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Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium: physiological rearrangement of tight junctions.

All epithelia slough dying cells but the consequences of this physiological process to epithelial barrier function is unknown. In mammalian small intestine absorptive cells are known to migrate from the villus base to the villus tip from which they slough. These villus tip extrusion zones are often envisioned as sites at which macromolecules could leak across the epithelium. However, only trace amounts of macromolecules cross this epithelium even though, based on known epithelial turnover rates, extrusion events occur millions of times daily. Here, we examine the characteristics of the epithelial barrier to macromolecular permeation at villus tip extrusion zones in rats and hamsters. Freeze-fracture, light and electron microscope studies reveal that extruding cells do not leave transient holes behind as they lift from the epithelium. Rather, as cells extrude, processes of adjacent cells extend under them. Moreover, tight junction elements proliferate between extruding cells and their neighbors and appear to move down the lateral margin of the extruding cell as it extends into the lumen. These observations suggest that newly formed junctional elements "zipper" the epithelium closed as extrusion proceeds thus preventing epithelial discontinuities from occurring. Correlative in vivo perfusion experiments using horseradish peroxidase as a macromolecular-tracer show that the above described dynamic alterations in tight junctions at extrusion sites are generally sufficient to prevent transepithelial leaks of macromolecules.

Animals↗

Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis.

Members of the newly identified claudin gene family constitute tight junction (TJ) strands, which play a pivotal role in compartmentalization in multicellular organisms. We identified oligodendrocyte-specific protein (OSP) as claudin-11, a new claudin family member, due to its sequence similarity to claudins as well as its ability to form TJ strands in transfected fibroblasts. Claudin-11/OSP mRNA was expressed in the brain and testis. Immunofluorescence microscopy with anti-claudin-11/OSP polyclonal antibody (pAb) and anti-neurofilament mAb revealed that in the brain claudin-11/OSP-positive linear structures run in a gentle spiral around neurofilament-positive axons. At the electron microscopic level, these linear structures were identified as the so-called interlamellar strands in myelin sheaths of oligodendrocytes. In testis, well-developed TJ strands of Sertoli cells were specifically labeled with anti-claudin-11/OSP pAb both at immunofluorescence and electron microscopic levels. These findings indicated that the interlamellar strands of oligodendrocyte myelin sheaths can be regarded as a variant of TJ strands found in many other epithelial cells, and that these strands share a specific claudin species, claudin-11/OSP, with those in Sertoli cells to create and maintain the repeated compartments around axons by oligodendrocytes.

Animals↗

Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase.

The intracellular mechanisms that mediate cytochalasin-induced increase in intestinal epithelial tight junction (TJ) permeability are unclear. In this study, we examined the involvement of myosin light chain kinase (MLCK) in this process, using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin B (Cyto B) (5 microg/ml) produced an increase in Caco-2 MLCK activity, which correlated with the increase in Caco-2 TJ permeability. The inhibition of Cyto B-induced MLCK activation prevented the increase in Caco-2 TJ permeability. Additionally, myosin-Mg(2+)-ATPase inhibitor and metabolic inhibitors (which inhibit MLCK induced actin-myosin contraction) also prevented the Cyto B-induced increase in Caco-2 TJ permeability. Cyto B caused a late-phase (15-30 min) aggregation of actin fragments into large actin clumps, which was also inhibited by MLCK inhibitors. Cyto B produced a morphological disturbance of the ZO-1 TJ proteins, visually correlating with the functional increase in Caco-2 TJ permeability. The MLCK and myosin-Mg(2+)-ATPase inhibitors prevented both the functional increase in TJ permeability and disruption of ZO-1 proteins. These findings suggested that Cyto B-induced increase in Caco-2 TJ permeability is regulated by MLCK activation.

Actin Cytoskeleton↗

Tumor necrosis factor alpha disrupts tight junction assembly.

