Tight junction formation is closely linked to the polar redistribution of intramembranous particles in aggregating MDCK epithelia

We have used freeze-fracture electron microscopy to investigate the relationship between the formation of the tight junction in the establishment of a differential distribution of intramembranous particles (IMPs) between the luminal and basolateral membranes of a canine kidney cell line (MDCK). This involves a characterization of the IMP distribution in these membranes in confluent monolayers of MDCK cells, in EGTA-dissociated cells, and in cells at various stages of reassociation. While normal confluent MDCK monolayer cultures exhibit tight junctions and an IMP differential distribution between the luminal and basolateral membranes, cultures dissociated with EGTA lose both formed tight junctional elements and the differential IMP distribution. We have also found that as tight junctions reform between reaggregating MDCK cells, intramembranous particles appear to rapidly redistribute with respect to them. An asymmetric distribution of these particles in the luminal and basolateral membranes is eventually achieved. Tight junction formation appears so closely linked to the genesis of IMP polarity that at early time points when only a string of tight junctional components spans the junctional zone, differential IMP distributions are seen. Thus, our dissociation studies suggest a close relationship between the integrity of the tight junction and the maintenance of IMP polarity between the luminal and basolateral membranes, while cell reassociation studies suggest that the tight junction may be functionally linked to the genesis of IMP polarity.     

Gap junction formation between normal and reaggregated endoderm cells ofXenopus laevis neurulae

Summary Using freeze-fracture electron microscopy and fluorescent dye injection we have analysed the contacts between cells of the deeper endoderm taken from neurulae ofXenopus laevis. Endodermal cells in situ have large 1.5 m diameter gap junctions composed of 8 nm P-face particles and corresponding E-face pits. Beside gap junctions, particle aggregates typical of desmosomal plaques are present but there are no tight junctions. The dissociation of endoderm into single cells involves profound structural alterations in the surface membrane including the complete disappearance of junctional structures among them gap junctions. The reaggregation of endoderm cells leads to the restoration of the surface membrane IMP (Intra Membrane Particle) pattern and, after ca. 30 min, to the establishment of functional pathways allowing for the intercellular transfer of fluorescent dye. Concomitantly gap junctions reappear. The observation that the dissociation and reaggregation of endodermal cells involves IMP alterations which go beyond the cell junctions themselves is discussed as an adaptation of the plasma membrane to changing environmental conditions.     

Development of Sertoli cell junctional specializations and the distribution of the tight-junction-associated protein ZO-1 in the mouse testis

Basally located tight junctions between Sertoli cells in the postpubertal testis are the largest and most complex junctional complexes known. They form at puberty and are thought to be the major structural component of the "blood-testis" barrier. We have now examined the development of these structures in the immature mouse testis in conjunction with immunolocalization of the tight-junction-associated protein ZO-1 (zonula occludens 1). In testes from 5-day-old mice, tight junctional complexes are absent and ZO-1 is distributed generally over the apicolateral, but not basal, Sertoli cell membrane. As cytoskeletal and reticular elements characteristic of the mature junction are recruited to the developing junctions, between 7 and 14 days, ZO-1 becomes progressively restricted to tight junctional regions. Immunogold labeling of ZO-1 on Sertoli cell plasma membrane preparations revealed specific localization to the cytoplasmic surface of tight junctional regions. In the mature animal, ZO-1 is similarly associated with tight junctional complexes in the basal aspects of the epithelium. In addition, it is also localized to Sertoli cell ectoplasmic specializations adjacent to early elongating, but not late, spermatids just prior to sperm release. Although these structures are not tight junctions, they do have a similar cytoskeletal arrangement, suggesting that ZO-1 interacts with the submembrane cytoskeleton. These results show that, in the immature mouse testis, ZO-1 is present on the Sertoli cell plasma membrane in the absence of recognizable tight junctions. In the presence of tight junctions, however, ZO-1 is found only at the sites of junctional specializations associated with tight junctions and with elongating spermatids.     

