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 A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells

1994 ◽  
Vol 124 (1) ◽  
pp. 101-115 ◽  
Author(s):  
A Zahraoui ◽  
G Joberty ◽  
M Arpin ◽  
JJ Fontaine ◽  
R Hellio ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Small Gtpase ◽  
Junctional Complex ◽  
Rab Gtpase ◽  
Cytoplasmic Vesicles ◽  
Microscopy Analysis ◽  
Polarized Epithelial Cells ◽  
Kidney Liver

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.

GENETIC SUSCEPTIBILITY TO IBD OPERATES VIA CONTROL OF APICAL JUNCTIONAL COMPLEXES

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S30-S30
Author(s):  
Isabelle Hébert-Milette ◽  
Chloé Lévesque ◽  
Guy Charron ◽  
John Rioux
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Intestinal Inflammation ◽  
Protein Localization ◽  
Junctional Complex ◽  
Epithelial Permeability ◽  
3D Cultures ◽  
Apical Junctional Complex ◽  
Junction Protein ◽  
The Impact

Abstract Introduction Intestinal permeability is increased in unaffected 1st degree relatives of patients with inflammatory bowel disease (IBD), and is considered a risk factor for the development of IBD, likely increasing the interactions between intestinal microorganisms and the immune system. We recently reported that C1orf106, a gene located within a genomic region associated with IBD, regulates epithelial permeability. We further demonstrated that a rare coding variant within C1orf106 (p.Y333F) decreases protein stability and that lower levels of C1orf106 protein leads altered stability of adherens junctions (AJ) and to an increase in epithelial permeability. Hypothesis In addition to altering AJ, we believe that C1orf106 is also involved in the regulation of tight junction (TJ) formation, which also impacts epithelial permeability. Objectives The objectives of the project are to (a) validate the impact of C1orf106 on tight junctions and (b) verify the impact of C1orf106 IBD-associated variants on intestinal barrier integrity. Results We observed that knocking down the expression of C1orf106 in Caco-2 cells leads to a number of phenotypes in human epithelial monolayer (2D) and spheroid (3D) cultures that are associated with alterations in TJs. Specifically, when studying the dynamic reformation of TJ in 2D cultures after transient withdrawal of calcium, which is required for TJ stability, we observed that lower levels of C1orf106 resulted in (1) decreased recovery of barrier function as measured by transepithelial electrical resistance (TEER); (2) an alteration of tight junction protein localization; and (3) thickening of the circumferential actin belt. Moreover, in 3D cultures, we observed an altered spheroid formation associated with impaired epithelial polarization. In addition, our preliminary studies of human induced pluripotent stem cell (hiPSC)-derived epithelial cultures support that Y333F heterozygotes also have altered structure and function of their tight junctions. Conclusion Our observations indicate an important role of C1orf106 in apical junctional complex (AJC) formation likely mediated by a regulation of the circumferential actin belt. This can affect other functions of AJC, like the establishment of cell polarity. AJC formation is important for epithelial repair after an injury and its dysregulation impairs the formation of an impermeable epithelial barrier, which likely facilitates the passage of microorganisms and the induction and maintenance of intestinal inflammation.

scholarly journals (Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit.

1989 ◽  
Vol 109 (3) ◽  
pp. 1057-1069 ◽  
Author(s):  
A Marxer ◽  
B Stieger ◽  
A Quaroni ◽  
M Kashgarian ◽  
H P Hauri
Keyword(s):  
Monoclonal Antibody ◽  
Small Intestine ◽  
Epithelial Cells ◽  
Brush Border ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Distal Colon ◽  
Proximal Colon ◽  
Beta Subunit ◽  
Cell Pole

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells (Quaroni, A., and K. J. Isselbacher. 1981. J. Natl. Cancer Inst. 67:1353-1362) was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit (Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush. 1985. Kidney Int. 28:899-913), was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.

scholarly journals Putative linear motifs mediate the trafficking to apical and basolateral membranes

2020 ◽  
Author(s):  
Laszlo Dobson ◽  
András Zeke ◽  
Levente Szekeres ◽  
Tamás Langó ◽  
Gábor Tusnády
Keyword(s):  
Epithelial Cells ◽  
Drug Transport ◽  
Basolateral Membrane ◽  
Transmembrane Proteins ◽  
Transport Processes ◽  
Membrane Domains ◽  
Protein Distribution ◽  
Basolateral Membranes ◽  
Linear Motifs ◽  
Cellular Components

AbstractCell polarity refers to the asymmetric organisation of cellular components in various cells. Epithelial cells are the best known examples of polarized cells, featuring apical and basolateral membrane domains. Despite huge efforts, the exact rules governing the protein distribution in such domains are still elusive. In this study we examined linear motifs accumulating in these parts and based on the results we prepared ‘Classical’ and Convolutional Neural Networks to classify human transmembrane proteins localizing into apical/basolateral membranes. Asymmetric expression of drug transporters results in vectorial drug transport, governing the pharmacokinetics of numerous substances, yet the data on how proteins are sorted in epithelial cells is very scattered. The provided dataset may offer help to experimentalists to characterize novel molecular targets to regulate transport processes more precisely.

