REVIEW J Am Soc Nephrol 10: 1337–1345, 1999

Transmembrane Proteins in the Tight Junction Barrier

ALAN S. FANNING, LAURA L. MITIC, and JAMES MELVIN ANDERSON Departments of Internal Medicine and Cell Biology, Yale School of Medicine, New Haven, Connecticut.

Abstract. Three types of transmembrane proteins have been edly, other members of this family have already been charac- identified within the tight junction, but it remains to be deter- terized without recognizing their relationship to tight junctions. mined how they provide the molecular basis for regulating the Junction adhesion molecule, the most recently identified tight paracellular permeability for water, solutes, and immune cells. junction component, is a member of the Ig superfamily and Several of these proteins localize specifically within the con- influences the paracellular transmigration of immune cells. A tinuous cell-to-cell contacts of the tight junction. One of these, plaque of cytoplasmic proteins under the junction may be occludin, is a cell adhesion molecule that has been demon- responsible for scaffolding the transmembrane proteins, creat- strated to influence ion and solute permeability. The claudins ing a link to the perijunctional actin cytoskeleton and trans- are a family of four-membrane spanning proteins; unexpect- ducing regulatory signals that control the paracellular barrier.

Tight junctions form continuous circumferential intercellular movement of solutes and water across the apical and basolat- contacts between epithelial cells and create a regulated barrier eral membranes. A specific profile of functionally distinct to the paracellular movement of water, solutes, and immune transporters and channels is distributed in a cell type-specific cells (1). They also provide a second type of barrier that manner on the apical and basolateral membranes and accounts contributes to cell polarity by limiting exchange of membrane for the unique transport characteristics of each epithelium and lipids between the apical and basolateral membrane domains. organ. Examples of specialization include the ability of the Numerous proteins within the junction have been identified renal collecting duct to resorb water in response to ADH and (Figure 1); however, our understanding of their functions re- the ability of the hepatocyte to transport bile acids. Transport mains limited. In this review, we will focus on insights gained rates are subject to a high degree of regulation and can be by the recent identification of three types of transmembrane turned off or on by second-messenger signaling pathways, proteins located at the junction. We consider how they might transmembrane voltage, pH, and many other factors. Transcel- provide a molecular explanation for some of the barrier’s lular transport is active, dependent either on hydrolysis of ATP physiologic properties, and how the cortical cytoskeletal net- or on the electro-osmotic gradient generated by basolaterally work may organize and regulate these proteins. The reader is positioned Naϩ/Kϩ-ATPase. In summary, transcellular trans- referred elsewhere for detailed reviews on the molecular ar- port has a very high degree of molecular specificity, is tightly chitecture of the tight junction (2–5), its physiologic properties regulated, and is variable among different epithelia. (6–10), and the very important role of the actin cytoskeleton in The paracellular component of transport is distinct from regulating structure and function (11,12). Several recent re- transcellular transport in several ways. It is a completely pas- views have focused on the implications of this work for normal sive process, resulting from paracellular dissipation of the renal physiology and changes in acute tubular ischemia (13– electro-osmotic gradients established by transcellular transport. 15). To understand the potential role of the transmembrane It also lacks the vast functional diversity and molecular dis- proteins, we begin with a brief review of the major physiologic crimination of the transcellular component. Paracellular trans- properties of the tight junction. port has two basic characteristics: (1) permeability – the mag- nitude of the barrier; and (2) permselectivity – the ability to Physiologic Characteristics of the Paracellular discriminate molecular size and ionic charge. In practice, most Pathway investigators quantify permeability by two complementary The net transepithelial transport across an epithelium is the techniques. The first is by measuring transepithelial electrical sum of two distinct components: transcellular and paracellular resistance (TER). This is a measure of the barrier to small ions transport. Transcellular transport results from the regulated (predominately Naϩ and ClϪ) in an experimentally applied electrical field in the bathing media. The second is by measur- ing the flux of tracer solutes, such as radiolabeled inulin or Received October 7, 1998. Accepted December 1, 1998. mannitol, which only traverse the epithelia through the inter- Correspondence to Dr. James M. Anderson, 1080 LMP, Department of Internal cellular space. Medicine, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520. Phone: 203-785-7312; Fax: 203-785-7273; E-mail: [email protected] Values for the paracellular element of electrical resistance in 1046-6673/1006-1337 natural epithelia vary enormously and with important func- Journal of the American Society of Nephrology tional consequences. For example, “leaky” mammalian proxi- Copyright © 1999 by the American Society of Nephrology mal renal tubules (6 to 7 ⍀ϫcm2) permit rapid paracellular 1338 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1337–1345, 1999

Figure 1. Model of the molecular interactions of proteins within the tight junction. Transmembrane proteins occludin, claudin(s), and junctional adhesion molecule (JAM) function to define barrier properties of the paracellular space. While presently unproven, they are shown as interacting in a homophilic manner. ZO-1, -2, and -3 are homologous, and all bind the cytoplasmic tail of occludin. ZO-1 is known to bind actin, AF-6, ZO-associated kinase (ZAK) fodrin, and ␣-catenin, although the last three proteins have yet to be localized to the junction by immunoelectron microscopy. Other proteins that have been localized to junctions but which presently lack functions and known molecular interactions include symplekin, cingulin, and 7H6. Several signaling proteins are found within the apical junction complex but are not unique to the tight junction.

