scholarly journals The Epithelial Cell Leak Pathway

2021 ◽  
Vol 22 (14) ◽  
pp. 7677
Author(s):  
Ashley Monaco ◽  
Ben Ovryn ◽  
Josephine Axis ◽  
Kurt Amsler
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Actin Filaments ◽  
Molecular Basis ◽  
Paracellular Permeability ◽  
Historical Background ◽  
Permeability Barrier ◽  
Junction Structure ◽  
Leak Pathway

The epithelial cell tight junction structure is the site of the transepithelial movement of solutes and water between epithelial cells (paracellular permeability). Paracellular permeability can be divided into two distinct pathways, the Pore Pathway mediating the movement of small ions and solutes and the Leak Pathway mediating the movement of large solutes. Claudin proteins form the basic paracellular permeability barrier and mediate the movement of small ions and solutes via the Pore Pathway. The Leak Pathway remains less understood. Several proteins have been implicated in mediating the Leak Pathway, including occludin, ZO proteins, tricellulin, and actin filaments, but the proteins comprising the Leak Pathway remain unresolved. Many aspects of the Leak Pathway, such as its molecular mechanism, its properties, and its regulation, remain controversial. In this review, we provide a historical background to the evolution of the Leak Pathway concept from the initial examinations of paracellular permeability. We then discuss current information about the properties of the Leak Pathway and present current theories for the Leak Pathway. Finally, we discuss some recent research suggesting a possible molecular basis for the Leak Pathway.

scholarly journals The Leak Pathway: A Pathway in Flux

2019 ◽  
Author(s):  
Ashley Monaco ◽  
Ben Ovryn ◽  
Josephine Axis ◽  
Kurt Amsler
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Molecular Mechanism ◽  
Actin Filaments ◽  
Molecular Basis ◽  
Paracellular Permeability ◽  
Permeability Barrier ◽  
Junction Structure ◽  
Leak Pathway

The epithelial cell tight junction structure is the site of the transepithelial movement of solutes and water between epithelial cells (paracellular permeability). Paracellular permeability can be divided into two distinct pathways, the Pore Pathway mediating the movement of small ions and solutes and the Leak Pathway mediating the movement of large solutes. Claudin proteins form the basic paracellular permeability barrier and mediate the movement of small ions and solutes via the Pore Pathway. The Leak Pathway remains less understood. Several proteins have been implicated in mediating the Leak Pathway, including occludin, ZO proteins, tricellulin, and actin filaments, but the proteins comprising the Leak Pathway remain unresolved. The properties of the Leak Pathway, such as its molecular mechanism, its regulation, and whether or not it has a potential size limit, remain controversial. This review will trace the evolution of the Leak Pathway concept from its origins, will discuss the current information about the properties of the Leak Pathway, and will discuss recent research suggesting a possible molecular basis for the Leak Pathway. Based on these findings, we propose a model for the molecular mechanism underlying the Leak Pathway and its regulation.

Localization of a novel 210 kDa protein in Xenopus tight junctions

1997 ◽  
Vol 110 (8) ◽  
pp. 1005-1012 ◽  
Author(s):  
C.S. Merzdorf ◽  
D.A. Goodenough
Keyword(s):  
Monoclonal Antibody ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Basolateral Membrane ◽  
Immunofluorescent Staining ◽  
Junctional Complex ◽  
Permeability Barrier ◽  
Membrane Domains ◽  
Kidney Liver

The tight junction is the most apical member of the intercellular junctional complex. It functions as a permeability barrier between epithelial cells and maintains the integrity of the apical and basolateral membrane domains. In order to study tight junctions in Xenopus laevis, a polyclonal antibody was raised which recognized Xenopus ZO-1. Monoclonal antibody 19B1 (mAb 19B1) was generated in rats using a crude membrane preparation from Xenopus lung as antigen. mAb 19B1 gave immunofluorescent staining patterns identical to those seen with anti-ZO-1 on monolayers of Xenopus A6 kidney epithelial cells and on frozen sections of Xenopus kidney, liver, and embryos. Electron microscopy showed that the 19B1 antigen colocalized with ZO-1 at the tight junction. Western blotting and immunoprecipitation demonstrated that ZO-1 is an approximately 220 kDa protein in Xenopus, while mAb 19B1 identified an approximately 210 kDa antigen on immunoblots. Immunoprecipitates of ZO-1 were not recognized by mAb 19B1 by western analysis. The solubility properties of the 19B1 antigen suggested that it is a peripheral membrane protein. Thus, the antigen recognized by the new monoclonal antibody 19B1 is not ZO-1 and represents a different Xenopus tight junction associated protein.

