Cut1/separase C-terminus affects spindle pole body positioning in interphase of fission yeast: pointed nuclear formation

BACKGROUND: The separase-securin complex is required for anaphase. Separase activated by securin destruction cleaves the cohesin subunit Scc1/Rad21 enriched in kinetochores. Fission yeast Cut1/separase resides in interphase cytoplasm and mobilizes to the spindle and the spindle pole bodies (SPBs) in mitosis, while Cut2/securin remains in the nucleus from interphase to metaphase, and temporarily locates at the short spindle. RESULTS: We here report a novel SPB-led dynamic nuclear movement in fission yeast, when the Cut1 C-terminal fragment is over-expressed. The tip of the pointed nucleus contained both SPB and centromeric DNA, and rapidly moved along the bundled cytoplasmic microtubules. The same pointed nucleus was produced when the human separase C-fragment was over-expressed. The pointed nuclear formation did not require the protease site of separase, but required the conserved C-terminus and a microtubule- and kinetochore-binding protein Mtc1/Alp14, a homologue of frog XMAP215 and budding yeast Stu2. The movement-inducing C-fragment should be cytoplasmic, as the pointed nucleus was abolished when the fragment contained the NLS (nuclear localization signal). CONCLUSIONS: Overproduced separase C-fragment abolishes correct SPB-positioning in interphase. Resulting pointed nuclear formation (alternatively called 'pigtail movement') requires cytoplasmic microtubules and Mtc1/Alp14.     

Shugoshin prevents cohesin cleavage by PP2ACdc55-dependent inhibition of separase

Chromosome segregation is triggered by separase, an enzyme that cleaves cohesin, the protein complex that holds sister chromatids together. Separase activation requires the destruction of its inhibitor, securin, which occurs only upon the correct attachment of chromosomes to the spindle. However, other mechanisms restrict separase activity to the appropriate window in the cell cycle because cohesin is cleaved in a timely manner in securin-deficient cells. We investigated the mechanism by which the protector protein Shugoshin counteracts cohesin cleavage in budding yeast. We show that Shugoshin can prevent separase activation independently of securin. Instead, PP2A^Cdc55 is essential for Shugoshin-mediated inhibition of separase. Loss of both securin and Cdc55 leads to premature sister chromatid separation, resulting in aneuploidy. We propose that Cdc55 is a separase inhibitor that acts downstream from Shugoshin under conditions where sister chromatids are not under tension.     

Bir1/Cut17 moving from chromosome to spindle upon the loss of cohesion is required for condensation, spindle elongation and repair

BACKGROUND: In mammals, proteins containing BIR domains (IAPs and survivin) are implicated in inhibiting apoptosis and sister chromatid separation. In the nematode, Bir1 is required for a proper localization of aurora kinase, which moves from the mitotic chromosome in metaphase to the spindle midzone in anaphase as a passenger. Fission yeast Bir1/Pbh1 is essential for normal mitosis. RESULTS: A temperature sensitive mutant cut17-275 exhibits the defect in condensation and spindle elongation at 36 degrees C, while securin is degraded. Gene cloning shows that the cut17+ gene is identical to bir1+/pbh1+. At 26 degrees C, cut17-275 is UV sensitive as the repair of DNA damage is severely compromised. Bir1/Cut17 is a nuclear protein in interphase, which is then required for recruiting condensin to the mitotic nucleus, and concentrates to form a discrete number of dots from prometaphase to metaphase. Once the chromatids are separated, Bir1/Cut17 no longer binds to kinetochores and instead moves to the middle of spindle. Chromatin immunoprecipitation suggested that Bir1/Cut17 associates with the outer repetitious centromere region in metaphase. Following the initiation of anaphase the protein switches from being a chromosomal protein to a spindle protein. This transit is stringently regulated by the state of sister chromatid cohesion proteins Mis4 and Rad21. Ark1, is an aurora kinase homologue whose mitotic distribution is identical to, and under the control of Bir1/Cut17. CONCLUSIONS: Bir1/Cut17 and Ark1 act as "passengers" but they may play a main role as a recruitment factor, essential for condensation, spindle elongation and DNA repair. Bir1/Cut17 should have roles both in mitotic and in interphase chromosome. The proper location of Ark1 requires Bir1/Cut17, and the mitotic localization of Bir1/Cut17 requires sister cohesion.     

