scholarly journals Apically Exposed, Tight Junction-Associated β1-Integrins Allow Binding and YopE-Mediated Perturbation of Epithelial Barriers by Wild-Type Yersinia Bacteria

2000 ◽  
Vol 68 (9) ◽  
pp. 5335-5343 ◽  
Author(s):  
Farideh Tafazoli ◽  
Anna Holmström ◽  
Åke Forsberg ◽  
Karl-Eric Magnusson
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Yersinia Pseudotuberculosis ◽  
Cell Structure ◽  
Wild Type ◽  
Filamentous Actin ◽  
Basolateral Membranes ◽  
Contact Points ◽  
Β1 Integrins

ABSTRACT Using polarized epithelial cells, primarily MDCK-1, we assessed the mode of binding and effects on epithelial cell structure and permeability of Yersinia pseudotuberculosis yadA-deficient mutants. Initially, all bacteria except the invasin-deficient (inv) mutant adhered apically to the tight junction areas. These contact points of adjacent cells displayed β1-integrins together with tight junction-associated ZO-1 and occludin proteins. Indeed, β1-integrin expression was maximal in the tight junction area and then gradually decreased along the basolateral membranes. Wild-type bacteria also opened gradually the tight junction to paracellular permeation of different-sized markers, viz., 20-, 40-, and 70-kDa dextrans and 45-kDa ovalbumin, as well as to their own translocation between adjacent cells in intimate contact with β1-integrins. The effects on the epithelial cells and their barrier properties could primarily be attributed to expression of the Yersinia outer membrane protein YopE, as the yopE mutant bound but caused no cytotoxicity. Moreover, the apical structure of filamentous actin (F-actin) was disturbed and tight junction-associated proteins (ZO-1 and occludin) were dispersed along the basolateral membranes. It is concluded that the Yersinia bacteria attach to β1-integrins at tight junctions. Via this localized injection of YopE, they perturb the F-actin structure and distribution of proteins forming and regulating tight junctions. Thereby they promote paracellular translocation of bacteria and soluble compounds.

Differential Integrin Expression Facilitates Isolation of Human Fetal Pancreatic Epithelial Cells

1994 ◽  
Vol 3 (4) ◽  
pp. 307-313 ◽  
Author(s):  
Fred Levine ◽  
Gillian M. Beattie ◽  
Alberto Hayek
Keyword(s):  
Epithelial Cells ◽  
Stromal Cells ◽  
Immunohistochemical Staining ◽  
Endocrine Cells ◽  
Yersinia Pseudotuberculosis ◽  
Affinity Binding ◽  
Β1 Integrin ◽  
Human Pancreas ◽  
High Affinity ◽  
Β1 Integrins

We have studied the expression of the β1 family of integrins in fetal and adult human pancreas. Immunohistochemical staining with a monoclonal anti-β1 antibody revealed that the epithelial cells of the human fetal pancreas express high amounts of β1 integrin, while the pancreatic stromal cells express substantially lower amounts. Islets of Langerhans from human adult pancreas also expressed high amounts of β1 integrin. Taking advantage of the extremely high affinity binding between the invasin protein of Yersinia pseudotuberculosis and many β1 integrins, we have been able to isolate highly enriched populations of fetal pancreatic epithelial cells. Epithelial-enriched cell populations retain the ability to differentiate into mature endocrine cells following transplantation into nude mice.

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.

Role of the β1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia

2002 ◽  
Vol 115 (13) ◽  
pp. 2669-2678 ◽  
Author(s):  
Anna Gustavsson ◽  
Annika Armulik ◽  
Cord Brakebusch ◽  
Reinhard Fässler ◽  
Staffan Johansson ◽  
...  
Keyword(s):  
Marked Reduction ◽  
Yersinia Pseudotuberculosis ◽  
Host Cells ◽  
Β1 Integrin ◽  
Complex Structures ◽  
Wild Type ◽  
Similar Reduction ◽  
Focal Complex ◽  
Β1 Integrins

Invasin of Yersinia pseudotuberculosis binds to β1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the β1-integrin-mediated internalization of Yersinia, a β1-integrin-deficient cell line, GD25, transfected with wild-type β1A, β1B or different mutants of the β1A subunit was used. Both β1A and β1B bound to invasin-expressing bacteria, but only β1A was able to mediate internalization of the bacteria. The cytoplasmic region of β1A, differing from β1B, contains two NPXY motifs surrounding a double threonine site. Exchanging the tyrosines of the two NPXYs to phenylalanines did not inhibit the uptake, whereas a marked reduction was seen when the first tyrosine (Y783) was exchanged to alanine. A similar reduction was seen when the two nearby threonines (TT788-9) were exchanged with alanines. It was also noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex structures was unable to mediate uptake of invasin-expressing bacteria.

