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PubMed1993

Localisation of tight junction protein cingulin is temporally and spatially regulated during early mouse development.

The molecular maturation of the tight junction in the mouse early embryo has been investigated by monitoring the distribution of cingulin, a 140 x 10(3) M(r) peripheral (cytoplasmic) membrane constituent of the junction, at different stages of development and in different experimental situations. Although tight junction formation does not begin until compaction at the 8-cell stage, cingulin is detectable in oocytes and all stages of cleavage, a factor consistent with our biochemical analysis of cingulin expression (Javed et al., 1992, Development 117, 1145-1151). Using synchronised egg and embryo stages and isolated cell clusters, we have identified three sites where cingulin is localised, the cytocortex, punctate cytoplasmic foci and tight junctions themselves. Cytocortical cingulin is present at the cumulus-oocyte contact site (both cell types), in unfertilised and fertilised eggs and in cleavage stages up to 16-cell morulae, particularly at microvillous domains on the embryo outer surface (eg. apical poles at compaction). Embryo manipulation experiments indicate that cortical cingulin is labile and dependent upon cell interactions and therefore is not merely an inheritance from the egg. Cingulin cytoplasmic foci are evident only in outer cells (prospective trophectoderm) from the 32-cell stage, just prior to cavitation, and decline from approx. 8 hours after cavitation has initiated. The appearance of these foci is insensitive to cycloheximide treatment and they colocalise with apically derived endocytic vesicles visualised by FITC-dextran, indicating that the foci represent the degradation of cytocortical cingulin by endocytic turnover. Cingulin is detectable at the tight junction site between blastomeres usually from the 16-cell stage, although earlier assembly occurs in a minority (up to 20%) of specimens. Cingulin assembly at the tight junction is sensitive to cycloheximide and is identifiable approx. 10 hours after cell adhesion is initiated and ZO-1 protein assembles. Collectively, our results indicate that (i) cingulin from nonjunctional sites does not contribute to tight junction assembly and (ii) the molecular maturation of the junction appears to occur progressively over at least two cell cycles.

Animals↗

PubMed1997

Time course of milk accumulation-induced opening of mammary tight junctions, and blood clearance of milk components.

Eight cows in early lactation were used to study the effect of milk accumulation on the state of mammary tight junctions and to examine alpha-lactalbumin as an indicator of tight junction permeability in vivo. During three successive periods, the cows were milked twice (4 days), once (6 days), and twice daily (4 days). Plasma lactose, alpha-lactalbumin, and milk sodium concentrations were used as indicators of tight junction permeability. Furthermore, four cows were used to study the clearance of lactose and alpha-lactalbumin from the blood. Milk yield during once-daily milking decreased by 15.4% (P < 0.001). All indicators of mammary tight junction patency increased (P < 0.05) transiently during once-daily milking and indicated that tight junctions opened after approximately 18 h. Plasma alpha-lactalbumin and lactose were highly correlated (r = 0.82, P < 0.001), indicating the suitability of plasma alpha-lactalbumin as an indicator of tight junction status in vivo. Clearance of alpha-lactalbumin and lactose from the blood was best described by a biexponential model. Elimination half-lives for lactose and alpha-lactalbumin were 44 and 40 min, respectively. This study showed that milk stasis during early established lactation induces tight junctions to switch to a leaky state after approximately 18 h and to revert to the closed state shortly after milking.

Animals↗

PubMed1995

Immunogold localization of actin in the testis and exocrine pancreas: spatial relationship with tight junctional strands.

The fracture-label technique was used in conjunction with a monoclonal antibody to actin and the phospholipase A2-colloidal gold (PLA2-CG) method to examine the spatial distribution of actin filaments in relation to the three-dimensional arrangement of tight junctional strands in rat testes and exocrine pancreatic acinar cells. The intimate association of actin filaments with tight junctional strands in the pancreas and testis was also illustrated by a double-labeling experiment in which freeze-fractured pancreas or testis was labeled with monoclonal antibody-protein A-gold (30 nm gold size) followed by incubation with a PLA2-CG complex (11 nm gold size). Freeze-fracture-exposed tight junctional strands in both testicular and exocrine pancreatic cells labeled by PLA2-CG complex, indicated the presence of phospholipids in these cylindrical membranous structures. Immunolabeling of freeze-fractured testes with a monoclonal antibody to actin revealed a narrow band of gold particles juxtaposed to the cytoplasmic aspect of the protoplasmic membrane halves decorated with parallel linear arrays of cylindrical tight junctional strands. Many of the gold particles representing actin antigenic sites were in direct contact with the cross-fractured tight junctional strands. Fracture-label preparations of exocrine pancreas labeled with the monoclonal anti-actin antibody also exhibited a similar labeling pattern at the apex of acinars cells where the tight junction complex is located. Double-labeling experiments revealed the simultaneous labeling of actin and phospholipids in the same fracture-label preparations. Digestion of testicular and pancreatic tissue samples in a free PLA2 solution prior to labeling with the monoclonal antibody or PLA2-CG complex removed not only the gold labeling previously seen over the tight junctional strands but also reduced drastically the immunolabeling for actin that was previously seen associated with the tight junction complex. Taken together, results of the present study showed that actin filaments are structural components of the tight junction strands and are connected to the cytoplasmic aspect of the latter structures. The interaction between this particular cytoskeletal element and the tight junction may be through the binding of a special domain of the actin filament to the phospholipids that partially make up the tight junctional complex.