reabsorption of most of the glomerular filtrate. Collecting ducts Experimental Problems in Measuring the Tight maintain electro-osmotic gradients used for transport and have Junction Barrier paracellular barriers of approximately 300 ⍀ϫcm2. The very Caution is required when using either TER or flux to char- “tight” epithelium of the bladder (6,000 to 300,000 ⍀ϫcm2) acterize the tight junction or changes in its sealing properties in can preserve the composition of either highly dilute or concen- response to experimental manipulations. Both methods are trated urine (reviewed in reference (6)). In contrast to the wide influenced by many factors other than those attributable spe- variation in permeability, the size cutoff for permselectivity is cifically to the molecular components of the tight junction (10). in a rather narrow range, 5 to 18 Å, for most natural epithelia Electrical resistance methods model an epithelial monolayer as and cultured epithelial monolayers (6,16). One interpretation of a circuit of parallel resistors composed of all the individual the variable resistance and fixed size is that the barrier includes transcellular and paracellular elements. Because the total re- a variable number of aqueous channels of predetermined size. sistance is a function of the inverse sums of individual resis- ϭ ϩ Charge selectively of the paracellular pathway also varies little tances, (1/Rtotal 1/R1 1/R2 ..), TER is dominated by among different epithelia and in most cases shows a slight elements with the lowest resistance. Since membrane resis- cation selectivity. For example, the relative ion permeabilities tance is generally much higher than intercellular resistance, it Ϫ for the dog proximal renal tubule for (PKϩ: PNaϩ : PCl ) are is usually safe to assume that a change in resistance after an (1.10 : 1.00 : 0.72), and for the rabbit ileum are (1.14 : 1.00 : experimental manipulation represents a change in the paracel- 0.55) (6). This small cation selectivity suggests that the para- lular pathway (16). Unfortunately, this method is excessively cellular pathway (the tight junction or lateral interspace) is sensitive to small regions with low resistance, such as crushed predominantly lined by negative charges. The very limited cells near the edge of a mounting apparatus or a patch of dead ability to discriminate charge is also consistent with the pres- cells within the monolayer. Another problem is that the lateral ence of large aqueous channels within the tight junction bar- interspace also contributes to the paracellular resistance, di- rier. This lack of paracellular discrimination can again be rectly by its length and inversely by its width. Both of these contrasted with the character of transcellular transport, for dimensions can be affected by seemingly trivial factors, such example, the exquisite molecular selectively of Kϩ channels, as changing culture media or the time a cultured epithelial amino acid transporters, or anion exchangers. monolayer has been in culture (1). J Am Soc Nephrol 10: 1337–1345, 1999 Transmembrane Proteins in the Tight Junction 1339

Permeability, measured by tracer flux, is linearly propor- paracellular barrier. This is seen following both experimental tional to the area across which diffusion occurs. Thus, it is manipulations of cultured epithelial monolayers and in disease theoretically less sensitive to trivial defects in the monolayer states in natural epithelia (2,20). If each row of intramembrane and more reliably reports changes induced in the junction by proteins creates a fixed resistive barrier positioned along the experimental manipulations. Flux has the added advantage that intercellular space, then the overall electrical resistance is small changes in the tight junction barrier can be distinguished predicted to be linearly proportional to the number of barriers. from defects in the monolayer by the ability of the monolayer In contrast, Claude noted that fibril number was actually pro- to maintain an upper size limit for diffusion. As future exper- portional to the logarithm of the paracellular resistance when iments attempt to define the specific contribution of transmem- comparing epithelia throughout the body (21). She proposed brane proteins to the tight junction barrier, the limitations of that the empirical data were better explained by a model that presently available methods should be taken into account. assumed the fibrils were not fixed barriers but contained indi- Consideration should be given to the less commonly used vidual channels that flicker open and closed (see reference (21) methods of alternating current impedance (17) and microelec- for mathematical justification). A solute molecule traversing trode surface scanning conductance (18) or, ideally, a combi- the junction would need to move in a stepwise manner along a nation of several complementary methods. series of intercellular spaces enclosed by fibrils. Passage be- tween each fibril-enclosed space would require an open chan- Structure–Function Correlation nel (Figure 2). The molecular basis for this behavior remains to Claude and Goodenough (19) were the first to point out a be determined. In addition, there are exceptions to this simple correlation between the ultrastructure of tight junctions and structure–function correlation, suggesting that other factors their permeability as defined experimentally in natural epithe- also contribute to defining the magnitude of the barrier. lia. In transmission electron micrographic images, the tight junction appears as several points of intercellular membrane Transmembrane Components of the contact at the apical end of the lateral interspace. In freeze Tight Junction fracture images, these contacts correspond to fibril-like rows of The discussion above supports the existence of both sealing intramembrane particles that form a continuous branching net- and channel elements within the tight junction, both of which work around the cell (Figure 2). Evidence that the fibrils are may be regulated. Many tight junction proteins have now been the sites of resistance comes from numerous observations in identified (Figure 1), and most of these are located in the which disruption of the fibrils correlates with loss of the cytoplasmic plaque under cell–cell contacts. Some of these are unique to the tight junction, whereas others are also located elsewhere in the cell. Several categories of transmembrane proteins have been identified that appear to be unique to tight junctions. Because they extend into the paracellular space, they are candidates for creating the seal and channels. Most exper- imental evidence addresses the role of occludin, the first of these proteins to be identified.