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation

1997 ◽  
Vol 273 (4) ◽  
pp. C1378-C1385 ◽  
Author(s):  
Jerrold R. Turner ◽  
Brian K. Rill ◽  
Susan L. Carlson ◽  
Denise Carnes ◽  
Rachel Kerner ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Inhibitors ◽  
Tight Junction Regulation ◽  
Tight Junction Permeability ◽  
Mlc Phosphorylation

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 ± 5% fall in transepithelial resistance (TER) ( P< 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 ± 2% ( P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 μM ML-7 or 40 μM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 ( P< 0.01), which was inhibited by ML-9 ( P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa ( P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.

Airway epithelial tight junctions and binding and cytotoxicity ofPseudomonas aeruginosa

1999 ◽  
Vol 277 (1) ◽  
pp. L204-L217 ◽  
Author(s):  
Alfred Lee ◽  
Dar Chow ◽  
Brian Haus ◽  
Wanru Tseng ◽  
David Evans ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junctions ◽  
Propidium Iodide ◽  
Time Course ◽  
Living Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Basolateral Membranes ◽  
Free Edges

The role of tight junctions in the binding and cytoxicity of Pseudomonas aeruginosato apical or basolateral membranes of lung airway epithelial cells was tested with fluorescence microscopy on living cells. Binding of noncytotoxic P. aeruginosa strain O1 was assessed with P. aeruginosa that expressed green fluorescent protein. Binding of cytotoxic P. aeruginosa strain 6206 was assessed with FITC-labeled P. aeruginosa; cytotoxicity was determined from nuclear uptake of the impermeant dye propidium iodide. The role of direct contact of P. aeruginosa to epithelial cells was tested with filters with small (0.45-μm) or large (2.0-μm) pores. High transepithelial resistance ( Rt) Calu-3 and cultured bovine tracheal monolayers ( Rt> 1,000 Ω ⋅ cm2) bound P. aeruginosa very infrequently (<1 P. aeruginosa/100 cells) at the apical membrane, but P. aeruginosabound frequently to cells near “free edges” at holes, wounds, islands, and perimeters; cytotoxicity required direct interaction with basolateral membranes. Wounded high Rtepithelia showed increased P. aeruginosa binding and cytotoxicity at the free edges because basolateral membranes were accessible to P. aeruginosa, and dead and living cells near the wound bound P. aeruginosa similarly. Compared with high Rtepithelia, low RtCFT1 ( Rt= 100–200 Ω ⋅ cm2) and EGTA-treated Calu-3 monolayers were 25 times more susceptible to P. aeruginosa binding throughout the monolayer. Cytotoxicity to CFT1 cells (throughout the confluent monolayer, not only at the free edge) occurred after a shorter delay (0.25 vs. 2.0 h) and then five times faster than to Calu-3 cells, indicating that the time course of P. aeruginosa cytotoxicity may be limited by the rate of gaining access through tight junctions and that this occurred faster in low Rtthan in high Rtairway epithelia. Cytotoxicity appeared to occur in a sequential process that led first to a loss of fura 2 and a later uptake of propidium iodide. P. aeruginosa bound three times more frequently to regions between cells (tight junctions?) than to cell membranes of low RtCFT1 cells.

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 CRB3 Binds Directly to Par6 and Regulates the Morphogenesis of the Tight Junctions in Mammalian Epithelial Cells

2004 ◽  
Vol 15 (3) ◽  
pp. 1324-1333 ◽  
Author(s):  
Céline Lemmers ◽  
Didier Michel ◽  
Lydie Lane-Guermonprez ◽  
Marie-Hélène Delgrossi ◽  
Emmanuelle Médina ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Transmembrane Protein ◽  
Direct Interaction ◽  
Extracellular Domain ◽  
Cytoplasmic Domain ◽  
Epithelial Morphogenesis ◽  
Neurotrophin Receptor ◽  
Tight Junction Formation

