Requirement of ZO-1 for the formation of belt-like adherens junctions during epithelial cell polarization

The molecular mechanisms of how primordial adherens junctions (AJs) evolve into spatially separated belt-like AJs and tight junctions (TJs) during epithelial polarization are not well understood. Previously, we reported the establishment of ZO-1/ZO-2-deficient cultured epithelial cells (1[ko]/2[kd] cells), which lacked TJs completely. In the present study, we found that the formation of belt-like AJs was significantly delayed in 1(ko)/2(kd) cells during epithelial polarization. The activation of Rac1 upon primordial AJ formation is severely impaired in 1(ko)/2(kd) cells. Our data indicate that ZO-1 plays crucial roles not only in TJ formation, but also in the conversion from "fibroblastic" AJs to belt-like "polarized epithelial" AJs through Rac1 activation. Furthermore, to examine whether ZO-1 itself mediate belt-like AJ and TJ formation, respectively, we performed a mutational analysis of ZO-1. The requirement for ZO-1 differs between belt-like AJ and TJ formation. We propose that ZO-1 is directly involved in the establishment of two distinct junctional domains, belt-like AJs and TJs, during epithelial polarization.     

Involvement of the Interaction of Afadin with ZO-1 in the Formation of Tight Junctions in Madin-Darby Canine Kidney Cells

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with β- and α-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-ΔPR1–2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.     

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)     

Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment

The adherens junction (AJ) and tight junction (TJ) are key regulators of epithelial polarity and barrier function. Loss of epithelial phenotype is accompanied by endocytosis of AJs and TJs via unknown mechanisms. Using a model of calcium depletion, we defined the pathway of internalization of AJ and TJ proteins (E-cadherin, p120 and beta-catenins, occludin, JAM-1, claudins 1 and 4, and ZO-1) in T84 epithelial cells. Proteinase protection assay and immunocytochemistry revealed orchestrated internalization of AJs and TJs into a subapical cytoplasmic compartment. Disruption of caveolae/lipid rafts did not prevent endocytosis, nor did caveolin-1 colocalize with internalized junctional proteins. Furthermore, AJ and TJ proteins did not colocalize with the macropinocytosis marker dextran. Inhibitors of clathrin-mediated endocytosis blocked internalization of AJs and TJs, and junctional proteins colocalized with clathrin and alpha-adaptin. AJ and TJ proteins were observed to enter early endosomes followed by movement to organelles that stained with syntaxin-4 but not with markers of late and recycling endosomes, lysosomes, or Golgi. These results indicate that endocytosis of junctional proteins is a clathrin-mediated process leading into a unique storage compartment. Such mechanisms may mediate the disruption of intercellular contacts during normal tissue remodeling and in pathology.     

Differential behavior of E-cadherin and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity

At the initial stage of cell-cell contact of epithelial cells, primordial spot-like junctions are formed at the tips of thin cellular protrusions radiating from adjacent cells, where E-cadherin and ZO-1 are precisely coconcentrated (Yonemura et al., 1995, J. Cell Sci. 108:127-142). In fully polarized epithelial cells, E-cadherin and ZO-1 are completely sorted into belt-like adherens junctions (AJ) and tight junctions (TJ), respectively. Here we examined the behavior of occludin, an integral membrane protein consisting of TJ, during the establishment of epithelial cell polarity. Using confocal immunofluorescence microscopy, we quantitatively compared the spatial relationship of occludin/ZO-1 with that of E-cadherin/ZO-1 during epithelial cellular polarization by replating or wounding cultured mouse epithelial cells (MTD1-A). At the initial stage of cell-cell contact, E-cadherin and ZO-1 appeared to be simultaneously recruited to the primordial form of spot-like junctions at the tips of cellular processes which showed no concentration of occludin. Then, as cellular polarization proceeded, occludin was gradually accumulated at the ZO-1-positive spot-like junctions to form belt-like TJ, and in a complementary manner E-cadherin was sorted out from the ZO-1-positive spot-like junctions to form belt-like AJ. The molecular mechanism of TJ/AJ formation during epithelial cellular polarization is discussed with special reference to the roles of ZO-1.     

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.     

