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Molecular Physiology and Pathophysiology of Tight Junctions I. Tight junction structure and function: lessons from mutant animals and proteins

Laura L. Mitic, Christina M. Van Itallie, and James M. Anderson

Departments of Internal Medicine and Cell Biology, Yale School of Medicine, New Haven, Connecticut 06520-8019


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Tight junctions form the major paracellular barrier in epithelial tissues. Barrier-sealing properties are quite variable among cell types in terms of electrical resistance, solute and water flux, and charge selectivity. A molecular explanation for this variability appears closer following identification of the transmembrane proteins occludin and members of the claudin multigene family. For example, the human phenotype of mutations in claudin-16 suggests that it creates a channel that allows magnesium to diffuse through renal tight junctions. Similarly, a mouse knockout of claudin-11 reveals its role in formation of tight junctions in myelin and between Sertoli cells in testis. The study of other claudins is expected to elucidate their contributions to creating junction structure and physiology in all epithelial tissues.

claudin; occludin; paracellin-1; oligodendrocyte-specific protein


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THE MOVEMENT OF SOLUTES, ions, and water across epithelia occurs through both transcellular and paracellular routes. Transcellular transport, through specific membrane pumps and channels, actively generates the unique electroosmotic gradients and secretory fluids characteristic of each epithelium. Maintenance of these gradients is dependent on limiting back diffusion between cells through the paracellular pathway. In this way, the tightness of the paracellular barrier and its molecular selectivity contribute significantly to overall epithelial transport characteristics. The major barrier in the paracellular pathway is created by the tight junction. For much of the last century, investigators were aware that the sealing properties of tight junctions were variable and regulated (3), yet they lacked a molecular explanation. We focus here on recent insights provided by molecular and genetic study of the transmembrane barrier-forming proteins occludin and claudin and outline their important roles in forming the junction's structure and in defining its physiological characteristics. These transmembrane proteins are coupled to plaque proteins on the cytoplasmic surface of the junction; we briefly discuss this organization and its interaction with the actin cytoskeleton. Finally, we suggest an unexpected role for junctions in vesicle targeting and generation of apical-basal cell polarity.

Tight junctions appear by transmission electron microscopy as a series of focal contacts between the plasma membranes of adjacent cells. Freeze fracture electron microscopy reveals that these contacts correspond to continuous, branching fibrils of transmembrane particles that encircle the apical aspect of the lateral surface of each cell. Fibrils on one cell presumably interact with fibrils on an adjacent cell to close the paracellular space and define the paracellular permeability characteristics. These fibrils are now known to be formed by at least two types of tetra-spanning transmembrane proteins: occludin and different permutations of the 20 members of the claudin family.


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Identified in 1993 as a tight junction component and localized via immunogold freeze fracture microscopy to fibrils, occludin, a 65-kDa phosphoprotein, was originally thought to be the main sealing protein (Fig. 1) (26). It spans the membrane four times with cytoplasmic NH2 and COOH terminals and forms two extracellular loops that are composed mostly of glycine and tyrosine. Several lines of evidence support a functional role for occludin in defining the barrier. First, it is adhesive, as demonstrated by its ability to confer calcium-independent adhesion when transfected into occludin-null fibroblasts (27). Next, disruption of occludin interactions by the addition of peptides corresponding to sequences of the extracellular loops results in a drop in transepithelial electrical resistance, a measure of tight junction permeability (1). Mutation or overexpression of occludin in cultured cells has similarly been shown to affect permeability properties of both electrical resistance and flux of noncharged solutes (26). Thus occludin seemed a plausible candidate for the primary sealing protein. Yet it was unclear how a single protein could produce the great variability in permeability observed among different epithelia.


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Fig. 1.   Schematic model of the protein interactions at the tight junction. ZO-1 interacts with the transmembrane proteins occludin and claudin(s) as well as the cytoplasmic proteins zonula occludins (ZO)-2, ZO-3, actin, AF-6, the kinase ZAK, the transcription regulator ZO-1-associated nucleic acid-binding protein (ZONAB), and cingulin. Details of the other proteins illustrated can be found in review articles cited in the text. BAP-1, BAI-associated protein; PKC, protein kinase C; ASIP, atypical PKC isotype-specific interacting protein; VAP, vesicle-associated membrane protein (VAMP)-associated protein; JAM, junction adhesion molecule.

