Role and Mechanism of Microtubule Nucleation within the Mitotic Spindle

项目摘要 微管（MT）为基础的有丝分裂纺锤体是负责可靠的细胞器 在真核细胞分裂过程中染色体分离。这个过程中的失败与许多 癌的纺锤体组装由MT通过γ微管蛋白环复合物（gTuRC）成核引发 在中心体，染色质和纺锤体本身，但它是未知的，如何gTuRC定位和特异性 在那里激活。最近鉴定的八亚基蛋白复合物Augmin将gTuRC定位于纺锤体 因此，它代表了第一个定义的gTuRC效应子。我近期的研究目标 目的是了解Augmin在MT产生中的作用机制及其在染色体中的确切作用 分离机制和其他非中心体成核位点。通过使用跨学科的 我的长期目标是阐明MT成核是如何局部激活和协调的。 自从我作为生命科学研究基金会的HHMI研究员来到加州大学旧金山分校以来， Augmin在减数分裂纺锤体组装中的功能，纯化的天然和重组Augmin以及 gTuRC，从而开发了独特的分子工具来研究MT体外成核。在此，我建议（i） 通过在体外重构MT成核并对其进行分析，确定Augmin和gTuRC如何产生MT 通过荧光显微镜（导师罗恩瓦尔博士）动态地观察。(ii)我会调查目前未知的 Augmin及其MT结合复合物的结构，以了解它如何激活MT成核， 分子水平使用电子显微镜（导师大卫Agard博士）和X射线晶体学（主要 博士技术，独立阶段）。(iii)通过将荧光Augmin添加到Xenopus纺锤体中， 鉴定和量化纺锤体组装途径（独立阶段）期间的MT生成事件。 这些结果试图回答主要的悬而未决的问题，何时，何地，以及如何成核的MT 构成自组装主轴，并可能是相关的MT成核在相间。 为了实现这些目标，我需要学习高分辨率光学显微镜和电子显微镜。 这将补充我在细胞生物学，生物化学和X射线晶体学方面的培训，并为我做好准备， 从任何必要的角度研究复杂的大分子系统，如有丝分裂纺锤体。基于 严格的职业发展计划，我发现优秀的指导团队将支持我， 扩展我的个人和实验室管理技能，为成功完成美国求职做准备 领导一个研究实验室结合最先进的设备，互动的精神， 优秀的职业培训活动，UCSF提供了最佳的环境，指导阶段，这 研究提案。K99/R 00奖将为我提供获得必要技能的机会， 过渡到一个独立的终身职位。MT成核的分子机理 将为理解生物学的基本过程铺平道路。

Project Summary The microtubule(MT)-based mitotic spindle is the cellular apparatus responsible for reliable chromosome segregation during eukaryotic cell division. Failure in this process is associated with many cancers. Spindle assembly is initiated by MT nucleation through the gamma tubulin ring complex (gTuRC) at centrosomes, chromatin and the spindle itself, yet it is unknown how gTuRC is localized and specifically activated there. The recently identified eight-subunit protein complex Augmin localizes gTuRC to spindle MTs for MT generation, and thus represents the first defined gTuRC effector. My immediate research goal is to understand the mechanism of Augmin in MT generation, and its exact role in the chromosome segregation machinery and other noncentrosomal nucleation sites. By employing an interdisciplinary approach, my long-term goal is to elucidate how MT nucleation is locally activated and coordinated. Since I arrived at UCSF as an HHMI Fellow of the Life Science Research Foundation, I characterized Augmin's function in meiotic spindle assembly, purified both native and recombinant Augmin as well as gTuRC, and thus developed unique molecular tools to study MT nucleation in vitro. Here, I propose to (i) determine how Augmin and gTuRC generate MTs by reconstituting MT nucleation in vitro and analyzing it dynamically by fluorescence microscopy (mentor Dr. Ron Vale). (ii) I will investigate the currently unknown structures of Augmin and its MT-bound complexes to understand how it activates MT nucleation at a molecular level using electron microscopy (mentor Dr. David Agard) and X-ray crystallography (major technique of Ph.D., independent phase). (iii) By adding fluorescent Augmin to Xenopus spindles, I will identify and quantify MT generation events during the spindle assembly pathway (independent phase). These results seek to answer the major unresolved questions of when, where and how MTs are nucleated to constitute the self-assembling spindle, and is likely to be relevant for MT nucleation during interphase. To achieve these aims, I will need to learn high-resolution light microscopy and electron microscopy. This will complement my training in cell biology, biochemistry and X-ray crystallography and prepare me to study complex macromolecular systems, such as the mitotic spindle, from any angle necessary. Based on a rigorous career development plan, the outstanding mentoring team I have found will support me in expanding my personal and lab management skills in preparation to complete a successful U.S. job search and lead a research laboratory. Combined with state-of-the-art equipment, an interactive spirit, and excellent career training activities, UCSF provides the optimal environment for the mentored phase of this research proposal. The K99/R00 award will provide me the opportunity to acquire the necessary skills to transition into an independent tenure-track position. Elucidating the molecular mechanism of MT nucleation will pave the way to understanding a fundamental process in biology.