Cellular actin and junction formation during reaggregation of adult rat hepatocytes into epithelial cell sheets

Aggregation of adult rat hepatocytes, isolated by the collagenase perfusion technique, was studied by ultrastructural methods and the indirect immunofluorescence technique using anti-actin antibodies. In a primary culture the cells rapidly made contact with each other by filopodia-like structures, as seen by scanning electron microscopy. In a few hours this led to stable adhesion of the cells. No identifiable junction formation occurred during the first 17 h in culture. Within 48 h the cells had formed epithelial cell sheets with junctional complexes consisting of tight junctions, bile canaliculus-like structures and zonula adhaerens-type junctions. The distribution of cytoplasmic actin fluorescence remained homogenous in the contacting cells during the first 24 h in culture, as seen with anti-actin antibodies by the indirect immunofluorescence technique. The first short, fluorescent actin filaments appeared in the periphery of the developing lamellipodia of the spreading cell islands. In organized epithelial cell sheets these filaments were seen as long fibres ending at the perinuclear region of the marginal cells. In the submarginal cells fluorescent actin fibres were distinct at the junctional regions of the cells. This filamentous fluorescence seemed to extend throughout the entire cell sheet in an organized manner and correspond to the apical layer of parallel microfilaments seen in transmission electron microscopy. Our results suggest that filamentous actin plays a role in the contact-induced spreading of the cells and in the maintenance of the internal organization of the epithelial cell sheets.