Figure 2

GFP-R-Ras localizes to discrete plasma membrane domains. Cos-7 cells were transfected with GFP-R-Ras constructs, and the localization of each to the plasma membrane determined. Plasma membrane localization was noted in 79% of GFP-R-Ras(wt) (A, n≥100 cells for each) and in 91% of constitutively active GFP-R-Ras(38V) (B) but not in cells transfected with dominant negative GFP-R-Ras(41A) (C, < 5% of cells). In all three transfectants, there was a variable amount of perinuclear fluorescence which included small, dot-like vesicles positive for GFP-R-Ras (arrows in C). In addition, both wt and 38V-expressing cells contained medium- and large-sized vesicles (≥0.4 μm). (D) 3× zoomed view of dashed box in (A) highlights the appearance of plasma membrane fluorescence (arrowheads) and the assortment of vesicle and endosomal compartment sizes (arrows). (E) A cell transfected with GFP-R-Ras(38V) was co-stained with 4 μg/mL Alexa Fluor-555 cholera toxin B (top panel) to reveal co-localization of R-Ras with ganglioside-GM1 in ruffles (arrowheads). The lack of ruffle formation in GFP-R-Ras(41A) cells was independently confirmed by co-staining with cholera toxin B where ruffles were observed in 0 of the 26 cells imaged. (F) Western blot for GFP-R-Ras shows that protein levels were the same in GFP-R-Ras-(wt), -(38V) and -(41A) transfected cells in spite of the dramatic differences in localization of each isoform. (G) The number of endocytic compartments per cell was measured in >100 cells for each of the transfected constructs (bars). The percentage of cells that contained GFP-R-Ras localized to ruffles was quantified (triangles).