BACKGROUND: We have previously shown an increase in intestinal permeability and a corresponding decrease in the expression of tight junction (TJ) proteins in the in testines of patients with Crohn's disease (CD). Tumor necrosis factor-alpha (TNFalpha) has been implicated in the inflammatory process of CD and its suppression has therapeutic benefit. ZO-1, occludin, and the claudins are key proteins in the TJ. HYPOTHESIS: TNFalpha disrupts the TJ. METHODS: MDCK cells were incubated with TNFalpha (0-100 ng/ml) for 5 days. Qualitative evaluation of the TJ was done with monoclonal antibody to ZO-1 detected by an immunofluorescence. Duplicate cells were lysed and ZO-1, occludin, and claudin-1 amount determined by western blot. RESULTS: Immunofluorescent staining of MDCK cells for ZO-1 showed TJ structural disruption with increasing amount of TNFalpha characterized by fragmented staining of ZO-1. There were no significant differences in quantitation of ZO-1 or occludin in the MDCK cells for all TNFalpha concentrations. There was a significant decrease in the amount of claudin-1 with increasing concentration of TNFalpha. CONCLUSIONS: (1) MDCK TJs are qualitatively disrupted by TNFalpha. (2) This disruption is not because of a decrease in cell number, lack of cell layer confluency, or a decrease in the amount of ZO-1 or occludin. (3) The amount of claudin-1 present in the cell is decreased with increasing amounts of TNFalpha suggesting that the lack of claudin-1 may cause a relocation of ZO-1 away from the TJ. (4) This rearrangement may play a role in the increased intestinal permeability seen in CD and other diseases.

Animals↗

cAMP perturbs inter-Sertoli tight junction permeability barrier in vitro via its effect on proteasome-sensitive ubiquitination of occludin.

Throughout spermatogenesis, inter-Sertoli tight junctions (TJs) that constitute the blood-testis barrier must be disassembled and reassembled to permit the timely movement of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment of the seminiferous epithelium. However, the mechanism and the participating molecules that regulate the bioavailability of TJ proteins are entirely unknown. Using Sertoli cell culture, it was shown that there was an increase in occludin level, concomitant with a reduction of an E3 ubiquitin ligase, Itch, at the time when inter-Sertoli TJs were assembled. By co-immunoprecipitation, occludin was shown to associate with Itch at the TJs. A novel interaction between Itch and UBC4 (an ubiquitin-conjugating enzyme) was identified. When TJs were disrupted by dibutyryl-cAMP (db-cAMP), an increase in protein levels of Itch and UBC4 along with a significant reduction in endogenous occludin was detected. These results seemingly suggest that the interaction of Itch and UBC4 on occludin is potentially involved in regulating Sertoli TJ dynamics. Addition of a proteasome inhibitor, MG-132, into Sertoli cells cultured with db-cAMP blocked the db-cAMP-induced occludin loss in vitro. Accumulations of ubiquitin-conjugated and Itch-conjugated occludin were detected in Sertoli cells cultured in the presence of both MG-132 and db-cAMP. These results suggest that MG-132 prevented db-cAMP-induced TJ disruption by altering the rate of occludin degradation. Taken collectively, the results reported herein support the notion that db-cAMP-induced TJ disruption was mediated by an induction of Itch protein expression, which in turn triggered the ubiquitination of occludin resulting in TJ disruption.

Animals↗

Regulation of Sertoli cell tight junction dynamics in the rat testis via the nitric oxide synthase/soluble guanylate cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G signaling pathway: an in vitro study.

Nitric oxide (NO) synthase (NOS) catalyzes the oxidation of L-arginine to NO. NO plays a crucial role in regulating various physiological functions, possibly including junction dynamics via its effects on cAMP and cGMP, which are known modulators of tight junction (TJ) dynamics. Although inducible NOS (iNOS) and endothelial NOS (eNOS) are found in the testis and have been implicated in the regulation of spermatogenesis, their role(s) in TJ dynamics, if any, is not known. When Sertoli cells were cultured at 0.5-1.2 x 10(6) cells/cm(2) on Matrigel-coated dishes or bicameral units, functional TJ barrier was formed when the barrier function was assessed by quantifying transepithelial electrical resistance across the cell epithelium. The assembly of the TJ barrier was shown to associate with a significant plummeting in the levels of iNOS and eNOS, seemingly suggesting that their presence by producing NO might perturb TJ assembly. To further confirm the role of NOS on the TJ barrier function in vitro, zinc (II) protoporphyrin-IX (ZnPP), an NOS inhibitor and a soluble guanylate cyclase inhibitor, was added to the Sertoli cell cultures during TJ assembly. Indeed, ZnPP was found to facilitate the assembly and maintenance of the Sertoli cell TJ barrier, possibly by inducing the production of TJ-associated proteins, such as occludin. Subsequent studies by immunoprecipitation and immunoblotting have shown that iNOS and eNOS are structurally linked to TJ-integral membrane proteins, such as occludin, and cytoskeletal proteins, such as actin, vimentin, and alpha-tubulin. When the cAMP and cGMP levels in these ZnPP-treated samples were quantified, a ZnPP-induced reduction of intracellular cGMP, but not cAMP, was indeed detected. Furthermore, 8-bromo-cGMP, a cell membrane-permeable analog of cGMP, could also perturb the TJ barrier dose dependently similar to the effects of 8-bromo-cAMP. KT-5823, a specific inhibitor of protein kinase G, was shown to facilitate the Sertoli cell TJ barrier assembly. Cytokines, such as TGF-beta and TNF-alpha, known to perturb the Sertoli cell TJ barrier, were also shown to stimulate Sertoli cell iNOS and eNOS expression dose dependently in vitro. Collectively, these results illustrate NOS is an important physiological regulator of TJ dynamics in the testis, exerting its effects via the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway.

