Tight Junction Dynamics in the Frog Urinary Bladder

In a previous study in frog skin (Castro et al., J. Memb. Biol. 134:15-29, 1993), it was shown that TJs experimentally disrupted by a selective deposition of BaSO₄ could be re-sealed upon addition of Ca²⁺to the apical solution; in the absence of apical Ca²⁺, the normal Ca²⁺ activity of the Na₂SO₄-Ringer's bathing the basolateral side was not able to induce TJ resealing. We now show that apical Ca²⁺also activates the TJ sealing mechanism in frog urinary bladders. Three known procedures were utilized to increase TJ permeability, all in the absence of apical Ca²⁺: (i) exposure to high positive transepithelial clamping potentials; (ii) exposure of the apical surface to hypertonic solutions; and (iii) selective deposition of BaSO₄ in the TJs. The resealing of the TJs was promoted by raising the concentration of Ca²⁺ in the apical solution. This effect of Ca²⁺ is not impaired by the presence of Ca²⁺ channel blockers (nifedipine, verapamil, Mn²⁺ or Cd²⁺) in the apical solution, indicating that junction resealing does not depend on Ca²⁺ entering the cells through the apical membrane. TJ resealing that occurs in response to raised apical Ca²⁺ most likely results from a direct effect of Ca²⁺, entering the disrupted TJs from the apical solution and reaching the zonula adhaerens Ca²⁺ receptors (E-cadherins). Protein kinase C (PKC) must play a significant role in the control of TJ assembly in this tight epithelia since the PKC inhibitor (H7) and the activator (diC8) markedly affect TJ recovery after disruption by apical hypertonicity. H7 treated tissues show marked recuperation of conductance even in the absence of apical Ca²⁺. In contrast, diC8 prevents tissue recuperation which normally occurs after addition of Ca²⁺ to the apical solution.     

Hypoglycosylated E-cadherin promotes the assembly of tight junctions through the recruitment of PP2A to adherens junctions

Epithelial cell-cell adhesion is controlled by multiprotein complexes that include E-cadherin-mediated adherens junctions (AJs) and ZO-1-containing tight junctions (TJs). Previously, we reported that reduction of E-cadherin N-glycosylation in normal and cancer cells promoted stabilization of AJs through changes in the composition and cytoskeletal association of E-cadherin scaffolds. Here, we show that enhanced interaction of hypoglycosylated E-cadherin-containing AJs with protein phosphatase 2A (PP2A) represents a mechanism for promoting TJ assembly. In MDCK cells, attenuation of cellular N-glycosylation with siRNA to DPAGT1, the first gene in the N-glycosylation pathway, reduced N-glycosylation of surface E-cadherin and resulted in increased recruitment of stabilizing proteins γ-catenin, α-catenin, vinculin and PP2A to AJs. Greater association of PP2A with AJs correlated with diminished binding of PP2A to ZO-1 and claudin-1 and with increased pools of serine-phosphorylated ZO-1 and claudin-1. More ZO-1 was found in complexes with occludin and claudin-1, and this corresponded to enhanced transepithelial resistance (TER), indicating physiological assembly of TJs. Similar maturation of AJs and TJs was detected after transfection of MDCK cells with the hypoglycosylated E-cadherin variant, V13. Our data indicate that E-cadherin N-glycans coordinate the maturity of AJs with the assembly of TJs by affecting the association of PP2A with these junctional complexes.     

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.     

Tight Junction Dynamics in the Frog Urinary Bladder

In a previous study in frog skin (Castro et al., J. Memb. Biol. 134:15–29, 1993), it was shown that TJs experimentally disrupted by a selective deposition of BaSO₄ could be re-sealed upon addition of Ca²⁺to the apical solution; in the absence of apical Ca²⁺, the normal Ca²⁺ activity of the Na₂SO₄-Ringer's bathing the basolateral side was not able to induce TJ resealing. We now show that apical Ca²⁺also activates the TJ sealing mechanism in frog urinary bladders. Three known procedures were utilized to increase TJ permeability, all in the absence of apical Ca²⁺: (i) exposure to high positive transepithelial clamping potentials; (ii) exposure of the apical surface to hypertonic solutions; and (iii) selective deposition of BaSO₄ in the TJs. The resealing of the TJs was promoted by raising the concentration of Ca²⁺ in the apical solution. This effect of Ca²⁺ is not impaired by the presence of Ca²⁺ channel blockers (nifedipine, verapamil, Mn²⁺ or Cd²⁺) in the apical solution, indicating that junction resealing does not depend on Ca²⁺ entering the cells through the apical membrane. TJ resealing that occurs in response to raised apical Ca²⁺ most likely results from a direct effect of Ca²⁺, entering the disrupted TJs from the apical solution and reaching the zonula adhaerens Ca²⁺ receptors (E-cadherins). Protein kinase C (PKC) must play a significant role in the control of TJ assembly in this tight epithelia since the PKC inhibitor (H7) and the activator (diC8) markedly affect TJ recovery after disruption by apical hypertonicity. H7 treated tissues show marked recuperation of conductance even in the absence of apical Ca²⁺. In contrast, diC8 prevents tissue recuperation which normally occurs after addition of Ca²⁺ to the apical solution.     

Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABalphaC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.     

The interaction of JRAB/MICAL-L2 with Rab8 and Rab13 coordinates the assembly of tight junctions and adherens junctions

The assembly of tight junctions (TJs) and adherens junctions (AJs) is regulated by the transport of integral TJ and AJ proteins to and/or from the plasma membrane (PM) and it is tightly coordinated in epithelial cells. We previously reported that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) mediated the endocytic recycling of an integral TJ protein occludin and the formation of functional TJs. Here, we investigated the role of Rab13 and JRAB/MICAL-L2 in the transport of other integral TJ and AJ proteins claudin-1 and E-cadherin to the PM by using a Ca(2+)-switch model. Although knockdown of Rab13 specifically suppressed claudin-1 and occludin but not E-cadherin transport, knockdown of JRAB/MICAL-L2 and expression of its Rab13-binding domain (JRAB/MICAL-L2-C) inhibited claudin-1, occludin, and E-cadherin transport. We then identified Rab8 as another JRAB/MICAL-L2-C-binding protein. Knockdown of Rab8 inhibited the Rab13-independent transport of E-cadherin to the PM. Rab8 and Rab13 competed with each other for the binding to JRAB/MICAL-L2 and functionally associated with JRAB/MICAL-L2 at the perinuclear recycling/storage compartments and PM, respectively. These results suggest that the interaction of JRAB/MICAL-L2 with Rab8 and Rab13 coordinates the assembly of AJs and TJs.     

Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction

We have investigated the mechanisms by which the epithelial apicolateral junctional complex (AJC) is generated during trophectoderm differentiation in the mouse blastocyst using molecular, structural and functional analyses. The mature AJC comprises an apical tight junction (TJ), responsible for intercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membrane contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single 'intermediate' complex formed following embryo compaction at the 8-cell stage in which the TJ-associated peripheral membrane protein, ZO-1alpha- isoform, was co-localized with both alpha- and beta-catenin. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1alpha- and other TJ proteins (ZO-1alpha+ isoform, occludin, cingulin) co-localized in an apical TJ which was separate from a subjacent E-cadherin/catenin zonula adherens complex. Thin-section electron microscopy confirmed that a single zonula adherens-like junctional complex present at the AJC site following compaction matured into a dual TJ and zonula adherens complex at the blastocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed that a functional TJ seal was established coincident with blastocoel formation. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates from the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1alpha- co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm differentiation and likely has functional importance in blastocyst formation. Moreover, we propose a role for rab13 in the specification of the AJC site and the formation and segregation of the TJ.     

Assembly of epithelial tight junctions is negatively regulated by Par6

Epithelial cells display apical-basal polarity, and the apical surface is segregated from the basolateral membranes by a barrier called the tight junction (TJ). TJs are constructed from transmembrane proteins that form cell-cell contacts-claudins, occludin, and junctional adhesion molecule (JAM)-plus peripheral proteins such as ZO-1. The Par proteins (partitioning-defective) Par3 and Par6, plus atypical protein kinase C (aPKC) function in the formation or maintenance of TJs and more generally in metazoan cell polarity establishment. Par6 contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain and binds the small GTPase Cdc42. Here, we show that Par6 inhibits TJ assembly in MDCK II epithelial cells after their disruption by Ca(2+) depletion but does not inhibit adherens junction (AJ) formation. Transepithelial resistance and paracellular diffusion assays confirmed that assembly of functional TJs is delayed by Par6 overexpression. Strikingly, the isolated, N-terminal fragment of PKCzeta, which binds Par6, also inhibits TJ assembly. Activated Cdc42 can disrupt TJs, but neither a dominant-negative Cdc42 mutant nor the CRIB domain of gammaPAK (p21-activated kinase), which inhibits Cdc42 function, observably inhibit TJ formation. These results suggest that Cdc42 and Par6 negatively regulate TJ assembly in mammalian epithelial cells.     

