Occldin and the Functions of Tight Junctions

Occludin and the Functions of Tight Junctions Karl Matter and Maria S. Balda Department of Cell Biology, University of Geneva, Geneva, Switzerland

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The tight junction or zonula occludens is the most apical structure of the epithelial junctional complex. Tight junctions form semipermeable intercellular diffusion barriers that control paracellular diffusion in a regulated manner. This intercellular junction also acts as an intramembrane fence that prevents the intermixing of apical and basolateral lipids in the exocytoplasmic leaflet of the plasma membrane. Moreover, evidence suggests that tight junction components participate in the regulation of cell growth and differentiation. Occludin was the first identified transmembrane protein of this intercellular junction and received much attention since its molecular characterization.This review discusses experiments that were done with occludin and how they influenced our current thinking of the molecular functioning of tight junctions. KEY WORDS: Tight junctions, Zonula occludens, Occludin, Paracellular permeability, Epithelial polarity, Phosphorylation, ZO-1.

I. Introduction Individual cells in epithelial sheets are interconnected by a set of specialized intercellular junctions that together form the epithelial junctional complex (Farquhar and Palade, 1963). Tight junctions are the most apical of these intercellular structures and form a border between the apical and the basolateral cell surface domains. In endothelia, tight junctions have a comparable morphology and similar functions as in epithelia, but their position relative to the other intercellular junctions can vary (Bowman et al., 1991; Rubin, 1992). Because high-molecular-weight tracers added to the basal side of epithelial sheets can diffuse freely along the paracellular space until they reach the level of tight junctions, they were soon recognized as the intercellular International Review of Cytology, Vol. I86 0074-7696199$25.00

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structure responsible for the sealing of the paracellular pathway in both epithelia and endothelia (Cereijido, 1991).Tight junctions are not an absolute diffusion barrier, however, but are semipermeable and restrict diffusion in a manner that depends on the charge and the molecular weight of the tracer (Lindemann and Solomon, 1962; Wright and Diamond, 1968; van 0 s et al., 1974; Moreno and Diamond, 1975; Cereijido et al., 1978; Reuss, 1991).Physiological studies with tracers of different sizes and charges demonstrated that the paracellular pathway behaves like having aqueous pores with a diameter of 3-4 nm that have a negatively charged internal surface. Furthermore, paracellular permeability is regulated by different physiological and pathological stimuli (Bentzel et al., 1991; Madara, 1988). The use of occludin, the first identified transmembrane component of tight junctions (Furuse et al., 1993), as a molecular tool to manipulate tight junction functions has demonstrated that this protein is involved in the sealing as well as in the selective permeability of the paracellular diffusion barrier and that these two parameters can be dissociated from each other (Balda et al., 1996b; McCarthy et al., 1996). Tight junctions are involved in the polarized organization of the epithelial cell surface by restricting the diffusion of lipids within the exoplasmic leaflet of the plasma membrane (Dragsten et al., 1981; van Meer and Simons, 1986). Although this fence function of tight junctions is clearly involved in lipid polarity, its importance for protein polarity is unclear. Occludin participates in the formation of this intramembrane diffusion barrier (Balda et al., 1996b). In addition to these classical functions of tight junctions, a series of observations suggest that tight junctions also participate in the regulation of cell growth and differentiation (Balda and Matter, 1998). Although none of these experiments suggests a direct role for occludin, a possible involvement is suggested by its direct interaction with ZO-1 (Furuse et al., 1994), a peripheral membrane protein that is homologous to a Drosophila tumor suppressor and that localizes to the nucleus in growing cells (Gottardi et al., 1996). A possible role of tight junction components in nuclear functions is also suggested by symplekin, a peripheral tight junction component that is also present in the nucleus (Keon et al., 1996), and by another new tight junction associated protein, the 2.1 antigen, that colocalizes with ZO1 at tight junctions and in nuclei of growing MDCK cells, (M.S. Balda and K. Matter, unpublished observation). Until the discovery of occludin, all known tight junction associated proteins were peripheral membrane proteins (Gumbiner, 1993).Three of these proteins, ZO-1, 20-2, and p130, are associated tightly with each other (Stevenson et al., 1986; Gumbiner et al., 1991; Balda et al., 1993). ZO-1 and 2 0 - 2 belong to a family of proteins that also include disc large A, a Drosophila tumor suppressor, and PSD-95 or SAP-90, a synaptic protein

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(Jesaitis and Goodenough, 1994; Willott et al., 1993; Tsukita et al., 1993; Woods and Bryant, 1993). The homologous portion is built up by an SH3 domain, a domain with similarities to yeast guanylate kinase, and three PDZ domains (PSD-95/DlgA/ZO-l homology domain). In its unique Cterminal half, ZO-1 contains an actin-binding domain and several alternatively spliced domains (Itoh et al., 1997; Anderson and Van Itallie, 1995). The presence of the (Y domain, one of those alternatively spliced domains, has been shown to correlate with junctional plasticity (Balda and Anderson, 1993). Moreover, ZAK (ZO-1-associated kinase), a serine protein kinase, binds to the SH3 domain of ZO-1 and phosphorylates a region C-terminal to this domain (Balda et al., 1996a), suggesting that ZO-1 is a central component of the signal transduction machinery associated with tight junctions. This possibility is also supported by experiments that showed that the tight junction associated ras target AF-6 interacts with ZO-1 (Yamamoto et al., 1997). Other possible regulatory components either associated with or at least localized closely to tight junctions include small GTPases of the rab family (e.g., rab 13, rab 3B, rab 8; Zahraoui et al., 1994; Weber et al., 1994; Huber et al., 1993). Although rabs are generally involved in vesicular transport, their function in tight junctions has not been determined. Several other peripheral membrane proteins are associated with tight junctions (cingulin, 7H6, BG9, and a 210-kDa protein from Xenopus; Citi et al., 1988; Zhong et al., 1993; Chapman and Eddy, 1989; Merzdorf and Goodenough, 1997), but neither their primary structure nor their function has been determined.

