Fig. 5. EGF stimulation induces vesicle accumulation in cells expressing an ArfGAP mutant of GIT1. (A) Schematic representation of the GFP-tagged GIT1 constructs utilized in this study: p95-GFP including the full length GIT1; p95-C2-GFP including residues 229-740 of GIT1; p95-C-GFP, including residues 346-740 of GIT1, and GFP alone. ANK, ankyrin repeats; SHD, Spa2 homology domain; LZ, leucine zipper; PBS, paxillin binding subdomain. (B,C) A431 cells were stimulated for the indicated times with 100-200 ng/ml of EGF: representative frames from time-lapse observation of cells expressing p95-C2-GFP (B) and p95-C-GFP (C); Bar, 10 µm. Arrowheads in B indicate newly formed GFP-positive structures following EGF stimulation. Arrows in C indicates growth factor-induced lamellipodial extensions. Bar, 10 µm. (D) Frames from time-lapse observation of cells expressing p95-C2-GFP. Arrowheads and arrows indicate two examples of fusion between GFP-positive structures during stimulation with EGF. Bar, 5 µm. (E) Cells expressing p95-C2-GFP (n=24 with EGF; n=21 without EGF), p95-C-GFP (n=18), p95-GFP (n=24), or GFP alone (n=24) were analyzed by time-lapse digital analysis, and scored after 40 minutes for the formation of GFP-labelled vesicle during stimulation with EGF. Bars represent the percentages of cells with EGF-induced vesicles.