Molecular Interactions and Dynamics of the Yeast SPB Core Architecture

中心体组织双极有丝分裂纺锤体，这是染色体分离所必需的， 在人类中，中心体的不稳定性导致染色体不稳定和癌症。酵母中心体（纺锤极 体，SPB）与人类中心体共享许多组分和调节因子。细胞周期蛋白磷酸化- 依赖性激酶和Mpsl激酶是中心体和SPB组装和活性所必需的。在这 在PPG项目中，我们将确定核心SPB组件的结构特征和磷酸化事件 在其组装、功能和交换中发挥重要作用。确定重要的功能域 和残基，我们将基于两个重要的信息产生SPB组分中的突变文库。 资源：第一个是Ivan Rayment（项目3）关于全长SPB组件或特定的结构数据。 域.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 将测试SPB蛋白在复制时组装成新SPB和组装成现有SPB的能力。 SPB在有丝分裂期间当SPB扩展时（模仿中心体成熟）。还将对它们进行评估， 与其他SPB组分的相互作用以及通过电子检测的结构扰动 显微镜和电子断层扫描。候选蛋白激酶、磷酸酶和其他调节剂将被 筛选特定SPB组分的功能。预计这项工作将扩大收集 SPB调节分子。最后，尽管有证据表明，组件交换进出SPB期间， 细胞周期，很少有人知道的机制，稳态。我们将首先确定SPB的范围 野生型细胞中的大小动态和组分交换，使用电子断层扫描和荧光 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 个别SPB组件的特定功能，以他们的交流，并试图确定交易 监管部门这第三个目标将涉及与戴维斯（项目2）就类似的SPB动力学问题密切合作 和重塑。总之，我们将提供SPB组分的体内分析， 在电子显微镜和断层扫描中与PPG联系在一起。我们将提供大量的体内分析， 从结构研究和分子模拟中推导出SPB组分行为的假设。几 SPB组分和调节剂具有人类直系同源物，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粒周围物质（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.