II. Structural Aspects A. Structure of Occludin Occludin was identified as an antigen recognized by monoclonal antibodies generated against a chicken liver plasma membrane fraction enriched in intercellular junctions (Furuse et al., 1993). Using these monoclonal antibodies, as well as subsequently generated polyclonal antibodies, occludin was localized to tight junctions of various epithelial and endothelial cell, suggesting that it is a universal component of this intercellular junction (Fallon et al., 1995; Aaku Saraste et al., 1996; Balda et al., 1996b; AndoAkatsuka et al., 1996; Hirase et al., 1997; Kimura et al., 1997; Saitou et al., 1997; Wong and Gumbiner, 1997). Figure 1 shows confocal sections, taken at the level of tight junctions, of polarized Madin-Darby canine kidney (MDCK) cells that were double labeled with a polyclonal anti-occludin antibody and a monoclonal antibody to ZO-1, a 220-kDa protein of the

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FIG. 1 Immunofluorescence micrographs showing colocalization of occludin and 20-1 in kidney epithelial cells. Mature MDCK cells in a monolayer that were grown for 1 week on polycarbonate filters, fixed, and permeabilized with ethanol/acetone (Balda ef aL, 1996b). Cells were then processed for double immunofluorescence stairing using a polyclonal antioccludin antibody and a monoclonal anti-ZO-1 antibody. Confocal xy sections are shown through the region of the monolayer that contains tight junctions. Note the precisely matching signals for occludin (A) and ZO-1 (B).

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submembrane cytoskeleton of tight junctions (Fig. 1: A, occludin; B, ZO1). Note that the two stainings overlap precisely. Occludin is a transmembrane protein of 60 to 65 kDa. Based on its sequence, occludin was predicted to span the membrane four times and to expose both termini to the cytosol, resulting in two extracellular loops (Furuse et al., 1993). This predicted membrane topology (shown schematically in Fig. 2) is supported by several different experiments. First, it was shown that the C-terminal domain of occludin interacts with ZO-1, a protein that associates with the cytosolic face of the membrane (Furuse et al., 1994). Second, probes specific for the extracellular loops have access to occludin in intact cells (Van Itallie and Anderson, 1997; Wong and Gumbiner, 1997). Third, N-linked glycosylation sites introduced into the predicted extracellular loops become glycosylated efficiently (Matter and Balda, 1998). Occludin has been cloned from different species ranging from chicken to human and, at first glance, exhibits a surprisingly high interspecies diversity (Ando-Akatsuka et al., 1996). Nevertheless, there are domains that are well conserved, such as the areas close to both termini, suggesting that they possess conserved functions. Extracellular domains show only a low degree of conservation but, interestingly, have a rather particular amino acid com-

arrangement of occludin within tight junctions

regulation of paracellular permeability interaction with 20-1 basolateraltargetlng arrangement of occludin within tight junctions

FIG. 2 A schematic representation of the topology of occludin in a lipid bilayer as proposed by Furuse and colleagues (1993). This predicted membrane topology has now been confirmed experimentally. Functions known to be mediated by cytoplasmic domains are also indicated.

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position. The first extracellular loop, for instance, consists of about 60% tyrosines and glycines in all analyzed species, even though a simple sequence alignment suggests a poor conservation. This poor positional but high compositional conservation suggests that the function of the extracellular domains depends on the overall composition and the resulting physicochemical properties and not simply on the linear sequence.

6. Occludin and Morphology of Tight Junctions In electron micrographs of thin sections, tight junctions appear at the apical end of the leteral membrane as very close contacts between the plasma membranes of neighboring cells (Fig. 3A). Depending on the sample preparation and embedding technique used, focal contacts can be seen where the outer leaflets of the two neighboring cell membranes are in apparent continuity (Farquhar and Palade, 1963). In freeze-fracture replicas (Fig. 3B), tight junctions appear as net-like meshworks of intramembrane fibrils that completely encircle the cells (Staehelin, 1973; Madara, 1991). Freezefracture studies from different tissues revealed that if both fracture faces are studied, these intramembrane strands are continuous (i.e., grooves in one fracture face correspond to particles in the other fracture face). These intramembrane strands are thought to represent the focal contacts seen in thin sections. The biochemical composition of these strands has been debated for many years but is still unclear. The two opposing models of tight junctions are based on different opinions about the composition of these strands. The lipid model predicts that intramembrane strands represent inverted lipid micelles and that the close contacts seen in thin sections represent hemifusions (Kachar and Reese, 1982; Wegener and Gall, 1996). The opposing and currently most widely accepted model, the protein model, assumes that the intramembrane strands represent rows of tightly lined up transmembrane proteins (Gumbiner, 1993). In the protein model, cell-cell interaction is therefore mediated by neighboring rows of transmembrane proteins that bind to each other and, in the lipid model, the main adhesive force comes from hydrophobic interactions among the lipid tails. Because of the discovery of a transmembrane protein associated with tight junctions, the lipid model has to be extended so that unconventional lipid structures and transmembrane proteins form the junction together. In this case, one of the functions of the transmembrane proteins could be to stabilize energetically unfavorable lipid structures. As a transmembrane protein, occludin could theoretically be important for tight junction morphology. Occludin associates with intramembrane strands seen in freeze-fracture replicas as well as focal contacts detected in

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FIG. 3 Electron micrographs of tight junctions. (A) Thin-sectioned MDCK cells. Filter-grown MDCK cells in a monolayer were fixed and then processed for electron microscopy using EponlAraldite. The position of tight junctions at the apical end of the lateral membrane is marked with an arrow and the desmosome with a star. Bar: 250 nm. (Courtesy of Dr. Denise Huber, Geneva). (B) Intramembrane strands of tight junctions. MDCK cells were fixed, freeze-fractured and analyzed by electron microscopy. A section of freeze-fracture replica shows the network of intramembrane strands, visible as rows of particles at the level of tight junctions. Bar: 100 nm. (Courtesy of Dr. Marcelino Cereijido, Mexico.)