This quandary may now be explained by the discovery of a family of proteins called claudins, named from the Latin claudere, "to close" (26). Their existence was suggested by the observation that embryonic stem cells from which occludin was deleted by homologous recombination retained a paracellular barrier (20). Claudins were identified by standard biochemical methods on the basis of the assumption that undiscovered transmembrane proteins would cofractionate with occludin. They share a similar membrane topology with occludin even though they are significantly smaller proteins, ~22 kDa, and their two extracellular loops and intracellular NH2 and COOH terminals are significantly shorter. At present, 20 claudins can be identified in the GenBank database. They range in sequence identity from 12.5% to 69.7% and appear to group into subfamilies, which may indicate similarities in function. Some claudins are clustered on human chromosomes 3, 7, 16, and 21 (Fig. 2).


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Fig. 2.   The relative mRNA or protein levels of individual claudins are compared among tissues. 0 indicates no detectable expression, and blank positions denote a lack of reported data. Sequence data can be obtained at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov), except for claudin-19, which has not been released.

Claudins possess several functional characteristics consistent with a role in barrier formation. They show an intrinsic ability to polymerize into linear fibrils, as shown by the extensive networks resulting from the transfection of a single type of claudin into claudin-null fibroblasts (6). This is in contrast to occludin, which forms only short fragments of strands. When occludin is transfected into claudin-expressing fibroblasts, it is recruited into the claudin fibrils, suggesting that claudins are the major element driving fibril formation. The claudins exhibit stronger adhesion than occludin (14), indicating that they can form the transcellular contacts presumably required to seal the intercellular space. Claudins display varied tissue distribution, which is consistent with the idea that differential expression might explain the variable permeability observed among different tissues. Only the tissue distribution is known for some claudins, whereas the cell type expression patterns are known for others. The latter can be strikingly restricted; for example, claudin-5 is reportedly expressed only in endothelial tight junctions (19) and thus is expressed in all tissues (Fig. 2). Similarly, narrow expression patterns of claudins-11 and -16 are described below. Although a detailed description for most claudins has not yet been reported, the available preliminary observations are summarized in Fig. 2.


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In addition to the characteristics cited above, several lines of direct experimental evidence provide convincing support for claudin's role in creating the tight junction's physiological barrier. Inai et al. (10) reported that when claudin-1 is overexpressed by transfection in cultured MDCK monolayers the electrical resistance increases severalfold above that of control cell lines. A coincident decrease in the paracellular flux of 4- and 40-kDa FITC-labeled dextrans was observed. This is in contrast to an intriguing result from several laboratories showing that when occludin is overexpressed the electrical resistance increases and, paradoxically, the flux also increases (1). Both claudin-3 and -4 are receptors for a cytotoxic enterotoxin (CPE) produced by the bacterium Clostridium perfringens. A COOH terminal fragment of the toxin binds claudin but is not cytotoxic. When MDCK cells are exposed to the noncytotoxic fragment of CPE, the fragment binds and results in removal of claudin-4 from the cell surface (MDCK I cells do not express claudin-3), whereas other claudins remain at the junction (23). Loss of claudin-4 from the cell surface coincides with dramatic changes in the morphology of tight junction fibrils. Freeze fracture electron micrographs reveal partially disintegrated fibrils and an incomplete strand network. The remaining network is presumably formed by CPE-insensitive claudins and occludin. CPE-treated monolayers exhibit significantly reduced electrical resistances (23), suggesting that removal of claudin-4 disrupts fibril organization and increases junction permeability.

Characterization of the first known inherited disease of tight junctions adds strong support to the idea that claudins confer specific selectivity properties to paracellular transport. With the use of positional cloning, human mutations in paracellin-1 (claudin-16) were shown to be responsible for a rare magnesium wasting syndrome called renal hypomagnesemia with hypercalciuria and nephrocalcinosis (21). Affected individuals display massive urinary magnesium loss, leading to hypomagnesemia and seizures at an early age. The defect cannot be corrected by oral magnesium administration. The pathological phenotype can be rationalized by assuming that claudin-16 creates a magnesium-permissive channel within the tight junction. Magnesium homeostasis is principally regulated in the kidney, where reabsorption from the glomerular filtrate occurs in the thick ascending limb of Henle. Resorption occurs only through the paracellular route and is driven by a positive electrical gradient within the tubule with respect to the interstitial space. Interestingly, the level of paracellular permeability may not be fixed since flux can be regulated over a very wide range in response to serum magnesium levels. Paracellin-1/claudin-16 expression in the kidney is restricted to the thick ascending limb, i.e., where magnesium is resorbed. The simplest interpretation of these finding is that paracellin-1/claudin-16 creates a magnesium-selective channel through the junctions in the thick ascending limb of Henle and, when absent, magnesium remains in the tubule and is lost in the urine. The channel-like activity created by paracellin-1/claudin-16 may actually be more broadly selective for divalent cations and not exclusive for magnesium, since affected individuals also demonstrate hypercalciuria. They may not develop hypocalcemia because, unlike magnesium, calcium loss can be compensated by several hormonally controlled mechanisms that increase intestinal absorption (vitamin D) and bone metabolism (parathyroid hormone).

