Figure 1.

Interaction of Grb2-CFP and YFP-Shc in endosomes induced by EGF. (A) Grb2-CFP and Shc-YFP were transiently expressed in A-431 cells. YFP, CFP, and FRET images were acquired before and after incubation of cells with 200 ng/ml EGF-Rh for 20 min at 37°C. EGF-Rh was detected using Cy3 filter channel. FRET^C was calculated as described in MATERIALS AND METHODS, and is presented as pseudocolor intensity-modulated image (FRET^C/YFP). Bar, 10 μm. On the graph, the apparent efficiencies of energy transfer between Grb2-CFP and YFP-Shc, E_a, calculated for multiple subregions of the images are shown. Left, averaged values of E_a are presented. Right, E_a values obtained for individual endosomes were plotted against mean intensities of YFP in each endosome. A.l.u.f.i., arbitrary linear units of fluorescence intensity. (B) Wild-type YFP-Shc and mutant YFP-Shc-3F (Y239F, Y240F, and Y317F mutations) were coexpressed with Grb2-CFP or Grb2R86A-CFP mutant in A-431 cells. The incubation with EGF-Rh and FRET imaging was performed as described in A. Arrows point to an example of the endosomes that contain EGF-Rh and YFP-Shc but lack Grb2R86A-CFP in the cell that expresses low levels of YFP-Shc. Bar, 10 μm.