Functional proflling of intestinal tight junctions in vitro highlights mechanistic differences between tight junction modulators+

Transcranial direct current stimulation (tDCS) is a non-invasive physical therapy to treat many psychiatric disorders and to enhance memory and cognition in healthy individuals. Our recent studies showed that tDCS with the proper dosage and duration can transiently enhance the permeability (P) of the blood-brain barrier (BBB) in rat brain to various sized solutes. Based on the in vivo permeability data, a transport model for the paracellular pathway of the BBB also predicted that tDCS can transiently disrupt the endothelial glycocalyx (EG) and the tight junction between endothelial cells. To confirm these predictions and to investigate the structural mechanisms by which tDCS modulates P of the BBB, we directly quantified the EG and tight junctions of in vitro BBB models after DCS treatment. Human cerebral microvascular endothelial cells (hCMECs) and mouse brain microvascular endothelial cells (bEnd3) were cultured on the Transwell filter with 3 μm pores to generate in vitro BBBs. After confluence, 0.1–1 mA/cm2 DCS was applied for 5 and 10 min. TEER and P to dextran-70k of the in vitro BBB were measured, HS (heparan sulfate) and hyaluronic acid (HA) of EG was immuno-stained and quantified, as well as the tight junction ZO-1. We found disrupted EG and ZO-1 when P to dextran-70k was increased and TEER was decreased by the DCS. To further investigate the cellular signaling mechanism of DCS on the BBB permeability, we pretreated the in vitro BBB with a nitric oxide synthase (NOS) inhibitor, L-NMMA. L-NMMA diminished the effect of DCS on the BBB permeability by protecting the EG and reinforcing tight junctions. These in vitro results conform to the in vivo observations and confirm the model prediction that DCS can disrupt the EG and tight junction of the BBB. Nevertheless, the in vivo effects of DCS are transient which backup its safety in the clinical application. In conclusion, our current study directly elucidates the structural and signaling mechanisms by which DCS modulates the BBB permeability.

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Enteroaggregative Escherichia coli Disrupts Epithelial Cell Tight Junctions

Infection and Immunity ◽

10.1128/iai.00580-10 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4958-4964 ◽

Cited By ~ 57

Author(s):

Maura C. Strauman ◽

Jill M. Harper ◽

Susan M. Harrington ◽

Erik Juncker Boll ◽

James P. Nataro

Keyword(s):

Escherichia Coli ◽

Tight Junction ◽

Tight Junctions ◽

Cell Model ◽

Barrier Dysfunction ◽

T84 Cells ◽

E Coli ◽

Eaec Strain ◽

Epithelial Barrier Dysfunction

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is responsible for inflammatory diarrhea in diverse populations, but its mechanisms of pathogenesis have not been fully elucidated. We have used a previously characterized polarized intestinal T84 cell model to investigate the effects of infection with EAEC strain 042 on tight junction integrity. We find that infection with strain 042 induces a decrease in transepithelial electrical resistance (TER) compared to uninfected controls and to cells infected with commensal E. coli strain HS. When the infection was limited after 3 h by washing and application of gentamicin, we observed that the TER of EAEC-infected monolayers continued to decline, and they remained low even as long as 48 h after the infection. Cells infected with the afimbrial mutant strain 042aafA exhibited TER measurements similar to those seen in uninfected monolayers, implicating the aggregative adherence fimbriae II (AAF/II) as necessary for barrier dysfunction. Infection with wild-type strain 042 induced aberrant localization of the tight junction proteins claudin-1 and, to a lesser degree, occludin. EAEC-infected T84 cells exhibited irregular shapes, and some cells became elongated and/or enlarged; these effects were not observed after infection with commensal E. coli strain HS or 042aafA. The effects on tight junctions were also observed with AAF/I-producing strain JM221, and an afimbrial mutant was similarly deficient in inducing barrier dysfunction. Our results show that EAEC induces epithelial barrier dysfunction in vitro and implicates the AAF adhesins in this phenotype.

