Neither Pim nor Thr share significant sequence similarities with known proteins, and their biochemical function is not known. However, the indistinguishable phenotypes resulting from null mutations in pim and thr suggest that the corresponding gene products might function in a complex. Therefore, Pim-Thr complex formation was analyzed by coimmunoprecipitation. Extracts were prepared from embryos carrying transgenes (gpim-myc or gthr-myc), allowing expression of either Pim protein with a carboxy-terminal extension of six myc epitope copies or Thr protein with a carboxy-terminal extension of 10 myc epitope copies under the control of the corresponding genomic promoters. These myc-tagged proteins are functional because the transgenes can rescue pim and thr mutants, respectively. Anti-myc immunoprecipitates of Pim-myc were found to contain Thr. Conversely, immunoprecipitates of Thr-myc contained Pim. Coimmunoprecipitation experiments also indicated that the Pim-Thr complex does not contain multiple copies of Pim and Thr. In case of complexes with multiple copies, Pim-myc and Thr-myc immunoprecipitates would be expected to contain wild-type Pim and Thr, respectively. However, the products expressed from the endogenous loci were not coimmunoprecipitated by the myc-tagged transgene products (Leismann, 2000).
Pim-myc is cleared from mitotic cells after the metaphase-anaphase transition, similar to Cyclin B (Stratmann, 1996). The mitotic degradation of Cyclin B and other mitotic regulators is dependent on the presence of a destruction box motif in the amino-terminal region. Drosophila Cyclin B lacking this destruction box cannot be degraded during mitosis and blocks exit from mitosis. Although Pim does not have a motif that fits the RX(A or V or L)LGXXXN consensus sequence of mitotic destruction boxes, it contains the related sequence KKPLGNLDN. To determine whether this sequence variant can function as a destruction box, a mutant Cyclin B protein was expressed in Drosophila embryos that had this Pim motif instead of the Cyclin B destruction box. The Pim motif confers mitotic instability indistinguishable from wild-type Cyclin B and does not result in a mitotic arrest. When the Pim motif is mutated from KKPLGNLDN to AKPAGNLDA (dba), it is no longer able to functionally replace the destruction box in Cyclin B (Leismann, 2000).
To determine whether the KKPLGNLDN sequence is required for Pim degradation during mitosis, the dba mutation was introduced into a pim transgene (UAS-pimdba-myc). The mutant Pimdba-myc protein product was found to be stable during mitosis, while wild-type Pim-myc expressed from an analogous transgene (UAS-pim-myc) is degraded normally (Leismann, 2000).
Sister-chromatid separation in mitosis requires proteolytic cleavage of a cohesin subunit. Separase, the corresponding protease, is activated at the metaphase-to-anaphase transition. Activation involves proteolysis of an inhibitory subunit, securin, following ubiquitination mediated by the anaphase-promoting complex/cyclosome. In Drosophila, the securin Pimples (Pim) associates not only with Separase (Sse), but also with an additional protein, Three rows (Thr). Thr is cleaved after the metaphase-to-anaphase transition. Thr cleavage occurs only in functional SSE complexes and in a region that matches the separase cleavage-site consensus. Mutations in this region abolish mitotic Thr cleavage. These results indicate that Thr is cleaved by Sse. Expression of noncleavable Thr variants results in cold-sensitive maternal-effect lethality. This lethality can be suppressed by a reduction of catalytically active Sse levels, indicating that Thr cleavage inactivates Sse complexes. Thr cleavage is particularly important during the process of cellularization, which follows completion of the last syncytial mitosis of early embryogenesis, suggesting that Drosophila separase has other targets in addition to cohesin subunits (Herzig, 2002).
Immunolabeling has revealed that Thr is partially degraded after the metaphase-to-anaphase transition, similar to Pim. However, the mitotic degradation of Pim and Thr is mechanistically and functionally distinct. Mitotic degradation of Pim is dependent on the presence of a destruction box (D-Box) and on Fizzy-APC/C, which promotes ubiquitination and subsequent degradation by the proteasome. This Pim degradation presumably leads to activation of Sse. In contrast, Thr does not seem to contain a functional D-box, and mitotic degradation of Thr is dependent on Sse. The initial Thr cleavage event is followed by degradation of the C-terminal cleavage product. Furthermore, rather than activating Sse as in the case of Pim degradation, Thr cleavage contributes to inactivation of Sse (Herzig, 2002).
