REGULATION OF ACTIN POLYMERIZATION IN CELL-FREE SYSTEMS BY GTP-GAMMA-S AND CDC42

We have established a cell-free system to investigate pathways that re gulate actin polymerization. Addition of GTP gamma S to lysates of pol ymorphonuclear leukocytes (PMNs) or Dictyostelium discoideum amoeba in duced formation of filamentous actin, The GTP gamma S appeared to act via a small G-protein, since it was active in lysates of D. discoideum mutants missing either the alpha(2)- or beta-subunit of the heterotri meric G-protein required for chemoattractant-induced actin polymerizat ion in living cells. Furthermore, recombinant Cdc42, but not Rho or Ra c, induced polymerization in the cell-free system. The Cdc42-induced i ncrease in filamentous actin required GTP gamma S binding and was inhi bited by a fragment of the enzyme PAK1 that hinds Cdc42. In a high spe ed supernatant, GTP gamma S alone was ineffective, but GTP gamma S-loa ded Cdc42 induced actin polymerization, suggesting that the response w as limited by guanine nucleotide exchange. Stimulating exchange by che lating magnesium, by adding acidic phospholipids, or by adding the exc hange factors Cdc24 or Dbl restored the ability of GTP gamma S to indu ce polymerization, The stimulation of actin polymerization did not cor relate with PIP2 synthesis.