scholarly journals NSP4 Enterotoxin of Rotavirus Induces Paracellular Leakage in Polarized Epithelial Cells

2001 ◽  
Vol 75 (3) ◽  
pp. 1540-1546 ◽  
Author(s):  
Farideh Tafazoli ◽  
Carl Q. Zeng ◽  
Mary K. Estes ◽  
Karl-Erik Magnusson ◽  
Lennart Svensson
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Electrical Resistance ◽  
Fluorescein Isothiocyanate ◽  
Transepithelial Electrical Resistance ◽  
Permeability Barrier ◽  
Filamentous Actin ◽  
Specific Effects ◽  
Polarized Epithelia ◽  
Polarized Epithelial Cells

ABSTRACT The nonstructural NSP4 protein of rotavirus has been described as the first viral enterotoxin. In this study we have examined the effect of NSP4 on polarized epithelial cells (MDCK-1) grown on permeable filters. Apical but not basolateral administration of NSP4 was found to cause a reduction in the transepithelial electrical resistance, redistribution of filamentous actin, and an increase in paracellular passage of fluorescein isothiocyanate-dextran. Significant effects on transepithelial electrical resistance were noted after a 20- to 30-h incubation with 1 nmol of NSP4. Most surprisingly, the epithelium recovered its original integrity and electrical resistance upon removal of NSP4. Preincubation of nonconfluent MDCK-1 cells with NSP4 prevented not only development of a permeability barrier but also lateral targeting of the tight-junction-associated Zonula Occludens-1 (ZO-1) protein. Taken together, these data indicate new and specific effects of NSP4 on tight-junction biogenesis and show a novel effect of NSP4 on polarized epithelia.

scholarly journals Differential Infection of Polarized Epithelial Cell Lines by Sialic Acid-Dependent and Sialic Acid-Independent Rotavirus Strains

2001 ◽  
Vol 75 (23) ◽  
pp. 11834-11850 ◽  
Author(s):  
Max Ciarlet ◽  
Sue E. Crawford ◽  
Mary K. Estes
Keyword(s):  
Sialic Acid ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Apical Cell ◽  
Paracellular Permeability ◽  
Apical Surface ◽  
Apical Cell Membrane ◽  
Basolateral Side ◽  
Epithelial Cell Lines

ABSTRACT Infection of epithelial cells by some animal rotaviruses, but not human or most animal rotaviruses, requires the presence ofN-acetylneuraminic (sialic) acid (SA) on the cell surface for efficient infectivity. To further understand how rotaviruses enter susceptible cells, six different polarized epithelial cell lines, grown on permeable filter membrane supports containing 0.4-μm pores, were infected apically or basolaterally with SA-independent or SA-dependent rotaviruses. SA-independent rotaviruses applied apically or basolaterally were capable of efficiently infecting both sides of the epithelium of all six polarized cell lines tested, while SA-dependent rotaviruses only infected efficiently through the apical surface of five of the polarized cell lines tested. Regardless of the route of virus entry, SA-dependent and SA-independent rotaviruses were released almost exclusively from the apical domain of the plasma membrane of polarized cells before monolayer disruption or cell lysis. The transepithelial electrical resistance (TER) of cells decreased at the same time, irrespective of whether infection with SA-independent rotaviruses occurred apically or basolaterally. The TER of cells infected apically with SA-dependent rotaviruses decreased earlier than that of cells infected basolaterally. Rotavirus infection decreased TER before the appearance of cytopathic effect and cell death and resulted in an increase in the paracellular permeability to [3H]inulin as a function of loss of TER. The presence of SA residues on either the apical or basolateral side was determined using a Texas Red-conjugated lectin, wheat germ agglutinin (WGA), which binds SA residues. WGA bound exclusively to SA residues on the apical surface of the cells, confirming the requirement for SA residues on the apical cell membrane for efficient infectivity of SA-dependent rotaviruses. These results indicate that the rotavirus SA-independent cellular receptor is present on both sides of the epithelium, but SA-dependent and SA-independent rotavirus strains infect polarized epithelial cells by different mechanisms, which may be relevant for pathogenesis and selection of vaccine strains. Finally, rotavirus-induced alterations of the epithelial barrier and paracellular permeability suggest that common mechanisms of pathogenesis may exist between viral and bacterial pathogens of the intestinal tract.

