Polarity and nucleation of microtubules in polarized epithelial cells.

Microtubules oriented in the apicobasal axis of columnar epithelial cells are arranged with a uniform polarity with minus ends toward the apical surface, suggesting that these cytoskeletal filaments might serve as a substrate for polarized movement of membrane vesicles within the cell. It is not known whether hepatocytes, a cuboidal epithelium in which transcellular transport is a requisite step in normal apical membrane biogenesis, contain microtubules arranged with a similar polarity. In the present study, we explore the question of microtubule polarity and possible mechanisms for nucleation in the epithelial cell lines WIF-B (hepatocyte), Caco-2 (intestine), and Madin-Darby canine kidney (MDCK). Caco-2 microtubules in the apicobasal axis had uniform polarity with minus ends nearest the apical surface. After cold and nocodazole-induced depolymerization, microtubule regrowth initiated in the apical region in all three cell types. The apex of WIF-B and Caco-2 cells contained two pools of gamma-tubulin: one associated with centrosomes and the other delocalized under the apical membrane. Non-centrosomal gamma-tubulin was present in complexes that sedimented between 10S and 29S; both forms could bind microtubules. The presence of both centrosomal and noncentrosomal gamma-tubulin in apical cytoplasm suggest multiple mechanisms by which microtubule nucleation might occur in epithelial cells.

Evidence that Fc gamma receptors in rabbit yolk sac endoderm do not depend upon an acid pH to effect IgG binding and transcytosis in vitro.

An in vitro culture system has been devised creating apical and basal compartments separated by rabbit visceral yolk sac (VYS) with an intact epithelium. Selective transcytosis and binding of heterologous IgG applied to the apical yolk sac endoderm (YSE) was demonstrated in vitro using double label immunofluorescence. Thus, whilst both human and bovine IgG could be detected in endosomes in YSE, only human IgG could be detected in the basement membrane and vascular mesenchyme. This mirrors what is found in vivo. The Fc fragment of human Ig was transcytosed but not the Fab fragment, indicating that Fc receptors were expressed in the cultured YSE. When VYS was previously chilled to 4 degrees C to prevent endocytosis and treated with rabbit serum albumin to prevent non-specific binding, human IgG, but not bovine IgG, became specifically bound to YSE apical plasma membrane; comparison of binding at pH 6.0, 7.3 (the average pH of rabbit uterine fluid) and 8.0 revealed no obvious difference. Pre-exposure of VYS for up to 5 min in monensin, followed by culture in monensin and immunoglobulin-containing medium, did not prevent the selective transcystosis of human IgG, suggesting that an acidic compartment may not be needed for transcytosis. An acid pH dependent Fc gamma receptor equivalent to that on suckling rat gut jejunal enterocyte plasma membranes could not be isolated from rabbit YSE following exposure of solubilized membrane to affinity matrix bound IgG at pH 6.0 and elution at pH 8.0. These results contradict a recent suggestion that Fc receptors on all IgG transcytosing epithelia require an acid pH to effect IgG binding and selective transcytosis.

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 majority (> 70%) of cells in confluent culture formed closed spaces with adjacent cells. These bile canalicular-like spaces (BC) accumulated fluorescein, a property of bile canaliculi in vivo. By indirect immunofluorescence, six plasma membrane (PM) proteins showed polarized distributions similar to rat hepatocytes in situ. Four apical PM proteins were concentrated in the BC membrane of WIF-B cells. Microtubules radiated from the BC (apical) membrane, and actin and foci of gamma-tubulin were concentrated in this region. The tight junction-associated protein ZO-1 was present in belts marking the boundary between apical and basolateral PM domains. We explored the functional properties of this boundary in living cells using fluorescent membrane lipid analogs and soluble tracers. When cells were incubated at 4 degrees C with a fluorescent analog of sphingomyelin, only the basolateral PM was labeled. In contrast, when both PM domains were labeled by de novo synthesis of fluorescent sphingomyelin from ceramide, fluorescent lipid could only be removed from the basolateral domain. These data demonstrate the presence of a barrier to the lateral diffusion of lipids between the PM domains. However, small soluble FITC-dextrans (4,400 mol wt) were able to diffuse into BC, while larger FITC-dextrans were restricted to various degrees depending on their size and incubation temperature. At 4 degrees C, the surface labeling reagent sNHS-LC-biotin (557 mol wt) had access to the entire PM, but streptavidin (60,000 mol wt), which binds to biotinylated molecules, was restricted to only the basolateral domain. Such differential accessibility of well-characterized probes can be used to mark each membrane domain separately. These results show that WIF-B cells are a suitable model to study membrane trafficking and targeting in hepatocytes in vitro.

Apical expression of an antigen common to rabbit yolk sac endoderm and kidney proximal tubule epithelium.

The tissue distribution, molecular weight, and biochemical nature of an antigen detected by a mouse monoclonal antibody designated 283D3 and raised against rabbit visceral yolk sac endodermal cells, has been investigated. The antigen is located on the luminal side of apical tubules and large sub-apical vesicles in rabbit yolk sac endoderm and proximal kidney tubule epithelial cells. It is expressed in a similar polarised fashion in epithelial cells lining the epididymis. Western blotting showed the antigen to comprise proteins of molecular weight 330-380 kDa. The antigen has been affinity purified from yolk sac and kidney and is predominantly protein in nature with a small percentage of N-linked carbohydrate. In terms of tissue distribution and molecular weight it has close similarity to Heymann nephritis antigen but differs in not being confined to coated pits. Its function is not known, but the association with endocytic elements implies a possible role in non-specific protein absorption.