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thin sections (Fujimoto, 1995; Furuse et al., 1993,1996) and, in baculovirusmediated expression experiments in insect cells, occludin was shown to accumulate in intracellular multilammelar structures where it appears to form transmembrane particles (Furuse et al., 1996). Additionally, stable MDCK cell lines transfected with wild-type chicken occludin were found to exhibit a slight increase in the number of intramembrane strands (Balda et al., 1996b;McCarthy et al., 1996).Thus, occludin is involved either directly in the formation of these strands or in the regulation of strand formation. If occludin is the main structural component of intramembrane strands, one would expect that major alterations in the distribution of occludin dramatically affect the junctional morphology in freeze-fracture replicas. In MDCK cells, stable expression of an N-terminally epitope-tagged and C-terminally truncated occludin mutant causes a dominant negative effect on the distribution of endogenous occludin (Balda et al., 1996b);the transfected mutant and endogenous occludin colocalize in patches along the junction (Fig. 4:A, transfected mutant occludin; B, endogenous occludin). This rather dramatic redistribution of occludin is not paralleled by a disruption of the intramembrane strands but only in a slight decrease in the total number of strands at high expression levels (Balda et al., 1996b). Although this does not exclude that occludin contributes to the substance of the intramembrane strands, it clearly excludes that occludin is the principal or even the only component of these strands. Because increased expression of wild-type occludin can induce slight increases in the total number of intramembrane strands (Balda et al., 1996b; McCarthy et al., 1996) but the distribution of occludin does not directly affect their continuity, occludin is rather a regulatory than a structural component of these structures. Interestingly, no increase in the number of intramembrane strands was observed when an N-terminally, epitopetagged, full-length occludin was expressed, suggesting that the N-terminal domain is important for the regulation of the formation of intramembrane strands (Balda et al., 1996b). Similar to the appearance in freeze-fracture replicas, disruption of the continuous distribution of occludin does not affect the appearance of tight junctions in electron micrographs of thin sections (Balda et al., 1996b) and the depletion of occludin from tight junctions of Xenopus A6 cells does not affect gross morphology (Wong and Gumbiner, 1997). Thus, occludin does not appear to be of primary morphological importance.

C. Occludin and Cell-Cell Interaction Occludin is clearly a part of intercellular complexes. If MDCK cells expressing the just-described, discontinuously distributed truncated occludin mu-

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FIG. 4 Immunofluorescence micrographs showing patching of occludin in transfected MDCK cells. Filter-grown MDCK cells producing an N-terminally epitope-tagged and C-terminally truncated mutant of occludin were processed for double immunofluorescence using a monoclonal antibody specific for the N-terminal epitope and a polyclonal antibody recognizing the C-terminal domain of endogenous occludin. Confocal xy sections through the junctional region of the monolayer show the discontinuous staining patterns of transfected (A) and endogenous occludin (B).

tant are cocultured with wild-type MDCK cells, endogenous and transfected occludin exhibit a normal continuous distribution along heterologues junctions formed by a wild-type and a transfected cell (Balda et al., 1996b).

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Thus, wild-type cells can correct the distribution of occludin in transfected cells if they are interacting directly with each other. Although this indicates that occludin is a component of intercellular complexes, this result does not allow conclusions about the composition of this complex (i.e., homoor heteromeric), the nature of the interaction (i.e., homo- or heterotypic), or whether occludin is involved directly or indirectly in the intercellular interaction. A direct involvement of occludin in intercellular interactions is suggested by the finding that ectopic expression of occludin in certain fibroblasts confers adhesiveness (Van Itallie and Anderson, 1997). Because increase in adhesiveness, measured in a cell-cell aggregation assay in suspension in the absence of calcium, can be quenched by the addition of peptides corresponding to sequences within the first extracellular loop of occludin, it was proposed that this extracellular domain participates directly in cell-cell adhesion (Van Itallie and Anderson, 1997). Whether this is due to homotypic interactions between occludin molecules of neighboring cells, to hydrophobic interactions mediated by the unusually hydrophobic extracellular domains of occludin, or to an indirect interaction with another cellular component is not clear. Interestingly, the increase in adhesiveness was only observed in fibroblasts in suspension that form cadherin-based adherens junctions under normal culture conditions, but not in fibroblasts that never form such structures. Thus, occludin-mediated adhesiveness requires the presence of one or several other junctional components. The missing component in nonadhesive fibroblasts is not ZO-1, a peripheral membrane protein of tight junctions that interacts with the C-terminal cytoplasmic domain of occludin (Furuse et al., 1994), as it is expressed in both types of cells. In cadherin-positive fibroblasts, transfected occludin clusters and colocalizes with endogenous ZO-1 in adherens junctions without having obvious morphological effects (Van Itallie and Anderson, 1997). As mentioned earlier, experiments in A6 epithelial cells showed that occludin can be depleted from the junction without significantly affecting the morpology of the cells (Wong and Gumbiner, 1997). Thus, the participation of occludin in intercellular complexes apparently does not reflect a morphologically important adhesive property, but rather a characteristic involved in other functions of tight junctions such as, for instance, paracellular permeability.

111. Biogenesis of Tight Junctions and Targeting of Occludin A. De Novo Assembly of Tight Junctions Biogenesis of tight junctions can be studied in two ways: de n o w assembly of tight junctions as it occurs during early development or integration of

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a newly synthesized component into preexisting junctions. To study de novo assembly, investigators most often made use of the calcium switch model (Cereijido et al., 1978; Gonzilez-Mariscal et al., 1990). In this experimental system, epithelial cells, usually MDCK cells, are seeded on permeable supports at low calcium concentrations that do not allow the formation of intercellular junctions. The addition of calcium then triggers cell-cell adhesion and the formation of electrically tight monolayers. The de novo formation of tight junctions on the addition of calcium is a complex process that is paralleled by dramatic changes of the cellular architecture and, hence, requires not only proper assembly of intercellular junctions, but also extensive remodeling of the cytoskeleton (Meza et al., 1980). Calcium appears to act primarily by activating E-cadherin-mediated cell-cell adhesion; the assembly of intercellular junctions on the addition of calcium can be inhibited by the addition of anti-E-cadherin antibodies (Behrens et al., 1985; Gumbiner et al., 1988). The induction of calciumdependent cell-cell adhesion then triggers a network of signaling pathways that include G-proteins, phospholipase C, protein kinases C and A, and calmodulin (Balda et al., 1991) and lead to the assembly of intercellular juntions. The function of E-cadherin-dependent cell-cell adhesion appears to be primarily to trigger signaling as the block of anti-E-cadherin antibodies can be overcome by CAMP(Behrens et al., 1985) and diC8, a diacylglycerol analogue (Balda et al., 1993). The latter compound is even able to stimulate partial assembly of tight junctions in the presence of low extracellular calcium (Balda et al., 1993). Protein kinases and protein phosphorylation are receiving much attention from investigators interested in the regulation of tight junction assembly (Anderson and Van Itallie, 1995); protein kinases are involved in assembly (Balda et al., 1991; Nigan et al., 1991) as well as disassembly (Citi, 1992) of tight junctions. Even the maintenance of functional tight junctions requires carefully balanced kinase activities as overstimulation can induce the loss of functional junctions without changing calcium concentrations (Ojakian, 1981;Mullin and O’Brien, 1986;Rosson et al., 1997).Although many studies imply an involvement of protein kinase C and a mix of protein kinase C isoforms from brain added to immunoprecipitated ZO-1 results in some phosphorylation in vitro (Stuart and Nigan, 1995), it is not known whether protein kinase C directly phosphorylates tight junction proteins in vivo or activates other kinases, such as the one associated with ZO-1 (which is distinct from protein kinase C& Balda et al., 1996a). Additionally, the protein kinase C isoform that localizes to the lateral membrane (protein kinase C& Stuart and Nigan, 1995; Dodane and Kachar, 1996) is not activated by phorpol esters and diacylglycerol (Dekker and Parker, 1994), excluding it as the direct target of these drugs in the aforementioned experiments.

