Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination

Vascular endothelium in vivo appears to function as a polarized epithelium. To determine whether cellular polarity exists at the level of the plasma membrane, we have examined cultured endothelial monolayers for evidence of differential distribution of externally disposed plasmalemmal proteins at apical and basal cell surfaces. Lactoperoxidase beads were used to selectively label the apical surfaces of confluent endothelial monolayers, the total surfaces of nonenzymatically resuspended cells, and the basal surfaces of monolayers inverted on poly-L-lysine-coated coverslips, while maintaining greater than 98% viability in all samples. Comparison of the SDS PAGE radioiodination patterns obtained for each surface revealed a number of specific bands markedly enriched on either apical or basal surface. This polarized distribution involved membrane- associated as well as integral membrane proteins and was observed in several strains of bovine aortic endothelial cells, as well as in both primary and passaged human umbilical vein endothelial cells. In contrast, two morphologically nonpolarized cell types, bovine aortic smooth muscle and mouse peritoneal macrophages, did not display differential localization of integral membrane proteins. Polarized distribution of integral membrane proteins was established before the formation of a confluent monolayer. When inverted (basal-side-up) monolayers were returned to culture, the apical-side-up pattern was reexpressed within a few days. These results demonstrate that cell surface-selective expression of plasmalemmal proteins is an intrinsic property of viable endothelial cells in vitro. This apical/basal asymmetry of membrane structure may provide a basis for polarized endothelial functions in vivo.     

Isolation of frog urinary bladder plasma membranes with polycation coated beads

It is now generally accepted that the increase in water permeability induced by antidiuretic hormone (ADH) in responsive epithelia is accompanied by the insertion of specific structures in the apical membrane of epithelial cells. There are strong indications that these particles, probably proteic in nature, represent water channels. In order to evaluate the nature and role of such proteins, plasma membranes were isolated by the affinity chromatography technique. The method is based on the firm attachment of the external face of the membrane to polycations covalently bound to the surface of polyacrylamide beads, followed by shearing of the rest of the cells. Maximal binding of epithelial cells to beads was achieved in a medium of low ionic strength and pH 5.2 (i.e. sucrose-MES buffer). By this procedure plasma membranes were obtained from both cAMP-stimulated cells and control cells. Membranes isolated on beads were enriched in the activity of typical membrane marker enzymes (LAP; H+ ATPase; Na+, K+ ATPase) with respect to a whole cell homogenate, whereas contamination of plasma membrane fraction by endoplasmic reticulum, lysosomes, and mitochondria was relatively low. Analysis by SDS polyacrylamide gel electrophoresis showed an interesting difference between cAMP-treated and control samples.     

Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase in rat parotid gland

Distribution of (Na+,K+)ATPase on the cell membranes of acinar and duct cells of rat parotid gland was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. In acinar cells, ATPase was localized predominantly on the basolateral plasma membranes. A small but significant amount of (Na+,K+)ATPase was, however, detected on the luminal plasma membranes, especially on the microvillar region of the acinar cells; the surface density on the luminal membrane was approximately one third of that on the basolateral membranes. In duct cells, many gold particles were found on the basolateral membrane, especially along the basal infoldings of the plasma membranes, whereas no significant gold particles were found on the luminal plasma membranes, suggesting unilateral distribution of ATPase in duct cells. We suggest that in acinar cells sodium ion is not only transported paracellularly but is also actively transported intracellularly into the luminal space by the (Na+,K+)ATPase located on the luminal plasma membranes, and that water is passively transported to the luminal space to form a plasma-like isotonic primary saliva, while in the duct cells the same ion is selectively re-absorbed intracellularly by (Na+,K+)ATPase found in abundance along the many infoldings of the basal plasma membranes, thus producing the hypotonic saliva.     

