A dominant mutant of occludin disrupts tight junction structure and function
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ABSTRACT The tight junction is the most apical intercellular junction of epithelial cells and forms a diffusion barrier between individual cells. Occludin is an integral membrane protein specifically associated with the tight junction which may contribute to the function or regulation of this intercellular seal. In order to elucidate the role of occludin at the tight junction, a full length and an N-terminally truncated murine occludin construct, both FLAG-tagged at the N terminus, were stably introduced into the murine epithelial cell line CSG 120/7. Both constructs were correctly targeted to the tight junction, as defined by colocalization with another tight junction protein, ZO-1. The construct lacking the N terminus and extracellular domains of occludin was found to exert a dramatic effect on tight junction integrity. Cell monolayers failed to develop an efficient permeability barrier, as demonstrated by low transcellular electrical resistance values and an increased paracellular flux to small molecular mass tracers. Furthermore, gaps were found to have been induced in the P-face associated tight junction strands, as visualized by freeze-fracture electron microscopy. These findings demonstrate an important role for the N-terminal half of occludin in tight junction assembly and maintaining the barrier function of the tight junction.Keywords:
Occludin
Paracellular transport
Claudin
Septate junctions
Barrier function
Adherens junction
Epithelial tissues form a selective barrier via direct cell–cell interactions to separate and establish concentration gradients between the different compartments of the body. Proper function and formation of this barrier rely on the establishment of distinct intercellular junction complexes. These complexes include tight junctions, adherens junctions, desmosomes, and gap junctions. The tight junction is by far the most diverse junctional complex in the epithelial barrier. Its composition varies greatly across different epithelial tissues to confer various barrier properties. Thus, epithelial cells rely on tightly regulated transcriptional mechanisms to ensure proper formation of the epithelial barrier and to achieve tight junction diversity. Here, we review different transcriptional mechanisms utilized during embryogenesis and disease development to promote tight junction assembly and maintenance of intercellular barrier integrity. We focus particularly on the Grainyhead‐like transcription factors and ligand‐activated nuclear hormone receptors, two central families of proteins in epithelialization.
Adherens junction
Septate junctions
Barrier function
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ABSTRACT The morphology and molecular composition of inter-cellular adherens junctions have most frequently been described in epithelial cells and the fascia adhaerens of the intercalated disc. A group of cytoplasmic molecules is known to be associated with adherens junctions. The intercellular bond is mediated by cadherins which bridge the cells by homophilic binding. Recently, endothelial cells have also been shown to form intercellular junctions of the adherens-type. However, they are morphologically less distinct and little is known about their molecular components. In this study we report the localization of some adherens junction components in intact microvessels of the blood-brain barrier in the rat. We used antibodies raised against α -actinin, vinculin, zyxin, cadherin (antipan-cadherin antibody) and A-CAM (N-cadherin) in immunohistochemical experiments at light and electron microscopical levels. Microvessel walls reacted positively for all antigens throughout postnatal development. All antigens were localised, though not necessarily exclusively, to interendothelial junctions. At the ultrastructural level, pan-cadherin reactivity was present throughout the entire length of the cleft. These results could mean that in blood-brain barrier endothelial cells the complex tight junction is embedded in an adherens junction which occupies the entire length of the cleft.
Adherens junction
Septate junctions
Immunoelectron microscopy
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The organization of tissues depends on intercellular junctions that connect individual cells to each other. In sheets of epithelial cells the junctions contain different components like adherens junctions or tight junctions in an asymmetric distribution along the cell-cell contacts. Tight junctions are located at the most apical region of cell junctions, act as a regulatable barrier for small solutes, and separate the apical membrane domain from the basolateral membrane domain. For a long time, the mechanisms that underly the formation of tight junctions and the development of apico-basal membrane polarity in epithelial cells have been poorly understood. Recently, strong evidence has been provided which implicates a conserved set of cell polarity proteins--the PAR proteins--in this process. Here we discuss the mechanisms by which PAR proteins regulate the formation of cell junctions with a special emphasis on vertebrate epithelial cells.
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The skin is an indispensable barrier which protects the body from the uncontrolled loss of water and solutes as well as from chemical and physical assaults and the invasion of pathogens. In recent years several studies have suggested an important role of intercellular junctions for the barrier function of the epidermis. In this review we summarize our knowledge of the impact of adherens junctions, (corneo)-desmosomes and tight junctions on barrier function of the skin.
Adherens junction
Septate junctions
Barrier function
Epidermis (zoology)
Skin Barrier
Desmosome
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The structure and development of junctional complexes during redifferentiation of chick pigmented epithelial cells in clonal culture have been studied with TEM and colloidal lanthanum. The mature junctional complex consists of a zonula adherens which is usually surmounted by one or more macular gap junctions of varying length. Tight junctions (zonulae occludentes) appear to surround the gap junctions and extend into the zonula adherens. Punctate intermediate junctions appear first. As differentiation progresses, these extend to form fasciae and zonulae adherentes. Focal membrane fustions are found both within and above the developing adherens junctions; gap junctions appear to form adjacent to the latter structures. Colloidal lanthanum passes through the junctional regions between cells in the outer zone of the colony but is stopped by those of the differentiated cells, suggesting that these cells are sealed by fasciae or zonulae occludentes. During redifferentiation, groups of cells undergo slow, coordinated contractions which appear to be involved in developing the differentiated cell shape. These begin shortly after the formation of the junctional complexes and are most active during junctional complex maturation. Once cellular junctional complex differentiation is well established, the contractions cease. The possible roles of the different junctions in the development of cellular shape are discussed.
