STRUCTURE AND FUNCTION OF CYTOPLASMIC MOTORS

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The goal of this project is to understand the distribution and functions of cytoplasmic motors in the axon of neurons. This information is intended to lead to an understanding, at the molecular level, of fast and slow axonal transport as well as the cytoplasmic organization in the axon. Our results showing that kinesin is more tightly bound to organelle surfaces than dynein have led to elaboration of the simple idea that direction of transport depends on anterograde vesicles binding kinesin and retrograde vesicles binding dynein. Since an organelle could retain its particular kinesins while it is being transported down the axon, we propose that up or down regulation of kinesin determines the direction of transport. New types of myosin motors has been discovered attached to the surfaces of squid axonal organelles, and it now appears that there are at least two species of myosin also involved in organelle transport. We have developed antibodies to both of these myosins, and both myosins are associated with organelles. New studies of the organization of the actin substrates for these motors in the axon has showed that parallel actin filaments intertwine with the microtubule bundles, and that they have mixed polarities, suggesting that they might carry organelles both to and from the microtubule bundles. We made a start on defining the mechanism of slow transport. Negatively charged macromolecule assemblies injected into the squid giant axon move in the anterograde direction at rates up to 0.5 um/sec. Of particular interest is that neurofilament proteins as well as actin and microtubule fragments move anterogradely, and that all movements appear to be along some type of intracellular tract. This in vitro assay should make it possible to define the motors for such movements. The bacterial flagellar motor in E. coli has been studied as another example of a motor system than can switch direction of translocation. We are completing a structural study of the cytoplasmic representation of this motor which is expected to provide new information on the