Fig. 1. EGF induces membrane ruffling and internalization of E-cadherin-GFP. (A) MCF-7 cells were fixed and labeled for F-actin using Alexa Fluor-488-conjugated Phalloidin either (a) without or (b) with prior EGF stimulation for 1 hour. Arrows indicate stimulation of actin-rich membrane ruffles in response to EGF. (B) MCF-7 cells stably overexpressing GFP-tagged E-cadherin (hE-GFP-MCF-7 cells) were imaged live every 12 seconds over a 1-hour treatment period with EGF using a TILLvision live imaging microscope. An example of E-cad-GFP-positive membrane ruffling is depicted in cropped movie frames 150-157 (30:00-31:24 minutes) of the boxed region of interest in a. Arrows depict ruffling membranes. See accompanying Movie 1 in supplementary material. (C) hE-GFP-MCF-7 cells were stimulated with EGF and imaged live. See accompanying Movie 2 in supplementary material. The diagram pinpoints the macropinocytosis event and cell features depicted in the movie and the subsequent intensity-coloured still frames show the main steps in the internalization of surface hE-cad-GFP (green arrow) into a macropinosome (white arrow) and its recycling (green arrowhead) back to the surface in a region of cell-cell contact. Bars, 20 µm for a and b; 2 µm for cropped movie frames.