Occludin Occludin is an approximately 65-kD type II transmembrane protein composed of four transmembrane domains, two extra- cellular loops, and a large C-terminal cytosolic domain (22) (Figure 3). This topology has been confirmed by antibody accessibility studies (23). The extracellular loops are chemi- cally quite distinctive, particularly the first, which contains approximately 65% tyrosine and glycine residues. Although the presence of alternating tyrosine and glycine residues is conserved in all five animal species presently cloned, the functional significance of this peculiar sequence is unclear (24). Figure 2. Solute molecules (X) attempting to traverse the tight junc- When observed by immuno-freeze fracture electron micros- tion encounter a series of fibril barriers (composed of occludin and copy, occludin is concentrated directly within the tight junction claudin). Passage across each fibril requires encountering an open fibrils (24). Interestingly, immunofluorescence localization re- channel (E) among a continuous barrier of closed channels (F). Channels open and close with a defined probability. The molecule at veals an additional minor pool of occludin along the lateral the far right has successfully passed through all of the fibril-enclosed membrane that is more easily extracted in nonionic detergents, spaces. Such a hypothetical model explains the logarithmic, rather less phosphorylated, and not assembled into fibrils (25,26). than linear, relationship between the number of tight junction fibrils Conceivably, the lateral pool represents a reservoir of subunits and net paracellular permeability. available for dynamic regulated expansion of junctional com- 1340 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1337–1345, 1999

Figure 3. The three known transmembrane proteins of the tight junction. Occludin and claudin(s) lack sequence homology but are both type II, four-membrane spanning proteins located specifically within the cell-to-cell contacts of the junction. JAM is a type I protein and a member of the Ig superfamily of single spanning proteins. Occludin and JAM are known to confer cell–cell adhesion. plexity. The capacity for rapid increases in fibril number was immunofluorescence staining and immunoblotting. Not sur- demonstrated in experiments predating the discovery of occlu- prising, forced expression of mutated occludin can produce a din. These include the observation that when trypsin is applied dominant-negative effect on barrier properties. For example, to the basolateral surface of cultured Madin-Darby canine expression in Xenopus embryos of truncated occludin con- kidney (MDCK) cells there follows a dramatic and rapid in- structs lacking the large C-terminal cytoplasmic domain results crease in fibril number and increase in transmonolayer electri- in increased permeability of tight junctions to a biotin tracer cal resistance (27). The role of occludin phosphorylation and molecule (28). However, expression of similar constructs in functional significance of the extrajunctional occludin in fibril MDCK cells resulted in increases in TER similar to the in- dynamics deserves investigation. Although the junctional pool crease seen with expression of full-length occludin (29,30). appears to be more phosphorylated, there is no proven causal Although overexpression of C-terminal truncated occludin relationship with assembly, at this time. in MDCK monolayers increases the electrical barrier, it also Occludin is a Ca2ϩ-independent intercellular adhesion mol- disrupts the intramembrane barrier and allows diffusion of ecule (23). When expressed in fibroblasts, which lack endog- lipids between the apical and basolateral membrane domains enous occludin, it confers adhesiveness in proportion to the (29). Dissociation of the intercellular (gate) and intramembrane level of occludin expressed and the adhesiveness is blocked by (fence) barriers was previously observed to occur in MDCK peptides corresponding to either of the two extracellular loops. monolayers in response to acute ATP depletion (32). However, It remains to be determined whether occludin is a homotypic in the latter situation the membrane polarity barrier remains adhesion molecule or has a yet unidentified counter-receptor. after the paracellular barrier is lost. Hypothetically, the two Occludin is also capable of lateral oligomerization through barriers could be based on lateral and cell–cell adhesive con- side-to-side associations, perhaps within the membrane bilayer tacts, respectively, and these are differentially affected in the (28). transfection and ATP-depletion experiments. Given its adhesive properties, occludin might create a para- A major surprise was the finding that while overexpression cellular barrier by polymerizing laterally in the membrane to of occludin in cultured MDCK cells increases the electrical create a continuous line of adhesion between cells. Several barrier, it simultaneously decreases the barrier to noncharged experiments are at least partly consistent with this model. For solutes like mannitol and dextrans (29,30). This suggests that example, when occludin levels are increased in cultured mono- the barriers for charged and noncharged species are somehow layers of MDCK cells through transfection, this results in an different. This seemingly paradoxical effect is even more pro- increase in the number of fibrils and an increase in transmono- nounced when an occludin construct lacking the C-terminal layer electrical resistance (29,30). Conversely, a decrease in cytoplasmic domain is expressed in cultured monolayers (29). transmonolayer electrical resistance and an increase in perme- Whatever the molecular explanation, occludin appears to be ability to tracer molecules can be induced by adding a synthetic more than a barrier-forming glue, and may instead act more peptide corresponding to the second extracellular loop of oc- like the regulated channel protein predicted by Claude (21). A cludin to monolayers of Xenopus A6 cells (31). The soluble greater understanding of occludin function will require detailed peptide is presumably interfering with occludin’s