Tight junction biogenesis in the early Xenopus embryo

Tight junctions (TJs) perform a critical role in the transport functions and morphogenetic activity of the primary epithelium formed during Xenopus cleavage. Biogenesis of these junctions was studied by immunolocalization of TJ-associated proteins (cingulin, ZO-1 and occludin) and by an in vivo biotin diffusion assay. Using fertilized eggs synchronized during the first division cycle, we found that membrane assembly of the TJ initiated at the animal pole towards the end of zygote cytokinesis and involved sequential incorporation of components in the order cingulin, ZO-1 and occludin. The three constituents appeared to be recruited from maternal stores and were targeted to the nascent TJ site by different pathways. TJ protein assembly was focused precisely to the border between the oolemma-derived apical membrane and newly-inserted basolateral membrane generated during cytokinesis and culminated in the formation of functional TJs in the two-cell embryo, which maintained a diffusion barrier. New membrane formation and the generation of cell surface polarity therefore precede initiation of TJ formation. Moreover, assembly of TJ marker protein precisely at the apical-basolateral membrane boundary was preserved in the complete absence of intercellular contacts and adhesion. Thus, the mechanism of TJ biogenesis in the Xenopus early embryo relies on intrinsic cues of a cell autonomous mechanism. These data reveal a distinction between Xenopus and mammalian early embryos in the origin and mechanisms of epithelial cell polarization and TJ formation during cleavage of the egg.     

Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions

We characterized the role of the E-cadherin adhesion system in the formation of epithelial tight junctions using the calcium switch model. In MDCK cells cultured in low (micromolar) calcium levels, the tight junctional protein Zonula Occludens-1 (ZO-1) is distributed intracellularly in granular clusters, the larger of which codistribute with E-cadherin. Two hours after activation of E-cadherin adhesion by transfer to normal (1.8 mM) calcium levels, ZO-1 dramatically redistributed to the cell surface, where it localized in regions rich in E-cadherin. Immunoprecipitation with ZO-1 antibodies of extracts from cells kept in low calcium and 2 h after shifting to 1.8 mM Ca2+ demonstrated the association of ZO-1 with alpha-, beta-, and gamma- catenins. E-cadherin was not detected in the ZO-1 immunoprecipitates but it was found in beta-catenin immunoprecipitates that excluded ZO-1, suggesting that the binding of ZO-1 to catenins may weaken the interaction of these proteins with E-cadherin. Immunofluorescence and immunoelectron microscopy confirmed a close association of beta-catenin and ZO-1 at 0 and 2 h after Ca2+ switch. 48 h after Ca2+ switch, upon complete polarization of the epithelium, most of the ZO-1 had segregated from lateral E-cadherin and formed a distinct, separate apical ring. The ZO-1-catenin complex was not detected in fully polarized monolayers. MDCK cells permanently transformed with Moloney sarcoma virus, which expresses low levels of E-cadherin, displayed clusters of cytoplasmic ZO-1 granules and very little of this protein at the cell surface. Upon transfection with E-cadherin into Moloney sarcoma virus-MDCK cells, ZO-1 redistributed to E-cadherin-rich lateral plasma membrane but later failed to segregate into mature tight junctions. Our experiments suggest that catenins participate in the mobilization of ZO-1 from the cytosol to the cell surface early in the development of tight junctions and that neoplastic transformation may block the formation of tight junctions, either by decreasing the levels of E-cadherin or by preventing a late event: the segregation of tight junction from the zonula adherens.     

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.     

Relative contribution of cell contact pattern, specific PKC isoforms and gap junctional communication in tight junction assembly in the mouse early embryo

In mouse early development, cell contact patterns regulate the spatial organization and segregation of inner cell mass (ICM) and trophectoderm epithelium (TE) during blastocyst morphogenesis. Progressive membrane assembly of tight junctional (TJ) proteins in the differentiating TE during cleavage is upregulated by cell contact asymmetry (outside position) and suppressed within the ICM by cell contact symmetry (inside position). This is reversible, and immunosurgical isolation of the ICM induces upregulation of TJ assembly in a sequence that broadly mimics that occurring during blastocyst formation. The mechanism relating cell contact pattern and TJ assembly was investigated in the ICM model with respect to PKC-mediated signaling and gap junctional communication. Our results indicate that complete cell contact asymmetry is required for TJ biogenesis and acts upstream of PKC-mediated signaling. Specific inhibition of two PKC isoforms, PKCdelta and zeta, revealed that both PKC activities are required for membrane assembly of ZO-2 TJ protein, while only PKCzeta activity is involved in regulating ZO-1alpha+ membrane assembly, suggesting different mechanisms for individual TJ proteins. Gap junctional communication had no apparent influence on either TJ formation or PKC signaling but was itself affected by changes of cell contact patterns. Our data suggest that the dynamics of cell contact patterns coordinate the spatial organization of TJ formation via specific PKC signaling pathways during blastocyst biogenesis.     

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.     

