Neurons extend axons over long distances during the development of the nervous system and also during regeneration following injury. The establishment of a highly organized array of microtubules is essential for the growth and maintenance of the axon. Each microtubule within the axon has a consistent lattice structure and is oriented with its assembly-favored """"""""plus-end"""""""" distal to the cell body of the neuron. In typical nonneuronal cells, microtubule organization and lattice structure are regulated by nucleation and attachment of the microtubules to a structure near the nucleus termed the centrosome. Axonal microtubules are not attached to the centrosome, however, raising the question as to how their structure and organization are regulated. Recent studies suggest a model in which microtubules destined for the axon are nucleated at the centrosome, where they aquire their consistent lattice structure, and then released for transport into the axon. A molecular motor protein is hypothesized to transport the microtubules specifically with their plus-ends-leading, thus establishing the uniformly plus-end-distal polarity pattern of the axonal microtubule array. If this model is correct, it should be possible to identify the relevant motor protein. It should also be possible to identify other important components of the cellular machinery that transports axonal microtubules. In this grant application, experiments are proposed to test the hypothesis that cytoplasmic dynein is the relevant motor protein and that it transports microtubules by generating forces against the actin filament network within the neuron. Additional experiments are aimed at determining whether microtubule-associated proteins such as tau play a role in microtubule transport by mediating the interactions between microtubules, actin filaments, and cytoplasmic dynein. These studies will involve a battery of cell and molecular biological assays on cultured rat sympathetic neurons and nonneuronal cells. The results of these studies will provide new insights into the mechanisms by which axons grow, and will assist in the development of strategies for dealing with pathologies of the nervous system involving microtubules.
Showing the most recent 10 out of 56 publications