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Similarly, protein kinase A is also involved in the regulation of tight junctions (Bentzel et al., 1991; Balda et af., 1991), but does not localize to tight junctions in MDCK cells. Furthermore, it also does not phosphorylate the C-terminal cytoplasmic domain of occludin, which is involved in the regulation of occludin (see later), in in vitro assays (Cordenonsi et al., 1997; M.S. Balda and K. Matter, unpublished observation). Moreover, calciuminduced differences in the phosphorylation of peripheral membrane proteins of tight junctions are controversial (Balda et al., 1993; Citi and Denisenko, 1995; Howarth and Stevenson, 1995; Stuart and Nigan, 1995). Like most of the peripheral tight junction proteins, occludin is also a phosphoprotein and, in vivo, appears to be primarily phosphorylated on serine and threonine residues (Sakakibara et af., 1997; Cordenonsi et af., 1997).Data on phosphorylation of occludin during de novo assembly of tight junctions are controversial. In the Ca switch model, the phosphorylation of occludin is induced if calcium is added to MDCK cells (Sakakibara et al., 1997). In early development of Xenopus, however, the induction of tight junction formation is paralleled by an apparent dephosphorylation of occludin (Cordenonsi et al., 1997). It is difficult to compare the two sets of experiments as neither changes in specific phosphate content nor phosphorylation sites were determined. Because de n o w formation of tight junctions can be paralleled by occludin phosphorylation or dephosphorylation, however, the phosphorylation state of occludin may not be relevant for junction assembly but rather for regulating the function(s) of occludin once junctions are assembled. The kinase(s) that phosphorylates occludin in vivo has not been identified. Nevertheless, a protein kinase binds to the C-terminal domain of occludin (Balda et al., 1998). This enzyme has not yet been identified, but its biochemical features are distinct from those of CDC2 and casein kinase 11, two kinases that can phosphorylate occludin fusion proteins if added as purified proteins to an in vitro assay (Cordenonsi et al., 1997).

6. Integration of Newly Synthesized Components into Existing Tight Junctions

The integration of newly synthesized components into assembled tight junctions under steady-state conditions is an intriguing but rarely studied problem. Because tight junctions form the border between the apical and the basolateral cell surface domains in epithelial cells, their biogenesis could involve either one or both of the two cell surface domains. Interestingly, certain experimental conditions (e.g., treatment with a calcium ionophore or with proteases) can induce the appearance of intramembrane strands in

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the lateral membrane, suggesting that the biogenesis of tight junctions involves the lateral membrane (Bentzel et al., 1991; Polak-Charcon, 1991). However, MDCK cells grown in low calcium concentrations contain vacuolar structures that contain apical membrane components and are also positive for ZO-1, a cytoplasmic component of tight junctions (Vega-Salas et al., 1988). Hence, it would be feasible that tight junction biogenesis is connected directly to apical membrane biogenesis. The discovery of occludin offered the possibility to study the transport pathway of a transmembrane protein to tight junctions. In polarized epithelial cells, apical and basolateral plasma membrane proteins are transported together to and through the Golgi apparatus after biosynthesis in the endoplasmic reticulum (Rodriguez-Boulan and Powell, 1992; Matter and Mellman, 1994). Upon arrival in the trans-Golgi network, proteins are sorted into pathways that mediate transport to specific cell surface domains. The exact routes taken by apical and basolateral membrane proteins depend on the protein as well as on the epithelial cell type studied. In MDCK cells, apical and basolateral membrane proteins are generally sorted directly to their corresponding cell surface domain. Basolateral targeting has been associated with distinct cytoplasmic targeting determinants, and apical targeting appears to be mediated by different types of sorting determinants, including particular transmembrane domains and luminal carbohydrates (Simons and Ikonen, 1997). The targeting of occludin to tight junctions is a conserved process as ectopically expressed chicken occludin integrates into tight junctions of epithelial cells from different origins, ranging from Xenopus to human (Furuse et al., 1994; Balda et al., 1996b; McCarthy et af., 1996; Chen et al., 1997). It soon became clear that the C-terminal domain of occludin is important for the transport of occludin to tight junctions in transiently transfected epithelial cells (Furuse et al., 1994). In stably transfected MDCK cells as well as microinjected Xenopus embryos (Chen et al., 1997),truncated occludin mutants are still transported to tight junctions but, at least in MDCK cells, at a reduced efficiency, resulting in an intracellular and a junctional pool of transfected occludin (Balda et al., 1996b; Gut et al., 1998). Because the deleted C-terminal domain of occludin contains a sorting signal (see later) and transfected occludin oligomerizes with endogenous occludin, it appears likely that transfected mutant occludin is dragged to the cell surface and tight junctions by endogenous occludin (Chen et al., 1997; Matter and Balda, 1998). The C-terminal domain of occludin is sufficient to mediate basolateral transport of a reporter protein, indicating that it contains a basolateral targeting determinant and suggesting that transport of occludin to tight junctions involves passage through the basolateral membrane (Matter and Balda, 1998). This possibility is also supported by the lateral accumulation

