Competition of solid and fluid liposomes for binding and metabolism with lipids from the cell surface

The competitive behavior of solid vs. fluid liposomes in liposome-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding and transfer experiments have demonstrated that: solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substance; fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; fluid liposomes that escape lysis dissociate from the cell taking away cell lipid molecules. No lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface.     

Lipid-cell interactions: Liposome adsorption and cell-to-liposome lipid transfer are mediated by the same cell-surface sites

The competitive behavior of solid vs. fluid liposomes in liposome-to-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding, transfer and ³¹P-NMR experiments have demonstrated that: (i) solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substances; (ii) fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; (iii) fluid liposomes that escape lysis dissociate from the cell, taking away cell lipid molecules. The latter process underlies the mechanism of cell-to-fluid liposome lipid transfer. In contrast, no lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface. Moreover, cell-induced aggregation of both fluid and solid freshly added liposomes is also inhibited by preincubation of the cells with either solid or fluid liposomes. Thus, different types of interaction of both fluid and solid liposomes with the cell are mediated by the same (or closely related) sites on the cell surface.     

Interaction of epithelial cells with the edges of fluid and solid lipid films

Capping of Concanavalin A (Con A) on the surface of epithelial cells near the cell-cell contacts has been compared with that in the regions of cell contacts with the edges of lipid films. If the lipids are in "fluid" state, Con A is capped likely as on the free edges of epithelial sheets, while contacts with the edge of solid lipid film inhibit capping of Con A as do cell-cell contacts. The same is true for capping of liposomes adsorbed on the surface of epithelial cells. We suppose that solid rather than fluid domains in plasma membranes may play a significant role in establishing cell-cell contacts.     

The distribution of solid liposomes of different size on the surface of epithelial cells

Interaction of large multilamellar and small sonicated liposomes with epithelial sheets in culture has been studied. Liposomes were comprised of dipalmitoyl- or distearoylphosphatidylcholine and were solid at 37 degrees. Sodium fluorescein was entrapped in liposomes and their localization on the cell surface was studied by fluorescence microscopy. The distribution of liposomes depends on their size. Large liposomes adhere uniformly all over the surface of the sheet, which proved to be nonadhesive either for other cells or for inert particles. Small liposomes adhere mostly along the margins of the sheet. When the cells are partly detached from each other by mild EDTA (ethylene diamine tetraacetic acid) treatment, the newly accessible cell margins become capable of binding small liposomes. The binding of both types of liposomes is trypsin-sensitive; however large liposomes, bound along the edges of the cellular sheet, are removed slower than those adhering to the inner parts of the sheet. Competition experiments show that large and small liposomes bind to the same sites on the surface of the sheet. Liposome-acceptor proteins are suggested to exist on the surface of epithelial cells. We suppose that these proteins are concentrated along the cell margins and are involved in cell-cell contacts.     

Microtubular system in cultured mouse epithelial cells

Previous observations had shown that colcemid does not affect locomotion of epithelial cells. Nevertheless, cultured mouse kidney cells forming epithelial sheets were found to contain a well-developed microtubular system sensitive to colcemid. The orientation of microtubules in the epithelial cells was not correlated with the stable or active state of the cell edges. It is suggested that microtubular system of the epithelial cells forming coherent sheets, in contrast to that of individually moving fibroblasts, is not essential for stabilization of the lateral cell edges.     

Vesicular stomatitis virus binds and fuses with phospholipid domain in target cell membranes

Fusion of vesicular stomatitis virus with some cells (HELR 66, KB, and human erythrocytes, both intact and trypsinized) and liposomes made of various natural and synthetic lipids was studied with spin-labeled phospholipid. Binding of virus was assayed separately with radiolabeled and spin-labeled virus. Binding to cells and liposomes was small at neutral pH but enhanced at acidic pHs. Fusion with cells and liposomes was negligibly small at neutral pH but greatly activated at acidic pHs lower than 6.5. Activation of fusion occurred at lower pH values than enhancement of binding. Fusion occurred rapidly and efficiently, reaching a plateau at 50-80% after 3 min at 37 degrees C. Binding and fusion with cells were enhanced by pretreatment of cells with trypsin. Binding to liposomes was dependent on the head group of the phospholipid, stronger to phosphatidylserine than to phosphatidylcholine, but not much dependent on the acyl chain composition. On the other hand, cis-unsaturated acyl chains were required for the efficient fusion, but there was only a small, if any, requirement for the head group. Cholesterol enhanced the fusion further. High fusion efficiency with cis-unsaturated phospholipids cannot be ascribed to the membrane fluidity but may be related to higher tail-to-head volume ratios. Possible mode of interaction of viral G glycoprotein with phospholipid is discussed. The virus cell entry mechanism is suggested as binding to the phospholipid domain in the cell surface membranes, endocytosis, and followed by fusion with the phospholipid domain in endosomes upon acidification.     

