CAREER: Single Molecule Studies to Dissect the Mechanochemistry of Cytoplasmic Dynein

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Scientific Merit:Cytoplasmic dynein is a dimeric motor protein responsible for nearly all minus-end directed transport along microtubules in eukaryotic cells. Dynein forms a large dimeric complex and it differs significantly from kinesin motor protein in origin, structure, mechanochemistry and regulation. In comparison with kinesin, whose structure and mechanism are increasingly well understood, we know relatively little about dynein mechanism. The major barriers to studying dynein's motility were the lack of a recombinant expression system and a paucity of tools for observing the movement and conformational states of single motors with sufficient spatial and temporal resolution. The goal of this project is to elucidate the fundamental mechanism of cytoplasmic dynein motility by using state-of-the-art single molecule methods in vitro. Three outstanding questions will be addressed about dynein motility: 1) How is movement of the two motor domains coordinated to maintain high processivity 2) How does ATP hydrolysis in the ring lead to unidirectional motion and which AAA sites are responsible for dynein motility 3) What is the structural basis for dynein's minus-end directionality? Dissecting the mechanism of dynein motility will significantly advance understanding of the way dynein functions in diverse cellular processes.Broader Impacts: This project will also include the following educational and outreach components: 1) Establish an educational outreach program for high school students from underrepresented areas of Oakland, CA. 2) Organize summer research opportunities and science fairs in local public schools to attract young generations to science. 3) Develop a new multidisciplinary graduate level course in biophysical imaging methods. 4) Develop a new curriculum with designated emphasis in Biophysics for Physics majors.