Mechanistic analysis of microtubule nucleation

该项目旨在定义决定新微管如何从其构建块--αβ-微管蛋白亚单位--形成的分子规则。微管是一种动态的聚合物组件，它组织细胞内部，在细胞分裂过程中调节遗传物质的忠实分离，并为基于运动的运输提供轨道。这项研究将集中在微管成核，这是一个鲜为人知的过程，αβ-微管单位通过它自组装来启动新的微管。该项目将揭示和量化单个αβ-微管蛋白构建块的结构和生化特性如何决定微管成核的速度和形成新微管之前的寡聚中间体序列，以及不同物种的αβ-微管蛋白中这些特性的差异。该项目还强调了教育影响。参与该项目的研究生将接受包括定量生物化学和计算建模在内的跨学科培训。与当地中小学的外展伙伴关系将把从事科学的兴奋带给代表性不足和经济困难的学生，同时为教师和他们的学生提供高科技但相对便宜的数字显微镜，以促进基于好奇心的学习和发现。这项提议的具体研究目标是建立对微管成核的分子理解。微管的动态性质来源于单个αβ-微管蛋白亚基的生化性质及其在聚合物组装中的相互作用。MT成核-从未聚合的亚基形成新的聚合物-是一种鲜为人知的行为，对构建MT网络至关重要，而且它受到细胞因素的高度调控。这项研究将使用计算和实验方法来解决三个具体目标：(I)在αβ-微管蛋白GTP酶活性受到抑制的简化环境中发展对微管成核的机理的了解；(Ii)使用蒙特卡罗模拟来创建更准确和更具普遍性的描述，以说明以一种能够解释GTP酶活性的方式控制MT成核的生化机制；(Iii)使用蛋白质工程来组装和捕获定义的αβ-微管蛋白低聚物，否则这些寡聚体将太不稳定而无法使用，并将这些低聚物用于新型的结构、生化和力学研究。所有工作将使用两个不同的模型系统，重组酵母和人αβ-微管蛋白，提供对微管成核和αβ-微管蛋白生化是否以及如何在不同物种之间差异的洞察。这项工作由分子生物物理和细胞动力学与功能簇联合资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This project seeks to define the molecular rules that determine how a new microtubule forms from its building blocks - the αβ-tubulin protein subunits. Microtubules are dynamic polymeric assemblies that organize the insides of cells, mediate faithful segregation of the genetic material during cell division, and provide tracks for motor-based transport. The research will focus on microtubule nucleation, the poorly understood process by which αβ-tubulin units self-assemble to initiate a new microtubule. The project will reveal and quantify how structural and biochemical properties of the individual αβ-tubulin building blocks determine the rate of microtubule nucleation and the sequence of oligomeric intermediates that precede formation of a new microtubule, and how these vary in αβ-tubulins from different species. The project also emphasizes an educational impact. Graduate students working on the project will receive interdisciplinary training that includes quantitative biochemistry and computational modeling. Outreach partnerships with local elementary and middle schools will bring the excitement of doing science to underrepresented and economically disadvantaged students, while providing teachers and their pupils access to high-tech, but relatively inexpensive, digital microscopes to facilitate curiosity-based learning and discovery. The specific research objective of this proposal is to create a molecular understanding of microtubule nucleation. Dynamic properties of microtubules derive from the biochemical properties of individual αβ-tubulin subunits and their interactions within the polymeric assembly. MT nucleation – the formation of a new polymer from unpolymerized subunits – is a poorly understood behavior that is critical for building MT networks and it is highly regulated by cellular factors. The research will use computational and experimental approaches to address three specific goals: (i) to develop a mechanistic understanding of microtubule nucleation in a simplified setting where αβ-tubulin GTPase activity has been suppressed, (ii) to use Monte Carlo simulations to create a more exact and generalizable description of the biochemical mechanisms that control MT nucleation in a way that can account for the effects of GTPase activity, (iii) to use protein engineering to assemble and trap defined oligomers of αβ-tubulin that would otherwise be too unstable to work with, and to use these oligomers for new kinds of structural, biochemical, and mechanistic studies. All work will be carried out using two different model systems, recombinant yeast and human αβ-tubulin, providing insight into whether and how microtubule nucleation and αβ-tubulin biochemistry vary across species.This works is jointly funded by the Molecular Biophysics and Cellular Dynamics and Function Clusters of MCB.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.