SPATIAL CONTROL OF PATHWAYS MEDIATING ACTIN DYNAMICS

The motility of crawling cells requires cyclical actin polymerization transients. The location of these transients within the cell cortex determines the direction of cell locomotion. Polymerization transients can be induced by extracellular stimuli such as EGF and requires the mobilization of free barbed ends. Barbed ends nucleate the polymerization of actin which is believed to supply the force for protrusion of the cell membrane. Current evidence indicates that barbed ends are produced by (a) cofilin-mediated severing of pre-existing filaments, (b) Arp 2/3- mediated de novo assembly of barbed ends, or a combination of these mechanisms. We propose to test the involvement of cofilin in the generation of barbed ends at the leading edge by preparing caged variants of the catalytic domain of LIM kinase. We will also prepare inhibitors of endogenous LIM-kinase that can be uncaged in a spatially and temporally controlled manner. We propose to test the involvement of the Arp 2/3 complex in the generation of barbed ends at the leading edge by preparing dominant negative variants of the WASP-family proteins that stimulate the activity of Arp 2/3. We will also prepare antibodies against Arp 2/3 subunits that can block the activity of the complex in vivo. These reagents will be used in a variety of ways in cells to define the importance of the above mechanisms and their interactions in vivo.