Single-molecule diffusion measurements of H-Ras at the plasma membrane of live cells reveal microdomain localization upon activation

Recent studies show that the partitioning of the small GTPase H-Ras in different types of membrane microdomains is dependent on guanosine 5′-triphosphate (GTP)-loading of H-Ras. Detailed knowledge about the in vivo dynamics of this phenomenon is limited. In this report, the effect of the activation of H-Ras on its microdomain localization was studied by single-molecule fluorescence microscopy. Individual human H-Ras molecules fused to the enhanced yellow fluorescent protein (eYFP) were imaged in the dorsal plasma membrane of live mouse cells and their diffusion behavior was analyzed. The diffusion of a constitutively inactive (S17N) and constitutively active (G12V) mutant of H-Ras was compared. Detailed analysis revealed that for both mutants a major, fast-diffusing population and a minor, slow-diffusing population were present. The slow-diffusing fraction of the active mutant was confined to 200 nm domains, which were not observed for the inactive mutant. In line with these results we observed that the slow-diffusing fraction of wild-type H-Ras became confined to 200 nm domains upon insulin-induced activation of wild-type H-Ras. This activation-dependent localization of H-Ras to 200 nm domains, for the first time directly detected in live cells, supports the proposed relationship between H-Ras microdomain localization and activation.