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: 8225469
• 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 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8225469
• 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. ) 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.

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期间负责可靠染色体分离的细胞器。这个过程的失败与许多癌症有关。纺锤体组装是由MT成核通过γ微管蛋白环复合物（gTuRC）在中心体，染色质和纺锤体本身，但它是未知的gTuRC是如何定位和特异性激活。最近鉴定的八亚基蛋白复合物Augmin将gTuRC定位于纺锤体MT以产生MT，因此代表了第一个定义的gTuRC效应子。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研究员来到加州大学旧金山分校，我描述了Augmin在减数分裂纺锤体组装中的功能，纯化了天然和重组Augmin以及gTuRC，从而开发了独特的分子工具来研究MT体外成核。在这里，我建议（i）通过在体外重建MT成核并通过荧光显微镜动态分析来确定Augmin和gTuRC如何产生MT（导师罗恩维尔博士）。(ii)我将研究Augmin及其MT结合复合物的目前未知结构，以了解它如何使用电子显微镜（导师大卫阿加德博士）和X射线晶体学（博士的主要技术，独立阶段）。(iii)通过添加荧光Augmin的非洲爪蟾纺锤体，我将确定和量化MT生成事件的纺锤体组装途径（独立阶段）。这些结果试图回答的主要悬而未决的问题时，在哪里以及如何MT成核构成的自组装主轴，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。基于严格的职业发展计划，我找到的优秀导师团队将支持我扩展个人和实验室管理技能，为成功完成美国求职和领导研究实验室做好准备。结合国家的最先进的设备，互动的精神，和优秀的职业培训活动，UCSF提供了最佳的环境，指导阶段的研究建议。K99/R00奖将为我提供机会，获得必要的技能，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中的基本过程铺平道路。) 公共卫生相关性：为了分裂，细胞需要将其染色体均等地分离成两个子细胞，这一过程由基于微管的有丝分裂纺锤体协调。这一过程的失败对细胞是有害的，也是癌症的主要原因。Augmin蛋白复合物在纺锤体微管的形成中起重要作用，阐明其确切的功能和机制具有重要的医学意义，并可作为肿瘤治疗的新靶点。