Junctions in the central nervous system of the cat

Interendothelial membrane contacts in different segments of brain blood vessels were investigated by the freeze-etching technique. The study demonstrated that the endothelial cells of the pre- and postcapillary segments were coupled by elaborate zonulae occludentes. These tight junction formations encompassed gap junctions of different sizes and distribution. The globules of the pre- and postcapillary tight junctions revealed a great fragility which led to an atypical distribution of the sealing elements. In the "typical" brain capillaries the endothelial cells were connected by continuous tight junctions. In contrast to the structural continuity of these formations the fenestrated segments of capillaries in the choroid plexus and area postrema demonstrated discontinuous fasciae occludentes. They were composed of rows of individual particles which should be regarded primarily as focal in nature.     

Determination of apical membrane polarity in mammary epithelial cell cultures: The role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions

The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. We have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembraneous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. In such cultures the boundary between apical and basal domains was observed at the point of cell contact with the substratum. Immunocytochemical analysis of these cell-substratum contacts revealed the absence of a characteristic basement membrane containing laminin, collagen (IV), and heparan sulfate proteoglycan. However, serum-derived vitronectin was associated with the basal cell surface and the cells were shown to express the vitronectin receptor on their basolateral membranes. Additionally, treatment of cultures with antibodies against the vitronectin receptor caused cell detachment. We suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS-O while colchicine and acrylamide did not. We hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.     

Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells

Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junctions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently.     

Occludin 蛋白与细胞间紧密连接关系及其临床意义

细胞间紧密连接(tight junctions)广泛存在于上皮细胞及内皮细胞之间,其作用是保持细胞间结构的完整性,确保其功能的正常发挥,紧密连接上有很多种蛋白,occludin蛋白是其中主要蛋白之一,occludin蛋白的结构发生变化会导致紧密连接结构及功能的改变,而紧密连接结构与功能的紊乱是很多临床疾病共同的病理生理学特点,如肿瘤、中风及炎症性肺疾病。Occludin蛋白的结构及功能的改变受很多机制的调控,本文主要对occludin蛋白的结构、功能、调控机制及其与紧密连接之间的关系进行叙述。     

Tight Junctions of the Blood–Brain Barrier

1. The blood–brain barrier is essential for the maintainance and regulation of the neural microenvironment. The blood–brain barrier endothelial cells comprise an extremely low rate of transcytotic vesicles and a restrictive paracellular diffusion barrier. The latter is realized by the tight junctions between the endothelial cells of the brain microvasculature, which are subject of this review. Morphologically, blood–brain barrier-tight junctions are more similar to epithelial tight junctions than to endothelial tight junctions in peripheral blood vessels.2. Although blood–brain barrier-tight junctions share many characteristics with epithelial tight junctions, there are also essential differences. However, in contrast to tight junctions in epithelial systems, structural and functional characteristics of tight junctions in endothelial cells are highly sensitive to ambient factors.3. Many ubiquitous molecular constituents of tight junctions have been identified and characterized including claudins, occludin, ZO-1, ZO-2, ZO-3, cingulin, and 7H6. Signaling pathways involved in tight junction regulation comprise, among others, G-proteins, serine, threonine, and tyrosine kinases, extra- and intracellular calcium levels, cAMP levels, proteases, and TNF. Common to most of these pathways is the modulation of cytoskeletal elements which may define blood–brain barrier characteristics. Additionally, cross-talk between components of the tight junction– and the cadherin–catenin system suggests a close functional interdependence of the two cell–cell contact systems.4. Recent studies were able to elucidate crucial aspects of the molecular basis of tight junction regulation. An integration of new results into previous morphological work is the central intention of this review.     

