Muscarinic acetylcholine receptor-mediated tight junction opening is involved in epiphora in late phase of submandibular gland transplantation

Submandibular gland (SMG) autotransplantation is an effective therapy for treating severe dry eye syndrome. However, epiphora occurs in more than 40% of patients 6 months after operation. We previously found that muscarinic acetylcholine receptor (mAChR) plays a crucial role in regulating SMG secretion partially through the modulation on tight junction (TJ)-based paracellular pathway. Therefore, the present study aimed to investigate the possible involvement of mAChR and TJ in a rabbit long-term model of SMG transplantation. We found that SMG secretion was significantly increased on postoperative days 90 and 180, which imitated epiphora observed in the patients with SMG transplantation. Although the mRNA expression and fluorescence intensity of M1 and M3 mAChR subtypes were reversed to control levels on postoperative days 30, 90, and 180, the content of β-arrestin2, but not β-arrestin1, was gradually decreased after transplantation, which suggests that mAChR may be hypersensitive in late phase of SMG transplantation. The width of acinar TJs was enlarged and fluorescence intensity of F-actin in peri-apicolateral membranes were remarkably increased on postoperative days 90 and 180. Topical treatment with atropine gel significantly reduced SMG secretion, TJ width, as well as F-actin intensity in peri-apicolateral membranes on postoperative days 180. Moreover, in a perfused rabbit SMG, carbachol increased salivary secretion, enlarged TJ width, and induced F-actin rearrangement, whereas these responses were inhibited by atropine pretreatment. Taken together, our findings suggest that the hypersensitive mAChR may contribute to epiphora in late phase of SMG transplantation through modulating TJ-based paracellular permeability.     

Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins

ZO-1, ZO-2, and ZO-3, which contain three PDZ domains (PDZ1 to -3), are concentrated at tight junctions (TJs) in epithelial cells. TJ strands are mainly composed of two distinct types of four-transmembrane proteins, occludin, and claudins, between which occludin was reported to directly bind to ZO-1/ZO-2/ZO-3. However, in occludin-deficient intestinal epithelial cells, ZO-1/ZO-2/ZO-3 were still recruited to TJs. We then examined the possible interactions between ZO-1/ZO-2/ZO-3 and claudins. ZO-1, ZO-2, and ZO-3 bound to the COOH-terminal YV sequence of claudin-1 to -8 through their PDZ1 domains in vitro. Then, claudin-1 or -2 was transfected into L fibroblasts, which express ZO-1 but not ZO-2 or ZO-3. Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited. When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1. Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions. In good agreement, PDZ1 and PDZ2 domains of ZO-1/ZO-2/ZO-3 were also recruited to claudin-based TJs, when introduced into cultured epithelial cells. The possible molecular architecture of TJ plaque structures is discussed.     

Distribution of Tight Junction Proteins in Adult Human Salivary Glands

Tight junctions (TJs) are an essential structure of fluid-secreting cells, such as those in salivary glands. Three major families of integral membrane proteins have been identified as components of the TJ: claudins, occludin, and junctional adhesion molecules (JAMs), plus the cytosolic protein zonula occludens (ZO). We have been working to develop an orally implantable artificial salivary gland that would be suitable for treating patients lacking salivary parenchymal tissue. To date, little is known about the distribution of TJ proteins in adult human salivary cells and thus what key molecular components might be desirable for the cellular component of an artificial salivary gland device. Therefore, the aim of this study was to determine the distribution of TJ proteins in human salivary glands. Salivary gland samples were obtained from 10 patients. Frozen and formalin-fixed paraffin-embedded sections were stained using IHC methods. Claudin-1 was expressed in ductal, endothelial, and ∼25% of serous cells. Claudins-2, -3, and -4 and JAM-A were expressed in both ductal and acinar cells, whereas claudin-5 was expressed only in endothelial cells. Occludin and ZO-1 were expressed in acinar, ductal, and endothelial cells. These results provide new information on TJ proteins in two major human salivary glands and should serve as a reference for future studies to assess the presence of appropriate TJ proteins in a tissue-engineered human salivary gland. (J Histochem Cytochem 56:1093–1098, 2008)     

Establishment and characterization of cultured epithelial cells lacking expression of ZO-1

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.     

Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis.     

Dynamics of tight and adherens junctions under EGTA treatment

The dynamics of tight junctions (TJs) and adherens junctions (AJs) under EGTA treatment were investigated in Madin Darby canine kidney (MDCK) cells. Detailed information about the behavior of TJ and AJ proteins during the opening and resealing of TJs and AJs is still scarce. By means of the "calcium chelation" method, the distribution and colocalization of junctional proteins were studied with confocal laser scanning microscopy using a deconvolution algorithm for high-resolution images. Colocalization was analyzed for pairs of the following proteins: ZO-1, occludin, claudin-1, E-cadherin and F-actin. Significant differences were found for the analyzed pairs in control cells compared to EGTA-treated cells with respect to the position of the colocalization maxima within the cell monolayers as well as with respect to the amount of colocalized voxels. Under EGTA treatment, colocalization for ZO-1/occludin, ZO-1/claudin-1, claudin-1/occludin, E-cadherin/occludin and E-cadherin/claudin-1 dropped below 35% of the control value. Only for the ZO-1/E-cadherin pair, the amount of colocalized voxels increased and a shift to a more basal position was observed. During the opening of TJs and AJs, ZO-1 colocalized with E-cadherin in the lateral membrane region, whereas in controls, ZO-1 colocalized with occludin and claudin-1 in the junctional complex. The combination of deconvolution with colocalization analysis of confocal data sets offers a powerful tool to investigate the spatial relationship of TJ and AJ proteins during assembly and disassembly of cell-cell contacts.     

Disruption of endothelial barrier function is linked with hyposecretion and lymphocytic infiltration in salivary glands of Sjögren's syndrome

Sjögren's syndrome (SS) is an inflammatory autoimmune disease that causes hyposecretion in salivary glands. Endothelial tight junctions (TJs) play crucial roles in salivation and barrier function of blood vessels. However, whether the alteration of endothelial TJs were involved in pathogenesis of SS was still unknown. Here, the ultrastructure and function of endothelial TJs in submandibular glands (SMGs) were detected by transmission electron microscopy and in vivo paracellular permeability assay in different aged NOD mouse model for SS. CFSE-labeled lymphocytes were injected into tail vein to trace the infiltration, while claudin-5 expression and distribution were detected by immunofluorescence, qRT-PCR, and western blot. Results showed that the stimulated salivary flow rate was gradually decreased and lymphocytic infiltration was found as age increased in 12- and 21-week-old NOD mice, but not 7-week-old NOD mice. Blood vessels were dilated, while endothelial TJ width and paracellular tracer transport were increased in 12-week-old NOD mice. Moreover, the injected CFSE-labeled lymphocytes were observed in SMGs of 12-week-old NOD mice. Claudin-5 level was increased and relocalized from the apical portion of neighboring endothelial cells to lateral membranes and cytoplasm in 12-week-old NOD mice. Additionally, the alteration of claudin-5 expression and distribution was further confirmed in labial salivary glands and bilateral parotid glands from SS patients. In cultured human microvessel endothelial cell line (HMEC-1), IFN-γ stimulation significantly increased claudin-5 expression. Taken together, we identified that the endothelial TJ barrier was disrupted and contributed to the development of salivary hyposecretion and lymphocytic infiltration in SS.     

Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin

ZO-2, a member of the MAGUK family, was thought to be specific for tight junctions (TJs) in contrast to ZO-1, another MAGUK family member, which is localized at TJs and adherens junctions (AJs) in epithelial and nonepithelial cells, respectively. Mouse ZO-2 cDNA was isolated, and a specific polyclonal antibody was generated using corresponding synthetic peptides as antigens. Immunofluorescence microscopy with this polyclonal antibody revealed that, similarly to ZO-1, in addition to TJs in epithelial cells, ZO-2 was also concentrated at AJs in nonepithelial cells such as fibroblasts and cardiac muscle cells lacking TJs. When NH2-terminal dlg-like and COOH-terminal non-dlg-like domains of ZO-2 (N-ZO-2 and C-ZO-2, respectively) were separately introduced into cultured cells, N-ZO-2 was colocalized with endogenous ZO-1/ZO-2, i.e. at TJs in epithelial cells and at AJs in non-epithelial cells, whereas C-ZO-2 was distributed along actin filaments. Consistently, occludin as well as alpha catenin directly bound to N-ZO-2 as well as the NH2-terminal dlg-like portion of ZO-1 (N-ZO-1) in vitro. Furthermore, immunoprecipitation experiments revealed that the second PDZ domain of ZO-2 was directly associated with N-ZO-1. These findings indicated that ZO-2 forms a complex with ZO-1/occludin or ZO-1/alpha catenin to establish TJ or AJ domains, respectively.     

Oncogenic Raf-1 disrupts epithelial tight junctions via downregulation of occludin

Occludin is an integral membrane protein of the epithelial cell tight junction (TJ). Its potential role in coordinating structural and functional events of TJ formation has been suggested recently. Using a rat salivary gland epithelial cell line (Pa-4) as a model system, we have demonstrated that occludin not only is a critical component of functional TJs but also controls the phenotypic changes associated with epithelium oncogenesis. Transfection of an oncogenic Raf-1 into Pa-4 cells resulted in a complete loss of TJ function and the acquisition of a stratified phenotype that lacked cell-cell contact growth control. The expression of occludin and claudin-1 was downregulated, and the distribution patterns of ZO-1 and E-cadherin were altered. Introduction of the human occludin gene into Raf-1-activated Pa-4 cells resulted in reacquisition of a monolayer phenotype and the formation of functionally intact TJs. In addition, the presence of exogenous occludin protein led to a recovery in claudin-1 protein level, relocation of the zonula occludens 1 protein (ZO-1) to the TJ, and redistribution of E-cadherin to the lateral membrane. Furthermore, the expression of occludin inhibited anchorage-independent growth of Raf-1-activated Pa-4 cells in soft agarose. Thus, occludin may act as a pivotal signaling molecule in oncogenic Raf- 1-induced disruption of TJs, and regulates phenotypic changes associated with epithelial cell transformation.     

Papillomavirus E6 oncoprotein up-regulates occludin and ZO-2 expression in ovariectomized mice epidermis

We have studied the expression of the tight junction proteins (TJ) occludin, claudin-1 and ZO-2 in the epidermis of female mice. We observed a peak of expression of these proteins at postnatal day 7 and a decrease in 6 week-old mice to values similar to those found in newborn animals. We explored if the expression of the E6 oncoprotein from high-risk human papilloma virus type 16 (HPV16) in the skin of transgenic female mice (K14E6), altered TJ protein expression in a manner sensitive to ovarian hormones. We observed that in ovariectomized mice E6 up-regulates the expression of occludin and ZO-2 in the epidermis and that this effect was canceled by 17β-estradiol. Progesterone instead induced occludin and ZO-2 over-expression. However, the decreased expression of occludin and ZO-2 induced by 17β-estradiol in the epidermis was not overturned by E6 or progesterone. In addition, we employed MDCK cells transfected with E6, and observed that ZO-2 delocalizes from TJs and accumulates in the cell nuclei due to a decrease in the turnover rate of the protein. These results reinforce the view of 17β-estradiol and E6 as risk factors for the development of cancer through effects on expression and mislocalization of TJ proteins.     

Disruption of epithelial tight junctions is prevented by cyclic nucleotide-dependent protein kinase inhibitors

Tight junctions (TJs), the most apical of the intercellular junctions, prevent the passage of ions and molecules through the paracellular pathway. Intracellular signalling molecules are likely to be involved in the regulation of TJ integrity. In order to specifically investigate the role of protein kinase A (PKA) in the maintenance of epithelial TJ integrity, calcium-switch experiments were performed, in which calcium was removed from EpH4 and MDCK culture medium, in the absence or presence of the PKA inhibitors H-89 or HA-1004. Removal of calcium from the culture media of the epithelial cells resulted in disruption of the TJs, characterised by a loss of membrane association of the TJ-associated proteins occludin, ZO-1 and ZO-2, by a loss of TJ strands, by a marked decrease in the transepithelial electrical resistance and by a dramatic increase in the transepithelial permeability to tracers. The association of occludin, ZO-1 and ZO-2 with the actin cytoskeleton is not affected. In contrast, when the removal of calcium was performed in the presence of either the PKA inhibitor H-89 or HA-1004, all barrier characteristics were preserved. Our data indicate that following the removal of calcium from the culture medium of epithelial cells in vitro, PKA is activated and subsequently is involved in the disruption of TJs.     

