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PubMed1998

Backleak, tight junctions, and cell- cell adhesion in postischemic injury to the renal allograft.

Postischemic injury in recipients of 3-7-d-old renal allografts was classified into sustained (n = 19) or recovering (n = 20) acute renal failure (ARF) according to the prevailing inulin clearance. Recipients of optimally functioning, long-standing allografts and living donors undergoing nephrectomy served as functional (n = 14) and structural controls (n = 10), respectively. Marked elevation above control of fractional clearance of dextrans of graded size was consistent with transtubular backleak of 57% of filtrate (inulin) in sustained ARF. No backleak was detected in recovering ARF. To explore a structural basis for backleak, allograft biopsies were taken intraoperatively, 1 h after reperfusion in all recipients, and again on day 7 after transplant in a subset (n = 10). Electron microscopy revealed disruption of both apical and basolateral membranes of proximal tubule cells in both sustained and recovering ARF, but cell exfoliation and tubule basement membrane denudation were negligible. Histochemical analysis of membrane-associated adhesion complexes confirmed an abnormality of proximal but not distal tubule cells, marked in sustained ARF but not in recovering ARF. Staining for the zonula occludens complex (ZO-1) and adherens complex (alpha, beta, and gamma catenins) revealed diminished intensity and redistribution of each cytoskeletal protein from the apico-lateral membrane boundary. We conclude that impaired integrity of tight junctions and cell-cell adhesion in the proximal tubule provides a paracellular pathway through which filtrate leaks back in sustained allograft ARF.

Acute Kidney Injury↗
PubMed2006

Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions.

Although paracellin-1 (PCLN-1) is known to have a crucial role in the control of Mg2+ reabsorption in the kidney, the molecular pathways involved in the regulation of PCLN-1 have not been clarified. We used FLAG-tagged PCLN-1 to investigate these pathways further, and found that PCLN-1 is phosphorylated at Ser217 by protein kinase A (PKA) under physiological conditions in Madin-Darby canine kidney (MDCK) cells. PCLN-1 expression decreased Na+ permeability, resulting in a decrease in the transepithelial electrical resistance (TER). By contrast, PCLN-1 enhanced transepithelial Mg2+ transport. PKA inhibitors, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and myristoylated protein kinase A inhibitor 14-22 amide PKI, and an adenylate cyclase inhibitor, 2',5'-dideoxy adenosine (DDA), reduced the phosphoserine level of PCLN-1. The inhibitory effect of DDA was rescued by 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP). PKA and adenylate cyclase inhibitors decreased transepithelial Mg2+ transport and TER. Dephosphorylated PCLN-1 moved from detergent-insoluble to soluble fractions and was dissociated from ZO-1. A fusion protein of PCLN-1 with glutathione-S-transferase revealed that Ser217 was phosphorylated by PKA. Phosphorylated PCLN-1 was localized in the tight junction (TJ) along with ZO-1, whereas dephosphorylated PCLN-1 and the S217A mutant were translocated into the lysosome. The degradation of dephosphorylated PCLN-1 and S217A mutant was inhibited by chloroquine, a specific lysosome inhibitor. Thus, the PKA-dependent phosphorylation of Ser217 in PCLN-1 is essential for its localization in the TJ and transepithelial Mg2+ transport.

Animals↗
PubMed1998

Seminal fluid factor increases the resistance of the tight junctional complex of cultured human cervical epithelium CaSki cells.

