ZO-1 Stabilizes the Tight Junction Solute Barrier through Coupling to the Perijunctional Cytoskeleton

ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin.     

The Ras Target AF-6 Interacts with ZO-1 and Serves as a Peripheral Component of Tight Junctions in Epithelial Cells

The dynamic rearrangement of cell–cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell–cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell–cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell–cell contacts and found that AF-6 accumulated at the cell–cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell–cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell–cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell–cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell–cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.     

Calcitonin Receptor-Zonula Occludens-1 Interaction Is Critical for Calcitonin-Stimulated Prostate Cancer Metastasis

The role of neuroendocrine peptide calcitonin (CT) and its receptor (CTR) in epithelial cancer progression is an emerging concept with great clinical potential. Expression of CT and CTR is frequently elevated in prostate cancers (PCs) and activation of CT–CTR axis in non-invasive PC cells induces an invasive phenotype. Here we show by yeast-two hybrid screens that CTR associates with the tight junction protein Zonula Occludens-1 (ZO-1) via the interaction between the type 1 PDZ motif at the carboxy-terminus of CTR and the PDZ3 domain of ZO-1. Mutation of either the CTR C-PDZ-binding motif or the ZO-1-PDZ3 domain did not affect binding of CTR with its ligand or G-protein-mediated signaling but abrogated destabilizing actions of CT on tight junctions and formation of distant metastases by orthotopically implanted PC cells in nude mice, indicating that these PDZ domain interactions were pathologically relevant. Further, we observed CTR-ZO-1 interactions in PC specimens by proximity ligation immunohistochemistry, and identified that the number of interactions in metastatic PC specimens was several-fold larger than in non-metastatic PC. Our results for the first time demonstrate a mechanism by which PDZ-mediated interaction between CTR and ZO1 is required for CT-stimulated metastasis of prostate cancer. Since many receptors contain PDZ-binding motifs, this would suggest that PDZ-binding motif-adaptor protein interactions constitute a common mechanism for cancer metastasis.     

Zona Occludens-2 Is Critical for Blood–Testis Barrier Integrity and Male Fertility

Tight junction integral membrane proteins such as claudins and occludin are tethered to the actin cytoskeleton by adaptor proteins, notably the closely related zonula occludens (ZO) proteins ZO-1, ZO-2, and ZO-3. All three ZO proteins have recently been inactivated in mice. Although ZO-3 knockout mice lack an obvious phenotype, animals deficient in ZO-1 or ZO-2 show early embryonic lethality. Here, we rescue the embryonic lethality of ZO-2 knockout mice by injecting ZO-2(−/−) embryonic stem (ES) cells into wild-type blastocysts to generate viable ZO-2 chimera. ZO-2(−/−) ES cells contribute extensively to different tissues of the chimera, consistent with an extraembryonic requirement for ZO-2 rather than a critical role in epiblast development. Adult chimera present a set of phenotypes in different organs. In particular, male ZO-2 chimera show reduced fertility and pathological changes in the testis. Lanthanum tracer experiments show a compromised blood–testis barrier. Expression levels of ZO-1, ZO-3, claudin-11, and occludin are not apparently affected. ZO-1 and occludin still localize to the blood–testis barrier region, but claudin-11 is less well restricted and the localization of connexin-43 is perturbed. The critical role of ZO-2 for male fertility and blood–testis barrier integrity thus provides a first example for a nonredundant role of an individual ZO protein in adult mice.     

Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin

Junctional adhesion molecule (JAM) is an integral membrane protein that has been reported to colocalize with the tight junction molecules occludin, ZO-1, and cingulin. However, evidence for the association of JAM with these molecules is missing. Transfection of Chinese hamster ovary cells with JAM (either alone or in combination with occludin) resulted in enhanced junctional localization of both endogenous ZO-1 and cotransfected occludin. Additionally, JAM was coprecipitated with ZO-1 in the detergent-insoluble fraction of Caco-2 epithelial cells. A putative PDZ-binding motif at the cytoplasmic carboxyl terminus of JAM was required for mediating the interaction of JAM with ZO-1, as assessed by in vitro binding and coprecipitation experiments. JAM was also coprecipitated with cingulin, another cytoplasmic component of tight junctions, and this association required the amino-terminal globular head of cingulin. Taken together, these data indicate that JAM is a component of the multiprotein complex of tight junctions, which may facilitate junction assembly.     