extracellular structure–function analysis of mutant forms of occludin ex- contacts. This effect requires several days, and loss of the pressed in cultured cells or transgenic animals. barrier coincides with loss of occludin as detected by both Since its identification, multiple lines of evidence suggested J Am Soc Nephrol 10: 1337–1345, 1999 Transmembrane Proteins in the Tight Junction 1341 that occludin was not the only integral membrane protein in the cytoplasmic protein ZO-1 has two major alternatively spliced tight junction. Overexpression of an occludin construct that isoforms; one is expressed in epithelial cells and the other in lacks the C-terminal cytosolic domain results in a dramatically endothelial cells (37,38). Another cytoplasmic protein, 7H6, discontinuous pattern of both the endogenous and exogenous appears to be restricted to epithelial cell tight junctions (39). occludin polypeptides, visualized at the light microscopic level Presumably, these structural differences will be found to un- (29). Despite this, there is no parallel effect on the number and derlie functional diversity. organization of tight junction fibrils visualized by freeze frac- Several claudins were previously cloned and characterized ture methods at the ultrastructural level. Furthermore, although without the investigators’ realizing that they were part of the the addition of a synthetic peptide corresponding to the second tight junction. Among these is claudin-4, previously referred to extracellular loop to cultured A6 cells results in the disappear- as the murine Clostridium perfringens enterotoxin receptor ance of occludin from the plasma membrane, it does not appear (CPE-R), which is 46.9% identical to claudin-1 (40). The to alter gross cell morphology or affect the distribution of other CPE-R is the high-affinity receptor for an enterotoxin produced proteins of the apical junction complex, such as ZO-1 (31). The by the intestinal pathogen Clostridium perfringens (41). Al- most direct implication for the existence of a second barrier though the mechanism of action is not fully known, binding of protein came with the observation that embryonic stem cells, toxin to the CPE-R results in formation of cytotoxic pores in from which occludin was deleted through homologous recom- the plasma membrane (42). It is not clear whether the CPE-R bination, could still establish tight junctions with characteristic is a component of the pore or simply serves as a receptor for freeze fracture fibrils, and a paracellular barrier to low molec- the toxin. A more distant branch of the family is represented by ular weight tracers (33). oligodendrocyte-specific protein (OSP) (43), which is 33.8% identical to claudin-1. This claudin was originally character- Claudin ized as a component of myelin in the mouse central nervous Two additional integral membrane proteins of the junction system (43). Interestingly, rows of freeze fracture fibrils are were recently identified by direct biochemical fractionation of found in Schwann cell membranes where they spiral around junction-enriched membranes from chicken liver (34). After axons. Although the subcellular location of OSP in Schwann removal of peripherally attached proteins with guanidine fol- cells is unknown, it is tempting to speculate that OSP is the lowed by sonication and stepwise sucrose density gradient glial equivalent of epithelial claudin. Located in fibrils, it could centrifugation, claudin-1 and claudin-2 (from the Latin clau- function to maintain the electrically resistive wrapping re- dere meaning to close) were found to copurify with occludin as quired to accelerate electrical conduction along the nerve. a broad approximately 22-kD gel band on sodium dodecyl Clearly, much remains to be learned about the claudins, their sulfate-polyacrylamide gel electrophoresis. Peptide microse- role in forming variable barrier properties, and their potential quencing permitted cloning of these two closely related pro- role in diseases affecting the barrier. teins from a mouse cDNA library. The deduced sequences predict four transmembrane helices, two short extracellular Junction Adhesion Molecule loops, and short cytoplasmic N- and C-termini (Figure 3). The recently identified junctional adhesion molecule (JAM) Despite topologies similar to that of occludin, they share no is a novel member of the Ig superfamily and localizes to the sequence homology. Subsequently, six more claudin gene tight junctions of both epithelial and endothelial cells (44). The products were identified based on homology to sequences in resolution of ultrastructural localization is presently inadequate the expressed sequence tagged database (35). These have now to determine whether JAM is located specifically within the all been cloned and have been shown to localize within tight fibrils with occludin and the claudins, but it is at least limited junction fibrils, as determined by immunogold freeze fracture to the membrane regions containing fibrils. A cDNA encoding labeling (35). No further functional data are available; how- JAM predicts an approximately 32-kD type I transmembrane ever, given that a barrier remains in the absence of occludin, protein with two extracellular Ig-like domains and two sites for they must be considered candidates for the primary seal-form- N-glycosylation (Figure 3). Experimental evidence suggests ing elements of the extracellular space. Consistent with this that JAM can mediate both homotypic adhesion and influence role, when either claudin-1 or -2 is expressed in fibroblasts, monocyte transmigration. When JAM is expressed in COS these proteins are capable of assembling into long branching cells, which do not have tight junctions and do not normally fibrils reminiscent of their organization in the tight junction of express this protein, it accumulates at sites of cell–cell contact epithelial cells. In contrast, occludin has a limited ability to and induces cell–cell adhesion. Addition of a monoclonal self-organize into fibrils in transfected fibroblasts, but will join antibody against JAM to the media bathing cultured endothe- the fibrils when claudin is