Formation of tight junction strands by expression of claudin-1 mutants in their ZO-1 binding site in MDCK cells

To investigate the formation mechanism of tight junctions (TJs), we constructed three claudin-1 mutants which varied in their COOH-termini and expressed them in MDCK cells under the control of doxycycline. The differences between these constructs are that a putative ZO-1 binding sequence (KDYV) at the COOH-terminus of claudin-1 was deleted (DeltaCmyc) or present (1CLmyc and DeltaCmycYV), or that a myc-epitope was added at the COOH-terminus (1CLmyc and DeltaCmyc) or inserted just before the KDYV sequence (DeltaCmycYV). All three constructs caused the formation of aberrant TJ strands along the lateral plasma membranes. However, when their expression levels were reduced by adding 0.2 ng/ml doxycycline, they were located at apical TJs and colocalized with ZO-1, even in the KDYV-deleted construct. These results suggest that, although the addition of the myc-epitope at or near the COOH-terminus of claudin-1 interfered with the binding to ZO-1 and induced aberrant TJ strand formation, endogenous claudins which could bind to ZO-1 might recruit these deformed claudin-1s expressed at a low level to apical TJs. A calcium switch assay revealed that claudin-1 was transported to cadherin-based cell-cell contacts where ZO-1 had already accumulated, and was then concentrated at apical TJs together with ZO-1. Crosslinking between claudin-1 and the perijunctional actin ring through ZO-1 may be necessary for TJ strands to be localized or retained at apical TJs.     

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.     

A novel role for integrin-linked kinase in epithelial sheet morphogenesis

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca(2+) triggers formation of cell-cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca(2+) treatment of keratinocytes induces rapid (6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell-cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/beta-catenin to cell borders, precluding Ca(2+)-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell-cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell-cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.     

Multi-PDZ domain protein 1 (MUPP1) is concentrated at tight junctions through its possible interaction with claudin-1 and junctional adhesion molecule.

Claudins, most of which end in valine at their COOH termini, constitute tight junction (TJ) strands, suggesting that TJ strands strongly attract PDZ-containing proteins. Indeed, ZO-1, -2, and -3, each of which contains three PDZ domains, were shown to directly bind to claudins. Using the yeast two-hybrid system, we identified ZO-1 and MUPP1 (multi-PDZ domain protein 1) as binding partners for the COOH terminus of claudin-1. MUPP1 has been identified as a protein that contains 13 PDZ domains, but it has not been well characterized. In vitro binding assays with recombinant MUPP1 confirmed the interaction between MUPP1 and claudin-1 and identified PDZ10 as the responsible domain for this interaction. A polyclonal antibody specific for MUPP1 was then generated. Immunofluorescence confocal microscopy as well as immunoelectron microscopy with this antibody revealed that in polarized epithelial cells MUPP1 was exclusively concentrated at TJs. Furthermore, in vitro binding and transfection experiments showed that junctional adhesion molecule, another TJ adhesion molecule, also bound to the PDZ9 domain of MUPP1. These findings suggested that MUPP1 is concentrated at TJs in epithelial cells through its binding to claudin and junctional adhesion molecule and that it may function as a multivalent scaffold protein that recruits various proteins to TJs.     

Normal establishment of epithelial tight junctions in mice and cultured cells lacking expression of ZO-3, a tight-junction MAGUK protein

ZO-1, ZO-2, and ZO-3 are closely related MAGUK family proteins that localize at the cytoplasmic surface of tight junctions (TJs). ZO-1 and ZO-2 are expressed in both epithelia and endothelia, whereas ZO-3 is exclusively expressed in epithelia. In spite of intensive studies of these TJ MAGUKs, our knowledge of their functions in vivo, especially those of ZO-3, is still fragmentary. Here, we have generated mice, as well as F9 teratocarcinoma cell lines, that do not express ZO-3 by homologous recombination. Unexpectedly, ZO-3(-/-) mice were viable and fertile, and rigorous phenotypic analyses identified no significant abnormalities. Moreover, ZO-3-deficient F9 teratocarcinoma cells differentiated normally into visceral endoderm epithelium-like cells in the presence of retinoic acid. These cells had a normal epithelial appearance, and the molecular architecture of their TJs did not appear to be affected, except that TJ localization of ZO-2 was upregulated. Suppression of ZO-2 expression by RNA interference in ZO-3(-/-) cells, however, did not affect the architecture of TJs. Furthermore, the speed with which TJs formed after a Ca(2+) switch was indistinguishable between wild-type and ZO-3(-/-) cells. These findings indicate that ZO-3 is dispensable in vivo in terms of individual viability, epithelial differentiation, and the establishment of TJs, at least in the laboratory environment.     

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.     

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.