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

微管相互作用中心体是一种非膜结合的亚细胞器，在细胞分裂和其他细胞过程中起着至关重要的作用。它作为动物细胞的主要微管组织中心，通过对微管的影响，参与了许多基本的细胞过程，包括囊泡运输、细胞运动、mRNA定位和细胞分裂过程中的染色体分离。尽管中心体对细胞生物学很重要，经过一个多世纪的研究，中心体的功能、结构和组成的许多方面仍然未知。由中心体形成的微管需要伽马微管蛋白，它是微管蛋白超家族中高度保守和普遍存在的成员。关于伽马微管蛋白的许多长期存在的问题仍有待回答，包括伽马微管蛋白如何在微管形成和稳定中发挥作用。这个问题的核心是了解伽马微管蛋白如何相互作用以及如何与微管的亚单位相互作用。这个小的探索性研究拨款项目代表了一组集中的高风险实验，旨在了解伽马微管蛋白是如何相对于微管的长轴排列的。存在两种可能性：或者伽马微管蛋白可以排列在微管末端的环中，或者，或者，伽马微管蛋白可以形成垂直于该环并与微管壁的二聚体(α和β)微管蛋白亚基对齐的线性排列。在这两种模型中，伽马微管蛋白和α、β微管蛋白二聚体之间的分子接触相差90度：肩并肩与头尾相连。为了区分这些模型，将对伽马微管蛋白和微管亚基之间形成的分子接触进行深入分析。利用裂解酵母作为模型系统，并极大地扩展了以前的工作，分子遗传学方法将与体内和体外实验相结合，试图特异地确定伽马微管蛋白的结构特征，这些结构特征对于它与微管的相互作用和它在微管形成中的作用是重要的。为了实现这一目标，将在S.pombe伽马微管蛋白基因中引入突变。然后，将在体内和体外测试这些突变对伽马-微管蛋白复合体组装及其成核能力的影响。智力价值：如果成功，这项研究的完成将从分子细节上提供伽马-微管蛋白复合体如何工作以及它如何影响微管形成的机制图。更广泛地说，这些结果最终将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停滞、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：这项研究包括通过跨学科合作加强科学基础设施的活动，这些合作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接受过高级培训的科学家。对本科生、研究生和研究生研究助理(包括代表性不足的少数民族和妇女)进行的现代细胞生物学技术培训将是该项目不可或缺的一个方面。该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高级研究人员，他们本身还不是独立的教师，通过指导经验较少的研究人员和本科生，获得有价值的教学和指导技能。

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.