OBJECTIVE: To determine the effects of human seminal fluid on cervical paracellular resistance. DESIGN: Experimental study. SETTING: Healthy volunteers in an academic research environment; cultures of human CaSki cells on filters, with phenotypic characteristics of the endocervix. PATIENT(S): Healthy men donating sperm to a sperm bank. INTERVENTION(S): Seminal fluid was obtained as the discarded fluid from ejaculates. MAIN OUTCOME MEASURE(S): Changes in transepithelial electrical resistance across CaSki cells on filters were determined in an Ussing chamber from successive measurements of the short-circuit current and the transepithelial potential difference. Changes in the dilution potential (and hence in the ratio of Cl- to Na+ mobilities) were determined after lowering the NaCl concentration in the luminal solution. RESULT(S): Seminal fluid increased transepithelial electrical resistance acutely (t1/2, 2 minutes), reversibly, and in a dose-related manner (ED50, 1%). The effect of seminal fluid was abolished when the extracellular calcium level was lowered, and the increase in transepithelial electrical resistance correlated with a decrease in the ratio Cl- to Na+ mobilities, indicating an increase in the resistance of the tight junctional complex. The increase in transepithelial electrical resistance in response to seminal fluid was nonadditive to that of sn-1,2-dioctanoyl diglyceride (a stable diacylglyceride and activator of protein kinase C), and it was abolished by prolonged preincubation with the phorbol ester phorbol 12-myristate 13-acetate (to downregulate protein kinase C) or with staurosporin (to inhibit protein kinase C), suggesting that seminal fluid acts through a protein kinase C-dependent mechanism. Slower (t1/2, 3.3 minutes) increases in transepithelial electrical resistance occurred when seminal fluid was added only to the luminal or the subluminal solution. Treatment with pertussis toxin, adenosine triphosphatase, or trypsin had no effect on the changes in transepithelial electrical resistance. Seminal fluid increased cytosolic calcium, but changes in cytosolic calcium are not important for the increases in transepithelial electrical resistance, suggesting that the effect of seminal fluid is not receptor-mediated. Preliminary studies indicate that the factor(s) in seminal fluid that increases transepithelial electrical resistance is a labile, low molecular weight (< 10 kd) lipid. CONCLUSION(S): Seminal fluid may regulate cervical mucus production in vivo by modulating endocervical permeability.

Calcium↗
PubMed2002

Role of IL-10 in hepatocyte tight junction alteration in mouse model of experimental colitis.

BACKGROUND: A variety of hepatobiliary abnormalities have been described in patients with chronic inflammatory bowel diseases (IBDs). The purpose of this study was to investigate the role of endogenous IL-10 in alteration of hepatocyte TJ paracellular barrier and in the rapid transcytotic vesicular pathway modification associated with intestinal inflammation. MATERIALS AND METHODS: To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). When compared to DNBS-treated IL-10 wild-type (IL-10WT) mice, DNBS-treated IL-10 knock-out mice (IL-10KO) mice experienced a higher rate of the extent and severity of the histological signs of colon injury. RESULTS: Colon and liver levels of the pro-inflammatory cytokines tumour necrosis factor, interleukin-1 beta and interleukin-6 were also greatly enhanced in IL-10KO mice in comparison to wild-type mice. Liver histology from IL-10KO and IL-10WT did not show any parenchymal and portal tract inflammation at 4 days after DNBS administration. Serum total bilirubin and Alanine aminotransferase, were significantly increased in DNBS-IL-10KO mice vs. DNBS-IL-10KO mice. Therefore, we found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the livers from DNBS-treated IL-10WT mice; lanthanum accumulated throughout the junctional area up to the most apical region bordering the lumen. Absence of a functional IL-10 gene in IL-10KO mice resulted in a significant augmentation of apical diffusion of lanthanum after DNBS-induced colitis. Immunofluorescent labelling of frozen liver sections from DNBS-IL10KO mice, immunolocalization for and claudin-1 and ZO-1 resulted in a significant alteration in the localization of the immunosignals for claudin-1 and ZO-1 after DNBS administration in comparison with DNBS-IL10WT. CONCLUSION: In conclusion, we suggest that the absence of IL-10 may represent an important pathophysiological mechanism of hepatobiliary injuries and cholestasis observed in patients with IBD.

Alanine Transaminase↗
PubMed2006

The tight junction proteins claudin-7 and -8 display a different subcellular localization at Henle's loops and collecting ducts of rabbit kidney.

BACKGROUND: The tight junction (TJ) regulates the passage of ions and molecules through the paracellular pathway. In multicellular organisms, epithelial sheets function as a barrier between a variety of environments and the internal media. Therefore, TJs are required to control the passage of diverse molecules in different epithelia. The mammalian nephron constitutes a particularly relevant model of this diversity, since the paracellular transport in this organ is significantly different along the various tubular segments. Here, we have analysed the distribution of claudins-7 and -8 in Henle's loops and collecting ducts isolated from rabbit kidneys. METHODS: Renal segments were manually isolated from newborn and adult rabbit kidneys and processed for immunofluorescence. The distribution of claudins-7 and -8 was studied by confocal microscopy. RESULTS: The localization of claudins-7 and -8 along Henle's loops and collecting ducts is remarkably different. While claudin-8 displays a clear cell border distribution in Henle's segment, claudin-7 shows a non-specific cytosolic staining. Moreover, in the collecting ducts, claudin-8 localizes at the TJ region, while claudin-7 shows a basolateral staining. This pattern is present from the newborn stage. The distribution of claudins along the mammalian kidney has been found to vary in different mammalian species. Accordingly, in the rabbit, we have found the expression of claudin-8 at the descending and ascending thin limbs of Henle, a distribution that differs from that found in the mouse by others. CONCLUSION: In the rabbit Henle's loop, claudin-8 is present at the cellular borders of the descending and ascending thin limbs, while claudin-7 displays no specific labelling. Instead, at the collecting duct, both claudins are present but exhibit a different subcellular distribution.