Identification of a novel protein, LYRIC, localized to tight junctions of polarized epithelial cells

Tight junctions (TJ) are multiprotein complexes that function to regulate paracellular transport of molecules through epithelial and endothelial cell layers. Many new tight junction-associated proteins have been identified in the past few years, and their functional roles and interactions have just begun to be elucidated. In this paper, we describe a novel protein LYsine-RIch CEACAM1 co-isolated (LYRIC) that is widely expressed and highly conserved between species. LYRIC has no conserved domains that would indicate function and does not appear to be a member of a larger protein family. Data from analysis of rat and human tissue sections and cell lines show that LYRIC colocalizes with tight junction proteins ZO-1 and occludin in polarized epithelial cells, suggesting that LYRIC is part of the tight junction complex. LYRIC dissociates from ZO-1 when junctional complexes are disrupted, and as tight junctions reform, ZO-1 relocalizes before LYRIC. These results suggest that LYRIC is most likely not a structural component required for TJ formation, but rather is recruited during the maturation of the tight junction complex.     

Zonula occludens-1 and -2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized epithelia

The structure and function of both adherens (AJ) and tight (TJ) junctions are dependent on the cortical actin cytoskeleton. The zonula occludens (ZO)-1 and -2 proteins have context-dependent interactions with both junction types and bind directly to F-actin and other cytoskeletal proteins, suggesting ZO-1 and -2 might regulate cytoskeletal activity at cell junctions. To address this hypothesis, we generated stable Madin-Darby canine kidney cell lines depleted of both ZO-1 and -2. Both paracellular permeability and the localization of TJ proteins are disrupted in ZO-1/-2-depleted cells. In addition, immunocytochemistry and electron microscopy revealed a significant expansion of the perijunctional actomyosin ring associated with the AJ. These structural changes are accompanied by a recruitment of 1-phosphomyosin light chain and Rho kinase 1, contraction of the actomyosin ring, and expansion of the apical domain. Despite these changes in the apical cytoskeleton, there are no detectable changes in cell polarity, localization of AJ proteins, or the organization of the basal and lateral actin cytoskeleton. We conclude that ZO proteins are required not only for TJ assembly but also for regulating the organization and functional activity of the apical cytoskeleton, particularly the perijunctional actomyosin ring, and we speculate that these activities are relevant both to cellular organization and epithelial morphogenesis.     

Evidence for a functional interaction between cingulin and ZO-1 in cultured cells

Cingulin, a protein component of the submembrane plaque of tight junctions (TJ), contains globular and coiled-coil domains and interacts in vitro with several TJ and cytoskeletal proteins, including the PDZ protein ZO-1. Overexpression of Xenopus cingulin in transfected Xenopus A6 cells resulted in the disruption of endogenous ZO-1 localization, suggesting that cingulin functionally interacts with ZO-1. Glutathione S-transferase pull-down experiments showed that a conserved ZO-1 interaction motif (ZIM) at the NH(2) terminus of cingulin is required for cingulin-ZO-1 interaction in vitro. An NH(2)-terminal region of cingulin, containing the ZIM, was sufficient, when fused to coiled-coil sequences, to target transfected cingulin to junctions. However, deletion of the ZIM did not abolish junctional localization of transfected cingulin in A6 cells, suggesting that cingulin can be recruited to TJ through multiple protein interactions. Interestingly, the ZIM was required for cingulin recruitment into ZO-1-containing adherens junctions of Rat-1 fibroblasts, indicating that cingulin junctional recruitment does not require the molecular context of TJ. Cingulin coiled-coil sequences enhanced the junctional accumulation of expressed cingulin head region in A6 cells, but purified recombinant cingulin did not form filaments under physiological conditions in vitro, suggesting that the cingulin coiled-coil domain acts primarily by promoting dimerization.     

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.     

Adaptors, junction dynamics, and spermatogenesis

Adaptors are component proteins of junctional complexes in all epithelia, including the seminiferous epithelium of the mammalian testis. They recruit other regulatory and structural proteins to the site of both anchoring junctions (such as cell-cell actin-based adherens junctions [AJs], e.g., ectoplasmic specialization [ES] and tubulobulbar complex, which are both testis-specific cell-cell actin-based AJ types, and cell-cell intermediate filament-based desmosome-like junctions) and tight junctions (TJ). Furthermore, adaptors per se can be substrates and/or activators of kinases or phosphatases. As such, the integrity of cell junctions and the regulation of junction dynamics during spermatogenesis rely on adaptors for their ability to recruit and link different junctional components to the same site and to tether transmembrane proteins at both anchoring and TJ sites to the underlying cytoskeletons, such as the actin filaments, intermediate filaments, and microtubules. These protein-protein interactions are possible because adaptors are composed of conserved protein binding domains, which allow them to link to more than one structural or signaling protein, recruiting multi-protein complexes to the same site. Herein, we provide a timely review of adaptors recently found at the sites of AJ (e.g., ES) and TJ. In addition, several in vivo models that can be used to delineate the function of adaptors in the testis are described, and the role of adaptors in regulating junction dynamics pertinent to spermatogenesis is discussed.     