Crumbs is an apical transmembrane protein crucial for epithelial morphogenesis in Drosophila melanogaster embryos. A protein with all the characteristics for a Crumbs homologue has been identified from patients suffering from retinitis pigmentosa group 12, but this protein (CRB1) is only expressed in retina and some parts of the brain, both in human and mouse. Here, we describe CRB3, another Crumbs homologue that is preferentially expressed in epithelial tissues and skeletal muscles in human. CRB3 shares the conserved cytoplasmic domain with other Crumbs but exhibits a very short extracellular domain without the EGF- and laminin A-like G repeats present in the other Crumbs. CRB3 is localized to the apical and subapical area of epithelial cells from the mouse and human intestine, suggesting that it could play a role in epithelial morphogenesis. Indeed, expression of CRB3 or of a chimera containing the extracellular domain of the neurotrophin receptor p75NTR and the transmembrane and cytoplasmic domains of CRB3 led to a slower development of functional tight junctions in Madin-Darby canine kidney cells. This phenotype relied on the presence of CRB3 four last amino acids (ERLI) that are involved in a direct interaction with Par6, a regulator of epithelial polarity and tight junction formation. Thus, CRB3, through its cytoplasmic domain and its interactors, plays a role in apical membrane morphogenesis and tight junction regulation.

scholarly journals Regulation of Tight Junctions in Upper Airway Epithelium

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Takashi Kojima ◽  
Mitsuru Go ◽  
Ken-ichi Takano ◽  
Makoto Kurose ◽  
Tsuyoshi Ohkuni ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Airway Epithelium ◽  
Barrier Function ◽  
Upper Airway ◽  
Mucosal Barrier ◽  
Upper Respiratory Tract ◽  
Junction Molecules ◽  
Human Nasal Epithelial Cells

The mucosal barrier of the upper respiratory tract including the nasal cavity, which is the first site of exposure to inhaled antigens, plays an important role in host defense in terms of innate immunity and is regulated in large part by tight junctions of epithelial cells. Tight junction molecules are expressed in both M cells and dendritic cells as well as epithelial cells of upper airway. Various antigens are sampled, transported, and released to lymphocytes through the cells in nasal mucosa while they maintain the integrity of the barrier. Expression of tight junction molecules and the barrier function in normal human nasal epithelial cells (HNECs) are affected by various stimuli including growth factor, TLR ligand, and cytokine. In addition, epithelial-derived thymic stromal lymphopoietin (TSLP), which is a master switch for allergic inflammatory diseases including allergic rhinitis, enhances the barrier function together with an increase of tight junction molecules in HNECs. Furthermore, respiratory syncytial virus infection in HNECsin vitroinduces expression of tight junction molecules and the barrier function together with proinflammatory cytokine release. This paper summarizes the recent progress in our understanding of the regulation of tight junctions in the upper airway epithelium under normal, allergic, and RSV-infected conditions.

Human junction adhesion molecule regulates tight junction resealing in epithelia

2000 ◽  
Vol 113 (13) ◽  
pp. 2363-2374 ◽  
Author(s):  
Y. Liu ◽  
A. Nusrat ◽  
F.J. Schnell ◽  
T.A. Reaves ◽  
S. Walsh ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Adhesion Molecule ◽  
Transmembrane Protein ◽  
Extracellular Domain ◽  
Cell Junctions ◽  
Human Neutrophils ◽  
Epithelial Barrier ◽  
Cell Cell

Epithelial cells form a highly selective barrier and line many organs. The epithelial barrier is maintained by closely apposed cell-cell contacts containing tight junctions, the regulation of which is incompletely understood. Here we report the cloning, tissue localization and evidence for a role in epithelial barrier regulation of an immunoglobulin superfamily member that likely represents the human homolog of murine junction adhesion molecule (JAM). Analysis of the primary structure of human JAM, cloned from T84 epithelial cells, predicts a transmembrane protein with an extracellular domain that contains two IgV loops. Monoclonal antibodies generated against the putative extracellular domain were reactive with a 35–39 kDa protein from both T84 epithelial cells and human neutrophils. By immunofluorescence, JAM mAbs labeled epithelial cells from intestine, lung, and kidney, prominently in the region of tight junctions (co-localization with occludin) and also along lateral cell membranes below the tight junctions. Flow cytometric studies confirmed predominant JAM expression in epithelial cells but also revealed expression on endothelial and hematopoietic cells of all lineages. Functional studies demonstrated that JAM specific mAbs markedly inhibited transepithelial resistance recovery of T84 monolayers after disruption of intercellular junctions (including tight junctions) by transient calcium depletion. Morphologic analysis revealed that, after disassembly of cell-cell junctions, anti-JAM inhibition of barrier function recovery correlated with a loss of both occludin and JAM, but not ZO-1, in reassembling tight junction structure. Reassembly of the major adherens junction component E-cadherin was not affected by JAM specific mAbs. Our findings suggest that JAM plays an important role in the regulation of tight junction assembly in epithelia. Furthermore, these JAM-mediated effects may occur by either direct, or indirect interactions with occludin.

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