CONCENTRATION-DEPENDENT EFFECTS OF CYTOCHALASIN-D ON TIGHT JUNCTIONS AND ACTIN-FILAMENTS IN MDCK EPITHELIAL-CELLS

The effects of different concentrations of the actin-disrupting drug c ytochalasin D on tight junction permeability and distribution of actin filaments in MDCK epithelial cells were examined. Consistent with pre vious studies, 2 mug/ml cytochalasin D caused a significant decrease i n transepithelial resistance, indicative of an increase in tight junct ion permeability. Surprisingly, increasing concentrations of cytochala sin D caused progressively smaller decreases in transepithelial resist ance. The effects of cytochalasin D were reversible. Light microscopic analysis utilizing rhodamine-conjugated phalloidin demonstrated two d istinct populations of actin filaments in MDCK cells: an apical periph eral ring of actin, presumably associated with the zonula adherens, an d larger actin bundles more basally situated. When treated with 2 mug/ ml cytochalasin D, both actin populations were severely disrupted and cells became flattened. Actin in the apical ring aggregated along cell boundaries, and these aggregates co-localized with similarly disrupte d focal accumulations of the tight junction-associated protein ZO-1. T he basal actin filament bundles also reorganized into focal aggregates . Increasing concentrations of cytochalasin D caused gradually less pe rturbation of the apical actin ring, consistent with the transepitheli al resistance observations. However, the basal actin bundles were disr upted at all concentrations of cytochalasin D tested, demonstrating th at the two actin populations are differentially sensitive to cytochala sin D and that apical actin filaments are more important in the regula tion of tight junction permeability. Finally, treatment of cells with cytochalasin D inhibited the decrease in transepithelial resistance in duced by the chelation of extracellular Ca2+. This indicates that the opening of tight junctions caused by removal of Ca2+ requires a functi onal actin cytoskeleton.