Junction formation between cultured normal rat hepatocytes an ultrastructural study on the presence of cholesterol and the structure of developing tight-junction strands

In cultures of normal rat hepatocytes, isolated by collagenase perfusion, formation of junctions started 4-7 h after seeding. The junctional complexes were localized at two preferential sites of the contiguous membrane: apically and on both sides of open spaces with microvilli at some distance from the upper surface. The domains of early contact formation are characterized by a flattening of the membranes, a decrease in intercellular space, a depletion of intramembrane particles (IMPs) and a concentration of electron-dense and of fine fibrillar material in the cytoplasm immediately adjacent to the membrane. We were able to demonstrate the presence of cholesterol in these domains by formation of cholesterol-filipin complexes that deformed the membrane. However, membrane-deformation in these domains was often inhibited. This inhibition appeared to be due to the presence of pepsin-sensitive material, since full deformation was induced after mild proteolysis. The arrangement of IMPs into small gap junctions and short tight-junction strands is synchronous in the membranes of the neighbouring cells. The tight junctions are composed of two strands arranged in a slightly offset configuration. The possibility that the strands are lipidic in nature is not excluded.