title: Single-molecule imaging analysis of Ras activation in living cells.
abstract: A single-molecule fluorescence resonance energy transfer (FRET) method has been developed to observe the activation of the small G protein Ras at the level of individual molecules. KB cells expressing H- or K-Ras fused with YFP (donor) were microinjected with the fluorescent GTP analogue BodipyTR-GTP (acceptor), and the epidermal growth factor-induced binding of BodipyTR-GTP to YFP-(H or K)-Ras was monitored by single-molecule FRET. On activation, Ras diffusion was greatly suppressed/immobilized, suggesting the formation of large, activated Ras-signaling complexes. These complexes may work as platforms for transducing the Ras signal to effector molecules, further suggesting that Ras signal transduction requires more than simple collisions with effector molecules. GAP334-GFP recruited to the membrane was also stationary, suggesting its binding to the signaling complex. The single-molecules FRET method developed here provides a powerful technique to study the signal-transduction mechanisms of various G proteins.
authors.affiliation: Department of Biological Science and Institute for Advanced Research, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Hideji
authors.initials: H
authors.lastname: Murakoshi
authors.forename: Ryota
authors.initials: R
authors.lastname: Iino
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Takahiro
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Chika
authors.initials: C
authors.lastname: Ohshima
authors.forename: Akihiko
authors.initials: A
authors.lastname: Yoshimura
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kusumi
mesh_terms: D001896:Boron Compounds / Q000737:chemistry; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D004735:Energy Transfer; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D015398:Signal Transduction; D018631:ras Proteins / Q000378:metabolism*
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D020690:GTPase-Activating Proteins; D018631:ras Proteins