Animals↗
PubMed1966

Electron microscopy: tight junctions in synapses of the chick ciliary ganglion.

Examination of the synaptic membranes in calyciform endings of the chick ciliary ganglion has shown tight junctions selectively located on axon hillocks of postsynaptic neurons. Observations of similar membrane fusion in other junctions that involve electrotonic transmission suggest the possibility of identifying electrotonic junctions by electron microscopy.

Animals↗
PubMed1991

Cingulin, a specific protein component of tight junctions, is expressed in normal and neoplastic human epithelial tissues.

Cingulin is a 140-kd protein localized on the cytoplasmic face of avian tight junctions. The expression of cingulin in human normal and neoplastic colonic tissue has been investigated with an antiserum against chicken cingulin. Human cingulin shares its apparent molecular mass and localization with avian cingulin. In normal colonic epithelium, villous adenomas, and differentiated adenocarcinomas, cingulin staining is observed in the junctional region of the polarized cells lining the surface, the crypts, and the glandular lumina. In poorly differentiated adenocarcinomas, labeling also is observed at the interface between cancer tissue and stroma, or in clumps of malignant cells, forming a pattern that highlights the presence of small, compressed lumina. The cingulin content of four adenocarcinomas, estimated by immunoblotting and densitometry, was higher than that of the normal tissue (150% to 230%). Cingulin was detected in a metastasis from a colon adenocarcinoma but not in nonepithelial tissues and neoplasias, suggesting that cingulin may be a useful marker in the characterization of colonic and probably other epithelial neoplasias.

Adenocarcinoma↗
PubMed2000

The transient increase of tight junction permeability induced by bryostatin 1 correlates with rapid downregulation of protein kinase C-alpha.

The role of PKC-alpha in altered epithelial barrier permeability following the activation of PKC by TPA (12-O-tetradecanoyl phorbol 13-acetate) and bryostatin 1 in LLC-PK1 cells was investigated in this study. Like TPA, bryostatin 1 binds to and activates PKC but unlike TPA, it is not a tumor promoter. TPA at 10(-7) M induced a sustained 95% decrease in transepithelial electrical resistance (R(t)) across LLC-PK1 epithelial cell sheets, while 10(-7) M bryostatin 1 caused only a 30% decrease in R(t), which spontaneously reversed after 5 h. Simultaneous exposure of cell sheets to 10(-7) M TPA and 10(-7) M bryostatin 1 blunted the increase in epithelial permeability observed with 10(-7) M TPA alone. Co-incubation of cell sheets with bryostatin 1 and MG-132, a proteasomal inhibitor, caused a further decrease in R(t) at the 6-h time point and inhibited the recovery in R(t) seen with bryostatin 1 alone at this time point. TPA caused a rapid translocation of PKC-alpha from the cytosol to the membrane of the cell where it remained elevated. Bryostatin 1 treatment resulted in a slower translocation of PKC-alpha from the cytosol to the membrane and a much more rapid downregulation of PKC-alpha, with disappearance from this compartment after only 6 h. The classical PKC inhibitor Go6976 prevented the decrease in R(t) seen with TPA. Treatment of cells with TPA and bryostatin 1 resulted in a PKC-alpha translocation and downregulation profile which more closely resembled that seen with bryostatin 1 alone. Co-incubation of cells with MG-132 and bryostatin 1 caused a slower downregulation of PKC-alpha from the membrane fraction. Bryostatin 1 treatment of cells expressing a dominant/negative form of PKC-alpha resulted in a slower and less extensive decrease in R(t) compared to the corresponding control cells. For both TPA and bryostatin 1, the level of PKC-alpha in the membrane-associated fraction of the treated cells correlated closely with increased transepithelial permeability. Due to its transient effect on tight junction permeability, bryostatin 1 offers a novel pharmacological tool to investigate junctional physiology.

Animals↗
PubMed1996

The SH3 domain of the tight junction protein ZO-1 binds to a serine protein kinase that phosphorylates a region C-terminal to this domain.

