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.     

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.     

Tight junctions of human keratinocytes in primary culture: a freeze-fracture study

Human skin explants obtained from 2- to 5-yr-old patients with harelips were cultured in NCTC 168 medium at 37 degrees C, in a humidified atmosphere containing 5% CO2 in air. After a 2-week incubation period, the newly grown cells were studied with special reference to tight junctions by freeze-fracture electron microscopy. Many completely formed tight junctions were observed between the uppermost living cells of migrating epithelium, and fragmented tight junctions were seen between the lower layer cells. The tight junctions in the uppermost cells developed so well that they formed a belt-like network consisting of two to six rows of strands. This observation may suggest that human keratinocytes have the ability to produce tight junctions perfectly enough to serve as a barrier, although no complete tight junctions were formed in situ.     

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.     

Tight junctions of dissociated and reaggregated embryonic lung cells

Treatment of embryonic lung tissue with trypsin resulted in clustering of intramembrane particles (IMP) and gradual disassembly of tight junctions. In dissociated single cells kept in trypsin-free medium, IMP are randomly distributed but degradation of tight junctions continue. Vesicles containing tight junction elements were observed within the cytoplasm. It is therefore assumed that tight junctions may be degraded in two ways: breakdown of elements to IMP, and endocytosis. In cells reaggregated by rotation tight junctions reassembled only in hystotypic aggregates. Cycloheximide which interferes with histotypic reaggregation prevents the reassembly of tight junctions.     

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。     

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.     

星形胶质细胞可以诱导内皮细胞系紧密连接的形成

为探索星形胶质细胞在血脑屏障内皮细胞紧密连接形成中的重要意义,通过内皮细胞系ECV304与星形胶质细胞体外接触共培养的方法,采用电镜及内皮细胞紧密连接的银染观察星形胶质细胞对内皮细胞系紧密连接的诱导作用。运用Millipore-ERS系统检测紧密连接的功能状况。结果发现,星形胶质细胞可以诱导内皮细胞系形成广泛而连续的紧密连接并产生较高的跨内皮阻抗(transendothelial electrical resistance,TER),于第10d可达321．3Ωcm^2。提示,星形胶质细胞可以诱导ECV304细胞产生紧密连接。同时,ECV304细胞与星形胶质细胞的体外共培养可以作为研究血脑屏障紧密连接结构与功能的一种可靠而简便的体外实验方法。     

Intercellular junctions between human odontoblasts. A freeze-fracture study after demineralization

Intercellular junctions in the odontoblastic layer have been studied with a freeze-fracture technique. Children's tooth germs were fixed, sliced and demineralized. Samples of the pulpodentinal border were routinely prepared for freeze-fracture. Three kinds of intercellular junctions were detected between human odontoblast cell bodies: gap junctions, desmosomes and tight junctions. Numerous gap junctions are responsible for intercellular communication at different levels of the cell bodies. Focal tight junctions, parallel to the axis of the cell, and desmosomes are sites of cell-to-cell adhesion between lateral plasma membranes. At the distal end of the cell bodies, junctional complexes consist of zonular tight junctions and gap junctions. These zonular tight junctions, never before described between odontoblasts, contribute to the pseudo-epithelial organization of the odontoblastic layer. They constitute a predentin-pulp barrier, the permeability of which must be studied to establish their role in relation to dentin formation.     

Development of Sertoli cell junctions in vitro--a freeze-fracture study

Seminiferous tubules of 1-day-old rats were maintained in organ culture for up to 40 days. Five classes of intercellular junctions between Sertoli cells were observed by the freeze-fracture method as the tissue aged: (a) typical gap junctions; (b) focal tight junctions; (c) macular tight junctions; (d) meandering tight junctions; and (e) extensive tight junctions. The relative proportions of these types of Sertoli cell junctions were quantitated as the organ cultures progressed. The junctional structures observed and classified in organ culture were identical to those seen in vivo, but the timing of their appearance and/or disappearance, as well as their relative proportions, was different from that observed in the developing animal. Extensive tight junctions, with numerous parallel strands, were observed in the 40-day cultures; however, their oblique orientation with respect to the myoid layer was in contrast to the parallel orientation observed in vivo.     

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.     

