Preimplantation Mouse Embryos Depend on Inhibitory Phosphorylation of Separase To Prevent Chromosome Missegregation

Abstract

Separase is a critical protease that catalyzes the cleavage of sister chromatid cohesins to allow the separation of sister chromatids in the anaphase. Its activity must be inhibited prior to the onset of the anaphase. Two inhibitory mechanisms exist in vertebrates that block the protease activity. One mechanism is through binding and inhibition by securin, and another is phosphorylation on Ser1126 (in humans [Ser1121 in mice]). These two mechanisms are largely redundant. However, phosphorylation on Ser1121 is critical for the prevention of premature sister separation in embryonic germ cells. As a result, Ser1121-to-Ala mutation leads to depletion of germ cells in development and subsequently to infertility in mice. Here, we report that the same mutation also causes embryogenesis failure between the 8-and 16-cell stages in mice. Our results indicate a critical role of separase phosphorylation in germ cell development as well as in early embryogenesis. Thus, deregulation of separase may be a significant contributor to infertility in humans.Sister chromatids are held together by a multisubunit complex called cohesin composed of . To separate the sister chromosomes, cohesin complexes are removed in a two-step process. First, cohesins on chromosome arms are removed by Plk1-and Aurora B-mediated phosphorylation before the anaphase (4,8,19,20,31,35). Second, the centromere-localized cohesins, which are protected by Sgo and PP2A from phosphorylation-mediated removal (13,21,28,29,32), are cleaved by a protease called separase at the onset of the anaphase (33, 34). Prior to the anaphase, separase is inhibited by securin and by phosphorylation, which is most likely catalyzed by cyclin B1/Cdk1. phosphorylation by cyclin B1/Cdk1 per se is not inhibitory to separase. Rather, the phosphorylation allows the binding of cyclin B1/Cdk1 as an inhibitor to separase (5). Two phosphorylation sites in separase, Ser1126 and Thr1326 (Ser1121 and Thr1321 in mice, respectively), that are important for the inhibition have been identified (30). Activation of separase depends on the function of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), since both securin and cyclin B1 are substrates of APC/C (1-3, 15,16,23,25,27,36). Given that APC/C is inhibited by the spindle assembly checkpoint, separation of sister chromatids therefore cannot occur until the checkpoint is satisfied. Thus, the spindle assembly checkpoint prevents premature sister separation and ensures chromosomal stability.Missegregation of chromosomes has dire consequences. It causes genetic imbalances that may transform cells and lead to cancer development in somatic tissues. In germ lines, missegregation in either meiosis I, mainly manifested as nondisjunction of homologue chromosomes, or meiosis II, manifested as premature sister chromatid separation, will generate aneuploid gametes, directly affecting the fecundity of an organism (26,37). Although the molecular mechanisms underlying chromosome segregation errors in meiosis are still not clear (6), deregula...

Protein phosphatase 5 is a negative regulator of separase function during cortical granule exocytosis inC. elegans

“…This persistent phosphorylation might affect enzymatic activity, protein-protein interactions, or subcellular localizations of proteins in the SEP-1 pathway. There are examples of phosphorylation playing such roles for many components of the separase pathway including cohesin, Cdc27 and separase itself (Huang et al, 2007;Huang et al, 2009;Kudo et al, 2009). Our finding that the reduced activity of pph-5 restores CG localization of SEP-1 in sep-1(ax110) embryos suggests that the subcellular localization of SEP-1 to CGs might be regulated by phosphorylation.…”

Positive and Negative Regulation of Vertebrate Separase by Cdk1-Cyclin B1 May Explain Why Securin Is Dispensable

“…In some organisms such as yeast, the Cdk1-cyclin B1-dependent separase regulation appears to have been totally lost during evolution. However, as mouse genetics teach us, this form of anaphase control remains essential in mammals until today (19,20). …”

“…Although Cdk1-cyclin B1 seems able to always compensate for the loss of securin, securin cannot always substitute for Cdk1-cyclin B1 in separase regulation. For unknown reasons, murine early embryonic and post-migratory primordial germ cells express only little securin and fully rely on Cdk1-cyclin B1-dependent control of separase (19,20). Despite the importance of this securin-independent control of anaphase, it remains enigmatic whether Cdk1-cyclin B1, similar to securin, might exert not only a negative but simultaneously also a positive effect on separase.…”

Identification of miRNAs associated with the follicular–luteal transition in the ruminant ovary

“…In addition, Dicer1 hypomorphism was associated with impaired corpus luteum (CL) function resulting from defects in angiogenesis (Otsuka et al 2008). Genome-wide miRNA expression has also been examined in whole ovaries from mice (Ro et al 2007, Mishima et al 2008, cattle (Hossain et al 2009, Huang et al 2009, Tripurani et al 2010 and pig . While these studies identified novel and ovarian-specific miRNA species, comparisons across different developmental stages were not performed.…”