ZO-1 is a tight junction phosphoprotein partially homologous to a tumor suppressor in Drosophila. The homologous region contains an SH3 domain with an unidentified function. Using fusion proteins containing the SH3 domain and various N- and C-terminal sequences, we tested for association of a kinase with this protein domain in extracts of MDCK cells. We show that the SH3 domain of ZO-1 binds a serine protein kinase that phosphorylates a region immediately C-terminal to the SH3 domain. This kinase associates specifically with the SH3 domain of ZO-1 and appears to be also associated with junctional complexes extracted from MDCK cells.

Animals↗
PubMed1988

Change in structure of tight junctions between epithelial cells in the uterine tube of the rat during early pregnancy.

Freeze-fracture electron microscopy has been used to study tight junctions of epithelial cells in the rat fallopian tube. At oestrus, junctions were found to consist of strands parallel to the surface whereas at day 6 of pregnancy, junctions were deeper and extensively interconnected. We consider the involvement of ovarian hormones and the cross-tissue significance of our findings.

Animals↗
PubMed1995

Basolateral but not apical application of protease results in a rapid rise of transepithelial electrical resistance and formation of aberrant tight junction strands in MDCK cells.

In the presence of Ca2+, application of trypsin to the basolateral surface of confluent MDCK cell monolayers with formed tight junctions (TJ), induces the formation of basolaterally oriented aberrant TJ strands. Induction of aberrant TJ strands is accompanied by an increase in transepithelial electrical resistance (TER), up to 90%, which upon addition of trypsin inhibitor is maintained for up to 1 h. Thereafter TER returns slowly to baseline values. Under similar conditions, application of trypsin to the apical surface has little or no effect on either TER or the number of aberrant TJ strands. Confocal microscopy of monolayers, immunostained for ZO-1, revealed that this TJ associated cytoplasmic protein, extended below the TJ along the basolateral surface following brief exposure to trypsin. Removing Ca2+ after treatment of the monolayer with basolaterally applied trypsin resulted, after 20 min, in the increased partitioning of TJ particles onto the E fracture face, of both normal and aberrant TJ strands. Like the TJ strands themselves, therefore, aberrant strands may be linked to cytoskeletal elements. Aberrant TJ strands do not form when monolayers, maintained in low Ca2+ medium, are exposed to trypsin, suggesting that under these conditions TJ precursors, and/or trypsin-sensitive proteins regulating TJ strand assembly, are sequestered in a vesicular compartment that is inaccessible to exogenous trypsin. Prolonged exposure of the apical surface of an established, polarized epithelium with intact TJ to trypsin, had little effect on TJ integrity and did not induce aberrant strands.

Animals↗
PubMed1995

Localization of the tight junction protein gene TJP1 to human chromosome 15q13, distal to the Prader-Willi/Angelman region, and to mouse chromosome 7.

The gene encoding the tight junction (zonula occludens) protein, TJP1, was mapped to human chromosome 15q13 by fluorescence in situ hybridization (FISH) using a cDNA probe. The Jackson Laboratory backcross DNA panel derived from the cross (C57BL/6JEi x SPRET/Ei) F1 females x SPRET/Ei males was used to map the mouse Tjp1 to chromosome 7 near position 30 on the Chromosome Committee Map, a region with conserved homology to human chromosome 15q13. FISH studies on metaphases from patients with the Prader-Willi (PWS) or the Angelman syndrome (AS) showed that TJP1 maps close but distal to the PWS/AS chromosome region.

Angelman Syndrome↗
PubMed1986

1-Naphthylisothiocyanate-induced permeability of hepatic tight junctions to proteins.

We have studied the early action of 1-naphthylisothiocyanate (ANIT) in relation to its effect on the permeability barrier formed by hepatic tight junctions. Materials having different Mr values [inulin (5000), horseradish peroxidase (HRP) (40,000), ovalbumin (also 40,000) and pig gamma-globulin (IgG) (160,000)] were individually pulsed, within 1 min, into perfused rat livers operating under single-pass conditions. In untreated rats, a small peak of HRP and ovalbumin and a comparatively larger peak of inulin were observed in the bile at 7 min. In rats treated with ANIT, with increasing duration of ANIT treatment the inulin peak increased proportionally, whereas the HRP and ovalbumin peaks remained unchanged until after 10 h of ANIT exposure; gamma-globulin was not detected in the 7 min bile sample until after 14 h of ANIT treatment. Bile flow in all rats remained approximately the same until after 14 h of ANIT pretreatment, when substantial bile-flow reduction was observed. Phenobarbitone pretreatment increased the effect of ANIT and massively elevated the first HRP peak; it also shortened the time (to 4 h) at which the increase in permeability to this protein was observed. In contrast, the first HRP peak was virtually abolished in rats that had received the mixed-function-oxidase inhibitor SKF 525A. These experiments suggest that (i) ANIT progressively increased the permeability of the junctional barrier before the reduction in bile flow, (ii) the ANIT-increased permeability change seems to be inversely dependent upon the Mr of the infused proteins, and (iii) metabolites of ANIT were involved in the development of the junctional permeability change.