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of a mutant occludin that contains the entire C-terminal cytoplasmic domain but does not integrate efficiently into tight junctions because of modified extracellular loops (Balda et al., 1998; Matter and Balda, 1998). Moreover, small amounts of wild-type occludin can be detected in the lateral membrane of chicken intestinal epithelial cells (Sakakibara et al., 1997) and MDCK cells expressing chicken occludin at high levels (Balda et al., 1998). These observations suggest that the assembly of tight junctions in polarized epithelial monolayers occurs from the basolateral membrane. Removal of the C-terminal domain of occludin, which contains the basolateral sorting signal, does not result in an efficient apical transport of occludin but rather in an accumulation of a fraction of the protein in the Golgi complex (Balda et al., 1996b; Gut et al., 1998). This was surprising as removal or inactivation of basolateral targeting determinants generally results in efficient apical transport but not in intracellular accumulation (Matter and Mellman, 1994). If a mutant with N-linked glycosylation sites was studied, a posttranslational modification that can mediate apical sorting (Simons and Ikonen, 1997), deletion of the C-terminal domain converted the laterally accumulating protein into an efficiently apically transported membrane protein (Gut et al., 1998). Thus, basolateral sorting of occludin is not only secured by a cytoplasmic basolateral sorting determinant but also by the absence of apical sorting signals. Once newly synthesized occludin arrived in the basolateral membrane, the extracellular loops of occludin appear to become important for the integration of occludin into tight junctions. If monolayers of Xenopus A6 cells are incubated with a peptide corresponding to the second extracellular loop of occludin, junctions become slowly depleted from occludin (Wong and Gumbiner, 1997). Because this process takes several days in mature monolayers, depletion of occludin from tight junctions may occur by preventing newly synthesized occludin from entering the junction. An involvement of extracellular loops in the accumulation of occludin in tight junctions is also supported by mutations in the extracellular loops that cause inefficient accumulation of the protein in tight junctions (Balda et al., 1998; Matter and Balda, 1998). The mechanism by which the extracellular domains mediate accumulation in tight junction is not clear, but it may be due to intercellular interactions that immobilize it at the junctions, as occludin has certain adhesive properties (Van Itallie and Anderson, 1997) and is a component of an intercellular complex (Balda et al., 1996b). Because the two terminal cytoplasmic domains are important for the distribution of occludin within the junction (Balda et al., 1998), it could also be that interactions occurring in the extracellular domains stimulate the cytoplasmic domains to interact with the submembrane cytoskeleton. ZO-1 may participate in this process (Furuse et al., 1994), but it is apparently not absolutely required (Ohsugi

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et al., 1997). Activation of such cytoplasmic interactions could involve alterations in phosphorylation or, alternatively, conformational changes in the cytoplasmic domains due to the formation of higher order oligomeric structures.

IV. Paracellular Permeability A. Transepithelial Eletrical Resistance versus Selective Paracellular Permeability The paracellular permeability barrier function of tight junctions is generally determined by measuring transepithelial electrical resistance and/or paracellular flux of soluble tracers such as [3H]mannitol,horseradish peroxidase, inulin, or fluorescent dextran. Neither transepithelial electrical resistance nor paracellular flux, despite its name, depends only on junctional permeability but also on transcellular characteristics. To make matters worse, the two parameters reflect different functional properties and, therefore, do not necessarily develop in parallel. The electrical resistance of an epithelial monolayer is a function of the transcellular resistance (the sum of the resistances of the apical and the basolateral membrane) and the paracellular electrical resistance (Reuss, 1991). Because the transcellular and the paracellular pathways are parallel to each other, the reciprocal value of the transepithelial electrical resistance is the sum of reciprocal values of the transcellular and the paracellular reistance. Thus, transepithelial electrical resistance can never be larger than either one of the two single resistors (i.e., transcellular and paracellular electrical resistance) and, if the two are not in a similar range, primarily reflects the more conductive (less resistant) route. As an example, the transepithelial electrical resistance of low resistance MDCK cells is around 70 SZ cm2, whereas the transcellular resistance is larger by several orders of magnitude (Gonzhlez-Mariscal et al., 1989). Therefore, transepithelial electrical resistance of this cell line essentially reflects paracellular resistance and increases in this parameter are due to increases in paracellular resistance. Decreases in transepithelial electrical resistance can be due to lower paracellular resistance as well as to drastic decreases in transcellular resistance. Similar to transepithelial electrical resistance, the transport of soluble tracers such as mannitol and dextrans across epithelial sheets occurs along a transcellular route (i.e., transcytosis) and by passive transport along a concentration gradient through the paracellular route. In a given epithelium, the relative contributions of the two pathways to the total transepithe-

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lial flux depend on the physical properties of the tracer. This is primarily due to the size and charge selectivity of the paracellular pathway (Lindemann and Solomon, 1962; Wright and Diamond, 1968; van 0 s et al., 1974; Moreno and Diamond, 1975; Cereijido et al., 1978). Small neutral tracers are therefore used to measure paracellular permeability. As an example, only the transepithelial flux of low- and medium-molecular-weight tracers, but not of high-molecular-weight compounds, can be increased and decreased by the expression of different mutants of a tight junction protein in MDCK cells without affecting fluid-phase transcytosis (Balda et al., 1996b, 1998). Transepithelial electrical resistance (or conductivity, its reciprocal value) is an instantaneous measurement that reflects the current crossing an epithelium at a given moment; therefore, this parameter reflects the actual tightness or the degree of sealing of the monolayer. In contrast, paracellular flux is measured over an interval of time (one to several hours) and thus reflects the transport capacity of the monolayer that may not need be due to continuously open pathways from one side of the monolayer to the other. Hence, the paracellular flux of small and medium size tracers is a measurement of the selective permeability of the junction. These considerations are of course only valid in well-established and mature monolayers. In immature or compromised monolayers with not fully formed junctions, transepithelial electrical resistance will be low (high conductivity) and paracellular permeability will be high (and not selective!).

B. Occludin and Selective Paracellular Permeability Occludin is a component of the semipermeable paracellular diffusion barrier. Expression of chicken occludin in MDCK cells was shown to result in two- to fivefold increases in transepithelial electrical resistance as well as in a slightly increased number of intramembrane strands (Balda et al., 1996b; McCarthy et al., 1996). The increase in transepithelial electrical resistance is not a direct consequence of the higher number of intramembrane strands as expression of an N-terminally epitope-tagged occludin results in the same increases in transepithelial electrical resistance as the untagged protein but does not affect the number of intramembrane strands (Balda et al., 1996b). This supports previous findings that indicated that the number of intramembrane strands does not necessarily reflect transepithelial electrical resistance (Martinez-Palomo and Erlij, 1975; Stevenson et al., 1988; GonzBlez-Mariscal et al., 1989). The concentration of occludin appears to be an important parameter that influences the sealing of the paracellular barrier as measured by transepithelial electrical resistance. This is not only suggested by the stable

