Paracellular Leakage Research Articles

Molecular mechanisms of endothelial hyperpermeability in sepsis

There is endothelial hyperpermeability in sepsis,trauma,ischaemia-reperfusion injury,acute respiratory distress syndrome and thrombosis,while an important mechanism underlying this process is increased paracellular leakage of plasma fluid and protein because of cytoskeleton contration under the role of histamine, thrombin,vascular endothelial growth factor and activated neutrophil. Structural changes initiate with agonist-receptor binding,followed by activation of intracellular signalling molecules,then phosphorylate alter the hypermeability of cell-cell adhesion. Targeting key signalling molecules that mediate endothelial-junction-cytoskeleton dissociation demonstrates a therapeutic potential to improve vascular barrier function during inflammatory injury. Key words: Endothelial cell; Microvascular permeability; Signal transduction; Sepsis

Structural and immunological barriers of the skin as potential therapeutic targets

Skin is the structure that covers our body and protects it from not only the entry of pathogens or allergens but also from the leakage of water, solutes or nutrients. These outside-in and inside-out skin barrier functions are dependent on the epidermis, a stratified epithelial cellular sheet. While mucus covers the epidermis in fish and amphibian tadpoles, terminally differentiated cornified cellular sheets called stratum corneum (SC) constitute the outermost epidermal barrier in amphibian adults, reptiles, birds and mammals. Beneath the mucus or SC, apical paracellular spaces of epidermal cells are sealed with tight junctions (TJs) that limit paracellular leakage of water and electrolytes to maintain fluid homeostasis. We applied time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) imaging technology to analyze the SC in skin sections, and found that the SC consisting of three layers of distinct functional properties. Under the barriers of the SC and TJ, antigen-presenting dendritic cells called Langerhans cells (LCs) distribute within the epidermis. LCs elongate their dendrites to penetrate through epidermal TJs upon activation and uptake antigens from extra-TJ environment. During antigen uptake, new TJs are formed between keratinocytes and LC dendrites to maintain the integrity of epidermal TJ barriers. To understand the epidermal barrier system and its deficiency observed in human skin diseases, we need to re-evaluate human epidermal barrier as a composite barrier consisting of SC and TJs and to investigate the molecular mechanism and immunological consequences of the extra-TJ antigen uptake activity of LCs.

Claudin‐5 increases alveolar permeability in alcoholic lung syndrome by destabilizing claudin‐18/zonula occludens‐1 interactions (716.4)

We found that alcohol abuse increases the severity of acute respiratory distress syndrome (ARDS) by impairing alveolar epithelial cell (AEC) tight junctions. In a rat model of chronic alcohol ingestion, decreased AEC barrier function was accompanied by increased claudin‐5 (cldn‐5) and claudin‐18 (cldn‐18) expression. Immunofluorescence of model type I AECs cultured on Transwell permeable supports showed that paracellular leak in cells from alcohol fed rats was accompanied by disrupted tight junction morphology. We then used Stochastic Optical Reconstruction Microscopy (STORM) which increases the resolution of immunofluorescence to 20 nm. STORM revealed that alcohol caused a significant decrease in co‐localization between AEC cldn‐18 and ZO‐1 along with increased co‐localization between cldn‐18 and cldn‐5. Thus, cldn‐5 binding to cldn‐18 inhibits binding to ZO‐1 which destabilizes tight junctions. We found that shRNA targeting to decrease cldn‐5 expression improved AEC tight junction morphology and function. Conversely, transfecting cells to increase expression of YFP‐tagged cldn‐5 altered tight junction morphology and impaired barrier function. Thus, increased cldn‐5 was necessary and sufficient to inhibit cldn‐18 assembly and function. These data suggest that targeting cldn‐5 may provide a therapeutic strategy to improve barrier function in ARDS.Grant Funding Source: Emory URC, NIH‐NIAAA, NIH‐NHLBI, DAAD

