The goal of this project is the characterization of the mechanism(s) by which thyroid hormone regulates actin polymerization and, thus, affects the integrity of the actin cytoskeleton in cells of the Central Nervous System. Because alterations in polymerization of the actin cytoskeleton would significantly affects the complex interactions between the cytoskeletal network and the extracellular matrix, resulting in attenuation of neuronal growth and development, studies of the interactions of T4 with the actin cytoskeleton will provide insight into thyroid hormone's role in the growth and differentiation of neurons. The model system to be examined is the T4- dependent regulation of type II iodothyronine 5'-deiodinase (5'D-II) in cultured, cAMP-stimulated glial cells. T4 modulates levels of this important cerebrocortical enzyme by decreasing the biological half-life of 5'D-II through a novel, extra-nuclear mode of action. Disruption of the microfilaments blocks this T4-mediated action, indicating that the actin cytoskeleton is a likely site for regulation by thyroid hormone. 5'D-II can be affinity labeled by a T4 derivative and, thus, can be visualized in the cell using immunocytochemical and histochemical techniques. The morphological characterization of the interactions between T4, actin and 5'D-II will be accomplished during the first phase of this project. The second phase of the project will involve the identification of the mediator(s) of thyroid hormone's effects on the cytoskeleton. Actin and T4 affinity chromatography will be used to isolate potential actin- binding proteins which will then be evaluated in a cell free system to asses their ability to affect actin polymerization. Protein(s) that bind to filaments actin and T4 and that demonstrate a T4-modulated effect on actin polymerization will be characterized further with 2D SDS-PAGE and peptide mapping and compared to information available on actin-binding proteins previously described. The identification and characterization of the mechanism(s) by which T4 interacts with the cytoskeleton, and thereby regulates the turnover of a short-lived membrane protein, will then allow the study of the more fundamental questions regarding thyroid hormone's influence on, and participation in, the growth of developing neurons and the subsequent establishment of cell-cell and cell-substratum attachments.
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