Inhibitory phosphorylation of separase is essential for genome stability and viability of murine embryonic germ cells

Activity of separase, a cysteine protease that cleaves sister chromatid cohesin at the onset of anaphase, is tightly regulated to ensure faithful chromosome segregation and genome stability. Two mechanisms negatively regulate separase: inhibition by securin and phosphorylation on serine 1121. To gauge the physiological significance of the inhibitory phosphorylation, we created a mouse strain in which Ser1121 was mutated to Ala (S1121A). Here we report that this S1121A point mutation causes infertility in mice. We show that germ cells in the mutants are depleted during development. We further demonstrate that S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death. Our results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells.     

Securin is not required for cellular viability, but is required for normal growth of mouse embryonic fibroblasts

Sister chromatid separation depends on the release of cohesion by the activity of Esp1, a member of the caspase family [1, 2]. In budding yeast, Esp1p is kept inactive by its association with Pds1p, until the onset of anaphase, when Pds1p is ubiquitinated by the APC/Cdc20 complex [3--5] and subsequently degraded by the 26S proteasome. Pds1 is not an essential gene in budding yeast, but is required for cell cycle arrest prior to anaphase in response to the disruption of spindle structures [6, 7]. Thus, Pds1 mutant yeast cells display precocious sister chromatid separation in the presence of nocodazole [6]. Mammalian orthologs of yeast Esp1 and Pds1, separin and securin, have been identified [8], and, as anticipated, a nondegradable mutant form of securin inhibits sister separation when added to mitotic Xenopus egg extracts [8]. Securin was also independently identified as PTTG (pituitary tumor transforming gene), a gene overexpressed in pituitary tumors [9]. The relationship between its overexpression in tumors and its control of sister chromatid cohesion remains ill defined. To explore securin function in mammals, we took a targeted gene disruption approach in mice. Here, we report that securin is neither essential for cell viability nor required for spindle checkpoint function, and mice lacking securin are viable and apparently normal, but mouse embryonic fibroblasts lacking securin grow abnormally in culture.     

Separase is Required at Multiple Pre-Anaphase Cell Cycle Stages in Human Cells

The yeast separase proteins Esp1 and Cut1 are required for loss of sister chromatid cohesion that occurs at the moment of anaphase onset. Circumstantial evidence has linked human separase to centromere separation at anaphase, but a direct test that the role of this enzyme is functionally conserved with the yeast proteins is lacking. Here we describe the effects of separase depletion from human cells using RNA interference. Surprisingly, HeLa cells lacking separase are delayed or arrest at the G2/M phase transition. This arrest is not likely due to the activation of a known checkpoint control, but may be a result of a failure to construct a mitotic chromosome. Without separase, cells also have a prolonged prometaphase, perhaps resulting from defects in spindle assembly or dynamics. In cells that reach mitosis, sister arm resolution and separation are perturbed, whereas in anaphase cells sister centromeres do appear to separate. These data indicate that separase function is not restricted to anaphase initiation and that its role in promoting loss of sister chromatid cohesion might be preferentially at arms but not centromeres.     

Requirement of chromatid cohesion proteins rad21/scc1 and mis4/scc2 for normal spindle-kinetochore interaction in fission yeast

BACKGROUND: Proteins conserved from yeast to human hold two sister chromatids together. The failure to form cohesion in the S phase results in premature separation of chromatids in G2/M. Mitotic kinetochores free from microtubules or the lack of tension are known to activate spindle checkpoint. RESULTS: The loss of chromatid cohesion in fission yeast mutants (mis4-242 and rad21-K1) leads to the activation of Mad2- and Bub1-dependent checkpoint, possibly due to a diminished microtubule-kinetochore interaction. Bub1, a checkpoint kinase, localizes briefly at early mitotic kinetochores in wild-type, whereas the cohesion mutation greatly increases the duration of kinetochore localization. Bub1 is bound to the central centromere region of mitotic cells. These cohesion mutants are hypersensitive to a tubulin poison and are synthetic lethal with dis1 and bir1/cut17, which are defective in microtubule-kinetochore interaction. The formation of specialized centromere chromatin containing CENP-A does not require cohesion. Dominant-negative noncleavable Rad21 fails to activate checkpoint but blocks sister chromatid separation and full spindle elongation in anaphase. CONCLUSIONS: Mis4 and Rad21 (budding yeast Scc2 and Scc1 homologs, respectively) act in establishing the normal spindle-kinetochore interaction in early mitosis and inhibit sister chromatid separation until the cleavage of Rad21 in anaphase. Checkpoint directly or indirectly monitors the states of cohesion in early mitosis. Full spindle extension occurs with unequal nuclear division in cohesion mutants in the absence of Mad2.     

Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast.

At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.     

Proteasome Activity is Required for Centromere Separation Independently of Securin Degradation in Human Cells

Loss of centromere cohesion during anaphase in human cells is regulated by the spindle assembly checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). APC-Cdc20 adds ubiquitin chains to securin inducing its destruction by the proteasome and these events correlate with the loss of sister chromatid cohesion and the onset of anaphase. But whether securin destruction is necessary and sufficient for anaphase initiation is not clear. Therefore, we asked if proteasome activity is needed for anaphase onset in human cells that lack securin. We find that even in the absence of securin, a metaphase block with cohered sister centromeres can be enforced in the absence of proteasome activity. Therefore, other targets of the proteasome must be degraded to allow anaphase onset.     

The budding yeast PP2ACdc55 protein phosphatase prevents the onset of anaphase in response to morphogenetic defects

Faithful chromosome transmission requires establishment of sister chromatid cohesion during S phase, followed by its removal at anaphase onset. Sister chromatids are tethered together by cohesin, which is displaced from chromosomes through cleavage of its Mcd1 subunit by the separase protease. Separase is in turn inhibited, up to this moment, by securin. Budding yeast cells respond to morphogenetic defects by a transient arrest in G2 with high securin levels and unseparated chromatids. We show that neither securin elimination nor forced cohesin cleavage is sufficient for anaphase in these conditions, suggesting that other factors contribute to cohesion maintainance in G2. We find that the protein phosphatase PP2A bound to its regulatory subunit Cdc55 plays a key role in this process, uncovering a new function for PP2A(Cdc55) in controlling a noncanonical pathway of chromatid cohesion removal.     

Shugoshin: guardian spirit at the centromere

A recently emerging protein family, shugoshin, plays a crucial role in the centromeric protection of cohesin, which is responsible for sister chromatid cohesion. This is especially important at the first meiotic division, where cohesin is cleaved by separase only along chromosome arms while the centromeric cohesin must be preserved. In vertebrate cells, arm cohesion is largely lost during prophase and prometaphase in order to facilitate sister chromatid resolution, whereas centromeric cohesion is preserved until the bipolar attachment of sister chromatids is established. Vertebrate shugoshin plays an essential role in protecting centromeric cohesin from prophase dissociation. In yeast, shugoshin also has a crucial role in sensing the loss of tension at kinetochores and in generating the spindle checkpoint signal.     

Regulation of human separase by securin binding and autocleavage

BACKGROUND: Sister chromatid separation is initiated by separase, a protease that cleaves cohesin and thereby dissolves sister chromatid cohesion. Separase is activated by the degradation of its inhibitor securin and by the removal of inhibitory phosphates. In human cells, separase activation also coincides with the cleavage of separase, but it is not known if this reaction activates separase, which protease cleaves separase, and how separase cleavage is regulated.RESULTS: Inhibition of separase expression in human cells by RNA interference causes the formation of polyploid cells with large lobed nuclei. In mitosis, many of these cells contain abnormal chromosome plates with unseparated sister chromatids. Inhibitor binding experiments in vitro reveal that securin prevents the access of substrate analogs to the active site of separase. Upon securin degradation, the active site of full-length separase becomes accessible, allowing rapid autocatalytic cleavage of separase at one of three sites. The resulting N- and C-terminal fragments remain associated and can be reinhibited by securin. A noncleavable separase mutant retains its ability to cleave cohesin in vitro.CONCLUSIONS: Our results suggest that separase is required for sister chromatid separation during mitosis in human cells. Our data further indicate that securin inhibits separase by blocking the access of substrates to the active site of separase. Securin proteolysis allows autocatalytic processing of separase into a cleaved form, but separase cleavage is not essential for separase activation.     