Actins↗

PubMed1991

Freeze-fracture observations on normal and abnormal human perineurial tight junctions: alterations in diabetic polyneuropathy.

Perineurial cells in the human sural nerve possess tight junctions which in freeze-fracture replicas are seen to be composed of networks of branching and anastomosing P face strands and E face grooves. Isolated circular tight junctions (maculae occludentes) may represent attachment devices between adjacent perineurial lamellae. At the overlapping margins of the cells, a belt-like tight junction (zonula occludens) encircles the cells and is believed to comprise a paracellular diffusion barrier. As the permeability of the perineurium has been found to be altered in diabetic polyneuropathy, the zonulae occludentes have been studied. In freeze-fracture replicas from cases of diabetic polyneuropathy a mixed population of structurally normal and abnormal junctions was observed. In some, the strands were abnormally curved with reduced numbers of intersections, the intervening plasma membrane displaying prominent P face concavities and E face convexities. At other sites, the junctions were severely disorganized and represented by fragmented and isolated strands with few intersections and numerous free ends. These abnormalities resemble changes that have been produced experimentally in epithelial tight junctions by osmotic damage. The possibility is considered that similar mechanisms could result in the alterations of the perineurial tight junctions in diabetic polyneuropathy and account for its impaired permeability barrier properties.

Adult↗

PubMed2005

Segment-specific expression of tight junction proteins, claudin-2 and -10, in the rat epididymal epithelium.

Tight junctions are known to be related to the variance by segment of the luminal fluid microenvironment of the epididymis. We examined the expression of claudins that regulate the paracellular permeability of ions, solutes, and water through tight junctions in the rat epididymis. RT-PCR analysis showed that claudin-2 and -10 were expressed in a segment-specific manner among fifteen claudins examined. Immunofluorescence microscopy demonstrated that both claudins showed developmental stage- and segment-specific subcellular localizations in the epididymal epithelium. Claudin-2 was detected principally in the apical junctional region in the initial segment at postnatal day 7 and week 10 but lost its expression in all the distal segments including the caput, corpus, and cauda. Claudin-10 was detected principally in the apical junctional region in all segments on postnatal day 7 but only in the initial segment--especially along the entire lateral membrane--at 10 weeks. In freeze fracture electron microscopy, well-developed intramembranous particle strands were observed on the P face and complementary grooves with a few particles on the E face at 10 weeks. Many particles were occasionally observed on the grooves in the E face in the initial segment and distal caput. Tight junction strands were continuous and impermeable to lanthanum nitrate on postnatal day 7, suggesting the establishment of functional tight junctions. These segment-specific expressions of claudin-2 and -10 may contribute to creating the specific luminal fluid microenvironment which is necessary for the transport, maturation, and storage of spermatozoa.

Animals↗

PubMed1996

The inter-Sertoli cell tight junctions in germ cell-free seminiferous tubules from prenatally irradiated rats: a freeze-fracture study.

The tight junctions between Sertoli cells were examined by freeze-fracture in 3-month-old prenatally irradiated rats, whose seminiferous tubules are devoid of germ cells. The replicas from irradiated tubules show elaborate interdigitations of the lateral membranes of Sertoli cells and very extensive tight junctions. These junctions are characterized by a great number of continuous parallel or complex interweaving strands of intramembranous particles, preferentially associated with E fracture faces. The presence of highly cross-linked tight junctional strands is compatible with an epithelium deprived of germ cells, with a reduced need for flexibility. Anomalous ectoplasmic specializations, consisting of groups of cisternae arranged perpendicularly to the lateral surface, are found in the irradiated tubules. These structures may be involved in a storage mechanism of redundant lateral membrane resulting from the elimination of germ cells. Typical gap junctions, intercalated between the tight junctional strands, are larger and more frequently found in treated animals than in controls. These findings indicate that a very tight permeability barrier seems to be established in the irradiated testis even in the absence of germ cells. Thus, the formation and maintenance of Sertoli tight junctions do not appear to be directly dependent on the presence of germ cells. Nevertheless, the alterations detected in the tight junction architecture and in the ectoplasmic specializations indicate that maturing germ cells probably contribute to the functional organization of the blood-testis barrier in the normal testis.

