Time-lapse recording of KRas activation on the late endosomal compartment. (A and B) COS-1 cells were transfected with Raichu-KRas (A) or Raichu-Pak-Rho (B). Starved cells were treated with 20 nM B-A1 overnight and then with 100 ng/ml EGF for 50 min. In the top panels, representative ratio images of YFP/CFP at the indicated time points after EGF stimulation are shown. In the bottom panels, representative images of CFP–Raichu-KRas (A) and CFP–Raichu-Pak-Rho (B) indicate localization of the probes at each corresponding FRET image frame. All images in A and B were obtained every 5 min with a time-lapse epifluorescent microscope. High FRET efficiency can be observed at the PM (arrowheads) and on endosomal structures (arrows) between 25 and 45 min in Raichu-KRas–expressing cells only. (C) Quantification of FRET (FRET ratio of YFP/CFP) efficiency on the endocytic structures from at least six independent experiments was plotted for each time point by measuring the increase over the basal activity, which was averaged over 10 min before EGF addition. (D) Time-lapse video microscopy of COS-1 cells expressing GFP-KRas treated with B-A1 and with 100 ng/ml EGF-TRITC for 1.5 h. Confocal section images were collected every 15 s. Endosomal fusion of KRas-positive vesicles (green channel) harboring internalized EGF (red channel) is observed (arrows). Arrowheads point to PM-localized EGF-TRITC. Endosomal diameters d1 and d2 are 2.6 µm and 3.4 µm, respectively. Data are means ± SEM. Bars: (A and B) 20 µm; (D) 2 µm.