Fission yeast APC/cyclosome subunits, Cut20/Apc4 and Cut23/Apc8, in regulating metaphase-anaphase progression and cellular stress responses

BACKGROUND: The 20S cyclosome/APC complex promotes metaphase-anaphase transition by ubiquitinating its specific substrates such as mitotic cyclins and anaphase inhibitor Cut2/Pds1/securin. The complex has been shown to contain more than 10 proteins in budding yeast and frog. In fission yeast, however, only five (Cut4, Cut9, Nuc2, Apc10, Hcn1) have been identified. RESULTS: More than five hundred temperature-sensitive mutants were screened for identifying those defective in mitotic anaphase. Fifty-five showed the cut (cell untimely torn) phenotype or metaphase-arrest phenotypes, 27 of them locating at new loci. Their extracts were run in sucrose gradient centrifugation, and four showed alterations in the sedimentation profiles. The gene products of cut20+ and cut23+ were thus identified. Phenotypes of cut20-100 mutant highly resemble cut4-533 in many ways: they are hypersensitive to canavanine and CdCl2, and suppressed by PKA-inactivating regulators, cAMP-dependent phosphodiesterase and PKA regulatory subunits. Cut20 interacts closely with Cut4 in the assembly process of cyclosome. But cut20 mutant differs from cut4, as a novel gene stw1+ suppresses cut20 mutant but not cut4. cut23-194 mutant cells are sterile and blocked at metaphase, but does not show sensitivity to the stress and cAMP. TPR repeat-containing Cut23 may not be the stable component of APC/cyclosome, and its level significantly fluctuates during cell cycle. Cut23 may be ubiquitinated and degraded in a cell cycle dependent fashion. CONCLUSIONS: We identified two new subunits of fission yeast cyclosome/APC complex. Our observations indicate that cyclosome components are divided into several subgroups with distinctly different roles.     

Faithful anaphase is ensured by Mis4, a sister chromatid cohesion molecule required in S phase and not destroyed in G1 phase

The loss of sister chromatid cohesion triggers anaphase spindle movement. The budding yeast Mcd1/Scc1 protein, called cohesin, is required for associating chromatids, and proteins homologous to it exist in a variety of eukaryotes. Mcd1/Scc1 is removed from chromosomes in anaphase and degrades in G₁. We show that the fission yeast protein, Mis4, which is required for equal sister chromatid separation in anaphase is a different chromatid cohesion molecule that behaves independent of cohesin and is conserved from yeast to human. Its inactivation in G₁ results in cell lethality in S phase and subsequent premature sister chromatid separation. Inactivation in G₂ leads to cell death in subsequent metaphase–anaphase progression but missegregation occurs only in the next round of mitosis. Mis4 is not essential for condensation, nor does it degrade in G₁. Rather, it associates with chromosomes in a punctate fashion throughout the cell cycle. mis4 mutants are hypersensitive to hydroxyurea (HU) and UV irradiation but retain the ability to restrain cell cycle progression when damaged or sustaining a block to replication. The mis4 mutation results in synthetic lethality with a DNA ligase mutant. Mis4 may form a stable link between chromatids in S phase that is split rather than removed in anaphase.     

Degradation of Drosophila PIM regulates sister chromatid separation during mitosis

Drosophila Pimples (PIM) and Three rows (THR) are required for sister chromatid separation in mitosis. PIM accumulates during interphase and is degraded rapidly during mitosis. This degradation is dependent on a destruction box similar to that of B-type cyclins. Nondegradable PIM with a mutant destruction box can rescue sister chromatid separation in pim mutants but only when expressed at low levels. Higher levels of nondegradable PIM, as well as overexpression of wild-type PIM, inhibit sister chromatid separation. Moreover, cells arrested in mitosis before sister chromatid separation (by colcemid or by mutations in fizzy/CDC20) fail to degrade PIM. Thus, although not related by primary sequence, PIM has intriguing functional similarities to the securin proteins of budding yeast, fission yeast, and vertebrates. Whereas these securins are known to form a complex with separins, we show that PIM associates in vivo with THR, which does not contain the conserved separin domain.     

Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase

Cohesin complex acts in the formation and maintenance of sister chromatid cohesion during and after S phase. Budding yeast Scc1p/Mcd1p, an essential subunit, is cleaved and dissociates from chromosomes in anaphase, leading to sister chromatid separation. Most cohesin in higher eukaryotes, in contrast, is dissociated from chromosomes well before anaphase. The universal role of cohesin during anaphase thus remains to be determined. We report here initial characterization of four putative cohesin subunits, Psm1, Psm3, Rad21, and Psc3, in fission yeast. They are essential for sister chromatid cohesion. Immunoprecipitation demonstrates stable complex formation of Rad21 with Psm1 and Psm3 but not with Psc3. Chromatin immunoprecipitation shows that cohesin subunits are enriched in broad centromere regions and that the level of centromere-associated Rad21 did not change from metaphase to anaphase, very different from budding yeast. In contrast, Rad21 containing similar cleavage sites to those of Scc1p/Mcd1p is cleaved specifically in anaphase. This cleavage is essential, although the amount of cleaved product is very small (<5%). Mis4, another sister chromatid cohesion protein, plays an essential role for loading Rad21 on chromatin. A simple model is presented to explain the specific behavior of fission yeast cohesin and why only a tiny fraction of Rad21 is sufficient to be cleaved for normal anaphase.     

