PM injury by SLO segregates BCRs from CTB-labeled lipid rafts at the cell surface and during BCR internalization. (A–C) Live-cell imaging of BCR and CTB internalization. B cells were incubated at 4°C with AF546 F(ab′)2 anti–mouse IgM and AF488-CTB. Time-lapse confocal images were acquired at 37°C for 10 min in the presence or absence of SLO. Shown are representative images at 10 min (A), a kymograph generated from time-lapse images along the blue line (B), and the mean colocalization rate (± SD) of BCR and CTB staining at 10 min (C), from three independent experiments. Bar, 5 µm. (D–G) TEM analysis of BCRs and CTB in B cells treated with or without SLO. B cells were incubated with gold anti–mouse IgM (18 nm, 10 µg/ml) and biotin-CTB (2 µg/ml) plus gold-streptavidin (10 nm, 2 µg/ml; arrows and arrowheads) at 4°C, and then with or without SLO at 37°C for 1 or 5 min. The percentages of CTB gold particles internalized into vesicles containing BCR gold particles at 5 min (D and E) and in the vicinity (<30 nm) of BCR gold particles at the PM (F and G) were determined in individual cell profiles. Shown are representative images (D and F) and the mean percentage (E and G) from ≥16 randomly selected cell profiles and two individual experiments. Bar, 100 nm. ****, P < 0.0001.