cotransfected (36). lial cells inhibits both spontaneous and chemokine-induced Interestingly, the eight known claudins show strikingly dif- transmigration of monocytes. This observation suggests that ferent mRNA expression patterns among different tissues, sug- JAM might function in normal transmigration across epithelial gesting that they may contribute to functional differences monolayers, perhaps by providing an adhesive contact required among different epithelia (35). Consistent with the idea that for monocytes to find the intercellular pathway (analogous to a differential expression of junction proteins may have func- homing receptor), or by transducing a signal within the endo- tional implications, other biochemical differences among tight thelial cell to open the junctional space. Previous studies have junctions have already been documented. For example, the demonstrated that a large flux of immune cells can traverse 1342 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1337–1345, 1999 such a monolayer without decreasing the transepithelial elec- contacts (C. Van Itallie, unpublished results), suggesting that trical resistance (45). This raises the possibility that JAM, or a interaction with ZO-1 is required for localization to specific similar protein, creates a seal with the immune cell as it passes sites on the plasma membrane. Consistent with this idea is the through the junction. JAM is the first molecular component of observation that when this domain is fused to the nonjunctional tight junctions that has been demonstrated to have a direct role protein connexin-32, the chimeric protein becomes positioned in the regulation of immune cell transport across the paracel- precisely within junctional fibrils (62). In contrast, a C-termi- lular seal and should provide important insights into the inter- nal truncated occludin is still recruited into tight junctions in action between immune cells, epithelia, and endothelia. stably transfected MDCK lines, suggesting that lateral interac- tions with full-length occludin are also sufficient to promote localization (29). Binding to ZO-1 is therefore sufficient but Assembly and Scaffolding of the Paracellular not necessary to assemble occludin into tight junctions, and the Sealing Proteins ZO-1/occludin link may be responsible for fine-tuning struc- For the transmembrane proteins to provide a paracellular tural aspects of the junction, such as creation of continuous seal, they must be properly targeted to and positioned at the fibrils. Although there is presently no evidence suggesting how tight junction. Assembly, scaffolding, and regulation of the claudin or JAM localizes to the junction, it is noteworthy that paracellular seal are presumably accomplished by the cytosolic both proteins have C-terminal motifs predicted to bind PDZ plaque of proteins associated with the inner surface of tight domains like those found in the ZO proteins. junction contacts (Figure 1). Among the presumed structural proteins are ZO-1 (46), ZO-2 (47,48), ZO-3 (49), cingulin (50), Perijunctional Actin 7H6 (39), and symplekin (51). The last three have no known Throughout the cell, actin filaments are known to serve both function, binding interactions, or informative amino acid se- structural and dynamic roles. Presumably, this is also true for quence homologies. Several presumed signaling proteins have the thick band of bipolar actin filaments positioned under the also been localized at the resolution of light microscopy to the apical junction complex. Individual filaments can be observed apical junction complex, including several heterotrimer G- in ultrastructural images to terminate at the membrane proteins (52,53), an isoform of PKC (53), vertebrate homologs (53,63,64) directly at the points where occludin and claudin are of the yeast secretory mutants Sec6 and Sec8 (54), and the focused into fibrils. ZO-1 binds to actin filaments, although it nonreceptor tyrosine kinases src and yes (55). The Ras-binding has not been resolved whether this is direct or through a linking protein AF6 (18) and ZA-kinase (56) both bind to ZO-1 but protein. ZO-1 also binds the cytoplasmic tail of occludin have not yet been immunolocalized at the ultrastructural level. (58,61,65,66) and thus is presently the best candidate for The potential role of intracellular signaling pathways in regu- coupling both structural and dynamic properties of perijunc- lating junctional properties has been frequently reviewed tional actin to the paracellular barrier. (2,57), although the specific molecular targets controlling bar- Several studies suggest that the ability of the tight junction rier function remain largely unknown. to form a seal is dependent on the structural organization of Occludin binds directly to ZO-1 (58), ZO-2 (B. R. Steven- actin. Treatment of cultured epithelial cells with the actin- son, unpublished observation), and ZO-3 (49), and there is disrupting drug cytochalasin causes a decrease in the size and evidence that the interaction with ZO-1 recruits and organizes complexity of tight junction fibrils (67–70). There is also occludin at the junction. ZO-1, like its homologues ZO-2 and considerable correlation between experimentally induced ZO-3, is a member of the MAGUK protein family (membrane- changes in permeability and actin filament organization. For associated guanylate kinase homologues) (48,49,59). Other example, experimentally reducing cellular ATP levels results members of the MAGUK family are clearly responsible for in altered tight junction structure, decreased transepithelial tethering transmembrane protein at specific sites of cell–cell electrical resistance, and an increased association of tight junc- contact or to specific cell membrane domains. Coupling is tion proteins like ZO-1 and ZO-2 with the actin cytoskeleton typically based on interaction between a protein binding do- (71,72). Similarly, changes in paracellular permeability in- main in the MAGUK protein, called a PDZ domain, and a short duced by the Vibrio cholerae toxin ZOT are also associated specific peptide motif at the extreme C terminus of the trans- with a reorganization of