Zonula Occludens-1, Occludin and E-cadherin Expression and Organization in Salivary Glands with Sj?gren’s Syndrome

Sjögren’s syndrome (SS) is a chronic inflammatory autoimmune disorder that causes secretory dysfunction of the salivary glands leading to dry mouth. Previous studies reported that tight junction (TJ) proteins are down-regulated and lose polarity in human minor salivary glands with SS, suggesting that TJ structure is compromised in SS patients. In this paper, we utilized the NOD/ShiLtJ mouse with the main goal of evaluating this model for future TJ research. We found that the organization of apical proteins in areas proximal and distal to lymphocytic infiltration remained intact in mouse and human salivary glands with SS. These areas looked comparable to control glands (i.e., with no lymphocytic infiltration). TJ staining was absent in areas of lymphocytic infiltration coinciding with the loss of salivary epithelium. Gene expression studies show that most TJs are not significantly altered in 20-week-old NOD/ShiLtJ mice as compared with age-matched C57BL/6 controls. Protein expression studies revealed that the TJ proteins, zonula occludens-1 (ZO-1), occludin, claudin-12, as well as E-cadherin, do not significantly change in NOD/ShiLtJ mice. Our results suggest that ZO-1, occludin and E-cadherin are not altered in areas without lymphocytic infiltration. However, future studies will be necessary to test the functional aspect of these results.     

Changes in the distribution of ZO-1, occludin,and claudins in the rat uterine epithelium during the estrous cycle

During the estrous cycle, the endometrium epithelium experiences marked cellular structural changes. For fertilization to proceed, maintenance of an adequate uterine environment by ovarian hormones is essential. Epithelial cells lining the uterine lumen are associated with each other by tight junctions (TJs), which regulate the passage of ions and molecules through the paracellular pathway. The aim of the present study was to assess by confocal immunofluorescence the distribution pattern of the TJ proteins ZO-1, occludin, and claudins 1–7 in the rat uterus during the estrous cycle. Our results reveal that on proestrus, the day when mating takes place, ZO-1, occludin, and claudins 1 and 5 are located in the TJs, while claudins 3 and 7 display a basolateral distribution. In contrast, on metestrus day, when no sexual mating occurs and the uterine lumen is devoid of secretions, none of these proteins were detected in the TJ region, and only a diffuse cytosolic staining was observed for some of the proteins. On estrus and diestrus days, an intermediate situation was encountered, since ZO-1 localized in the TJs, whereas occludin was no longer detectable in the TJs. The distribution of claudins during these stages varied from the lowermost portion of the basolateral membrane to its apex. In conclusion, the results show that the protein composition of TJs present in the luminal epithelial cells of the uterus changes during the different days of the estrous cycle, and suggest that the expression of TJ proteins participates in providing an adequate environment for a successful fertilization.This work was supported by grants PAPIIT (IN210902, IX228504) and PAIP (6190-08) from the National Autonomous University of Mexico (UNAM), and by grants G34511-M and 37846-N from the Mexican National Council on Science and Technology (CONACYT).     