Animals↗

Tight junctions: closing in on the seal.

The claudins have recently been identified as a large family of transmembrane proteins located at tight junctions between epithelial cells; they create the paracellular diffusion barrier and, surprisingly, may also confer channel-like selectivity for passage of solutes through the tissue barrier.

Animals↗

[An analysis of the structural characteristics of the tight junction of the enterocytes of the rat small intestine during nutrient absorption (immunoelectron microscopic research)].

To analyze structural changes of intercellular relationships of the enterocytes during glycine, glucose, and triolein absorption the structural and immunocytochemical methods of electron microscopy were used. The study was carried out on the proximal part of the rat small intestine in acute and chronic experiments. In the acute ones glucose or glycine solutions (both of 10 and 40 mM) or triolein emulsion (0.5%) were inserted into the isolated small intestinal segments for 20 min. In chronic experiments the isolated loop of the small intestine was perfused with glucose solution (40 mM). Then the corresponding pieces of the intestinal tissue were fixed for structural and immunocytochemical studies. Rarely (in 1% of all cases), and only in chronic experiments, structural changes in the tight junctions--"blisters" and dilatations--were found. At the same time the analysis of the spatial distribution of actin filaments showed that during glucose and glycine absorption the antiactin tracers were found not only within microvilli and on the "root" filaments but also in vicinity of the tight junction and between terminal filaments. The results obtained agree with the hypothesis about the possibility of paracellular transport of some nutrients induced by sodium-dependent transport of glucose and glycine.

Amino Acids↗

CaCo-2 cells treated with Clostridium perfringens enterotoxin form multiple large complex species, one of which contains the tight junction protein occludin.

The previous model for the action of Clostridium perfringens enterotoxin (CPE) proposed that (i) CPE binds to host cell receptor(s), forming a small ( approximately 90 kDa) complex, (ii) the small complex interacts with other eucaryotic protein(s), forming a large ( approximately 160 kDa) complex, and (iii) the large complex triggers massive permeability changes, thereby inducing enterocyte death. In the current study, Western immunoblot analysis demonstrated that CPE bound to CaCo-2 human intestinal cells at 37 degrees C forms multiple large complex species, with apparent sizes of approximately 200, approximately 155, and approximately 135 kDa. These immunoblot experiments also revealed that occludin, an approximately 65-kDa tight junction protein, is present in the approximately 200-kDa large complex but absent from the other large complex species. Immunoprecipitation studies confirmed that occludin physically associates with CPE in large complex material and also indicated that occludin is absent from small complex. These results strongly suggest that occludin becomes associated with CPE during formation of the approximately 200-kDa large complex. A postbinding association between CPE and occludin is consistent with the failure of rat fibroblast transfectants expressing occludin to bind CPE in the current study. Those occludin transfectants were also insensitive to CPE, strongly suggesting that occludin expression is not sufficient to confer CPE sensitivity. However, the occludin-containing, approximately 200-kDa large complex may contribute to CPE-induced cytotoxicity, because nontoxic CPE point mutants did not form any large complex species. By showing that large complex material is comprised of several species (one containing occludin), the current studies indicate that CPE action is more complicated than previously appreciated and also provide additional evidence for CPE interactions with tight junction proteins, which could be important for CPE-induced pathophysiology.

Animals↗

Tight junctions and paracellular permeability in cultured bovine corneal endothelial cells.

