Analysis of Ras activation in living cells with GFP-RBD.

Several genetically encoded fluorescent biosensors for Ras family GTPases have been developed that permit spatiotemporal analysis of the activation of these signaling molecules in living cells. We describe here the use of the simplest of these probes, the Ras binding domain (RBD) of selected effectors fused with green fluorescent protein (GFP) or one of its spectral mutants. When expressed in quiescent cells, these probes are distributed homogeneously through the cytosol and nucleoplasm. On activation of their cognate GTPases on membranes, they are recruited to these compartments, and activation can be scored by redistribution of the probe. The advantage of this system is its simplicity: the probes are genetically encoded and can easily be constructed with standard cloning techniques, and the readout of activation requires only standard epifluorescence or confocal microscopy. The disadvantage of the system is that only rarely are Ras-related GTPases expressed at high enough levels to permit detection of the activation of the endogenous proteins. In general, the method requires overexpressing untagged, wild-type versions of the GTPase of interest. However, we describe a FRET-based method called bystander FRET developed to detect endogenous proteins that can be used to validate the results obtained by overexpressing Ras proteins. By use of this technique, we and others have uncovered important new features of the spatiotemporal regulation of Ras and related GTPases.