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© The Rockefeller University Press, 0021-9525/1998//1919 $5.00
The Journal of Cell Biology, Volume 143, Number 7, , 1998 1919-1930

Regular Articles

Visualization and Molecular Analysis of Actin Assembly in Living Cells



Dorothy A. Schafer*, Matthew D. Welch§, Laura M. Machesky||, Paul C. Bridgman{ddagger}, Shelley M. Meyer*, and John A. Cooper*

* Department of Cell Biology and Physiology and {ddagger} Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110; § Molecular and Cell Biology, University of California, Berkeley, California 94702; and || School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

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 flourescent 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.

Key Words: actin assembly • Arp2/3 complex • capping protein • cell motility • Rho family GTPase

Abbreviations used in this paper: BDM, butanedione monoxime; CP, capping protein; GFP, green fluorescent protein; PI 3,4,5-P3, phosphatidylinositol 3,4,5-triphosphate; PI 4,5-P2, phosphatidylinositol 4,5-biphosphate; PI 5-kinase, phosphatidylinositol 5-kinase.


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