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

1998 ◽  
Vol 275 (3) ◽  
pp. C798-C809 ◽  
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.

scholarly journals Cingulin unfolds ZO-1 and organizes myosin-2B and γ-actin to mechanoregulate apical and tight junction membranes

2020 ◽  
Author(s):  
Ekaterina Vasileva ◽  
Florian Rouaud ◽  
Domenica Spadaro ◽  
Wenmao Huang ◽  
Adai Colom ◽  
...  
Keyword(s):  
Tight Junction ◽  
Actin Filaments ◽  
Mdck Cells ◽  
Cell Junctions ◽  
Detectable Effect ◽  
Myosin Isoforms ◽  
Apical Surface ◽  
Dependent Manner ◽  
Membrane Tension

SUMMARYHow junctional proteins regulate the mechanics of the plasma membrane and how actin and myosin isoforms are selectively localized at epithelial cell-cell junctions is poorly understood. Here we show by atomic force indentation microscopy, immunofluorescence analysis and FLIM membrane tension imaging that the tight junction (TJ) protein cingulin maintains apical surface stiffness and TJ membrane tortuosity and down-regulates apico-lateral membrane tension in MDCK cells. KO of cingulin in MDCK, mCCD and Eph4 cells results in a decrease in the juxta-membrane accumulation of labeling for cytoplasmic myosin-2B (NM2B), γ-actin, phalloidin and ARHGEF18, but no detectable effect on myosin-2A (NM2A) and β-actin. Loss of paracingulin leads to weaker mechanical phenotypes in MDCK cells, correlating with no detectable effect on the junctional accumulation of myosins and actins. Cingulin and paracingulin form biomolecular condensates, bind to the ZU5 domain of ZO-1, and are recruited as clients into ZO-1 condensates in a ZU5-dependent manner. Cingulin binding to ZO-1 promotes the unfolding of ZO-1, as determined by interaction with DbpA in cells lacking ZO-2 and in vitro. Cingulin promotes the accumulation of a pool of ZO-1 at the TJ and is required in a ZU5-dependent manner for the recruitment of phalloidin-labelled actin filaments into ZO-1 condensates, suggesting that ZU5-cingulin interaction promotes ZO-1 interaction with actin filaments. Our results indicate that cingulin tethers the juxta-membrane and apical branched γ-actin-NM2B network to TJ to modulate ZO-1 conformation and the TJ assembly of a pool of ZO-1 and fine-tune the distribution of forces to apical and TJ membranes.

scholarly journals Differential regulation of junctional complex assembly in renal epithelial cell lines

2003 ◽  
Vol 285 (1) ◽  
pp. C102-C111 ◽  
Author(s):  
Shobha Gopalakrishnan ◽  
Mark A. Hallett ◽  
Simon J. Atkinson ◽  
James. A. Marrs
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Rho Gtpase ◽  
Mdck Cells ◽  
Adherens Junction ◽  
Stress Fiber ◽  
Cell Junctions ◽  
Junctional Complex ◽  
Complex Assembly ◽  
E Cadherin

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and β-catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner.

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

2006 ◽  
Vol 74 (11) ◽  
pp. 6075-6084 ◽  
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.

scholarly journals ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

1998 ◽  
Vol 141 (1) ◽  
pp. 199-208 ◽  
Author(s):  
Julie Haskins ◽  
Lijie Gu ◽  
Erika S. Wittchen ◽  
Jennifer Hibbard ◽  
Bruce R. Stevenson
Keyword(s):  
Amino Acid ◽  
Tight Junction ◽  
Molecular Mass ◽  
Mdck Cells ◽  
Cdna Libraries ◽  
Pdz Domains ◽  
Large Tumor ◽  
Junction Protein ◽  
Discs Large

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ∼130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.

Increased Tyr phosphorylation of ZO-1 during modification of tight junctions between glomerular foot processes

1995 ◽  
Vol 268 (3) ◽  
pp. F514-F524 ◽  
Author(s):  
H. Kurihara ◽  
J. M. Anderson ◽  
M. G. Farquhar
Keyword(s):  
Tight Junction ◽  
Tyrosine Phosphorylation ◽  
Tight Junctions ◽  
Mesangial Cells ◽  
Basal Membrane ◽  
Cell Junctions ◽  
Phosphorylated Proteins ◽  
Cell Matrix ◽  
Rapid Changes ◽  
And Control

