RHO-DEPENDENT AND RAC-DEPENDENT ASSEMBLY OF FOCAL ADHESION COMPLEXES AND ACTIN-FILAMENTS IN PERMEABILIZED FIBROBLASTS - AN ESSENTIAL ROLE FOR EZRIN RADIXIN/MOESIN PROTEINS/

The small GTPases Rho and Rac regulate actin filament assembly and the formation of integrin adhesion complexes to produce stress fibers and lamellipodia, respectively, in mammalian cells. Although numerous can didate effecters that might mediate these responses have been identifi ed using the yeast two-hybrid and affinity purification techniques, th eir cellular roles remain unclear. We now describe a biological assay that allows components of the Rho and Rac signaling pathways to be ide ntified. Permeabilization of serum-starved Swiss 3T3 cells with digito nin in the presence of guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S) induces both actin filament and focal adhesion complex assembly thr ough activation of endogenous Rho and Rac. These responses are lost wh en GTP gamma S is added 6 min after permeabilization, but can be recon stituted using concentrated cytosolic extracts. We have achieved a 10, 000-fold purification of the activity present in pig brain cytosol and protein sequence analysis shows it to contain moesin. Using recombina nt proteins, we show that moesin and its close relatives ezrin and rad ixin can reconstitute stress fiber assembly, cortical actin polymeriza tion and focal complex formation in response to activation of Rho and Rac.