Sealing junctions in a number of arachnid tissues

The junctions present in the central nervous system (CNS), midgut, silk gland and venom gland of arachnids have been investigated. Special care was taken to try to locate tight junctions in tissue other than CNS but they were not found in any of the other tissues. The detailed structure of the junctions present are discussed. The tight junctions present in CNS are somewhat different in appearance and fracturing behaviour to most vertebrate tight junctions and closely resemble only those found in Urochordates (a non-vertebrate chordate). The two types of septate junctions found in the other tissues belong to the pleated septate and smooth septate classes but show some interesting differences. It appears probable that the septate junctions in Arachnida, Merostomata and Myriopoda have different fracturing properties from those found in other arthropods. The finding that only septate junctions are present in most arachnid tissues, although tight junctions are present in CNS, is discussed in the context of the sealing function of septate junctions in invertebrate tissues.     

Tight junction development between cultured hepatoma cells: Possible stages in assembly and enhancement with dexamethasone

Freeze-fracture and thin-section methods were used to study tight junction formation between confluent H4-II-E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: (1) “formation zones” (smooth P-fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; (2) linear strands and aggregates of 9–11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E-face depressions; (3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specialization may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 × 10^?6 M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.     

PERMEABILITY OF SERTOLI CELL TIGHT JUNCTIONS TO LANTHANUM AFTER LIGATION OF DUCTUS DEFERENS AND DUCTULI EFFERENTES

The permeability of Sertoli cell tight junctions to lanthanum administered during fixation has been compared in rats after ligation of the ductus deferens and after ligation of the ductuli efferentes. In both control and vasoligated testes, lanthanum penetrated only short distances into the Sertoli cell tight junctions before stopping abruptly. The tight junction, consisting of numerous pentalaminar fusions of contiguous Sertoli cell membranes, prevented diffusion of lanthanum into the adluminal compartment of the seminiferous epithelium. In rats with ligated ductuli efferentes, lanthanum completely permeated many Sertoli cell tight junctions and occupied intercellular spaces of the adluminal compartment. In spite of their newly acquired permeability to lanthanum, tight junctions retained characteristic ultrastructural features, including numerous membrane fusions. When lanthanum-filled tight junctions were sectioned en face, membrane fusions appeared as pale lines in lakes of electron-opaque tracer. These linearly extensive fasciae occludentes occasionally ended blindly, suggesting that lanthanum may have traversed the junction by diffusing around such incomplete barriers. The increased permeability of Sertoli cell tight junctions after efferent ductule ligation, which caused rapid testicular weight gain followed by atrophy, indicates that tight junctions are sensitive to enforced retention of testicular secretions inside the seminiferous tubules. The apparent normalcy of Sertoli cell tight junctions after vasoligation, which had no effect on testis weight, supports the view that blockage of testicular secretions distal to the epididymis is relatively innocuous.     

Assembly of tight junction is regulated by the antagonism of conventional and novel protein kinase C isoforms

Apparently conflicting observations indicated that protein kinase C both may block and support the assembly of tight junctions. We therefore tested the hypothesis that different isoenzymes antagonistically affect tight junction proteins and function. Thus, by using specific inhibitors we investigated the involvement of conventional and novel protein kinase C of kidney tubule cells in tight junction assembly. In low Ca2+ medium, the application of pan-protein kinase C inhibitor GF-109203X blocked the formation of tight junctions induced by protein kinase C agonist diacyglycerol. G?6976, inhibitor of conventional protein kinase C, promoted the formation of tight junctions and occludin phosphorylation in cells cultivated in low Ca2+ medium and attenuated the disruption of tight junction complex induced by the switch to low Ca2+ medium. In addition, G?6976 accelerated the occludin phosphorylation and the formation of tight junction barrier during assembly of tight junctions induced by Ca2+ re-addition. This phosphorylation was accompanied by accelerated occludin incorporation into newly forming tight junctions and by reducing the paracellular permeability. In contrast, inhibitor of novel protein kinase C rottlerin blocked the occludin phosphorylation and the formation of tight junction barrier, both caused by re-addition of normal Ca2+ medium. It is concluded that the conventional protein kinase C alpha participates in tight junction disassembly while the novel protein kinase C epsilon plays a role in tight junction formation of kidney epithelial cells. The discovered antagonism contributes to a better understanding of the regulation of the structure and function of tight junctions and hence to that of the epithelial barrier.     

