Molecular mechanism of centrosome separation in budding yeast meiosis

 描述(申请人提供)：芽期酵母减数分裂中中心体分离的分子机制通常被称为纺锤体极体(SPB)，芽期酵母中心体与动物中心体共享结构成分，并且在功能上等同于动物中心体。就像人类细胞中的中心体一样，酵母中复制的SPB被一种称为“半桥”的蛋白质结构连接在一起，但这种连接是如何维持并及时溶解以进行纺锤体组装的，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聚集和分离的调节。在减数分裂过程中，当重组发生时，复制的SPB被拴在延长的G2期；此外，SPB在减数分裂II之前复制，这与DNA复制是分离的。因此，SPB的凝聚和分离过程是细胞分裂过程中的关键调控步骤。以前的工作表明，半桥的切割可能允许SPB分离。我们的实验室最近证明，正确分离SPB需要半桥亚单位的翻译后修饰，并且减数分裂特异的蛋白保护SPB的凝聚力。因此，我们假设我们确定的半桥亚单位是蛋白质切割的位置，这是由翻译后修饰和减数分裂的“守护”蛋白调节的。我们将实现三个特定的目标来验证这一假说：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阶段保持，以及(3)SPB凝聚力如何为纺锤体组装而溶解。在第一个目标下，将确定介导SPB凝聚力的半桥上的蛋白质相互作用网络。在第二个目标下，将确定保护SPB凝聚力的“守护”蛋白质的特征。在第三个目标下，申请人实验室生成的SPB减数分裂磷酸蛋白质组图将指导分离SPB所需的蛋白质磷酸化的研究。这项研究有望揭示SPB凝聚和分离的分子机制，该机制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性，因此这项拟议的工作有望为哺乳动物中心体分离的调控提供见解，从而有助于阐明人类染色体错误分离和非整倍体的原因。

 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.