According to this proposal, degradation of Pim should precede Thr cleavage, since these two events would define a window of Sse activity. Thr cleavage should not occur too fast after Pim degradation so that Sse can cleave its other targets. Thr cleavage therefore might be regulated (for instance, by Scc1 cleavage fragments) or might not lead to Sse inactivation immediately. Sse inactivation might occur only once Thr cleavage fragments have been removed. Alternatively, Sse might cleave its substrates with different kinetics. Fast and efficient Scc1 cleavage may be followed by less efficient and slower Thr cleavage (Herzig, 2002).
It is emphasized that there is no direct evidence for this proposal from biochemical separase activity assays. The assay developed for human separase in the Xenopus extract system does not work for Drosophila Sse complexes for unknown reasons. Perhaps activation of Drosophila Sse complexes is only possible in a particular cellular context, for instance, on the mitotic spindle or at the kinetochore. Consistent with this proposal, only a fraction of Pim and Thr is degraded during mitosis in Drosophila embryos, and a slight enrichment of Pim and Thr on mitotic spindles, similar to securin and separase in yeast, can be visualized with appropriate fixation procedures in the syncytial blastoderm (Herzig, 2002).
Even without biochemical evidence, the data strongly support the notion that Thr is cleaved by Sse. Cleavage occurs at a conserved separase-cleavage consensus sequence. Substitution of a single arginine by an aspartate within this region abolishes cleavage, as previously observed for cleavage of yeast and human Scc1 by separase. Furthermore, mitotic Thr cleavage requires functional Sse complexes, since Thr is neither cleaved in pim mutants, nor in Sse complexes containing nonfunctional Thr mutants, nor in cells arrested in the mitotic checkpoint, when Sse is inactive (Herzig, 2002).
To investigate whether the putative Drosophila securin Pim can bind to Sse, the yeast two-hybrid system was used. A strong interaction between Pim and Sse was seen. Interestingly, a mutant Pim protein with a small internal deletion (amino acids 110-114) failed to bind to Sse. This deletion was identified in pim2, a Drosophila allele that results in an amorphic phenotype (Stratmann, 1996). Whereas Pim2 fails to interact with Sse, it binds to a Thr fragment (amino acids 1-933) just like wild-type Pim. The deletion of amino acids 110-114 therefore abolishes specifically the binding to Sse and does not result in destabilization or complete misfolding of the mutant Pim2 protein. It is concluded that the interaction of Pim and Sse is likely to be functionally significant (Jäger, 2001).
The behavior of Pim2 suggested that different domains of Pim mediate the binding to Sse and Thr. To evaluate this notion, Pim fragments were analyzed in additional two-hybrid experiments. Like other securin proteins, Pim is composed of a basic N-terminal and an acidic C-terminal domain. The N-terminal domain interacts with Sse, whereas an interaction with Thr 1-933 is barely detectable. Conversely, the C-terminal domain interacts with Thr 1-933, but not with Sse. These results strongly indicate that Sse and Thr bind to different Pim domains (Jäger, 2001).
Experiments with Sse fragments indicate that Pim binds to the N-terminal regions of Sse. The interaction of Pim with full-length Sse or with a Sse fragment comprising amino acids 1-467 appears to be stronger than with a shorter Sse fragment (amino acids 1-247). It is assumed that region 1-247 of Sse is sufficient for Pim binding, but the region 248-467 of Sse further strengthens this interaction. Clearly, the conserved C-terminal region of Sse is not required for the interaction with Pim (Jäger, 2001).
Experiments with Thr fragments indicate that Pim binds to the N-terminal region of Thr. Region 1-476 of Thr is sufficient for Pim binding. An interaction between Pim and the full-length Thr protein was not observed. The considerable size of full-length Thr 1-1379 might preclude expression of sufficiently high levels and/or entry into the nucleus (Jäger, 2001).
Finally, tests were performed for a direct interaction between Thr and Sse. The Thr fragment 1-933 interacts with full-length Sse, Sse 1-247, and Sse 1-467. These results therefore raise the possibility that Thr and Pim bind to the same region of Sse and thus might be competing in vivo. Moreover, the interactions between Thr and Sse appear to be weaker than the interactions between Pim and Sse, since in the former case, only one of the two reporter genes was activated. It was not possible to define the region in Thr that mediates Sse binding in more detail, because the shorter constructs Thr 1-476, Thr 208-933, and Thr 477-933 failed to bind to Sse (Jäger, 2001).
