Molecular Analysis of Kinetochore Function

项目摘要/摘要 我的实验室的目标是确定准确的细胞分裂发生的分子机制。我们的 努力集中在核心细胞分裂机制上，包括大分子动粒和 以微管为基础的有丝分裂纺锤体。尽管纺锤体和动粒具有核心重要性，但 它们许多活动的分子基础仍然不完全清楚。我们寻求产生一个连贯的 多个动粒成分和纺锤体相关蛋白单独作用的分子模型 并以一种综合的方式指导忠实的染色体分离。为了进行染色体分离， 动点必须稳定地与每个染色体上的一个位置相关联，构建一个大的大分子 组装，与来自双极有丝分裂纺锤体的动态微管聚合物形成牢固的相互作用，以及 这些活动必须受到精确的调控，以确保高保真地进行染色体分离。在……里面 除了这些分子参与者在积极分裂细胞方面的功能外，细胞分裂机制必须 也在不同的生理条件下被差异调节，例如在减数分裂细胞期间 分裂或在静止细胞中发生的持续细胞周期停滞期间。要分析这些关键字 问题，我们的工作结合了人类细胞、细胞生物学的功能遗传学方法 蛋白质定位与动力学、亲和纯化及蛋白质组学鉴定方法的研究 相互作用和修饰，以及生化重组。在我们最近的工作中，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， 它改变了我们系统地定义人类基因对细胞分裂和 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， 重点关注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 在非分裂细胞状态下发生的细胞变化的基础，如静止和衰老， 3)采用大规模的细胞生物学和功能遗传学方法来分析细胞分裂。

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.