Neurons require axonal transport to establish and maintain axons and synapses. Cytoplasmic dynein is the motor for fast retrograde transport of membranous organelles and viruses from the axon terminus to the cell body. Dynein is also implicated in the transport of microtubules and neurofilaments along the axon in slow transport. Dynein is a large protein complex and very little is known about how it functions. This application will investigate the role of different isoforms of subunits in the cargo-binding domain in dynein binding to organelles, cytoskeletal filaments, and other proteins. Recent evidence suggests that the dynein light chains are important for dynein binding to specific proteins.The first three aims focus on the functional role of the tctex1 family. (1) Do the two members of the family, tctex1 and rp3, interact with different proteins? Yeast two-hybrid and protein binding assays will be used to identify rp3 binding partners. (2) Do the two light chains identify dynein populations with different roles in cells? Mutated forms of each of the light chains will be used in a dominant-negative approach to determine if expression of a mutated light chain disrupts a subset of dynein-based motility. (3) Biochemical analyses and in vivo co-expression methods will be used to test the hypothesis that the light chains are necessary for the association of intermediate chains in the dynein cargo-binding domain. Dynein is in the anterograde fast and slow axonal transport components and the two populations of dynein are composed of different intermediate chains.
The fourth aim focuses on the roles of the two anterograde pools of dynein. Epitope-tagged intermediate chains will be transfected into cultured cells and immunocytochemistry will be used to determine if dyneins with different intermediate chains are bound to different organelles or cytoskeletal structures. Live cell imaging of intermediate chain-GFP fusion proteins will be used to determine if the two pools of dynein have different anterograde and retrograde motility properties. These studies will contribute valuable information about the mechanism of axonal transport, a neuronal process important for axon growth and regeneration.