Microtubule Integrity Is Necessary for the Epithelial Barrier Function of Cultured Thyroid Cell Monolayers

Vectorial transport in the thyroid epithelium requires an efficient barrier against passive paracellular flux, a role which is principally performed by the tight junction (zonula occludens). There is increasing evidence that tight junction integrity is determined by integral and peripheral membrane proteins which interact with the cell cytoskeleton. Although the contribution of the actin cytoskeleton to tight junction physiology has been intensively studied, less is known about possible interactions with microtubules. In the present study we used electrophysiological and immunohistochemical approaches to investigate the contribution of microtubules to the paracellular barrier in cultured thyroid cell monolayers which displayed a high transepithelial electrical resistance (6000-9000 ohm · cm²). Colchicine (1 μM) caused a progressive fall in electrical resistance to <10% of baseline after 6 h and depolarization of the transepithelial electrical potential difference consistent with a significant increase in paracellular permeability. The effect of colchicine on TER was not affected by agents which inhibit the major apical conductances of thyroid cells but was reversed upon removal of the drug. Immunofluorescent staining for tubulin combined with confocal laser scanning microscopy demonstrated that thyroid cells possessed a dense microtubule network extending throughout the cytoplasm which was destroyed by colchicine. Colchicine also produced changes in the localization of the tight junction-associated protein, ZO-1: its normally continuous junctional distribution was disrupted by striking discontinuities and the appearance of many fine strands which extended into the cytoplasm. A similar disruption in E-cadherin staining was also observed, but colchicine did not affect the distribution of vinculin associated with adherens junctions nor the integrity of the perijunctional actin ring. We conclude that microtubules are necessary for the functional and structural integrity of tight junctions in this electrically tight, transporting epithelium.     

Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption

Oxidants such as monochloramine (NH(2)Cl) decrease epithelial barrier function by disrupting perijunctional actin and possibly affecting the distribution of tight junctional proteins. These effects can, in theory, disturb cell polarization and affect critical membrane proteins by compromising molecular fence function of the tight junctions. To examine these possibilities, we investigated the actions of NH(2)Cl on the distribution, function, and integrity of barrier-associated membrane, cytoskeletal, and adaptor proteins in human colonic Caco-2 epithelial monolayers. NH(2)Cl causes a time-dependent decrease in both detergent-insoluble and -soluble zonula occludens (ZO)-1 abundance, more rapidly in the former. Decreases in occludin levels in the detergent-insoluble fraction were observed soon after the fall of ZO-1 levels. The actin depolymerizer cytochalasin D resulted in a decreased transepithelial resistance (TER) more quickly than NH(2)Cl but caused a more modest and slower reduction in ZO-1 levels and in occludin redistribution. No changes in the cellular distribution of claudin-1, claudin-5, or ZO-2 were observed after NH(2)Cl. However, in subsequent studies, the immunofluorescent cellular staining pattern of all these proteins was altered by NH(2)Cl. The actin-stabilizing agent phalloidin did not prevent NH(2)Cl-induced decreases in TER or increases of apical to basolateral flux of the paracellular permeability marker mannitol. However, it partially blocked changes in ZO-1 and occludin distribution. Tight junctional fence function was also compromised by NH(2)Cl, observed as a redistribution of the alpha-subunit of basolateral Na(+)-K(+)-ATPase to the apical membrane, an effect not found with the apical membrane protein Na(+)/H(+) exchanger isoform 3. In conclusion, oxidants not only disrupt perijunctional actin but also cause redistribution of tight junctional proteins, resulting in compromised intestinal epithelial barrier and fence function. These effects are likely to contribute to the development of malabsorption and dysfunction associated with mucosal inflammation of the digestive tract.     

