Comparative Analysis of Cohesin, Shugoshin and APC/C in Mitosis versus Meiosis

在女性减数分裂I期间同源染色体的错误分布是人类流产和智力低下的主要遗传原因。然而，基因组单倍体化的分子机制仍然知之甚少。我们建议研究染色体周期的三个关键参与者的新功能和调控：内聚蛋白（一种保持姐妹染色单体配对的环状蛋白质复合物），内聚蛋白（一种内聚保护蛋白）和APC/C（一种多亚基泛素连接酶或内聚拮抗剂，触发染色体分离）。我们之前已经表明，内聚蛋白和shugoshin也在中心体中起作用，这可能解释了中心体周期通常与染色体周期协调的原因。现在，我们将通过电子显微镜精细绘制内聚蛋白的中心体定位，试图确定其底物，并研究在精母细胞中中心粒复制是如何与DNA复制解耦的。此外，我们将确定shugoshin变异在染色体或中心体上的互斥功能的决定因素，并研究shugoshin在减数分裂I后如何失活以允许减数分裂II中的二体分离。扩展最近的工作，我们将揭示必不可少的肽基脯氨酸顺式反式异构酶Pin1如何参与纺锤体组装检查点以限制APC/C活性的分子机制。最后，我们将从减数分裂的不同阶段分离APC/C，比较亚基组成和底物特异性，从而通过调节这个关键的E3连接酶的活性来获得减数分裂时间的新见解。

Erroneous distribution of homologous chromosomes during female meiosis I is the major genetic cause of miscarriage and mental retardation in humans. Yet, the molecular mechanisms of genome haploidization remain poorly understood. We propose to study novel functions and regulations of three key players of the chromosome cycle: Cohesin, a ring-shaped protein complex that keeps sister chromatids paired, shugoshin, a protector of cohesion, and the APC/C (anaphase-promoting complex or cyclosome), a multi-subunit ubiquitin ligase and antagonist of cohesion which triggers chromosome segregation. We have previously shown that cohesin and shugoshin function also at centrosomes, which might explain why the centrosome cycle is usually coordinated with the chromosome cycle. Now, we will fine-map cohesin's centrosomal localization by electron microscopy, try to identify its substrate and investigate how centriole duplication is uncoupled from DNA replication in spermatocytes. Furthermore, we will identify determinants of the mutual exclusive function of shugoshin variants at chromosomes or centrosomes and investigate how shugoshin is inactivated after meiosis I to allow for the separation of dyads in meiosis II. Extending recent work, we will uncover the molecular mechanism of how the essential peptidyl-prolyl cis-trans isomerase Pin1 contributes to the spindle assembly checkpoint to restrict APC/C activity. Finally, we will isolate APC/C from different phases of meiosis, compare subunit compositions and substrate specificities and thereby gain new insight into the timing of meiosis by regulated activity of this crucial E3 ligase.