A non-proteolytic function of separase links the onset of anaphase to mitotic exit

Separase is a protease that triggers chromosome segregation at anaphase onset by cleaving cohesin, the chromosomal protein complex responsible for sister chromatid cohesion. After anaphase, cells exit from mitosis; that is, they complete downregulation of cyclin-dependent kinase activity, undergo cytokinesis and enter G1 of the next cell cycle. Here we show that separase activation at the onset of anaphase is sufficient to promote release from the nucleolus and activation of the budding yeast phosphatase, Cdc14, a key step in mitotic exit. The ability of separase to activate Cdc14 is independent of its protease function but may involve promoting phosphorylation of the Cdc14 inhibitor Net1. This novel separase function is coregulated with its proteolytic activity by the separase inhibitor securin. This helps to explain the coupling of anaphase and mitotic exit--after securin degradation at anaphase onset, separase cleaves cohesin to trigger chromosome segregation and concurrently uses a non-proteolytic mechanism to initiate mitotic exit.     

Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast

At the onset of anaphase, a caspase-related protease (separase) destroys the link between sister chromatids by cleaving the cohesin subunit Scc1. During most of the cell cycle, separase is kept inactive by binding to an inhibitory protein called securin. Separase activation requires proteolysis of securin, which is mediated by an ubiquitin protein ligase called the anaphase-promoting complex. Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle. Though no longer regulated by this mitotic surveillance mechanism, sister separation remains tightly cell cycle regulated in yeast mutants lacking securin. We show here that the Polo/Cdc5 kinase phosphorylates serine residues adjacent to Scc1 cleavage sites and strongly enhances their cleavage. Phosphorylation of separase recognition sites may be highly conserved and regulates sister chromatid separation independently of securin.     

Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase

Sister chromatid cohesion is essential for cell viability. We have isolated a novel temperature-sensitive lethal mutant named eso1-H17 that displays spindle assembly checkpoint-dependent mitotic delay and abnormal chromosome segregation. At the permissive temperature, the eso1-H17 mutant shows mild sensitivity to UV irradiation and DNA-damaging chemicals. At the nonpermissive temperature, the mutant is arrested in M phase with a viability loss due to a failure to establish sister chromatid cohesion during S phase. The lethal M-phase arrest phenotype, however, is suppressed by inactivation of a spindle checkpoint. The eso1(+) gene is not essential for the onset and progression of DNA replication but has remarkable genetic interactions with those genes regulating the G(1)-S transition and DNA replication. The N-terminal two-thirds of Eso1p is highly homologous to DNA polymerase eta of budding yeast and humans, and the C-terminal one-third is homologous to budding yeast Eco1p (also called Ctf7p), which is required for the establishment of sister chromatid cohesion. Deletion analysis and determination of the mutation site reveal that the function of the Eco1p/Ctf7p-homologous domain is necessary and sufficient for sister chromatid cohesion. On the other hand, deletion of the DNA polymerase eta domain in Eso1p increases sensitivity to UV irradiation. These results indicate that Eso1p plays a dual role during DNA replication. The C-terminal region acts to establish sister chromatid cohesion, and the N-terminal region presumably catalyzes translesion DNA synthesis when template DNA contains lesions that block regular DNA replication.     

Reduced Mad2 expression keeps relaxed kinetochores from arresting budding yeast in mitosis

Chromosome segregation depends on the spindle checkpoint, which delays anaphase until all chromosomes have bound microtubules and have been placed under tension. The Mad1-Mad2 complex is an essential component of the checkpoint. We studied the consequences of removing one copy of MAD2 in diploid cells of the budding yeast, Saccharomyces cerevisiae. Compared to MAD2/MAD2 cells, MAD2/mad2Δ heterozygotes show increased chromosome loss and have different responses to two insults that activate the spindle checkpoint: MAD2/mad2Δ cells respond normally to antimicrotubule drugs but cannot respond to chromosomes that lack tension between sister chromatids. In MAD2/mad2Δ cells with normal sister chromatid cohesion, removing one copy of MAD1 restores the checkpoint and returns chromosome loss to wild-type levels. We conclude that cells need the normal Mad2:Mad1 ratio to respond to chromosomes that are not under tension.     