Restoration of tight junction structure and barrier function by down-regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells

In the Madin-Darby canine kidney epithelial cell line, the proteins occludin and ZO-1 are structural components of the tight junctions that seal the paracellular spaces between the cells and contribute to the epithelial barrier function. In Ras-transformed Madin-Darby canine kidney cells, occludin, claudin-1, and ZO-1 were absent from cell-cell contacts but were present in the cytoplasm, and the adherens junction protein E-cadherin was weakly expressed. After treatment of the Ras-transformed cells with the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059, which blocks the activation of mitogen-activated protein kinase (MAPK), occludin, claudin-1, and ZO-1 were recruited to the cell membrane, tight junctions were assembled, and E-cadherin protein expression was induced. Although it is generally believed that E-cadherin-mediated cell-cell adhesion is required for tight junction assembly, the recruitment of occludin to the cell-cell contact area and the restoration of epithelial cell morphology preceded the appearance of E-cadherin at cell-cell contacts. Both electron microscopy and a fourfold increase in the transepithelial electrical resistance indicated the formation of functional tight junctions after MEK1 inhibition. Moreover, inhibition of MAPK activity stabilized occludin and ZO-1 by differentially increasing their half-lives. We also found that during the process of tight junction assembly after MEK1 inhibition, tyrosine phosphorylation of occludin and ZO-1, but not claudin-1, increased significantly. Our study demonstrates that down-regulation of the MAPK signaling pathway causes the restoration of epithelial cell morphology and the assembly of tight junctions in Ras-transformed epithelial cells and that tyrosine phosphorylation of occludin and ZO-1 may play a role in some aspects of tight junction formation.     

Angulin-1 seals tricellular contacts independently of tricellulin and claudins

Tricellular tight junctions (tTJs) are specialized tight junctions (TJs) that seal the intercellular space at tricellular contacts (TCs), where the vertices of three epithelial cells meet. Tricellulin and angulin family membrane proteins are known constituents of tTJs, but the molecular mechanism of tTJ formation remains elusive. Here, we investigated the roles of angulin-1 and tricellulin in tTJ formation in MDCK II cells by genome editing. Angulin-1–deficient cells lost the plasma membrane contact at TCs with impaired epithelial barrier function. The C terminus of angulin-1 bound to the TJ scaffold protein ZO-1, and disruption of their interaction influenced the localization of claudins at TCs, but not the tricellular sealing. Strikingly, the plasma membrane contact at TCs was formed in tricellulin- or claudin-deficient cells. These findings demonstrate that angulin-1 is responsible for the plasma membrane seal at TCs independently of tricellulin and claudins.     

Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels

The three connexins expressed in the ocular lens each contain PDZ domain-binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell-cell communication in HeLa cells, whereas the addition of a seven-amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain-binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.     

ZO-1 controls endothelial adherens junctions,cell–cell tension,angiogenesis, and barrier formation

Intercellular junctions are crucial for mechanotransduction, but whether tight junctions contribute to the regulation of cell–cell tension and adherens junctions is unknown. Here, we demonstrate that the tight junction protein ZO-1 regulates tension acting on VE-cadherin–based adherens junctions, cell migration, and barrier formation of primary endothelial cells, as well as angiogenesis in vitro and in vivo. ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin–VE-cadherin complex. Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation. ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF. ZO-1 is thus a central regulator of VE-cadherin–dependent endothelial junctions that orchestrates the spatial actomyosin organization, tuning cell–cell tension, migration, angiogenesis, and barrier formation.     

Association of ARVCF with zonula occludens (ZO)-1 and ZO-2: binding to PDZ-domain proteins and cell-cell adhesion regulate plasma membrane and nuclear localization of ARVCF

ARVCF, an armadillo-repeat protein of the p120(ctn) family, associates with classical cadherins and is present in adherens junctions, but its function is poorly understood. Here, we show that ARVCF interacts via a C-terminal PDZ-binding motif with zonula occludens (ZO)-1 and ZO-2. ARVCF and ZO-1 partially colocalize in the vicinity of the apical adhesion complex in polarized epithelial Madin-Darby canine kidney cells. ARVCF, ZO-1, and E-cadherin form a complex and are recruited to sites of initial cell-cell contact in sparse cell cultures. E-cadherin binding and plasma membrane localization of ARVCF require the PDZ-binding motif. Disruption of cell-cell adhesion releases ARVCF from the plasma membrane and an increased fraction of the protein localizes to the nucleus. Nuclear localization of ARVCF also requires the PDZ-binding motif and can be mediated by the PDZ domains of ZO-2. Thus, the interaction of ARVCF with distinct PDZ-domain proteins determines its subcellular localization. Interactions with ZO-1 and ZO-2, in particular, may mediate recruitment of ARVCF to the plasma membrane and the nucleus, respectively, possibly in response to cell-cell adhesion cues.     

Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly

The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.     

Expression and function of tight junction associated molecules in human breast tumor cells is not affected by the Ras-MEK1 pathway.

Constitutive activation of Ras or Ras-mediated signaling pathways is one of the initial steps during tumorigenesis that promotes neoplastic transformation. Recently it was reported that in Ha-Ras overexpressing MDCK cells the tight junction proteins claudin-1, occludin and ZO-1 were absent at cell-cell contact sites but present in the cytoplasm. Inhibition of MEK1 activity recruited all three proteins to the cell membrane leading to a restoration of the tight junction barrier function in MDCK cells. In order to evaluate the relevance of the MEK1 pathway in tight junction regulation in breast cancer cells, we investigated the effect ofMEK1 inhibition on expression of claudin-1, occludin and ZO-1 in natively claudin-1 expressing T47-D cells (low Ras activity), claudin-1 negative MCF-7 cells (elevated Ras activity) as well as two retroviral claudin-1 transduced MCF-7 daughter cell lines with prominent membrane and cytoplasmic claudin-1 dominant homing, respectively. Although we effectively blocked phosphorylation of MAPKs ERK-1 and ERK-2 using the selective MEK1 inhibitor PD98059, no quantitative changes of mRNA or protein levels of claudin-1, occludin and ZO-1 could be detected in all cell lines investigated. Furthermore, immnfluorescence analysis of claudin-1 revealed that inhibition of the MAPK pathway did not alter th e subcellular cytoplasmic distribution of claudin-1 to be more membrane specific. Finally, the diffusion barrier properties of tight junctions as analyzed by transepithelial resistance (TER) or paracellular flux analysis of 3 and 40 kDa dextran of tight junctions were not altered in the claudin-1 positive T47-D and the MCF-7 cell lines. Our findings indicate that the proposed involvement of the Ras-MEK-ERK pathway is likely not involved in the dysregulated tight junction formation in breast tumor cells and indicates that elevated activity of Ras might not be of general importance for the disruption of tight junction structures in breast tumors.     

The coxsackievirus and adenovirus receptor interacts with the multi-PDZ domain protein-1 (MUPP-1) within the tight junction

The coxsackievirus and adenovirus receptor (CAR) is a component of the epithelial cell tight junction. In a yeast two-hybrid screen we identified the multi-PDZ domain protein MUPP1 as an interaction partner for the CAR cytoplasmic domain. CAR and MUPP1 were found to colocalize at the tight junction, to coprecipitate from epithelial cells, and to interact in vitro. The interaction was found to specifically involve the PDZ-binding motif within the CAR C terminus and MUPP1 PDZ domain 13. In transfected cells, CAR recruited MUPP1 to cell-cell contacts. The inhibition of CAR expression with small interfering RNA inhibited MUPP1 localization to the tight junction. The results indicated that CAR interacts with MUPP1 and is involved in MUPP1 recruitment to the tight junction.     

Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1

Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell–cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell–cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell–cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell–cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1.     

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

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

Nuclear localization of the tight junction protein ZO-2 in epithelial cells

The tight junction constitutes the major barrier to solute and water flow through the paracellular space of epithelia and endothelia. It is formed by transmembrane proteins and submembranous molecules such as the MAGUKs ZOs. We have previously found that several MAGUKs, including those of the tight (ZO-1, ZO-2, and ZO-3) and septate junction (tamou and Dlg), contain one or two nuclear sorting signals located at their first PDZ and GK domains. Now we show that these proteins also contain a nuclear export signal and focus our study on the nuclear membrane shuttling of ZO-2. In sparse cultures this molecule concentrates at the nucleus in clusters, where it partially colocalizes with splicing factor SC35. Nuclear staining diminishes as the monolayer acquires confluence through a process sensitive to the nuclear export inhibitor leptomycin B. Nuclear localization can be induced by impairing cell-cell contacts, by mechanical injury. ZO-2 that shuttles from the cell periphery into the nucleus is not newly synthesized but originates from a preexistent pool. The movement of this protein is mediated by the actin cytoskeleton.