To assess Sse function, mutant alleles were generated. Starting with a P-element insertion within a neighboring gene (CG17334), a small deficiency [Df(3L)SseA] was isolated by male recombination. A molecular breakpoint analysis indicates that this deficiency deletes Sse and parts of the neighboring genes, CG17334 and sif. Interestingly, this deficiency fails to complement the recessive lethal mutation l(3)13m-281. This mutation has been shown to affect mitotically proliferating cells in a way that might be expected for a mutation in Sse (Gatti, 1989). Sequence analysis of the Sse region isolated from the l(3)13m-281 chromosome has revealed a deletion of four bases between the positions encoding the invariant histidine and cysteine residues, resulting in a frame shift followed by a premature translational stop. The product encoded by l(3)13m-281, therefore, is expected to lack part of the conserved C-terminal separase domain, including the invariant cysteine residue believed to be involved in catalysis. Thus, l(3)13m-281 is presumably a null allele of Sse and will be designated as Sse13m in the following (Jäger, 2001).
Previous phenotypic analyses (Gatti, 1989) have indicated that Sse13m homozygotes die at the larval-pupal boundary. In Sse13m larvae at third instar wandering stage, imaginal discs were found to be abnormally small and the mitotic index in the brains was strongly reduced. Moreover, the few mitotic figures observed in larval brain squashes were reported to contain endoreduplicated chromosomes with bundles of two, four, or eight times the normal number of sister chromatid arms. Phenotypic analyses with Sse13m/Df(3L)SseA and Df(3L)SseA/Df(3L)SseA larvae confirmed these findings. These larvae showed identical phenotypes to Sse13m homozygotes (Jäger, 2001).
No mitotic abnormalities could be detected in embryos of Sse mutants, suggesting that the maternal Sse contribution is sufficient to allow normal cell divisions throughout embryogenesis. However, in brains of Sse13m/Df(3L)SseA early second instar larvae, the number of phosphorylated histone H3 (PH3)-positive cells was reduced to ~40% of the number of PH3-positive cells in sibling brains. PH3 is an excellent marker for cells during the early mitotic stages until anaphase. Whereas mutants and siblings show very similar fractions of prophase and metaphase figures in these PH3-positive cells, anaphase figures were almost completely absent from mutant brains. It is concluded, therefore, that the maternal Sse contribution is no longer sufficient to support normal anaphase when mitotic proliferation of postembryonic brain neuroblasts resumes during the second instar stage, although entry into mitosis still occurs. If Sse mutants specifically fail in separating sister chromatids, a rapid accumulation of chromosomes with twice the number of normal arms (diplo-chromosomes), as well as more extensively endoreduplicated chromosomes, is expected. Analysis of squash preparations of second instar larval brains fully confirmed this expectation. A total of 95% of the mitotic cells contained either diplo-chromosomes or more extensively endoreduplicated chromosomes, whereas none of the mitotic cells of siblings contained endoreduplicated chromosomes. In addition to the presence of endoreduplicated chromosomes, mitotic figures in mutants frequently contained aneuploid numbers of chromosomes. However, polyploid figures with normal chromosomes were never observed, indicating that Sse mutants have no primary defect in cytokinesis (Jäger, 2001).
In third instar wandering stage larvae, Sse mutant brains contained very few PH3-positive cells. The PH3-positive cells were abnormally large and had very high levels of DNA. No normal anaphase and telophase figures could be detected in mutants, and squashes revealed highly abnormal and severely endoreduplicated chromosomes, as described previously (Gatti, 1989). The progressive depletion of mitotic cells with increasing age suggests that most of the mitotically proliferating neuroblasts eventually die in Sse mutants, presumably as a consequence of hyper- and aneuploidy (Jäger, 2001).
To show that the endoreduplicated unseparated chromosomes in Sse13m larvae result from the loss of Sse+ function and not from linked second-site mutations, rescue experiments were performed. One copy of the gSse transgene with a 10-kb genomic DNA fragment encompassing only the Sse gene rescued the phenotype of Sse13m homozygotes to full viability and fertility. Moreover, the cytological defects of Sse13m mutants could be prevented by a combination of da-GAL4 and UAS-HA-Sse. All these findings show that Sse is an essential gene required for sister chromatid separation during mitosis. Moreover, the Sse gene product is likely to function as a cysteine protease, since it was found that da-GAL4 mediated expression of UAS-HA-SseC497S, in which the codon for the putative catalytic cysteine residue was mutated into a serine codon was unable to rescue Sse mutants (Jäger, 2001).
Cdk1-CycB plays a key role in regulating many aspects of cell-cycle events, such as cytoskeletal dynamics and chromosome behavior during mitosis. To investigate how Cdk1-CycB controls the coordination of these events, a dosage-sensitive genetic screen was performed that was based on the observations that increased maternal CycB (four extra gene copies) leads to higher Cdk1-CycB activity in early Drosophila embryos, delays anaphase onset, and generates a sensitized non-lethal phenotype at the blastoderm stage (defined as six cycB phenotype). Mutations in the gene three rows (thr) enhance, while mutations in pimples (pim, encoding Drosophila Securin) or separase (Sse) suppress, the sensitized phenotype. In Drosophila, both Pim and Thr are known to regulate Sse activity, and activated Sse cleaves a Cohesin subunit to initiate anaphase. Compared with the six cycB embryos, reducing Thr in embryos with more CycB further delays the initiation of anaphase, whereas reducing either Pim or Sse has the opposite effect. Furthermore, nuclei move slower during cortical migration in embryos with higher Cdk1-CycB activity, whereas reducing either Pim or Sse suppresses this phenotype by causing a novel nuclear migration pattern. Therefore, the genetic screen has identified all three components of the complex that regulates sister chromatid separation, and these observations indicate that interactions between Cdk1-CycB and the Pim-Thr-Sse complex are dosage sensitive (Ji, 2005).