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The Unique-5 and -6 Motifs of ZO-1 Regulate Tight Junction Strand Localization and Scaffolding Properties

Molecular Biology of the Cell ◽

10.1091/mbc.e06-08-0764 ◽

2007 ◽

Vol 18 (3) ◽

pp. 721-731 ◽

Cited By ~ 91

Author(s):

Alan S. Fanning ◽

Brent P. Little ◽

Christoph Rahner ◽

Darkhan Utepbergenov ◽

Zenta Walther ◽

...

Keyword(s):

Tight Junction ◽

Tight Junctions ◽

Cultured Cells ◽

Variable Region ◽

Binding Studies ◽

Vitro Binding ◽

Complex Protein ◽

Regulatory Functions ◽

Maguk Proteins

The proper cellular location and sealing of tight junctions is assumed to depend on scaffolding properties of ZO-1, a member of the MAGUK protein family. ZO-1 contains a conserved SH3-GUK module that is separated by a variable region (unique-5), which in other MAGUKs has proven regulatory functions. To identify motifs in ZO-1 critical for its putative scaffolding functions, we focused on the SH3-GUK module including unique-5 (U5) and unique-6 (U6), a motif immediately C-terminal of the GUK domain. In vitro binding studies reveal U5 is sufficient for occludin binding; U6 reduces the affinity of this binding. In cultured cells, U5 is required for targeting ZO-1 to tight junctions and removal of U6 results in ectopically displaced junction strands containing the modified ZO-1, occludin, and claudin on the lateral cell membrane. These results provide evidence that ZO-1 can control the location of tight junction transmembrane proteins and reveals complex protein binding and targeting signals within its SH3-U5-GUK-U6 region. We review these findings in the context of regulated scaffolding functions of other MAGUK proteins.

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A novel in vitro platform for the study of SN38-induced mucosal damage and the development of Toll-like receptor 4-targeted therapeutic options

Experimental Biology and Medicine ◽

10.1177/1535370216640932 ◽

2016 ◽

Vol 241 (13) ◽

pp. 1386-1394 ◽

Cited By ~ 5

Author(s):

Hannah R Wardill ◽

Rachel J Gibson ◽

Ysabella ZA Van Sebille ◽

Kate R Secombe ◽

Richard M Logan ◽

...

Keyword(s):

Tight Junction ◽

Tight Junctions ◽

Mucosal Damage ◽

Toll Like Receptor ◽

Tight Junction Proteins ◽

T84 Cells ◽

Toll Like Receptor 4 ◽

Transmission Electron ◽

Junction Proteins

Tight junction and epithelial barrier disruption is a common trait of many gastrointestinal pathologies, including chemotherapy-induced gut toxicity. Currently, there are no validated in vitro models suitable for the study of chemotherapy-induced mucosal damage that allow paralleled functional and structural analyses of tight junction integrity. We therefore aimed to determine if a transparent, polyester membrane insert supports a polarized T84 monolayer with the phenotypically normal tight junctions. T84 cells (passage 5–15) were seeded into either 0.6 cm2, 0.4 µm pore mixed-cellulose transwell hanging inserts or 1.12 cm2, 0.4 µm pore polyester transwell inserts at varying densities. Transepithelial electrical resistance was measured daily to assess barrier formation. Immunofluoresence for key tight junction proteins (occludin, zonular occludens-1, claudin-1) and transmission electron microscopy were performed to assess tight junction integrity, organelle distribution, and polarity. Reverse transcription-polymerase chain reaction was performed to determine expression of toll-like receptor 4 (TLR4). Liquid chromatography was also conducted to assess SN38 degradation in this model. Polyester membrane inserts support a polarized T84 phenotype with functional tight junctions in vitro. Transmission electron microscopy indicated polarity, with apico-laterally located tight junctions. Immunofluorescence showed membranous staining for all tight junction proteins. No internalization was evident. T84 cells expressed TLR4, although this was significantly lower than levels seen in HT29 cells ( P = .0377). SN38 underwent more rapid degradation in the presence of cells (−76.04 ± 1.86%) compared to blank membrane (−48.39 ± 4.01%), indicating metabolic processes. Polyester membrane inserts provide a novel platform for paralleled functional and structural analysis of tight junction integrity in T84 monolayers. T84 cells exhibit the unique ability to metabolize SN38 as well as expressing TLR4, making this an excellent platform to study clinically relevant therapeutic interventions for SN38-induced mucosal damage by targeting TLR4.

