Vaccinia virus preferentially enters polarized epithelial cells through the basolateral surface.

The uptake of vaccinia virus in polarized epithelial cells was studied to determine whether the site of entry was confined to either the apical or the basolateral membrane. Virus infection was monitored with a recombinant vaccinia virus carrying the luciferase reporter gene. Using cell lines MDCK and MDCK-D11, a clonal line with high transepithelial electrical resistance, we determined that vaccinia virus preferentially enters through the basolateral membrane. The possibility that there is a polarized cell surface distribution of vaccinia virus receptors which may be involved in systemic poxvirus infections is discussed.     

Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.     

RalA but not RalB enhances polarized delivery of membrane proteins to the basolateral surface of epithelial cells

RalA and RalB constitute a family of highly similar (85% identity) Ras-related GTPases. Recently, active forms of both RalA and RalB have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. However, we show here that only active RalA enhances the rate of delivery of E-cadherin and other proteins to their site in the basolateral membrane of MDCK cells, consistent with RalA being a regulator of exocyst function. One reason for this difference is that RalA binds more effectively to the exocyst complex than active RalB does both in vivo and in vitro. Another reason is that active RalA localizes to perinuclear recycling endosomes, where regulation of vesicle sorting is thought to take place, while active RalB does not. Strikingly, analysis of chimeras made between RalA and RalB reveals that high-affinity exocyst binding by RalA is due to unique amino acid sequences in RalA that are distal to the common effector-binding domains shared by RalA and RalB. Moreover, these chimeras show that the perinuclear localization of active RalA is due in part to its unique variable domain near the C terminus. This distinct localization appears to be important for RalA effects on secretion because all RalA mutants tested that failed to localize to the perinuclear region also failed to promote basolateral delivery of E-cadherin. Interestingly, one of these inactive mutants maintained binding to the exocyst complex, suggesting that RalA binding to the exocyst is necessary but not sufficient for RalA to promote basolateral delivery of membrane proteins.     

The building blocks for basolateral vesicles in polarized epithelial cells

After the discovery of basolateral sorting signals for polarized delivery in epithelial cells in the early 1990s, it was only about a decade later that the epithelial-cell-specific sorting adaptor AP-1B was discovered. AP-1B decodes a subclass of basolateral sorting signals and localizes to the recycling endosomes as opposed to the trans-Golgi network, suggesting that this is its major site of action. Furthermore, AP-1B does not simply select its cargo but also facilitates the recruitment of the exocyst complex needed for subsequent fusion with the plasma membrane. This review discusses our current knowledge of AP-1B function in cargo sorting to the basolateral membrane and its impact on our understanding of the similarities and differences between AP-1B-minus fibroblasts and AP-1B-positive epithelial cells.     

The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells

The mouse and human Zip5 proteins are members of the ZIP family of metal ion transporters. In this study, we present evidence that mouse Zip5 is a zinc uptake transporter that is specific for Zn(II) over other potential metal ion substrates. We also show that, unlike many other mammalian ZIP proteins, the endocytic removal of mZip5 from the plasma membrane is not triggered by zinc treatment. Thus, the activity of mZip5 does not appear to be down-regulated by zinc repletion. Zip5 expression is restricted to many tissues important for zinc homeostasis, including the intestine, pancreas, liver, and kidney. Zip5 is similar in sequence to the Zip4 protein, which is involved in the uptake of dietary zinc. Co-expression of Zip4 and Zip5 in the intestine led to the hypothesis that these proteins play overlapping roles in the uptake of dietary zinc across the apical membrane of intestinal enterocytes. Surprisingly, however, we found that mZip5 localizes specifically to the basolateral membrane of polarized Madin-Darby canine kidney cells. These observations suggest that Zip5 plays a novel role in polarized cells by carrying out serosal-to-mucosal zinc transport. Furthermore, given its expression in tissues important to zinc homeostasis, we propose that Zip5 plays a central role in controlling organismal zinc status.     

