J. Cell Biol.
© The Rockefeller University Press
0021-9525/97/07/363/12 $2.00
Volume 138, Number 2, July 28, 1997 363-374

Regulation of Actin Polymerization in Cell-free Systems by GTPS and Cdc42

Sally H. Zigmond,^* Michael Joyce,^* Jane Borleis,^§ Gary M. Bokoch, and Peter N. Devreotes^§

^* Biology Department, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018;  Department of Immunology and Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037; and ^§ Department of Biological Chemistry, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205

We have established a cell-free system to investigate pathways that regulate actin polymerization. Addition of GTPS to lysates of polymorphonuclear leukocytes (PMNs) or Dictyostelium discoideum amoeba induced formation of filamentous actin. The GTPS appeared to act via a small G-protein, since it was active in lysates of D. discoideum mutants missing either the ₂- or -subunit of the heterotrimeric G-protein required for chemoattractant-induced actin polymerization in living cells. Furthermore, recombinant Cdc42, but not Rho or Rac, induced polymerization in the cell-free system. The Cdc42-induced increase in filamentous actin required GTPS binding and was inhibited by a fragment of the enzyme PAK1 that binds Cdc42.

In a high speed supernatant, GTPS alone was ineffective, but GTPS-loaded Cdc42 induced actin polymerization, suggesting that the response was limited by guanine nucleotide exchange. Stimulating exchange by chelating magnesium, by adding acidic phospholipids, or by adding the exchange factors Cdc24 or Dbl restored the ability of GTPS to induce polymerization. The stimulation of actin polymerization did not correlate with PIP₂ synthesis.

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