CAREER: Understanding Microtubule Dynamics using Biochemically-defined Tubulin Mutants to Integrate Structure, Biochemistry, and Kinetics

智力优势：本研究的目标是通过使用新开发的酵母α - β -微管蛋白的机械定义突变体进行一系列以前不可能的实验，统一微管动力学的结构，生化和动力学观点。微管是α - β -微管蛋白的动态聚合物，可以在生长和收缩之间切换。微管细胞骨架对真核细胞至关重要，是染色体分离和细胞内组织所必需的。微管的动力学对其功能至关重要，并且源于单个微管亚基的性质及其在MT晶格内的相互作用。聚合和解聚合的微管末端以及微管的核心都显示出不同的特征几何形状，这是由于α - β -微管蛋白的不同构象造成的，这必然会影响微管蛋白的生物化学：微管蛋白相互作用。我们对微管动力学的分子机制了解很多，但重要的问题仍未得到解答：α - β -微管蛋白的默认构象是什么，它是否取决于核苷酸状态，α - β -微管蛋白的不同构象如何影响微管蛋白：微管蛋白相互作用和聚合物端构型？了解结构、生物化学和聚合物动力学是如何相互关联的一直是具有挑战性的，因为很少有实验室研究重组α - β -微管蛋白，因此很少有原子结构和基于结构的诱变的力量几乎没有被利用。本研究将：(i)使用酵母和动物MT动力学的比较分析来发现和探索它们不同聚合动力学的生化起源，（ii）通过实验和计算分析生物化学定义的突变体如何干扰MT动力学，确定MT端构型和GTP/GDP分布与灾难的关系，以及（iii）通过量化MT晶格外的α - β -微管蛋白自身相互作用来统一MT动力学的结构和生化观点。通过确定β -微管蛋白的新结构。微管动力学是一个具有挑战性的前沿问题，它测试了我们将“一次一个分子”的生物化学和结构观点与跨越不同长度和时间尺度的集体行为的低分辨率测量相结合的能力。只有结合不同的方法，我们才有希望理解单个蛋白质的结构和生化特性之间的联系，以及它们集体相互作用产生的复杂行为。这项工作的成功完成将代表我们从分子角度理解这种基本和复杂行为的能力的重大进步。更广泛的影响：该项目有两个主要的教育目标：(i)在一所公立K-3小学开发一个以显微镜为重点的、基于探究的教育项目，该小学的学生群体几乎完全由未被充分代表的少数民族组成；（ii）建立一个1对1的指导项目，鼓励和促进来自未被充分代表的群体的德克萨斯大学西南分校研究生申请博士前奖学金。K-3教育活动将为服务不足的人口提供使用数字显微镜进行“现场”实验的机会，这是一种先进但相对便宜的技术。在UT西南社区志愿者的指导下，学生们将以小组为单位，收集自己的样本（来自家庭和/或学校的熟悉的自然或人造物体），在显微镜下研究它们，并讨论他们的结果。基本上，他们将以一种强调观察和思考的方式来实践科学，这是令人兴奋和引人入胜的，这将补充更多传统的教科书驱动的教学方法。奖学金指导活动旨在促进来自代表性不足群体的研究生的进步，采用导师配对的方法，提供个性化的一对一指导，帮助他们确定感兴趣的研究主题，并围绕该主题制定建议。

Intellectual Merit: The goal of this research is to unify structural, biochemical, and kinetic views of microtubule dynamics by performing a series of previously impossible experiments using newly developed, mechanistically defined mutants of yeast alpha beta-tubulin. Microtubules are dynamic polymers of alpha beta-tubulin that switch between growing and shrinking. The microtubule cytoskeleton is essential to eukaryotic cells and is required for chromosome segregation and intracellular organization. The dynamics of microtubules are essential for their function, and derive from the properties of individual tubulin subunits and their interactions within the MT lattice. Polymerizing and depolymerizing microtubule ends, and the core of the microtubule, all show distinct characteristic geometries that result from different conformations of alpha beta-tubulin and that must inevitably affect the biochemistry of tubulin:tubulin interactions. Much is known about the molecular mechanisms of microtubule dynamics, but important questions remain unanswered: what is the default conformation of alpha beta-tubulin, does it depend on nucleotide state, and how do the different conformations of alpha beta-tubulin affect tubulin:tubulin interactions and polymer end configurations? Understanding how structure, biochemistry, and polymer kinetics interrelate has been challenging because very few labs work with recombinant alpha beta-tubulin and as a result there are few atomic structures and the power of structure-based mutagenesis has barely been tapped. This research will: (i) use comparative analysis of yeast and animal MT dynamics to discover and explore the biochemical origins of their differing polymerization dynamics, (ii) determine how MT end configurations and GTP/GDP distributions relate to catastrophe by experimentally and computationally analyzing how biochemically defined mutants perturb MT dynamics, and (iii) unify structural and biochemical views of MT dynamics by quantifying alpha beta-tubulin self interactions outside the MT lattice, and by determining new structures of alpha beta-tubulin. Microtubule dynamics is a challenging frontier problem that tests our ability to integrate "one molecule at a time" views of biochemistry and structure with lower resolution measurements of collective behavior spanning different length and time scales. Only by combining diverse approaches can we hope to understand the connections between the structural and biochemical properties of individual proteins and the complex behavior that emerges from their collective interactions. Successful completion of this work will represent a major advance in our ability to understand this essential and complex behavior in molecular terms.Broader Impact: This project has two primary educational goals: (i) to develop a microscopy-focused, inquiry-based educational program at a public K-3 elementary school with a student body almost entirely composed of underrepresented minorities and (ii) to establish a 1-on-1 mentoring program to encourage and facilitate UT Southwestern graduate students from underrepresented groups to apply for predoctoral fellowships. The K-3 educational activities will provide an underserved population access to 'live' experiments using digital microscopy, an advanced but relatively inexpensive technology. Working in small groups, and guided by volunteers from the UT Southwestern community, the students will collect their own samples (familiar natural or manmade objects from home and/or school), study them in the microscope, and discuss their results. Basically, they will do hands-on science in a way that emphasizes observation and thinking, that is exciting and engaging, and that will complement more traditional textbook-driven, didactic approaches. The fellowship mentoring activities aim to foster the advancement of graduate students from underrepresented groups using a mentor-matching approach that will provide personalized, one-on-one guidance to help them identify a research topic of interest and formulate a proposal around it.