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transfection experiments described earlier, but also by the finding that the amount of endogenously expressed occludin correlates with the monolayer tightness in different types of endothelia (Hirase et al., 1997). Moreover, incubation of Xenopus A6 cells (which develop monolayers of high electrical resistance) with a peptide corresponding to the amino acid sequence of the second extracellular loop of occludin results in large decreases of transepithelial electrical resistance and almost complete depletion of occludin from tight junctions after long incubation times (Wong and Gumbiner, 1997). Although of these experiments suggest that occludin is an important functional component of the paracellular barrier, it is not clear how occludin participates in this tight junction function. Disruption of the continuous distribution of occludin in MDCK cells by transfecting an N-terminally epitope-tagged and C-terminally truncated mutant of occludin does not result in decreased transepithelial electrical resistance but in a two- to threefold increase (Balda et al., 1996b). Because a continuous junctional ring is apparently not required for electrical resistance, occludin can at least not be the only structural component of the paracellular seal. If occludin participates as a structural component in the sealing of the junction, the distribution of the other component(s) must be very flexible to be able to easily cover the gaps caused by the discontinuous distribution of occludin. Observations that the presence of occludin affects the electrical resistance of the junction in a concentration-dependent manner but that the distribution of occludin within the junction is of secondary importance suggest that occludin is a regulatory component of the tight junctional seal. The stable expression of wild-type chicken occludin in MDCK cells results in increased transepithelial electrical resistance but, at high expression levels, also in increased paracellular permeability (Balda et al., 1996b; McCarthy et al., 1996). Moreover, cells expressing an N-terminally, epitopetagged occludin mutant that lacks the C-terminal cytoplasmic domain exhibit even larger increases in paracellular flux (Balda et al., 1996b). Because increased paracellular flux occurs in a size-selective manner (Balda et al., 1996b), occludin is not only involved in the sealing of tight junctions, but also in selective paracellular permeability. A direct involvement of occludin in paracellular diffusion pathway is further supported by the inhibition of selective paracellular diffusion by the stable expression of dominantnegative mutants of occludin in MDCK cells (Balda et al., 1998). The C-terminal cytoplasmic domain of occludin appears to be important for the regulation of selective paracellular permeability. This does not occur via clustering of occludin, as deletion of the C-terminal domain causes similar increases in selective paracellular permeability and transepithelial electrical resistance in the presence or absence of the N-terminal epitope but a discontinuous distribution only if the N-terminal cytoplasmic domain

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is blocked by the ectopic epitope (Balda et al., 1998). The importance of the C-terminal cytoplasmic domain is also supported by experiments in which microinjection of mRNAs coding for different C-terminally truncated occludin mutants into Xenopus embryos was found to cause increased paracellular permeability if at least the last 119 amino acids were deleted (Chen et al., 1997). Whether this increase is due to increased paracellular permeability or a disrupted seal was not tested. In fact, it is possible that overexpression of mutant, or even wild-type, occludin causes defects in the paracellular seal in more dynamic systems by retarding junction assembly. The easiest way one can think of how the C-terminal cytoplasmic domain of occludin is involved in the regulation of paracellular permeability involves binding to other components as permeability is higher if this part of occludin is deleted. This is also supported by the analysis of different clones of transfected MDCK cells expressing chicken occludin. Figure 5 shows that all clones exhibited at least as much or more transepithelial electrical resistance than wild-type cells. However, clones exhibited a biphasic response if paracellular flux of [3H]mannitol was assayed. With slight increases in transepithelial electrical resistance (and occludin expression; not shown), paracellular permeability decreased and then started to increase to reach levels twice as high as wild-type cells at large increases in transepi-

A A

A

A

04 0

1 2 3 4 5 Transepithelial electrical resistance (normalized to wild-type MDCK)

FIG. 5 Graphs showing transepithelial electrical resistance and paracellular permeability of MDCK cells producing chicken occludin. MDCK cells were transfected with a cDNA coding for chicken occludin. Seven clones homogeneously expressing the chicken occludin gene were grown and analyzed without (0) or with (A) induction of higher expression levels by sodium butyrate by measuring transepithelial electrical resistance and paracellular flux of [3H]mannitol. All values were normalized to nontransfected MDCK cells. Note the initial decrease in permeability.

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thelial electrical resistance. This suggests that the regulation of paracellular permeability via the C-terminal domain of occludin is achieved by a mechanism that is saturated by high levels of occludin in transfected cells. Thus, selective paracellular permeability might be controlled by a regulated interaction between the C-terminal domain of occludin and a component of the submembrane coat of tight junctions as, for instance, ZO-1. Analysis of different epithelial tissues revealed an exponential relationship between the number of intramembrane strands in freeze-fracture replicas and transepithelial electrical resistance (Claude and Goodenough, 1973; Claude, 1978). Therefore, it was proposed that tight junctions consist of a series of diffusion barriers that contain channels that fluctuate between an opened and a closed state (Claude, 1978). Because transepithelial electrical resistance values do not fluctuate, these channels would have to be compartmentalized by a network such as the one formed by intramembrane strands, which are thought to represent these diffusion barriers (Cereijido et al., 1989). While this is an attractive model and the channels could explain many of the results obtained with occludin, there are several exceptions to the direct exponential relationship between electrical resistance and the number of intramembrane strands (Martinez-Palomo and Erlij, 1975; Stevenson et al., 1988; Gonzalez-Mariscal et al., 1989). The occludin experiments are also not in agreement with a direct relationship between the number of strands and the electrical resistance as increases in transepithelial electrical resistance were not always found to be paralleled by additional intramembrane strands. Therefore, if intramembrane strands represent diffusion barriers, it cannot be simply their number that determines transepithelial electrical resistance but also their composition. In the channel model, paracellular permeability can be controlled by the number of channels and by regulating the time they are open. Occludin could therefore be involved either in forming the channels or in regulating their opening. Because short deletions in the extracellular loops of occludin result in reduced paracellular permeability, it could be that these loops are involved directly in the selective permeability of the junction (Balda et al., 1998). Nevertheless, these deletion mutants are integrated inefficiently into tight junctions, and similarly reduced levels of permeability can be observed when mutant and chimeric occludin proteins are expressed that do not visibly accumulate in tight junctions, suggesting that in the case of loop deletions, the inhibitory effect could also be due to the protein not incorporated into the junction. Such a dominant-negative effect could be due to preventing other components required for paracellular permeability (e.g., one or several components that form those hypothetical fluctuating channels) to reach tight junctions. The component that is prevented from reaching the junction is neither ZO-1 nor, at least in those cases where it could

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be tested, endogenous occludin. According to this scenario, occludin could be either a subunit or a regulator of such a channel. These considerations then lead to a model of tight junctions in which the transepithelial electrical resistance is determined by three factors: the number of strands, the specificresistance of the strands (which is determined by their composition and might be regulated as well), and the number of open channels per compartment of the newtork. Selective paracellular permeability is meditated by these aqueous channels and can be regulated by changing the number of open channels or the open probability.