The slit diaphragms between the glomerular epithelial foot processes represent a variant of the tight junction that are rapidly replaced by typical tight junctions after perfusion with protamine sulfate (PS). To investigate the mechanism of signaling involved, tyrosine phosphorylation of glomerular proteins was analyzed in newborn, PS-treated, and control rats using antiphosphotyrosine immunoglobulin G. In glomeruli of normal adults, phosphotyrosine (Ptyr) staining was confined largely to mesangial cells by immunofluorescence, whereas in newborn and PS-treated rats, the Ptyr signal was dramatically increased in the glomerular epithelium. By immunogold labeling, it was found that newly phosphorylated proteins were concentrated along the newly formed tight junctions (cell-cell junctions) and the basal membrane of the foot processes (cell-matrix junctions). By immunoblotting, several prominent bands were detected with anti-Ptyr in glomerular lysates of controls; in PS-treated rats, additional bands were detected at 225, 180, and 100 kDa. The 225-kDa protein was identified as ZO-1 by immunoprecipitation with anti-ZO-1 followed by immunoblotting with anti-Ptyr. These findings indicate that ZO-1 is one of the targets for tyrosine phosphorylation after PS treatment. They indicate that phosphorylation of tight junction and other proteins occurs during the formation of tight junctions in glomeruli under circumstances where there are rapid changes in epithelial cell shape.

scholarly journals Rab13 regulates PKA signaling during tight junction assembly

2004 ◽  
Vol 165 (2) ◽  
pp. 175-180 ◽  
Author(s):  
Katja Köhler ◽  
Daniel Louvard ◽  
Ahmed Zahraoui
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Inhibitory Effect ◽  
Cell Junctions ◽  
Unknown Mechanism ◽  
Epithelial Tight Junctions ◽  
Actin Remodelling ◽  
Cell Cell

The GTPase Rab13 regulates the assembly of functional epithelial tight junctions (TJs) through a yet unknown mechanism. Here, we show that expression of the GTP-bound form of Rab13 inhibits PKA-dependent phosphorylation and TJ recruitment of the vasodilator-stimulated phosphoprotein, an actin remodelling protein. We demonstrate that Rab13GTP directly binds to PKA and inhibits its activity. Interestingly, activation of PKA abrogates the inhibitory effect of Rab13 on the recruitment of vasodilator-stimulated phosphoprotein, ZO-1, and claudin1 to cell–cell junctions. Rab13 is, therefore, the first GTPase that controls PKA activity and provides an unexpected link between PKA signaling and the dynamics of TJ assembly.

scholarly journals Direct Current Stimulation Disrupts Endothelial Glycocalyx and Tight Junctions of the Blood-Brain Barrier in vitro

2021 ◽  
Vol 9 ◽  
Author(s):  
Yifan Xia ◽  
Yunfei Li ◽  
Wasem Khalid ◽  
Marom Bikson ◽  
Bingmei M. Fu
Keyword(s):  
Endothelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Blood Brain Barrier ◽  
Direct Current ◽  
Endothelial Glycocalyx ◽  
Microvascular Endothelial Cells ◽  
Bbb Permeability

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.

Regulation of human epithelial tight junction proteins by Lactobacillus plantarum in vivo and protective effects on the epithelial barrier

2010 ◽  
Vol 298 (6) ◽  
pp. G851-G859 ◽  
Author(s):  
Jurgen Karczewski ◽  
Freddy J. Troost ◽  
Irene Konings ◽  
Jan Dekker ◽  
Michiel Kleerebezem ◽  
...  
Keyword(s):  
Tight Junction ◽  
Lactobacillus Plantarum ◽  
Transmembrane Protein ◽  
Intestinal Barrier ◽  
Protective Effects ◽  
Epithelial Tight Junction ◽  
Vitro Model ◽  
Probiotic Mechanisms

Lactobacillus plantarum , a commensal bacterium of humans, has been proposed to enhance the intestinal barrier, which is compromised in a number of intestinal disorders. To study the effect of L. plantarum strain WCFS1 on human barrier function, healthy subjects were administered L. plantarum or placebo in the duodenum for 6 h by means of a feeding catheter. The scaffold protein zonula occludens (ZO)-1 and transmembrane protein occludin were found to be significantly increased in the vicinity of the tight-junction (TJ) structures, which form the paracellular seal between cells of the epithelium. In an in vitro model of the human epithelium, L. plantarum induced translocation of ZO-1 to the TJ region; however, the effects on occludin were minor compared with those seen in vivo. L. plantarum was shown to activate Toll-like receptor 2 (TLR2) signaling, and treatment of Caco-2 monolayers with the TLR2 agonist Pam3-Cys-SK4(PCSK) significantly increased fluorescent staining of occludin in the TJ. Pretreatment of Caco-2 monolayers with L. plantarum or PCSK significantly attenuated the effects of phorbol ester-induced dislocation of ZO-1 and occludin and the associated increase in epithelial permeability. Our results identifying commensal bacterial stimulation of TLR2 in the gut epithelium as a regulator of epithelial integrity have important implications for understanding probiotic mechanisms and the control of intestinal homeostasis.

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