Adherens junction
Septate junctions
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Oral mucosa
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Adherens junction
Septate junctions
Lateral plate mesoderm
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Renal tubular segments are specialized structures with selective functions defined by the specificity of highly differentiated epithelial cells. All polarized epithelial cells in tubular structures have distinct apical and basal plasma membrane domains as well as lateral surfaces that connect sister cells by specialized cellular junctions. The basal membrane of epithelial cells adhere to the extracellular matrix primarily by integrins and syndecans, whereas the lateral surfaces of the epithelial cells interact with each other through lateral cellular junctions. Among these junctions, the apical tight junction complex controls paracellular transport, the adherens junctions localized below the tight junctions allow cells to adhere to each other, and gap junctions often found below the adherens junctions allow intercellular exchange of ions and small molecules.1,2 The tight junctions are composed of members of the occludin, claudin, and junctional adhesion molecule families. They are transmembrane proteins that connect two adjacent cells through interactions with their extracellular domains and link to respective actin cytoskeleton by their cytoplasmic domains. The major functions of the tight junctions are to form a barrier that separates the apical and basolateral aspects of epithelial cells. The degree of "tightness" of this barrier is determined by the expression of specific members of tight junction proteins during differentiation.3 As tubules elongate, epithelial cells are thought to switch from a proliferative phenotype, where many of the molecules forming cellular junctions are not expressed, to a highly differentiated cell type with well-defined adherens and tight junction complexes. How the formation of these complexes is signaled is one of the key unanswered questions in epithelial cell biology. A key component of tight junctions is the adaptor protein zona occludens 1 (ZO-1). This scaffolding molecule interacts with multiple other proteins, including actin, claudins, occludins, α-catenin, signaling proteins, and the Y-box transcription factor ZONAB. The interaction with ZONAB is particularly interesting because this transcription factor is a member of a family of DNA-binding proteins that regulate the expression of genes involved in proliferation, including cyclins, PCNA, and Erb receptors. ZONAB shuttles between tight junctions and the nucleus, and its transcriptional activity is inversely correlated with ZO-1 expression.3 When polarized renal MDCK cells become confluent, they express more ZO-1, resulting in increased ZONAB localization to the tight junctions and decreased levels of ZONAB in the cytoplasm and nucleus.4 This finding suggests that ZONAB interactions with ZO-1 are critical components of a tight junction–associated signal transduction pathway regulating the transition of epithelial cells from a proliferative to a differentiated phenotype. The article by Lima et al.5 in this issue of JASN shows in vivo and in vitro that ZONAB and ZO-1 are important in determining when proximal renal tubular cells stop proliferating and start differentiating into specialized cells. An inverse relationship between ZONAB expression and apical proximal epithelial differentiation is observed during kidney development. In proximal tubular cells in vitro, decreased ZONAB expression occurs concomitantly with increased expression of apical endocytic receptors (megalin) and maturation of primary cilium. Interestingly, ZONAB overexpression prevents polarization and differentiation of these cells while promoting a proliferative phenotype. Thus, these novel findings show the levels of ZO-1 and the localization of ZONAB, a transcription factor that normally interacts with ZO-1 and multiple other nuclear and cytoplasmic proteins, are key modulators defining when epithelial cells along proximal tubules switch from a proliferative (nuclear/cytoplasmic ZONAB) to differentiated (ZO-1–bound ZONAB) state. These observations add to a growing literature showing that cellular junctions actively modulate epithelial phenotype. The mechanism of cellular function regulated by tight junctions mirrors those attributed to the E-cadherin–mediated adherens junctions in the process of epithelial-to-mesenchymal transition. In addition to mediating the physical association of epithelial cells, E-cadherin regulates cell proliferation and migration by its interactions with β-catenin. When E-cadherin binds β-catenin, the nuclear translocation and the transcription activity of β-catenin are prevented.6 In contrast, decreased E-cadherin expression, as observed in the course of tumor metastasis or epithelial-to-mesenchymal transition, leads to increased translocation of β-catenin to the nucleus followed by increased expression of genes involved in cell survival, proliferation, or migration.7–9 This pathway plays a key role in development, organ fibrosis, and tumorogenesis in multiple tissues, including the kidney.10–13 Tight junctions and adherens junctions not only mediate similar signaling pathways, such as the ZO-1/ZONAB and the E-cadherin/β-catenin–dependent pathways, but also link to each other by interactions with common scaffolding proteins such as α-catenin and the actin cytoskeleton.3 Thus, these different cell–cell junctional complexes might act as sensors of specific physical stimuli leading to cooperation with each other in the regulation of fundamental epithelial cell functions such as proliferation, polarity, and differentiation. Understanding how these pathways are regulated and intersect with each other may very well help us to define how cell–cell contact of tubular epithelial cells modulates their ability to switch from proliferative to highly differentiated phenotypes. Disclosures None.
Adherens junction
Septate junctions
Paracellular transport
Claudin
Epithelial polarity
Occludin
Cell–cell interaction
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Septate junctions
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Septate junctions
Paracellular transport
Barrier function
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