Small Synthetic Peptides Homologous to Segments of the First External Loop of Occludin Impair Tight Junction Resealing

This study shows that resealing of opened tight junctions (TJs) is impaired by interaction with oligopeptides homologous to the external domain of chick occludin. The experiments were carried out with confluent A6 cell monolayers grown on collagen supports under stable transepithelial electrical resistance (TER). The monolayers were bathed on the apical side with a 75 mm KCl solution and on the basolateral side by NaCl-Ringer's solution. TJ opening was induced by basolateral Ca²⁺ removal and was characterized by a marked drop of TER. The reintroduction of Ca²⁺ triggered junction resealing as indicated by an elevation of TER to control values. Custom-made peptides SNYYGSGLSY (corresponding to the residues 100 to 109) and SNYYGSGLS (residues 100 to 108), homologous to segments of the first external loop of chick occludin molecule, impaired junction resealing when the peptides were included in the apical bathing fluid (concentrations in the range of 0.5 to 1.5 mg/ml). Peptide removal from the apical solution usually triggered a slow recovery of TER, indicating a slow recovery of the TJ seal. Changes in localization of ZO-1, a cytoplasmic protein that underlies the membrane at the TJs, were evaluated immunocytochemically following Ca²⁺ removal and reintroduction. The presence or absence of the oligopeptides showed no influence on the pattern of change of ZO-1 localization. These observations support the hypothesis that the TJ seal results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, our results show that small peptides homologous to segments of the occludin first external loop can be used as specific reagents to manipulate the permeability of tight junctions. Received: 4 December 1998/Revised: 22 January 1999     

Effect of cell polarization on hepatitis C virus entry

The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.     

Activation of TLR2 enhances tight junction barrier in epidermal keratinocytes

The epidermis has developed physical and immunological barriers that prevent infiltration of deleterious chemicals and pathogens. As a first step to understanding the relationship between these barriers, we investigated whether TLR2 activation functionally alters tight junctions (TJs) in cultured human keratinocytes. Stimulation with peptidoglycan, a ligand for TLR2, elevated the TJ-associated barrier in the space of 3 h. The increase in TJ-associated barrier function due to peptidoglycan stimulation was suppressed by the knockdown of TLR adaptor MyD88 or the pretreatment with TLR2-neutralizing Ab, indicating that TLR2 activation enhanced TJ-associated barrier. One and 3 h after peptidoglycan stimulation, expression levels of the TJ proteins occludin, claudin-1, claudin-4, and ZO-1 were unchanged. However, immunoprecipitation studies demonstrated that the association of phospho-atypical protein kinase Cζ/ι, crucial for TJ biogenesis, with occludin was increased. Significantly, inhibition of atypical protein kinase Cζ/ι activity completely blocked the immediate elevation of the TJ-associated barrier. Finally, peptidoglycan was applied to the stratum corneum surface of a human skin equivalent, and the TJ barrier was evaluated. In the space of 3 h after the stimulation, the amount of intercellular tracer in the stratum corneum incubated from the dermal side was reduced, indicating that the TJ barrier is strengthened via TLR2 activation. Taken together, our findings indicated that infiltration of pathogens into the epidermis immediately enhanced TJ function via TLR2 signaling. Furthermore, the dynamically controlled TJs in skin are considered fundamental in preventing further invasion of pathogens and maintaining cutaneous barrier homeostasis.     

Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells

Infection of intestinal epithelial cells with enteropathogenic Escherichia coli (EPEC) disrupts tight junction (TJ) architecture and barrier function. The aim of this study was to determine the impact of EPEC on TJ protein interactions and localization. Human intestinal epithelial cells (T84) were infected for 1, 3 or 6 h with EPEC. To probe the TJ protein-protein interactions, co-immunoprecipitations were performed. The associations between ZO-1, occludin and claudin-1 progressively decreased after infection. Corresponding morphological changes were analysed by immunofluorescence confocal microscopy. Tight junction proteins progressively lost their apically restricted localization. Freeze-fracture electron microscopy revealed the appearance of aberrant strands throughout the lateral membrane that contained claudin-1 and occludin as determined by immunogold labelling. These structural alterations were accompanied by a loss of barrier function. Mutation of the gene encoding EspF, important in the disruption of TJs by EPEC, prevented the disruption of TJs. Tight junction structure normalized following eradication of EPEC with gentamicin and overnight recovery. This is the first demonstration that a microbial pathogen can cause aberrant TJ strands in the lateral membrane of host cells. We speculate that the disruption of integral and cytoplasmic TJ protein interactions following EPEC infection allows TJ strands to form or diffuse into the lateral plasma membrane.