In vivo imaging of signal transduction cascades with probes based on Förster Resonance Energy Transfer (FRET).
Genetically encoded FRET probes enable us to visualize a variety of signaling events such as protein phosphorylation and G-protein activation in living cells. This unit focuses on FRET probes wherein both the donor and acceptor are fluorescence proteins and incorporated into a single molecule, i.e., a unimolecular probe. Advantages of these probes lie in their easy loading into cells, simple acquisition of FRET images, and clear evaluation of data. We have developed FRET probes for Ras-superfamily GTPases, designated Ras and interacting protein chimeric unit (Raichu) probes. We hereby describe strategies to develop Raichu-type FRET probes, procedures for their characterization, and acquisition and processing of images. Although improvements upon FRET probes are still based on trial-and-error, we provide practical tips for their optimization and briefly discuss the theory and applications of unimolecular FRET probes.
Nakamura
Takeshi
T
Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
Matsuda
Michiyuki
M
D001671:Biochemistry / Q000295:instrumentation / Q000379:methods*; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C430804:Raichu-Ras protein; D020558:GTP Phosphohydrolases; D018631:ras Proteins