Degradation of tight junction proteins in meprin betatransfected kidney cells subjected to hypoxia

Tight junctions prevent paracellular leakage in epithelial cells. Meprins, metalloproteinases that are abundantly expressed in the brush border membranes of proximal kidney tubules, have been implicated in the pathology of ischemia reperfusion (IR) induced kidney injury. Disruption of the meprin [beta] gene, or pretreatment with the meprin inhibitor, actinonin, both protect mice from IR‐induced renal injury. The mechanism by which meprins cause kidney injury is not fully understood. The goal of the current study was to determine if hypoxia activates meprins leading to degradation of the tight junction proteins. Meprin β transfected MDCK cells were depleted of oxygen by exposure to cobalt chloride. Non‐transfected MDCK cells were used as controls. Proteins were extracted and fractionated into cytosolic‐, nuclear‐ and membrane‐enriched fractions. Western blot analysis was used to quantify the levels and fragmentation of occludin and E‐cadherin in the membrane‐enriched protein fractions. A time‐dependent degradation of both E‐cadherin and occludin was observed in the cobalt chloride treated meprin β transfected cells but not in non‐transfected control cells. This suggests that hypoxia triggers activation of meprin B, leading to degradation of the two tight junction proteins. Meprin degradation of tight junction proteins may be partly responsible for the renal injury observed in IR.

Transepithelial antigen delivery in the small intestine

The intestinal epithelium is a dynamic barrier protecting the body from the multitudes of luminal micro-organisms present in the gut. However, this barrier is not impermeable and mechanisms exist that allow small amounts of antigen to traverse the epithelium in controlled manner to maintain tolerance and to mount immune responses. This review will summarize our current understanding of how luminal antigens traverse the small intestine epithelium without disrupting the epithelial barrier and how these antigen delivery pathways might influence the resulting immune responses. Recent findings have revealed four pathways for transepithelial antigen delivery in the absence of barrier disruption. We propose that during homeostasis, antigen introduced through microfold cells induces immunoglobulin A responses, antigen delivered by goblet cell-associated antigen passages contributes to peripheral tolerance, and antigen delivered by paracellular leak initiates immune responses in the mesenteric lymph node. In contrast, dendritic cell transepithelial dendrites may play an important role in host protection during pathogen infection, but do not appear to play a role in antigen capture by lamina propria dendritic cells in the steady state. These observations indicate that the route by which antigen crosses the epithelium directs the outcome of the subsequent immune response.

Zinc Supplementation Modifies Tight Junctions and Alters Barrier Function of CACO-2 Human Intestinal Epithelial Layers

Zinc deficiency is known to result in epithelial barrier leak in the GI tract. Precise effects of zinc on epithelial tight junctions (TJs) are only beginning to be described and understood. Along with nutritional regimens like methionine-restriction and compounds such as berberine, quercetin, indole, glutamine and rapamycin, zinc has the potential to function as a TJ modifier and selective enhancer of epithelial barrier function. The purpose of this study was to determine the effects of zinc-supplementation on the TJs of a well-studied in vitro GI model, CACO-2 cells. Barrier function was assessed electrophysiologically by measuring transepithelial electrical resistance (Rt), and radiochemically, by measuring transepithelial (paracellular) diffusion of 14C-D-mannitol and 14C-polyethyleneglycol. TJ composition was studied by Western immunoblot analyses of occludin, tricellulin and claudins-1 to -5 and -7. Fifty- and 100-μM zinc concentrations (control medium is 2 μM) significantly increase Rt but simultaneously increase paracellular leak to D-mannitol. Claudins 2 and 7 are downregulated in total cell lysates, while occludin, tricellulin and claudins-1, -3, -4 and -5 are unchanged. Claudins-2 and -7 as well as tricellulin exhibit decreased cytosolic content as a result of zinc supplementation. Zinc alters CACO-2 TJ composition and modifies TJ barrier function selectively. Zinc is one of a growing number of "nutraceutical" substances capable of enhancing epithelial barrier function, and may find use in countering TJ leakiness induced in various disease states.

