34] Microinjection of Rho and Rac into quiescent Swiss 3T3 cells

Abstract

Publisher Summary This chapter describes the microinjection of Rho and Rac into quiescent Swiss 3T3 cells. Microinjection is a technique that rapidly introduces macromolecules into cells, thereby allowing analysis of the immediate cellular changes occurring in response to the injected substance. Microinjection has proved important in defining the functions of several small Ras-related GTP-binding proteins. The microinjection of H-Ras has shown that it rapidly stimulates membrane ruffling and pinocytosis. An inhibitor of Rho function, which is the bacterial exoenzyme C3 transferase that ribosylates Rho proteins, inhibits growth factor-induced stress fiber formation when injected into Swiss 3T3 cells, and ribosylated Rho acts as a dominant inhibitor of Rho function when injected. Rac, on the other hand, stimulates membrane ruffling when injected, and, in addition, a dominant negative inhibitor of Rac function, N17Rac1, inhibits growth factor-induced membrane ruffling.Rho proteins stimulate the formation of stress fibers and focal adhesions following microinjection into quiescent Swiss 3T3 cells.