Actin cross-linking proteins interact with actin filaments to form either randomly associated networks, or filament bundles. A 30,000 dalton protein present in the filopodia of Dictyostelium organizes actin filaments into bundles in vitro, and this bundling activity is inhibited by elevation of the concentrations of free calcium or magnesium. The primary amino acid sequences of this protein will be deduced by isolation and sequencing of the cDNA encoding the protein. The results will be used to identify regions of the primary sequence of the protein that may potentially interact with actin and with calcium and magnesium ions. Functionally significant regions of the molecular will be identified directly by studies of the activity of proteolytic fragments of the 30,000 dalton protein, and by studies of the regions of the 30,000 dalton protein and actin that are trapped in direct association by chemical cross-linking reagents. A quantitative assay for the orientation of actin filaments in cross-linked actin networks will be developed. Careful studies of the structures formed in mixtures of actin with different cross-linking proteins may help to reveal differences in the activities of the multiple actin cross-linking proteins present in cells. Actin monomer binding proteins are believed to regulate the self- assembly of actin. A novel actin monomer binding protein will be purified from Dictyostelium discoideum, and its structure and interaction with actin will be dissected. Antisera specific for the protein will be elicited in rabbits, and used to determine the quantity and distribution of this protein in Dictyostelium amoebae, and to determine the presence of related proteins in other species. Knowledge of the novel actin monomer binding protein and the 30,000 dalton protein provide a foundation for future use of biochemical and genetic approached to determine the contributions of these proteins to control of actin filaments in cells, and the molecular mechanisms of essential cellular processes such as locomotion, phagocytosis, and cytokinesis. Cell movements such as phagocytosis, locomotion, and cytokinesis are essential to the life of eucaryotic cells. Actin filaments contribute to the control of cell shape and generation of force for cell movements. The properties of actin in cells must be explained from a knowledge of the structure and chemistry of purified actin, and of the properties of actin binding proteins. The presence of similar actin binding proteins in invertebrate and vertebrate cells provides strong evidence for the significance of these proteins as regulators of the behavior of actin in the cytosol, and motivates detailed inquiry.
