Mutations in the ESP1 gene of Saccharomyces cerevisiae disrupt normal cell-cycle control and cause many cells in a mutant population to accumulate extra spindle pole bodies. To determine the stage at which the esp1 gene product becomes essential for normal cell-cycle progression, synchronous cultures of ESP1 mutant cells were exposed to the nonpermissive temperature for various periods of time. The mutant cells retained viability until the onset of mitosis, when their viability dropped markedly. Examination of these cells by fluorescence and electron microscopy showed the first detectable defect to be a structural failure in the spindle. Additionally, flow cytometric analysis of DNA content demonstrated that massive chromosome missegregation accompanied this failure of spindle function. Cytokinesis occurs despite the aberrant nuclear division, which often results in segregation of both spindle poles to the same cell. At later times, the missegregated spindle pole bodies enter a new cycle of duplication, thereby leading to the accumulation of extra spindle pole bodies within a single nucleus. The DNA sequence predicts a protein product similar to those of two other genes that are also required for nuclear division: the cut1 gene of Schizosaccharomyces pombe and the bimB gene of Aspergillus nidulans (McGrew, 1992).
The separation of sister chromatids in anaphase is followed by spindle disassembly and cytokinesis. These events are governed by the anaphase-promoting complex (APC), which triggers the ubiquitin-dependent proteolysis of key regulatory proteins: anaphase requires the destruction of the anaphase inhibitor the yeast securin Pds1, whereas mitotic exit requires the destruction of mitotic cyclins and the inactivation of Cdk1. Pds1 is not only an inhibitor of anaphase, but also blocks cyclin destruction and mitotic exit by a mechanism independent of its effects on sister chromatid separation. Pds1 is also required for the mitotic arrest and inhibition of cyclin destruction that occurs after DNA damage. Even in anaphase cells, where Pds1 levels are normally low, DNA damage stabilizes Pds1 and prevents cyclin destruction and mitotic exit. Pds1 blocks cyclin destruction by inhibiting its binding partner Esp1. Mutations in ESP1 delay cyclin destruction; overexpression of ESP1 causes premature cyclin destruction in cells arrested in metaphase by spindle defects and in cells arrested in metaphase and anaphase by DNA damage. The effects of Esp1 are dependent on Cdc20 (an activating subunit of the APC) and on several additional proteins (Cdc5, Cdc14, Cdc15, Tem1) that form a regulatory network governing mitotic exit. It is speculated that the inhibition of cyclin destruction by Pds1 may contribute to the ordering of late mitotic events by ensuring that mitotic exit is delayed until after anaphase is initiated. In addition, the stabilization of Pds1 after DNA damage provides a mechanism to delay both anaphase and mitotic exit while DNA repair occurs (Tinker-Kulberg, 1999).
Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin. Attachment of sister kinetochores to the mitotic spindle during mitosis generates forces that would immediately split sister chromatids were it not opposed by cohesion. Cohesion is essential for the alignment of chromosomes in metaphase but must be abolished for sister separation to start during anaphase. In the budding yeast Saccharomyces cerevisiae, loss of sister-chromatid cohesion depends on a separating protein (separin) called Esp1 and is accompanied by dissociation from the chromosomes of the cohesion subunit Scc1. Esp1 causes the dissociation of Scc1 from chromosomes by stimulating its cleavage by proteolysis. A mutant Scc1 is described that is resistant to Esp1-dependent cleavage and which blocks both sister-chromatid separation and the dissociation of Scc1 from chromosomes. The evolutionary conservation of separins indicates that the proteolytic cleavage of cohesion proteins might be a general mechanism for triggering anaphase (Uhlmann, 1999).
In eukaryotic cells, replicated DNA strands remain physically connected until their segregation to opposite poles of the cell during anaphase. This 'sister chromatid cohesion' is essential for the alignment of chromosomes on the mitotic spindle during metaphase. Cohesion depends on the multisubunit cohesin complex, which possibly forms the physical bridges connecting sisters. Proteolytic cleavage of cohesin's Sccl subunit at the metaphase to anaphase transition is essential for sister chromatid separation and depends on a conserved protein called separin. Separin is a cysteine protease related to caspases that alone can cleave Sccl in vitro. Cleavage of Sccl in metaphase arrested cells is sufficient to trigger the separation of sister chromatids and their segregation to opposite cell poles (Uhlmann, 2000).