Increasing maternal Cdk1-CycB activity leads to defective mitoses, indicating a disruption in the coordination between the nuclear and cytoplasmic cycle (Ji, 2002). Nevertheless, these embryos develop to adults. Also, higher Cdk1-CycB activity causes shorter microtubules, and longer metaphase but shorter anaphase (Ji, 2002). These observations suggest that a slight delay of anaphase initiation may result in slightly disrupted coordination between nuclear and cytoplasmic events, such as chromatid separation and microtubule dynamics. Thus, in the six cycB genetic background, mutations that worsened the defect in coordination were identified as enhancers, whereas mutations that rectified the defects were identified as suppressors (Ji, 2002) (Ji, 2005).
Indeed, further reducing maternal thr by one copy in embryos with higher Cdk1-CycB activity leads to an even greater delay of anaphase onset, resulting in more frequent and severe nuclear defects. It is proposed 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, 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, but not in the wild-type (two cycB) background (Ji, 2005).
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, would lead to slightly earlier activation of Sse than in six cycB embryos, and thus correct the timing of anaphase initiation (Ji, 2005).
Alternatively, both Pim and CycB need to be degraded to initiate anaphase -- 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'. 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. 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 (Ji, 2005).
Both scenarios could explain why reducing Pim in embryos with higher Cdk1-CycB normalizes anaphase onset. 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 (Ji, 2005).
How can the dominant effect of the pim2 allele be explained? Since Pim2 can still bind to Thr even though it does not bind to Sse, Pim2 may inhibit Thr by titrating it into an ineffective Pim2-Thr complex that cannot recruit Sse. 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 (Ji, 2005).
It has been 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. 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 (Ji, 2005).
If the hypothesis that levels of Thr and Pim affect the onset of anaphase by modifying Sse activity is correct, Sse is expected to be an enhancer. However, both the amorphic allele sse13m and the deficiency Df(3L)SseA are suppressors. This presents a challenge. Two scenarios are proposed to explain this unexpected result. If cleavage of Thr by Thr-Sse inactivates Sse, it is speculated 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 does increasing Thr-Sse levels (i.e., Separase activity), helping to overcome the inhibitory effect of a higher Cdk1-CycB activity on sister chromatid separation. The 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, suggesting that the onset of anaphase is defined by the cleavage efficiency of Drad21/Scc1 (Ji, 2005).
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. (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. It is still an open question whether Separase has additional substrates. Although it is not known whether similar mechanisms also occur in Drosophila, it is possible that the suppressive effect observed by reducing Sse may be caused by affecting the exit of mitosis through other Sse targets (Ji, 2005).
Reducing either Pim or Sse restores the microtubule morphology in interphase, but not in metaphase. 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. 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. In Drosophila, both Sse and Pim co-localize with spindle microtubules. Furthermore, components of APC/C, such as CDC16 and CDC27, co-immunoprecipitate with microtubules in Drosophila embryos. Finally, Securin co-localizes with mitotic spindles in HeLa cells (Ji, 2005).
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 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. If Sse levels affect Pim degradation, reducing either Pim or Sse could have similar effects on CycB degradation. Thus it is speculated 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 (Ji, 2005).
To understand why reducing either Pim or Sse leads 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; this pushes nuclei to the cortex, where the microfilament network is denser than in the interior. 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 (Ji, 2005).
In embryos with more Cdk1-CycB, microtubules become less stable (Ji, 2002). This may generate a weaker force to push nuclei to the cortex, resulting in the slower and less direct nuclear movement that was observed. When either Pim or Sse is further reduced, microtubule morphology is restored in early interphase. 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. 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 suggested was a model in which microtubule and microfilament networks antagonistically interact with each other, and in which Cdk1-CycB activity negatively affects this interaction in early Drosophila embryos (Ji, 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, dramatic global cytoplasmic movements are also observed in pim1/four cycB and sse13m/four cycB embryos during the nuclear cortical migration. Thus, an increased microtubule network and the less dense microfilament matrix might account for accelerated nuclear movements (Ji, 2005).
This genetic screen has identified modifiers of the six cycB phenotype (Ji, 2002). The studies have documented an interplay between Cdk1-CycB, microtubules and microfilaments. This study reports 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), it is found that the six cycB phenotype is restored. 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 (Ji, 2005).
Progress in developmental genetics requires the functional analyses of genes, which is best addressed by the description of pleiotropic phenotypes. 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, the term 'heterosis combined with epistasis' is suggested to describe the microtubule phenotype. Such a mechanism may have a selective advantage and therefore might occur in other slightly deleterious genetic combinations (Ji, 2005).
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date revised: 25 October 2014
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