Hormonal regulation of the polarized function and distribution of Na/H exchange and Na/HCO3 cotransport in cultured mammary epithelial cells

The time course for development of polarized function and morphological distribution of pH regulatory mechanisms has been examined in a mouse mammary epithelial cell line (31EG4). Monolayers grown on permeable supports had tight junctions when grown 3-4 days in the presence of the lactogenic hormones dexamethasone (D, a synthetic glucocorticoid) and insulin (I), or in D, I, and prolactin (P), but there were no tight junctions in the absence of D. Microspectrofluorimetry of the pH- sensitive dye BCECF was used to measure pH (pHi) in cells mounted in a two-sided perfusion chamber to distinguish pH regulatory activity at the apical and basolateral membranes. Na/H exchange was assayed as the Na-dependent, amiloride-sensitive component of pHi recovery from an acid load induced by a pulse of NH3/NH4-containing solution. When monolayers were grown 3-4 d in the presence of P, D, and I, Na/H exchange was restricted to the basolateral membrane. In contrast, in the absence of P, Na/H exchange was present on both the apical and basolateral membranes. After 5-6 days, in the presence or absence of P, Na/H exchange was present only on the basolateral membrane. An antibody to the NHE-1 isoform of the Na/H exchanger was used to determine its morphological distribution. In all hormone conditions the antibody recognized a protein of approximately 110 kD (Western blot), and confocal immunofluorescence microscopy of this antibody and of an anti- ZO-1 (the marker of the tight junctions) antibody showed that the morphological distribution of the Na/H exchanger was similar to the functional distribution under all hormonal treatments. In addition, a putative Na/HCO3 cotransport system was monitored as a Na-dependent, amiloride-insensitive pHi recovery mechanisms that was inhibited by 200 microM H2DIDS. After treatment with D+I (but not with I alone) cotransport appeared exclusively on the basolateral membrane, and the polarized expression of this transporter was not altered by P. We conclude that when mammary cells are grown in D+I-containing media, the Na/H exchanger is expressed initially (i.e., after 3-4 d) on both the apical and basolateral membranes and later (5-6 d) on only the basolateral membrane. P (in the presence of D+I) selectively speeds this polarization, which is determined by polarized distribution of the exchanger to the apical and/or basal membrane and not by the activation and/or inactivation of transporters.(ABSTRACT TRUNCATED AT 400 WORDS)     

Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton

In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.     

Synthesis of basement membrane proteins by rat mammary epithelial cells

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.     

Involvement of the membrane-cytoskeleton in development of epithelial cell polarity

The generation of cell surface polarity in transporting epithelial cells occurs in three distinct stages that involve cell-cell recognition and adhesion, cell surface remodelling to form biochemically and functionally distinct cell surface domains, and development of vectorial function. A widely used model system to study mechanisms involved in these stages is the Madin-Darby canine kidney (MDCK) cell line. Under appropriate growth conditions, MDCK cells develop in similar stages into polarized, multicellular epithelial structures. Analysis of membrane-cytoskeletal proteins ankyrin and fodrin during development of MDCK cell surface polarity shows that they gradually assemble into an insoluble protein complex on the basal-lateral membrane domain upon cell-cell adhesion, concomitantly with the redistribution of Na+,K(+)-ATPase, a marker protein of the basal-lateral membrane. Biochemical analysis shows that ankyrin, fodrin occur in a complex with Na+,K(+)-ATPase and the cell adhesion molecule uvomorulin in MDCK cells. A model is presented in which assembly of membrane-cytoskeletal complexes at sites of uvomorulin-induced cell-cell contact causes a remodelling of the cell surface distribution of specific membrane proteins which, in turn, contributes to the generation of epithelial cell surface polarity.     

Crag regulates epithelial architecture and polarized deposition of basement membrane proteins in Drosophila

The polarized architecture of epithelia relies on an interplay between the cytoskeleton, the trafficking machinery, and cell-cell and cell-matrix adhesion. Specifically, contact with the basement membrane (BM), an extracellular matrix underlying the basal side of epithelia, is important for cell polarity. However, little is known about how BM proteins themselves achieve a polarized distribution. In a genetic screen in the Drosophila follicular epithelium, we identified mutations in Crag, which encodes a conserved protein with domains implicated in membrane trafficking. Follicle cells mutant for Crag lose epithelial integrity and frequently become invasive. The loss of Crag leads to the anomalous accumulation of BM components on both sides of epithelial cells without directly affecting the distribution of apical or basolateral membrane proteins. This defect is not generally observed in mutants affecting epithelial integrity. We propose that Crag plays a unique role in organizing epithelial architecture by regulating the polarized secretion of BM proteins.     