Epstein-Barr virus infection of polarized epithelial cells via the basolateral surface by memory B cell-mediated transfer infection

Epstein Barr virus (EBV) exhibits a distinct tropism for both B cells and epithelial cells. The virus persists as a latent infection of memory B cells in healthy individuals, but a role for infection of normal epithelial is also likely. Infection of B cells is initiated by the interaction of the major EBV glycoprotein gp350 with CD21 on the B cell surface. Fusion is triggered by the interaction of the EBV glycoprotein, gp42 with HLA class II, and is thereafter mediated by the core fusion complex, gH/gL/gp42. In contrast, direct infection of CD21-negative epithelial cells is inefficient, but efficient infection can be achieved by a process called transfer infection. In this study, we characterise the molecular interactions involved in the three stages of transfer infection of epithelial cells: (i) CD21-mediated co-capping of EBV and integrins on B cells, and activation of the adhesion molecules, (ii) conjugate formation between EBV-loaded B cells and epithelial cells via the capped adhesion molecules, and (iii) interaction of EBV glycoproteins with epithelial cells, with subsequent fusion and uptake of virions. Infection of epithelial cells required the EBV gH and gL glycoproteins, but not gp42. Using an in vitro model of normal polarized epithelia, we demonstrated that polarization of the EBV receptor(s) and adhesion molecules restricted transfer infection to the basolateral surface. Furthermore, the adhesions between EBV-loaded B cells and the basolateral surface of epithelial cells included CD11b on the B cell interacting with heparan sulphate moieties of CD44v3 and LEEP-CAM on epithelial cells. Consequently, transfer infection was efficiently mediated via CD11b-positive memory B cells but not by CD11b–negative naïve B cells. Together, these findings have important implications for understanding the mechanisms of EBV infection of normal and pre-malignant epithelial cells in vivo.     

Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells

Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm.     

Targeting of the SF/HGF receptor to the basolateral domain of polarized epithelial cells

Scatter Factor, also known as Hepatocyte Growth Factor (SF/HGF), has pleiotropic functions including direct control of cell-cell and cell- substrate adhesion in epithelia. The subcellular localization of the SF/HGF receptor is controversial. In this work, the cell surface distribution of the SF/HGF receptor was studied in vivo in epithelial tissues and in vitro in polarized MDCK monolayers. A panel of monoclonal antibodies against the beta chain of the SF/HGF receptor stained the basolateral but not the apical surface of epithelia lining the lumen of human organs. Radiolabeled or fluorescent-tagged anti- receptor antibodies selectively bound the basolateral cell surface of MDCK cells, which form a polarized monolayer sealed by intercellular junctions, when grown on polycarbonate filters in a two-chamber culture system. The receptor was concentrated around the cell-cell contact zone, showing a distribution pattern overlapping with that of the cell adhesion molecule E-cadherin. The basolateral localization of the SF/HGF receptor was confirmed by immunoprecipitation after domain selective cell surface biotinylation. When cells were fully polarized the SF/HGF receptor became resistant to non-ionic detergents, indicating interaction with insoluble component(s). In pulse-chase labeling and surface biotinylation experiments, the newly synthesized receptor was found exclusively at the basolateral surface. We conclude that the SF/HGF receptor is selectively exposed at the basolateral plasma membrane domain of polarized epithelial cells and is targeted after synthesis to that surface by direct delivery from the trans-Golgi network.     

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells

Overexpressionof the epidermal growth factor receptors (EGFR) in polarized kidneyepithelial cells caused them to appear in high numbers at both thebasolateral and apical cell surfaces. We utilized these cells to lookfor differences in the regulation and signaling of apical vs.basolateral EGFR. Apical and basolateral EGFR were biologically activeand mediated EGF-induced cell proliferation to similar degrees.Receptor downregulation and endocytosis were less efficient at theapical surface, resulting in prolonged EGF-induced tyrosine kinaseactivity at the apical cell membrane. Tyrosine phosphorylation of EGFRsubstrates known to mediate cell proliferation, Src-homologous andcollagen protein (SHC), extracellularly regulated kinase 1 (ERK1), andERK2 could be induced similarly by activation of apical or basolateralEGFR. Focal adhesion kinase was tyrosine phosphorylated more bybasolateral than by apical EGFR; however, -catenin was tyrosinephosphorylated to a much greater degree following the activation ofmislocalized apical EGFR. Thus EGFR regulation and EGFR-mediatedphosphorylation of certain substrates differ at the apical andbasolateral cell membrane domains. This suggests that EGFRmislocalization could result in abnormal signal transduction andaberrant cell behavior.