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Detection of the tight junction-associated protein ZO-1 in astrocytes and other nonepithelial cell types

AJP Cell Physiology ◽

10.1152/ajpcell.1992.262.2.c461 ◽

1992 ◽

Vol 262 (2) ◽

pp. C461-C469 ◽

Cited By ~ 93

Author(s):

A. G. Howarth ◽

M. R. Hughes ◽

B. R. Stevenson

Keyword(s):

Tight Junction ◽

Tight Junctions ◽

Primary Cultures ◽

Fibroblast Cell ◽

Cell Types ◽

Dermal Fibroblasts ◽

Cell Contact ◽

Cell Periphery ◽

Cell Extracts

ZO-1 is a high molecular mass phosphoprotein peripherally associated with the cytoplasmic surface of tight junctions in epithelial and endothelial cells. We report here that ZO-1 is also present in several nonepithelial cell types in vitro that are not believed to form tight junctions, including primary cultures of astrocytes, Schwann cells, and dermal fibroblasts and the C6 glioma, S-180 (sarcoma), and P3 myeloma cell lines. Immunoblots of cell extracts probed with a ZO-1-specific monoclonal antibody reveal a single band that comigrates with ZO-1 from rodent epithelial cells at 225 kDa. In addition, these cells contain a single mRNA species of identical size to that previously reported for ZO-1 in epithelial tissues, as determined by Northern blots probed with a partial ZO-1 cDNA. Immunofluorescence microscopy demonstrates diverse ZO-1 distributions in these cells. In astrocytes, identified by the presence of glial fibrillary acidic protein, ZO-1 is localized at discrete sites of cell-cell contact as well as within the cell cytoplasm. In contrast, S-180 cells display diffuse staining at the cell periphery and within the cytoplasm. Dermal fibroblasts show no staining above background, although ZO-1 was detected on immunoblots of fibroblast cell extracts. Immunofluorescence staining of frozen sections of mouse brain demonstrates no detectable ZO-1 immunoreactivity outside blood vessels where endothelial cell tight junctions of the blood-brain barrier are located. These studies suggest that, although ZO-1 is found to be associated with the tight junction, it has a broader distribution than previously recognized.

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Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.3.c798 ◽

1998 ◽

Vol 275 (3) ◽

pp. C798-C809 ◽

Cited By ~ 110

Author(s):

Shobha Gopalakrishnan ◽

Narayan Raman ◽

Simon J. Atkinson ◽

James A. Marrs

Keyword(s):

