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.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.     

Rnd3/RhoE induces tight junction formation in mammary epithelial tumor cells

Glucocorticoid hormones stimulate adherens and tight junction formation in Con8 mammary epithelial tumor cells through a multistep process in which the membrane organization of structural apical junction proteins and tight junction sealing is controlled by specific signal transduction components. We have previously shown that dexamethasone stimulation of apical junction formation requires down-regulation of the small GTPase RhoA. Here we identified Rnd3/RhoE, a GTPase-deficient Rho family member and RhoA antagonist, as a key regulator of apical junction dynamics. Exogenously expressed Rnd3/RhoE co-localized with actin at the cell periphery and induced the localization of the adherens junction protein beta-catenin and the tight junction protein ZO-1 to sites of cell-cell contact, and led to the formation of highly sealed tight junctions. Treatment with glucocorticoids was not required to achieve complete apical junction remodeling. Consistent with Rnd3/RhoE acting as an antagonist of RhoA, expression of Rnd3/RhoE rescued the disruptive effects of constitutively active RhoA on apical junction organization. Our results demonstrate a new role for the Rho family member Rnd3/RhoE in regulating the assembly of the apical junction complex and tight junction sealing.     

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4alpha-induced epithelial polarization

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization.     

Tjp3/zo-3 is critical for epidermal barrier function in zebrafish embryos

TJP3/ZO-3 is a scaffolding protein that tethers tight junction integral membrane proteins to the actin cytoskeleton and links the conserved Crumbs polarity complex to tight junctions. The physiological function of TJP3/ZO-3 is not known and mice lacking TJP3/ZO-3 show no apparent phenotype. Here we show that Tjp3/Zo-3 is a component of tight junctions present in the enveloping cell layer of zebrafish embryos. Silencing tjp3/zo-3 using morpholinos leads to edema, loss of blood circulation and tail fin malformations in the embryos. The ultrastructure of tight junctions of the enveloping cell layer is disrupted, without affecting the asymmetric distribution of plasma membrane proteins. Morphants show a loss of the epidermal barrier, as assessed by an increased permeability of the enveloping cell layer to low molecular weight tracers and a higher sensitivity of the embryos to osmotic stress. Subjecting wild-type embryos to osmotic stress mimicks the morphant phenotype, consistent with the phenotype being a direct consequence of failed osmoregulation. Thus, Tjp3/Zo-3 is critical for barrier function of the enveloping cell layer and osmoregulation in early stages of zebrafish development.     

Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell–cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.     

PICK-1: a scaffold protein that interacts with Nectins and JAMs at cell junctions

Nectin adhesion molecules are involved in the early steps of cell junction formation. Later during the polarisation process, Nectins are components of epithelial adherens junctions where they are indirectly associated with the E-cadherin/Catenins complex via the adaptator AF-6. To have a better understanding of Nectin-based cell junctions, we looked for some new Nectins' partners. We demonstrate that the scaffold molecule PICK-1, involved in the clustering of junctional receptors in synaptic junctions, interacts directly with Nectins in a PSD-95/Dlg/ZO-1 domain-dependent manner and is localised at adherens junctions in epithelial cells. Finally, we observed that protein interacting with C-kinase-1 (PICK-1) also interacts directly with the junctional adhesion molecules, and we suggest that PICK-1 could be involved in the regulation of both adherens and tight junctions in epithelial cells.     

Distinct Effects of Protein Kinase C on the Barrier Function at Different Developmental Stages

We show here, that activation of protein kinase C by the phorbol ester PMA improves barrier function in colon carcinoma (HT 29) cells. By contrast, in canine kidney (MDCK I) cells it caused increased permeability and opening of tight junctions; the latter has also been noticed in other studies. Thus, with PMA confluent HT 29 cells responded with a reduced passage of 330 kDa sodium fluorescein, increased transepithelial electrical resistance, and a change in the cell shape of the HT 29 cells from an irregular to a regular, hexagonal form. Confocal imaging revealed parallel distinct changes in the staining of occludin and caludin-1, viz. a translocation from cytoplasmic clusters to apical cell–cell contacts. Interestingly, in both cell lines protein kinase A activation caused a decreased in the threonine phosphorylation of occludin that correlated with tight junction assembly in HT 29 cells and tight junction disassembly in MDCK I cells. We conclude that protein kinase C regulation of the epithelial barrier involves specific molecular mechanisms and achieves distinct effects at different developmental stages.     

Epithelial cell-cell junctions and plasma membrane domains

Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally, compositionally, and functionally distinct surface domains. Here we consider the (in)dependence of epithelial cell polarisation and the function of smaller plasma membrane domains (e.g. adherens junctions, gap junctions, tight junctions, apical lipid rafts, caveolae, and clathrin-coated pits) in the development and maintenance of cell surface polarity. Recent evidence of cross-talk and/or overlap between the different cell-cell junction components and alternate functions of junction components, including gene expression regulation, are discussed in the context of cell surface polarity.     

Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery

The epithelial and endothelial barriers of the human body are major obstacles for drug delivery to the systemic circulation and to organs with unique environment and homeostasis, like the central nervous system. Several transport routes exist in these barriers, which potentially can be exploited for enhancing drug permeability. Beside the transcellular pathways via transporters, adsorptive and receptor-mediated transcytosis, the paracellular flux for cells and molecules is very limited. While lipophilic molecules can diffuse across the cellular plasma membranes, the junctional complexes restrict or completely block the free passage of hydrophilic molecules through the paracellular clefts. Absorption or permeability enhancers developed in the last 40 years for modifying intercellular junctions and paracellular permeability have unspecific mode of action and the effective and toxic doses are very close. Recent advances in barrier research led to the discovery of an increasing number of integral membrane, adaptor, regulator and signalling proteins in tight and adherens junctions. New tight junction modulators are under development, which can directly target tight or adherens junction proteins, the signalling pathways regulating junctional function, or tight junction associated lipid raft microdomains. Modulators acting directly on tight junctions include peptides derived from zonula occludens toxin, or Clostridium perfringens enterotoxin, peptides selected by phage display that bind to integral membrane tight junction proteins, and lipid modulators. They can reversibly increase paracellular transport and drug delivery with less toxicity than previous absorption enhancers, and have a potential to be used as pharmaceutical excipients to improve drug delivery across epithelial barriers and the blood-brain barrier.     

Regulation of Tight Junction Assembly and Epithelial Polarity by a Resident Protein of Apical Endosomes

The establishment of tight junctions and cell polarity is an essential process in all epithelia. Endotubin is an integral membrane protein found in apical endosomes of developing epithelia when tight junctions and epithelial polarity first arise. We found that the disruption of endotubin function in cells in culture by siRNA or overexpression of the C‐terminal cytoplasmic domain of endotubin causes defects in organization and function of tight junctions. We observe defects in localization of tight junction proteins, reduced transepithelial resistance, increased lanthanum penetration between cells and reduced ability of cells to form cysts in three‐dimensional culture. In addition, in cells overexpressing the C‐terminal domain of endotubin, we observe a delay in re‐establishing the normal distribution of endosomes after calcium switch. These results suggest that endotubin regulates trafficking of polarity proteins and tight junction components out of the endosomal compartment, thereby providing a critical link between a resident protein of apical endosomes and tight junctions.     

Loss of tight junction barrier function and its role in cancer metastasis

As the most apical structure between epithelial and endothelial cells, tight junctions (TJ) are well known as functioning as a control for the paracellular diffusion of ions and certain molecules. It has however, become increasingly apparent that the TJ has a vital role in maintaining cell to cell integrity and that the loss of cohesion of the structure can lead to invasion and thus metastasis of cancer cells. This article will present data showing how modulation of expression of TJ molecules results in key changes in TJ barrier function leading to the successful metastasis of a number of different cancer types.     

Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions

Using a mutant hepatocyte cell line in which E-cadherin and beta-catenin are completely depleted from the cell surface, and, consequently, fail to form adherens junctions, we have investigated adherens junction requirement for apical-basolateral polarity development and polarized membrane trafficking. It is shown that these hepatocytes retain the capacity to form functional tight junctions, develop full apical-basolateral cell polarity, and assemble a subapical cortical F-actin network, although with a noted delay and a defect in subsequent apical lumen remodeling. Interestingly, whereas hepatocytes typically target the plasma membrane protein dipeptidyl peptidase IV first to the basolateral surface, followed by its transcytosis to the apical domain, hepatocytes lacking E-cadherin-based adherens junctions target dipeptidyl peptidase IV directly to the apical surface. Basolateral surface-directed transport of other proteins or lipids tested was not visibly affected in hepatocytes lacking E-cadherin-based adherens junctions. Together, our data show that E-cadherin/beta-catenin-based adherens junctions are dispensable for tight junction formation and apical lumen biogenesis but not for apical lumen remodeling. In addition, we suggest a possible requirement for E-cadherin/beta-catenin-based adherens junctions with regard to the indirect apical trafficking of specific proteins in hepatocytes.     

'Putting the squeeze' on the tight junction: understanding cytoskeletal regulation

The apical perijunctional actomyosin ring of epithelia is structurally associated with the tight junction. The functional association between the tight junction and the perijunctional actomyosin ring was initially described in studies using pharmacological agents that disrupt microfilaments. More recently, this interaction has been studied in physiological, pathophysiological, and molecular models of tight junction regulation. These studies have demonstrated the central role of actomyosin contraction in tight junction regulation. With the identification of novel tight junction proteins and characterization of their protein:protein interactions comes the promise of detailed understanding of the molecular interactions that mediate tight junction regulation.     

Tight junctions and the modulation of barrier function in disease

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease.     

Microtubules regulate disassembly of epithelial apical junctions

Background  

Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion.     

Increased IP3/Ca signaling compensates depletion of LET-413/DLG-1 in C. elegans epithelial junction assembly

The let-413/scribble and dlg-1/discs large genes are key regulators of epithelial cell polarity in C. elegans and other systems but the mechanism how they organize a circumferential junctional belt around the apex of epithelial cells is not well understood. We report here that IP₃/Ca²⁺ signaling is involved in the let-413/dlg-1 pathway for the establishment of epithelial cell polarity during the development in C. elegans. Using RNAi to interfere with let-413 and dlg-1 gene functions during post-embryogenesis, we discovered a requirement for LET-413 and DLG-1 in the polarization of the spermathecal cells. The spermatheca forms an accordion-like organ through which eggs must enter to complete the ovulation process. LET-413- and DLG-1-depleted animals exhibit failure of ovulation. Consistent with this phenotype, the assembly of the apical junction into a continuous belt fails and the PAR-3 protein and microfilaments are no longer localized asymmetrically. All these defects can be suppressed by mutations in IPP-5, an inositol polyphosphate 5-phosphatase and in ITR-1, an inositol triphosphate receptor, which both are supposed to increase the intracellular Ca²⁺ level. Analysis of embryogenesis revealed that IP₃/Ca²⁺ signaling is also required during junction assembly in embryonic epithelia.     

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.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.