The overall goal of this proposal is to understand the structure and function of the neuronal cytoskeleton. The proposed studies are designed to give us a basic knowledge of the function of the neuronal cytoskeleton in the normal CNS. Towards this end, we will use a wide variety of techniques to study the expression, assembly, phosphorylation and interactions of a number of neuronal cytoskeletal proteins. Ultimately, we hope that this information will help us understand why and how the neuronal cytoskeleton is perturbed by certain neurological diseases, such as Alzheimer's disease and amyotropic lateral sclerosis and by exposure to neurotoxins, such as aluminum and acrylamide.
The specific aims of this proposal are 1) to study the interactions and assembly of the three neurofilament proteins using purified enzymatic and cyanogen bromide derived fragments of each neurofilament triplet molecule to determine the role of phosphorylation of these three proteins in the neurofilament assembly; 2) to identify and isolate the kinase and/or phosphatase responsible for the phosphorylation of the neurofilament proteins; 3) to continue our studies on the interactions of neurofilaments and microtubules, in order to define which microtubule associated proteins (MAPs) are involved in these interactions; 4) to study the importance of the cytoskeletal associated ATPase, Beta-internexin in cytoskeleton-mediated cytoplasmic transport in neurons and cultured cells and 5) to obtain cDNA probes for the high molecular weight neurofilament proteins, NF-M and NF-H, which will be used to study the expression and function of these proteins and to study the assembly of various modified proteins. The cDNA clones will be placed in expression vectors and their distribution will be studied in cells, which do not normally contain neurofilaments. These studies will allow us to determine what structures they form in the cell and what their function might be.
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