Noninvasive Monitoring of Glucose Levels: Is Exhaled Breath the Answer?

Monitoring of blood glucose levels is clinically important in the management of diseases affecting insulin secretion and resistance, most notably diabetes mellitus and cystic fibrosis. Typically, blood glucose monitoring is an invasive technique that may cause distress and discomfort, particularly in the pediatric population. Development of noninvasive methods of monitoring blood glucose is therefore indicated, particularly for use in children. Using respiratory fluids (the liquid present in the lumen of the airways and alveoli) to estimate blood glucose levels indirectly is one potential method. Glucose concentrations in respiratory fluids are typically low, maintained by the equilibrium between paracellular leakage of glucose from the lung interstitium and active cotransport of glucose by epithelial cells. Measurement of glucose in respiratory fluid by collection of exhaled breath condensate is therefore a potentially clinically useful method of estimating blood glucose levels if it can be shown that there is good agreement between these values. This article reviews the research in this area.

Co‐regulation of Transcellular and Paracellular Leak Across Microvascular Endothelium By Dynamin and Rac

Increased permeability of the endothelium is the hallmark of inflammation. Leakage can occur between (paracellular) or through (transcytosis) endothelial cells, yet little is known about whether these pathways are linked. We investigated whether transcytosis and paracellular leakage are co‐regulated. Using molecular and pharmacologic approaches, we inhibited transcytosis of albumin in primary human microvascular endothelium and measured paracellular permeability. Blockade of transcytosis induced an increase in paracellular leak that was not explained by decreases in caveolin‐1 or by increased activity of nitric oxide synthase. The effect required caveolin‐1 but occurred in cells depleted of clathrin, indicating that it was not due to the general inhibition of endocytosis. Inhibiting transcytosis by blocking dynamin increased paracellular leak concomitantly with the loss of cortical actin from the plasma membrane and the displacement of active Rac from the plasmalemma. Importantly, inhibition of paracellular leak by sphingosine‐1‐phosphate, which activates Rac and induces cortical actin, caused a significant increase in transcytosis of albumin in vitro and in an ex vivo whole lung model. Dominant‐negative Rac diminished albumin uptake by endothelia. Thus, transcytosis and paracellular permeability are co‐regulated through a signalling pathway linking dynamin, Rac and actin.

Hydrogen peroxide selectively increases paracellular leak pathway permeability of renal epithelial cells

Increased paracellular solute flux is observed in several renal pathogenic processes characterized by oxidative stress. We undertook a study to examine the effect of H2O2 treatment, as a model of oxidative stress, on paracellular permeability of two renal epithelial cell lines, LLC‐PK1 and MDCK. Both the pore pathway (small ions; TransEpithelial Resistance (TER)) and leak pathway (large solutes; calcein flux) were monitored. MDCK monolayer calcein flux was much lower than LLC‐PK1 monolayer calcein flux. In contrast, MDCK monolayer TER was about 2‐fold lower than LLC‐PK1 monolayer TER. Sublethal H2O2 produced a concentration‐dependent increase in calcein flux in both renal cell lines. In contrast, TER only decreased at H2O2 concentrations that increased cell death. H2O2 treatment did not produce a marked change in total contents of multiple tight junction proteins nor in their distributions between Triton X‐100‐soluble and –insoluble fractions, although treatment may have produced a modest decrease in Triton X‐100‐insoluble ZO‐2 content. Surprisingly, occludin overexpression impaired and occludin knockdown modestly enhanced the ability of H2O2 to increase transepithelial calcein flux. H2O2 treatment produced a transient activation of src Family Kinases (SFKs). SFK inhibitors, but not inactive analogs, attenuated the H2O2‐induced increase in calcein flux. (Supported by institutional funds)

