DESCRIPTION (provided by applicant): Molecular motors drive the active transport of organelles and vesicles along the cellular cytoskeleton. This transport is required for normal cellular function in higher eukaryotes, but is critically important in highly polarized cells such s neurons. Neurons extend axons that can reach up to 1 meter in length. Axons must be continually supplied with proteins and organelles from the cell body~ clearance of aging proteins and dysfunctional organelles are also required to maintain cellular homeostasis. Thus, axonal transport driven by the coordinated activities of cytoplasmic dynein and kinesin motors is essential, and mutations in the motors that drive this transport cause neurodegeneration. Here we focus on understanding the molecular coordination of dynein and kinesin motors driving axonal transport, using the synergistic approaches of live cell imaging and in vitro reconstitution with single molecule resolution to understand the mechanisms involved. We will focus on three specific aims: (1) how does dynactin activate dynein-driven transport? We hypothesize that the binding of dynein to dynactin activates the motor by enhancing recruitment to the microtubule and inducing a shift from diffusive movement to processed motility. Dynactin is a required activator for most dynein-driven functions in the cell. Importantly, however, it is still not clear how dynactin activates dynein. Using newly developed tools and approaches, we will test three specific hypotheses: (i) binding to dynactin enhances recruitment of dynein to the microtubule~ (ii) binding to dynactin enhances dynein's processivity~ and (iii) dynein regulatory proteins dynactin and Lis1 synergistically activate dynein during long-distance axonal transport. (2) What are the mechanisms regulating the engagement of dynein-driven retrograde transport? We hypothesize that there is a spatially-specific mechanism for retrograde transport initiation in the neuron, involving the ordered recruitment of microtubule plus-end binding proteins, dynactin and dynein that is required for normal neuronal function. We will test this hypothesis by addressing the following questions: (i) is binding to CLIP170 necessary and sufficient to explain the recruitment of dynactin to dynamic microtubule plus-ends leading to retrograde transport initiation? And, (ii) what regulates retrograde transport initiation in the neuron? (3) What are he mechanisms coordinating bidirectional organelle transport along the axon? We hypothesize that scaffolding proteins regulate dynein and kinesin motors bound to cargo, and that motors are clustered on the vesicle to facilitate this coordination. We will use live imaging in primary neurons as well as structural approaches including super-resolution microscopy and cryoEM to examine the role of the scaffolding protein JIP1, and the clustering of motors on axonal transport vesicles. Together, these approaches should provide important new information on the mechanisms of motor function during organelle transport. As disruption of these mechanisms leads to neurodegeneration, continued progress will provide new insights into the pathogenesis of neurodegenerative disease and offer new opportunities for targeted therapies.

PUBLIC HEALTH RELEVANCE: The active movement of proteins, vesicles, and organelles along the extended axons of neurons is called axonal transport, and is driven by the molecular motors dynein and kinesin. This transport is essential to maintain healthy neurons, which have axons that can extend for a meter. Opposing dynein and kinesin motors bind to organelles undergoing transport along the axon~ their motor activities must be tightly coordinated to achieve specific and directed transport in the neuron. As defects in axonal transport cause neurodegeneration and contribute to the pathogenesis of diseases such as Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's disease), it is essential to understand the mechanisms regulating molecular motor function.