V. Occludin and Cell Surface Polarity Tight junctions form a morphological as well as functional barrier between the apical and the basolateral cell surface domains. Experiments with fluorescent lipids and lipid probes demonstrated that tight junctions form an intramembrane diffusion barrier, often called fence, that prevents the intermixing of apical and basolateral lipids in the outer leaflet of the plasma membrane (Dragsten et al., 1981; van Meer and Simons, 1986). The stable expression of mutant occludin in MDCK cells showed that occludin is also involved in this function of tight junctions. Disruption of the continuous junctional ring formed by occludin by expression of the aforementioned N-terminally epitope-tagged and Cterminally truncated occludin results in cell lines unable to efficiently maintain fluorescently labeled sphingomyelin in the cell surface domain into which it has been inserted (Balda et al., 1996b). In contrast to selective paracellular permeability, expression of a C-terminally truncated mutant lacking the N-terminal epitope, a protein that forms a continuous junctional ring, is not sufficient to disrupt the intramembrane fence, indicating that the paracellular permeability and restriction of lipid diffusion do not rely on the same features of occludin (Balda et al., 1998). The integrity of the intramembrane diffusion barrier thus correlates with the continuous distribution of occludin, suggesting that the continuous junctional organization of occludin, seen by immunofluorescence, is important for the fence function of tight junctions. At least some of the properties of tight junctions responsible for the intramembrane and the paracellular diffusion barriers are different as cell lines unable to maintain a fluorescent lipid in a specific cell surface domain still exhibit higher transepithelial electrical resistance than wild-type cells. The opposite result was obtained by short times of ATP depletion that result in a loss of transepithelial electrical resistance but not in disruption of the intramembrane diffusion barrier (Mandel et al., 1993). In this system,

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disruption of the fence function of tight junctions requires longer times of energy depletion and correlates with fragmentation of the intramembrane strands and disruption of the junctional ring of ZO-1, a peripheral membrane protein (Bacallao et al., 1994). Whereas ZO-1 is able to interact with the C-terminal cytoplasmic domain of occludin in vitro (Furuse et al., 1994), it is not known how ATP depletion affects the distribution of occludin. Nevertheless, disruption of the intramembrane fence by expression of mutant occludin only results in a minimal effect on the distribution of ZO-1 and no fragmentation of the intramembrane strands was observe, indicating that disruption of intramembrane strands is not required for disrupting the intramembrane diffusion fence (Balda et af., 1996b). These data do not exclude an involvement of intramembrane strands in the fence function of tight junctions. Because the discontinuous distribution of occludin is paralleled by slightly fewer intramembrane strands, it could be that the number of strands determines the efficiency of the intramembrane diffusion fence, and because occludin is associated directly with intramembrane strands (Fujimoto, 1995;Furuse et al., 1996), a discontinuous distribution of occludin in the presence of continuous intramembrane stands must lead to heterogeneous strands (i.e., areas with and areas without occludin). It would therefore be possible that only zones of strands containing occludin are able to act as efficient intramembrane diffusion barriers. Disruption of the continuous junctional ring of occludin causes a deficiency in the restriction of apical/basolateral lipid diffusion, but no defects in protein polarity could be detected, even though different types of proteins were studied (Balda etal., 1996b). Although this could be due to incomplete disruption of the intramembrane diffusion barrier, this indicates that the fence function of tight junctions is at least more critical for lipid than for protein polarity. This does not exclude an involvement of tight junctions in protein polarity as the extracellular domains of transmembrane proteins are often rather bulky and are therefore unable to efficiently cross tight junctions because of the paracellular barrier. Additionally, restriction of membrane protein diffusion can also be achieved by interactions between their cytosolic domains and cytoskeletal components, and certain membrane proteins are distributed in a polarized manner in the absence of tight junctions (Nelson, 1992; Vega-Salas et al., 1987).

VI. Regulation of Occludin The paracellular diffusion barrier of tight junctions not only has different properties from one epithelium to another, but is regulated by a large variety of different parameters and conditions: developmental changes; the

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cell cycle; physiologicalfactors such as hormones and vitamins; pathological conditions; and the passage of other cells as during the migration of leukocytes or in the maturation of spermatocytes and environmental conditions such as osmolarity, pH, ionic strength, and mechanical tension (Cereijido et al., 1988;Madara, 1988;Schneeberger and Lynch, 1992;Hirsch and Noske, 1993; Rahner et al., 1996). Thus, the tight junction has to be regarded as a very dynamic structure that is regulated by different mechanisms. Because the regulatory mechanisms involved in the assembly and disassembly of tight junctions were already discussed briefly earlier, this section focuses on possible mechanisms involved in the regulation of occludin function in fully assembled and functional junctions. As mention previously, occludin is involved in both the sealing and the selective permeability of tight junctions. Although it is impossible to make definitive conclusions about the precise role of occludin in these processes, it appears that the primary function of occludin is a regulatory one. The C-terminal cytoplasmic domain of occludin is instrumental for these functions of occludin: expression of mutant occludin lacking this domain results in increased selective paracellular permeability (Balda et al., 1996b) and expression of a chimeric membrane protein containing only the C-terminal domain of occludin (which does not go to tight junctions) causes increased transepithelial electrical resistance at high expression levels (Balda et al., 1998). The C-terminal domain of occludin was demonstrated to bind to ZO-1 in vitro (Furuse et al., 1994). Although it had originally been proposed that this interaction is involved in targeting of occludin (Furuse et al., 1994), it is conceivable that this interaction is of importance for the regulation of the functions of occludin. Because the deletion of the C-terminal domain of occludin results in higher levels of selective paracellular permeability, it could be that this is due to the loss of the interaction with ZO-1. Because the C-terminal half of ZO-1 contains an actin-binding site, ZO-1 might serve a bridge to link occludin do the cytoskeleton. Occludin and, thereby, paracellular permeability could then be regulated by contraction or relaxation of the cytoskeleton and/or by regulating the interaction with ZO-1. Such a model is supported by the effects of myosin light-chain kinase activity on paracellular permeability (Hecht et al., 1996;Turner et al., 1997). The C-terminal cytoplasmic domain of occludin is large and ZO-1 might therefore not be the only interacting protein. Nevertheless, ZO-1 is an attractive candidate to serve as a regulatory component as it is known to occur in a complex with other proteins (Gumbiner et al., 1991; Balda et al., 1993),can interact with at least one kinase (Balda et al., 1996a; M. S. Balda and K. Matter, unpublished observation), and contains three PDZ and one SH3 domain (Willott et al., 1993), motifs known to act as protein-binding modules that often interact with signaling proteins (Musacchio et al., 1992;

