Transmembrane proteins of tight junctions

Tight junctions contribute to the paracellular barrier, the fence dividing plasma membranes, and signal transduction, acting as a multifunctional complex in vertebrate epithelial and endothelial cells. The identification and characterization of the transmembrane proteins of tight junctions, claudins, junctional adhesion molecules (JAMs), occludin and tricellulin, have led to insights into the molecular nature of tight junctions. We provide an overview of recent progress in studies on these proteins and highlight their roles and regulation, as well as their functional significance in human diseases.     

Transmembrane proteins of tight junctions

Tight junctions from a morphological and functional boundary between the apical and basolateral cell surface domains of epithelia and endothelia, and regulate selective diffusion along the paracellular space. Two types of four-span transmembrane proteins, occludin and claudins, as well as the single-span protein JAM are associated with tight junctions. The functional analysis of these proteins starts to reveal how they are involved in the functions of tight junctions, which of their domains are important for these functions, and how they interact with each other to form the junctional diffusion barriers.     

1-Naphthylisothiocyanate-induced permeability of hepatic tight junctions to proteins.

We have studied the early action of 1-naphthylisothiocyanate (ANIT) in relation to its effect on the permeability barrier formed by hepatic tight junctions. Materials having different Mr values [inulin (5000), horseradish peroxidase (HRP) (40,000), ovalbumin (also 40,000) and pig gamma-globulin (IgG) (160,000)] were individually pulsed, within 1 min, into perfused rat livers operating under single-pass conditions. In untreated rats, a small peak of HRP and ovalbumin and a comparatively larger peak of inulin were observed in the bile at 7 min. In rats treated with ANIT, with increasing duration of ANIT treatment the inulin peak increased proportionally, whereas the HRP and ovalbumin peaks remained unchanged until after 10 h of ANIT exposure; gamma-globulin was not detected in the 7 min bile sample until after 14 h of ANIT treatment. Bile flow in all rats remained approximately the same until after 14 h of ANIT pretreatment, when substantial bile-flow reduction was observed. Phenobarbitone pretreatment increased the effect of ANIT and massively elevated the first HRP peak; it also shortened the time (to 4 h) at which the increase in permeability to this protein was observed. In contrast, the first HRP peak was virtually abolished in rats that had received the mixed-function-oxidase inhibitor SKF 525A. These experiments suggest that (i) ANIT progressively increased the permeability of the junctional barrier before the reduction in bile flow, (ii) the ANIT-increased permeability change seems to be inversely dependent upon the Mr of the infused proteins, and (iii) metabolites of ANIT were involved in the development of the junctional permeability change.     

Functional analysis of tight junctions

Epithelial and endothelial cells are joined to each other via a set of intercellular junctions that differ in their morphological appearance, composition, and function. The tight junction or zonula occludens is the intercellular junction that regulates diffusion between cells and therefore allows endothelia and epithelia to form cellular barriers that separate compartments of different composition. This intercellular gate formed by tight junctions is not only highly regulated but is size- and ion-selective and, hence, represents a semipermeable diffusion barrier. In epithelia, tight junctions form a morphological and functional border between the apical and basolateral cell surface domains. They directly contribute to the maintenance of cell surface polarity by forming a fence that prevents apical/basolateral diffusion of lipids in the outer leaflet of the plasma membrane. Here we describe a set of assays that allow the analysis of tight junctions to determine their integrity and functional state.     

Cytokine regulation of tight junctions

Epithelial and endothelial tight junctions act as a rate-limiting barrier between an organism and its environment. Continuing studies have highlighted the regulation of the tight junction barrier by cytokines. Elucidation of this interplay is vital for both the understanding of physiological tight junction regulation and the etiology of pathological conditions. This review will focus on recent advances in our understanding of the molecular mechanisms of tight junctions modulation by cytokines.     