Role of the kinetochore protein Ndc10 in mitotic checkpoint activation in Saccharomyces cerevisiae

Mitotic checkpoints delay cell cycle progression in response to alterations in the mitotic apparatus, thus ensuring correct chromosome segregation. While improper spindle orientation activates the Bub2/Bfa1-dependent checkpoint in budding yeast, delaying exit from mitosis, lack of bipolar kinetochore-microtubule attachment activates a signal transduction cascade that prevents both anaphase onset and exit from mitosis by inhibiting the Cdc20/APC (Anaphase Promoting Complex)-mediated proteolysis of securin and inactivation of mitotic cyclin-dependent kinases (CDKs), respectively. Proteolysis of the securin Pdsl is necessary to liberate the separase Esp1, which then triggers sister chromatid separation, whereas inactivation of mitotic CDKs is a prerequisite for exit from mitosis and for starting a new round of DNA replication in the next cell cycle. In budding yeast, this latter checkpoint response involves the proteins Mad1, 2, 3, Bub1 and Bub3, whose vertebrate counterparts localize to unattached kinetochores. Mutations that alter other kinetochore proteins result in mitotic checkpoint activation, while the ndc10-1 mutation not only impairs kinetochore function, but also disrupts the checkpoint response, indicating a role for Ndc10 in this process. Here we present evidence that Ndc10 is not part of the Bub2/Bfa1-dependent pathway, and its role in the checkpoint response might also be different from that of the other Mad and Bub proteins. Indeed, Ndc10, unlike other mitotic checkpoint proteins, is not required for the mitotic block induced by overexpression of the Mpsl protein kinase, which is implicated in mitotic checkpoint control. Furthermore, the delay in mitotic exit caused by non-degradable Pds1, which does not require Mad and Bub proteins, depends on Ndc10 function. We propose that a pathway involving Ndc10 might monitor defects in the mitotic apparatus independently of the Mad and Bub proteins. Since the Espl separase is required for exit from mitosis in both ndc10-1 and nocodazole-treated mad2delta cells, the two signal transduction cascades might ultimately converge on the inactivation of Esp1.     

Spindle formation, chromosome segregation and the spindle checkpoint in mammalian oocytes and susceptibility to meiotic error

The spindle assembly checkpoint (SAC) monitors attachment to microtubules and tension on chromosomes in mitosis and meiosis. It represents a surveillance mechanism that halts cells in M-phase in the presence of unattached chromosomes, associated with accumulation of checkpoint components, in particular, Mad2, at the kinetochores. A complex between the anaphase promoting factor/cylosome (APC/C), its accessory protein Cdc20 and proteins of the SAC renders APC/C inactive, usually until all chromosomes are properly assembled at the spindle equator (chromosome congression) and under tension from spindle fibres. Upon release from the SAC the APC/C can target proteins like cyclin B and securin for degradation by the proteasome. Securin degradation causes activation of separase proteolytic enzyme, and in mitosis cleavage of cohesin proteins at the centromeres and arms of sister chromatids. In meiosis I only the cohesin proteins at the sister chromatid arms are cleaved. This requires meiosis specific components and tight regulation by kinase and phosphatase activities. There is no S-phase between meiotic divisions. Second meiosis resembles mitosis. Mammalian oocytes arrest constitutively at metaphase II in presence of aligned chromosomes, which is due to the activity of the cytostatic factor (CSF). The SAC has been identified in spermatogenesis and oogenesis, but gender-differences may contribute to sex-specific differential responses to aneugens. The age-related reduction in expression of components of the SAC in mammalian oocytes may act synergistically with spindle and other cell organelles' dysfunction, and a partial loss of cohesion between sister chromatids to predispose oocytes to errors in chromosome segregation. This might affect dose-response to aneugens. In view of the tendency to have children at advanced maternal ages it appears relevant to pursue studies on consequences of ageing on the susceptibility of human oocytes to the induction of meiotic error by aneugens and establish models to assess risks to human health by environmental exposures.     