Characterization of human immunodeficiency virus type 1 gp120 binding to liposomes containing galactosylceramide.

Human immunodeficiency virus type 1 (HIV-1) infects some cell types which lack CD4, demonstrating that one or more alternative viral receptors exist. One such receptor is galactosylceramide (GalCer), a glycosphingolipid distributed widely in the nervous system and in colonic epithelial cells. Using a liposome flotation assay, we found that the HIV-1 surface glycoprotein, gp120, quantitatively bound to liposomes containing GalCer but not to liposomes containing phospholipids and cholesterol alone. Binding was saturable and was inhibited by preincubating liposomes with anti-GalCer antibodies. We observed less efficient binding of gp120 to liposomes containing lactosylceramide, glucosylceramide, and galactosylsulfate, whereas no binding to liposomes containing mixed gangliosides, psychosine, or sphingomyelin was detected. Binding to GalCer was rapid, largely independent of temperature and pH, and stable to conditions which remove most peripheral membrane proteins. By contrast, gp120 bound to lactosylceramide could be removed by 2 M potassium chloride or 3 M potassium thiocyanate, demonstrating a less stable interaction. Removal of N-linked oligosaccharides on gp120 did not affect binding efficiency. However, as previously observed for CD4 binding, heat denaturation of gp120 prevented binding to GalCer. Finally, binding was critically dependent on the concentration of GalCer in the target membrane, suggesting that binding to glycolipid-rich domains occurs and that GalCer conformation may be important for gp120 recognition.     

Fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density

Influenza virus gains access to the cytoplasm of its host cell by means of a fusion event between viral and host cell membrane. Fusion is mediated by the envelope glycoprotein hemagglutinin (HA) and is triggered by low pH. To learn how many hemagglutinin trimers are necessary to cause membrane fusion, we have used two NIH 3T3 fibroblast cell lines that express HA protein at different surface densities. On the basis of quantitations of the number of HA trimers per cell and the relative surface areas of the two cell lines, the HAb-2 cells have a 1.9-fold higher plasma membrane surface density than the GP4F cells. The membrane lateral diffusion coefficient and the mobile fraction for HA is the same for both cell lines. A Scatchard analysis of the binding of glycophorin-bearing liposomes to the cells showed 1700 binding sites for the GP4F cells and 3750 binding sites for the HAb-2 cells, with effectively the same liposome-cell binding constant, about 7 x 10(10) M-1. Binding was specific for glycophorin on the liposomes and HA expressed on the cells. A competition experiment employing toxin-containing and empty liposomes allowed us to quantitate the number of liposomes that fused per cell, which was a small constant fraction of the number of bound liposomes. For the HAb-2 cells, about 1 in every 70 bound liposomes fused and for the GP4F cells about 1 in every 300 bound liposomes fused. Hence, the HAb-2 cells showed 4.4 times more fusion per bound liposome, even though the surface density of HA was only 1.9 times greater. We conclude the following: (i) One HA trimer is not sufficient to induce fusion. (ii) The HA bound to glycophorin is not the HA that induces fusion. That is, even though each HA has a binding and a fusion function, those functions are not performed by the same HA trimer.     

Simultaneous interaction of monoclonal antibody-targeted liposomes with two receptors on K562 cells

We have investigated the interaction of targeted liposomes with human erythrocytes, and K562 cells, a human leukemic line which expresses both glycophorin A and Fc receptors. Liposomes conjugated to monoclonal anti-human glycophorin A bind to human erythrocytes in 80-fold greater amounts than liposomes conjugated to a non-specific monoclonal antibody. Binding is inhibited by soluble anti-glycophorin but not by its Fab fragment. In contrast, binding of antibody-conjugated liposomes to K562 cells is very high irrespective of the specificity of the antibody. Liposomes conjugated to a nonspecific monoclonal antibody interact with K562 cells via an Fc receptor, and binding is inhibited by soluble human IgG. Liposomes conjugated to anti-human glycophorin A interact with K562 cells via an Fc receptor and glycophorin A. Binding is not inhibited by either human IgG or anti-glycophorin Fab alone. Binding is only partially inhibited by anti-glycophorin, or by human IgG in the presence of anti-glycophorin Fab, and completely inhibited only by human IgG in the presence of anti-glycophorin. Simultaneous binding of targeted liposomes to two cell membrane antigens is therefore partially resistant to inhibition by single soluble ligands even when they are present in large excess. We conclude that simultaneous binding to more than one receptor may be of considerable advantage for in vivo applications of targeted liposomes.     