Taken together, the results of the two-hybrid experiments show that any two of these three proteins, Pim, Thr, and Sse can interact with one another, independent of the third (Jäger, 2001).
To confirm that Pim, Thr, and Sse interact in vivo, coimmunoprecipitation experiments were performed. Two antibodies against Sse were raised and affinity purified. In embryo extracts, these antibodies detect a prominent band at 75 kD, which comigrates with Sse translated in vitro. This band was also detected in immunoprecipitates that were isolated with an anti-myc antibody from extracts of embryos expressing Pim or Thr fused with myc epitope tags. Control experiments indicated that coimmunoprecipitation of Sse with Pim-myc and Thr-myc is specific. This specific association was also observed when the antibodies against Sse were used for immunoprecipitation followed by immunoblotting with anti-myc. Taken together, these results clearly show that Sse associates in vivo with Pim and Thr (Jäger, 2001).
The interaction between Thr and Sse observed in the yeast two-hybrid experiments was weak, raising the possibility that it might not be sufficiently strong to allow formation of Thr-Sse complexes in vivo. Pim might therefore be required to bring Thr and Sse together. To evaluate whether Thr-Sse complexes can be formed in the absence of Pim in vivo, gUAS-thr-myc (which allow expression of myc-tagged products under the control of the normal genomic regulatory regions) and UAS-HA-Sse were expressed in late embryos when endogenous Pim and Thr levels are very low. After precipitation of HA-Sse with an anti-HA antibody, Thr-myc as well as minor amounts of Pim were detected in the immunoprecipitates. Quantitative immunoblotting revealed that Thr-myc is present in an at least fivefold molar excess over Pim in these immunoprecipitates. Because Thr does not form oligomers (Leismann, 2000), it is concluded that Thr and Sse can associate in the absence of Pim (Jäger, 2001).
To investigate whether Pim is able to bind to Sse in the absence of Thr in vivo, a complementary experiment was performed by expressing UAS-HA-Sse and UAS-pim-myc in late embryos. Surprisingly, in contrast to the results of the yeast two-hybrid experiments, only minor amounts of Pim-myc could be coprecipitated with Sse. However, simultaneous coexpression of gUAS-thr-myc, UAS-HA-Sse, and UAS-pim-myc results in the recovery of significant Pim levels. It is estimated that <10% of Pim is precipitated in the absence of Thr when compared with the amount precipitated in the presence of Thr. Significantly, Pim2-myc does not form a stable complex with HA-Sse, even in the presence of Thr. These results indicate that Pim, Thr, and Sse form a trimeric complex in vivo, and that Sse is not sufficient to recruit Pim in the absence of Thr. This notion was supported by the behavior of a Thr deletion mutant (Thr 445-1379-myc), which was lacking the region required for the two-hybrid interaction with Pim. When this mutant protein was expressed from a transgene under the control of the normal thr regulatory region and immunoprecipitated from embryo extracts, almost no coimmunoprecipitation of Pim was detected. However, Sse was readily detected in the immunoprecipitates, confirming that Thr and Sse can form a complex without Pim. Quantitative immunoblotting experiments indicated that Sse associates with Thr 445-1379-myc with at least 50% efficiency when compared with its binding to full-length Thr, whereas binding of Pim to Thr 445-1379-myc was reduced to <5%. On the basis of the results of two-hybrid experiments, in which Sse interacts strongly with Pim, the Sse present in the Thr 445-1379-myc immunoprecipitates would be expected to result in coimmunoprecipitation of Pim as well. However, the absence of Pim in the immunoprecipitates suggests that Pim cannot join Sse and Thr in a trimeric complex when it is not bound by Thr. Control experiments with Thr-myc versions that contained the N-terminal Pim-binding region show that coimmunoprecipitation of Pim along with Sse can be readily detected in these cases (Jäger, 2001).
