FIG. 5.
The Rho-GAP domain inhibits the association between GFP-N-WASP and Cdc42. Cells were injected with GFP-N-WASP and WT Cdc42-HA, with or without the Rho-GAP domain. Lifetime analysis and confocal imaging of the cells were carried out with different microscopes, and the images were aligned. Some between-cell (n = 5) heterogeneity in FRET efficiency for the GFP-N-WASP/anti-HA Fab-Cy3 (WT Cdc42) pair was observed in the absence of the RICH-1 Rho-GAP domain, but there was a clear partial reduction in FRET efficiency when the Rho-GAP domain was coexpressed, as demonstrated in the efficiency histogram (bottom panel). FRET efficiency = 1 − τda/τd, where τda is the pixel-by-pixel fluorescence lifetime of the donor in the presence of acceptor and τd is the average lifetime of the donor in the absence of acceptor (unstained controls in the upper panels).