Fig. 6. H36 and PM4C4 proteins are excluded from macropinocytic cups. (A) Macropinocytosis in living cells expressing CRAC-GFP. Pictures of closed macropinosomes as well as pictures of macropinocytic cups at 6 and 12 seconds before closure are shown. Schematic representations of the criteria used to identify macropinocytic cups are shown on the right of each corresponding series of pictures. w: width, h: height. U-shaped CRAC-GFP structures designate structures where either the width is smaller than the height (top drawing) or where the width at the aperture is smaller than the width at the bottom of the cup (bottom drawing). 82% of U-shaped CRAC-GFP structures observed by live microscopy closed within 20 seconds and thus represented bona fide macropinocytic cups. (B,C) Cells expressing CRAC-GFP were fixed and processed for total immunofluorescence using different antibodies. (B) Relative intensity for H36, PM4C4, H72 and H161 staining in macropinocytic cups. H36 and PM4C4 were depleted in macropinocytic cups, H72 and H161 were not. (C) Macropinocytic cups in cells stained with H36 or H72 antibodies (white), and a pseudopod stained with H36. H36 was not depleted in the pseudopod. Graphs on the right represent the relative intensity of staining in cups or pseudopods compared with plasma membrane staining. Each graph is the average of a line scanning (8 µm long) done on the membrane of four cups or four pseudopods. The region scanned is delimited by the two red squares while the red arrows indicate the middle of the cups and of the pseudopods. Bars, 4 µm.