The functional importance of claudins in forming fibrils was recently demonstrated in claudin-11 knockout mice. Claudin-11 (also known as oligodendrocyte-specific protein or OSP) expression is restricted in the adult to the myelin sheaths of oligodendrocytes in the central nervous system, Sertoli cells in the testis (18), the organ of Corti, the choroid plexus, and the collecting ducts in the kidney (8). Coincidentally, some of these junctions are organized as parallel fibrils deficient in cross-links, suggesting that claudin-11 forms unbranched fibrils. Knockout mice show a complete loss of tight junction fibrils in Sertoli and central nervous system myelin cells, suggesting that claudin-11 is directly responsible for the intramembranous particles and is the only claudin expressed in these junctions (8). The phenotype of claudin-11-null mice includes male sterility and neurological defects consistent with slowing of neuronal conduction times in the central nervous system (8). Presumably, these defects result from loss of the tight junction barrier in the testis and central nervous system oligodendrocytes, respectively. A fascinating morphological finding in the null animals is that although fibrils are absent, there are linear particle-free membrane depressions in their place, suggesting that the cytoplasmic proteins are correctly localized and physically distort the membrane.

Together, the diverse tissue distributions of claudins, their ability to form continuous adhesive fibrils, and the phenotypes of the paracellin-1/claudin-16 mutations in humans and the claudin-11 knockout mice strongly suggest that they are the primary proteins responsible for the physiological and structural paracellular barrier. It is expected, although not proven, that each claudin possesses selective permeability properties and that the permutations of claudins expressed in each cell define its paracellular properties.


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The observation that several claudins can exist in the same junction and are not obviously segregated raises questions about the nature of their interactions both within a single fibril and between cells. It seems likely that claudins (and occludin) organize into higher-order structures, such as oligomers. Freeze fracture micrographs indicate that fibrils are composed of particles ~10 nm in diameter, which is similar to the diameter of gap junction connexons. Connexons consist of hexamers of four-span transmembrane proteins similar in size to claudins and occludin. By analogy, it is likely that the 10-nm tight junction particles consist of more than one claudin or occludin molecule. When a claudin and occludin are coexpressed in fibroblasts, claudin recruits occludin to fibrils (6), but this could be mediated by either cooligomerization or interactions with cytoplasmic proteins. Might different types of claudins oligomerize in the membrane bilayer to form heteromultimers or are interactions homotypic? Combinatorial association of specific claudins may provide an additional way to modulate permeability properties.

Although understanding lateral oligomerization requires further study, recent work by Furuse et al. (7) demonstrated that claudins on one cell can associate heterotypically with claudins on another cell. Using claudin-null L cells as a system for investigating exogenously transfected claudins in the absence of tight junctions, these authors demonstrated that claudin-1 and -3 and claudin-2 and -3 cocluster across cells but that claudin-1 and -2 do not (7). Thus of the 20 known claudins some may interact transcellularly with each other, whereas others may not. It is unclear how such contacts would create a pore in the paracellular space. Perhaps formation of a pore requires two oligomers on opposite cells to contact, with the pore forming between the two. Future mutational and structural studies should resolve how these proteins contact their partners on opposing cells.


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A dense cytoplasmic network of proteins has been described at tight junctions. These proteins and their interactions appear to be involved in scaffolding the transmembrane proteins and coupling them to cytoplasmic regulation and, unexpectedly, to vesicle targeting and cell polarity. The COOH terminals of both occludin and claudins bind to a family of highly related cytoplasmic proteins called ZOs (ZO-1, ZO-2, ZO-3) (5, 11). ZOs are part of the membrane-associated guanylate kinase (MAGUK) superfamily, whose members are characterized by one or more postsynaptic protein-95/discs large/zonula occludens-1 (PSD-95/DLG/ZO-1; PDZ) domains, an src homology (SH-3) domain, and an enzymatically inactive guanylate kinase-like (GUK) domain. All three domains function as protein-binding modules, giving other MAGUKs a well-documented role in organizing proteins at the plasma membrane (4). Claudins bind to the first PDZ domain of ZOs (11), whereas occludin's interaction with ZO-1 has been mapped to a region encompassing the GUK domain.

