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，这对于染色体分离和缺陷至关重要 中心体中的细菌会导致人类染色体不稳定和癌症。酵母中心体（纺锤体 Body (SPB) 与人类中心体共享许多组件和调节器。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 的组装和活性至关重要。在这个 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，我们将根据两个重要的信息生成 SPB 成分的突变库 资源：第一个是 Ivan Rayment（项目 3）关于全长 SPB 组件或特定组件的结构数据 域。第二个是我们的酵母 SPB 磷酸化蛋白质组中体内定位的磷酸化位点。变异的 SPB 蛋白将测试其在复制时组装成新 SPB 和现有 SPB 的能力。 有丝分裂期间 SPB 扩张（模仿中心体成熟）。他们还将接受评估 与其他 SPB 成分的相互作用受损以及电子分析的结构扰动 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 筛选特定 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，尽管有证据表明组件在 SPB 中交换 人们对细胞周期的稳态机制知之甚少。我们首先确定 SPB 的范围 使用电子断层扫描和荧光技术观察野生型细胞的尺寸动态和成分交换 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 各个 SPB 组件的特定特征及其交换并尝试识别交易 监管机构。第三个目标将涉及与 Davis（项目 2）在 SPB 动态的类似问题上密切合作 和改造。总的来说，我们将提供 SPB 成分的体内分析，发挥我们的优势 PPG 的电子显微镜和断层扫描技术。我们将提供大量的体内分析来进行测试 来自结构研究和分子建模的 SPB 成分行为假设。几个 SPB 成分和调节剂具有人类直系同源物，并且对酵母的研究对于 了解它们在中心体周围材料（PCM）中的组装和功能。