Neurons extend axons over long distances to reach their target tissues during normal development and also during regeneration following injury. A growing body of evidence indicates that the growth and maintenance of the axon is dependent upon its cytoskeletal structure, and in particular of its microtubules. Microtubules are filamentous proteins that have important functions in generating and maintaining cellular architecture, regulating motile events in the cell, and in organizing the cytoplasm to carry out essential metabolic processes. Within the axon, microtubules are uniformly oriented with their """"""""plus"""""""" ends distal to the cell body, and this organization is essential for directing organelle traffic in the axon. Unfortunately, the mechanisms by which the microtubule array of the axon is elaborated and organized are poorly understood, and a matter of some controversy. In particular, there has been disagreement with regard to the sites where axonal microtubules originate, and the relative contributions of microtubule assembly and transport to the growth of the axon. This application represents the continuation of efforts to address these questions. Recent indirect evidence suggests that axonal microtubules originate at the centrosome, and are then released for translocation into and down the axon. If this is correct, then there is a shift during transit from a large number of short microtubules to a smaller number of very long microtubules within the axon. With regard to the organization of these microtubules, an attractive possibility is that the translocation of microtubules through the cytoplasm occurs exclusively with plus-ends leading, thereby establishing the uniform polarity orientation of microtubules within the axon. In this application, experiments are proposed that will evaluate the role of the centrosome in generating axonal microtubules, and determine the relative contributions of microtubule transport and assembly to axon growth. Other experiments will specifically address whether the transport properties of axonal microtubules can account for their plus-end-distal polarity orientation. Finally, efforts will be directed toward measuring in very fine detail the lengths of individual microtubules throughout the axon. Collectively, the information derived from these studies will resolve important issues about axonal microtubules, and thereby provide new insights into the mechanisms by which the axonal microtubule array is elaborated and organized. Information of this kind is essential for understanding the cell biology of the neuron, and for elucidating the causes and potential cures for neuropathologies that involve axonal degeneration and/or retardation of axon growth.
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