Animals↗

PubMed1996

Disturbed structural interactions between microfilaments and tight junctions in rat hepatocytes during extrahepatic cholestasis induced by common bile duct ligation.

Microfilaments in epithelial cells are important for the structural and functional integrity of tight junctions. In the present study, we examined the relationship between microfilaments and tight junctions in hepatocytes of rat liver following common bile duct ligation (CBDL) for up to 2 weeks. Actin filaments and tight junctions were studied by fluorescence microscopy using 7-nitrobenzene-2-oxa-1,3-diazole phallacidin (NBD-ph) and an anti-ZO-1 antibody, respectively. Double-stained sections were examined with confocal laser scanning microscopy (CLSM). Electron microscopy was applied for the assessment of structural alterations in microfilaments and in tight junctions with detergent-extraction and freeze-fracture preparations. Our results showed that F-actin was present at the entire plasma membrane of hepatocytes in control liver, whereas CBDL increased the amount of F-actin mainly at the bile canalicular and lateral plasma membranes. Simultaneously, the immunofluorescence of ZO-1 underwent striking changes, i.e., from a uniform to an irregular staining pattern with various fluorescence intensities. CLSM demonstrated a colocalization of ZO-1 and F-actin in control liver and its deterioration in CBDL liver. Electron microscopy showed marked alterations of microfilaments and tight junctions due to CBDL. It is concluded that actin filaments are intimately associated with tight junctions in normal hepatocytes. CBDL impairs this association by progressively diminishing the structural interaction between F-actin and ZO-1, which may in turn lead to functional disturbances of tight junctions.

Actin Cytoskeleton↗

PubMed1979

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.

Animals↗

PubMed1999

Copper treatment alters the permeability of tight junctions in cultured human intestinal Caco-2 cells.

The effects of copper on tight-junction permeability were investigated in human intestinal Caco-2 cells, monitoring transepithelial electrical resistance and transepithelial passage of mannitol. Apical treatment of Caco-2 cells with 10-100 microM CuCl(2) (up to 3 h) produced a time- and concentration-dependent increase in tight-junction permeability, reversible after 24 h in complete medium in the absence of added copper. These effects were not observed in cells treated with copper complexed to L-histidine [Cu(His)(2)]. The copper-induced increase in tight-junction permeability was affected by the pH of the apical medium, as was the apical uptake of (64)CuCl(2), both exhibiting a maximum at pH 6.0. Treatment with CuCl(2) produced a concentration-dependent reduction in the staining of F actin but not of the junctional proteins zonula occludens-1, occludin, and E-cadherin and produced ultrastructural alterations to microvilli and tight junctions that were not observed after treatment with up to 200 microM Cu(His)(2) for 3 h. Overall, these data point to an intracellular effect of copper on tight junctions, mediated by perturbations of the F actin cytoskeleton.

Actins↗

PubMed1995

Regulated assembly of tight junctions by protein kinase C.

We have previously shown that protein phosphorylation plays an important role in the sorting and assembly of tight junctions. We have now examined in detail the role of protein kinases in intercellular junction biogenesis by using a combination of highly specific and broad-spectrum inhibitors that act by independent mechanisms. Our data indicate that protein kinase C (PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibited development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, was paralleled by a delay in the sorting of the tight-junction protein, zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction were not detectably affected, as determined by immunocytochemical analysis. In addition, ZO-1 was phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevented approximately 85% of the phosphorylation increase. Furthermore, in vitro measurements indicate that ZO-1 may be a direct target of PKC. Moreover, membrane-associated PKC activity more than doubled during junction assembly, and immunocytochemical analysis revealed a pool of PKC zeta that appeared to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins was detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may be occludin. The mass of this complex and the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were not PKC dependent.

Animals↗

PubMed2006

Evidence that tight junctions are disrupted due to intimate bacterial contact and not inflammation during attaching and effacing pathogen infection in vivo.