perijunctional actin (73). Finally, membrane protein (60). However, in the case of ZO-1, the activation of small GTP binding proteins of the rho family interaction with occludin is not mediated by a PDZ domain trigger a marked reorganization of perijunctional actin (74), (61). Assuming this is also true for ZO-2 and ZO-3, then the which coincides with a dramatic increase in permeability of nine total PDZ domains present in ZO-1, -2, and -3 are avail- tight junctions (75). Because such manipulations result in able to recruit and coordinate presently unknown proteins widespread alterations to the actin cytoskeleton, these obser- under the junctional contact. vations must be interpreted with caution. However, they sug- The mechanisms responsible for occludin targeting have gest that overall cytoskeletal organization is crucial to tight also been examined in numerous transfection studies. When junction integrity and could be coupled through ZO-1 and full-length occludin is expressed in fibroblasts that normally do regulated by cellular signaling pathways. not contain tight junctions, it is targeted to sites of cell–cell A dynamic role for the perijunctional actin ring is supported contact that contain ZO-1 (23). Occludin lacking the C-termi- by its ability to undergo circumferential contraction (17,76). nal ZO-1 binding domain fails to target to these cell–cell Contraction may be coupled to physiologically relevant sig- J Am Soc Nephrol 10: 1337–1345, 1999 Transmembrane Proteins in the Tight Junction 1343 nals. For example, studies in both isolated tissues and cultured 7. Spring KR: Routes and mechanism of fluid transport by epithe- cells have demonstrated that activation of the sodium/glucose lia. Annu Rev Physiol 60: 105–119, 1998 transporter leads to cytoskeletal contraction and a concurrent 8. Denker BM, Nigam SK: Molecular structure and assembly of the increase in paracellular permeability (17,77,78). In cultured tight junction. Am J Physiol 274: F1–F9, 1998 cells, this has been shown to occur in concert with the phos- 9. Cerijido M, Valdes J, Shoshani L, Contreras RG: Role of tight junctions in establishing and maintaining cell polarity. Annu Rev phorylation of myosin light chain, which is known to activate Physiol 60: 161–177, 1998 nonmuscle myosin contractility (79). In addition, Hecht et al. 10. Yap AS, Mullin JM, Stevenson BR: Molecular analyses of tight (80) have shown that the introduction of a constitutively acti- junction physiology: Insights and paradoxes. J Membr Biol 163: vated myosin light chain kinase into cultured MDCK cells 159–167, 1998 results in a contraction of the cortical cytoskeleton. This is 11. Madara JL: Regulation of the movement of solutes across tight associated with a decreased TER and increased paracellular junctions. Annu Rev Physiol 60: 143–159, 1998 flux (80). These observations suggest that the force generated 12. Molitoris BA: Ischemia-induced loss of epithelial polarity: Po- by actomyosin contraction can somehow be transmitted to the tential role of the actin cytoskeleton. Am J Physiol 260: F769– tight junction and cause a transient physical change in the F778, 1991 paracellular space. Such changes could be based on changes in 13. Molitoris BA, Falk SA, Dahl R: Ischemic-induced loss of epi- cell–cell or lateral organization of occludin and claudin, me- thelial polarity: Role of the tight junction. J Clin Invest 84: 1334–1339, 1989 diated through ZO-1. 14. Molitoris BA: New insights into the cell biology of ischemic acute renal failure. J Am Soc Nephrol 1: 1263–1270, 1991 Future Directions 15. Weinberg JM: The cell biology of ischemic renal injury. Kidney Much work remains to define the individual contributions of Int 39: 476–500, 1991 occludin, the claudins, and JAM in regulating paracellular 16. Reuss L: Tight junction permeability to ions and water. In: Tight permeability, permselectivity, and immune cell transmigration. Junctions, edited by Cereijido M, Boca Raton, CRC Press, 1991, In addition, there may also be other uncharacterized transmem- p49 brane proteins within the tight junction. For example, does the 17. Madara JL, Pappenheimer JR: The structural basis for physio- large combinatorial diversity of their expression of claudins logical regulation of paracellular pathways in intestinal epithelia. lead to physiologic differences among cell types? Organizing J Membr Biol 100: 149–164, 1987 18. Gitter AH, Bertog M, Schulzke J-D, Fromm M: Measurement of the transmembrane proteins is obviously a key aspect in de- paracellular epithelial conductivity by conductance scanning. fining the barrier, and the role of plaque proteins in assembly Pflu¨gers Arch 434: 830–840, 1997 and scaffolding requires more study. Finally, it may now be 19. Claude P, Goodenough DA: Fracture faces of zonulae occluden- possible to address whether junctions are physiologically reg- tes from “tight” and “leady” epithelia. J Cell Biol 58: 390–400, ulated in an acute manner to control transepithelial transport. 1973 Identification of transmembrane components of the tight junc- 20. Madara JL, Parkos C, Colgan S, Nusrat A, Atisook K, Kaoutzani tion has provided new tools with which to examine the molec- K, Kaoutzani P: The movement of solutes and cells across tight ular basis for paracellular permeability. Cytoplasmic signaling junctions. Ann NY Acad Sci 664: 47–60, 1992 pathways can clearly affect the barrier, and we are now set to 21. Claude P: Morphological factors influencing transepithelial per- reinterpret many past experiments in terms of the effects of meability: A model for the resistance of the zonula occludens. these pathways on the transmembrane proteins. J Membr Biol 39: 219–232, 1978 22. Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S: Occludin: A novel integral membrane protein localizing at Acknowledgments tight junctions. J Cell Biol 123: 1777–1788, 1993 The authors are supported by National Institutes of Health Grants 23. Van Itallie CM, Anderson JM: Occludin confers adhesiveness DK45134, DK38979, DK34989, and CA66263. We thank Dr. Chris- when expressed in fibroblasts. J Cell Sci 110: 1113–1121, 1997 tina M. Van Itallie for valuable suggestions. 