Molecular mechanism of centrosome separation in budding yeast meiosis

 描述（由申请人提供）：芽殖酵母减数分裂中中心体分离的分子机制通常称为纺锤体极体（SPB），芽殖酵母中心体与动物中心体共享结构组分，并且在功能上等同于动物中心体。像人类细胞中的中心体一样，酵母中复制的SPB被称为“半桥”的蛋白质结构所束缚，但这种连接如何维持并及时溶解以进行纺锤体组装在分子水平上尚不清楚。本项目的目的是利用芽殖酵母减数分裂中独特的SPB循环作为模型来确定中心体凝聚和分离的调节。在减数分裂期间，当重组发生时，重复的SPB被束缚在延长的G2期;此外，SPB在减数分裂II之前重复，这与DNA复制解偶联。因此，SPB的凝聚和分离过程是细胞分裂过程中的关键调节步骤。以前的工作表明，分裂的半桥可能允许SPB分离。我们的实验室最近证明，翻译后修饰的半桥亚基是必要的适当SPB分离，并减数分裂特异性蛋白质保护SPB的凝聚力。因此，我们假设，我们确定的半桥亚基是蛋白质切割的网站，这是由翻译后修饰和减数分裂的“监护人”蛋白。为了验证这一假设，我们将实现三个具体目标：我们将确定（1）半桥蛋白如何建立SPB凝聚力，（2）SPB凝聚力如何在G2期维持，以及（3）SPB凝聚力如何溶解以进行纺锤体组装。在第一个目标下，将确定介导SPB内聚的半桥处的蛋白质相互作用网络。在第二个目标下，保护SPB凝聚力的“守护”蛋白将被表征。在第三个目标下，在申请人的实验室中产生的减数分裂SPB磷酸化蛋白质组图谱将指导SPB分离所需的蛋白质磷酸化的研究。该研究有望揭示SPB凝聚和分离的分子机制，SPB凝聚和分离与纺锤体组装和染色体分离相协调。由于酵母中的四个半桥亚基中有三个在人类细胞中具有已知的同源物，因此预计所提出的工作将为哺乳动物中心体分离的调节提供见解，从而有助于阐明人类染色体错误分离和非整倍性的原因。

 DESCRIPTION (provided by applicant): Molecular mechanism of centrosome separation in budding yeast meiosis often referred to as the spindle pole body (SPB), the budding yeast centrosome shares structural components with, and is functionally equivalent to, the animal centrosome. Like centrosomes in human cells, duplicated SPBs in yeast are tethered by a proteinaceous structure, called the "half-bridge", but how this linkage is maintained and then dissolved in a timely manner for spindle assembly is unclear at the molecular level. The objective of this project is to use the unique SPB cycle in budding yeast meiosis as a model to determine the regulation of centrosome cohesion and separation. During meiosis, duplicated SPBs are tethered for an extended G2 phase when recombination occurs; in addition, SPBs reduplicate before meiosis II, which is uncoupled from DNA replication. The process of SPB cohesion and separation is thus a key regulatory step during cell division. Previous works indicate that cleavage of the half-bridge likely permits SPB separation. Our laboratory recently demonstrated that posttranslational modification of a half-bridge subunit is required for proper SPB separation, and that a meiosis-specific protein protects SPB cohesion. We therefore hypothesize that the half-bridge subunit we identified is the site of protein cleavage, which is regulated by posttranslational modification and by the meiotic "guardian" protein. Three specific aims will be fulfilled to test this hypothesis: We will determine (1) how half-bridge proteins establish SPB cohesion, (2) how SPB cohesion is maintained during the G2 phase, and (3) how SPB cohesion is dissolved for spindle assembly. Under the first aim, the protein-interaction network at the half-bridge that mediates SPB cohesion will be determined. Under the second aim, the "guardian" protein that protects SPB cohesion will be characterized. Under the third aim, a meiotic SPB phosphoproteome map, generated in the applicant's lab, will guide the study of protein phosphorylation required for SPB separation. The proposed research is expected to reveal the molecular mechanism of SPB cohesion and separation, which is coordinated with spindle assembly and chromosome segregation. Because three of the four half-bridge subunits in yeast have known homologs in human cells, the proposed work is expected to provide insights into the regulation of mammalian centrosome separation and thereby contribute to elucidating the causes of chromosome missegregation and aneuploidy in humans.