In Saccharomyces cerevisiae, the metaphase-anaphase transition is initiated by the anaphase-promoting complex-dependent degradation of Pds1, whereby Esp1 is activated to promote sister chromatid separation. Although this is a fundamental step in the cell cycle, little is known about the regulation of Esp1 and how loss of cohesion is coordinated with movement of the anaphase spindle. Esp1 has a novel role in promoting anaphase spindle elongation. The localization of Esp1 to the spindle apparatus, analyzed by live cell imaging, is regulated in a manner consistent with a function during anaphase B. The protein accumulates in the nucleus in G2 and is mobilized onto the spindle pole bodies and spindle midzone at anaphase onset, where it persists into midanaphase. Association with Pds1 occurs during S phase and is required for efficient nuclear targeting of Esp1. Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding. In agreement, Pds1 interacts with the spindle at the metaphase-anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells. Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction (Jensen, 2001).
Sister chromatid separation at the metaphase-to-anaphase transition is induced by the proteolytic cleavage of one of the cohesin complex subunits. This process is mediated by a conserved protease called separase. Separase is associated with its inhibitor, securin, until the time of anaphase initiation, when securin is degraded in an anaphase-promoting complex/cyclosome (APC/C)-dependent manner. In budding yeast securin/Pds1 not only inhibits separase/Esp1, but also promotes its nuclear localization. The molecular mechanism and regulation of this nuclear targeting are presently unknown. Pds1 is a substrate of the cyclin-dependent kinase Cdc28. Phosphorylation of Pds1 by Cdc28 is important for efficient binding of Pds1 to Esp1 and for promoting the nuclear localization of Esp1. These results uncover a previously unknown mechanism for regulating the Pds1-Esp1 interaction and shed light on a novel role for Cdc28 in promoting the metaphase-to-anaphase transition in budding yeast (Agarwal, 2002).
Sister chromatid separation and segregation at anaphase onset are triggered by cleavage of the chromosomal cohesin complex by the protease separase. Separase is regulated by its binding partner securin in two ways: securin is required to support separase activity in anaphase; and, at the same time, securin must be destroyed via ubiquitylation before separase becomes active. The molecular mechanisms underlying this dual regulation of separase by securin are unknown. In budding yeast, securin supports separase localization. Separase enters the nucleus independently of securin, but securin is required and sufficient to cause accumulation of separase in the nucleus, where its known cleavage targets reside. Securin also ensures that separase gains full proteolytic activity in anaphase. Securin, while present, directly inhibits the proteolytic activity of separase. Securin prevents the binding of separase to its substrates. It also hinders the separase N terminus from interacting with and possibly inducing an activating conformational change at the protease active site 150 kDa downstream at the protein's C terminus. It is concluded that securin inhibits the proteolytic activity of separase in a 2-fold manner. While inhibiting separase, securin is able to promote nuclear accumulation of separase and help separase to become fully activated after securin's own destruction at anaphase onset (Hornig, 2002).
Separases are large proteins of 150-230 kDa in different species. An is exception, Drosophila, in which the separase function seems to have split in two, with the protein Three rows functioning as the separase N-terminus and the protein Separase functioning as the N-terminase. Separases have been initially identified as Esp1 in budding yeast and Cut1 in fission yeast. A C-terminal region, spanning ca. 50 kDa, is conserved in all species and has been called 'separase domain'. The second half of this separase domain harbors the conserved cysteine and histidine residues of the protease active site and has been predicted to adopt the fold of CD clan proteases. The large regions N-terminal of the separase domain do not show obvious conservation between species. The contribution of these extended N termini to separase function has remained unclear. In fission yeast, N-terminal sequences are required for the function of separase and have been implicated in its nuclear localization, and more central sequences have been implicated in the possible cytoplasmic retention of the protein. Also in fission yeast, as well as in budding yeast, N-terminal regions are thought to be the sites of interaction with separase's binding partner, securin (Hornig, 2002 and references therein).
