Large Ca²⁺ and cholesterol-dependent endosomes are induced by plasma membrane injury. (A) EM showing endosomes formed in NRK cells 4 min after exposure to SLO in the presence of Ca²⁺ and BSA-gold. Arrows point to gold particles. (bottom right) Clathrin-coated vesicle found in the same cells. (right) Quantification shows ∼13-fold less BSA-gold–containing vesicles in cells permeabilized by SLO without Ca²⁺. The data represent the mean ± SD (error bars). **, P < 0.0001 (unpaired t test). (B) EM showing similar endosomes 4 min after scrape wounding HeLa cells. Short arrows, BSA-gold particles; long arrow, clathrin-coated vesicle. (right) Quantification shows less BSA-gold–containing vesicles in cells scraped without Ca²⁺ or in Ca²⁺ after cholesterol extraction with MβCD. The data represent the mean ± SD (error bars). *, P < 0.005; **, P < 0.0001 (unpaired t test). (C) Cholesterol depletion inhibits wound repair. HeLa cells were treated with MβCD and analyzed by FACS after scrape wounding (gray fill, PI added during wounding). Bars, 200 nm.