Visualization and Molecular Analysis of Actin Assembly in Living Cells

• Published in: The Journal of cell biology 1998-12, Vol.143 (7), p.1919-1930
• Main Authors: Schafer, Dorothy A., Welch, Matthew D., Machesky, Laura M., Bridgman, Paul C., Meyer, Shelley M., Cooper, John A.
• Format: Article
• Language: English
• Subjects: Actin Cytoskeleton - metabolism; Actin depolymerizing factors; Actins; Actins - metabolism; Animals; Antibodies; cdc42 GTP-Binding Protein, Saccharomyces cerevisiae; Cell Cycle Proteins - antagonists & inhibitors; Cell Cycle Proteins - physiology; Cell Membrane - ultrastructure; Cell motility; Cell Movement; Cells; Cellular biology; Fibroblasts; Fibroblasts - cytology; GTP-Binding Proteins - antagonists & inhibitors; GTP-Binding Proteins - physiology; Macropodidae; Microfilaments; Phosphatidylinositols; Phosphoprotein Phosphatases - antagonists & inhibitors; Physiological regulation; Polymerization; Protein Kinase Inhibitors; Proteins; rac GTP-Binding Proteins; Recombinant Fusion Proteins - metabolism; Regular; rhoA GTP-Binding Protein; Tetradecanoylphorbol Acetate - pharmacology
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.143.7.1919

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green fluorescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM-kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP-CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.