Claudin-1 involved in neonatal ichthyosis sclerosing cholangitis syndrome regulates hepatic paracellular permeability

Neonatal ichthyosis and sclerosing cholangitis (NISCH) syndrome is a liver disease caused by mutations of CLDN1 encoding Claudin-1, a tight-junction (TJ) protein. In this syndrome, it is speculated that cholestasis is caused by Claudin-1 absence, leading to increased paracellular permeability and liver injuries secondary to paracellular bile regurgitation. We studied the role of claudin-1 in hepatic paracellular permeability. A NISCH liver and polarized rat cell lines forming TJs, the hepatocellular Can 10 and the cholangiocellular normal rat choloangiocyte (NRC), were used. In contrast to NRC, Can 10 does not express claudin-1. Can 10 cells were transfected with a plasmid encoding Claudin-1, and stable Claudin-1-expressing clones were isolated. Claudin-1 expression was silenced by transfection with short interfering RNA in Can 10 clones and with short hairpin RNA in NRC. Claudin-1 expression was evaluated by quantitative reverse-transcriptase polymerase chain reaction, immunoblotting, and immunolocalization. Paracellular permeability was assessed by fluorescein isothiocyanate-dextran passage in both lines and by transepithelial resistance measurements in NRC. In the NISCH liver, Claudin-1 was not detected in hepatocytes or cholangiocytes. In Claudin-1 expressing Can 10 clones, Claudin-1 was localized at the TJ and paracellular permeability was decreased, compared to parental Can 10 cells, this decrease correlating with claudin-1 levels. Silencing of Claudin-1 in Can 10 clones increased paracellular permeability to a level similar to that of parental cells. Similarly, we observed an increase of paracellular permeability in NRC cells silenced for claudin-1 expression. Defect in claudin-1 expression increases paracellular permeability in polarized hepatic cell lines, supporting the hypothesis that paracellular bile leakage through deficient TJs is involved in liver pathology observed in NISCH syndrome.

Mucosal barrier defects in gastric intestinal metaplasia: in vivo evaluation by confocal endomicroscopy

Helicobacter pylori infection and intestinal metaplasia (IM) are associated with gastric cancer. An impaired gastric mucosal barrier could be involved in this carcinogenesis. To evaluate laser confocal laser endomicroscopy (CLE) for in vivo functional imaging of mucosal barrier defects in patients with IM. Prospective, controlled study. A tertiary-care academic center. This study involved patients with IM of the gastric mucosa who underwent CLE for surveillance. Specific IM mucosa and non-IM mucosa in patients were identified by CLE, and targeted biopsy samples were taken for histopathology and electron microscopy. Post-CLE assessment of paracellular fluorescein leakage was devised and validated by electron microscopy. We also evaluated the effect of H pylori eradication on the mucosal barrier. Forty-two patients were included. Of non-IM samples, the paracellular permeability was significantly increased in H pylori-positive samples compared with H pylori-negative controls (54 ± 31% vs 3 ± 6%, P < .05). Of IM samples, the permeability was significantly increased in both H pylori-negative and H pylori-positive samples (67 ± 34% and 72 ± 28% vs 3 ± 6%, both P < .05). The results of post-CLE assessment correlated well with the electron microscopy findings (R(2) 0.834, P < .0001). After the eradication of H pylori, the paracellular barrier dysfunction of non-IM mucosa was significantly improved as shown by electron microscopy and CLE (both P < .001). However, there was no significant change in IM mucosa. Single-center study. CLE allows functional imaging of mucosal barrier defects. Gastric IM is associated with an impaired paracellular barrier irrespective of H pylori eradication.

