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In meiosis, two specialized cell divisions allow the separation of paired chromosomes first, then of sister chromatids. Separase removes the cohesin complex holding sister chromatids together in a stepwise manner from chromosome arms in meiosis I, then from the centromere region in meiosis II. Using mouse oocytes, our study reveals that cyclin A2 promotes entry into meiosis, as well as an additional unexpected role; namely, its requirement for separase-dependent sister chromatid separation in meiosis II. Untimely cyclin A2-associated kinase activity in meiosis I leads to precocious sister separation, whereas inhibition of cyclin A2 in meiosis II prevents it. Accordingly, endogenous cyclin A is localized to kinetochores throughout meiosis II, but not in anaphase I. Additionally, we found that cyclin B1, but not cyclin A2, inhibits separase in meiosis I. These findings indicate that separase-dependent cohesin removal is differentially regulated by cyclin B1 and A2 in mammalian meiosis., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Proteolytic activity of separase is required for chiasma resolution during meiosis I in mouse oocytes. Rec8, the meiosis-specific alpha-kleisin subunit of cohesin, is a key target of separase in yeast. Is the equivalent protein also a target in mammals? We show here that separase cleaves mouse Rec8 at three positions in vitro but only when the latter is hyper-phosphorylated. Expression of a Rec8 variant (Rec8-N) that cannot be cleaved in vitro at these sites causes sterility in male mice. Their seminiferous tubules lack a normal complement of 2 C secondary spermatocytes and 1 C spermatids and contain instead a high proportion of cells with enlarged nuclei. Chromosome spreads reveal that Rec8-N expression has no effect in primary spermatocytes but produces secondary spermatocytes and spermatids with a 4 C DNA content, suggesting that the first and possibly also the second meiotic division is abolished. Expression of Rec8-N in oocytes causes chromosome segregation to be asynchronous and delays its completion by 2-3 hours during anaphase I, probably due to inefficient proteolysis of Rec8-N by separase. Despite this effect, chromosome segregation must be quite accurate as Rec8-N does not greatly reduce female fertility. Our data is consistent with the notion that Rec8 cleavage is important and probably crucial for the resolution of chiasmata in males and females.

Sister chromatids are held together by the ring-shaped cohesin complex, which likely entraps both DNA-double strands in its middle. This tie is resolved in anaphase when separase, a giant protease, becomes active and cleaves the kleisin subunit of cohesin. Premature activation of separase and, hence, chromosome missegregation are prevented by at least two inhibitory mechanisms. Although securin has long been appreciated as a direct inhibitor of separase, surprisingly its loss has basically no phenotype in mammals. Phosphorylation-dependent binding of Cdk1 constitutes an alternative way to inhibit vertebrate separase. Its importance is illustrated by the premature loss of cohesion when Cdk1-resistant separase is expressed in mammalian cells without or with limiting amounts of securin. Here, we demonstrate that crucial inhibitory phosphorylations occur within a region of human separase that is also shown to make direct contact with the cyclin B1 subunit of Cdk1. This region exhibits a weak homology to Saccharomyces cerevisiae Cdc6 of similar Cdk1 binding behavior, thereby establishing phosphoserine/threonine-mediated binding of partners as a conserved characteristic of B-type cyclins. In contrast to the Cdc6-like domain, the previously identified serine 1126 phosphorylation is fully dispensable for Cdk1 binding to separase fragments. This suggests that despite its in vivo relevance, it promotes complex formation indirectly, possibly by inducing a conformational change in full-length separase.

The onset of anaphase is triggered by the activation of a site-specific protease called separase. Separase cleaves the chromosomal cohesins holding the duplicated sister chromatids together, allowing sisters to simultaneously separate and segregate to opposite ends of the cell before division. Activated separase cleaves not only cohesin, but also itself; however, the biological significance of separase self-cleavage has remained elusive. Before anaphase, separase is inhibited by at least two mechanisms. The first involves the binding of securin, whereas the second requires the phosphorylation-dependent binding of cyclin-dependent kinase 1 (Cdk1)/cyclin B1. Because securin and Cdk1/cyclin B1 interact with separase in a mutually exclusive manner, the degradation of both these inhibitors plays an important role in activating separase at anaphase. Here we identify a new separase interacting partner, a specific subtype of the heterotrimeric protein phosphatase 2A (PP2A). PP2A associates with separase through the B' (B56) regulatory subunit and does so independently of securin and cyclin B1 binding. The association of PP2A with separase requires a 55-amino acid domain closely juxtaposed to separase autocleavage sites. Strikingly, mutation of these cleavage sites increases PP2A binding, suggesting that separase cleavage disrupts the interaction of PP2A with separase. Furthermore, expression of a non-cleavable separase, but not a non-cleavable mutant that cannot bind PP2A, causes a premature loss of centromeric cohesion. Together these observations provide a new mechanistic insight into a physiological function for separase self-cleavage.

Separase not only triggers anaphase of meiosis I by proteolytic cleavage of cohesin on chromosome arms, but in vitro vertebrate separase also acts as a direct inhibitor of cyclin-dependent kinase 1 (Cdk1) on liberation from the inhibitory protein, securin. Blocking separase-Cdk1 complex formation by microinjection of anti-separase antibodies prevents polar-body extrusion in vertebrate oocytes. Importantly, proper meiotic maturation is rescued by chemical inhibition of Cdk1 or expression of Cdk1-binding separase fragments lacking cohesin-cleaving activity.

Chromosome segregation in mitosis and meiosis is triggered by activation of a large protease, separase. While it has been known for some time that the anaphase inhibitor securin regulates separase activity recent work shows that this is only half the story. In vertebrates Cdk1-dependent inhibition of separase represents a second, securin-independent branch of anaphase regulation. Furthermore, an unanticipated ability of separase to inhibit Cdk1 suggests additional, nonproteolytic functions of separase.

After the degradation of its inhibitor securin, separase initiates chromosome segregation during the metaphase-to-anaphase transition by cleaving cohesin. Here we present a density map at a resolution of 25 A of negatively stained separase-securin complex. Based on labeling data and sequence analysis, we propose a model for the structure of separase, consisting of 26 ARM repeats, an unstructured region of 280 residues and two caspase-like domains, with securin binding to the ARM repeats.

Cleavage of the ring-like cohesin complex by separase triggers segregation of sister chromatids in anaphase. This simplistic model has recently been extended by exciting discoveries on three levels: regulation of anaphase by posttranslational modifications and the cohesin protector shugoshin; non-proteolytic roles of separase; and cohesin-independent linkage of sister chromatids.

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, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that 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, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.