The goal of this project is to understand how membrane bounded organelles move in neuronal process during fast axonal transport. Two ATPases, kinesin and dynein, are proposed to be the motor proteins the movement of membrane bonded organelles along microtubules in fast axonal transport. It is believed that kinesin is the motor for organelle transport in the anterograde direction (from the cell body to the synaptic terminal) while 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 remains to be explained. One basic missing component is an understanding of how membrane associated dynein is transported to the synaptic terminal without interfering with anterograde movement. It is reasonable to propose that the motor activity of dynein is regulated. At least two mechanisms can be proposed to account for such regulation:(1) a posttranslational modification of dynein could turn it on and off as necessary; or (2) specific regulatory polypeptides cold modulated the functional state of dynein, will be identified which reflect the different functional states of neuronal cytoplasmic dynein, will be tested by characterizing anterograde and retrograde dynein from sciatic nerve blocks and other axonal transport model systems. To determine if the observed differences contribute to the regulation of the motor activity of dynein, the functional properties of the different forms of dynein will be measured, including ATPase activity. The functional significance of observed differences in the phosphorylation of anterograde and whole cells dynein polypeptides will be measured. The association of dynein with membrane bounded organelles will be studied in vivo and in vitro. Antibodies to mammalian brain cytoplasmic dynein will be used as inhibitory probes of the function of dynein in the extruded axoplasm model system.