Tight Junction ◽

Tight Junctions ◽

Rho Gtpase ◽

Mdck Cells ◽

Atp Depletion ◽

Cell Junctions ◽

Mutant Proteins ◽

Vitro Model ◽

The Relationship

Tight junctions control paracellular permeability and cell polarity. Rho GTPase regulates tight junction assembly, and ATP depletion of Madin-Darby canine kidney (MDCK) cells (an in vitro model of renal ischemia) disrupts tight junctions. The relationship between Rho GTPase signaling and ATP depletion was examined. Rho inhibition resulted in decreased localization of zonula occludens-1 (ZO-1) and occludin at cell junctions; conversely, constitutive Rho signaling caused an accumulation of ZO-1 and occludin at cell junctions. Inhibiting Rho before ATP depletion resulted in more extensive loss of junctional components between transfected cells than control junctions, whereas cells expressing activated Rho better maintained junctions during ATP depletion than control cells. ATP depletion and Rho signaling altered phosphorylation signaling mechanisms. ZO-1 and occludin exhibited rapid decreases in phosphoamino acid content following ATP depletion, which was restored on recovery. Expression of Rho mutant proteins in MDCK cells also altered levels of occludin serine/threonine phosphorylation, indicating that occludin is a target for Rho signaling. We conclude that Rho GTPase signaling induces posttranslational effects on tight junction components. Our data also demonstrate that activating Rho signaling protects tight junctions from damage during ATP depletion.

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Luminal nutrients alter tight-junction permeability in the rat jejunum: an in vivo perfusion model

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y93-125 ◽

1993 ◽

Vol 71 (10-11) ◽

pp. 835-839 ◽

Cited By ~ 46

Author(s):

D. C. Sadowski ◽

J. B. Meddings

Keyword(s):

Molecular Weight ◽

Tight Junction ◽

High Molecular Weight ◽

Tight Junctions ◽

Polyethylene Glycol 400 ◽

Ussing Chambers ◽

Perfusion Model ◽

Tight Junction Permeability

The regulation of tight-junction permeability between enterocytes has been studied using in vitro perfused loops, Ussing chambers, and cultured cell monolayers. In this communication we demonstrate the ability of an in vivo perfusion model to monitor tight-junction permeability and respond appropriately to physiological luminal stimuli. By using the highly charged anionic ferrocyanide molecule, water flux could be accurately assessed in the rat, and the luminal clearance of high molecular weight dextrans could be used to probe the opening and closing of the paracellular pathway. By utilizing two different molecular weight dextrans markers simultaneously, each conjugated with a different fluorophore, we were able to calculate luminal clearances of these compounds by fluorometric techniques in the presence of luminal nutrients that have previously been demonstrated to open intercellular tight junctions. In the absence of luminal nutrients or the presence of a non-nutrient sugar such as mannitol, clearance of these compounds was negligible. However, with the addition of either D-glucose or L-alanine, clearance of both high molecular weight markers increased dramatically. Thus, opening of tight junctions between enterocytes appears to be a physiological event that occurs in vivo under conditions likely to be found in the lumen. Polyethylene glycol 400 (PEG-400) clearance did not correlate well with the clearance of either dextran marker, suggesting that this probe utilizes a different permeation pathway and may not be appropriate to quantify the nutrient-regulatable pathway observed with the former probes.Key words: intestinal permeability, glucose transport, paracellular transport.

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Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2000.279.4.g757 ◽

2000 ◽

Vol 279 (4) ◽

pp. G757-G766 ◽

Cited By ~ 105

Author(s):

Kathleen G. Dickman ◽

Scott J. Hempson ◽

Joseph Anderson ◽

Scott Lippe ◽

Liming Zhao ◽

...

Keyword(s):

Tight Junction ◽

Tight Junctions ◽

Intestinal Cells ◽

Metabolic Dysfunction ◽

Rotavirus Infection ◽

Severe Diarrhea ◽

Consistent Feature ◽

And Function ◽

Junction Structure

Rotaviruses infect epithelial cells of the small intestine, but the pathophysiology of the resulting severe diarrhea is incompletely understood. Histological damage to intestinal epithelium is not a consistent feature, and in vitro studies showed that intestinal cells did not undergo rapid death and lysis during viral replication. We show that rotavirus infection of Caco-2 cells caused disruption of tight junctions and loss of transepithelial resistance (TER) in the absence of cell death. TER declined from 300 to 22 Ω · cm2between 8 and 24 h after infection and was accompanied by increased transepithelial permeability to macromolecules of 478 and 4,000 Da. Distribution of tight junction proteins claudin-1, occludin, and ZO-1 was significantly altered during infection. Claudin-1 redistribution was notably apparent at the onset of the decline in TER. Infection was associated with increased production of lactate, decreased mitochondrial oxygen consumption, and reduced cellular ATP (60% of control at 24 h after infection), conditions known to reduce the integrity of epithelial tight junctions. In conclusion, these data show that rotavirus infection of Caco-2 intestinal cells altered tight junction structure and function, which may be a response to metabolic dysfunction.