scholarly journals Correlation of occludin protein mobility with paracellular leak pathway permeability in renal epithelia

2018 ◽  
Author(s):  
Josephine Axis ◽  
Alexander L. Kolb ◽  
Robert L. Bacallao ◽  
Kurt Amsler
Keyword(s):  
Tight Junction ◽  
Ischemia Reperfusion Injury ◽  
Mdck Cells ◽  
Ischemia Reperfusion ◽  
Paracellular Permeability ◽  
Tight Junction Protein ◽  
Protein Mobility ◽  
Junction Protein ◽  
Leak Pathway

ABSTRACTStudies have demonstrated regulation of the epithelial paracellular permeability barrier, the tight junction, by a variety of stimuli. Recent studies have reported a correlation between changes in paracellular permeability, particularly paracellular permeability to large solutes (leak pathway), and mobility of the tight junction protein, occludin, in the plane of the plasma membrane. This had led to the hypothesis that changes in occludin protein mobility are causative for changes in paracellular permeability. Using a renal epithelial cell model system, MDCK, we examined the effect of various manipulations on both leak pathway permeability, monitored as the paracellular movement of a fluorescent molecule (calcein), and occludin protein mobility, monitored through fluorescence recovery after photobleaching. Our results indicate that knockdown of the associated tight junction protein, ZO-1, increases baseline leak pathway permeability, whereas, knockdown of the related tight junction protein, ZO-2, does not alter baseline leak pathway permeability. Knockdown of either ZO-1 or ZO-2 decreases the rate of movement of occludin protein but only knockdown of ZO-2 protein alters the percent of occludin protein that is mobile. Further, treatment with hydrogen peroxide increases leak pathway permeability in wild type MDCK cells and in ZO-2 knockdown MDCK cells but not in ZO-1 knockdown MDCK cells. This treatment decreases the rate of occludin movement in all three cell lines but only alters the mobile fraction of occludin protein in ZO-1 knockdown MDCK cells. Finally, we examined the effect of renal ischemia/reperfusion injury on occludin protein mobility in vivo.Ischemia/reperfusion injury both increased the rate of occludin mobility and increased the fraction of occludin protein that is mobile. These results indicate that, at least in our cell culture and in vivo model systems, there is no consistent correlation between paracellular leak pathway permeability and occludin protein mobility.

scholarly journals A Synthetic Peptide Corresponding to the Extracellular Domain of Occludin Perturbs the Tight Junction Permeability Barrier

1997 ◽  
Vol 136 (2) ◽  
pp. 399-409 ◽  
Author(s):  
Vivian Wong ◽  
Barry M. Gumbiner
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Adherens Junction ◽  
Extracellular Domain ◽  
Integral Membrane Protein ◽  
Epithelial Cell Line ◽  
Permeability Barrier ◽  
Junction Protein ◽  
Tight Junction Permeability

Occludin, the putative tight junction integral membrane protein, is an attractive candidate for a protein that forms the actual sealing element of the tight junction. To study the role of occludin in the formation of the tight junction seal, synthetic peptides (OCC1 and OCC2) corresponding to the two putative extracellular domains of occludin were assayed for their ability to alter tight junctions in Xenopus kidney epithelial cell line A6. Transepithelial electrical resistance and paracellular tracer flux measurements indicated that the second extracellular domain peptide (OCC2) reversibly disrupted the transepithelial permeability barrier at concentrations of < 5 μM. Despite the increased paracellular permeability, there were no changes in gross epithelial cell morphology as determined by scanning EM. The OCC2 peptide decreased the amount of occludin present at the tight junction, as assessed by indirect immunofluorescence, as well as decreased total cellular content of occludin, as assessed by Western blot analysis. Pulse-labeling and metabolic chase analysis suggested that this decrease in occludin level could be attributed to an increase in turnover of cellular occludin rather than a decrease in occludin synthesis. The effect on occludin was specific because other tight junction components, ZO-1, ZO-2, cingulin, and the adherens junction protein E-cadherin, were unaltered by OCC2 treatment. Therefore, the peptide corresponding to the second extracellular domain of occludin perturbs the tight junction permeability barrier in a very specific manner. The correlation between a decrease in occludin levels and the perturbation of the tight junction permeability barrier provides evidence for a role of occludin in the formation of the tight junction seal.

