Title: Coordination of molecular motor activity in intracellular transport and assembly of cytoskeletal architecture. P.I. ? Richard J. McKenney Research Summary Intracellular transport is essential for cellular homeostasis in eukaryotes. Much of this process is carried out by molecular motors that convert the chemical energy from ATP hydrolysis into motion along the actin and microtubule cytoskeletal networks. Decades of research has uncovered structural and molecular details that explain how many of these motors move along their filament tracks in isolation. In the cellular milieu, most of these motors act in concert with complex regulatory machinery that links them to their respective cargos, modulates their motile properties, and dictates spatiotemporal activity. How individual motor output is controlled by this machinery is currently not clear and difficult to dissect in the complex environment of the cell. In addition, many cargos are moved simultaneously by motors of opposite polarity, in a process called bidirectional transport. How individual motors are recruited to cargo, activated, and integrated with other classes of motors presents a large challenge to the field. Further, the activities of disparate motors are harnessed to build and maintain critical cytoskeletal structures such as the mitotic spindle, cilium, and cleavage furrow. How motor and regulatory activities are coordinated to drive the self-assembly of such structures is currently a significant barrier to understanding normal and diseased cellular physiology. This application seeks to develop novel assays and tools to study the complexity of motor recruitment and regulation, bidirectional transport of cargos, and the self-assembly of cytoskeletal structures driven by motors and associated molecules. Our approach to combine biochemistry and single- molecule analysis towards in vitro reconstitutions that test molecular function, and translate our findings into in vivo systems that test hypotheses generated by these reconstitutions, will open up fruitful long-term avenues of research. We propose to: 1) Reconstitute and study the recruitment, regulation, and motility of cytoplasmic dynein and kinesin motors bound to membranous cargo through the endogenous Rab GTPase machinery that is known to link these motors to endocytic vesicles and mitochondria in cells, and 2) Reconstitute and study functions of dynein and kinesin motors that drive the self-assembly of the mitotic spindle. These broad goals build and expand on our expertise and previous work in dissecting the regulatory mechanisms of the cytoplasmic dynein motor, and aim to provide powerful new tools useful towards dissecting complex motor function. Our work will illuminate basic molecular and cell biological principles that drive cellular homeostasis and provide insight into the pathological mechanisms that arise from molecular motor malfunction.