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.

项目总结/摘要 我的实验室的目标是确定精确的细胞分裂发生的分子机制。我们 努力集中在核心细胞分裂机制，包括大分子动粒和 基于微管的有丝分裂纺锤体。尽管纺锤体和动粒至关重要， 其许多活性的分子基础仍然不完全清楚。我们力求建立一个连贯的 多个动粒组分和纺锤体相关蛋白如何单独作用的分子模型 并以整合的方式指导忠实的染色体分离。为了使染色体分离发生， 动粒必须与每条染色体上的单个位点稳定结合，构建一个大分子 组装，与来自双极有丝分裂纺锤体的动态微管聚合物形成稳健的相互作用，以及 这些活性必须被精确地调节以确保染色体分离以高保真度发生。在 除了这些分子参与者在活跃的细胞分裂中的功能外，细胞分裂机制必须 在不同的生理条件下，例如在细胞减数分裂期间， 分裂或在静止细胞中发生的持续细胞周期停滞期间。分析这些关键 问题，我们的工作结合了人类细胞的功能遗传学方法，细胞生物学方法， 蛋白质定位和动力学研究，亲和纯化和蛋白质组学方法鉴定蛋白质 相互作用和修饰，以及生化重组。对于我们最近的工作，我们还 实施了使用基于Cas9的光学筛选来分析细胞表型的大规模方法， 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， 其他核心细胞过程 在接下来的5年里，我们的实验室将研究人类细胞分裂的基本机制， 重点是三个相互关联的目标：1）分析染色体分离的分子基础，2）定义染色体分离的分子基础。 在非分裂细胞状态期间发生的细胞变化的基础，例如静止和衰老， 以及3）进行大规模细胞生物学和功能遗传学方法以分析细胞分裂。