Ras Diffusion and Interactions with the Plasma Membrane Measured by FRAP Variations

Ras proteins are non-integral membrane proteins, which bind to the plasma membrane by virtue of farnesylation and palmitoylation or a positively charged polybasic cluster at their C-terminus. Their membrane interactions and/or localization to membrane microdomains, which play important roles in signaling, are regulated by their lateral diffusion at the plasma membrane and their ability to exchange between the membrane and the cytoplasm (binding/unbinding kinetics). Here, using N-Ras as an example, we describe the use of variations of fluorescence recovery after photobleaching (FRAP) to measure the dynamics of the association of N-Ras with the plasma membrane of living cells and their dependence on several parameters (cholesterol, clustering of raft proteins, and palmitoylation/depalmitoylation).