Bipolar assembly of caveolae in retinal pigment epithelium

Caveolae and their associated structural proteins, the caveolins, are specialized plasmalemmal microdomains involved in endocytosis and compartmentalization of cell signaling. We examined the expression and distribution of caveolae and caveolins in retinal pigment epithelium (RPE), which plays key roles in retinal support, visual cycle, and acts as the main barrier between blood and retina. Electron microscopic observation of rat RPE, in situ primary cultures of rat and human RPE and a rat RPE cell line (RPE-J) demonstrated in all cases the presence of caveolae in both apical and basolateral domains of the plasma membrane. Caveolae were rare in RPE in situ but were frequent in primary RPE cultures and in RPE-J cells, which correlated with increased levels in the expression of caveolin-1 and -2. The bipolar distribution of caveolae in RPE is striking, as all other epithelial cells examined to date (liver, kidney, thyroid, and intestinal) assemble caveolae only at the basolateral side. This might be related to the nonpolar distribution of both caveolin-1 and 2 in RPE because caveolin-2 is basolateral and caveolin-1 nonpolar in other epithelial cells. The bipolar localization of plasmalemmal caveolae in RPE cells may reflect specialized roles in signaling and trafficking important for visual function. caveolin; raft microdomains; membrane traffic; normal rat kidney     

An ion-transporting ATPase encodes multiple apical localization signals

Epithelial cells accumulate distinct populations of membrane proteins at their two plasmalemmal domains. We have examined the molecular signals which specify the differential subcellular distributions of two closely related ion pumps. The Na,K-ATPase is normally restricted to the basolateral membranes of numerous epithelial cell types, whereas the H,K-ATPase is a component of the apical surfaces of the parietal cells of the gastric epithelium. We have expressed full length and chimeric H,K-ATPase/Na,K-ATPase cDNAs in polarized renal proximal tubular epithelial cells (LLC-PK1). We find that both the alpha and beta subunits of the H,K-ATPase encode independent signals that specify apical localization. Furthermore, the H,K-ATPase beta-subunit possesses a sequence which mediates its participation in the endocytic pathway. The interrelationship between epithelial sorting and endocytosis signals suggested by these studies supports the redefinition of apical and basolateral as functional, rather than simply topographic domains.     

Scanning electron microscopy of isolated epithelium of the murine gastrointestinal tract: morphology of the basal surface and evidence for paracrinelike cells

By using the method of Bjerknes and Cheng, isolated murine gastrointestinal epithelial sheets were prepared for scanning electron microscopy. Examination of isolated epithelium from fundic stomach revealed numerous branched gastric glands. Parietal cells were easily detected bulging from the basal surface of the glandular epithelium. The basal surface membrane of parietal cells appeared smooth, with only sparse microvilluslike projections, whereas adjacent glandular cells had numerous 1- to 2-micron fingerlike projections which interdigitated laterally with similar processes from adjacent cells. Occasionally, paracrinelike cells having long cytoplasmic processes ranging from 10 to 20 micron in length were observed on the basal epithelial surface of the stomach and the colon, but not the small intestine. In isolated intestinal epithelia, the basal surface of crypt epithelial cells showed extensive cytoplasmic interdigitations, but no distinct morphology permitting recognition of individual cell types. Various stages of intestinal crypt bifurcation were seen. Craterlike spaces in the basal surface of crypt epithelium, presumably due to migrating leukocytes, were also numerous. Examination of the luminal surface of the isolated intestinal epithelium revealed that intimate associations between epithelium and mucosal-associated microorganisms were maintained, thus suggesting that minimal alterations in surface morphology were incurred by epithelial isolation. These observations on epithelial structure suggest that isolated gastrointestinal epithelia may be well suited for physiological studies of epithelial function and interactions with the microbial flora.     

