Amoeboid cells of Dictyostelium discoideum are chemotactic to a variety of agents including extracellular cAMP. As in other chemotactic amoeboid cells, such as leukocytes and metastatic tumor cells, stimulation of Dictyostelium amoebae with chemoattractant elicits a burst of actin polymerization. The location and timing of this polymerization response helps determine subsequent cell polarity, and creates part of the force for pseudopod extension. In the previous funding period we identified and purified a protein, aginactin, that may be involved in regulating chemoattractant induced actin polymerization in cells by uncapping the barbed ends of cytoskeleton associated actin filaments. In this renewal we propose to characterize aginactin at the molecular level. Aginactin is a cytosolic isoform of the HSC-70 family of proteins. We will identity which members of the HSC-70 family have aginactin-like capping activity through the use of antibodies that specifically crossreact with the subset of HSC-70s that have capping activity and by protein biochemistry. We will analyze the domain structure of aginactin to identity phosphorylation, actin binding and capping domains in the protein to gain insights into the mechanism of capping and its regulation. The localization of aginactin in situ will be determined relative to F-actin and the free barbed ends of actin filaments particularly immediately before and after chemotactic stimulation that causes uncapping of barbed ends. To determine the function of aginactin in vivo we will prepare gene disruption, replacement and/or antisense constructs that can be used to disrupt or lower the expression of, or overexpress aginactin in vivo. Phenotypes of transformants prepared by these methods will be analyzed using established biochemical assays, fluorescence microscopy and computer assisted video microscopy to detect even subtle differences compared to wild type and control cells. The combined approaches of protein chemistry, bacterial expression and molecular genetics in Dictyostelium will provide a definitive analysis of the structure and function of aginactin.
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