Fig. 8. (A) Cholesterol is required for the TPA-induced localization of Rac 1 to the free surface of the cells. (a-c) Serum-starved cells were untreated or incubated with or without 5 mM mßCD for 30 minutes at 37°C prior to addition of 0.1 µM TPA. After 10 minutes at 37°C the cells were fixed, permeabilized and stained with an antibody against Rac 1. Arrows in b indicate localization of Rac 1 to the free surface, where ruffling and macropinocytosis takes place. In d the cells were first incubated with 0.1 µM TPA for 10 minutes to localize Rac 1 to the cell periphery, followed by a 5 minute pulse with 15 mM mßCD to remove cholesterol. The cells were then processed to visualize endogenous Rac 1. (B) Dominant-negative Rac 1T17N inhibits TPA-induced actin reorganization required for membrane ruffling. Serum-starved A431 cells transiently transfected with wild-type Rac 1 (a,b) or Rac 1T17N (c,d) were incubated with 0.1 µM TPA for 10 minutes and processed for indirect immunofluorescence. Rac 1 was labeled with a mouse antibody against six amino acids in the EE-epitope, and F-actin was labeled with rhodamine-phalloidin. Arrows in `a' indicate localization of Rac 1 to the free surface (compare with c), while arrows in b indicate actin associated with ruffles in both transfected and untransfected cells. It should be noted that in d peripheral actin staining (arrows) is observed only in the cells not transfected with the dominant-negative mutant.