Effects of extracellular calcium depletion on membrane topography and occluding junctions of mammary epithelial cells in culture.

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Abstract
Ca2+ dependence of occluding junction structure and permeability, well documented in explanted or cultured epithelial sheets, presumably reflects inherent control mechanisms. As an approach to identification of these mechanisms, we induced disassembly of zonulae occludentes in confluent monolayers of mouse mammary epithelial cells by exposure to low concentrations of the chelators, EGTA or sodium citrate. Stages in disassembly were monitored during treatment by phase-contrast microscopy and prepared for transmission and scanning electron microscopy. Cellular response included several events affecting occluding junctions: (a) Centripetal cytoplasmic contraction created tension on junction membranes and displaced intramembrane strands along lines determined by the axis of tension. (b) Destabilization of junction position, probably through increased membrane fluidity, augmented tension-induced movement of strands, resulting in fragmentation of the junction belt. (c) Active ruffling and retraction of freed peripheral membranes remodeled cell borders to produce many filopodia, distally attached by occluding-junction fragments to neighboring cell membranes. Filopodia generally persisted until mechanically ruptured, when endocytosis of the junction and adhering cytoplasmic bleb ensued. Junction disassembly thus resulted from mechanical tensions generated by initial centripetal contraction and subsequent peripheral cytoskeletal activity, combined with destabilization of the junction's intramembrane strand pattern.