COREQUIREMENT OF SPECIFIC PHOSPHOINOSITIDES AND SMALL GTP-BINDING PROTEIN CDC42 IN INDUCING ACTIN ASSEMBLY IN XENOPUS EGG EXTRACTS

Both phosphoinositides and small GTP-binding proteins of the Rho famil y have been postulated to regulate actin assembly in cells. We have re constituted actin assembly in response to these signals in Xenopus ext racts and examined the relationship of these pathways. We have found t hat GTP gamma S stimulates actin assembly in the presence of endogenou s membrane vesicles in low speed extracts, These membrane vesicles are required, but can be replaced by lipid vesicles prepared from purifie d phospholipids containing phosphoinositides. Vesicles containing phos phatidylinositol (4,5) bisphosphate or phosphatidylinositol (3,4,5) tr isphosphate can induce actin assembly even in the absence of GTP gamma S. RhoGDI, a guanine-nucleotide dissociation inhibitor for the Rho fa mily, inhibits phosphoinositide-induced actin assembly, suggesting the involvement of the Rho family small G proteins. Using various dominan t mutants of these G proteins, we demonstrate the requirement of Cdc42 for phosphoinositide-induced actin assembly. Our results suggest that phosphoinositides may act to facilitate GTP exchange on Cdc42, as wel l as to anchor Cdc42 and actin nucleation activities. Hence, both phos phoinositides and Cdc42 are required to induce actin assembly in this cell-free system.