SGER: Molecular Analysis of the Gamma-Tubulin:Microtubule Interaction

中心体是一种非膜结合的亚细胞细胞器，在细胞分裂和其他细胞过程中起着至关重要的作用。它是动物细胞中主要的微管组织中心，通过对微管的影响，中心体参与了许多基本的细胞过程，包括细胞分裂过程中的囊泡运输、细胞运动、mRNA定位和染色体分离。尽管中心体对细胞生物学的重要性和一个多世纪的研究，中心体的功能、结构和组成的许多方面仍然未知。中心体的微管成核需要γ -微管蛋白，这是微管蛋白超家族中高度保守且普遍存在的成员。关于γ -微管蛋白的许多长期存在的问题仍有待回答，包括γ -微管蛋白如何在微管形成和稳定中起作用。这个问题的核心是了解γ -微管如何相互作用以及与微管的亚基相互作用。这个探索性研究项目的小额赠款代表了一组重点的风险实验，旨在了解微管的长轴是如何排列的。存在两种可能性：要么-微管蛋白可以排列成一个环，“盖住”微管的末端，要么-微管蛋白可以形成一个垂直于环的线性排列，并与微管壁的二聚体（α和β）微管蛋白亚基对齐。在这两种模型中，微管蛋白与α、β微管蛋白二聚体之间的分子接触度分别为肩对肩与头对尾，相差90度。为了区分这些模型，将对γ -微管蛋白和微管亚基之间形成的分子接触进行深入分析。利用分裂酵母（Schizosaccharomyces pombe）作为模型系统，极大地扩展了先前的工作，分子遗传学方法将与体内和体外试验相结合，试图明确确定γ -微管蛋白的结构特征，这些特征对其与微管的相互作用及其在微管形成中的功能至关重要。为了实现这一目标，将突变引入S. pombe γ -微管蛋白基因。这些突变对γ -微管蛋白复合物组装及其成核能力的影响将在体内和体外进行分析。智力优势：如果成功，这项研究的完成将提供，在分子细节上，一个关于γ -微管复合物如何工作以及它如何影响微管形成的机制图片。更广泛地说，这些结果将最终有助于阐明中心体如何参与微管的形成和组织。由于中心体缺陷会导致细胞周期阻滞、细胞死亡和基因组不稳定，这项研究将有助于我们了解影响细胞和发育生物学许多领域的基本细胞过程。更广泛的影响：这项研究包括通过涉及在细胞和分子生物学、生物化学、生物物理学和数学方面受过高级培训的科学家的跨学科合作来加强科学基础设施的活动。对本科生、研究生和研究生的研究助理（包括未被充分代表的少数民族和妇女）进行现代细胞生物学技术培训将是该项目的一个组成部分。这个项目的一个重要组成部分和更广泛的影响是吸引和培训未来的教师，因为它将包括更多的资深研究人员，他们自己还不是独立的教师，通过监督经验不足的研究人员和本科生，获得宝贵的教学和指导技能。

SGER: Molecular analysis of the gamma-tubulin:microtubule interactionThe centrosome is a non-membrane-bound subcellular organelle that has a crucial function in cell division and other cellular processes. It serves as the major microtubule-organizing center of animal cells and through its influence on microtubules, the centrosome is involved in many fundamental cellular processes including vesicular traffic, cell motility, mRNA localization, and chromosome segregation during cell division. Despite its importance to cell biology and more than a century of scrutiny, many aspects of centrosome function, structure, and composition remain unknown. Microtubule nucleation by the centrosome requires gamma-tubulin, a highly conserved and ubiquitous member of the tubulin superfamily. Numerous long-standing questions about gamma-tubulin remain to be answered, including how gamma-tubulin functions in microtubule formation and stabilization. Central to this question is an understanding of how gamma-tubulins interact with each other and with the subunits of the microtubule. This Small Grant for Exploratory Research project represents a focused set of risky experiments aimed at understanding how gamma-tubulins are arranged with respect to the long axis of the microtubule. Two possibilities exist: either gamma-tubulins could be arranged in a ring that 'caps' the end of the microtubule, or, alternatively, gamma-tubulins could form a linear arrangement that is perpendicular to the ring and aligns with the dimeric (alpha and beta) tubulin subunits of the microtubule wall. The molecular contacts made between gamma-tubulin and the alpha, beta tubulin dimers differ by 90 degrees in these two models: shoulder-to-shoulder vs head-to-tail, respectively. To distinguish between these models, an in-depth analysis of the molecular contacts formed between gamma-tubulin and the microtubule subunits will be undertaken. Using the fission yeast, Schizosaccharomyces pombe, as a model system and greatly extending previous work, molecular genetic approaches will be combined with in vivo and in vitro assays to try to specifically determine the structural features of gamma-tubulin that are important for its interaction with the microtubule and for its function in microtubule formation. To achieve this goal, mutations will be introduced into the S. pombe gamma-tubulin gene. The effect of these mutations on the assembly of the gamma-tubulin complex and its nucleation capacity will then be assayed in vivo and in vitro. Intellectual Merit: If successful, completion of this research will provide, in molecular detail, a mechanistic picture of how the gamma-tubulin complex works and how it affects microtubule formation. More broadly, the results would ultimately help elucidate how centrosomes participate in the formation and organization of microtubules. Because centrosome defects can result in cell cycle arrest, cell death, and genomic instability, this research will contribute to our knowledge of fundamental cellular processes that impact many areas of cell and developmental biology. Broader Impact: This research includes activities that enhance scientific infrastructure via cross-disciplinary collaborations that involve scientists with advanced training in cell and molecular biology, biochemistry, biophysics, and mathematics. Training in modern cell biology techniques of undergraduate, graduate, and post-graduate research assistants (including underrepresented minorities and women) will be an integral aspect of the project. A significant component and broader impact of this project is to engage and train future faculty, as it will include the opportunity for more senior researchers who are not yet independent faculty members themselves to obtain valuable teaching and mentoring skills by supervising less experienced researchers and undergraduate students.