It is widely accepted that tight junctions are altered during infections by attaching and effacing (A/E) pathogens. These disruptions have been demonstrated both in vitro and more recently in vivo. For in vivo experiments, the murine model of A/E infection with Citrobacter rodentium is the animal model of choice. In addition to effects on tight junctions, these bacteria also colonize the colon at high levels, efface colonocyte microvilli, and cause hyperplasia and inflammation. Although we have recently demonstrated that tight junctions are disrupted by C. rodentium, the issue of direct effects of bacteria on epithelial cell junctions versus the indirect effects of inflammation still remains to be clarified. Here, we demonstrate that during the C. rodentium infections, inflammation plays no discernible role in the alteration of tight junctions. The distribution of the tight junction proteins, claudin-1, -3, and -5, are unaffected in inflamed colon, and junctions appear morphologically unaltered when viewed by electron microscopy. Additionally, tracer molecules are not capable of penetrating the inflamed colonic epithelium of infected mice that have cleared the bacteria. Finally, infected colonocytes from mice exposed to C. rodentium for 14 days, which have high levels of bacterial attachment to colonocytes as well as inflammation, have characteristic, altered claudin localization whereas cells adjacent to infected colonocytes retain their normal claudin distribution. We conclude that inflammation plays no discernible role in tight junction alteration during A/E pathogenesis and that tight junction disruption in vivo appears dependent only on the direct intimate attachment of the pathogenic bacteria to the cells.

Animals↗

PubMed1988

Alterations in uterine epithelial tight junction structure during the oestrous cycle and implantation in the pig.

The structure of the pig uterine epithelium tight junction has been studied using freeze-fracture methods during oestrus and implantation. Compared with non-pregnant non-cyclic pigs, in both pregnancy and during the cycle the number of intersections per unit area of the tight junction ridges increases to a maximum 16 days after ovulation. There was no significant alteration in the depth or number of ridges in the tight junction band. The only significant difference between tight junctions in cyclic and pregnant pigs was a brief increase in the depth during the cycle (12-15 days after ovulation). The number of intersections is thought to be a far more important measure than depth in determining the permeability of the tight junction. It therefore seems unlikely that changes in tight junction structure play any direct role in the rescue of the corpus luteum or in establishing pregnancy but merely reflect the alterations in progesterone concentrations. However, the decrease in uterine permeability indicated by the increase in intersection frequency has been reported for other species and may reflect an important change in the uterine milieu in preparation for a possible pregnancy, apparently induced by alterations in progesterone concentration.

Animals↗

PubMed1994

The structure of the tricellular region of endothelial tight junctions of pulmonary capillaries analyzed by freeze-fracture.

This study provides the first description of tight junction organization in the tricellular regions of pulmonary capillary endothelial cells. Three important characteristics of tight junction organization at these corners were observed. First, endothelial tight junctions are discontinuous at these corners where an intramembranous gap which averages 27.4 +/- 2.3 (SE) nm in width and 1.1 + 0.17 (SE) micron in length was observed to cross the depth of the junctional complex and endothelium. Second, the depth of the tricellular region of endothelial tight junctions at these corners was made possible by an overlapping flap provided by one of the three cells, the flap cell, which covers the remaining two adjacent cells. Third, the observations in this study demonstrated that the tricellular regions of endothelial tight junctions are oriented parallel to the plane of the endothelium rather than perpendicular as in epithelium. A model of this organization, based upon freeze-fracture replicas of 16 tricellular regions, thin section data, and scanning electron microscopic data of perfusion fixed guinea pig lungs is provided.

Animals↗

PubMed2006

Growing axons in fish optic nerve are accompanied by astrocytes interconnected by tight junctions.

Mammalian astrocytes are in general interconnected by gap but not by tight junctions and play an ambiguous and controversially discussed role in central nervous system regeneration. At different neuroanatomical sites, fish astrocytes are interconnected by tight junctions and desmosomes and are involved in the successful regeneration of lesioned fiber tracts. In fish, newly generated retinal ganglion cells continuously grow new axons to the optic tectum but the interrelationship between glial tight junctions and axonal growth is undefined so far. We therefore investigated the occurrence of tight junctional structures and molecules within the ribbon-shaped optic nerve of a teleost fish (Astatotilapia burtoni) and found a predominant expression of zonula occludens protein-1 and claudin-1 in astrocytes where axons of new ganglion cells are assembled retinotopically within the optic nerve. This may support a previously formulated hypothesis according to that different properties of astrocytic membranes could be responsible for different glio-neuronal interactions which in turn may determine the micro-environmental conditions of growing axons.

Animals↗

PubMed2005

Heterogeneity in expression and subcellular localization of tight junction proteins, claudin-10 and -15, examined by RT-PCR and immunofluorescence microscopy.

