PROJECT SUMMARY Microtubule-associated motor proteins couple the energy derived from ATP hydrolysis to mechanical work, which is then used to power intracellular movements associated with cell division, migration, and transport of cellular cargoes. There are two classes of microtubule-associated motors, cytoplasmic dynein-1 (dynein) and kinesins. These motors move in opposite directions on the microtubule track: dynein moves toward the microtubule minus- end, which is typically near the nucleus and kinesin moves toward the microtubule plus-end. While there are over forty kinesins, there is only one dynein that promotes cytoplasmic cargo trafficking. Dynein is highly regulated and interacts with a vast network of proteins that modulate its function and activity. However, molecular mechanisms of how many dynein binding proteins regulate dynein activity is largely unknown. Understanding how dynein is regulated is a pressing need as mutation in dynein or its regulators is associated with many neurodevelopmental and neurodegenerative diseases. This proposal seeks to decipher the rules that govern dynein-mediated cellular trafficking using an interdisciplinary approach combining proteomics, biochemistry, structural biology, single molecule fluorescence microscopy, and fixed and live cell imaging. We will focus on two overarching goals. The first goal is to determine the function of a new class of dynein regulatory proteins that our lab has discovered. These novel regulators associate with kinesin, dynein, and known dynein regulators and promote the formation of focal adhesions. By determining the function of these new regulators, we will determine the role of microtubule-based transport in focal adhesion assembly and dynamics. The second goal is to determine the molecular mechanism of how the dynein regulatory protein, Nudel functions. Here, we will focus on determining how Nudel effects the kinetics of dynein activation and dynein’s ability to bind to and move on the microtubule track. Completion of this goal is a pressing need, as Nudel dysfunction is associated with microcephaly, epilepsy, and schizophrenia. Together, the results of this work will reveal novel mechanisms of dynein regulation, which has broad implications in human health and disease.