1-Naphthylisothiocyanate↗
PubMed2005

Loss of the tight junction protein claudin 4 correlates with histological growth-pattern and differentiation in advanced gastric adenocarcinoma.

The expression of E-cadherin located in the adherens junction varies with disruption of glandular morphology and loss of differentiation. It has been suggested that the loss of tight junction function is related to tumor differentiation, but little is known about the roles of major proteins, such as claudin 4 and ZO-1, in gastric cancer. The aims of this study were to examine the differences in expression of E-cadherin, claudin 4, and ZO-1 proteins according to the pathological and clinical features of advanced gastric cancer. The expression of E-cadherin, claudin 4, and ZO-1 was analyzed immuno-histochemically using formalin-fixed, paraffin-embedded tissues obtained from 49 patients who underwent radical resection for advanced gastric cancer. Western blot analysis and RT-PCR were performed in representative tumors of the diffuse or intestinal type. Immunostaining for E-cadherin, claudin 4, and ZO-1 was reduced in 69, 69, and 37% of cancers, respectively. Similar patterns of expression were noted for E-cadherin and claudin 4, but ZO-1 expression differed. According to the Lauren classification, the reduced expression of E-cadherin and claudin 4 was more frequent in diffuse than intestinal type tumors (p<0.001). The reduced expression of E-cadherin and claudin 4 correlated with poor differentiation (p<0.05). Western blot analysis and RT-PCR also showed decreased claudin 4 expression in diffuse type tumors and poorly-differentiated adenocarcinoma. The reduced expression of claudin 4 and E-cadherin correlates with disruption of glandular structure and loss of differentiation, which suggests that the dysfunction of claudin 4 may play a role in the disruption of cell-to-cell adhesion in diffuse type gastric cancer and in a loss of differentiation.

Adenocarcinoma↗
PubMed1988

Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells.

ZO-1, originally identified by mAb techniques, is the first protein shown to be specifically associated with the tight junction. Here we describe and compare the physical characteristics of ZO-1 from mouse liver and the Madin-Darby canine kidney (MDCK) epithelial cell line. The ZO-1 polypeptide has an apparent size of 225 kD in mouse tissues and 210 kD in canine-derived MDCK cells as determined by SDS-PAGE/immunoblot analysis. ZO-1 from both sources is optimally solubilized from isolated plasma membranes by either 6 M urea or high pH conditions; partial solubilization occurs with 0.3 M KCl. The nonionic detergents, Triton X-100 and octyl-beta-D-glucopyranoside, do not solubilize ZO-1. These solubility properties indicate that ZO-1 is a peripherally associated membrane protein. ZO-1 was purified to electrophoretic homogeneity from [35S]methionine metabolically labeled MDCK cells by a combination of gel filtration and immunoaffinity chromatography. Purified ZO-1 has an s20,w of 5.3 and Stokes radius of 8.6 nm. These values suggest that purified ZO-1 is an asymmetric monomeric molecule. Corresponding values for mouse liver ZO-1, characterized in impure protein extracts, were 6 s20,w and 9 nm. ZO-1 was shown to be a phosphoprotein in MDCK cells metabolically labeled with [32P]orthophosphate; analysis of phosphoamino acids from purified ZO-1 revealed only phosphoserine. ZO-1 epitope number was determined by Scatchard analysis of competitive and saturable binding of two different 125I-mAbs to SDS-solubilized proteins from liver and MDCK cells immobilized on nitrocellulose. Saturation binding occurs at 26 ng mAb/mg liver and 63 ng/mg of MDCK cell protein. This is equivalent to 30,000 ZO-1 molecules per MDCK cell assuming a single epitope/ZO-1 molecule.

Animals↗
PubMed1994

Tight junctions of the human corneal endothelium: morphological and electrophysiological features.

