytoskeletal stability.
Progress in developmental genetics requires the functional analyses of genes, which is best addressed by the description of pleiotropic phenotypes. We observed that increasing Cdk1-CycB in combination with decreasing Pim or Sse almost completely corrects the onset of anaphase and normalizes nuclear distribution at cycle 10. What is not expected and could only be observed by combining live analysis with data from fixed embryos is that microtubule configuration is corrected to wild type in interphase but not metaphase, and that a novel nuclear cortical migration pattern appears. Because this phenotype is only observed in combination with excessive Cdk1-CycB, we suggest using the term `heterosis combined with epistasis' to describe the microtubule phenotype. Such a mechanism may have a selective advantage and therefore might occur in other slightly deleterious genetic combinations.
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ACKNOWLEDGMENTS |
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Footnotes |
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