Methionine Restriction and Modification of Epithelial Tight Junction Barrier Function and Permeability

It is well known that caloric restriction leads to an increased longevity by forestalling age-related diseases. Dietary restriction of methionine also renders similar benefits. In this review, we report studies on the effect of methionine restriction on epithelial barrier function in a renal epithelial LLC-PK1 cell, a rodent gastrointestinal model and a clinical trial using Crohn's patients. In LLC-PK1 cell culture, a reduction of culture medium methionine by 80% resulted in altered tight junctional claudin composition with improved epithelial barrier function as exemplified by increased trans- epithelial electrical resistance and decreased paracellular leak to 14 C-D-mannitol. In addition to small but significant reductions in plasma and intracellular colonocyte methionine levels, dietary methionine restriction tightens intestinal epithelium and reduces mannitol permeability in rat colonic, but not ileal tissue. Crohn's patients on methionine-restricted diet reported improved symptoms as demonstrated by a reduced CDAI index, but this diet was not able to significantly reduce the plasma methionine level in patients recruited. An increased urinary lactulose/mannitol ratio was also the result of the regimen in Crohn's patients, suggesting an increased, rather than decreased ileal leakage. Overall, our results showed that reduction in dietary intake of methionine is promising in improving at least some epithelial barriers and this may be through altering tight junctional protein compositions. Methionine restriction might result in contrasting effects depending on the tissue type and possible disease conditions, thus wider range of studies are required.

Transepithelial leak in Barrett's esophagus patients: The role of proton pump inhibitors

To determine if the observed paracellular sucrose leak in Barrett's esophagus patients is due to their proton pump inhibitor (PPI) use. The in vivo sucrose permeability test was administered to healthy controls, to Barrett's patients and to non-Barrett's patients on continuous PPI therapy. Degree of leak was tested for correlation with presence of Barrett's, use of PPIs, and length of Barrett's segment and duration of PPI use. Barrett's patients manifested a near 3-fold greater, upper gastrointestinal sucrose leak than healthy controls. A decrease of sucrose leak was observed in Barrett's patients who ceased PPI use for 7 d. Although initial introduction of PPI use (in a PPI-naïve population) results in dramatic increase in sucrose leak, long-term, continuous PPI use manifested a slow spontaneous decline in leak. The sucrose leak observed in Barrett's patients showed no correlation to the amount of Barrett's tissue present in the esophagus. Although future research is needed to determine the degree of paracellular leak in actual Barrett's mucosa, the relatively high degree of leak observed with in vivo sucrose permeability measurement of Barrett's patients reflects their PPI use and not their Barrett's tissue per se.

A Dual-Tracer Method for Differentiating Transendothelial Transport from Paracellular Leakage in Vivo and in Vitro

Inflammation-induced impaired function of vascular endothelium may cause leakage of plasma proteins that can lead to edema. Proteins may leave the vascular lumen through two main paracellular and transcellular pathways. As the first involves endothelial cell (EC) junction proteins and the second caveolae formation, these two pathways are interconnected. Therefore, it is difficult to differentiate the prevailing role of one or the other pathway during pathology that causes inflammation. Here we present a newly developed dual-tracer probing method that allows differentiation of transcellular from paracellular transport during pathology. This fluorescence-based method can be used in vitro to test changes in EC layer permeability and in vivo in various animal vascular preparations. The method is based on comparison of low molecular weight molecule (LMWM) transport to that of high molecular weight molecule (HMWM) transport through the EC layer or the vascular wall during physiological and pathological conditions. Since the LMWM will leak through mainly the paracellular and HMWM will move through paracellular (when gaps between the ECs are wide enough) and transcellular pathways, the difference in transport rate (during normal conditions and pathology) of these molecules will indicate the prevailing transport pathway involved in overall protein crossing of vascular wall. Thus, the novel approach of assessing the transport kinetics of different size tracers in vivo by intravital microscopy can clarify questions related to identification of target pathways for drug delivery during various pathologies associated with elevated microvascular permeability.

