Visualization and Molecular Analysis of Actin Assembly in Living Cells

doi:10.1083/jcb.143.7.1919

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J. Cell Biol., Volume 143, Number 7, December 28, 1998 1919-1930

Visualization and Molecular Analysis of Actin Assembly in Living Cells

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

^* Department of Cell Biology and Physiology and 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 assemblyin vitro. To understand how these proteins regulate the dynamicsof actin filament assembly in a motile cell, we visualized theirdistribution in living fibroblasts using green flourescent protein(GFP) tagging. Both proteins were concentrated in motile regionsat the cell periphery and at dynamic spots within the lamella.Actin assembly was required for the motility and dynamics of spotsand 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 theselocations. Inhibition of the Rho family GTPase rac1, and to alesser extent cdc42 and RhoA, blocked motility at the cell peripheryand the formation of spots. Increased expression of phosphatidylinositol5-kinase promoted the movement of spots. Increased expressionof LIM-kinase-1, which likely inactivates cofilin, decreased thefrequency of moving spots and led to the formation of aggregatesof GFP-CP. We conclude that spots, which appear as small projectionson the surface by whole mount electron microscopy, represent sitesof actin assembly where local and transient changes in the corticalactin cytoskeleton take place.

Key words: actin assembly; Arp2/3 complex; capping protein; cell motility; Rho family GTPase

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