The function of tight junctions in maintaining differences in lipid composition between the apical and the basolateral cell surface domains of MDCK cells.

Tight junctions in epithelial cells have been postulated to act as barriers inhibiting lateral diffusion of lipids and proteins between the apical and basolateral plasma membrane domains. To study the fence function of the tight junction in more detail, we have fused liposomes containing the fluorescent phospholipid N-Rh-PE into the apical plasma membrane of MDCK cells. Liposome fusion was induced by low pH and mediated by the influenza virus hemagglutinin, which was expressed on the apical cell surface after viral infection. Redistribution of N-Rh-PE to the basolateral surface, monitored at 0 degree C by fluorescence microscopy, appeared to be dependent on the transbilayer orientation of the fluorescent lipids in the plasma membrane. Asymmetric liposomes containing over 85% of the N-Rh-PE in the external bilayer leaflet, as shown by a phospholipase A2 assay, were generated by octyl beta-D-glucoside dialysis. When these asymmetric liposomes were fused with the apical plasma membrane, fluorescent lipid did not move to the basolateral side. Symmetric liposomes which contained the marker in both leaflets were obtained by freeze-thawing asymmetric liposomes or by reverse-phase evaporation. Upon fusion of these with the apical membrane, redistribution to the basolateral membrane occurred immediately. Redistribution could be observed with asymmetric liposomes only when the tight junctions were opened by incubation in a Ca2+-free medium. During the normal experimental manipulations the tight junctions remained intact since a high trans-epithelial electrical resistance was maintained over the cell monolayer. We conclude that the tight junction acts as a diffusion barrier for the fluorescent phospholipid N-Rh-PE in the exoplasmic leaflet of the plasma membrane but not in the cytoplasmic leaflet.     

Characterization of high-density lipoprotein binding and cholesterol efflux in cultured mouse adipose cells

Binding of high-density lipoproteins to cultured mouse Ob1771 adipose cells was studied, using labeled human HDL3, mouse HDL and apolipoprotein AI- or AII-containing liposomes. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 18, 42, 30 and 3.4 micrograms/ml, whereas the maximal binding capacities were found to be 160, 100, 90 and 21 ng/mg of cell protein. Apoprotein AI not inserted into liposomes did not bind. The binding of 125I-HDL3 was competitively inhibited by apolipoprotein AI-containing liposomes greater than mouse HDL greater than HDL3. The binding of 125I-labeled apolipoprotein AI- and 125I-labeled apolipoprotein AII-containing liposomes was competitively inhibited by HDL3, apolipoprotein AI- and apolipoprotein AII-containing liposomes. Dimyristoylphosphatidylcholine liposomes containing or not cholesterol did not interfere with the binding of labeled HDL3 or apolipoprotein-containing liposomes. Binding studies on crude membranes of Ob1771 adipose cells revealed the presence of intracellular binding sites for LDL and HDL3. Thus, adipose cells have specific binding sites for apolipoprotein E-free HDL and apolipoprotein AI (or AII) is the ligand for these binding sites. Long-term exposure of adipose cells to LDL cholesterol as a function of LDL concentration led to an accumulation of cellular unesterified cholesterol. This process was saturable and reversible as a function of time and concentration by exposure to HDL3 or apolipoprotein AI-containing liposomes, whereas apolipoprotein AII-containing liposomes did not promote any cholesterol efflux. Since long-term exposure of adipose cells to LDL and HDL3 did not affect the number of apolipoprotein B,E receptors and apolipoprotein E-free binding sites, respectively, it appears that adipose cells do not show efficient cholesterol homeostasis and thus could accumulate or mobilize unesterified cholesterol.     

E-cadherin mediated lateral interactions between neighbor cells necessary for collective migration

Collective cell movement is critical in pathological processes such as wound healing and cancer invasion. It entails complex interactions between adjacent cells and between cells-extracellular matrices. Most studies measure the migration patterns and force propagation by placing cells on flat, patterned substrates. The cooperative behavior resulting from cell-cell interactions is not well understood. We have developed a multi-channel microfluidic device that has junctional protein E-cadherin coated onto the sidewalls of the channels that enables the cells’ lateral interactions with their neighbors to be studied. Our study reveals that epithelial cells rely on lateral E-cadherin-based adhesions to maintain the cohesion of the group. Cells move faster in narrower channels, but the average velocity along the channels is reduced in E-cadherin coated channels versus non-adhesive channels. We have directly measured the forces in the cross-linking protein, alpha-actinin, using FRET sensors during cell migration, and found that higher tension exists at the cell edges adjacent to the walls coated with E-cadherin, the implication being E-cadherin transmits the shear forces but does not provide a driving force for this migration.     