Intramembrane specializations of cultured bovine corneal endothelial cells were studied with thin section and freeze-fracture electron microscopy and related to the paracellular permeability and the transendothelial resistance (Rt) of the monolayers. The following intercellular junctions were found: single and discontinuous networks of tight junctions (TJ) which girdle the apico-lateral cell perimeter incompletely, gap junctions, and membrane undulations suggesting intermediate junctions. The macromolecular tracer ruthenium red penetrated into the lateral intercellular space beyond the level of the incomplete belt of TJ. Rt of these monolayers was 20.9 +/- 1.0 omega.cm2. Protamine induced a reversible increase of Rt to 118 +/- 5% of its control value. We conclude that incomplete belts of TJ may be the morphological counterpart of the high paracellular permeability of this monolayer and functionally and morphologically resemble those of their native endothelium. Cultured corneal endothelial cells are an excellent model for studying the influence of incomplete belts of TJ on paracellular permeability of cells.

Animals↗

Epithelial transport of drugs in cell culture. VIII: Effects of sodium dodecyl sulfate on cell membrane and tight junction permeability in human intestinal epithelial (Caco-2) cells.

This study demonstrates how the common pharmaceutical wetting agent sodium dodecyl sulfate (SDS) increases the absorption of drugs and peptides across the human intestinal epithelium. First, an assay that could follow the reversible and irreversible time-dependent effects of SDS on the permeability of Caco-2 cell monolayers with high reproducibility was developed. SDS (0.40 mM) exposure for 20 min resulted in reversible absorption enhancement of mannitol (M(r), 182 g/mol), 1-deamino-8-D-arginine-vasopressin (M(r), 1071 g/mol), and polyethylene glycol (M(r), 4000 g/mol). A longer (2 h) exposure to SDS resulted in irreversible absorption enhancement. Second, transepithelial electrical resistance measurements (TEER) together with fluorescence and transmission electron microscopy were used to study the effects of SDS on epithelial integrity, cell membranes, intracellular calcium concentration, cytoskeleton, and tight junctions. The effect of SDS (0.40 mM) on epithelial integrity was immediate. A significant decrease in transepithelial electrical resistance measurements was obtained with 1 min after exposure to SDS that was concomitant with increases in the permeability of the apical cell membranes and intracellular calcium concentration. SDS shortened the microvilli of the cells and produced apical (but not basolateral) membrane wounds, actin disbandment, disorganization of the terminal web, and structural separation of the tight junctions. The absorption enhancement was not reduced after repair of the apical cell membranes, indicating that SDS enhances drug and peptide absorption across the intestinal epithelium by the paracellular pathway.

Calcium↗

Electron microscopic observations on the maintenance of the tight junction during cell division in the epithelium of the mouse small intestine.

Dividing epithelial cells in the mouse small intestine were examined by thin-section electron microscopy with special attention given to the mode of cytokinesis. As the columnar epithelial cells entered mitosis in the crypt, they became rounded, maintaining their junctional complexes with neighboring cells while detaching themselves from the basal lamina. In such rounded cells the mitotic apparatus was formed with its long axis parallel to the luminal surface. Replicated centrioles moved down from the apical region to locate themselves lateral to the nucleus, where they served as the poles of the mitotic spindle. During mitosis the cell retained microvilli on its luminal surface, though the terminal web became much thinner. At telophase the formation of a cleavage furrow proceeded asymmetrically from the basal side alone, and thus the contractile ring which was prominent at the base of the furrow, merged with the terminal web. Eventually, an intercellular bridge with a midbody was formed on the luminal surface. The space in the furrow was occupied by the flattened cytoplasmic processes of the neighboring cells. The tight junction was also seen on the basolateral surface of the intercellular bridge with the underlying neighboring cells. At very late telophase the intercellular bridge was disconnected from the neighboring cells and protruded into the lumen. These observations have led us to propose a mode by which the simple columnar epithelium maintain the tight junctional seal during cell division in the crypt of the small intestinal epithelium.

Animals↗

Is cadmium chloride-induced inter-sertoli tight junction permeability barrier disruption a suitable in vitro model to study the events of junction disassembly during spermatogenesis in the rat testis?