Thr 1-478-myc does not form complexes with Pim in vivo, whereas Thr 1-476 and Pim associate in yeast two-hybrid experiments. This discrepancy might result from different positions of the fused tags. Whereas in the two-hybrid experiments, the GAL4-binding domain was fused to the N terminus, the 10 myc epitope tags were fused to the C terminus of the Thr fragment analyzed in Drosophila embryos. The C-terminal myc tags, therefore, might interfere with Pim binding to Thr 1-478-myc (Jäger, 2001).
The C-terminal fragment Thr 932-1379-myc does not bind Pim or Sse, as expected from the yeast two-hybrid analysis. This fragment is present at very low levels in the embryo extracts as determined for four independent transgenic lines. This low abundance is presumably due to protein instability, since all genomic Thr constructs contained identical 5'- and 3'-noncoding regulatory sequences. No coimmunoprecipitation of Pim or Sse was detected when loading was adjusted to compensate for the low abundance of Thr 932-1379-myc (Jäger, 2001).
PIM transcripts are detected in embryos, third instar larvae, and pupae. By in situ hybridization, PIM transcripts are seen distributed throughout the embryo up to embyronic cycle 16. At later stages they become restricted to the developing nervous system and appear to be correlated with mitotic proliferation (Stratmann, 1996).
Mutations in the Drosophila genes pimples and three rows result in a defect of sister chromatid separation during mitosis. As a consequence, cytokinesis is also defective. However, cell cycle progression including the mitotic degradation of cyclins A and B is not blocked by the failure of sister chromatid separation, and as a result, metaphase chromosomes with twice the normal number of chromosome arms still connected in the centromeric region are observed in the following mitosis. pimples encodes a novel protein that is rapidly degraded in mitosis. These observations suggest that Pimples and Three rows act during mitosis to release the cohesion between sister centromeres (Stratmann, 1996).
Mutations in the Drosophila genes three rows (thr), fizzy (fzy), and pimples (pim) block chromosome segregation in mitosis. fzy mutations also block cyclin degradation and affected cells become permanently arrested in metaphase. Mutations in pim and thr prevent chromatid separation but proteolysis of mitotic cyclins occurs normally and cells leave mitosis. Since it has been shown that active cdc2 is required to maintain the arrest seen in fzy embryos it was determined if pim+ and thr+ were also required. By constructing double-mutant combinations of the three genes it has been established that the fzy arrest persists in the absence of either pim or thr and that there is no synergistic interaction between pim and thr (Philp, 1997).
Nondegradable mutant Pimdba-myc does not block the mitotic degradation of Cyclin A and Cyclin B, indicating that it does not inhibit the APC/C-dependent degradation pathway. Interestingly, however, Pimdba-myc does block sister chromatid separation. In UAS-pimdba-myc-expressing embryos, only abnormal, decondensing metaphase plates are observed in the regions without Cyclin B labeling instead of anaphase and telophase figures, which are abundant in those regions of control embryos that have degraded Cyclin B and thus have progressed beyond the metaphase-anaphase transition. Early mitotic figures (prophase and metaphase) are normal in UAS-pimdba-myc-expressing embryos, and tubulin labeling has revealed the presence of mitotic spindles. The observation that congression of mitotic chromosomes into the metaphase plate occurs normally indicates that Pimdba-myc does not interfere with spindle function (Leismann, 2000).
The nos-GAL4-GCN4-bcd3'UTR transgene used in these experiments to drive UAS-pimdba-myc expression results in a graded expression with a maximum at the anterior pole of the embryo. Whereas an apparently complete block of sister chromatid separation occurs in regions with high levels of expression, only a partial inhibition is observed in regions with lower expression levels. In these regions, aberrant anaphase and telophase figures with chromatin bridges are frequent (Leismann, 2000).
To confirm that high levels of UAS-pimdba-myc expression abolish sister chromatid separation specifically and not other processes during cell cycle progression, mitotic chromosomes from UAS-pimdba-myc I.1; UAS-pimdba-myc III.1/da-GAL4 were analyzed in embryos after treatment with the microtubule destabilizing drug colcemid (demecolcine) during the stage of mitosis 16. In these embryos, sister chromatid separation appears to be inhibited completely during mitosis 15 which follows after the onset of da-GAL4-driven UAS-transgene expression. Thus, after nondisjunction of sister chromatids during mitosis 15 and re-replication during S phase 16, diplochromosomes would be expected to be present during the colcemid-arrested mitosis 16. In fact, whereas only normal mitotic chromosomes were observed in control embryos; mitotic cells with a normal number of chromosomes that had twice as many arms than normal chromosomes were present in the UAS-pimdba-myc-expressing embryos. The presence of these diplochromosomes demonstrates that UAS-pimdba-myc expression specifically blocks sister chromatid separation (Leismann, 2000).
