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.
Lommerse
Piet H M
PH
Department of Biophysics, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands.
Snaar-Jagalska
B Ewa
BE
Spaink
Herman P
HP
Schmidt
Thomas
T
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001703:Biophysics / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D004058:Diffusion; D005347:Fibroblasts / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D011905:Genes, ras / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D015233:Models, Statistical; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014162:Transfection; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D007328:Insulin; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D006160:Guanosine Triphosphate; D018631:ras Proteins