The corneal endothelium controls the hydration and nutrition of the avascular corneal stroma. To analyze the role of the tight junctions (TJ) for these functions, we examined human corneal endothelium by thin-section and freeze-fracture electron microscopy and by impedance analysis. On thin sections, tannic acid was seen to mark the external leaflet of the lateral plasma membrane also beyond the location of the TJ, indicating a significant macromolecular porosity of the TJ. On freeze-fracture images, the TJ surrounded the entire apicolateral plasma membrane but were found to be focally incomplete, suggesting a nonhomogeneous seal of the lateral intercellular space. Impedance analysis revealed a very leaky endothelial layer with a transendothelial resistance of 9.0 +/- 1.4 omega cm2. These findings are consistent with the hypothesized "pump-leak" model of the corneal endothelium: the TJ allow an effective dehydration of the corneal stroma, whereas the interruptions in the TJ network may be the morphological correlate for the passage of nutrients into the corneal stroma. Our data corroborate the assignment of the corneal endothelium to the group of "very leaky epithelia" that exhibit high transport rates of water and solutes against only minimal osmotic gradients.

Cell Membrane↗
PubMed2002

Interferon-beta counteracts inflammatory mediator-induced effects on brain endothelial cell tight junction molecules-implications for multiple sclerosis.

To elucidate mechanisms of endothelial cell (EC) dysfunction in CNS inflammatory responses and beneficial effects of interferon-beta (IFN-gamma) in multiple sclerosis (MS), we analyzed effects of individual and combinations of soluble inflammatory mediators on the intracellular localization of the EC tight junction-associated molecules zonula occludens-1 and -2 (ZO-1 and ZO-2) in human brain ECs. The cytoplasm in the majority of cells in control EC cultures was clear; ZO-1 and ZO-2 were localized peripherally near sites of cell contact and associated with submembranous cytoplasmic filaments. H2O2 induced reversible time- and concentration-dependent translocation of ZO-1 and ZO-2 to a random distribution within EC cytoplasm and retraction of EC borders. For low concentrations, these effects were accompanied by less prominent submembranous filaments but not by evidence of cytotoxicity, increased cell death or altered amounts of ZO-1. Tumor necrosis factor-beta induced similar alterations but interferon-y did not. Co-treatment with either cytokine increased H2O2 effects whereas IFN-beta reversed H2O2-induced effects. In control white matter samples, EC cytoplasm was clear and ZO-1 was located on cell borders. In inflammatory/demyelinating lesions, EC ZO-1 was diffuse, indicating that the alterations induced in vitro mimic those in active MS lesions. These findings suggest that in MS patients, IFN-beta treatment may counteract inflammatory mediator effects on CNS EC tight junction molecules, thereby preserving EC barrier function.

Cell Line↗
PubMed1993

Cytochemical evidence for the presence of phospholipids in epithelial tight junction strands.

Previous freeze-fracture experiments using either glutaraldehyde-fixed and cryoprotected specimens or unfixed rapid-frozen samples led to the proposal that cylindrical strands of the tight junction (TJ) observed in freeze-fracture preparations are inverted cylindrical micelles made up of membrane lipids and, possibly, membrane proteins. However, no one has yet been able to directly label the structural fibrils of the TJ. To test the hypothesis that TJ strands observed on freeze-fracture preparations are composed at least partially of lipids, we have combined the phospholipase A2-gold and the fracture-label techniques for localization of phospholipids. Phospholipase A2, purified from bee venom, was adsorbed on gold particles and used for specific labeling of its substrate. Phospholipase A2-colloidal gold (PLA2-CG) complex was applied to freeze-fractured preparations of rat exocrine pancreatic cells and testicular Sertoli cells, both of which are known to have extensive TJ complexes on their plasma membranes. Fracture-label replicas of exocrine pancreatic cells revealed specific association of gold particles with TJ fibrils on the protoplasmic fracture-face of the plasma membrane. The majority of these gold particles were observed either directly on the top of the TJ fibrils or adjacent to these cylindrical structures. A high density of PLA2-CG labeling was also observed over the complementary exoplasmic fracture-face of the TJ complex. This intimate association of PLA2-CG labeling with the TJ is particularly evident in the Sertoli cell plasma membrane, where rows of gold particles were observed to be superimposed on parallel arrays of cylindrical strands of the TJ complex. The present findings provide direct cytochemical evidence to support the hypothesis that cylindrical TJ strands observed in freeze-fracture preparations contain phospholipids.

Animals↗