Securin can have a separase cleavage site by substitution mutations in the domain required for stabilization and inhibition of separase

Securin-separase complex is required for sister chromatid separation. Securin degrades in an APC/cyclosome dependent manner. Separase is activated on the destruction of securin and cleaves cohesin. Fission yeast securin/Cut2 required for proper separase localization has the motifs for destruction and separase-binding at the N- and C-termini, respectively. We report here the third essential domain, which becomes toxic when the 76-amino acid fragment (81-156) in the middle is overproduced. The fragment inhibits separase, while separase is recruited normally and securin is destroyed. It may interfere with separase activation after destruction of securin. If the 127DIE129 stretch is substituted for AIA, the fragment toxicity and the full-length function are abolished. Interestingly, Cut2 is cleaved in a separase dependent manner if the cleavage consensus is introduced following the DIE sequence. This finding is consistent with the proposed model that the DIE region may mimic the cleavage site of separase and inhibit the activation of separase. Evidence for physical interaction between the fragment and separase is provided. A temperature sensitive mutation cut1-K73 isolated by its specific resistance to the fragment toxicity resides in the superhelical region of separase, suggesting that the catalytic site and the helical region in separase may cooperate for activation.     

A brute force postgenome approach to identify temperature-sensitive mutations that negatively interact with separase and securin plasmids

BACKGROUND: The fission yeast Schizosaccharomyces pombe separase/Cut1 and securin/Cut2 are required for anaphase-specific activation of proteolysis that leads to proper sister chromatid separation. We intended to identify ts (temperature sensitive) strains whose growth was inhibited by multicopy plasmid pCUT1 or pCUT2 at the permissive temperature. RESULTS: After a one-by-one transformation of 1015 randomly isolated ts strains, 18 transformants that retarded in colony formation at the permissive or semipermissive temperature were isolated. Six of them, in the absence of pCUT1 or pCUT2, produced mitotic phenotypes with condensed chromosomes at the restrictive temperature. Gene cloning established that these mutants were defective in either the subunits (Cut9, Cut23, Cut20 or Apc10) of APC (anaphase promoting complex)/cyclosome or Cut8, a regulator for 26S proteasome localization. The inhibitory effect of separase against APC/cyclosome mutations was abolished when the catalytic site mutation C1730A was introduced and overproduced, indicating that inhibition needs an active separase. Securin/Cut2 overproduction also caused a negative effect on these mutants. Surprisingly, the phenotypes of cut9 and cut23 in the presence of pCUT1 or pCUT2 were not the mitotic arrest, and they were strikingly different depending on pCUT1 or pCUT2. CONCLUSIONS: This study shows the functional link between separase/Cut1 and APC/cyclosome in a separase activity-dependent manner. The negative effect of active separase overproduction on APC/cyclosome mutations is possibly due to the direct inhibition of APC/cyclosome. In addition, the manner of the inhibition by high copy securin and separase plasmids were quite different each other and did not result in the mitotic block.     

Spo13 protects meiotic cohesin at centromeres in meiosis I

In the absence of Spo13, budding yeast cells complete a single meiotic division during which sister chromatids often separate. We investigated the function of Spo13 by following chromosomes tagged with green fluorescent protein. The occurrence of a single division in spo13Delta homozygous diploids depends on the spindle checkpoint. Eliminating the checkpoint accelerates meiosis I in spo13Delta cells and allows them to undergo two divisions in which sister chromatids often separate in meiosis I and segregate randomly in meiosis II. Overexpression of Spo13 and the meiosis-specific cohesin Rec8 in mitotic cells prevents separation of sister chromatids despite destruction of Pds1 and activation of Esp1. This phenotype depends on the combined overexpression of both proteins and mimics one aspect of meiosis I chromosome behavior. Overexpressing the mitotic cohesin, Scc1/Mcd1, does not substitute for Rec8, suggesting that the combined actions of Spo13 and Rec8 are important for preventing sister centromere separation in meiosis I.     