Two novel peripheral membrane proteins, pasin 1 and pasin 2, associated with Na+,K(+)-ATPase in various cells and tissues

Purification of pig kidney Na+,K(+)-ATPase at low concentrations of SDS (0.5%) allowed copurification of several peripheral membrane proteins. Some of these associated proteins were identified as components of the membrane cytoskeleton. Here we describe two novel globular proteins of of Mr 77,000 (pasin 1) and Mr 73,000 (pasin 2) which copurify and coimmunoprecipitate with Na+,K(+)-ATPase and can be stripped off Na+,K(+)-ATPase microsomes by 1 M KCl. Pasin 1 and pasin 2 were detected by immunoblot analysis in various cells and tissues including erythrocytes and platelets. Immunostaining revealed colocalization of pasin 1 and Na+,K(+)-ATPase along the basolateral cell surface of epithelial cells of kidney tubules and parotid striated ducts (titers of pasin 2 antibodies were too weak for immunocytochemistry). In erythrocytes, pasin 1 and pasin 2 are minor components bound to the cytoplasmic surface of the plasma membrane. Pasin 1 showed the same electrophoretic mobility as protein 4.1b. However, both proteins have different isoelectric points (pasin 1, pI 6; protein 4.1, pI 7), different chymotryptic fragments, and are immunologically unrelated. Short pieces of sequence obtained from pasin 1 and pasin 2 were not found in any other known protein sequence. The occurrence of pasin 1 and pasin 2 in diverse cells and tissues and their association with Na+,K(+)-ATPase suggests a general role of these proteins in Na+,K(+)- ATPase function.     

Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia

In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K(+)-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)- ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K(+)-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal- lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)- dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K(+)-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B- cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular sorting and polarized cell surface delivery of (Na+,K+)ATPase, an endogenous component of MDCK cell basolateral plasma membranes

Madin Darby Canine Kidney (MDCK) cells grown on polycarbonate filters in a two-chamber culture system were used to study the postsynthetic sorting of the alpha-subunit of the (Na+,K+)ATPase, an important native protein of the MDCK cell basolateral plasmalemmal domains. The N-azidobenzoyl derivative of ouabain (NAB-ouabain) and anti-ouabain antibodies were used in pulse labeling experiments to monitor the arrival of newly synthesized molecules of (Na+,K+)ATPase at the apical and basolateral cell surfaces. The results show that newly synthesized alpha-subunits bind NAB-ouabain and become substrates for immunoprecipitation only when this compound is present in the basolateral chamber. No more than 10% of the (Na+,K+)ATPase synthesized during the pulse period could appear at the apical surface without being detected by our assay. Thus, sorting of this native protein is effected intracellularly prior to its direct insertion into the basolateral plasmalemmal domain. Passage through an acidic compartment is not required for proper sorting.     

A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity

In polarized Madin-Darby canine kidney (MDCK) epithelial cells, ankyrin, and the alpha- and beta-subunits of fodrin are components of the basolateral membrane-cytoskeleton and are colocalized with the Na+,K+-ATPase, a marker protein of the basolateral plasma membrane. Recently, we showed with purified proteins that the Na+,K+-ATPase is competent to bind ankyrin with high affinity and specificity (Nelson, W. J., and P. J. Veshnock. 1987. Nature (Lond.). 328:533-536). In the present study we have sought biochemical evidence for interactions between these proteins in MDCK cells. Proteins were solubilized from MDCK cells with an isotonic buffer containing Triton X-100 and fractionated rapidly in sucrose density gradients. Complexes of cosedimenting proteins were detected by analysis of sucrose gradient fractions in nondenaturing polyacrylamide gels. The results showed that ankyrin and fodrin cosedimented in sucrose gradient. Analysis of the proteins from the sucrose gradient in nondenaturing polyacrylamide gels revealed two distinct ankyrin:fodrin complexes that differed in their relative electrophoretic mobilities; both complexes had electrophoretic mobilities slower than that of purified spectrin heterotetramers. Parallel analysis of the distribution of solubilized Na+,K+-ATPase in sucrose gradients showed that there was a significant overlap with the distribution of ankyrin and fodrin. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the alpha- and beta-subunits of the Na+,K+-ATPase colocalized with the slower migrating of the two ankyrin:fodrin complexes. The faster migrating ankyrin:fodrin complex did not contain Na+,K+-ATPase. These results indicate strongly that the Na+,K+-ATPase, ankyrin, and fodrin are coextracted from whole MDCK cells as a protein complex. We suggest that the solubilized complex containing these proteins reflects the interaction of the Na+,K+-ATPase, ankyrin, and fodrin in the cell. This interaction may play an important role in the spatial organization of the Na+,K+-ATPase to the basolateral plasma membrane in polarized epithelial cells.     