Association between adherens junctions and tight junctions via Rap1 promotes barrier protective effects of oxidized phospholipids

Previous studies showed that cyclopenthenone-containing products resulting from oxidation of a natural phospholipid, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibit potent barrier-protective effects in the in vitro and in vivo models of lung endothelial cell (EC) barrier dysfunction, and these effects are associated with enhancement of peripheral actin cytoskeleton, cell-cell and cell-substrate contacts driven by activation of Rac and Cdc42 GTPases. Rap1 GTPase is another member of small GTPase family involved in control of cell-cell interactions; however, its involvement in EC barrier-protective effects by OxPAPC remains unknown. This study examined a role of Rap1 in regulation of OxPAPC-induced interactions in adherens junctions (AJ) and tight junctions (TJ) as a novel mechanism of EC barrier preservation in vitro and in vivo. Immunofluorescence analysis, subcellular fractionation, and co-immunoprecipitation assays indicate that OxPAPC promoted accumulation of AJ proteins: VE-cadherin, p120-catenin, and β-catenin; and TJ proteins: ZO-1, occludin, and JAM-A in the cell membrane, and induced novel cross-interactions between AJ and TJ protein complexes, that were dependent on OxPAPC-induced Rap1 activation. Inhibition of Rap1 function suppressed OxPAPC-mediated pulmonary EC barrier enhancement and AJ and TJ interactions in vitro, as well as inhibited protective effects of OxPAPC against ventilator-induced lung injury in vivo. These results show for the first time a role of Rap1-mediated association between adherens junctions and tight junction complexes in the OxPAPC-induced pulmonary vascular EC barrier protection.     

RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin

Epithelial intercellular junctions regulate cell-cell contact and mucosal barrier function. Both tight junctions (TJs) and adherens junctions (AJs) are regulated in part by their affiliation with the F-actin cytoskeleton. The cytoskeleton in turn is influenced by Rho family small GTPases such as RhoA, Rac1, and Cdc42, all of which constitute eukaryotic targets for several pathogenic organisms. With a tetracycline-repressible system to achieve regulated expression in Madin-Darby canine kidney (MDCK) epithelial cells, we used dominant-negative (DN) and constitutively active (CA) forms of RhoA, Rac1, and Cdc42 as tools to evaluate the precise contribution of each GTPase to epithelial structure and barrier function. All mutant GTPases induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. TJ proteins occludin, ZO-1, claudin-1, claudin-2, and junctional adhesion molecule (JAM)-1 were dramatically redistributed in the presence of CA RhoA or CA Cdc42, whereas only claudins-1 and -2 were redistributed in response to CA Rac1. DN Rac1 expression also induced selective redistribution of claudins-1 and -2 in addition to JAM-1, whereas DN Cdc42 influenced only claudin-2 and DN RhoA had no effect. AJ protein localization was unaffected by any mutant GTPase, but DN Rac1 induced a reduction in E-cadherin detergent solubility. All CA GTPases increased the detergent solubility of claudins-1 and -2, but CA RhoA alone reduced claudin-2 and ZO-1 partitioning to detergent-insoluble membrane rafts. We conclude that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms and that perturbations in barrier function reflect any imbalance in active/resting GTPase levels rather than simply loss or gain of GTPase activity. epithelium; tight junctions; paracellular permeability; Madin-Darby canine kidney cells     

Larazotide acetate promotes tight junction assembly in epithelial cells

Tight junctions (TJ) control paracellular permeability and apical-basolateral polarity of epithelial cells. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. TJ formation is dependent on E-cadherin-mediated cell-cell adhesion and actin rearrangement, and is regulated by the Rho family GTPase and aPKC signaling pathways. Larazotide acetate, an 8-mer peptide and TJ modulator, inhibits TJ disassembly and dysfunction caused by endogenous and exogenous stimuli in intestinal epithelial cells. Here, we examined the effect of larazotide acetate on de novo TJ assembly using 2 different model systems. In MDCK cells, larazotide acetate promoted TJ assembly in a calcium switch assay. Larazotide acetate also promoted actin rearrangement, and junctional distribution of zonula occludens-1 (ZO-1), occludin, claudins, and E-cadherin. Larazotide acetate promoted TJ maturation and decreased paracellular permeability in "leaky" Caco-2 cells. Taken together, our data indicate that larazotide acetate enhances TJ assembly and barrier function by promoting actin rearrangement and redistribution of TJ and AJ proteins.     