The events of germ cell movement during spermatogenesis are composed of intermittent phases of junction disassembly and reassembly. Although primary Sertoli cells cultured in vitro can be used to study junction reassembly, an in vitro model to study the events of junction disassembly is still lacking. We have assessed whether the CdCl(2)-induced inter-Sertoli tight junction (TJ) permeability barrier disruption in vitro can fill this gap. When Sertoli cells (1.2 x 10(6) cells/cm(2)) were cultured on Matrigel-coated bicameral units to allow the assembly of inter-Sertoli TJs, it was manifested by a steady rise in transepithelial electrical resistance across the Sertoli cell epithelia. Exposure of these cells on day 1 (i.e. 24 h after their isolation) to CdCl(2) at 5-10 microM for 8 h could perturb the inter-Sertoli TJ assembly dose dependently without any apparent cytotoxicity. Likewise, when cells were exposed to CdCl(2) (0.1-5 microM) on day 4 for 8 h after inter-Sertoli TJs were already assembled, CdCl(2) also perturbed the maintenance of inter-Sertoli TJ permeability barrier dose dependently without signs of cell cytotoxicity. Although the perturbed inter-Sertoli TJs were not capable of resealing even after the removal of CdCl(2), the presence of testosterone (T) at 1 x 10(-9) M allowed resealing of the inter-Sertoli TJ barrier after CdCl(2) was removed, whereas the presence of 2 x 10(-7) M testosterone even protected Sertoli cells from CdCl(2)-induced damage. More important, the reassembly of inter-Sertoli TJs after CdCl(2)-induced TJ disruption was accompanied by changes in cellular gene expression of occludin and urokinase plasminogen activator, which mimicked their patterns during inter- Sertoli TJ assembly in vitro without CdCl(2) treatment. Based on these results, it is apparent that CdCl(2)-induced inter-Sertoli TJ disassembly is a potential in vitro model to study the events of junction disassembly.

Animals↗

Role of nectin in organization of tight junctions in epithelial cells.

BACKGROUND: In polarized epithelial cells, cell-cell adhesion forms specialized membrane structures comprised of claudin-based tight junctions (TJs) and of E-cadherin-based adherens junctions (AJs). These structures are aligned from the apical to the basal side of the lateral membrane, but the mechanism of this organization remains unknown. Nectin is a Ca2+ independent immunoglobulin-like cell-cell adhesion molecule which localizes at AJs. Nectin is associated with E-cadherin through their respective cytoplasmic tail-binding proteins, afadin and catenins, and involved in the formation of AJs in cooperation with E-cadherin. We show here that nectin is also involved in the formation of TJs. RESULTS: During the formation of the junctional complex consisting of AJs and TJs in Madin-Darby canine kidney (MDCK) cells, claudin and occludin accumulated at the apical sites of the nectin-based cell-cell adhesion sites. This accumulation of claudin and occludin was inhibited by inhibitors acting on the trans interaction of nectin. The barrier function of TJs was also impaired by the nectin inhibitors. It has been shown that a phorbol ester promotes the formation of a TJ-like structure in an E-cadherin-independent manner. This phorbol ester-induced formation of the TJ-like structure was also inhibited by the nectin inhibitors. CONCLUSIONS: These results suggest a role of the nectin-afadin system in the organization of TJs as well as AJs in epithelial cells.

Animals↗

Effect of capsianoside, a diterpene glycoside, on tight-junctional permeability.

Previous work (Hashimoto et al., (1994) Biosci. Biotech. Biochem. 58, 1345) revealed that a sweet pepper extract enhanced the tight-junctional (TJ) permeability of a human intestinal Caco-2 cell monolayer. In the present study, the substance which modulated the TJ permeability was chromatographically purified from the extract. The active substances were identified as capsianosides A-F, diterpene glycosides. Treatment of the cells with capsianoside F, the most active compound, decreased the cellular G-actin content by 40% and increased the F-actin content by 16%. The effect of capsianoside F was significantly suppressed by disturbing the cytoskeletal structure with cytochalasin D at a low dose (50 ng/ml). These results suggest that capsianosides affected the cytoskeletal function by modulating the reorganization of actin filaments, by which the TJ structure and permeability were changed. The possible involvement of a PKC inhibition in the mechanism of an increase in TJ permeability is also suggested.

Actins↗

Transforming growth factor-beta3 perturbs the inter-Sertoli tight junction permeability barrier in vitro possibly mediated via its effects on occludin, zonula occludens-1, and claudin-11.