Antibodies to phospholipids and liposomes: binding of antibodies to cells

Binding of two monoclonal anti-liposome antibodies to the surface of cultured murine peritoneal macrophages was investigated by indirect immunofluorescence and enzyme-linked immunosorbent assay. Neither antibody bound to cultures of freshly explanted, nonadherent macrophages, but immunoreactivity was observed following cell adherence to tissue culture plastic. Fluorescent microscopic evaluation revealed heterogeneity in staining patterns of the antibodies on adherent cells. Binding both to viable and fixed adherent macrophages was observed even after a 10,000-fold dilution of antibody. Treatment of adherent macrophage cultures with trypsin increased antibody binding. Further treatment of trypsinized-macrophages with alkaline phosphatase or neuraminidase did not affect antibody binding, but phospholipase D and, to a greater extent, phospholipase C resulted in a marked decrease in cellular binding. The data indicate that antibodies produced against liposomes appear to bind to surface phospholipids of macrophages, but binding can be influenced by the physiological state of the macrophage and overlying cell surface proteins.     

Distribution of sialoglycoconjugates on acinar cells of the mammalian pancreas

Using the sialic acid-specific lectin, limulin (LPA; from Limulus polyphemus hemolymph), the distribution and nature of sialoglycoconjugates on the surface of rat pancreatic cells has been investigated. Binding of rhodaminated LPA (Rh-LPA) or horseradish peroxidase-conjugated LPA (HRP-LPA) to fixed-frozen sections of adult rat pancreas resulted in intense linear staining of the apical surface of acinar cells with fainter staining on the basal but not the lateral cell surfaces. LPA binding was specific in that it could be abolished by 1) pretreatment of tissue sections with neuraminidase or periodic acid; 2) competition with sialic acid; and 3) incubation in Ca2+ -free buffers. Pretreatment of sections with proteases abolished LPA binding to the apical surfaces of acinar cells and also enhanced LPA binding to the lateral cell surface. Lipid extraction of sections following protease treatment markedly reduced LPA binding to the acinar cell periphery. These results suggest that LPA binding sites on the acinar cell apical surface may be primarily sialoglycoproteins, while those on the basolateral surfaces may consist in part of gangliosides. Electron microscopy of collagenase-dispersed acini exposed to HRP-LPA confirmed binding of LPA to the basal plasmalemma and, in addition, revealed staining of basal lamina when present. LPA binding to the acinar cell surface was not affected by digestion of tissue sections with hyaluronidase, heparinase, collagenase, or 6 M guanidine-HCl. Control experiments indicated that rat pancreatic secretory proteins contain undetectable amounts of sialoglycoproteins and thus that the apical localization of LPA is not due to adherent secretory proteins. Islets of Langerhans were always uniformly and heavily stained with LPA conjugates; this staining was protease insensitive. Appearance of LPA binding sites was examined on embryonic pancreatic epithelia. At day 15 of gestation, Rh-LPA stained the entire periphery of the epithelial cells, including the lateral cell surface, although more intense staining was already noted on the apical surface. This pattern persisted through day 17 of gestation, but by day 19 an adult staining pattern was observed with loss of staining of the lateral cell surfaces.     

Lewis a blood group antigen of non-secretors: a receptor for candida blastospores

Abstract This study tested the hypothesis that the Lewis ^a blood group antigen found predominantly on the cells of non-secretors might be one of the receptors for Candida species. Binding of strain 3118C to epithelial cells from either secretor or non-secretor donors was not inhibited by treating the cells with anti-Lewis ^a or anti-Lewis ^b antisera. Binding of strain 3091 to non-secretor cell was inhibited by pretreating the cells with anti-Lewis ^a, but this was not observed for secretor cells.
The results suggest that Lewis ^a might be one of the receptors for some yeast strains.     

Desmosome dynamics in migrating epithelial cells requires the actin cytoskeleton

Re-modeling of epithelial tissues requires that the cells in the tissue rearrange their adhesive contacts in order to allow cells to migrate relative to neighboring cells. Desmosomes are prominent adhesive structures found in a variety of epithelial tissues that are believed to inhibit cell migration and invasion. Mechanisms regulating desmosome assembly and stability in migrating cells are largely unknown. In this study we established a cell culture model to examine the fate of desmosomal components during scratch wound migration. Desmosomes are rapidly assembled between epithelial cells at the lateral edges of migrating cells and structures are transported in a retrograde fashion while the structures become larger and mature. Desmosome assembly and dynamics in this system are dependent on the actin cytoskeleton prior to being associated with the keratin intermediate filament cytoskeleton. These studies extend our understanding of desmosome assembly and provide a system to examine desmosome assembly and dynamics during epithelial cell migration.     

