Amoeboid cells of Dictyostelium discoideum and rat cultured cells are chemotactic to a variety of agents including extracellular cAMP and EGF, respectively. Stimulation of cells 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. We have demonstrated that the initiation of actin polymerization transients that occur in response to cAMP stimulation of Dictyostelium and EGF stimulation of rat cells involves the severing of actin filaments. Furthermore, this severing appears to result from the switching on of a cofilin-like severing activity which accounts for the increase in barbed ends and filament number observed in cells after stimulation. The cofilin-induced actin polymerization transient appears to be terminated by a combination of capping by capping proteins and switching on of a gelsolin-like severing activity. We propose to study the role of cofilin in generating and severing in terminating the actin polymerization transients in cells. This will be done by controlled expression of cofilin in mutant cells transfected with gene disruption and antisense constructs. We will study the regulation of barbed end number, filament severing, F-actin content, filament number and filament length in situ in mutants expressing various levels of cofilin and severin, and correlate these effects with cell behavior quantitatively. The effects of under- and overexpression of cofilin and severin on the structure of the actin cytoskeleton and behavior of cells will be likewise correlated. This analysis is predicted to allow a complete description of the molecular interactions required for the actin polymerization transients seen in a variety of cells during hormones stimulation.
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