Molecular Interactions and Dynamics of the Yeast SPB Core Architecture

中心体组织两极有丝分裂纺锤体，这是染色体分离所必需的，也是缺陷 中心体的突变会导致人类染色体不稳定和癌症。酵母中心体(纺锤体极 Body，SPB)与人类中心体有许多共同的成分和调节因子。Cyclin的磷酸化- 依赖蛋白水解酶和丝裂原激活蛋白水解酶对中心体和SPB的组装和活性都是必不可少的。在这 在PPG项目中，我们将确定SPB核心组件的结构特征和磷酸化事件 它们在它们的组装、功能和交换中发挥着重要作用。确定重要的功能域 和残基，我们将基于两个重要的信息生成SPB组分的突变文库 资料来源：首先是Ivan Rayment(项目3)提供的关于SPB全长组件或特定组件的结构数据 域名。第二个是在我们的酵母SPB磷酸化蛋白质组中定位的磷酸化位点。突变的 将测试SPB蛋白在复制时组装成新的SPB和现有SPB的能力 在有丝分裂期间，当SPBS扩张时(模仿中心体成熟)。他们还将接受评估，以 与其他SPB组分的折衷相互作用和由电子分析的结构扰动 显微镜和电子断层扫描。候选的蛋白激酶、磷酸酶和其他调节剂将 已筛选具有特定SPB组件的功能。预计这项工作将扩大收藏范围 SPB调节分子。最后，尽管有证据表明部件在SPBS期间进出SPBS 在细胞周期方面，人们对动态平衡的机制知之甚少。我们将首先确定SPB的程度 用电子断层扫描和荧光技术研究野生型细胞的大小动态和成分交换 光漂白后恢复(FRAP)。然后我们将使用我们的突变体集合来评估 单个SPB组件的特定特征进行交换，并尝试识别反式作用 监管者。第三个目标将涉及与Davis(项目2)在SPB动力学的类似问题上密切合作 和改建。总体而言，我们将提供SPB组件的活体分析，发挥我们的优势 在电子显微镜和断层扫描到PPG。我们将贡献大量的体内分析来测试 SPB组分行为的假说源自结构研究和分子模拟。其中几个 SPB组分和调节器有人类同源基因，在酵母中的研究将是至关重要的 了解它们在中心体的中心三极周围物质(PCM)中的组装和功能。

Centrosomes organize the bipolar mitotic spindle, which is essential for chromosome segregation, and defects in centrosomes lead to chromosomal instability and cancer in humans. The yeast centrosome (Spindle Pole Body, SPB) shares many components and regulators with human centrosomes. Phosphorylation by Cyclin- dependent kinase and Mpsl kinase is essential for both centrosome and SPB assembly and activity. In this project in the PPG, we will determine structural features and phosphorylation events in core SPB components that play significant roles in their assembly, function and exchange. To ascertain important functional domains and residues, we will generate a library of mutations in SPB components based on two significant informational resources: the first is structural data from Ivan Rayment (Project 3) on full-length SPB components or particular domains. The second is phosphorylation sites mapped in vivo in our yeast SPB phosphoproteome. Mutated SPB proteins will be tested for their competence in assembly into new SPBs at duplication and into existing SPBs during mitosis when SPBs expand (mimicking centrosome maturation). They will also be evaluated for compromised interactions with other SPB components and for structural perturbations as assayed by electron microscopy and electron tomography. Candidate protein kinases, phosphatases and other regulators will be screened for function with particular SPB components. It is anticipated that this work will expand the collection of SPB regulatory molecules. Finally, despite evidence that components exchange in and out of SPBs during the cell cycle, little is known about the mechanism of homeostasis. We will first determine the extent of SPB size dynamics and component exchange in wild-type cells, using electron tomography and Fluorescent Recovery After Photobleaching (FRAP). We will then use our mutant collection to assess the contribution of particular features of individual SPB components to their exchange and attempt to identify trans-acting regulators. This third aim will involve working closely with Davis (Project 2) on similar issues of SPB dynamics and remodeling. Overall, we will provide an in vivo analysis of SPB components, bringing to bear our strengths in electron microscopy and tomography to the PPG. We will contribute substantial in vivo analyses to test hypotheses of SPB component behavior derived from structural studies and molecular modeling. Several of the SPB components and regulators have human orthologs and studies in yeast will be critical to understanding their assembly and function in the pericentriolar material (PCM) of centrosomes.