Co-Regulation of Transcellular and Paracellular Leak Across Microvascular Endothelium by Dynamin and Rac

Increased permeability of the microvascular endothelium to fluids and proteins is the hallmark of inflammatory conditions such as sepsis. Leakage can occur between (paracellular) or through (transcytosis) endothelial cells, yet little is known about whether these pathways are linked. Understanding the regulation of microvascular permeability is essential for the identification of novel therapies to combat inflammation. We investigated whether transcytosis and paracellular leakage are co-regulated. Using molecular and pharmacologic approaches, we inhibited transcytosis of albumin in primary human microvascular endothelium and measured paracellular permeability. Blockade of transcytosis induced a rapid increase in paracellular leakage that was not explained by decreases in caveolin-1 or increases in activity of nitric oxide synthase. The effect required caveolin-1 but was observed in cells depleted of clathrin, indicating that it was not due to the general inhibition of endocytosis. Inhibiting transcytosis by dynamin blockade increased paracellular leakage concomitantly with the loss of cortical actin from the plasma membrane and the displacement of active Rac from the plasmalemma. Importantly, inhibition of paracellular leakage by sphingosine-1-phosphate, which activates Rac and induces cortical actin, caused a significant increase in transcytosis of albumin in vitro and in an ex vivo whole-lung model. In addition, dominant-negative Rac significantly diminished albumin uptake by endothelia. Our findings indicate that transcytosis and paracellular permeability are co-regulated through a signaling pathway linking dynamin, Rac, and actin.

Paracellular versus Transcellular Intestinal Permeability to Gliadin Peptides in Active Celiac Disease

The intestinal permeability of undegraded α9-gliadin peptide 31-49 (p31-49) and 33-mer gliadin peptides is increased in active celiac disease. Two distinct transport pathways have been proposed: paracellular leakage through epithelial tight junctions and protected transcellular transport. To analyze the relative contribution of these pathways, we compared mucosa-to-serosa permeability of small and large permeability markers [ionic conductance (G), mannitol, 182 Da; horseradish peroxidase, 40 kDa] and gliadin peptides [33-mer (p56-88, 3900 Da), 19-mer (p31-49, 2245 Da; and p202-220, 2127 Da), and 12-mer (p57-68, 1453 Da)] in duodenal biopsy specimens mounted in Ussing chambers. The permeability of intact peptides was much higher for p31-49 or 33-mer than for horseradish peroxidase, p202-220, and p57-68. A positive correlation was observed between G, an index of paracellular diffusion of ions, and mannitol permeability. The absence of correlation between G and permeability to intact 33-mer or p31-49 did not favor paracellular diffusion of the peptides. Immunofluorescence studies indicated that 33-mer enters the early endosome antigen 1-positive compartment but escapes the lysosomal-associated protein 2-positive compartment. The results underline that mannitol and ionic conductance G cannot be considered markers of permeability to gliadin peptides. In active celiac disease, increases in transcellular permeability to intact gliadin peptides might be considered in treatment strategies aimed at controlling epithelial permeability to gluten.

GROWTH AND DEVELOPMENT SYMPOSIUM: Inflammation: Role in the etiology and pathophysiology of clinical mastitis in dairy cows1

Genetic selection for increased milk production in dairy cattle was not associated with an attenuated inflammatory response. The systemic and local inflammatory responses contribute to altered metabolism, reduced production performance, and increased cull rate of lactating dairy cows with clinical mastitis. More aggressive inflammatory responses were observed during the peripartum period when compared with cows in late lactation after an intramammary challenge with purified lipopolysaccharide. The epidemiology of clinical mastitis indicates that the greatest incidence is observed during the peripartum period; therefore, an enhanced inflammatory response with concomitant suppression in other immune responses may be involved in the etiology and severity of the clinical mastitis observed in peripartum cows. Milk production losses and compositional changes are observed among all mammary quarters from a cow with clinical mastitis, but the responses are more severe and sustained among infected quarters. The infected mammary quarters reflect both the systemic and local reactions, whereas uninfected quarters represent only the systemic response. The systemic effects of the inflammatory response include reduced DMI, hyperthermia, and changes in whole-body nutrient partitioning affecting mammary epithelial substrate availability, whereas local inflammatory effects include energetic requirements of the increased inflammatory leukocyte pool, decreased synthetic capacity of mammary epithelium independent of substrate availability, and paracellular leakage of milk components from the alveolar lumen into the extracellular fluid. Research has focused on improving host immunological defenses, attenuating the inflammatory response, or improving the resolution of the disease state to limit the deleterious effects during clinical mastitis. This paper highlights the role inflammation plays in the etiology and pathophysiology of clinical mastitis as well as potential management strategies to reduce or prevent those losses.

