ORGANELLE MOVEMENT IN FAST AXONAL TRANSPORT

University Of Virginia Charlottesville, Charlottesville VA

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Abstract

DESCRIPTION The neuronal cell body is the site of most axonal protein synthesis. Therefore, material necessary for the maintenance and growth of the axon must be transported from the cell body along the axon to the terminal. Thus, axonal transport is important for nerve growth and regeneration, and defects in axonal transport result in neurodegeneration and disease. The goal of this project is to understand one component of axonal transport, how membranous organelles, and their cargo, are transported along the axon in fast axonal transport. Two microtubule-based proteins are the motors for this movement. Kinesin is the motor for organelle transport in the anterograde direction (from the cell body to the axon terminal) while cytoplasmic dynein moves organelles in the retrograde direction, from the terminal to the cell body. However, many details about the coordination of anterograde and retrograde organelle movements remain to be explained. This proposal concentrates on the retrograde motor, cytoplasmic dynein. It has been found that distinct dynein complexes, which are distinguished by different isoforms of the dynein intermediate chain subunit (IC74), are transported along the axon in the anterograde direction in two different compartments. One pool of dynein is moving in slow axonal transport, and a second pool is associated with the membranous organelles moved by kinesin. The goal of this proposal is to understand the regulation of dynein and the relationship of these two anterograde pools of dynein to the retrograde dynein motor. Epitope tagged IC74 isoforms will be transfected into cultured cells and the identity of the IC74 isoforms which are associated with retrogradely moving organelles in vivo will be determined. The different dynein complexes will be purified and the functional properties of each complex will be determined, including ATPase activity, in vitro motility, and organelle binding. Differences in the phosphorylation state of the dynein complexes have been identified. The phosphorylation state of dynein will be manipulated in vitro and the role of phosphorylation in regulating the properties of dynein will be determined.