The finding that sister chromatid separation is inhibited by the nondegradable Pimdba-myc protein suggests that this process is dependent on mitotic Pim degradation. High levels of wild-type Pim resulting from overexpression, therefore, might inhibit sister chromatid separation equally. In fact, sister chromatid separation fails when two copies of the UAS-pim-myc transgene are expressed during the embryonic mitoses using the da-GAL4 or prd-GAL4 transgenes. Expression of one UAS-pim-myc copy does not inhibit sister chromatid separation. Quantitative immunoblotting experiments indicate that ubiquitous expression of two UAS-pim-myc copies with da-GAL4 results in about five-fold higher levels of expression compared to wild type. Although this level of overexpression inhibits sister chromatid separation, it does not interfere with mitotic cyclin destruction. Moreover, UAS-pim-myc overexpression in endoreduplicating salivary gland cells throughout late embryogenesis and larval development has no effect, whereas it results in severe phenotypic abnormalities in mitotically proliferating imaginal disc cells. Overexpression of wild-type pim, therefore, is not generally cytotoxic and inhibits sister chromatid separation specifically (Leismann, 2000).
Interestingly, the phenotype resulting from UAS-pimdba-myc and UAS-pim-myc overexpression is identical to the phenotype observed in mutant embryos lacking pim function (Stratmann, 1996. It appears, therefore, that both the accumulation of Pim during interphase as well as the subsequent degradation during mitosis are important for sister chromatid separation (Leismann, 2000).
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).
Faithful transmission of genetic information during mitotic divisions depends on bipolar attachment of sister kinetochores to the mitotic spindle and on complete resolution of sister-chromatid cohesion immediately before the metaphase-to-anaphase transition. Separase is thought to be responsible for sister-chromatid separation, but its regulation is not completely understood. Therefore, a screen was performed for genetic loci that modify the aberrant phenotypes caused by overexpression of the regulatory separase complex subunits Pimples/securin and Three rows in Drosophila. An interacting gene was found to encode a constitutive centromere protein. Characterization of its centromere localization domain revealed the presence of a diverged CENPC motif. While direct evidence for an involvement of this Drosophila Cenp-C homolog in separase activation at centromeres could not be obtained, in vivo imaging clearly demonstrated that it is required for normal attachment of kinetochores to the spindle (Heeger, 2005).
Overexpression of pim during Drosophila eye development results in an aberrant rough eye phenotype in adults. Although this phenotype has not been characterized at a cellular level, it is assumed that it results primarily from inhibition of sister-chromatid separation during mitotic divisions of eye imaginal disc cells, because pim overexpression during embryogenesis is known to have this effect. Moreover, analyses in salivary glands indicate that pim overexpression does not have obvious effects in cells progressing through endoreduplication cycles that lack mitotic divisions (Heeger, 2005).
When expressed during eye development, a mutant thr version lacking C-terminal sequences (thrDeltaC) results in a phenotype very similar to that caused by eye-specific pim overexpression. The effect of thrDeltaC is suppressed by concomitant expression of wild-type thr, suggesting that thrDeltaC acts in a dominant-negative manner. The severity of the aberrant phenotypes resulting from pim and thrDeltaC overexpression during eye development is correlated with transgene copy numbers (Heeger, 2005).
To identify loci interacting with pim and thr, a collection of chromosomal deficiencies was crossed into the backgrounds with the transgenes resulting in pim or thrDeltaC overexpression during eye development. Heterozygosity for the deficiency Df(3R)p-XT103, which deletes the chromosomal interval 85A-85C, was found to enhance the rough eye phenotypes caused by pim and thrDeltaC overexpression. Based on analyses with additional deficiencies, the interacting region could be narrowed down. Moreover, heterozygosity for the EMS-induced, recessive lethal mutation l(3)85Aaprl41, which had previously been mapped to this chromosomal region, was also found to enhance the aberrant eye phenotype resulting from pim and thrDeltaC overexpression. l(3)85Aaprl41 was determined to represent a mutant allele of CG31258 that interacts genetically with pim and thr (Heeger, 2005).
