Role and Mechanism of Microtubule Nucleation within the Mitotic Spindle

有丝分裂纺锤体内微管成核的作用和机制

• 批准号: 8411980
• 负责人: Sabine Petry
• 金额: $ 9万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8411980
• 关键词: Actins; Aneuploidy; Award; Back; Binding; Biochemistry; Bioinformatics; Biological Sciences; Biology; Books; Cancer Etiology; Cell division; Cell physiology; Cells; Cellular biology; Centrosome; Characteristics; Chromatin; Chromosome Segregation; Chromosomes; Complement; Complex; Cryoelectron Microscopy; Development Plans; Doctor of Philosophy; EB1 microtubule binding proteins; Electron Microscopy; Ensure; Environment; Equipment; Eukaryotic Cell; Event; Failure; Fluorescence Microscopy; Foundations; Gamma-Tubulin Ring; Generations; Genetic Materials; Goals; Growth; In Vitro; Individual; Interphase; Label; Laboratory Research; Lead; Learning; Length; Life; Macromolecular Complexes; Malignant Neoplasms; Maps; Medical; Meiosis; Mentors; Microscopy; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modeling; Molecular; Negative Staining; Occupations; Pathway interactions; Phase; Phenotype; Play; Plus End of the Microtubule; Positioning Attribute; Preparation; Process; Protein Complex Subunit; Recombinants; Research; Research Proposals; Resolution; Resources; Role; Site; Staging; Structure; System; Techniques; Text; Training; Training Activity; X-Ray Crystallography; Xenopus; base; cancer cell; cancer therapy; career; career development; daughter cell; egg; gamma Tubulin; insight; interdisciplinary approach; light microscopy; particle; polymerization; protein complex; reconstitution; reconstruction; segregation; skills; tomography; tool

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. )

描述(申请人提供)：基于微管(MT)的有丝分裂纺锤体是负责真核细胞分裂过程中可靠的染色体分离的细胞装置。这一过程中的失败与许多癌症有关。纺锤体组装是由MT通过中心体、染色质和纺锤体上的伽马微管蛋白环复合体(GTuRC)启动的，但gTuRC在那里是如何定位和特异性激活的尚不清楚。最近发现的八亚单位蛋白复合体Augmin将gTuRC定位于纺锤形的MT生成，从而代表了第一个已定义的gTuRC效应器。我目前的研究目标是了解Augmin在MT发生中的机制，以及它在染色体分离机制和其他非中心体成核部位中的确切作用。通过采用跨学科的方法，我的长期目标是阐明MT成核是如何在局部激活和协调的。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，我研究了Augmin在减数分裂纺锤体组装中的功能，纯化了天然和重组的Augmin以及gTuRC，从而开发了独特的分子工具来研究体外MT成核。在这里，我建议(I)通过在体外重建MT成核并用荧光显微镜动态分析它(导师Ron Vale博士)来确定Augmin和gTuRC是如何生成MT的。(Ii)我将使用电子显微镜(Mentor David Agard博士)和X射线结晶学(博士的主要技术，独立相)来研究Augmin及其MT结合络合物目前未知的结构，以了解它如何在分子水平上激活MT成核。(Iii)通过将荧光Augmin添加到非洲爪哇纺锤体中，我将识别和量化纺锤体组装过程中的MT生成事件(独立阶段)。这些结果试图回答尚未解决的主要问题，即MT在何时、何地和如何成核以构成自组装纺锤体，并且可能与MT在相间的成核有关。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射线结晶学方面的培训，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。基于严格的职业发展计划，我找到的优秀指导团队将支持我扩展个人和实验室管理技能，为完成在美国的成功求职和领导一个研究实验室做准备。加州大学旧金山分校结合最先进的设备、互动精神和出色的职业培训活动，为本研究提案的指导阶段提供了最佳环境。K99/R00奖项将让我有机会获得必要的技能，以过渡到一个独立的终身教职职位。阐明MT成核的分子机制将为理解生物学中的基本过程铺平道路。)