Visualization and molecular analysis of actin assembly in living cells

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 co mplex and capping protein (CP) regulate actin assembly in vitro. To under s tand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts usin g green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lam ella. Actin assembly was required for the motility and dynamics of spots an d for motility at the cell periphery. In permeabilized cells, rhodamine-act in assembled at the cell periphery and at spots, indicating that actin fila ment barbed ends were present at these locations. Inhibition of the Rho fam ily GTPase rad, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phos phatidylinositol 5-kinase promoted the movement of spots. Increased express ion of LIM-kinase-1, which likely inactivates cofilin, decreased the freque ncy of moving spots and led to the formation of aggregates of GFP-CP. We co nclude that spots, which appear as small projections on the surface by whol e mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.