Actin is a major cytoskeletal protein in neuronal cells, and is involved in a variety of motile functions such as growth cone motility, axonal transport and neurotransmitter release. In the cell, actin monomers and polymers exist in a dynamic equilibrium which is dependent on the interplay between actin binding proteins controlling the rate of actin nucleation, the availability of actin monomers and the length and number of filaments. Each class of regulatory proteins have been identified in the brain. Recently, thymosin beta4 (Tbeta4), a protein which is abundantly expressed in the brain, has been identified as an actin monomer binding protein which may play a significant role in maintaining a large proportion of the actin in the unpolymerized form. It suppresses spontaneous actin nucleation but allows actin to polymerize from uncapped actin nuclei. This provides a simple mechanism for coordinating spatial and temporal control of the actin polymerization cascade in cells. In this grant, we will examine the role of beta-thymosins in regulating actin assembly and disassembly in neurons. Because beta-thymosin function must be considered in the context of a multi-component system, we will also characterize, but in less detail, gelsolin, an actin severing and capping protein and profilin, an actin monomer binding protein. First, we will determine if Tbeta4 regulates growth cone motility and actin dynamics. The distribution of Tbeta4 in neurons will be examined. Tbeta4 will be microinjected into neurons and effects on growth cone motility and actin dynamics will be monitored. Second, to test the hypothesis that Tbeta4, which is induced in PC12 cells by nerve growth factor, promotes neurite extension by inducing actin depolymerization. Tbeta4 level in PC12 will be raised by bulk loading or transfection of an expression vector. Tbeta4 level will be decreased by bulk loading of anti-Tbeta4 antibody and incubation with antisense nucleotides. Third, determine if Tbeta4 is important for the regulation of actin dynamics in synaptosome and growth cone preparations. Fourth, identify the Tbeta4 actin binding site, which may also be present in several other actin binding proteins. The combined use of microscopic, biochemical and molecular biological approaches will provide a better understanding of how the actin cytoskeleton is maintained in several subdomains of the neuron. The combined use of microscopic, biochemical and molecular biological approaches will provide a better understanding of how the actin cytoskeleton is maintained in different subdomains of the neuron.
