By immunofluorescence and freeze fracture methods, we have studied the establishment of hepatic cell polarity in WIF-B9 cells, a subclone of the WIF-B rat hepatoma-derived hybrid cell line. As previously shown (Ihrke et al. (1993) J. Cell Biol. 123, 1761–1775; Shanks et al. (1994) J. Cell Sci. 107, 813–825), these cells are a suitable model for in vitro studies of various hepatic functions, particularly polarity: in confluent cultures, the majority of cells form bile canaliculus-like structures; membrane domains are settled, according to plasma membrane protein localization similar to rat hepatocytes in situ. We here report that the establishment of WIF-B9 cell polarity is a slow progressive biphasic phenomenon. During the first days of culture, the majority of cells do not make bile canaliculus-like structures. However, they display a polarity similar to that of simple epithelial cells: apical membrane proteins and villin are found at the cell apex; basolateral ones, excluded from this area, are expressed in the remaining membrane area; the tight junction-associated protein ZO-1 and actin are concentrated at the boundary of these two poles, whereas E-cadherin is present at the lateral pole just under the apex. With time in culture, the number of cells expressing this simple epithelial polarized phenotype decreases progressively and, after 10–15 days, depending on the plating density, nearly all the cells express the typical hepatic polarized phenotype. The expression of these two phenotypes is mutually exclusive. Freeze-fracture replicas of both types of polarized cells show either macula occludens, fascia occludens (simple epithelial polarity) or zonula occludens (hepatic polarity), associated with gap junctions. In this last case, two or three continuous strands are generally present all around the bile canaliculus-like structures.

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