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Mayer and Eck, 1995;Fanning and Anderson, 1996). Thus, it is conceivable that ZO-1 serves as submembrane adaptor that links regulatory components to occludin and tight junctions. Such a regulatory system may not only be used to regulate tight junctions, but also to transmit signals from the junction to the interior of the cells. Theoretically, any of the proteins known to interact with ZO-1 could regulate the binding of ZO-1 to occludin. Because both ZO-1 and occludin are phosphoproteins (Anderson et al., 1988; Cordenonsi et al., 1997; Sakakibara et al., 1997; Balda et al., 1998) and kinases have often been suggested to be involved in the regulation of paracellular permeability, it could be that this interaction is regulated by a protein kinase. A good candidate is ZAK, which binds to the SH3 domain of ZO-1 and, in vitro, phosphorylates ZO-1 on one or two serine residues just C-terminal to the SH3 domain (Balda et al., 1996a). Interestingly, ZAK also phosphorylates a fusion protein containing the C-terminal cytoplasmic domain of occludin (M. s. Balda and K. Matter, unpublished observation). A similar protein kinase activity coimmunoprecipitates with ZO-1 from solubilized MDCK cells. Although ZO-1 is phosphorylated on serine residues in vivo (Anderson et al., 1988), a careful analysis of the phosphorylation site and its physiological relevance has not yet been done. Because ZO-1 associates with catenin complex in cells that lack tight junctions (Rajasekaran et al., 1996), probably because it interacts with acatenin (Itoh et d.,1997), it could also be that ZAK is involved in functions of ZO-1 not related to the regulation of paracellular permeability. Because ZAK might be involved in the nuclear localization of ZO-1 (M. S. Balda and K. Matter, unpublished observation) and given the putative role of ZO-1 in cell growth and differentiation (Balda and Matter, 1998), it could be that occludin binding to ZO-1 is used to regulate the pool of ZO-1 at the tight junction. Another type of phosphorylation that could be involved in the regulation of the ZO-l/occludin interaction is tyrosine phosphorylation. Induction of tyrosine phosphorylation on ZO-1 in some cases disrupts (Staddon et aZ., 1995; Takeda et al., 1995) and in other cases induces junctions (Kurihara et al., 1995; Van Itallie et al., 1995). It is not clear whether this discrepancy is due to phosphorylation of different sites on ZO-1, to different levels of phosphorylation, or to phosphorylation of additional proteins. Moreover, occludin also becomes phosphorylated on tyrosine residues if MDCK monolayers are incubated with sodium vanadate to inhibit tyrosine phosphatases (M. S. Balda and K. Matter unpublished observation). Because this treatment causes tyrosine phosphorylation of tight junctional proteins and a loss of functional junction, as well as a general change in cellular morphology and detachment from the substrate (Volberg et al., 1992), it is difficult to judge the functional relevance of these observations.

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In mature MDCK monolayers, occludin is phosphorylated on serine and threonine residues (Cordenonsi et al., 1997; Sakakibara et al., 1997; Balda et al., 1998). Because most of these phosphorylation sites appear to be within the C-terminal cytoplasmic domain (Balda et al., 1998), it could be that phosphorylation of occludin governs the interaction with submembrane components such as ZO-1. The C-terminal cytoplasmic domain of occludin also interacts with a protein kinase that phosphorylates similar sites in v i m as those phosphorylated in vivo (M. S. Balda and K. Matter, in preparation). An involvement of phosphorylation in the regulation of occludin function at the junction rather than in assembly of the junction would also make it easier to reconcile the contradicting observations made on phosphorylation during de novo formation of tight junctions in different experimental systems (Cordenonsi et al., 1997; Sakakibara et al., 1997). Nevertheless, to determine the role(s) of occludin phosphorylation, it will be necessary to identify the interacting kinase and study the importance of specific phosphorylation sites for occludin function and tight junction physiology.

VII. Concluding Remarks The studies described clearly in this review indicate that occludin is a central component of tight junctions involved in both classical functions of tight junctions: selective paracellular permeability and restriction of apical/basolateral lipid diffusion. Although it is not clear how occludin participates in these functions, it appears that occludin forms or helps to form the intramembrane fence as a structural component and acts as a regulator of the semipermeable paracellular diffusion barrier. Because of the many parameters that influence paracellular permeability and the structural characteristics of this protein, occludin is likely to receive signals from the interior and from the exterior of the cells. The elucidation of molecular mechanisms and the identification of functional partners of occludin will require the discovery of additional tight junction transmembrane components that participate in the formation of the paracellular barrier and the selective paracellular diffusion pathway as well as submembrane components that are involved in regulating occludin and in signaling from occludin to regulate tight junctions and, perhaps, other cellular processes. Acknowledgments We thank Marcelino Cereijido and Denise Huber for electron micrographs and Andy Whitney for comments on the manuscript. K.M. is a fellow of the START (Swiss Talents in Academic

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Research and Teaching) program of the Swiss National Science Foundation (31-38794.93). Research in our laboratory is supported by the Swiss National Science Foundation and the Canton de Genttve.

Note added in proof The ZO-I-associated protein p130 (Balda et al., 1993) has now been shown to be homologous to ZO-1 and 2 0 - 2 and to localize to tight junctions in transfected MDCK cells; p130 has therefore been renamed 20-3 (Haskins ef al., 1998, J. Cell Biol. 141, 199-208). Occludin-deficient embryonic stem cells have now been generated and described to be able to form morphologically normal tight junctions (Saitou et al., 1998, J. Cell BioL, 141, 397-408), further supporting the conclusion that occludin is a regulatory rather than a structural component of tight junctions.

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