Molecular characterization of the tight junction protein ZO-1 in MDCK cells

Most of the information on the structure and function of the tight junction (TJ) has been obtained in MDCK cells. Accordingly, we have sequenced ZO-1 in this cell type, because this protein is involved in the response of the TJ to changes in Ca2+, phosphorylation, and the cytoskeleton. ZO-1 of MDCK cells comprises 6805 bp with a predicted open reading frame of 1769 amino acids. This sequence is 92 and 87% homologous to human and mouse ZO-1, respectively. Two nuclear sorting signals located at the PDZ1 and GK domains and 17 SH3 putative binding sites at the proline-rich domain were detected. We found two new splicing regions at the proline-rich region: beta had not been reported in human and mouse counterparts, and gamma, which was previously sequenced in human and mouse ZO-1, is now identified as a splicing region. The expression of different beta and gamma isoforms varies according to the tissue tested. With the information provided by the sequence, Southern blot, and PCR experiments we can predict a single genomic copy of MDCK-ZO-1 that is at least 13.16 kb long. MDCK-ZO-1 mRNA is 7.4 kb long. Its expression is regulated by calcium, while the expression of MDCK-ZO-1 protein is not.     

Freeze-fracture electron microscopic study of tight junction strands in HEK293 cells and MDCK II cells expressing claudin-1 mutants in the second extracellular loop

Claudins constitute tight junction (TJ) strands. In order to examine the function of the second extracellular loop (ECL2), we constructed 1CLΔFY and 1CLΔPL in which highly conserved amino acids, FY or PL, in the ECL2 of mouse claudin-1 were deleted. They were then tagged with either EGFP at the NH₂-terminus (EGFP1CLΔFY and EGFP1CLΔPL) or the myc-epitope at the COOH-terminus (1CLΔFYmyc and 1CLΔPLmyc). The expression of EGFP1CLΔFY and EGFP1CLΔPL in TJ-free HEK293 cells formed TJ strands resembling those formed by wild-type claudin-1. The expression of 1CLΔPLmyc in TJ-bearing MDCK II cells induced aberrant TJ strands in the lateral plasma membranes whose intramembranous particles were almost equally distributed in the P- and E-face. In contrast, 1CLΔFYmyc formed aggregates of short continuous strands which were frequently associated with vesicle-like structures. Coculture experiments with MDCK II cells showed that 1CLΔPLmyc was localized at heterotypic cell–cell junctions but 1CLΔFYmyc was not. These results suggest that changes in the TJ morphology due to the expression of either 1CLΔFYmyc or 1CLΔPLmyc may be caused by some factors specific to epithelial MDCK II cells including endogenous claudins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Formation of aberrant TJ strands by overexpression of claudin-15 in MDCK II cells

Claudins are one of the transmembrane proteins found at tight junctions (TJs); they constitute the backbone of TJ strands and comprise a multigene family. Claudins share a YV sequence at the COOH-termini, which is considered to be a ZO-binding motif. Overexpression of claudin-15 (15CL) or claudin-15 tagged with enhanced green fluorescent protein at the NH₂-terminus (EGFP-15CL) induced aberrant strands in MDCK II cells, even though claudin-15 has the ZO-binding motif. Morphometric analysis by freeze-fracture electron microscopy revealed that the mean number of apical TJ strands increased by 47% in EGFP-1CL-expressing cells, 21% in EGFP-15CL-expressing cells, and 28% in 15CL-expressing cells. The number of free-ended apical strands increased remarkably in EGFP-15CL- and 15CL-expressing cells, but not in EGFP-1CL-expressing cells. When MDCK cells expressing EGFP-1CL, EGFP-15CL or 15CL were co-cultured with parent MDCK cells, which express claudin-1 but not claudin-15, EGFP-15CL and 15CL could not be concentrated at the apical junctional region between the heterotypic cells, though EGFP-1CL could. These results suggest that not only binding to ZO-1, but also head-to-head compatibility between claudin species, is involved in organizing claudin proteins at the apical junctional region.     

The PIKfyve inhibitor YM201636 blocks the continuous recycling of the tight junction proteins claudin-1 and claudin-2 in MDCK cells

Tight junctions mediate the intercellular diffusion barrier found in epithelial tissues but they are not static complexes; instead there is rapid movement of individual proteins within the junctions. In addition some tight junction proteins are continuously being endocytosed and recycled back to the plasma membrane. Understanding the dynamic behaviour of tight junctions is important as they are altered in a range of pathological conditions including cancer and inflammatory bowel disease. In this study we investigate the effect of treating epithelial cells with a small molecule inhibitor (YM201636) of the lipid kinase PIKfyve, a protein which is involved in endocytic trafficking. We show that MDCK cells treated with YM201636 accumulate the tight junction protein claudin-1 intracellularly. In contrast YM201636 did not alter the localization of other junction proteins including ZO-1, occludin and E-cadherin. A biochemical trafficking assay was used to show that YM201636 inhibited the endocytic recycling of claudin-1, providing an explanation for the intracellular accumulation. Claudin-2 was also found to constantly recycle in confluent MDCK cells and treatment with YM201636 blocked this recycling and caused accumulation of intracellular claudin-2. However, claudin-4 showed negligible endocytosis and no detectable intracellular accumulation occurred following treatment with YM201636, suggesting that not all claudins show the same rate of endocytic trafficking. Finally, we show that, consistent with the defects in claudin trafficking, incubation with YM201636 delayed formation of the epithelial permeability barrier. Therefore, YM201636 treatment blocks the continuous recycling of claudin-1/claudin-2 and delays epithelial barrier formation.     

Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells.

Tight junctions (TJs) in endothelial cells are thought to determine vascular permeability. Recently, claudin-1 to -15 were identified as major components of TJ strands. Among these, claudin-5 (also called transmembrane protein deleted in velo-cardio-facial syndrome [TMVCF]) was expressed ubiquitously, even in organs lacking epithelial tissues, suggesting the possible involvement of this claudin species in endothelial TJs. We then obtained a claudin-6-specific polyclonal antibody and a polyclonal antibody that recognized both claudin-5/TMVCF and claudin-6. In the brain and lung, immunofluorescence microscopy with these polyclonal antibodies showed that claudin-5/TMVCF was exclusively concentrated at cell-cell borders of endothelial cells of all segments of blood vessels, but not at those of epithelial cells. Immunoreplica electron microscopy revealed that claudin-5/TMVCF was a component of TJ strands. In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries. In addition, in all other tissues we examined, claudin-5/TMVCF was specifically detected in endothelial cells of some segments of blood vessels, but not in epithelial cells. Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face-associated TJs. These findings indicated that claudin-5/TMVCF is an endothelial cell-specific component of TJ strands.     

Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells

The molecular chaperone protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat-denatured substrate protein was analyzed in vitro. Hsp78 alone was not able to mediate reactivation of firefly luciferase. Rather, efficient refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p. Bacterial DnaK/DnaJ/GrpE, which cooperates with the Hsp78 homolog, ClpB in Escherichia coli, could not substitute for the mitochondrial Hsp70 system. However, efficient Hsp78-dependent refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experiments, suggesting a specific functional interaction of both chaperone proteins. These findings establish the cooperation of Hsp78 with the Hsp70 machinery in the refolding of heat-inactivated proteins and demonstrate a conserved mode of action of ClpB homologs.     

Effect of FCCP on tight junction permeability and cellular distribution of ZO-1 protein in epithelial (MDCK) cells

The effect of the uncoupler of oxidative phosphorylation, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), on the tight junction of Madin-Darby canine kidney cells was examined. FCCP induced an abrupt decrease in the transepithelial electrical resistance of the confluent monolayers over a period of 20 s. When FCCP was withdrawn from the incubation medium, the monolayer resistance recovered to close to the original level in less than 2 h. Staining of the tight junction-associated protein ZO-1 showed that the changes in transepithelial electrical resistance were accompanied by a diffusing of the protein away from cell peripheries and a reconcentration to the tight junction areas following resistance recovery. Intracellular pH was decreased by FCCP on a similar time-scale with no obvious changes in ATP levels over this time-course. These data suggest that the uncoupler FCCP has a profound effect on tight junction permeability and cellular distribution of the tight junction protein ZO-1 in the epithelial cells and that it probably acts by breaking down proton gradients and altering intracellular pH.     

Limited contribution of claudin-5-dependent tight junction strands to endothelial barrier function

Members of the claudin family are involved in formation of barriers that control access to the paracellular space of epithelia. Likewise, endothelium-specific claudin-5 is involved in the function of the blood-brain barrier (BBB). Here, we assessed the role of claudin-5 in non-BBB endothelial barriers using lentiviral-driven overexpression and silencing of claudin-5 in its native environment of primary vascular endothelial cells. Effects were monitored using macromolecular tracers between 342Da and 40kDa. Measurements were made both in absence and presence of transmigrating leukocytes. Freeze-fracture preparations were analyzed for effects at the ultrastructural level. We show that overexpression of claudin-5 leads to formation of elaborate networks of junction strands, which are absent in untransduced endothelial cells. Concomitantly, a modest, non-size-selective enhancement of the barrier function was observed. In contrast, silencing of endogenous claudin-5 does not influence barrier function. The efficient sealing of the endothelium during diapedesis of monocytes or granulocytes is also claudin-5 independent. Collectively, these data provide evidence for a limited contribution of claudin-5 to the barrier function of human umbilical vein endothelial cells (HUVEC), implying that, unlike selective barriers in epithelia, the barrier of non-BBB endothelium seems largely independent of claudin-directed tight junction structures.     