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.

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.

Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells

1991 ◽  
Vol 260 (4) ◽  
pp. C691-C700 ◽  
Author(s):  
J. Bruno de Almeida ◽  
Jennifer L. Stow
Keyword(s):  
Epithelial Cells ◽  
Basement Membrane ◽  
Membrane Protein ◽  
Heparan Sulfate ◽  
Basolateral Membrane ◽  
Immunofluorescent Staining ◽  
Secretory Proteins ◽  
Microtubule Depolymerization ◽  
Microtubule Network ◽  
Microtubule Disruption

Basement membrane proteins such as the heparan sulfate proteoglycan (HSPG) are secreted in a polarized fashion from the basolateral membrane of epithelial cells. We have used the microtubule-disrupting drug colchicine to study the role of the microtubule network in directing constitutive secretion to the basolateral membrane of LLC-PK1 renal epithelial cells. Microtubule depolymerization induced by colchicine resulted in fragmentation and redistribution of fluorescently labeled trans-Golgi membranes. Increased immunofluorescent staining of HSPG was associated with these dispersed Golgi cisternae. The biosynthetic processing of HSPG was not significantly altered by the loss of microtubules or by the dispersal of the Golgi elements. The most striking effect of microtubule disruption was the loss of polarity of HSPG secretion. Immunoprecipitation studies showed that HSPG was secreted from both apical and basolateral surfaces of LLC-PK1 cells treated with colchicine, and a similar result was found for the delivery of laminin, another basement membrane protein. In contrast, there was no change in the distribution of an integral basolateral membrane protein, Na+-K+-ATPase, following colchicine treatment. Our results provide the first demonstration that microtubules are involved in the constitutive trafficking of basolateral secretory proteins. These data also suggest that there may be an inherent difference in the targeting or delivery of membrane and secretory proteins to the basolateral cell surface. polarized secretion; heparan sulfate proteoglycans; sorting; Golgi processing Submitted on July 10, 1990 Accepted on November 12, 1990

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 Connexin-Occludin Chimeras Containing the Zo-Binding Domain of Occludin Localize at Mdck Tight Junctions and Nrk Cell Contacts

1999 ◽  
Vol 146 (3) ◽  
pp. 683-693 ◽  
Author(s):  
Laura L. Mitic ◽  
Eveline E. Schneeberger ◽  
Alan S. Fanning ◽  
James Melvin Anderson
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Rat Kidney ◽  
Freeze Fracture ◽  
Binding Domain ◽  
Permeability Barrier ◽  
Cell Contacts ◽  
Cytoplasmic Proteins

Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, we created chimeras with the transmembrane portions of connexin 32. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localized within fibrils when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1–containing cell–cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again required only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family.

Structure, biochemistry, and assembly of epithelial tight junctions

1987 ◽  
Vol 253 (6) ◽  
pp. C749-C758 ◽  
Author(s):  
B. Gumbiner
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Lateral Diffusion ◽  
Junctional Complex ◽  
Epithelial Cell Layer ◽  
Dependent Cell ◽  
Freeze Fracture Electron Microscopy ◽  
Dynamic Structures

The zonula occludens (ZO), also referred to as the tight junction, forms the barrier to the diffusion of molecules and ions across the epithelial cell layer through the paracellular space. The level of electrical resistance of the paracellular pathway seems to depend on the number of strands in the ZO observed by freeze-fracture electron microscopy (EM). The ZO also forms the boundary between the compositionally distinct apical and basolateral plasma membrane domains because it is a barrier to the lateral diffusion of lipids and membrane proteins that reside in the extracytoplasmic leaflet of the membrane bilayer. In contrast to its appearance in transmission EM, the tight junction is not a fusion between the outer membrane leaflets of neighboring cells. Rather it consists of protein molecules, including the newly discovered protein ZO-1 and probably others, which bring the plasma membranes into extremely close apposition so as to occlude the extracellular space. Very little is known about the assembly of tight junctions, but several kinds of evidence suggest that they are very dynamic structures. Other elements of the epithelial junctional complex including the zonula adherens (ZA), the Ca2+-dependent cell adhesion molecule uvomorulin, or L-CAM, and actin filaments of the cytoskeleton may participate in the assembly of the ZO.

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