Cells permeabilized by SLO in the presence of Ca²⁺ undergo dynamin-2–independent endocytosis. (A) NRK cells exposed or not exposed to 250 ng/ml SLO were incubated for 4 min with TR dextran. In SLO/Ca²⁺, dextran is detected in multiple intracellular vesicles; without Ca²⁺, it enters the cytosol through SLO pores. (B) Dextran-containing vesicles (red) colocalize partially (arrows) or do not colocalize (arrowheads) with mAbs to Lamp-1 (LY1C6; green). Two independent confocal z sections are shown. (C) Most dextran-containing endosomes (red) colocalize with antibodies to EEA1 (green). Arrows indicate vesicles colocalizing with antibodies; arrowheads indicate one vesicle not colocalizing. (D) NRK cells expressing dynamin-2–GFP internalize transferrin (red; bottom left) or TR dextran after SLO/Ca²⁺ (red; top left). Cells expressing K44A dynamin-2–GFP exclude transferrin (bottom right; arrow) but still endocytose dextran after SLO/Ca²⁺ (top right; arrow). Transfected cells are outlined in green. WT, wild type. (E) FACS of PI staining (gated on GFP⁺ cells) shows that Ca²⁺-dependent resealing of SLO-permeabilized NRK cells is not inhibited by K44A dynamin-2–GFP. Dashed lines, no SLO; solid lines, SLO. Bars: (A and D) 5 μm; (B and C) 1 μm.