A possible reason why cells lacking securin show reduced separase function in budding yeast might be the incorrect localization of separase in the absence of securin. This has been studied in budding yeast strains overexpressing separase. To address this under more natural conditions, separase Esp1 was observed by virtue of myc epitopes that were added to the genomic copy of the ESP1 gene. In G1 cells, when securin is absent, about 50% of the cells showed a weak separase accumulation in the nucleus. At the G1/S transition, separase is enriched in the nucleus of over 80% of cells, and the nuclear accumulation further increases during the G2 and M period. All cells in early anaphase, when securin abruptly disappears, show a strong nuclear concentration of separase. In marked contrast, cells deleted of securin show a seeming exclusion of separase from the nucleus at all cell cycle stages. This shows that securin is required for accumulation of separase in the nucleus. Although securin disappears in anaphase and is absent in wild-type G1 cells, separase is still concentrated in the nuclei of some of these cells. In contrast, separase seems to be excluded from all nuclei of cells deleted of securin. The reason for this is unclear; it could be because separase leaves the nucleus rather slowly after securin has been destroyed. Alternatively, separase that was bound to securin in the previous metaphase might be in a different functional state compared to separase that has never seen securin (Hornig, 2002).
The cohesin subunit Scc1 is cleaved on time in budding yeast cells lacking securin, suggesting that a certain level of separase, sufficient to cleave Scc1, can reach the nucleus even in the absence of securin. To address this directly, separase was observed on chromatin spreads in which cytoplasmic components of the cell are washed away. Separase is seen associated with metaphase chromatin in wild-type cells, and to a lesser but still significant extent, it is seen on spreads from metaphase cells lacking securin. Separase is no longer chromatin-associated in anaphase. Instead, separase is visible at spindle poles and, in wild-type cells, also at the anaphase spindle. The association of a low level of separase with chromatin in the absence of securin indicates that separase can indeed enter the nucleus independently of securin (Hornig, 2002).
Securin binds and inactivates separase before cells enter S phase, but separase gradually accumulates in nuclei throughout G2, reaching maximum levels only in mitosis. This could mean that cell cycle-dependent events other than the presence of securin contribute to nuclear accumulation of separase. However, separase nuclear accumulation is unchanged when DNA replication is blocked using the replication inhibitor hydroxyurea or when all of the mitotic cyclins, Clb1-4, are inactivated. Alternatively, the presence of securin might be sufficient to cause nuclear accumulation of separase, but it might be a relatively slow process or require an excess of securin over separase. In this case, even G1 cells should accumulate nuclear separase if securin is ectopically expressed. To test this, cells lacking securin were arrested stably in G1 by pheromone treatment and then expression of securin was induced from the galactose-inducible GAL1 promoter. As soon as securin appeared in the nucleus, separase had redistributed and was also concentrated in the nucleus, reminiscent of cells in mitosis. This demonstrates that expression of securin in G1 is sufficient to cause nuclear accumulation of separase. Because levels of securin after expression from the GAL1 promoter were about 10-fold higher than endogenous levels in metaphase, an excess of securin might be sufficient to promote fast nuclear separase accumulation. Together, this suggests that separase can enter the nucleus independently of securin, but that the presence of securin is required and sufficient to cause nuclear concentration of separase (Hornig, 2002).
Only since the discovery of separase as a site-specific protease that cleaves the cohesive bond between sister chromatids is it possible to analyze separase regulation on a molecular level. Securin forms a tight complex with separase, and this directly inhibits the protease activity of separase. The architecture of the complex between separase and securin shows unexpected features. Securin binds to both N- and C-terminal regions of separase, and, by doing so, securin disrupts, like a molecular wedge, interactions within separase. Thereby, securin might prevent the separase N terminus from inducing an activating conformational change at the protease active site required to recognize or attack a substrate cleavage site. At the same time, this model also offers a possible explanation for securin's proposed chaperone function that enables efficient activation of separase. By bridging the separase N and C termini, which are separated from each other by over 1500 amino acids, securin might bring them in juxtaposition, preparing them for interaction upon securin destruction. It is currently not nknow whether the interaction within separase is truly intramolecular, i.e., whether the N terminus of the polypeptide folds back onto its own C terminus. The interaction might likewise happen between two molecules of separase in an intermolecular fashion, thereby forming separase dimers (Hornig, 2002).