Phosphorylation of the mitotic regulator Pds1/securin by Cdc28 is required for efficient nuclear localization of Esp1/separase

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. Here we show that 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. Our 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.     

Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage

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 Pds1, whereas mitotic exit requires the destruction of mitotic cyclins and the inactivation of Cdk1. We find that 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. We speculate 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.     

Localization of human SMC1 protein at kinetochores

Proper cohesion of sister chromatids is prerequisite for correct segregation of chromosomes during cell division. The cohesin multiprotein complex, conserved in eukaryotes, is required for sister chromatid cohesion. Human cohesin is composed of a stable heterodimer of the structural maintenance of chromosomes (SMC) family proteins, hSMC1 and hSMC3, and non-SMC components, hRAD21 and SA1 (or SA2). In yeast, cohesin associates with chromosomes from late G1 to metaphase and is required for the establishment and maintenance of both chromosome arm and centromeric cohesion. However, in human cells, the majority of cohesin dissociates from chromosomes before mitosis. Although it was recently shown that a small amount of hRAD21 localizes to the centromeres during metaphase, the presence of other cohesin components at the centromere has not been demonstrated in human cells. Here we report the mitosis-specific localization of hSMC1 to the kinetochores. hSMC1 is targeted to the kinetochore region during prophase concomitant with kinetochore assembly and remains through anaphase. Importantly, hSMC1 is targeted only to the active centromere on dicentric chromosomes. These results suggest that hSMC1 is an integral component of the functional kinetochore structure during mitosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spo13 regulates cohesin cleavage

A key aspect of meiotic chromosome segregation is that cohesin, the protein complex that holds sister chromatids together, dissociates from chromosome arms during meiosis I and from centromeric regions during meiosis II. The budding yeast protein Spo13 plays a key role in preventing centromeric cohesin from being lost during meiosis I. We have determined the molecular basis for the metaphase arrest obtained when SPO13 is overexpressed during the mitotic cell cycle. Overexpression of SPO13 inhibits anaphase onset by at least two mechanisms. First, Spo13 causes a transient delay in degradation of the anaphase inhibitor Pds1. Second, Spo13 inhibits cleavage of the cohesin subunit Scc1/Mcd1 or its meiosis-specific homolog, Rec8, by the separase Esp1. The finding that Spo13 did not prevent cleavage of another Esp1 substrate, Slk19, suggests that overexpression of SPO13 is sufficient to prevent cohesin cleavage by protecting specific substrates from separase activity.     

Meikin‐associated polo‐like kinase specifies Bub1 distribution in meiosis I

In meiosis I, sister chromatids are captured by microtubules emanating from the same pole (mono‐orientation), and centromeric cohesion is protected throughout anaphase. Shugoshin, which is localized to centromeres depending on the phosphorylation of histone H2A by Bub1 kinase, plays a central role in protecting meiotic cohesin Rec8 from separase cleavage. Another key meiotic kinetochore factor, meikin, may regulate cohesion protection, although the underlying molecular mechanisms remain elusive. Here, we show that fission yeast Moa1 (meikin), which associates stably with CENP‐C during meiosis I, recruits Plo1 (polo‐like kinase) to the kinetochores and phosphorylates Spc7 (KNL1) to accumulate Bub1. Consequently, in contrast to the transient kinetochore localization of mitotic Bub1, meiotic Bub1 persists at kinetochores until anaphase I. The meiotic Bub1 pool ensures robust Sgo1 (shugoshin) localization and cohesion protection at centromeres by cooperating with heterochromatin protein Swi6, which binds and stabilizes Sgo1. Furthermore, molecular genetic analyses show a hierarchical regulation of centromeric cohesion protection by meikin and shugoshin that is important for establishing meiosis‐specific chromosome segregation. We provide evidence that the meiosis‐specific Bub1 regulation is conserved in mouse.     

Cohesin release is required for sister chromatid resolution,but not for condensin-mediated compaction,at the onset of mitosis

The establishment of metaphase chromosomes is an essential prerequisite of sister chromatid separation in anaphase. It involves the coordinated action of cohesin and condensin, protein complexes that mediate cohesion and condensation, respectively. In metazoans, most cohesin dissociates from chromatin at prophase, coincident with association of condensin. Whether loosening of cohesion at the onset of mitosis facilitates the compaction process, resolution of the sister chromatids, or both, remains unknown. We have found that the prophase release of cohesin is completely blocked when two mitotic kinases, aurora B and polo-like kinase (Plx1), are simultaneously depleted from Xenopus egg extracts. Condensin loading onto chromatin is not affected under this condition, and rod-shaped chromosomes are produced that show an apparently normal level of compaction. However, the resolution of sister chromatids within these chromosomes is severely compromised. This is not because of inhibition of topoisomerase II activity that is also required for the resolution process. We propose that aurora B and Plx1 cooperate to destabilize the sister chromatid linkage through distinct mechanisms that may involve phosphorylation of histone H3 and cohesin, respectively. More importantly, our results strongly suggest that cohesin release at the onset of mitosis is essential for sister chromatid resolution but not for condensin-mediated compaction.     