Relationship between orthogonal arrays of particles and tight junctions as demonstrated in cells of the ventricular wall of the rat brain

Summary Ependymal cells in the ventricular wall and in several circumventricular organs of the rat were compared by means of freeze-fracturing. In principle, tight junctions and orthogonal arrays of particles (OAP) do not coexist in the cells bordering the ventricular wall: (1) Ordinary ependymal cells of the rat possess OAP and are devoid of tight junctions. (2) Epithelial cells of the rat choroid plexus are connected by tight junctions; OAP are lacking here. In some cases, however, tight junctions and OAP coexist in the same cell. In the boundary zone between choroid plexus and ependyma of the rat, the density of OAP is very low, whereas the tight junctions are well developed. In the subfornical and the subcommissural organ (SCO) of the rat both structures are poorly developed; in the SCO they occur segregated in different membranous areas. An overview of the literature confirms that tight junctions and OAP mostly exclude each other. The possibility that in astrocytes and ependymal cells tight junctions may have been replaced by OAP during phylogeny is briefly discussed.Dedicated to Professor A. Bohle on the occasion of his 65th birthdayPresent address: Dept. of Biol., Univ. of Oregon, Eugene, Oregon, 97403, USA     

Intercellular junctions in the corpora cardiaca of locusts

Summary The intercellular junctions in the corpora cardiaca of the locusts Schistocerca gregaria and Locusta migratoria were investigated by transmission electron microscopy. In the glandular lobes, complexes consisting of scalariform junctions and associated mitochondria, comparable to those previously observed in ion transporting epithelia, are formed between gland cells, and more rarely between gland cells and the neurons innervating them. Their structure and abundance are apparently unaffected by the stage of development or by the various experimental conditions employed. In the neural lobe, scalariform junctions form between glial cells and show close association with the endoplasmic reticulum. Gap junctions are present among glandular, neural and glial elements, and are formed between cells of the same type and of different types. Contacts resembling punctate tight junctions are widely distributed in the gland, but would be unlikely to form a barrier to diffusion. Septate junctions are formed exclusively between glial cells.     

Morphologic characteristics of intercellular junctions in early embryogenesis of mice

Dynamics concerning certain intercellular junctions have been followed during the preimplantation period of development in mouse embryos. The morphological analysis of the preimplantational embryos has demonstrated, that at the initial stages of cleavage (2-4 blastomeres) the cells make contacts by means of nonspecific junctions. Specialized intercellular junctions appear at the stage of 8 blastomeres and are presented as dotted tight and gap junctions. When the embryo is developing from the stage of 8 up to the stage of 16 blastomeres, certain connective complexes appear, consisting of dotted or cord-like tight and gap junctions. At the late morula stage, the external blastomeres in the apical part have contacts with each other by means of cingular tight junctions. In this place a connective complex might emerge; it is displayed as a tight junction and one or two gap junctions. At the blastocyst stage desmosomes and adhision zones appear. Between trophectodermal cells a connective complex arises; it is presented in the slice as a tight cingular junction, desmosomes (as a rule two) and an adhision zone. Between cells of the internal cellular mass the intercellular junctions are presented as dotted tight and gap junctions. Cells of the polar trophoectoderm and cells of the internal cellular mass could have contacts by means of gap and dotted tight junctions.     

Na-K-ATPase regulates tight junction permeability through occludin phosphorylation in pancreatic epithelial cells

Tight junctions are crucial for maintaining the polarity and vectorial transport functions of epithelial cells. We and others have shown that Na-K-ATPase plays a key role in the organization and permeability of tight junctions in mammalian cells and analogous septate junctions in Drosophila. However, the mechanism by which Na-K-ATPase modulates tight junctions is not known. In this study, using a well-differentiated human pancreatic epithelial cell line HPAF-II, we demonstrate that Na-K-ATPase is present at the apical junctions and forms a complex with protein phosphatase-2A, a protein known to be present at tight junctions. Inhibition of Na-K-ATPase ion transport function reduced protein phosphatase-2A activity, hyperphosphorylated occludin, induced rearrangement of tight junction strands, and increased permeability of tight junctions to ionic and nonionic solutes. These data suggest that Na-K-ATPase is required for controlling the tight junction gate function.     

Altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis

The dysfunction of alveolar barriers is a critical factor in the development of lung injury and subsequent fibrosis, but the underlying molecular mechanisms remain poorly understood. To clarify the pathogenic roles of tight junctions in lung injury and fibrosis, we examined the altered expression of claudins, the major components of tight junctions, in the lungs of disease models with pulmonary fibrosis. Among the 24 known claudins, claudin-1, claudin-3, claudin-4, claudin-7, and claudin-10 were identified as components of airway tight junctions. Claudin-5 and claudin-18 were identified as components of alveolar tight junctions and were expressed in endothelial and alveolar epithelial cells, respectively. In experimental bleomycin-induced lung injury, the levels of mRNA encoding tight junction proteins were reduced, particularly those of claudin-18. The integrity of the epithelial tight junctions was disturbed in the fibrotic lesions 14 days after the intraperitoneal instillation of bleomycin. These results suggest that bleomycin mainly injured alveolar epithelial cells and impaired alveolar barrier function. In addition, we analyzed the influence of transforming growth factor-β (TGF-β), a critical mediator of pulmonary fibrosis that is upregulated after bleomycin-induced lung injury, on tight junctions in vitro. The addition of TGF-β decreased the expression of claudin-5 in human umbilical vein endothelial cells and disrupted the tight junctions of epithelial cells (A549). These results suggest that bleomycin-induced lung injury causes pathogenic alterations in tight junctions and that such alterations seem to be induced by TGF-β.     

Junctions between interstitial cells of the renal medulla: A freeze-fracture study

Summary Cell junctions between interstitial cells of the renal medulla were studied in freeze-fracture replicas of kidneys from rat, rabbit, hamster and the tree-shrew Tupaia belangeri. In all species studied a composite type of intercellular junction was found comprising elements of tight junctions and irregular gap junctions of highly variable size and shape. The number of these junctions increased towards the tip of the papilla.Our findings suggest that the composite junctions observed play a role in the maintainance of the ladder-like arrangement of the interstitial cells in the inner zone. The existence of irregular gap junctions raises the possibility that the functions of the interstitial cells are coordinated, especially during alterations of the functional state of the kidney.Supported by the Deutsche Forschungsgemeinschaft     

Morphological changes in tight junctions of Necturus maculosus proximal tubules undergoing saline diuresis

Tight junctions between epithelial cells are believed to control the paracellular diffusion of substances across epithelia. Epithelia in which tight junctions are poorly developed display a higher paracellular electrical conductance, while those with extensive tight junctions show lower conductance values. We described here a particular epithelium, that of the proximal tubules of the Necturus kidney, in which the development of the tight junctions varies in parallel with a change of paracellular electrical conductance. In control conditions, tight junctions between epithelial cells of the proximal tubules are more developed than in tubules undergoing saline diuresis, a situation which increases the conductance across the paracellular shunt pathway.     

Separation of induction and expression of tight junction formation mediated by proteinases

The formation of tight junctions can be induced in the human adenocarcinoma cell line HT 29 by treatment with trypsin at 37°C. In contrast, after treatment of the cells with trypsin at low temperature (3°C), no tight junctions were observed. However, abundant formation of tight junctions occurred when cells were treated with trypsin at 3°C, washed with soybean trypsin inhibitor, and subsequently incubated at 37°C. Thus, this protocoi allows for the first time the temporal separation of the induction and assembly of tight junctions.     

Claudins in the tight junctions of stria vascularis marginal cells

In the mammalian cochlea, tight junctional strands are visible on freeze fracture images of marginal cells and other inner ear epithelia. The molecular composition of the strial tight junctions is, however, largely unknown. We investigated the expression of integral tight junction-proteins, claudin-1 to -4, and occludin, in stria vascularis of the guinea-pig cochlea, as compared to kidney. Western blot analysis revealed a strong expression of claudin-4 and occludin in strial tissue, and confocal immunofluorescence microscopy demonstrated their presence in the tight junctions of the marginal cells. In addition, a moderate level of claudin-3 and claudin-1 was detected and both were located in the marginal tight junctions. Claudins-1, -3, and -4 are characteristic of epithelia with low paracellular permeability and claudin-4 is known to restrict the passage of cations through epithelial tight junctions. In the marginal cells, these claudins appear to be responsible for the separation of the potassium-rich endolymph from the sodium-rich intrastrial fluid. In contrast, Western blot analysis and confocal microscopy demonstrated that the marginal cell epithelium does not contain claudin-2, which forms a cation-selective pore in tight junctions. Its absence indicates a cation-tight paracellular pathway in the marginal cells.     