These observations also go some way in explaining why separases are such large proteins. Sequences very close to the N terminus of the protein are required for protease activity at the protein's C terminus. What about the sequences in between? When fragments from the N and C termini of separase are coexpressed, they efficiently form complexes, but it is not possible to reconstitute protease activity from these fragments. This indicates that the intactness of the middle portion of separase is also important for the function of the protease. Consistent with this, when ten temperature-sensitive alleles of the fission yeast separase Cut1 were sequenced, eight were found to encode single amino acid changes in the middle of the protein. This might mean that this region also contributes to the proteolytic activity of separase (Hornig, 2002).
A seeming exception to the separase-securin architecture exists in Drosophila. Here, separase is much smaller, and the N terminus does not extend far beyond the conserved separase domain. However, it has recently been suggested that the Three rows protein in Drosophila, which is required for separase activity, might play the role of the separase N terminus. The pattern of interactions of Three rows with separase and the Drosophila securin Pimples is reminiscent of the interactions of the budding yeast's separase N terminus with its catalytic C terminus and securin. It is therefore suggested that the results for the activation of separase's protease activity and its inhibition by securin might be applicable to Drosophila as well, supporting a model in which Three rows activates separase by an interaction that is prevented by Pimples (Hornig, 2002).
Will this model of separase inhibition and activation also be applicable to vertebrates? Xenopus and human securin must be degraded for separase activation, and human securin inhibits human separase in vitro (I. Waizenegger and J.-M. Peters, personal communication to Hornig, 2002). Securins are poorly conserved between species on the amino acid level, but all contain equivalent clusters of charged residues. Therefore, while the primary amino acid sequence differs between securins and separases in different species, the overall structure and organization of these sequences begins to appear very similar. A distinct feature of human separase is that, after its activation by securin degradation, separase cleaves itself into two halves at a position upstream of the separase domain. Processed separase is still active to cleave cohesin. And, consistent with the idea that the N terminus might be required for proteolytic activity, it stays associated with the C terminus after cleavage (I. Waizenegger and J.-M. Peters, personal communication to Hornig, 2002). No evidence was detected for self-cleavage of separase in budding yeast. Another level of regulation of human separase is its inhibition by Cdk(CDC2)-dependent phosphorylation. This inhibition is effective even after securin is degraded. The phosphorylation takes place in the center of separase, and it will be interesting to see whether it influences an interaction between the N and C termini or inhibits separase by an alternative mechanism. In budding yeast, there is no evidence for Cdk-dependent inhibition of separase. Sister separation can proceed in the presence of high kinase activity; securin destruction is sufficient to promote anaphase onset, and separase appears to be no longer regulated in the absence of securin. Finally, whether human securin is also involved in recruiting separase to its places of action while securin is still keeping the protease inactive has yet to be addressed (Hornig, 2002).
It is concluded that the regulation of separase activity is of critical importance for cells to ensure faithful segregation of their genetic material. Separase is not only the trigger of sister chromatid segregation, but it orchestrates multiple mitotic events, including mitotic spindle stability and mitotic exit. A key regulator of separase, securin, acts to concentrate separase in the nucleus where separase cleaves its known targets in anaphase. While recruiting separase into the nucleus and preparing it for efficient activation, securin inhibits the protease activity of separase. Thereby, an accumulation of inactive separase is built up that can be suddenly unleashed after securin is destroyed at anaphase onset. Securin uses a double strategy to inhibit separase. Not only does securin prevent access of substrates to separase, but securin also seems to inhibit separase by preventing it from activating itself (Hornig, 2002).
