Axons contain an elaborate cytoskeleton that consists of microtubules, neurofilaments, and microfilaments. The cytoskeleton comprises an architectural framework that defines the external shape of the axon and also organizes the intracellular motility necessary to grow and maintain the axon. Thus, the mechanisms that establish the cytoskeleton in neurons contribute directly to the elaboration and maintenance of neuronal form and thereby function. This application proposes direct experiments on the dynamic processes that generate the cytoskeleton in growing axons. These experiments focus on microtubules, with the goal of defining the mechanisms that modulate microtubule assembly and dynamics in growing axons. These mechanisms are essential to the generation of the microtubule array required for axon elongation and to aspects of growth cone motility involved in axonal pathfinding. The proposed experiments focus on three proteins that are excellent candidates to modulate the properties of the microtubule array in growing axons; these proteins are tau, microtubule-associated protein 1b (MAP1b), and stable-tubule-only-polypeptide (STOP). All three proteins promote microtubule assembly and stability in vitro. We hypothesize that these proteins establish the dynamic behavior of microtubules required for the initiation and continued growth of the axon. To test this hypothesis, we propose to selectively inactivate these proteins using antibody injection approaches, or to introduce excess amounts of these proteins into neurons, and then to use high resolution video and fluorescence microscopy as well as electron microscopy to evaluate the consequences of these manipulations on axon structure, growth cone motility, and the organization, assembly dynamics, and stability of axonal microtubules. We have already made progress on tau and MAP1b functions in developing neurons using these approaches. Successful completion of the proposed experiments will define essential mechanisms involved in generating the microtubule arrangements required for the normal growth of axons. In addition, many pathologies of the nervous system are characterized by abnormalities of cytoskeletal organization. By defining normal mechanisms for generating and maintaining microtubule arrangements in neurons, the proposed research will contribute toward a better understanding of these pathologies.
| Slaughter, Theresa; Black, Mark M (2003) STOP (stable-tubule-only-polypeptide) is preferentially associated with the stable domain of axonal microtubules. J Neurocytol 32:399-413 |
| Roy, S; Coffee, P; Smith, G et al. (2000) Neurofilaments are transported rapidly but intermittently in axons: implications for slow axonal transport. J Neurosci 20:6849-61 |
| Tint, I; Slaughter, T; Fischer, I et al. (1998) Acute inactivation of tau has no effect on dynamics of microtubules in growing axons of cultured sympathetic neurons. J Neurosci 18:8660-73 |
