Figure 6: Adhesive exosome trails are left behind migrating cells

(a) Confocal image of mCherry-CD63 (red) and GFP-paxillin (green)-expressing HT1080 cell from a representative movie from n=17 movies on FN-coated (1 μg ml⁻¹) MatTek dishes. Arrows indicate co-localization of mCherry-CD63 with adhesions, whereas an arrowhead indicates localization of paxillin within endosomes. Magnification of boxed area is shown right. See also Supplementary Movie 5. (b) Image series from a representative epifluorescence movie (1 frame per 2 min, from n=10 movies) of a migrating HT1080 cell on FN-coated (1 μg ml⁻¹) MatTek dishes transiently transfected with pHLuorin-CD63 to show sites of exosome secretion. Note CD63 fluorescence in ruffles at the front of the cell (arrowheads) and in an adhesive trail left behind the cell. Zoom is brightened and shows adhesive trail. See also Supplementary Movie 6. (c) Image from a representative epifluorescence movie with a rapid frame rate (1 frame per 6 s, from n=20 movies) to capture the dynamics of pHLuorin-CD63 in protrusions. Filled arrowheads indicate fluorescence in ruffles, whereas open arrowhead points to adhesive trail. The arrow points to rear fluorescence near retracting fibres. Heat map-coloured intensity is shown right. Grayscale and coloured kymographs show protrusion over time across the perpendicular line drawn in heat map image. Note lack of enhanced fluorescence at flat protrusion membrane edge, which is outlined by drawn white lines. See also Supplementary Movie 7. Scale bars, 30 μm.