Induction of tight junctions in human connexin 32 (hCx32)-transfected mouse hepatocytes: connexin 32 interacts with occludin

Small gap junction plaques are associated with tight junction strands in some cell types including hepatocytes and it is thought that they may be closely related to tight junctions and the establishment of cell polarity. In order to examine roles of gap junctions in regulating expression and structure of tight junctions, we transfected human Cx32 cDNA into immortalized mouse hepatocytes (CHST8 cells) which lack endogenous Cx32 and Cx26. Immunocytochemistry revealed that endogenous integral tight junction protein occludin was strongly localized and was colocalized with Cx32 at cell borders in transfectants, whereas neither was detected in parental cells. In Northern blots, mRNAs encoding occludin and the other integral tight junction proteins, claudin-1 and -2, were induced in the transfectants compared to parental cells. In Western blots, occludin protein was increased in the transfectants compared to parental cells, and binding of occludin to Cx32 protein was demonstrated by immunoprecipitation. In freeze fracture of the transfectants, tight junction strands were more numerous and complex compared to parental cells, and small gap junction plaques appeared within induced tight junction strands. Nevertheless, no change in barrier function of tight junctions was observed. These results indicate that in hepatocytes, gap junction, and tight junction expression are closely coordinated, and that Cx32 may play a role in regulating occludin expression.     

Endocytosis of cell-cell junctions and spontaneous cell disaggregation in a cultured human ovarian adenocarcinoma (COLO 316)

Although cultured COLO 316 human ovarian adenocarcinoma cells are joined by extensive tight junctions and numerous desmosomes in confluent monolayers, viable cells may be spontaneously released into the nutrient medium. Intracytoplasmic vesicles containing tight junctional and desmosomal elements were identified in freeze-fracture and thin section preparations of the released cells and some vesicles exhibited structural signs of degradation. Possible mechanisms for tight junctional and desmosomal interiorization and the possible relationship between junctional interiorization and certain malignant behaviors are discussed.     

Cell surface modifications in the epithelium of rat ventral prostate during adaptation to in vitro conditions: An ultrastructural study

Summary Sequential changes in epithelial cells of collagenase-dissociated rat ventral prostate were studied by thin-section and freeze-fracture electron microscopy. Epithelial cells did not attach to the substrate for 48 h. Pelleted cells obtained 1, 24, and 48 h after dissociation were assigned to three categories depending on morphology and cellular associations. (a) Solitary epithelial cells degenerated as determined by extensive vacuolization in the cytoplasm and aggregation of intramembranous particles (IMP). (b) Epithelial clusters consisted of a homogeneous population of well-maintained, closely packed cells. Aggregation of IMP was minimal. Tight junctions that formed between cells at the periphery of the clusters appeared normal and provided an effective permeability barrier demonstrated by the exclusion of ruthenium red tracer. (c) Tissue fragments were comprised of varying combinations of epithelial, endothelial, and smooth muscle cells as well as fibroblasts and erythrocytes. Maintenance of tissue fragments was variable. Plasma membranes often displayed aggregated IMP and proliferated tight junctional strands. An effective permeability barrier was absent. After the 48 h “latent period”, epithelial cells in the clusters lost interdependence, disassociated from one another, and attached to the substrate. These isolated cells, which did not display aggregated IMP, retained the ability to form an effective permeability barrier upon reaching confluency. During the first 48 h, epithelial cells did not tolerate solitary existence, yet as participants in clusters they were well maintained. After this interval, they no longer required interactions with neighbors in order to survive. These results indicate that under our experimental conditions, an adaptation period is required by prostatic epithelial cells. The enhanced quality of maintenance associated with epithelial clusters suggests that control over the internal microenvironment, provided by a tight junctional barrier, may be important during the initial period of adaptation in vitro. This work was supported by funds from NIH Grants CA 26063, 29513, and CA 15776; National Cancer Institute; DHHS; and Charlton Fund, Tufts University School of Medicine (awarded to P. K.).