WIF-B CELLS - AN IN-VITRO MODEL FOR STUDIES OF HEPATOCYTE POLARITY

We have evaluated the utility of the hepatoma-derived hybrid cell line , WIF-B, for in vitro studies of polarized hepatocyte functions. The m ajority (>70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated f luorescein, a property of bile canaliculi in vivo. By indirect immunof luorescence, six plasma membrane (PM) proteins showed polarized distri butions similar to rat hepatocytes in situ. Four apical PM proteins we re concentrated in the BC membrane of WIF-B cells. Microtubules radiat ed from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction-associated protei n ZO-1 was present in belts marking the boundary between apical and ba solateral PM domains. We explored the functional properties of this bo undary in living cells using fluorescent membrane lipid analogs and so luble tracers. When cells were incubated at 4-degrees-C with a fluores cent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fl uorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the prese nce of a barrier to the lateral diffusion of lipids between the PM dom ains. However, small soluble FITC-dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC-dextrans were restricted to variou s degrees depending on their size and incubation temperature. At 4-deg rees-C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had a ccess to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral doma in. Such differential accessibility of well-characterized probes can b e used to mark each membrane domain separately. These results show tha t WIF-B cells are a suitable model to study membrane trafficking and t argeting in hepatocytes in vitro.