Analysis of the spatiotemporal activation of rho GTPases using Raichu probes

GFP-based FRET probes that can visualize local activity changes in Rho GTPases in living cells are now available for examining the spatio-temporal regulation of these proteins. We previously developed FRET probes for Rho (and Ras) GTPases and collectively designated them "Ras and interacting protein chimeric unit" (Raichu) probes. In this chapter, we describe the principles and strategies used to develop Raichu-type FRET probes for Rho-family GTPases. The procedures for characterizing candidate probes, setting up the imaging system, and image acquisition/processing are also explained. An optimal FRET probe should: (1) have a wide dynamic range (i.e., a high sensitivity); (2) demonstrate high fluorescence intensity (i.e., a high signal-to-noise ratio); (3) show target specificity; and (4) cause minimal perturbation of endogenous signaling cascades. Although improvements of FRET probes should be executed in a trial-and-error manner, we provide practical tips for their optimization. In addition, some experimental results are presented to illustrate the expanding number of fields for the application of Raichu-RhoA/Rac1/Cdc42, and the advantages and disadvantages of Raichu probes are discussed.