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

智力优势：这项研究的目标是统一的结构，生化和动力学微管动力学的观点，通过执行一系列以前不可能的实验，使用新开发的，机械定义的酵母α β-微管蛋白突变体。微管是α β微管蛋白的动态聚合物，在生长和收缩之间切换。微管细胞骨架是真核细胞所必需的，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，并且源自单个微管蛋白亚基的特性及其在MT晶格内的相互作用。聚合和解聚微管末端，以及微管的核心，都显示出不同的特征几何形状，这是由α β-微管蛋白的不同构象引起的，并且必然会影响微管蛋白的生物化学：微管蛋白相互作用。关于微管动力学的分子机制已经知道很多，但重要的问题仍然没有答案：什么是α β-微管蛋白的默认构象，它是否依赖于核苷酸状态，以及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末端构型？了解结构、生物化学和聚合物动力学如何相互关联一直是一个挑战，因为很少有实验室研究重组α β-微管蛋白，因此几乎没有原子结构，基于结构的诱变能力几乎没有被利用。这项研究将：（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物化学起源，（ii）通过实验和计算分析生物化学定义的突变体如何扰乱MT动力学来确定MT末端构型和GTP/GDP分布如何与灾难相关，和（iii）通过量化MT晶格外的α β-微管蛋白自身相互作用和确定α β-微管蛋白的新结构，统一MT动力学的结构和生物化学观点。微管动力学是一个具有挑战性的前沿问题，它测试我们将生物化学和结构的“一次一个分子”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，我们才有希望了解单个蛋白质的结构和生化特性与它们集体相互作用产生的复杂行为之间的联系。这项工作的成功完成将代表着我们在分子水平上理解这一基本而复杂的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）发展一个以显微镜为重点的，在一所公立K-3小学开展以探究为基础的教育方案，其学生群体几乎完全由代表性不足的少数民族组成，以及（ii）建立一个1对1指导计划，以鼓励和促进UT西南研究生从代表性不足的群体申请博士前奖学金。K-3教育活动将为服务不足的人群提供使用数字显微镜的“现场”实验，这是一种先进但相对便宜的技术。在小组工作，并从UT西南社区志愿者的指导下，学生将收集自己的样品（熟悉的自然或人造物体从家里和/或学校），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进行实践科学，这是令人兴奋和引人入胜的，这将补充更传统的教科书驱动的教学方法。研究金辅导活动的目的是促进来自代表性不足群体的研究生的进步，采用导师匹配的方法，提供个性化的一对一指导，帮助他们确定感兴趣的研究课题，并围绕该课题提出建议。

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.