Concentration-dependent effects of cytochalasin D on tight junctions and actin filaments in MDCK epithelial cells

1994 ◽  
Vol 107 (3) ◽  
pp. 367-375 ◽  
Author(s):  
B.R. Stevenson ◽  
D.A. Begg
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Actin Filaments ◽  
Mdck Cells ◽  
Microscopic Analysis ◽  
Cytochalasin D ◽  
Transepithelial Resistance ◽  
Actin Bundles ◽  
Filament Bundles ◽  
Tight Junction Permeability

The effects of different concentrations of the actin-disrupting drug cytochalasin D on tight junction permeability and distribution of actin filaments in MDCK epithelial cells were examined. Consistent with previous studies, 2 micrograms/ml cytochalasin D caused a significant decrease in transepithelial resistance, indicative of an increase in tight junction permeability. Surprisingly, increasing concentrations of cytochalasin D caused progressively smaller decreases in transepithelial resistance. The effects of cytochalasin D were reversible. Light microscopic analysis utilizing rhodamine-conjugated phalloidin demonstrated two distinct populations of actin filaments in MDCK cells: an apical peripheral ring of actin, presumably associated with the zonula adherens, and larger actin bundles more basally situated. When treated with 2 micrograms/ml cytochalasin D, both actin populations were severely disrupted and cells became flattened. Actin in the apical ring aggregated along cell boundaries, and these aggregates co-localized with similarly disrupted focal accumulations of the tight junction-associated protein ZO-1. The basal actin filament bundles also reorganized into focal aggregates. Increasing concentrations of cytochalasin D caused gradually less perturbation of the apical actin ring, consistent with the transepithelial resistance observations. However, the basal actin bundles were disrupted at all concentrations of cytochalasin D tested, demonstrating that the two actin populations are differentially sensitive to cytochalasin D and that apical actin filaments are more important in the regulation of tight junction permeability. Finally, treatment of cells with cytochalasin D inhibited the decrease in transepithelial resistance induced by the chelation of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Lipoxin A4prevents tight junction disruption and delays the colonization of cystic fibrosis bronchial epithelial cells byPseudomonas aeruginosa

2016 ◽  
Vol 310 (11) ◽  
pp. L1053-L1061 ◽  
Author(s):  
Gerard Higgins ◽  
Coral Fustero Torre ◽  
Jean Tyrrell ◽  
Paul McNally ◽  
Brian J. Harvey ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Cell Lines ◽  
Bronchial Epithelial Cell ◽  
Bacterial Invasion ◽  
Airway Epithelial ◽  
Bronchial Epithelial ◽  
Tight Junction Disruption

The specialized proresolution lipid mediator lipoxin A4(LXA4) is abnormally produced in cystic fibrosis (CF) airways. LXA4increases the CF airway surface liquid height and stimulates airway epithelial repair and tight junction formation. We report here a protective effect of LXA4(1 nM) against tight junction disruption caused by Pseudomonas aeruginosa bacterial challenge together with a delaying action against bacterial invasion in CF airway epithelial cells from patients with CF and immortalized cell lines. Bacterial invasion and tight junction integrity were measured by gentamicin exclusion assays and confocal fluorescence microscopy in non-CF (NuLi-1) and CF (CuFi-1) bronchial epithelial cell lines and in primary CF cultures, grown under an air/liquid interface, exposed to either a clinical or laboratory strains of P. aeruginosa. LXA4delayed P. aeruginosa invasion and transepithelial migration in CF and normal bronchial epithelial cell cultures. These protective effects of LXA4were inhibited by the ALX/FPR2 lipoxin receptor antagonist BOC-2. LXA4prevented the reduction in mRNA biosynthesis and protein abundance of the tight junction protein ZO-1 and reduced tight junction disruption induced by P. aeruginsosa inoculation. In conclusion, LXA4plays a protective role in bronchial epithelium by stimulating tight junction repair and by delaying and reducing the invasion of CF bronchial epithelial cells by P. aeruginsosa.

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