PP2A delays APC/C‐dependent degradation of separase‐associated but not free securin

The universal triggering event of eukaryotic chromosome segregation is cleavage of centromeric cohesin by separase. Prior to anaphase, most separase is kept inactive by association with securin. Protein phosphatase 2A (PP2A) constitutes another binding partner of human separase, but the functional relevance of this interaction has remained enigmatic. We demonstrate that PP2A stabilizes separase‐associated securin by dephosphorylation, while phosphorylation of free securin enhances its polyubiquitylation by the ubiquitin ligase APC/C and proteasomal degradation. Changing PP2A substrate phosphorylation sites to alanines slows degradation of free securin, delays separase activation, lengthens early anaphase, and results in anaphase bridges and DNA damage. In contrast, separase‐associated securin is destabilized by introduction of phosphorylation‐mimetic aspartates or extinction of separase‐associated PP2A activity. G2‐ or prometaphase‐arrested cells suffer from unscheduled activation of separase when endogenous securin is replaced by aspartate‐mutant securin. Thus, PP2A‐dependent stabilization of separase‐associated securin prevents precocious activation of separase during checkpoint‐mediated arrests with basal APC/C activity and increases the abruptness and fidelity of sister chromatid separation in anaphase.     

Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion

CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both exhibit genetic and physical ties to replication fork constituents. We find that absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint. The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion. We find that Ctf18p, an RFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of the budding yeast polymerase kappa, encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion. Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-C(CTF18) may be involved in a polymerase switch event that facilities sister chromatid cohesion. The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process.     

A role for the FEAR pathway in nuclear positioning during anaphase

In budding yeast, cells lacking separase function exit mitosis with an undivided nucleus localized to the daughter cell. Here we show that the inability to separate sister chromatids per se is not sufficient to cause the daughter preference. Rather, separase affects nuclear positioning as part of the Cdc14 early anaphase release (FEAR) pathway. The role of the FEAR pathway in nuclear positioning is exerted during anaphase and is not shared by the mitotic exit network. We find that the nuclear segregation defect in FEAR mutants does not stem from nonfunctional spindle poles or the absence of cytoplasmic microtubules. Instead, the concomitant inactivation of sister chromatid separation and the FEAR pathway uncovered a mother-directed force in anaphase that was previously masked by the elongating spindle. We propose that at anaphase onset, the FEAR pathway activates cytoplasmic microtubule-associated forces that facilitate chromosome segregation to the mother cell.     

Dynein participates in chromosome segregation in fission yeast

Background information. In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. For faithful segregation of sister chromatids, each sister kinetochore must attach to microtubules that extend to opposite poles (chromosome bi‐orientation). At the metaphase—anaphase transition, cohesion between sister chromatids is removed, and each sister chromatid is pulled to opposite poles of the cell by microtubule‐dependent forces. Results. We have studied the role of the minus‐end‐directed motor protein dynein by analysing kinetochore dynamics in fission yeast cells deleted for the dynein heavy chain (Dhc1) or the light chain (Dlc1). In these mutants, we found an increased frequency of cells showing defects in chromosome segregation, which leads to the appearance of lagging chromosomes and an increased rate of chromosome loss. By following simultaneously kinetochore dynamics and localization of the checkpoint protein Mad2, we provide evidence that dynein function is not necessary for spindle‐assembly checkpoint inactivation. Instead, we have demonstrated that loss of dynein function alters chromosome segregation and activates the Mad2‐dependent spindle‐assembly checkpoint. Conclusions. These results show an unexpected role for dynein in the control of chromosome segregation in fission yeast, most probably operating during the process of bi‐orientation during early mitosis.     

Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells

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 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. Our 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.