Truncation mutants of the tight junction protein ZO-1 disrupt corneal epithelial cell morphology

The tight junction is the most apical intercellular junction of epithelial cells and regulates transepithelial permeability through the paracellular pathway. To examine possible functions for the tight junction-associated protein ZO-1, C-terminally truncated mutants and a deletion mutant of ZO-1 were epitope tagged and stably expressed in corneal epithelial cell lines. Only full-length ZO-1 and one N-terminal truncation mutant targeted to cell borders; other mutants showed variable cytoplasmic distributions. None of the mutants initially disrupted the localization of endogenous ZO-1. However, long-term stable expression of two of the N-terminal mutants resulted in a dramatic change in cell shape and patterns of gene expression. An elongated fibroblast-like shape replaced characteristic epithelial cobblestone morphology. In addition, vimentin and smooth muscle actin expression were up-regulated, although variable cytokeratin expression remained, suggesting a partial transformation to a mesenchymal cell type. Concomitant with the morphological change, the expression of the integral membrane tight junction protein occludin was significantly down-regulated. The localizations of endogenous ZO-1 and another family member, ZO-2, were disrupted. These findings suggest that ZO-1 may participate in regulation of cellular differentiation.     

NaCl flux between apical and basolateral side recruits claudin-1 to tight junction strands and regulates paracellular transport

In multicellular organisms, epithelia separate and divide the internal environment maintaining appropriate conditions in each compartment. To maintain homeostasis in these compartments, claudins, major cell adhesion molecules in tight junctions (TJs), regulate movements of several substances through the paracellular pathway (barrier function). In this study, we investigated effects of the flux of several substances between apical and basolateral side on paracellular transport and TJ protein localization. NaCl flux from apical to basolateral side increased paracellular conductance (Gp) and recruited claudin-1 from lateral cell membrane to the apical end with the colocalization with occludin, one of the TJ proteins concentrated at TJ strands. Oppositely-directed flux of sucrose against NaCl flux inhibited these reactions and same directional flux of sucrose with NaCl enhanced the increase of Gp, whereas 10-kDa dextran inhibited these reactions regardless of the side of administration. Our present findings indicated that TJ protein localization and barrier function are regulated depending on the environmental differences between apical and basolateral side.     

CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice

Oligodendrocyte-specific protein (OSP)/claudin-11 is a recently identified transmembrane protein found in CNS myelin and testis with unknown function. Herein we demonstrate that Osp null mice exhibit both neurological and reproductive deficits: CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Freeze fracture reveals that tight junction intramembranous strands are absent in CNS myelin and between Sertoli cells of mutant mice. Our results demonstrate that OSP is the mediator of parallel-array tight junction strands and distinguishes this protein from other intrinsic membrane proteins in tight junctions. These novel results provide direct evidence of the pivotal role of the claudin family in generating the paracellular physical barrier of tight junctions necessary for spermatogenesis and normal CNS function.     

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.     

Phosphorylation of claudin-4 by PKCepsilon regulates tight junction barrier function in ovarian cancer cells

Claudin proteins belong to a large family of transmembrane proteins essential to the formation and maintenance of tight junctions (TJs). In ovarian cancer, TJ protein claudin-4 is frequently overexpressed and may have roles in survival and invasion, but the molecular mechanisms underlying its regulation are poorly understood. In this report, we show that claudin-4 can be phosphorylated by protein kinase C (PKC) at Thr189 and Ser194 in ovarian cancer cells and overexpression of a claudin-4 mutant protein mimicking the phosphorylated state results in the disruption of the barrier function. Furthermore, upon phorbol ester-mediated PKC activation of OVCA433 cells, TJ strength is decreased and claudin-4 localization is altered. Analyses using PKC inhibitors and siRNA suggest that PKCepsilon, an isoform typically expressed in ovarian cancer cells, may be important in the TPA-mediated claudin-4 phosphorylation and weakening of the TJs. Furthermore, immunofluorescence studies showed that claudin-4 and PKCepsilon are co-localized at the TJs in these cells. The modulation of claudin-4 activity by PKCepsilon may not only provide a mechanism for disrupting TJ function in ovarian cancer, but may also be important in the regulation of TJ function in normal epithelial cells.     

Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3

Defining how the molecular constituents of the tight junction interact is a prerequisite to understanding tight junction physiology. We utilized in vitro binding assays with purified recombinant proteins and immunoprecipitation analyses to define interactions between ZO-1, ZO-2, ZO-3, occludin, and the actin cytoskeleton. Actin cosedimentation studies showed that ZO-2, ZO-3, and occludin all interact directly with F-actin in vitro, indicating that actin is engaged in multiple interactions at the tight junction. Low speed sedimentation analyses demonstrated that neither ZO-2, ZO-3, nor occludin act as F-actin cross-linking proteins, and further evidence indicates that these proteins do not bind to actin filament ends. The binding interactions of ZO-2, ZO-3, and occludin were corroborated in vivo by immunofluorescence colocalization experiments which showed that all three proteins colocalized with actin aggregates at cell borders in cytochalasin D-treated Madin-Darby canine kidney cells. Exploration of other tight junction protein interactions demonstrated that ZO-2 binds directly to both ZO-1 and occludin. Contrary to previous beliefs, our immunoprecipitation results indicate that ZO-1, ZO-2, and ZO-3 exist in situ primarily as independent ZO-1.ZO-2 and ZO-1.ZO-3 complexes rather than a trimeric ZO-1.ZO-2.ZO-3 grouping. These studies elucidate direct binding interactions among tight junction-associated proteins, giving insight into their organization as a multimolecular structure.