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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...
Ras GTPases regulate cellular growth and differentiation and are modulated by myriad stimuli including growth factors, cytokines, antigens, and UV irradiation. Ras GTPases are molecular switches that are active when GTP-bound and inactive when GDP-bound. The ability of these GTPases to signal requires that the GTP-bound form engage downstream effectors, interactions that occur only on the cytosolic...
Recent advances in our understanding of the intracellular trafficking, membrane microenvironment, and subcellular sites of signaling of Ras have been driven by observations of GFP-tagged Ras in living cells. Here, we describe methods to gain further insight into the regulation of these events through the use of quantitative fluorescence microscopy. We focus on three techniques, fluorescence recovery...
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