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Fig. 2. Expression of constitutively active or dominant-negative R-Ras regulates the organization of the peripheral actin network and formation of filopodia. (A) Stable expression of constitutively active R-Ras promotes a strong, branched peripheral actin network. MCF10A cells stably transfected with control vector or constitutively active R-Ras^38V were plated on collagen I (30 µg/ml), fibronectin (20 µg/ml) or poly-L-lysine (0.01%) for 30 minutes. The cells were then fixed and stained with TRITC-phalloidin to visualize F-actin. Note the dense peripheral actin and loss of filopodia in R-Ras^38V-expressing cells. A bubble is present in the lower right corner of control p-lysine cells. (B) Dominant-negative R-Ras enhances filopodia formation in MCF10A cells. MCF10A cells were transiently co-transfected with pCMV-R-Ras^41A and GFP to identify transfected cells. 48 hours post-transfection, the cells were assayed for their ability to spread on fibronectin-coated coverslips for 45 minutes. Arrow indicates a cell transfected with R-Ras^41A. (C) R-Ras regulates membrane protrusion and filopodia formation. Cos7 cells were transiently transfected with GFP:R-Ras^Wt, GFP:R-Ras^38V or GFP:R-Ras^41A. 48 hours post-transfection, cells were detached and were allowed to adhere and spread on fibronectin (30 µg/ml) for 1 hour. Cells were then fixed and stained for F-actin. Overlay images of F-actin (red) and GFP-R-Ras (green) are shown. Note that GFP-R-Ras^wt localizes to leading edges, whereas GFP-R-Ras^41A is not found on the plasma membrane, and GFP-R-Ras^38V is localized more uniformly around the cells at the ruffling lamellipod. Representative phenotypes for each transfection are shown, and are labeled A, B and C. (D) Quantification of phenotypes shown in C. Note that expression of constitutively active R-Ras^38V dramatically enhances phenotype C, which has a strong peripheral actin network and no filopodia, whereas expression of dominant-negative R-Ras^41A promotes phenotype B, which has enhanced filopodia formation. Values represent the mean of three experiments ± s.d. Bars, 10 µm (A-C).

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