The role of electrical signals in murine corneal wound re-epithelialization

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.     

Binding of CTP: phosphocholine cytidylyltransferase to large unilamellar vesicles

We have studied the binding of CTP: phosphocholine cytidylyltransferase from HeLa cell cytosol to large unilamellar vesicles of egg phosphatidylcholine (PC) or HeLa cell phospholipids that contain various amounts of oleic acid. A fatty acid/phospholipid molar ratio exceeding 10% was required for CTP: phosphocholine cytidylyltransferase binding to liposomes. At a fatty acid/phospholipid molar ratio of 1; 85% of the cytosolic CTP: phosphocholine cytidylyltransferase was bound. The enzyme also bound to liposomes with at least 20 mol% palmitic acid, monoolein, diolein or oleoylacetylglycerol. Oleoyl-CoA did not promote enzyme binding to liposomes. Binding to oleate-PC vesicles was blocked by Triton X-100 but not by 1 M KCl, and was reversed by incubation of the vesicles with bovine serum albumin. Cytidylyltransferase bound to egg PC vesicles that contained 33 mol% oleic acid equally well at 4 degrees C and 37 degrees C. The enzyme also bound to dimyristoyl- and dipalmitoylphosphatidylcholine vesicles containing oleic acid at temperatures below the phase transition for these liposomes. Binding of the cytidylyltransferase to egg PC vesicles containing oleic acid, monoolein, oleoylacetylglycerol or diolein resulted in enzyme activation, as did binding to dipalmitoylPC-oleic acid vesicles. However, binding to egg PC-palmitic acid vesicles did not fully activate the transferase. Various mechanisms for cytidylyltransferase interaction with membranes are discussed.     

Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells

Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (protein-free lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH- dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions. Virosomes and rosettes, but not liposomes, bound to cells. Binding occurred preferentially to microvilli and was inhibited by added SFV; it increased with decreasing pH but was, in all cases, less efficient than intact virus. At 37 degrees C the cell surface-bound rosettes and virosomes were internalized via coated pits and coated vesicles. After a lag period of 45 min the protein components of the internalized ligands were degraded and appeared, as acid-soluble activity, in the medium. The uptake of rosettes and virosomes was found to be similar to the adsorptive endocytosis of SFV except that their average residence times on the cell surface were longer. The rosettes and the liposomes did not show low pH-induced membrane fusion activity. The virosomes, however, irrespective of the lipid compositions used, displayed hemolytic activity at mildly acidic pH and were able to fuse with the plasma membrane of cells with an efficiency of 0.25 that observed with intact viruses. Cell-cell fusion activity was not observed with any of the subviral components. The results indicated that subviral components possess some of the entry properties of the intact virus.     

The Herpes Simplex Virus gE-gI Complex Facilitates Cell-to-Cell Spread and Binds to Components of Cell Junctions

The herpes simplex virus (HSV) glycoprotein complex gE-gI mediates the spread of viruses between adjacent cells, and this property is especially evident for cells that form extensive cell junctions, e.g., epithelial cells, fibroblasts, and neurons. Mutants lacking gE or gI are not compromised in their ability to enter cells as extracellular viruses. Therefore, gE-gI functions specifically in the movement of virus across cell-cell contacts and, as such, provides a molecular handle on this poorly understood process. We expressed gE-gI in human epithelial cells by using replication-defective adenovirus (Ad) vectors. gE-gI accumulated at lateral surfaces of the epithelial cells, colocalizing with the adherens junction protein β-catenin but was not found on either the apical or basal plasma membranes and did not colocalize with ZO-1, a component of tight junctions. In subconfluent monolayers, gE-gI was found at cell junctions but was absent from those lateral surfaces not in contact with another cell, as was the case for β-catenin. Similar localization of gE-gI to cell junctions was observed in HSV-infected epithelial cells. By contrast, HSV glycoprotein gD, expressed using a recombinant Ad vectors, was found primarily along the apical surfaces of cells, with little or no protein found on the basal or lateral surfaces. Expression of gE-gI without other HSV polypeptides did not cause redistribution of either ZO-1 or β-catenin or alter tight-junction functions. Together these results support a model in which gE-gI accumulates at sites of cell-cell contact by interacting with junctional components. We hypothesize that gE-gI mediates transfer of HSV across cell junctions by virtue of these interactions with cell junction components.