C-Jun N-Terminal kinase mediates disassembly of apical junctions in model intestinal epithelia

Dynamic remodeling of intercellular junctions is a critical determinant of epithelial barrier function in both physiological and pathophysiological states. While the disassembly of epithelial tight junctions (TJ) and adherens junctions (AJ) has been well-described in response to pathogens and other external stressors, the role of stress-related signaling in TJ/AJ regulation remains poorly understood. The aim of this study was to define the role of stress-activated c-Jun N-terminal kinase (JNK) in disruption of intercellular junctions in model intestinal epithelia. We show that rapid AJ/TJ disassembly triggered by extracellular calcium depletion of T84 and SK-CO15 cell monolayers was accompanied by activation (phosphorylation) of JNK, and prevented by pharmacological inhibitors of JNK. The opposite process, TJ/AJ reassembly, was accelerated by JNK inhibition and suppressed by the JNK activator anisomycin. JNK1 but not JNK2 was found to colocalize with intercellular junctions, and siRNA-mediated down-regulation of JNK1 attenuated the TJ/AJ disruption caused by calcium depletion. JNK inhibition also blocked formation of characteristic contractile F-actin rings in calcium-depleted epithelial cells, suggesting that JNK regulates junctions by remodeling the actin cytoskeleton. In this role JNK acts downstream of the actin-reorganizing Rho-dependent kinase (ROCK), since ROCK inhibition abrogated JNK phosphorylation and TJ/AJ disassembly after calcium depletion. Furthermore, JNK acts upstream of F-actin-membrane linker proteins of the ERM (ezrin-radixin-moesin) family, but in a complex relationship yet to be fully elucidated. Taken together, our findings suggest a novel role for JNK in the signaling pathway that links ROCK and F-actin remodeling during disassembly of epithelial junctions.     

Glutamine regulates Caco-2 cell tight junction proteins

Intestinal epithelial tight junction (TJ) barrier dysfunction may lead to inflammation and mucosal injury. Glutamine (GLN) plays a role in maintenance of intestinal barrier function in various animal models and critically ill humans. Recent evidence from intestinal cell monolayers indicates that GLN maintains transepithelial resistance and decreases permeability. The mechanisms of these effects remain undefined. We hypothesized that GLN affects proteins involved in the intercellular junctional complex. GLN availability was controlled in Caco-2 monolayers by addition to the medium and treatment with methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Expression of TJ proteins, claudin-1, occludin, and zonula occluden (ZO)-1 was measured by immunoblotting. Localization of TJ proteins was evaluated by immunofluorescence light microscopy. Structure of TJ was determined by transmission electron microscopy (TEM). Deprivation of GLN decreased claudin-1, occludin, and ZO-1 protein expression and caused a disappearance of perijunctional claudin-1 and a reduction of occludin but had no effect on ZO-1. TEM revealed that MSO-treated cells in the absence of GLN formed irregular junctional complexes between the apical lateral margins of adjoining cells. These findings indicate that TJ protein expression and cellular localization in Caco-2 cell monolayers rely on GLN. This mechanism may similarly relate to GLN-mediated modulation of intestinal barrier function in stressed animals and humans.     

Dual roles of tight junction-associated protein, zonula occludens-1, in sphingosine 1-phosphate-mediated endothelial chemotaxis and barrier integrity

In this report, sphingosine-1-phosphate (S1P), a serum-borne bioactive lipid, is shown to activate tight-junction-associated protein Zonula Occludens-1 (ZO-1), which in turn plays a critical role in regulating endothelial chemotaxis and barrier integrity. After S1P stimulation, ZO-1 was redistributed to the lamellipodia and cell-cell junctions via the S1P1/G(i)/Akt/Rac pathway. Similarly, both endothelial barrier integrity and cell motility were significantly enhanced in S1P-treated cells through the G(i)/Akt/Rac pathway. Importantly, S1P-enhanced barrier integrity and cell migration were abrogated in ZO-1 knockdown cells, indicating ZO-1 is functionally indispensable for these processes. To investigate the underlying mechanisms, we demonstrated that cortactin plays a critical role in S1P-induced ZO-1 redistribution to the lamellipodia. In addition, S1P significantly induced the formation of endothelial tight junctions. ZO-1 and alpha-catenin polypeptides were colocalized in S1P-induced junctional structures; whereas, cortactin was not observed in these regions. Together, these results suggest that S1P induces the formation of two distinct ZO-1 complexes to regulate two different endothelial functions: ZO-1/cortactin complexes to regulate chemotactic response and ZO-1/alpha-catenin complexes to regulate endothelial barrier integrity. The concerted operation of these two ZO-1 complexes may coordinate two important S1P-mediated functions, i.e. migration and barrier integrity, in vascular endothelial cells.