抄録
A small G protein Ras plays key roles in many intracellular signalling pathways, but the mechanism by which activated Ras transmits its signal to downstream effectors has not been completely cleared. We addressed this problem by directly imaging this signal transfer process at the level of individual single molecules. By simultaneously observing single molecules of GFP-H-Ras and mRFP-Raf-1 at video rate, we found that upon H-Ras activation, it temporarily stops diffusion in the cell membrane (0.3–1 s), and to this immobilized activated GFP-H-Ras, Raf-1 is recruited. Their transient binding, which was confirmed by single-molecules FRET at video rate, typically lasts less than 1 s, during which Raf may be activated by Ras, a process totally different from random collisions of signalling molecules as often assumed. The transient immobilisation of activated Ras is likely caused by temporary formation of activated-Ras-induced signalling complexes including activated-Ras specific scaffolding proteins like SUR-8, which may stabilize the Ras-Raf binding, but not due to upstream/downstream signalling molecules or rafts/caveolae domains. [Jpn J Physiol 55 Suppl:S29 (2005)]