24. Fujimoto K: Freeze-fracture replica electron microscopy com- bined with SDS digestion for cytochemical labeling of integral References membrane proteins: Application to the immunogold labeling of 1. Cereijido M: Evolution of ideas on the tight junction. In: Tight intracellular junctional complexes. J Cell Sci 108: 3443–3449, Junctions, eduted bt Cereijido M, Boca Raton, CRC Press, 1992, 1995 p1 25. Sakakibara A, Furuse M, Saitou M, Ando-Akatsuka Y, Tsukita 2. Anderson JM, Van Itallie CM: Tight junctions and the molecular S: Possible involvement of phosphorylation of occludin in tight basis for regulation of paracellular permeability. Am J Physiol junction formation. J Cell Biol 137: 1393–1401, 1997 269: G467–G475, 1995 26. Cordenonsi M, Mazzon E, DeRigo L, Baraldo S, Meggio F, Citi 3. Balda MS, Matter K: Tight junctions. J Cell Sci 111: 541–547, S: Occludin dephosphorylation in early development of Xenopus 1998 laevis. J Cell Sci 110: 3131–3139, 1997 4. Tsukita S, Furuse M, Itoh M: Molecular dissection of tight 27. Lynch RD, Tkachuk-Ross LJ, McCormack JM, McCarthy KM, junctions. Cell Struct Funct 21: 381–385, 1996 Rogers RA, Schneeberger EE: Basolateral but not apical appli- 5. Mitic L, Anderson JM: Molecular architecture of tight junctions. cation of protease results in a rapid rise of transepithelial elec- Annu Rev Physiol 60: 121–141, 1998 trical resistance and formation of aberrant tight junction strands 6. Powell DW: Barrier function of epithelia. Am J Physiol 241: in MDCK cells. Eur J Cell Biol 66: 257–267, 1995 G275–G288, 1981 28. Chen Y, Merzdorf C, Paul DL, Goodenough DA: COOH termi- 1344 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1337–1345, 1999

nus of occludin is required for tight junction barrier function in intercellular junctions and modulates monocyte transmigration. early Xenopus embryos. J Cell Biol 138: 891–899, 1997 J Cell Biol 142: 117–127, 1998 29. Balda MS, Whitney JA, Flores C, Gonzalez S, Cereijido M, 45. Dejana E, Corada M, Lampugnani M-G: Endothelial cell-to-cell Matter K: Functional dissociation of paracellular permeability junctions. FASEB J 9: 910–918, 1995 and transepithelial electrical resistance and disruption of the 46. Stevenson BR, Silicano JD, Mooseker M, Goodenough DA: apical-basolateral intramembrane diffusion barrier by expression Identification of ZO-1: A high molecular weight polypeptide of a mutant tight junction membrane protein. J Cell Biol 134: associated with the tight junction (zonula occludens) in a variety 1031–1049, 1996 of epithelia. J Cell Biol 103: 755–766, 1986 30. McCarthy KM, Skare IB, Stankewich MD, Furuse M, Tsukita S, 47. Gumbiner B, Lowenkopf T, Apatira D: Identification of 160-kDa Rogers RA, Lynch RD, Schneeberger EE: Occludin is a func- polypeptide that binds to the tight junction protein ZO-1. Proc tional component of the tight junction. J Cell Sci 109: 2287– Natl Acad Sci USA 88: 3460–3464, 1991 2298, 1996 48. Jesaitis LA, Goodenough DA: Molecular characterization and 31. Wong V, Gumbiner B: Synthetic peptide corresponding to the tissue distribution of ZO-2, a tight junction protein homologous extracellular domain of occludin perturbs the tight junction per- to ZO-1 and the Drosophila discs-large tumor suppressor protein. meability barrier. J Cell Biol 136: 399–409, 1997 J Cell Biol 124: 949–961, 1994 32. Mandel LJ, Bacallao R, Zampighi G: Uncoupling of the molec- 49. Haskins J, Gu L, Wittchen ES, Hibbard J, Stevenson BR: ZO-3, ular “fence” and paracellular “gate” functions in epithelial tight a novel member of the MAGUK protein superfamily found at the junctions. Nature 361: 552–555, 1993 tight junction, interacts with ZO-1 and occludin. J Cell Biol 141: 33. Saitou M, Fujimoto K, Doi Y, Itoh M, Fujimoto T, Furuse M, Takano H, Noda T, Tsukita S: Occludin-deficient embryonic 199–208, 1998 stem cells can differentiate into polarized epithelial cells bearing 50. Citi S, Sabannay H, Jakes R, Geiger B, Kendrich-Jones J: Cin- tight junctions. J Cell Biol 141: 397–408, 1998 gulin, a new peripheral component of tight junctions. Nature 34. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S: Claudin-1 333: 272–275, 1988 and -2: Novel integral membrane proteins localizing at tight 51. Keon BH, Schafer S, Kuhn C, Grund C, Franke WW: Symplekin, junctions with no sequence similarity to occludin. J Cell Biol a novel type of tight junction plaque protein. J Cell Biol 134: 141: 1539–1550, 1998 1003–1018, 1996 35. Morita K, Furuse M, Fujimoto K, Tsukita S: Claudin multigene 52. Denker BM, Saha C, Khawaja S, Nigam SK: Involvement of a family encoding four-transmembrane domain protein compo- heterotrimeric G protein alpha subunit in tight junction biogen- nents of tight junction strands. Proc Natl Acad Sci USA 96: esis. J Biol Chem 271: 25750–25753, 1996 511–516, 1999 53. Dodane V, Kachar B: Identification of isoforms of G proteins 36. Furuse M, Sasaki H, Fujimoto K, Tsukita S: A single gene and PKC that colocalize with tight junctions. J Membr Biol 149: product, claudin-1 or -2, reconstitutes tight junction strands and 199–209, 1996 recruits occludin in fibroblasts. J Cell Biol 143: 391–401, 1998 54. Grindstaff KK, Yeaman C, Anandasabapathy N, Hsu S-C, Ro- 37. Fallon MB, Brecher A, Balda MS, Matter K, Anderson JM: driguez-Boulan E, Scheller RH, Nelson WJ: Sec6/8 complex is Altered expression and localization of the tight junction proteins recruited to cell-cell contacts and specifies transport vesicle occludin and ZO-1 after common bile duct ligation. Am J Physiol delivery to the basal-lateral membrane in epithelial cells. Cell 93: 260: C1057–C1062, 1995 731–740, 1998 38. Bhanot P, Brink M, Samos CH, Hsieh J-C, Wang Y, Macke JP, 55. Tsukita S, Oishi K, Akiyama T, Yamanashi Y, Yamamoto T: Andrew D, Nathans J, Nusse R: A new member of the frizzled Specific proto-oncogenic tyrosine kinases of src family are en- family from Drosophila functions as a Wingless receptor. Nature riched in cell-to-cell adherens junctions where the level of ty- 382: 225–230, 1996 rosine phosphorylation is elevated. J Cell Biol 113: 867–879, 39. Zhong Y, Saitoh T, Minase T, Sawada N, Enomoto K, Mori M: 1991 Monoclonal antibody 7H6 reacts with a novel tight junction- 56. Balda MS, Anderson JM, Matter K: The SH3 domain of the tight associated protein distinct from ZO-1, cingulin and ZO-2. J Cell junction protein ZO-1 binds to a serine protein kinase that Biol 120: 477–483, 1993 phosphorylates a region C-terminal to this domain. FEBS Lett 40. Anderson JM: Hepatocyte tight junctions in health and disease. 