Neurons extend axons over long distances to reach their target tissues during normal development and also during regeneration in response to injury. Axons contain an elaborate system of microtubules that provides the architectural framework of the axon, and organizes the cytoplasm within the axon to carry out essential mottle and metabolic processes. The elaboration of this microtubule framework is directly involved in the growth and maintenance of the axon. Microtubules are polymers of the protein tubulin, and several lines of evidence indicate that axon growth involves the assembly of new microtubules followed by their stabilization and incorporation into the stable cytoskeleton of the axon. No direct data exist which indicate where microtubule assembly and stabilization occur in growing axons and how these processes are regulated. Resolution of these issues is essential to a molecular explanation for the generation of the microtubule framework required to support the growth of the axon. The present application proposes experiments to address these issues. Indirect evidence indicates that microtubule assembly in the axon is regulated by discrete templates that nucleate tubulin assembly. One major goal of this application is to identify the microtubule nucleating structures of the axon and to determine their location within the axon. We shall also determine where in the axon newly assembled microtubules become stabilized, and how this stabilization occurs. These experiments involve direct visualization of newly assembled and stable microtubules of the axon using both quantitative immunofluorescence and immunoelectron microscopy. Successful completion of the proposed experiments will resolve several issues concerning the spatial regulation of microtubule assembly and stabilization during axon growth. This information will further current understanding of cellular and molecular processes that contribute to neuronal development and plasticity, and may provide clues to the cellular defects that underlie the cytoskeletal abnormalities associated with degenerative pathologies of the nervous system.
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