Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes.
GFP-based fluorescence resonance energy transfer (FRET) probes that visualize local activity-changes of Ras and Rho GTPases in living cells are now available for examining the spatio-temporal regulation of these proteins. This article describes principles and strategies to develop intramolecular FRET probes for Ras- and Rho-family GTPases. The procedure for characterizing candidate probes, and image acquisition and processing are also explained. An optimal FRET probe should have (i) a wide dynamic range (which means a high sensitivity), (ii) a high fluorescence intensity, (iii) target specificity, and (iv) a minimal perturbation to endogenous signaling cascades. Although an improvement of FRET probes should be executed in a trial-and-error manner, practical tips for optimization are provided here. In addition, we illustrate some applications of FRET probes for neuronal cells, which are composed of diverse subcellular compartments with different functions; thus, tools to decipher the dynamics of GTPase activity in each compartment have long been desired.
Nakamura
Takeshi
T
Department of Tumor Viology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita-shi, Japan. t-nakamu@biken.osaka-u.ac.jp
Aoki
Kazuhiro
K
Matsuda
Michiyuki
M
D000818:Animals; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020439:Growth Cones / Q000378:metabolism; D015335:Molecular Probes / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D011994:Recombinant Proteins; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D015335:Molecular Probes; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C430804:Raichu-Ras protein; D020558:GTP Phosphohydrolases; D018631:ras Proteins; D020741:rho GTP-Binding Proteins