Multifunctional strands in tight junctions

Tight junctions are one mode of cell-cell adhesion in epithelial and endothelial cellular sheets. They act as a primary barrier to the diffusion of solutes through the intercellular space, create a boundary between the apical and the basolateral plasma membrane domains, and recruit various cytoskeletal as well as signalling molecules at their cytoplasmic surface. New insights into the molecular architecture of tight junctions allow us to now discuss the structure and functions of this unique cell-cell adhesion apparatus in molecular terms.     

Structural integrity of hepatocyte tight junctions

The significance of discontinuities frequently found in freeze-fracture replicas of the tight junction was evaluated using complementary replicas of hepatocyte junctions from control and bile duct-ligated rats. An extensive analysis of complementary replicas using rotary platinum shadowing indicates that discontinuities in the protoplasmic (P) fracture face do not represent structural breaks in the tight- junctional network. In no case did P-face discontinuities correspond with interruptions in the groove network on the complementary extracellular (E) face. Quantitative analysis of replicas shows that P- face discontinuities result in part from "transfer" of material to the complementary E face (approximately 7% of the junctional length). However, many P-face discontinuities (7-30% of the junctional length) are matched only by a groove on the complementary E face. This finding demonstrates that a significant amount of material can be lost during freeze-fracture. An analysis of junctions from bile duct-ligated rats, which are known to have an increased paracellular permeability, shows comparable transfer and loss of material. However, the number of junctional elements and the tight-junction network density was significantly reduced by bile duct ligation. These observations indicate that discontinuities in tight-junctional elements result during the preparation of freeze-fracture replicas and are not physiologically important features of the junctional barrier. Variation in the number of elements provides the best explanation for observed differences in tight-junction permeability.     

Structural organization of the tight junctions

Tight junctions are the most apical organelle of the apical junctional complex and are primarily involved in the regulation of paracellular permeability and membrane polarity. Extensive research in the past two decades has identified not only the individual molecules of the tight junctions but also their mutual interactions, which are the focus of the present review article. While a complete map of the interactions among the tight junction molecules is probably far from being complete, the available evidence already allows outlining the general molecular architecture of the tight junctions. Here, with the aim of gaining deeper mechanistic understanding of tight junction assembly, regulation and function, we have subdivided the known molecular interactions into four major clusters that are centered on cell surface, polarity, cytoskeletal and signaling molecules.     

Structural organization of the tight junctions

Tight junctions are the most apical organelle of the apical junctional complex and are primarily involved in the regulation of paracellular permeability and membrane polarity. Extensive research in the past two decades has identified not only the individual molecules of the tight junctions but also their mutual interactions, which are the focus of the present review article. While a complete map of the interactions among the tight junction molecules is probably far from being complete, the available evidence already allows outlining the general molecular architecture of the tight junctions. Here, with the aim of gaining deeper mechanistic understanding of tight junction assembly, regulation and function, we have subdivided the known molecular interactions into four major clusters that are centered on cell surface, polarity, cytoskeletal and signaling molecules.     

Molecular physiology and pathophysiology of tight junctions. I. Biogenesis of tight junctions and epithelial polarity

The tight junction (TJ) was first noticed through its ability to control permeation across the paracellular route, but the homologies of its molecular components with peptides that participate in tumor suppression, nuclear addressing, and cell proliferation indicate that it may be involved in many other fundamental functions. TJs are formed by a dozen molecular species that assemble through PDZ and other protein-protein clustering promoting sequences, in response to the activation of E-cadherin. The TJ occupies a highly specific position between the apical and the basolateral domains. Its first molecular components seem to be delivered to such a position by addressing signals in their molecule and, once anchored, serve as a clustering nucleus for further TJ-associated molecules. Although in mature epithelial cells TJs and E-cadherin do not colocalize, a complex chain of reactions goes from one to the other that involves alpha-, beta-, and gamma-catenins, two different G proteins, phospholipase C, protein kinase C, calmodulin, mitogen-activated protein kinase, and molecules pertaining to the cytoskeleton, which keep the TJ sensitive to physiological requirements and local conditions (notably to Ca(2+)-dependent cell-cell contacts) throughout the life of the epithelium.     