Aerolysin From Aeromonas hydrophila Perturbs Tight Junction Integrity and Cell Lesion Repair in Intestinal Epithelial HT-29/B6 Cells

Aeromonads cause a variety of infections, including gastroenteritis, sepsis, and wound necrosis. Pathogenesis of Aeromonas hydrophila and its hemolysin has been characterized, but the mechanism of the epithelial barrier dysfunction is currently poorly understood. Human colon epithelial monolayers HT-29/B6 were apically inoculated with clinical isolates of A. hydrophila or with the secreted pore-forming toxin aerolysin. Epithelial resistance and permeability for several markers were determined in Ussing chambers, using 2-path impedance spectroscopy. The subcellular distribution of tight junction (TJ) and cytoskeleton proteins was analyzed by Western blotting and confocal laser-scanning microscopy. A. hydrophila infection induces chloride secretion with a small decrease in transcellular resistance. However, the major effect of A. hydrophila, mediated by its toxin aerolysin, was a sustained reduction of paracellular resistance by retracting sealing TJ proteins from the TJ strands. Aerolysin-treated monolayers showed increased paracellular permeability to FITC-dextran-4000 (0.104 ± 0.014 vs 0.047 ± 0.001 10(-6) cm/s in control; P < .05). Moreover, restitution of epithelial lesions was impaired. The effects were myosin light chain kinase (MLCK) dependent and mediated by intracellular Ca(2+) signaling. During Aeromonas infection, pore formation by aerolysin impairs epithelial integrity by promoting TJ protein redistribution and consequently affecting wound closure. Thus, Aeromonas-induced diarrhea is mediated by 2 mechanisms, transcellular secretion and paracellular leak flux.

Cell polarity-determining proteins Par-3 and PP-1 are involved in epithelial tight junction defects in coeliac disease

BackgroundEpithelial barrier defects are well known in coeliac disease, but the mechanisms are only poorly defined. It is unclear, whether barrier disturbance reflects upregulated epithelial transcytosis or paracellular leakage.ObjectiveTo characterise...

Analysis of protein dynamics within the septate junction reveals a highly stable core protein complex that does not include the basolateral polarity protein Discs large

Barrier junctions prevent pathogen invasion and restrict paracellular leakage across epithelial sheets. To understand how one barrier junction, the septate junction (SJ), is regulated in vivo, we used fluorescence recovery after photobleaching (FRAP) to examine SJ protein dynamics in Drosophila. Most SJ-associated proteins, including Coracle, Neurexin IV and Nervana 2, displayed similar, extremely immobile kinetics. Loss of any of these components resulted in dramatically increased mobility of all others, suggesting that they form a single, highly interdependent core complex. Immobilization of SJ core components coincided with formation of the morphological SJ but occurred after their known role in maintaining epithelial polarity, suggesting that these functions are independent. In striking contrast to the core components, the tumor suppressor protein Discs large was much more mobile and its loss did not affect mobility of core SJ proteins, suggesting that it is not a member of this complex, even though it colocalizes with the SJ. Similarly, disruption of endocytosis affected localization of SJ core components, but did not affect their mobility. These results indicate that formation of a stable SJ core complex is separable from its proper subcellular localization, and provide new insights into the complex processes that regulate epithelial polarity and assembly of the SJ.