399: 326–332, 1996 In: Progress in Liver Diseases, edited by Boyer JL, Philadelphia, 57. Denker BM, Nigam SK: Molecular structure and assembly of the Saunders, 1993, p 45 tight junction. Am J Physiol 274: F1–F9, 1998 41. Katahira J, Inoue N, Horiguchi Y, Matsuda M, Sugimoto N: Molecular cloning and functional characterization of the receptor 58. Furuse M, Itoh M, Hirase T, Nagafuchi F, Yonemura S, Tsukita for Clostridium perfringens enterotoxin. J Cell Biol 136: 1239– S: Direct association between occludin and ZO-1 and its possible 1247, 1997 involvement in the localization of occludin at tight junctions. 42. McClane BA: Clostridium perfringens enterotoxin acts by pro- J Cell Biol 127: 1617–1626, 1994 ducing small molecule permeability alterations in plasma mem- 59. Willott E, Balda MS, Fanning AS, Jameson B, Van Itallie C, branes. Toxicology 87: 43–67, 1994 Anderson JM: The tight junction protein ZO-1 is homologous to 43. Bronstein JM, Popper P, Micevych PD, Fraber DB: Isolation and the Drosophila discs-large tumor suppressor protein of septate characterization of a novel oligo dendrocyte-specific protein. junctions. Proc Natl Acad Sci USA 90: 7834–7838, 1993 Neurology 47: 778, 1996 60. Fanning AS, Anderson JM: PDZ domains and the formation of 44. Martin-Padura I, Lostaglio S, Schneemann M, Williams L, Ro- protein networks at the plasma membrane. In: Current Topics in mano M, Fruscella P, Panzeri C, Stoppacciaro A, Ruco L, Villa Microbiology and Immunology: Protein Modules in Signal A, Simmons D, Dejana E: Junctional adhesion molecule, a novel Transduction, edited by Pawson AJ, Heidelburg, Springer-Ver- member of the immunoglobulin superfamily that distributes at lag, 1998, p 209 J Am Soc Nephrol 10: 1337–1345, 1999 Transmembrane Proteins in the Tight Junction 1345

61. Fanning AS, Jameson B, Jesaitis LA, Anderson JM: The tight epithelial tissues. I. Rearrangement of the actin cytoskeleton. junction protein ZO-1 establishes a link between the transmem- J Cell Sci 107: 3301–3313, 1994 brane protein occludin and the actin cytoskeleton. J Biol Chem 72. Tsukamoto T, Nigam SK: Tight junction proteins form large 273: 29745–29753, 1998 complexes and associate with the cytoskeleton in an ATP deple- 62. Mitic L, Schneeberger EE, Fanning AS, Anderson JM: Interac- tion model for reversible junction assembly. J Biol Chem 272: tion with ZO-1 organizes connexin-occludin chimeras at tight 16133–16139, 1997 junctions [Abstract]. Mol Biol Cell 8: 205a, 1997 73. Fasano A, Florentini C, Gianfranco D, Kaper JB, Margaretten K, 63. Dehar S, Hannigan GE: Integrin cytoplasmic interactions and Ding X, Guandalini S, Comstock L, Goldblum GE: Zonula bidirectional transmembrane signalling. Curr Opin Cell Biol 8: occludens toxin (ZOT) modulates tight junctions through protein 657–669, 1996 kinase C-dependent actin reorganization in vitro. J Clin Invest 64. Madara JL: Intestinal absorptive cell tight junctions are linked to 96: 710–720, 1995 cytoskeleton. Am J Physiol 253: C171–C175, 1987 74. Nusrat A, Giry M, Turner JR, Colgan SP, Parkos CA, Carnes D, 65. Itoh M, Nagafuchi A, Moroi S, Tsukita S: Involvement of ZO-1 Lemichez E, Boquet P, Madara JL: Rho protein regulates tight in cadherin-based adhesion through its direct binding to a catenin junctions and perijunctional actin organization in polarized epi- and actin filaments. J Cell Biol 138: 181–192, 1997 thelia. Proc Natl Acad Sci USA 92: 10629–10633, 1995 66. Fugimoto K, Nagafuchi A, Tsukita S, Kuraoka A, Ohokuma A, 75. Jou T-S, Schneeberger EE, Nelson WJ. Structural and functional Shibata Y: Dynamics of connexins, E-cadherin, and a-catenin on regulation of tight junctions by RhoA, and Rac1 small GTPases. cell membranes during gap junction formation. J Cell Sci 110: J Cell Biol 142: 101–115, 1998 311–322, 1997 76. Keller TCS, Mooseker MS: Ca2ϩ-calmodulin dependent phos- 67. Bentzel CJ, Hainau B, Ho S, Hui SW, Edelman A, Anagnosto- phorylation of myosin, and its role in brush border contraction in poulos T, Benedetti EL: Cytoplasmic regulation of tight-junction vitro. J Cell Biol 95: 943–959, 1982 permeability: Effect of plant cytokinins. Am J Physiol 239: C75–C89, 1980 77. Pappenheimer JR: Physiological regulation of transepithelial im- 68. Madara JL, Barenberg D, Carlson S: Effects of cytochalasin D on pedance in the intestinal mucosa of rats and hamsters. J Membr occluding junctions of intestinal absorptive cells: Further evi- Biol 100: 136–148, 1987 dence that the cytoskeleton may influence paracellular perme- 78. Turner JR, Madara JL: Physiological regulation of intestinal ϩ ability and junctional charge selectivity. J Cell Biol 102: 2125– epithelial tight junctions as a consequence of Na( )-coupled 2136, 1986 nutrient transport. Gastroenterology 109: 1391–1396, 1995 69. Nybom P, Magnusson KE: Modulation of the junctional integrity 79. Turner JR, Rill BK, Carlson SL, Carnes D, Kerner R, Mrsny RJ, by low or high concentrations of cytochalasin B and dihydrocy- Madara JL: Physiological regulation of epithelial tight junctions tochalasin B is associated with distinct changes in F-actin and is associated with myosin light-chain phosphorylation. Am J ZO-1. Biosci Rep 16: 313–326, 1996 Physiol 4: C1378–C1385, 1997 70. Stevenson BR, Begg DA: Concentration-dependent effects of 80. Hecht G, Pestic L, Nikcevic G, Koutsouris A, Tripuraneni J, cytochalasin D on tight junctions and actin filaments in MDCK Lorimer DD, Nowak G, Guerriero V Jr, Elson EL, Lanerolle PD: epithelial cells. J Cell Sci 107: 367–375, 1994 Expression of the catalytic domain of myosin light chain kinase 71. Bacallao R, Garfinkel A, Monke S, Zampighi G, Mandel LJ: increases paracellular permeability. Am J Physiol 271: C1678– ATP depletion: A novel method to study junctional properties in C1684, 1996