Signalling to and from tight junctions

Tight junctions have long been regarded as simple barriers that separate compartments of different compositions, but recent research indicates that different types of signalling proteins and transduction pathways are associated with these junctions. They receive and convert signals from the cell interior to regulate junction assembly and function, and transmit signals to the cell interior to modulate gene expression and cell behaviour.     

Distinct claudins and associated PDZ proteins form different autotypic tight junctions in myelinating Schwann cells

The apposed membranes of myelinating Schwann cells are joined by several types of junctional specializations known as autotypic or reflexive junctions. These include tight, gap, and adherens junctions, all of which are found in regions of noncompact myelin: the paranodal loops, incisures of Schmidt-Lanterman, and mesaxons. The molecular components of autotypic tight junctions have not been established. Here we report that two homologues of Discs Lost-multi PDZ domain protein (MUPP)1, and Pals-associated tight junction protein (PATJ), are differentially localized in myelinating Schwann cells and associated with different claudins. PATJ is mainly found at the paranodal loops, where it colocalized with claudin-1. MUPP1 and claudin-5 colocalized in the incisures, and the COOH-terminal region of claudin-5 interacts with MUPP1 in a PSD-95/Disc Large/zona occludens (ZO)-1 (PDZ)-dependent manner. In developing nerves, claudin-5 and MUPP1 appear together in incisures during the first postnatal week, suggesting that they coassemble during myelination. Finally, we show that the incisures also contain four other PDZ proteins that are found in epithelial tight junctions, including three membrane-associated guanylate-kinase proteins (membrane-associated guanylate-kinase inverted-2, ZO-1, and ZO-2) and the adaptor protein Par-3. The presence of these different tight junction proteins in regions of noncompact myelin may be required to maintain the intricate cytoarchitecture of myelinating Schwann cells.     

Cell-cell junctions of dermal microvascular endothelial cells contain tight and adherens junction proteins in spatial proximity

Endothelial cell-cell contacts control the vascular permeability, thereby regulating the flow of solutes, macromolecules, and leukocytes between blood vessels and interstitial space. Because of specific needs, the endothelial permeability differs significantly between the tight blood-brain barrier endothelium and the more permeable endothelial lining of the non-brain microvasculature. Most likely, such differences are due to a differing architecture of the respective interendothelial cell contacts. However, while the molecules and junctional complexes of macrovascular endothelial cells and the blood-brain barrier endothelium are fairly well characterized, much less is known about the organization of intercellular contacts of microvascular endothelium. Toward this end, we developed a combined cross-linking and immunoprecipitation protocol which enabled us to map nearest neighbor interactions of junctional proteins in the human dermal microvascular endothelial cell line HMEC-1. We show that proteins typically located in tight or adherens junctions of epithelial cells are in the proximity in HMEC-1 cells. This contrasts with the separation of the different types of junctions observed in polarized epithelial cells and "tight" endothelial layers of the blood-brain barrier and argues for a need of the specific junctional contacts in microvascular endothelium possibly required to support an efficient transendothelial migration of leukocytes.     

Vertebrate-like tight junctions in the insect eye

Unequivocal vertebrate-like anastomosing tight junctions have been observed for the first time in insect tissues. In freeze-fractured replicas of dipteran compound eyes, the intercellular junctions between certain glial cells in regions distal to the optic neuropile display an extensive network of continuous intramembranous P face (PF) ridges. The intramembranous E face (EF) possesses a reticulum of grooves which occur in the depths of troughs and thereby produce a ‘quilted’ appearance. At PF/EF membrane face transitions, there is an obliteration of the intercellular space at points of membrane fusion; here the PF ridges and EF grooves appear in register and are therefore complementary. Although the septate junctions found here are patent, these tight junctions are occluding to lanthanum and appear to represent the blood-retinal barrier previously demonstrated electrophysiologically in insects. The existence and vertebrate-like structural complexity of these junctions in arthropods supports the concept of the universality of the membrane specializations that mediate cell-to-cell interactions.