     

Exocytotic pathways exist to both the apical and the basolateral cell surface of the polarized epithelial cell MDCK

Secretion of a foreign protein--chicken oviduct lysozyme--and of endogenous proteins was studied in the polarized epithelial Madin-Darby Canine Kidney (MDCK) cell line. Cell clones that secrete enzymatically active chicken lysozyme were generated by transforming the cells with lysozyme cDNA inserted in a SV40-pBR322 recombinant vector and a dominant selectable marker gene. The kinetics and polarity of lysozyme secretion from one transformed cell clone were studied using cell monolayers grown on nitrocellulose filters. Lysozyme was secreted into the apical and the basolateral medium, demonstrating the existence of direct transport pathways to each cell surface. Control experiments excluded the effects of monolayer leakiness, reabsorption, transepithelial transport, and depolarization. In contrast, the secretion of a set of endogenous proteins of MW 30-40 kd was found to be strictly apical showing that polarized secretion also occurs in this cell line. The latter group of proteins appear to be generated from larger precursor molecules by intracellular cleavage.     

The ammonium transporter RhBG: requirement of a tyrosine-based signal and ankyrin-G for basolateral targeting and membrane anchorage in polarized kidney epithelial cells

RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells.     

The ABCA1 transporter functions on the basolateral surface of hepatocytes

ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.     

Analysis of detergent-resistant membranes associated with apical and basolateral GPI-anchored proteins in polarized epithelial cells

Detergent-resistant membranes (DRMs) represent specialized membrane domains resistant to detergent extraction, which may serve to segregate proteins in a specific environment in order to improve their function. Segregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in DRMs has been shown to be involved in their sorting to the apical membrane in polarized epithelial cells. Nonetheless, we have shown that both apical and basolateral GPI-APs associate with DRMs. In this report we investigated the lipid composition of DRMs associated with an apical and a basolateral GPI-AP. We found that apical and basolateral DRMs contain the same lipid species although in different ratios. This specific lipid ratio is maintained after mixing the cells before lysis indicating that DRMs maintain their identity after Triton extraction.     

Recycling endosomes of polarized epithelial cells actively sort apical and basolateral cargos into separate subdomains

The plasma membranes of epithelial cells plasma membranes contain distinct apical and basolateral domains that are critical for their polarized functions. However, both domains are continuously internalized, with proteins and lipids from each intermixing in supranuclear recycling endosomes (REs). To maintain polarity, REs must faithfully recycle membrane proteins back to the correct plasma membrane domains. We examined sorting within REs and found that apical and basolateral proteins were laterally segregated into subdomains of individual REs. Subdomains were absent in unpolarized cells and developed along with polarization. Subdomains were formed by an active sorting process within REs, which precedes the formation of AP-1B-dependent basolateral transport vesicles. Both the formation of subdomains and the fidelity of basolateral trafficking were dependent on PI3 kinase activity. This suggests that subdomain and transport vesicle formation occur as separate sorting steps and that both processes may contribute to sorting fidelity.     

Vesicular stomatitis virus infects and matures only through the basolateral surface of the polarized epithelial cell line,MDCK

We have used Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters to study the polarity of virus infection and maturation. The cells form epithelia-like monolayers, which display high (>1000 Ω cm²) electrical resistance and a cuboidal morphology. Vesicular stomatitis virus (VSV) was found to infect the monolayer at least 100 times more efficiently when applied through the filter to the basolateral surface than when applied to the apical surface. The avian influenza, fowl plague virus (FPV), infected the monolayer through either the apical or basolateral surface. The polarity of virus budding was evaluated by harvesting virus from the two sides of the monolayer. More than 99% of released influenza hemagglutinin titre was found on the apical side of the filter, while more than 98% of budded VSV was found on the basal side. This polarity of budding was retained through 10 hr of viral infection, as was the polarity of surface expression of viral envelope proteins revealed by immunofluorescence. The strong preference of VSV for basolateral maturation is paralleled by an equally strong preference for infection through the basolateral membrane of this polar epithelial cell.     

