Fig. 6. (A) Schematic showing the differences in diffusion and domain localization between the inactive eYFP-H-Ras(N17) and active eYFP-H-Ras(V12) mutants. For both mutants the fast-diffusing fraction was freely diffusing (blue), but its size and diffusion coefficient were significantly smaller for the active H-Ras mutant than the inactive mutant. Also, the slow-diffusing fraction of the inactive mutant was freely diffusing (green), whereas it was confined in 200 nm domains for the active mutant (red). (B) Model depicting the effect of H-Ras(wt) activation on its diffusion characteristics. Before stimulation (left part) slow (25%, green) and fast (75%, blue) diffusing populations of H-Ras(wt) molecules were present, that were both freely diffusing. Activation of H-Ras(wt) by addition of insulin for 5 minutes resulted in the confinement of the slow-diffusing fraction in domains (red) with a diameter of 200 nm.