An interactive gene network for securin-separase, condensin, cohesin, Dis1/Mtc1 and histones constructed by mass transformation

The small genome of fission yeast Schizosaccharomyces pombe contains 4824 predicted genes and gene disruption suggests that approximately 850 are essential for viability. To obtain information on interactions among genes required for chromosome segregation, an approach called Strategy B was taken using mass transformation of the 1015 temperature-sensitive (ts) mutants that were made by random mutagenesis and transformed by plasmids carrying the genes for securin, separase, condensin, cohesin, kinetochore microtubule-binding proteins Dis1/Mtc1 or histones. Mutant strains whose phenotypes were either suppressed or inhibited by plasmids were selected. Each plasmid interacted positively or negatively with the average 14 strains. Identification of the mutant gene products by cloning revealed many hitherto unknown interactions. The interactive networks of segregation therefore may consist of genes with a variety of functions. For example, separase/Cut1 interacts with Cdc48/p97/VCP, which stabilizes securin and separase. Surprisingly, S. pombe cdc48 mutants displayed the mitotic phenotype highly similar to separase/cut1 mutants. This approach also provides a novel way of mutant isolation, resulting in two histone H2B strains and a cohesion mutant with a new phenotype.     

Releasing cohesin from chromosome arms in early mitosis: opposing actions of Wapl–Pds5 and Sgo1

The cohesin complex establishes sister chromatid cohesion during S phase. In metazoan cells, most if not all cohesin dissociates from chromatin during mitotic prophase, leading to the formation of metaphase chromosomes with two cytologically discernible chromatids. This process, known as sister chromatid resolution, is believed to be a prerequisite for synchronous separation of sister chromatids in subsequent anaphase. To dissect this process at a mechanistic level, we set up an in vitro system. Sister chromatid resolution is severely impaired upon depletion of Wapl from Xenopus egg extracts. Exogenously added human Wapl can rescue these defects and, remarkably, it can do so in a very short time window of early mitosis. A similar set of observations is made for Pds5, a factor implicated previously in the stabilization of interphase cohesion. Characteristic amino acid motifs (the FGF motifs) in Wapl coordinate its physical and functional interactions with Pds5 and cohesin subunits. We propose that Wapl and Pds5 directly modulate conformational changes of cohesin to make it competent for dissociation from chromatin during prophase. Evidence is also presented that Sgo1 plays a hitherto underappreciated role in stabilizing cohesin along chromosome arms, which is antagonized by the mitotic kinases polo-like kinsase (Plk1) and aurora B.     

Time course analysis of precocious separation of sister centromeres in budding yeast: continuously separated or frequently reassociated?

BACKGROUND: Sister kinetochores are bioriented toward the spindle poles in eukaryotic metaphase before chromosome segregation. In the budding yeast Saccharomyces cerevisiae, sister centromeres/kinetochores are separated in the early spindle, while the sister arms remain associated. Biorientation is thought to be established in this organism with precocious separation of sister centromeres in early stages of the cell cycle. It is not, however, settled whether this pre-anaphase separation is continuous or only transient and whether the transient separation has any physiological significance. RESULTS: Time-lapse observation of the behaviour of budding yeast centromeres in living cells was performed using GFP alone or in combination with CFP marking. Sixty-three per cent of the cell population showed permanent separation of centromeres for a long period of time from the small-budded stage to the onset of anaphase in the single-colour GFP-CEN construct. The remaining cell population (6 of 16) showed brief apparent reassociation of centromere signals before anaphase, but the frequency of the association was very low. In a time-lapse observation of the double-colour marked cells by GFP-CEN and CFP-SPB (the spindle pole body), the continuous separation of sister centromeres in the short medial spindle was firmly established. CONCLUSIONS: In the budding yeast, once sister centromeres separate, they rarely reassociate in pre-anaphase. Sister centromere cohesion at this stage appears to be irrelevant for normal chromosome segregation. Whether abundant cohesin in the centromere regions has any role in anaphase remains to be determined.