Fig. 4: Endocytosis of the inactive bent-closed α5β1 integrin via CLICs.
A Inactive bent-closed α5β1 integrin accumulates in clathrin-independent carriers (CLICs). Top: EM micrographs of HeLa cells that were incubated continuously for 9 min at 37 °C with HRP-coupled mAb13 (mAb13-HRP). Boxes show zooms of areas illustrating the accumulation of mAb13-HRP in typical crescent-shaped CLIC structures. Red stars indicate larger, likely endosomal compartments. Bottom left: Same experiments as above, for both mAb13-HRP or 9EG7-HRP antibodies (6 or 9 min uptake). Green insets: Zooms of CLICs. Orange insets: Zooms of endocytic vesicles. Scale bars = 1 µm in all global views, and 100 nm in the zoomed boxes. Bottom right: Quantification of endocytic structures: n = 97 (for mAb13) and n = 60 (for 9EG7) HRP-positive structures were counted. Four independent experiments. Means ± SEM, unpaired two-sided t-test; ns = P > 0.05, ****P < 0.0001. Effect of ciliobrevin D (CBD) on α5β1 integrin endocytosis. Quantification by confocal microscopy of mAb13 (B) or 9EG7 (C) uptake after incubation for 10 min at 37 °C with RPE-1 cells in the presence (+CBD) or absence (Ctrl) of CBD. Cells were pre-loaded or not for 30 min at 4 °C with exogenous Gal3 (200 nM). Note the strong inhibitory effect of CBD on mAb13 uptake. B Left graph, n = 48 (for Ctrl) and n = 60 (for CBD) cells; right graph, n = 40 cells per condition. C Left graph, n = 50 (for Ctrl) and n = 40 (for CBD) cells; right graph, n = 40 cells per condition. Representatives of three independent experiments. Means ± SEM, unpaired t-test; **P < 0.002, ****P < 0.0001. Scale bars = 10 µm. Nuclei in blue.
