Disruption of the actin cytoskeleton enhances endocytosis and resealing in cells permeabilized by SLO pores or mechanical wounds. (A) NRK cells treated or not treated with cytochalasin D (CD) were exposed or not exposed to 250 ng/ml SLO for 4 min in the presence of TR dextran (red). (B) Endocytosis was quantified with a trypan blue quenching FACS assay in HeLa (left) or NRK (right) cells labeled with WGA-FITC and wounded by SLO or by scraping. The percentage of cells with WGA-FITC protected from quenching is indicated, showing that cytochalasin D (red fill) enhances endocytosis. (C) EM of NRK cells treated or not treated with cytochalasin D and fixed 4 min after 200 ng/ml SLO or scraping in Ca²⁺. The increase in endosome number/size (clear vesicles; arrows) is apparent in cytochalasin D–treated cells. (D) Number of BSA-gold–containing endosomes in EM sections. The data represent the mean ± SD (error bars). *, P < 0.05; **, P < 0.005 (unpaired t test comparing Ca²⁺ and Ca²⁺/cytochalasin D samples). (E) Area occupied by vesicles containing BSA-gold in EM sections. Each symbol represents one vesicle, and the bars represent mean area ± SD. **, P < 0.005 (unpaired t test comparing Ca²⁺ and Ca²⁺/cytochalasin D samples). (F) FACS of PI staining after scraping (NRK) or SLO (HeLa). The percentage of cells excluding PI is indicated, showing enhanced resealing in cytochalasin D–treated cells (red fill). The green dashed lines indicate the gates used to distinguish injured (PI positive) from resealed (PI negative) cell populations. Bars, 5 μm.