Molecular Analysis of Kinetochore Function

着丝粒功能的分子分析

• 批准号: 10622233
• 负责人: Iain McPherson Cheeseman
• 金额: $ 79.37万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622233
• 关键词: Affinity Chromatography; Biochemical; Biological; Cell Cycle Arrest; Cell division; Cell physiology; Cells; Chromosome Segregation; Chromosomes; Congenital Abnormality; DNA; Defect; Diagnosis; Disease; Failure; Genes; Goals; Human; Individual; Interphase Cell; Kinetochores; Laboratories; Meiosis; Microtubule Polymerization; Microtubules; Mitosis; Mitotic spindle; Modification; Molecular; Molecular Analysis; Optics; Phenotype; Physiological; Proteins; Proteomics; Rod; Site; Structure; Work; defined contribution; genetic approach; genetic information; macromolecular assembly; molecular modeling; reconstitution; screening; senescence; tumor progression

Project Summary/Abstract The goal of my laboratory is to define the molecular mechanisms by which accurate cell division occurs. Our efforts focus on the core cell division machinery, including the macromolecular kinetochore and the microtubule-based mitotic spindle. Despite the central importance of the spindle and the kinetochore, the molecular basis for their many activities remains incompletely understood. We seek to generate a coherent molecular model for how the multiple kinetochore components and spindle-associated proteins act individually and in an integrated manner to direct faithful chromosome segregation. For chromosome segregation to occur, kinetochores must stably associate with a single site on each chromosome, build a large macromolecular assembly, form robust interactions with the dynamic microtubule polymers from a bipolar mitotic spindle, and these activities must be precisely regulated to ensure that chromosome segregation occurs with high fidelity. In addition to the function of these molecular players in actively dividing cells, the cell division machinery must also be differentially modulated across diverse physiological conditions, for example during meiotic cell divisions or during the persistent cell cycle arrest that occurs in quiescent cells. To analyze these key questions, our work uses a combination of functional genetics approaches in human cells, cell biological studies on protein localization and dynamics, affinity purification and proteomics approaches to identify protein interactions and modifications, and biochemical reconstitutions. For our recent work, we have also implemented large-scale approaches to analyzing cellular phenotypes using Cas9-based optical screening, which has transformed our ability to systematically define the contributions of human genes to cell division and other core cellular processes Over the next 5 years, our lab will investigate the fundamental mechanisms of cell division in human cells, focusing on three interrelated goals: 1) Analyze the molecular basis for chromosome segregation, 2) Define the basis for the cellular changes that occur during non-dividing cell states, such as quiescence and senescence, and 3) Conduct large-scale cell biological and functional genetics approaches to analyze cell division.

项目摘要/摘要 我实验室的目的是定义出现准确细胞分裂的分子机制。我们的 努力集中在核心细胞分裂机械上，包括大分子动物学和 基于微管的有丝分裂纺锤体。尽管主轴和动力学的重要性，但 他们的许多活动的分子基础尚未完全理解。我们试图产生连贯的 多个动力学成分和纺锤体相关蛋白如何单独起作用的分子模型 并以综合的方式指导忠实的染色体隔离。为了发生染色体隔离， 动物体必须稳定地与每个染色体上的一个位点缔合，建立一个大的大分子 组装，与来自双极有丝分裂主轴的动态微管聚合物形成强大的相互作用，并形成 必须精确调节这些活动，以确保染色体隔离以高忠诚发生。在 这些分子参与者在主动分裂的细胞中的功能增加，细胞分裂机械必须 也可以在不同的生理条件下进行差异调节，例如减数分裂细胞 分裂或在静止细胞中发生的持续细胞周期停滞。分析这些关键 问题，我们的工作结合了人类细胞中的功能遗传学方法，细胞生物学 蛋白质定位和动力学，亲和力纯化和蛋白质组学方法的研究以鉴定蛋白质 相互作用和修改以及生化的重构。对于我们最近的工作，我们也 实施了使用基于CAS9的光学筛选的大规模方法来分析细胞表型， 这改变了我们系统地定义人类基因对细胞分裂的贡献的能力和 其他核心细胞过程 在接下来的5年中，我们的实验室将研究人类细胞中细胞分裂的基本机制， 关注三个相互关联的目标：1）分析染色体隔离的分子基础，2）定义 在非分裂细胞状态（例如静止和衰老）期间发生的细胞变化的基础， 3）进行大规模细胞生物学和功能遗传学方法来分析细胞分裂。