Membrane domains of intestinal epithelial cells: distribution of Na+,K+- ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation

The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.     

Novel function of the cell adhesion molecule uvomorulin as an inducer of cell surface polarity

Na+,K(+)-ATPase has distinctly different distributions in mesenchymal cells, where it has an unrestricted distribution over the entire cell surface, compared with polarized epithelial cells, where it is restricted to the basal-lateral membrane domain. The generation of this restricted distribution is important in mesenchyme to epithelia conversion in development and the function of transporting epithelia, but the mechanisms involved are unknown. Here we show that expression of the epithelial CAM uvomorulin in transfected fibroblasts is sufficient to induce a redistribution of Na+,K(+)-ATPase to sites of uvomorulin-mediated cell-cell contacts, similar to that in polarized epithelial cells. This restricted distribution of Na+,K(+)-ATPase occurs in the absence of tight junctions but coincides with the reorganization of the membrane cytoskeleton. The results indicate a direct role for CAMs as inducers of cell surface polarity of selective cytoplasmic and membrane proteins.     

Polarized functions and permeability properties of rat epididymal epithelial cells in vitro.

Cultured rat caput and cauda epididymidal epithelial cells are shown to exhibit polarized properties characteristic of functioning epithelia. When grown on plastic substrates coated with reconstituted basement membrane, confluent monolayers of cells from both regions formed domes characteristic of other transporting epithelia. Immunocytochemical localization of three proteins characteristically associated with epithelial junctional complexes revealed that uvomorulin, zonula occludens 1 and cingulin were present in cultured epididymal epithelial cells and that their distribution was similar to that in the epididymal epithelium in vivo. These three molecules were not found in epididymal stromal cells. Cells from both regions growing in two compartment chambers developed an electrical resistance across the monolayer with a magnitude characteristic of high resistance epithelia. The optimal plating density of cells was 0.75 x 10(6) cells cm-2. The presence of reconstituted basement membrane on the filters did not affect the resistance of the cells. Inulin passage from basal to apical chambers was less than 2% over 24 h. These results show that several polarized functions of epididymal epithelial cells can be maintained in culture and that this type of culture system is useful for studying the function of the epididymis in vitro.     

Expression of functional domains of beta G-spectrin disrupts epithelial morphology in cultured cells

Spectrin is a major structural protein associated with the cytoplasmic surface of plasma membranes of many types of cells. To study the functions of spectrin, we transfected Caco-2 intestinal epithelial cells with a plasmid conferring neomycin resistance and encoding either actin-binding or ankyrin-binding domains of beta G-spectrin fused with beta-galactosidase. These polypeptides, in principle, could interfere with the interaction of spectrin with actin or ankyrin, as well as block normal assembly of alpha- and beta-spectrin subunits. Cells expressing the fusion proteins represented only a small fraction of neomycin-resistant cells, but they could be detected based on expression of beta-galactosidase. Cells expressing spectrin domains exhibited a progressive decrease in amounts of endogenous beta G- spectrin, although alpha-spectrin was still present. Beta G-spectrin- deficient cells lost epithelial cell morphology, became multinucleated, and eventually disappeared after 10-14 d in culture. Spectrin- associated membrane proteins, ankyrin and adducin, as well as the Na+,K(+)-ATPase, which binds to ankyrin, exhibited altered distributions in cells transfected with beta G-spectrin domains. E- cadherin and F-actin, in contrast to ankyrin, adducin, and the Na+,K(+)- ATPase, were expressed, and they exhibited unaltered distribution in beta G-spectrin-deficient cells. Cells transfected with the same plasmid encoding beta-galactosidase alone survived in culture as the major population of neomycin-resistant cells, and they exhibited no change in morphology or in the distribution of spectrin-associated membrane proteins. These results establish that beta G-spectrin is essential for the normal morphology of epithelial cells, as well as for their maintenance in monolayer culture.