Chromosome segregation during mitosis and meiosis is triggered by dissolution of sister chromatid cohesion, which is mediated by the cohesin complex. Mitotic sister chromatid disjunction requires that cohesion be lost along the entire length of chromosomes, whereas homolog segregation at meiosis I only requires loss of cohesion along chromosome arms. During animal cell mitosis, cohesin is lost in two steps. A nonproteolytic mechanism removes cohesin along chromosome arms during prophase, while the proteolytic cleavage of cohesin's Scc1 subunit by separase removes centromeric cohesin at anaphase. In Saccharomyces cerevisiae and Caenorhabditis elegans, meiotic sister chromatid cohesion is mediated by Rec8, a meiosis-specific variant of cohesin's Scc1 subunit. Homolog segregation in S. cerevisiae is triggered by separase-mediated cleavage of Rec8 along chromosome arms. In principle, chiasmata could be resolved proteolytically by separase or nonproteolytically using a mechanism similar to the mitotic 'prophase pathway'. Inactivation of separase in C. elegans has little or no effect on homolog alignment on the meiosis I spindle but prevents timely sister chromatid disjunction. It also interferes with chromatid separation during subsequent embryonic mitotic divisions but does not directly affect cytokinesis. Surprisingly, separase inactivation also causes osmosensitive embryos, possibly due to a defect in the extraembryonic structures, referred to as the 'eggshell'. It is concluded that separase is essential for homologous chromosome disjunction during meiosis I. Proteolytic cleavage, presumably of Rec8, might be a common trigger for the first meiotic division in eukaryotic cells. Cleavage of proteins other than REC-8 might be necessary to render the eggshell impermeable to solutes (Siomos, 2001).
Polarization of the one-cell C. elegans embryo establishes the animal's anterior-posterior (a-p) axis. Reduction-of-function anaphase-promoting complex (APC) mutations have been discovered that eliminate a-p polarity. The APC activator cdc20 is required for polarity. The APC excludes PAR-3 from the posterior cortex, allowing PAR-2 to accumulate there. The APC is also required for tight cortical association and posterior movement of the paternal pronucleus and its associated centrosome. Depletion of the protease separin, a downstream target of the APC, causes similar pronuclear and a-p polarity defects. It is proposed that the APC/separin pathway promotes close association of the centrosome with the cortex, which in turn excludes PAR-3 from the posterior pole early in a-p axis formation (Rappleye, 2002).
A vertebrate securin (vSecurin) was identified on the basis of its biochemical analogy to the Pds1p protein of budding yeast and the Cut2p protein of fission yeast. The vSecurin protein binds to a vertebrate homolog of yeast separins Esp1p and Cut1p and is degraded by proteolysis mediated by an anaphase-promoting complex in a manner dependent on a destruction motif. Furthermore, expression of a stable Xenopus securin mutant protein blocks sister-chromatid separation but does not block the embryonic cell cycle. The vSecurin proteins share extensive sequence similarity with each other but show no sequence similarity to either of their yeast counterparts. Human securin is identical to the product of the gene called pituitary tumor-transforming gene (PTTG), which is overexpressed in some tumors and exhibits transforming activity in NIH 3T3 cells. The oncogenic nature of increased expression of vSecurin may result from chromosome gain or loss, produced by errors in chromatid separation (Zou, 1999).
In yeast, anaphase depends on cohesin cleavage. How anaphase is controlled in vertebrates is unknown because vertebrate cohesins dissociate from chromosomes before anaphase. Residual amounts of the cohesin SCC1 remain associated with human centromeres until the onset of anaphase when a similarly small amount of SCC1 is cleaved. In Xenopus extracts, SCC1 cleavage depends on the anaphase-promoting complex and separin. Separin immunoprecipitates are sufficient to cleave SCC1, indicating that separin is associated with a protease activity. Separin activation coincides with securin destruction and partial separin cleavage, suggesting that several mechanisms regulate separin activity. It is proposed that in vertebrates, a cleavage-independent pathway removes cohesin from chromosome arms during prophase, whereas a separin-dependent pathway cleaves centromeric cohesin at the metaphase-anaphase transition (Waizenegger, 2000).
Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, it has been demonstrated that high CDC2 activity inhibits anaphase but not securin degradation. Separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, it has been shown that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. It is proposed that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate (Stemmann, 2001).
Cell division depends on the separation of sister chromatids in anaphase. In yeast, sister separation is initiated by cleavage of cohesin by the protease separase. In vertebrates, most cohesin is removed from chromosome arms by a cleavage-independent mechanism. Only residual amounts of cohesin are cleaved at the onset of anaphase, coinciding with its disappearance from centromeres. Two separase cleavage sites in the human cohesin subunit SCC1 have been identified and noncleavable SCC1 mutants have been conditionally expressed in human cells. The results indicate that cohesin cleavage by separase is essential for sister chromatid separation and for the completion of cytokinesis (Hauf, 2001).
Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila Mei-s332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin, and by inference its Drosophila homolog Mei-S332, prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. These results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin (Marston, 2004).
Faithful segregation of homologous chromosomes during the first meiotic division is essential for further embryo development. The question at issue is whether the same mechanisms ensuring correct separation of sister chromatids in mitosis are at work during the first meiotic division. In mitosis, sister chromatids are linked by a cohesin complex holding them together until their disjunction at anaphase. Their disjunction is mediated by Separase, which cleaves the cohesin. The activation of Separase requires prior degradation of its associated inhibitor, called securin. Securin is a target of the APC/C (Anaphase Promoting Complex/Cyclosome), a cell cycle-regulated ubiquitin ligase that ubiquitinates securin at the metaphase-to-anaphase transition and thereby targets it for degradation by the 26S proteasome. After securin degradation, Separase cleaves the cohesins and triggers chromatid separation, a prerequisite for anaphase. In yeast and worms, the segregation of homologous chromosomes in meiosis I depends on the APC/C and Separase activity. Yet, it is unclear if Separase is required for the first meiotic division in vertebrates because APC/C activity is thought to be dispensable in frog oocytes. Therefore, whether Separase activity is required for correct chromosome segregation in meiosis I in mouse oocytes was investigated. The results in mice are in accordance with studies in S. cerevisiae and C. elegans but are contrary to what has been shown in X. laevis. The APC/C is required for the activation of Separase by targetting its inhibitor, securin, for degradation by the 26S proteasome. It is proposed that the meiosis I-to-II transition in mouse oocytes also depends on APC/C activity, as has been shown in S. cerevisiae and C. elegans. In human oocytes, missegregation events in meiosis I are responsible for the generation of aneuploidies, which may lead to trisomies, malformations of the embryo, and spontaneous abortion. Therefore, it is of great importance to know the mechanisms controlling meiosis I. This study gives important insights into an understanding of the regulation of correct chromosome segregation in meiosis I in mammalian oocytes (Terret, 2003).
During meiosis, two rounds of chromosome segregation after a single round of DNA replication produce haploid gametes from diploid precursors. At meiosis I, maternal and paternal kinetochores are pulled toward opposite poles, and chiasmata holding bivalent chromosomes together are resolved by cleavage of cohesin's alpha-kleisin subunit (Rec8) along chromosome arms. This creates dyad chromosomes containing a pair of chromatids joined solely by cohesin at centromeres that had resisted cleavage. The discovery that centromeric Rec8 is protected from separase during meiosis I by shugoshin/MEI-S332 proteins that bind PP2A phosphatase suggests that phosphorylation either of separase or cohesin may be necessary for Rec8 cleavage. This study shows that multiple phosphorylation sites within Rec8 as well as two different kinases, casein kinase 1delta/epsilon (CK1delta/epsilon) and Dbf4-dependent Cdc7 kinase (DDK), are required for Rec8 cleavage and meiosis I nuclear division. Rec8 with phosphomimetic mutations is no longer protected from separase at centromeres and is cleaved even when the two kinases are inhibited. These data suggest that PP2A protects centromeric cohesion by opposing CK1delta/epsilon- and DDK-dependent phosphorylation of Rec8 (Katis, 2010).
It has been proposed that separase-dependent centriole disengagement at anaphase licenses centrosomes for duplication in the next cell cycle. This study tests whether such a mechanism exists in intact human cells. Loss of separase blocked centriole disengagement during mitotic exit and delayed assembly of new centrioles during the following S phase; however, most engagements were eventually dissolved. Polo-like kinase 1 (Plk1) was identified as a parallel activator of centriole disengagement. Timed inhibition of Plk1 mapped its critical period of action to late G2 or early M phase, i.e., prior to securin destruction and separase activation at anaphase onset. Crucially, when cells exited mitosis after downregulation of both separase and Plk1, centriole disengagement failed completely, and subsequent centriole duplication in interphase was also blocked. These results indicate that Plk1 and separase act at different times during M phase to license centrosome duplication, reminiscent of their roles in removing cohesin from chromosomes (Tsou, 2009).
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