Tight junctions regulate paracellular permeability, create the luminal fluid microenvironment of blood vessels and the digestive tract, and also form the protective barrier in the stratified epithelium including the epidermis. Claudins are the integral membrane proteins at tight junctions and form a multigene family composed of at least 24 members, but knowledge of the subcellular localization of each claudin is still fragmentary. We performed RT-PCR for fifteen claudin species to examine the mRNA expression in various mouse tissues, and focused on investigating the subcellular localization of claudin-10 and -15 by immunofluorescence microscopy in various rat tissues. Neither claudin-10 nor -15 was detected in vascular endothelial cells in most tissues, and these claudins were restricted to the vasa recta in the kidney medulla. Both claudins were also detected at apical tight junctions in the epithelium of the jejunum with no intensity gradients along the crypt-to-villus axis. However, both claudins were expressed only in the basal half of the crypt epithelium in the colon, showing obvious gradients along crypt-to-surface axis. Moreover, claudin-10 showed the ectopic subcellular localization where tight junction strands do not exist. Claudin-10 was detected along the entire lateral membranes of acinar cells in addition to the apical tight junctions in exocrine glands, and in the cytoplasm of basal cells in the stratified epithelium including the dorsal skin and cutaneous stomach. These heterogeneous distributions of claudin-10 and -15 in tissues may be related to the differences in paracellular permeability among tissues.

Animals↗

PubMed1999

Transmembrane proteins in the tight junction barrier.

Three types of transmembrane proteins have been identified within the tight junction, but it remains to be determined how they provide the molecular basis for regulating the paracellular permeability for water, solutes, and immune cells. Several of these proteins localize specifically within the continuous cell-to-cell contacts of the tight junction. One of these, occludin, is a cell adhesion molecule that has been demonstrated to influence ion and solute permeability. The claudins are a family of four-membrane spanning proteins; unexpectedly, other members of this family have already been characterized without recognizing their relationship to tight junctions. Junction adhesion molecule, the most recently identified tight junction component, is a member of the Ig superfamily and influences the paracellular transmigration of immune cells. A plaque of cytoplasmic proteins under the junction may be responsible for scaffolding the transmembrane proteins, creating a link to the perijunctional actin cytoskeleton and transducing regulatory signals that control the paracellular barrier.

Animals↗

PubMed2006

AMP-activated protein kinase regulates the assembly of epithelial tight junctions.

AMP activated protein kinase (AMPK), a sensor of cellular energy status in all eukaryotic cells, is activated by LKB1-dependent phosphorylation. Recent studies indicate that activated LKB1 induces polarity in epithelial cells and that this polarization is accompanied by the formation of tight junction structures. We wished to determine whether AMPK also contributes to the assembly of tight junctions in the epithelial cell polarization process. We found that AMPK is activated during calcium-induced tight junction assembly. Activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside facilitates tight junction assembly under conditions of normal extracellular Ca2+ concentrations and initiates tight junction assembly in the absence of Ca2+ as revealed by the relocation of zonula occludens 1, the establishment of transepithelial electrical resistance, and the paracellular flux assay. Expression of a dominant negative AMPK construct inhibits tight junction assembly in MDCK cells, and this defect in tight junction assembly can be partially ameliorated by rapamycin. These results suggest that AMPK plays a role in the regulation of tight junction assembly.

AMP-Activated Protein Kinases↗

PubMed1993

Two classes of tight junctions are revealed by ZO-1 isoforms.

The tight junction forms the intercellular barrier separating tissue compartments. The characteristics of this barrier are remarkably diverse among different epithelia and endothelia and are not explained by our limited knowledge of its molecular composition. Two isoforms of the 220-kDa tight junction protein ZO-1 result from alternative RNA splicing and differ by an internal 80-amino acid domain, termed alpha (E. Willott, M. S. Balda, M. Heintzman, B. Jameson, and J. M. Anderson. Am. J. Physiol. 262 (Cell Physiol. 31): C1119-C1124, 1992). Using antibodies specific for each isoform and double-labeled immunofluorescence microscopy, we observed that the ZO-1 alpha- isoform is restricted to junctions of endothelial cells and highly specialized epithelial cells of both seminiferous tubules (Sertoli cells) and renal glomeruli (podocytes); in contrast, the ZO-1 alpha+ isoform is expressed in cells of all other epithelia examined. Both immunoblotting and ribonuclease protection analysis confirmed this pattern of expression. This distribution does not correlate with differences in junctional resistance or ultrastructural complexity. Instead, we observe a correlation with junctional plasticity; ZO-1 alpha- is expressed in structurally dynamic junctions, whereas ZO-1 alpha+ is expressed in those which are less dynamic. This is the first molecular distinction among tight junctions and reveals a fundamental dichotomy with implications for how the paracellular barriers of endothelia and epithelia are regulated.

Amino Acid Sequence↗