Role and Mechanism of Microtubule Nucleation within the Mitotic Spindle

描述（由申请人提供）：基于微管（MT）的有丝分裂纺锤体是真核细胞分裂过程中负责可靠的染色体分离的细胞装置。这一过程的失败与许多癌症有关。纺锤体组装是由MT成核通过中心体、染色质和纺锤体本身的γ微管蛋白环复合物（gTuRC）启动的，但目前尚不清楚gTuRC是如何定位和特异性激活的。最近发现的8亚基蛋白复合物Augmin将gTuRC定位到纺锤体MT上，从而产生MT，因此代表了第一个确定的gTuRC效应物。我的近期研究目标是了解Augmin在MT产生中的机制，以及它在染色体分离机制和其他非中心体成核位点中的确切作用。通过采用跨学科的方法，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UCSF以来，我研究了Augmin在减数分裂纺锤体组装中的功能，纯化了原生和重组Augmin以及gTuRC，从而开发了独特的分子工具来研究MT体外成核。在这里，我建议(I)确定Augmin和gTuRC如何通过体外重建MT成核并通过荧光显微镜动态分析它来产生MT（导师Ron Vale博士）。（ii）我将使用电子显微镜（导师David Agard博士）和x射线晶体学（博士主要技术，独立相）研究目前未知的Augmin及其MT结合复合物的结构，以了解它如何在分子水平上激活MT成核。（iii）通过将荧光Augmin添加到爪蟾纺锤体中，我将识别和量化纺锤体组装途径（独立阶段）中的MT生成事件。这些结果试图回答MT在何时、何地以及如何成核以构成自组装纺锤体的主要未解决的问题，并可能与间期MT成核有关。为了达到这些目标，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射线晶体学方面的训练，并为我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）做好准备。根据严格的职业发展规划，我找到的优秀的导师团队将支持我扩展我的个人和实验室管理技能，为成功地完成在美国的求职和领导一个研究实验室做准备。结合最先进的设备、互动精神和优秀的职业培训活动，加州大学旧金山分校为本研究计划的指导阶段提供了最佳环境。K99/R00奖项将使我有机会获得必要的技能，以过渡到一个独立的终身职位。阐明MT成核的分子机制将为理解生物学的基本过程铺平道路。

DESCRIPTION (provided by applicant): 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. ) PUBLIC HEALTH RELEVANCE: In order to divide, a cell needs to equally segregate its chromosomes into two daughter cells, a process which is coordinated by the microtubule-based mitotic spindle. Failure in this process is detrimental for a cell and a leading cause for cancer. The protein complex Augmin plays an important role in generating spindle microtubules, and elucidating its exact function and mechanism is of medical importance and can be used as a new target for cancer therapy.