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

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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.