Axons and dendrites contain elaborate cytoskeletons that consist of microtubules (MTs), neurofilaments (NFs), and actin filaments. These structures comprise an architectural framework that defines the external shape of the axon and dendrite. Thus, the mechanisms that establish the cytoskeleton contribute directly to the elaboration and maintenance of neuronal form and thereby function. This application proposes direct experiments on the dynamic processes that establish the cytoskeleton in growing axons. This research focuses on MTs and NFs, which are linear polymers of tubulin and NF proteins, respectively. Tubulin and NF proteins are synthesized in the neuron soma and are then actively transported into the axon. We have recently developed a system for directly visualizing vectorial transport of NFs in living axons of cultured neurons. The major goal of this application is to dissect the mechanisms of this transport. Toward this end, we propose to (1) define the cytoskeletal elements required for NF transport (2) identify motor proteins that transport NFs, and (3) dissect tubulin/MT transport mechanisms in living neurons. All of this research will use time-lapse video microscopy to directly visualize cytoskeletal transport in living neurons. Cytoskeletal elements will be labeled by expression of green fluorescent protein (GFP) chimeras of tubulin and NF protein that are assembly competent and reliably mark MTs and NFs, respectively, for microscopic visualization. Transport mechanisms will be identified using manipulations that perturb putative transport systems. Much of the required methodology has already been developed and used to begin examining mechanisms of NF transport. The results obtained will be integrated with our previous results on cytoskeletal assembly and disassembly to define how polymer transport mechanisms coordinate with polymer assembly mechanisms to generate the cytoskeletal arrays required for axon growth. In addition, many pathologies of the nervous system are characterized by abnormalities of cytoskeletal organization. By defining normal mechanisms for establishing the neuronal cytoskeleton, the proposed research will contribute toward a better understanding of these pathologies.
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