Dependency of the Spindle Assembly Checkpoint on Cdk1 Renders the Anaphase Transition Irreversible

SummaryActivation of anaphase-promoting complex/cyclosome (APC/CCdc20) by Cdc20 is delayed by the spindle assembly checkpoint (SAC). When all kinetochores come under tension, the SAC is turned off and APC/CCdc20 degrades cyclin B and securin, which activates separase [1]. The latter then cleaves cohesin holding sister chromatids together [2]. Because cohesin cleavage also destroys the tension responsible for turning off the SAC, cells must possess a mechanism to prevent SAC reactivation during anaphase, which could be conferred by a dependence of the SAC on Cdk1 [3–5]. To test this, we analyzed mouse oocytes and embryos expressing nondegradable cyclin B together with a Cdk1-resistant form of separase. After biorientation and SAC inactivation, APC/CCdc20 activates separase but the resulting loss of (some) cohesion is accompanied by SAC reactivation and APC/CCdc20 inhibition, which aborts the process of further securin degradation. Cyclin B is therefore the only APC/CCdc20 substrate whose degradation at the onset of anaphase is necessary to prevent SAC reactivation. The mutual activation of tension sensitive SAC and Cdk1 creates a bistable system that ensures complete activation of separase and total downregulation of Cdk1 when all chromosomes have bioriented

Acute and rapid degradation of endogenous proteins by Trim-Away.

Protein depletion is a key approach to understanding the functions of a protein in a biological system. We recently developed the Trim-Away approach in order to rapidly degrade endogenous proteins without prior modification. Trim-Away is based on the ubiquitin ligase and Fc receptor TRIM21, which recognizes antibody-bound proteins and targets them for degradation by the proteasome. In a typical Trim-Away experiment, protein degradation is achieved in three steps: first, introduction of an antibody against the target protein; second, recruitment of endogenous or exogenous/overexpressed TRIM21 to the antibody-bound target protein; and third, proteasome-mediated degradation of the target protein, antibody and TRIM21 complex. Protein degradation by Trim-Away is acute and rapid, with half-lives of ~10-20 min. The major advantages of Trim-Away over other protein degradation methods are that it can be applied to any endogenous protein without prior modification; that it uses conventional antibodies that are widely available; and that it can be applied to a wide range of cell types, including nondividing primary human cells, for which other loss-of-function assays are challenging. In this protocol, we describe the detailed procedures for antibody preparation and delivery in mouse oocytes and cultured cells via microinjection and electroporation. In addition, we provide recommendations for antibody selection and validation, and for the generation of TRIM21-overexpressing cell lines for cases in which endogenous TRIM21 is limited. A typical Trim-Away experiment takes just a few hours.The research leading to these results received financial support from the Medical Research Council (MC_U105192711 and MC_U105181010), the Max Planck Society, the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 241548, European Research Council (ERC) Starting Grant no. 337415 and a Wellcome Trust Investigator Award

Oocyte Development, Meiosis and Aneuploidy

AbstractMeiosis is one of the defining events in gametogenesis. Male and female germ cells both undergo one round of meiotic cell division during their development in order to reduce the ploidy of the gametes, and thereby maintain the ploidy of the species after fertilisation. However, there are some aspects of meiosis in the female germline, such as the prolonged arrest in dictyate, that appear to predispose oocytes to missegregate their chromosomes and transmit aneuploidies to the next generation. These maternally-derived aneuploidies are particularly problematic in humans where they are major contributors to miscarriage, age-related infertility, and the high incidence of Down's syndrome in human conceptions. This review will discuss how events that occur in foetal oocyte development and during the oocytes’ prolonged dictyate arrest can influence meiotic chromosome segregation and the incidence of aneuploidy in adult oocytes

Multiple duties for spindle assembly checkpoint kinases in meiosis

Cell division in mitosis and meiosis is governed by evolutionary highly conserved protein kinases and phosphatases, controlling the timely execution of key events such as nuclear envelope breakdown, spindle assembly, chromosome attachment to the spindle and chromosome segregation, and cell cycle exit. In mitosis, the spindle assembly checkpoint (SAC) controls the proper attachment to and alignment of chromosomes on the spindle. The SAC detects errors and induces a cell cycle arrest in metaphase, preventing chromatid separation. Once all chromosomes are properly attached, the SAC-dependent arrest is relieved and chromatids separate evenly into daughter cells. The signaling cascade leading to checkpoint arrest depends on several protein kinases that are conserved from yeast to man. In meiosis, haploid cells containing new genetic combinations are generated from a diploid cell through two specialized cell divisions. Though apparently less robust, SAC control also exists in meiosis. Recently, it has emerged that SAC kinases have additional roles in executing accurate chromosome segregation during the meiotic divisions. Here, we summarize the main differences between mitotic and meiotic cell divisions, and explain why meiotic divisions pose special challenges for correct chromosome segregation. The less-known meiotic roles of the SAC kinases are described, with a focus on two model systems: yeast and mouse oocytes. The meiotic roles of the canonical checkpoint kinases Bub1, Mps1, the pseudokinase BubR1 (Mad3), and Aurora B and C (Ipl1) will be discussed. Insights into the molecular signaling pathways that bring about the special chromosome segregation pattern during meiosis will help us understand why human oocytes are so frequently aneuploid

Rec8-containing cohesin maintains bivalents without turnover during the growing phase of mouse oocytes

During female meiosis, bivalent chromosomes are thought to be held together from birth until ovulation by sister chromatid cohesion mediated by cohesin complexes whose ring structure depends on kleisin subunits, either Rec8 or Scc1. Because cohesion is established at DNA replication in the embryo, its maintenance for such a long time may require cohesin turnover. To address whether Rec8- or Scc1-containing cohesin holds bivalents together and whether it turns over, we created mice whose kleisin subunits can be cleaved by TEV protease. We show by microinjection experiments and confocal live-cell imaging that Rec8 cleavage triggers chiasmata resolution during meiosis I and sister centromere disjunction during meiosis II, while Scc1 cleavage triggers sister chromatid disjunction in the first embryonic mitosis, demonstrating a dramatic transition from Rec8- to Scc1-containing cohesin at fertilization. Crucially, activation of an ectopic Rec8 transgene during the growing phase of Rec8TEV/TEV oocytes does not prevent TEV-mediated bivalent destruction, implying little or no cohesin turnover for ≥2 wk during oocyte growth. We suggest that the inability of oocytes to regenerate cohesion may contribute to age-related meiosis I errors