Molecular Interactions and Dynamics of the Yeast SPB Core Architecture

酵母 SPB 核心架构的分子相互作用和动力学

• 批准号: 8668223
• 负责人: MARK WINEY
• 金额: $ 10.14万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8668223
• 关键词: Address; Affect; Amino Acids; Architecture; Behavior; Binding; Binding Sites; Biological Assay; Biological Testing; Body Size; C-terminal; Calmodulin; Cell Cycle; Cell Cycle Arrest; Cells; Centrosome; Chromosomal Instability; Chromosome Segregation; Chromosomes; Collection; Competence; Complement; Complex; Core Protein; Cyclin-Dependent Kinases; Data; Defect; Docking; Electron Microscopy; Event; Genes; Growth; Homeostasis; Human; In Vitro; Individual; Interphase; Label; Lead; Length; Libraries; Malignant Neoplasms; Maps; Methods; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modification; Molecular Models; Monitor; Mutate; Mutation; Names; Normal Cell; Orthologous Gene; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Photobleaching; Play; Ploidies; Post-Translational Protein Processing; Process; Protein Kinase; Proteins; Publishing; Recovery; Resources; Role; Saccharomyces cerevisiae; Saccharomycetales; Signal Transduction; Structure; Study models; Testing; Thick; Tubulin; Ursidae Family; Work; Yeasts; base; cell type; data modeling; design; electron tomography; gamma Tubulin; in vivo; interest; molecular dynamics; molecular modeling; mutant; neoplastic cell; spindle pole body; tomography

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.

中心体组织双极有丝分裂纺锤体，这对于染色体分离至关重要，缺陷 在中心体中导致人类染色体不稳定性和癌症。酵母中心体（主轴杆 Body，SPB）与人类的中心体共享许多组件和调节剂。细胞周期蛋白磷酸化 依赖性激酶和MPSL激酶对于中心体和SPB组装和活性至关重要。在这个 PPG中的项目，我们将确定核心SPB组件中的结构特征和磷酸化事件 该在其组装，功能和交换中起着重要作用。确定重要的功能域 残留物，我们将基于两个重要的信息，在SPB组件中生成一个突变库 资源：第一个是全长SPB组件的Ivan Rayment（项目3）的结构数据 域。第二个是在我们的酵母SPB磷酸蛋白酶中映射的磷酸化位点。突变 SPB蛋白将在重复时期和现有 SPB扩展时有丝分裂期间的SPB（模仿中心体成熟）。他们还将评估 与其他SPB组件的相互作用损害了，并且用于通过电子测定的结构扰动 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是 通过特定的SPB组件筛选功能。预计这项工作将扩大收藏 SPB调节分子。最后，尽管有证据表明组件在spb中交换 细胞周期对稳态的机理知之甚少。我们将首先确定SPB的程度 使用电子断层扫描和荧光的野生型细胞中的尺寸动力学和组件交换 光漂白后恢复（FRAP）。然后，我们将使用我们的突变集收集来评估 各个SPB组件的特定特征与其交换并尝试识别跨性别 监管机构。第三个目标将涉及与戴维斯（Project 2）在SPB Dynamics的类似问题上紧密合作 和改建。总体而言，我们将对SPB组件进行体内分析，从而实现我们的优势 在电子显微镜和PPG的断层扫描中。我们将在体内进行大量分析以测试 SPB成分行为的假设来自结构研究和分子建模。几个 SPB组件和调节剂具有人类直系同源物，并且在酵母中研究对 了解它们在中心体的周围三元素材料（PCM）中的组装和功能。