Throughout spermatogenesis, inter-Sertoli tight junctions (TJs) that create the blood-testis barrier in the rat must be disassembled and reassembled to permit the timely passage of preleptotene spermatocytes from the basal to the adluminal compartment of the seminiferous epithelium. However, the mechanism(s) and the participating molecules that regulate this event are largely unknown. Although there is no in vitro model to study the event and regulation of inter-Sertoli TJ disassembly, primary cultures of Sertoli cells in vitro can be used to study junction assembly. In this study, we sought to investigate whether cytokines are involved in the inter-Sertoli TJ assembly in vitro. Sertoli cells isolated from 20-day-old rats were cultured at a density of 0.5-1.2 x 10(6) cells/cm(2) on Matrigel-coated dishes or bicameral units for 8-9 days. The steady-state messenger RNA levels of basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-beta2, and TGF-beta3 at different time points were assessed by semiquantitative RT-PCR. In selected experiments, the assembly of inter-Sertoli TJs was monitored by transepithelial electrical resistance measurement. It was found that there was no change in the expression of basic fibroblast growth factor throughout the entire culture period. However, there was a 2-fold reduction in the expression of TGF-beta2 and TGF-beta3 at the time inter-Sertoli TJs were being assembled. On days 5-8, after the inter-Sertoli TJs had been assembled, the Sertoli cell steady-state messenger RNA levels of TGF-beta2 and TGF-beta3 increased by as much as 3- and 6-fold, respectively, when compared with Sertoli cells on days 1-3 when TJs were being assembled. Also, it was found that recombinant TGF-beta3 added to Sertoli cells cultured in vitro at 1.2 x 10(6) cells/cm(2) on Matrigel-coated bicameral units perturbed the inter-Sertoli TJ permeability barrier dose-dependently. Moreover, the presence of TGF-beta3 also inhibited the transient and/or basal expression of several TJ-associated proteins, which include occludin, zonula occludens-1, and claudin-11 when inter-Sertoli TJs were being assembled in vitro. These results suggest that TGF-beta plays a crucial role in regulating the complicated biochemical events of junction assembly in the testis.

Amino Acid Sequence↗

Development of blood-brain barrier tight junctions in the rat cortex.

The structural equivalent of the blood-brain barrier are the complex tight junctions (TJs) between endothelial cells of brain capillaries. In this study, we have quantitatively investigated by the freeze-fracture technique the modulation of the fine structure of TJs in blood-brain barrier endothelial cells during development of the rat cerebral cortex. The complexity of the TJ network as defined by fractal dimension, the integrity of TJ strands and the degree of TJ particle association to the protoplasmic leaflet of the membrane bilayer in percent of total TJ length were evaluated at embryonic days (E) 13, 15, 18, postnatal day (P) 1 and adult. We observed that the overall complexity of the TJ network and P-face association of TJ particles are significantly increased between E18 and P1. The increase in both of these TJ parameters in combination with the completed particle insertion starting from E18 is likely to reflect the process of transition to the mature state of the blood-brain barrier, which is characterized by high complexity of TJs and predominance of P-face association of TJ particles and correlated tightly with previous physiological measurements, e.g. transendothelial electrical resistance. Two populations of TJs differing in TJ particle density were distinguishable at E15 and E18, which indicates a non-linear asynchronous mechanism of TJ assembly. At E13, particle-free membrane specializations arranged in a TJ-like pattern strongly resembled TJ specific grooves and ridges. Similar results were obtained from cultures of brain endothelial cells in the presence of low calcium conditions, which suggests the involvement of the cadherin/catenin complex in TJ regulation. The particle-free 'TJ precursors' strongly indicate an established TJ associated cytoskeletal network before the TJ particles are present in their intra-junctional location.

Animals↗

Assembly of the tight junction: the role of diacylglycerol.

Extracellular Ca2+ triggers assembly and sealing of tight junctions (TJs) in MDCK cells. These events are modulated by G-proteins, phospholipase C, protein kinase C (PKC), and calmodulin. In the present work we observed that 1,2-dioctanoylglycerol (diC8) promotes the assembly of TJ in low extracellular Ca2+, as evidenced by translocation of the TJ-associated protein ZO-1 to the plasma membrane, formation of junctional fibrils observed in freeze-fracture replicas, decreased permeability of the intercellular space to [3H]mannitol, and reorganization of actin filaments to the cell periphery, visualized by fluorescence microscopy using rhodamine-phalloidin. In contrast, diC8 in low Ca2+ did not induce redistribution of the Ca-dependent adhesion protein E-cadherin (uvomorulin). Extracellular antibodies to E-cadherin block junction formation normally induced by adding Ca2+. diC8 counteracted this inhibition, suggesting that PKC may be in the signaling pathway activated by E-cadherin-mediated cell-cell adhesion. In addition, we found a novel phosphoprotein of 130 kD which coimmunoprecipitated with the ZO-1/ZO-2 complex. Although the assembly and sealing of TJs may involve the activation of PKC, the level of phosphorylation of ZO-1, ZO-2, and the 130-kD protein did not change after adding Ca2+ or a PKC agonist. The complex of these three proteins was present even in low extracellular Ca2+, suggesting that the addition of Ca2+ or diC8 triggers the translocation and assembly of preformed TJ subcomplexes.

Actins↗