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Phase separation of zonula occludens proteins drives formation of tight junctions

10.1101/589580 ◽

2019 ◽

Cited By ~ 2

Author(s):

Oliver Beutel ◽

Riccardo Maraspini ◽

Karina Pombo-Garcia ◽

Cécilie Martin-Lemaitre ◽

Alf Honigmann

Keyword(s):

Phase Separation ◽

Tight Junction ◽

Tight Junctions ◽

Cell Biology ◽

Active Phase ◽

Molecular Assembly ◽

Scaffolding Proteins ◽

Molecular Binding ◽

Membrane Bound

AbstractTight junctions are cell adhesion complexes that seal tissues and are involved in cell polarity and signalling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands is dependent on the two membrane associated scaffolding proteins ZO1 and ZO2. To understand how ZO proteins organize junction assembly, we performed quantitative cell biology andin vitroreconstitution experiments. We discovered that ZO proteins self-organize membrane attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight junction proteins including adhesion receptors, cytoskeletal adapters and transcription factors. Our results suggest that an active phase transition of ZO proteins into a condensed membrane bound compartment drives claudin polymerization and coalescence of a continuous tight junction belt.

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Evidence that Tight Junctions Are Disrupted Due to Intimate Bacterial Contact and Not Inflammation during Attaching and Effacing Pathogen Infection In Vivo

Infection and Immunity ◽

10.1128/iai.00721-06 ◽

2006 ◽

Vol 74 (11) ◽

pp. 6075-6084 ◽

Cited By ~ 63

Author(s):

Julian A. Guttman ◽

Fereshte N. Samji ◽

Yuling Li ◽

A. Wayne Vogl ◽

B. Brett Finlay

Keyword(s):

Tight Junction ◽

Tight Junctions ◽

Pathogenic Bacteria ◽

Bacterial Attachment ◽

Cell Junctions ◽

In Vivo Experiments ◽

Tracer Molecules ◽

Tight Junction Disruption

ABSTRACT It is widely accepted that tight junctions are altered during infections by attaching and effacing (A/E) pathogens. These disruptions have been demonstrated both in vitro and more recently in vivo. For in vivo experiments, the murine model of A/E infection with Citrobacter rodentium is the animal model of choice. In addition to effects on tight junctions, these bacteria also colonize the colon at high levels, efface colonocyte microvilli, and cause hyperplasia and inflammation. Although we have recently demonstrated that tight junctions are disrupted by C. rodentium, the issue of direct effects of bacteria on epithelial cell junctions versus the indirect effects of inflammation still remains to be clarified. Here, we demonstrate that during the C. rodentium infections, inflammation plays no discernible role in the alteration of tight junctions. The distribution of the tight junction proteins, claudin-1, -3, and -5, are unaffected in inflamed colon, and junctions appear morphologically unaltered when viewed by electron microscopy. Additionally, tracer molecules are not capable of penetrating the inflamed colonic epithelium of infected mice that have cleared the bacteria. Finally, infected colonocytes from mice exposed to C. rodentium for 14 days, which have high levels of bacterial attachment to colonocytes as well as inflammation, have characteristic, altered claudin localization whereas cells adjacent to infected colonocytes retain their normal claudin distribution. We conclude that inflammation plays no discernible role in tight junction alteration during A/E pathogenesis and that tight junction disruption in vivo appears dependent only on the direct intimate attachment of the pathogenic bacteria to the cells.

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