A PDZ-binding motif controls basolateral targeting of syndecan-1 along the biosynthetic pathway in polarized epithelial cells

The cell surface proteoglycan, syndecan-1, is essential for normal epithelial morphology and function. Syndecan-1 is selectively localized to the basolateral domain of polarized epithelial cells and interacts with cytosolic PDZ (PSD-95, discs large, ZO-1) domain-containing proteins. Here, we show that the polarity of syndecan-1 is determined by its type II PDZ-binding motif. Mutations within the PDZ-binding motif lead to the mislocalization of syndecan-1 to the apical surface. In contrast to previous examples, however, PDZ-binding motif-dependent polarity is not determined by retention at the basolateral surface but rather by polarized sorting prior to syndecan-1's arrival at the plasma membrane. Although none of the four known PDZ-binding partners of syndecan-1 appears to control basolateral localization, our results show that the PDZ-binding motif of syndecan-1 is decoded along the biosynthetic pathway establishing a potential role for PDZ-mediated interactions in polarized sorting.     

Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells

The cell surface proteoglycan on normal murine mammary gland mouse mammary epithelial cells consists of an ectodomain bearing heparan and chondroitin sulfate chains and a lipophilic domain that is presumed to be intercalated into the plasma membrane. Because the ectodomain binds to matrix components produced by stromal cells with specificity and high affinity, we have proposed that the cell surface proteoglycan is a matrix receptor that binds epithelial cells to their underlying basement membrane. We now show that the proteoglycan surrounds cells grown in subconfluent or newly confluent monolayers, but becomes restricted to the basolateral surface of cells that have been confluent for a week or more; Triton X-100 extraction distinguishes three fractions of cell surface proteoglycan: a fraction released by detergent and presumed to be free in the membrane, a fraction bound via a salt-labile linkage, and a nonextractable fraction; the latter two fractions co-localize with actin filament bundles at the basal cell surface; and when proteoglycans at the apical cell surface are cross- linked by antibodies, they initially assimilate into detergent- resistant, immobile clusters that are subsequently aggregated by the cytoskeleton. These findings suggest that the proteoglycan, initially present on the entire surface and free in the plane of the membrane, becomes sequestered at the basolateral cell surface and bound to the actin-rich cytoskeleton as the cells become polarized in vitro. Binding of matrix components may cross-link proteoglycans at the basal cell surface and cause them to associate with the actin cytoskeleton, providing a mechanism by which the cell surface proteoglycan acts as a matrix receptor to stabilize the morphology of epithelial sheets.     

v-SNARE cellubrevin is required for basolateral sorting of AP-1B-dependent cargo in polarized epithelial cells

The epithelial cell-specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B-dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking.     

Expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells.

Polarized epithelial cells exhibit apical (lumenal) and basolateral (serosal) membrane domains that are separated by circumferential tight junctions. In such cells, enveloped viruses that mature by budding at cell surfaces are released at particular membrane domains. We have used a vaccinia virus recombinant to investigate the site of surface expression of the human immunodeficiency virus type 1 envelope glycoprotein in Madin-Darby canine kidney cells. Cells were infected with the vaccinia virus recombinant, and surface expression of the glycoprotein was analyzed by indirect immunofluorescence, 125I-protein A binding, and immunoelectron microscopy. The glycoprotein appeared exclusively at the basolateral surface as early as 2 h postinfection and reached a maximum level at 8 h postinfection. The gp120 glycoprotein was found to be secreted efficiently into culture medium, and this secretion occurred exclusively at the basolateral surface.