Formation of tight junctions and desmosomes protects MDCK cells against hyperthermic killing

Cell density is known to modify the survival of mammalian cells exposed to elevated temperatures. We have examined the role that cell–cell contact plays in this phenomenon. The formation of cell–cell contact is carried out by cells' junctional complex, i.e., tight junctions, desmosomes, and gap junctions. Lack of formation of tight junctions and desmosomes, or their opening, could interfere with the functions and structures of cell membrane. Membrane damage is at least partially responsible for cell death at elevated temperatures. MDCK cells with high density plated in low calcium medium form confluent monolayers devoid of the formation of tight junctions and desmosomes but quickly assemble them after Ca²⁺ restoration. We used MDCK cells and the calcium switch technique to investigate effects of cell–cell contact and, independently, of cell density on hyperthermic cell killing. We found that MDCK cells that formed tight junctions and desmosomes were more resistant to hyperthermic treatment than those that did not. Blocking the formation pathway of tight junctions made cells sensitive to heat. Cells growing at lowdensity showed almost the same survival as did cells at high density in the absence of the formation of tight junctions and desmosomes. The results suggest that the formation of tight junctions and desmosomes play a more important role in determining hyperthermic response than does density per se. The formation of tight junctions and desmosomes appears to protect cells modestly against hyperthermic killing. © 1994 Wiley-Liss, Inc.     

Extracellular calcium and the organization of tight junctions in pancreatic acinar cells

In pancreatic lobules incubated in Ca²⁺-free Krebs-Ringer bicarbonate solution +0.5 mM EGTA tight junctions are first disarrayed and then break up into fasciae occludentes and small fibrillar fragments, which move laterally in the plane of the plasmalemma and often wind up around the gap junctions. The interruption of the continuity of tight junctions results in the disappearance of the difference in intramembranous particle density between the lateral and luminal regions of the plasmalemma. These results are consistent with the interpretation of tight junctions as dynamic structures, probably resulting from a specific polymerization of intramembranous particles and confirm that tight junctions might have a role in establishing and maintaining the regional differences of the plasmalemma.     

Knockout animals and natural mutations as experimental and diagnostic tool for studying tight junction functions in vivo

Two sides of functions of tight junctions; the barrier and the channel in the paracellular pathway are believed to be essential for the development and physiological functions of organs. Recent identification of molecular components of tight junctions has enabled us to analyze their functions by generating knockout mice of the corresponding genes. In addition, positional cloning has identified mutations in the genes of several components of tight junctions in hereditary diseases. These studies have highlighted in vivo functions of tight junctions.     

Development of sertoli cell junctions in vitro—A freeze-fracture study

Summary Seminiferous tubules of 1-day-old rats were maintained in organ culture for up to 40 days. Five classes of intercellular junctions between Sertoli cells were observed by the freeze-fracture method as the tissue aged: (a) typical gap junctions; (b) focal tight junctions; (c) macular tight junctions; (d) meandering tight junctions; and (e) extensive tight junctions. The relative proportions of these types of Sertoli cell junctions were quantitated as the organ cultures progressed. The junctional structures observed and classified in organ culture were identical to those seen in vivo, but the timing of their appearance and/or disappearance, as well as their relative proportions, was different from that observed in the developing animal. Extensive tight junctions, with numerous parallel strands, were observed in the 40-day cultures; however, their oblique orientation with respect to the myoid layer was in contrast to the parallel orientation observed in vivo. This study was supported by Grant 801D185 (Dr. Posalaky) from the Medical Education and Research Foundation, St. Paul-Ramsey Hospital.     

Knockout animals and natural mutations as experimental and diagnostic tool for studying tight junction functions in vivo

Two sides of functions of tight junctions; the barrier and the channel in the paracellular pathway are believed to be essential for the development and physiological functions of organs. Recent identification of molecular components of tight junctions has enabled us to analyze their functions by generating knockout mice of the corresponding genes. In addition, positional cloning has identified mutations in the genes of several components of tight junctions in hereditary diseases. These studies have highlighted in vivo functions of tight junctions.