The most recent gene model of CG31258 annotated by the Drosophila Genome Project agrees with an independent cDNA sequence analyses except for the position of the intron-exon junction at the start of exon 4. Initial database searches with the predicted amino acid sequence did not reveal statistically significant similarities to other genes until sequence traces of various Drosophilid genome projects were deposited in the public domain. The sequence comparisons with these Drosophilid orthologs in combination with the results of functional characterizations eventually allowed an identification of CG31258 as the Drosophila Cenp-C homolog. In the following, therefore, this gene is designated as Cenp-C, even though its identity was recognized only after and because of the functional characterization (Heeger, 2005).
Identification of Drosophila Cenp-C closes a prominent gap in the arguments for homologous centromere organization. Centromeric DNA sequences have evolved extremely rapidly and appear to have driven the coevolution of centromeric proteins during eukaryote evolution. The resulting low sequence similarity between centromeric proteins has effectively concealed the existence of a common set of constitutive eukaryotic centromere proteins. The first features demonstrated to be shared among fungal, plant, and animal centromeres were centromere-specific histone H3 variants. In addition to these Cenp-A homologs, only one further constitutive centromere component, Cenp-C, has so far been shown to be present in each of the three main eukaryotic branches. The findings of this study now also establish the existence of Drosophila Cenp-C, which had previously escaped detection by careful bioinformatics genome analyses because of its very limited sequence similarity. In combination with the recent identification of related Cenp-H-, Cenp-I-, and Mis12-like proteins in both vertebrates and yeast, these results provide strong support for the notion of a common set of constitutive centromere proteins. These proteins, which are centromeric throughout the cell cycle, appear to provide a foundation for kinetochore assembly and spindle attachment during mitosis by recruiting several distinct multisubunit complexes that also contain highly diverged proteins (Heeger, 2005).
The extensive sequence divergence characteristically observed among homologous eukaryotic centromere and kinetochore proteins is striking, especially in the light of their common fundamental cellular function. The average amino acid identity observed in a genome-wide comparison of D. melanogaster and D. pseudoobscura ortholog pairs is 77% and only 38% in case of the Cenp-C pair. Moreover, based on the ratio between radical charge mutations and conservative substitutions in D. melanogaster and D. pseudoobscura ortholog pairs, Cenp-C is one of 44 genes likely to have evolved under positive selection. Except for a few very restricted regions, comparison of D. melanogaster Cenp-C with the orthologs from D. erecta and D. yakuba, which are closer relatives than D. pseudoobscura and thus amenable to dN/dS analyses, did not reveal strong evidence for positive selection, in contrast to the recent findings in plant and mammalian lineages. However, it is pointed out that these dN/dS analyses ignore insertions and deletions (indels), which have occurred considerably more often during Cenp-C evolution in Drosophilids than in the mammalian lineage. Most of the indels are observed within the minimally conserved central regions of Drosophilid Cenp-C. Similar variabilities resulting from recurrent exon duplications have been observed in the central region of the plant Cenp-C genes (Heeger, 2005).
The adaptively evolving regions of mammalian Cenp-C have been shown to bind to DNA in vitro, consistent with the proposed coevolution of centromeric DNA and protein sequences. However, this DNA binding in vitro is not sequence-specific, suggesting that interactions with centromere-specific proteins contribute to centromere localization of Cenp-C. As in other organisms, Cid/Cenp-A is also required for centromere localization of Cenp-C in Drosophila. High-resolution light microscopy of mitotic chromosomes has indicated that human Cenp-C covers the poleward-oriented peripheral region of the Cenp-A-containing centromeric chromatin. Direct interactions between Cenp-A and Cenp-C have not yet been demonstrated in any organism. Attempts with yeast two-hybrid experiments were also unsuccessful (Heeger, 2005).
The CENPC motif has recently been identified as the only region conserved among the Cenp-C orthologs from fungi, plant, and animals. In Drosophilids, even this short motif of ~24 amino acids is not fully conserved in its C-terminal part. The results suggest that this CENPC motif is crucial for centromere localization. A single-point mutation affecting one of the invariant positions in the CENPC motif interferes with centromere localization of the C-terminal domain of Cenp-C in the transfection assay. This mutation was identified as the only missense mutation interfering with centromere localization after extensive random mutagenesis. Further experiments will reveal whether and how the CENPC motif contacts Cid/Cenp-A nucleosomes. It is emphasized, however, that also in Drosophila Cenp-C, other regions than the CENPC motif clearly contribute to efficient centromere localization. Centromere localization of the CN subregion (1009-1205), for instance, is only detected in the transfection assay in live but not in fixed cells, while centromere localization of the larger C region (1009-1411) is resistant to fixation (Heeger, 2005).