A ring of actin microfilaments, containing myosin II, underlies the apical junctional complex, and its contraction has been proposed to regulate paracellular permeability. For example, experimental disruption of actin with agents such as phalloidin and cytochalasin disrupt the barrier and change morphology of the junction fibrils (17). The ZO MAGUKs bind to actin (5, 29), suggesting that they mediate a direct link between the actin cytoskeleton and the sealing proteins.

Numerous other proteins (Fig. 1) have been localized to the cytoplasmic surface of the tight junction by light or electron microscopy, but additional studies are needed to fully define their functions. Many of these proteins interact with each other, and several proteins, such as the protein kinase C-lambda -interacting protein atypical PKC isotype-specific interacting protein (ASIP) (12) and the ras-binding protein acute lymphocytic leukemia fusion-6 (AF-6) (30), contain PDZ domains available for protein interactions. Thus a complex protein network underlies the transmembrane proteins. Cingulin, a 140- to 160-kDa phosphoprotein, has recently been shown to interact with directly with ZO-1 (2). Cingulin has structural similarity to myosin, allowing the intriguing speculation that it may interact with actin filaments, thereby providing another link to the actomyosin contractile network.

Symplekin (13) and 7H6 (32), 150-kDa and 155-kDa proteins, respectively, have both been localized to tight junctions by immunoelectron microscopic analysis, and symplekin shows an additional localization in the nucleus. Symplekin's possible involvement in mRNA polyadenylation (24) may explain the nuclear localization, although it is presently unclear how such a function relates to tight junction physiology.

Additional proteins localized at tight junctions, such as rab 3B (28), rab 13 (31), Sec 6/8 (9), and vesicle-associated membrane protein (VAMP)-associated protein (VAP)-33 (15), may be involved in vesicle transport processes. This provides a molecular link to early observations, suggesting that vesicle trafficking might occur through the apical junctional complex (16). In yeast, the Sec6/8 complex is part of a larger complex called the exocyst and is required for specific vesicle targeting to the bud tip (25). In streptolysin-O-permeabilized MDCK cells, antibodies against the mammalian homologue of Sec6/8 specifically inhibited delivery of low-density lipoprotein receptor-containing vesicles to the basolateral membrane, while not affecting the apical targeting of another protein (9). Thus Sec6/8 situated at the tight junction may be used as a directional marker for vesicle trafficking to the lateral membrane. Lapierre et al. (15) recently reported that occludin's COOH terminus binds VAP-33, a VAMP/synaptobrevin-binding protein, further implicating occludin or the junction in vesicle trafficking (22). Consistent with this, overexpression of VAP-33 results in relocation of occludin from the tight junction to the lateral cell surface (15). The authors postulate that VAP-33 may help target occludin to tight junctions, or it may function as a more general marker for vesicle trafficking.


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The working model of tight junction architecture in Fig. 1 illustrates numerous proteins and some of their documented interactions, but the functional properties of how they assemble and regulate the barrier are largely unknown. For example, how does this complex form at the membrane, and is it dynamically regulated to control paracellular permeability? How do interactions between the transmembrane proteins physically seal the paracellular space and bring about variable permeability properties? Are there inherited diseases of proteins other than claudin-16/paracellin-1? How many claudins exist, and are there additional types of fibril proteins? Is the tight junction involved in vesicle trafficking? Clearly, the rapid progress in tight junction research has produced exciting new results and has raised equally exciting questions for future research. We can soon expect a more detailed understanding of the barrier and its contribution to normal physiology and disease.


    ACKNOWLEDGEMENTS

We thank Dotty Franco for assistance in preparation of the manuscript.


    FOOTNOTES

We are supported by National Institute of Health Grants DK-45134 and DK-55389.

Address for reprint requests and other correspondence: J. M. Anderson, Section of Digestive Diseases, Dept. of Internal Medicine, 1080 LMP, Yale School of Medicine, 333 Cedar St., New Haven, CT 06520-8019.


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