A major focus of current work in the field of cell motility has been to elucidate the mechanisms responsible for actin polymerization transients and how they are regulated. In the context of chemotaxis this problem expands to include the spatial control of regulation of actin polymerization. There is structural and biochemical evidence that both severing by cofilin and nucleation by Arp2/3 complex are required for nucleation of actin filaments in vivo. This evidence suggests that these pathways operate either independently in different regions of the cell and/or that they can synergize to produce the amplified appearance of barbed ends, which occurs after EGF-stimulation. In this project we propose to distinguish and quantify the relative contributions of cofilin and Arp2/3 pathways to barbed end generation and protrusion during cell motility and EGF-stimulated lamellipod extension. To accomplish this goal we propose the following specific aims: 1. The preparation and use in vivo of caged cofilin to determine the function of cofilin in different regions of the cell; 2. Preparation and use in vivo of WASP- and Arp2/3 complex- directed stimulators and inhibitors to determine the contribution of these pathways to barbed end generation; 3. Use the reagents in 1 and 2 in vivo to determine the relationships between cofilin and the Arp2/3 complex in the regulation of actin nucleation in unstimulated and EGF-stimulated cells
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