The highest conservation among Drosophilid Cenp-C proteins is observed within the N-terminal third, which is neither required nor sufficient for normal centromere localization. Nevertheless, prolonged overexpression of this domain in proliferating eye and wing imaginal disc cells results in severe defects. The conserved N-terminal Cenp-C domains (R and DH) might bind to kinetochore proteins and titrate these away from the centromere when overexpressed. Biochemical and genetic characterizations in Saccharomyces cerevisiae and Caenorhabditis elegans have suggested that Cenp-C is not only associated with Cenp-A, but that it also recruits the next layer of kinetochore proteins, in particular the Mis12/Mtw1 and Ndc80 complexes, which remain to be identified in Drosophila (Heeger, 2005).
As in yeast, chicken, and mice, Cenp-C is also an essential gene in Drosophila. Antibody microinjection experiments in mammalian cells; RNA interference in C. elegans; and phenotypic analysis in yeast, and mutant Drosophila embryos demonstrate that Cenp-C is required for normal chromosome segregation during mitosis. In vivo imaging in Cenp-C mutant embryos discloses these defects in detail. Previously, in vivo imaging has also been applied in C. elegans CENP-C(RNAi) embryos. The formation of holocentric chromosomes and transient Cenp-C recruitment only during mitosis differentiates C. elegans from other metazoans like Drosophila and mammalian cells. Moreover, in contrast to the findings in C. elegans, chromosome congression into a central plane is still observed in the Drosophila Cenp-C mutants. Presumably, this chromosome congression reflects the function of residual maternally provided Cenp-C, which is still detectable at the stage of the analyses. Moreover, time-lapse analyses demonstrate that chromosome congression is not entirely normal in the Cenp-C mutant embryos. Metaphase plate formation is delayed and often does not lead to the highly ordered arrangement of all chromosomes characteristically observed before anaphase onset in wild type. Occasional chromosomes fail to achieve bipolar attachment in the Cenp-C mutants. These chromosomes do not segregate normally during anaphase. Cenp-C is, therefore, clearly required for normal attachment of kinetochores to the mitotic spindle (Heeger, 2005).
Evidently, the insufficiently attached chromosomes in Cenp-C mutant embryos are unable to inhibit the onset of anaphase, even though the mitotic spindle checkpoint appears to be at least partially functional in Cenp-C mutants at the analyzed stage. However, assembly of mitotic spindle checkpoint proteins might fail, particularly on the kinetochores of those chromosomes that do not attach correctly to the mitotic spindle (Heeger, 2005).
In principle, the chromosome segregation defects observed in the Cenp-C mutants might not only reflect impaired interactions between kinetochores and spindle. Segregation of sister chromatids to the spindle poles also depends on complete resolution of sister-chromatid cohesion at the metaphase-to-anaphase transition. This final separation of sister chromatids is thought to be achieved by separase-mediated cleavage of the Scc1/Rad21 subunit of those cohesin complexes that perdure in the pericentromeric region until the metaphase-to-anaphase transition. Several observations are consistent with the idea that a localized full activation of separase might be assisted by centromeric proteins. Accordingly, mutations in Cenp-C might reduce separase activity and thereby explain the genetic interactions with the regulatory separase subunits Pim/securin and Thr that led to the identification of Drosophila Cenp-C. No coimmunoprecipitation of Cenp-C with separase complex proteins was observed and no effects of Cenp-C mutations on Pim and Thr levels were observed. This analysis has, therefore, not exposed clear evidence for separase activation at centromeres. Nevertheless, Cenp-C is clearly required for normal chromosome attachment to the mitotic spindle, and thus the observed genetic interactions most likely reflect a summation of negative effects on the efficiency of sister-chromatid separation (by separase) and segregation (by the spindle). However, it is emphasized that the results certainly do not rule out a local activation of separase within the centromeric region (Heeger, 2005).
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date revised: 10 December 2005
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