Mechanistic analysis of microtubule nucleation

该项目旨在定义决定新微管如何从其构建模块-αβ-微管蛋白亚基形成的分子规则。微管是动态聚合物组件，其组织细胞内部，在细胞分裂期间介导遗传物质的忠实分离，并为基于马达的运输提供轨道。这项研究将集中在微管成核，这是一个知之甚少的过程，αβ-微管蛋白单元通过自组装启动一个新的微管。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速率和形成新微管之前的低聚中间体序列，以及这些在不同物种的αβ-微管蛋白中如何变化。该项目还强调教育影响。从事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。与当地小学和中学的外联伙伴关系将为代表性不足和经济困难的学生带来科学的兴奋，同时为教师和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。这项建议的具体研究目标是建立一个微管成核的分子理解。微管的动力学性质来源于单个αβ-微管蛋白亚基的生物化学性质及其在聚合物组装体内的相互作用。MT成核-从未聚合的亚基形成新的聚合物-是一种知之甚少的行为，对于构建MT网络至关重要，并且它受到细胞因子的高度调节。该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GTdR活性被抑制的简化环境中发展微管成核的机制理解，（ii）使用Monte Carlo模拟来创建以可以解释GTdR活性的影响的方式控制MT成核的生化机制的更精确和可概括的描述，（iii）利用蛋白质工程来组装和捕获αβ-微管蛋白的特定寡聚体，否则这些寡聚体将太不稳定而无法使用，并将这些寡聚体用于新类型的结构，生物化学和机制研究。所有工作将使用两种不同的模型系统进行，重组酵母和人αβ-微管蛋白，提供了对微管成核和αβ-微管蛋白的生物化学因物种而异。这项工作是由MCB的分子生物物理学和细胞动力学和功能群联合资助的。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值进行评估来支持和更广泛的影响审查标准。

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.