scholarly journals Loss of Occludin Affects Tricellular Localization of Tricellulin

2008 ◽  
Vol 19 (11) ◽  
pp. 4687-4693 ◽  
Author(s):  
Junichi Ikenouchi ◽  
Hiroyuki Sasaki ◽  
Sachiko Tsukita ◽  
Mikio Furuse ◽  
Shoichiro Tsukita
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Tight Junctions ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Polarized Epithelia ◽  
Cross Links ◽  
Exogenous Expression ◽  
Domain Protein ◽  
Zona Occludens

The tricellular tight junction (tTJ) forms at the convergence of bicellular tight junctions (bTJs) where three epithelial cells meet in polarized epithelia, and it is required for the maintenance of the transepithelial barrier. Tricellulin is a four transmembrane domain protein recently identified as the first marker of tTJ, but little is known about how tricellulin is localized at tTJs. As for the molecular mechanism of association of tricellulin with tight junctions (TJs), we found that tricellulin was incorporated into claudin-based TJs independently of binding to zona occludens-1. Unexpectedly, exogenous expression of tricellulin increased cross-links of TJ strands in the plasma membrane. As for the molecular mechanisms for localization of tricellulin at tricellular junctions, we found that knockdown of occludin caused mislocalization of tricellulin to bTJs, implying that occludin supports tricellular localization of tricellulin by excluding tricellulin from bTJs.

Effect of temperature on the assembly of tight junctions and on the mobility of lipids in membranes of HT29 cells

1990 ◽  
Vol 97 (1) ◽  
pp. 119-125
Author(s):  
E. Cohen ◽  
I. Ophir ◽  
Y.I. Henis ◽  
A. Bacher ◽  
Y. Ben Shaul
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Temperature Dependence ◽  
Tight Junctions ◽  
Membrane Lipids ◽  
Effect Of Temperature ◽  
Proteolytic Activation ◽  
Tight Junction Formation ◽  
Threshold Temperatures ◽  
Junction Formation

In the human colon adenocarcinoma cell line HT29, tight junctions can be induced by treatment with appropriate proteases or salt solutions. The temperature dependence of induced tight junction formation is characterized by a marked sigmoidal behavior. The different methods of induction used in this study were characterized by threshold temperatures ranging from 15 to 32 degrees C. Fluorescence photobleaching recovery measurements of the lateral diffusion of a fluorescent phospholipid probe in the cellular plasma membrane gave no evidence for a phase transition or for alteration in the organization of membrane lipids in lateral domains in the temperature range between 0 and 37 degrees C. Moreover, dynamic parameters of the probe in the plasma membrane did not change substantially on mild treatment with trypsin. Thus, the temperature dependence of tight junction formation is not dictated by the bulk properties of the cytoplasmic membrane lipids. The observed temperature dependence suggests that the assembly of tight junctions is a cooperative process, which may involve conformational rearrangement in a protein precursor subsequent to its proteolytic activation.

scholarly journals Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

2011 ◽  
Vol 437 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Suneet Jain ◽  
Takuya Suzuki ◽  
Ankur Seth ◽  
Geetha Samak ◽  
Radhakrishna Rao
Keyword(s):  
Protein Kinase ◽  
Tight Junction ◽  
Tight Junctions ◽  
Intercellular Junctions ◽  
Cell Monolayers ◽  
Terminal Domain ◽  
Tight Junction Regulation ◽  
Epithelial Tight Junctions ◽  
Zona Occludens

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin–Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.

Altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis

2012 ◽  
Vol 302 (2) ◽  
pp. L193-L205 ◽  
Author(s):  
Hiromitsu Ohta ◽  
Shigeki Chiba ◽  
Masahito Ebina ◽  
Mikio Furuse ◽  
Toshihiro Nukiwa
Keyword(s):  
Epithelial Cells ◽  
Pulmonary Fibrosis ◽  
Lung Injury ◽  
Tight Junction ◽  
Tight Junctions ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Alveolar Epithelial Cells ◽  
Altered Expression ◽  
Alveolar Epithelial

The dysfunction of alveolar barriers is a critical factor in the development of lung injury and subsequent fibrosis, but the underlying molecular mechanisms remain poorly understood. To clarify the pathogenic roles of tight junctions in lung injury and fibrosis, we examined the altered expression of claudins, the major components of tight junctions, in the lungs of disease models with pulmonary fibrosis. Among the 24 known claudins, claudin-1, claudin-3, claudin-4, claudin-7, and claudin-10 were identified as components of airway tight junctions. Claudin-5 and claudin-18 were identified as components of alveolar tight junctions and were expressed in endothelial and alveolar epithelial cells, respectively. In experimental bleomycin-induced lung injury, the levels of mRNA encoding tight junction proteins were reduced, particularly those of claudin-18. The integrity of the epithelial tight junctions was disturbed in the fibrotic lesions 14 days after the intraperitoneal instillation of bleomycin. These results suggest that bleomycin mainly injured alveolar epithelial cells and impaired alveolar barrier function. In addition, we analyzed the influence of transforming growth factor-β (TGF-β), a critical mediator of pulmonary fibrosis that is upregulated after bleomycin-induced lung injury, on tight junctions in vitro. The addition of TGF-β decreased the expression of claudin-5 in human umbilical vein endothelial cells and disrupted the tight junctions of epithelial cells (A549). These results suggest that bleomycin-induced lung injury causes pathogenic alterations in tight junctions and that such alterations seem to be induced by TGF-β.

scholarly journals Accumulation of milk increases the width of tight junctions in the epithelium of mouse mammary alveoli

2020 ◽  
Vol 65 (3) ◽  
Author(s):  
Natalia Kruglova ◽  
Irina Razgovorova ◽  
Salah Amasheh ◽  
Alexander Markov
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Molecular Mechanisms ◽  
Ultrastructural Level ◽  
Control Group ◽  
Apical Region ◽  
Mechanical Pressure ◽  
Secretory Epithelium

The study of the molecular mechanisms of maintaining the integrity of the epithelium during mechanical stress remains a relevant problem in the physiology of tissue barriers. A methodical approach has been applied which makes it possible to reproduce mechanical pressure on the apical region of cells in vivo and to study the participation and role of tight junctions in maintaining the integrity of the epithelial structure. Mammary gland tissue specimens from lactating control mice and animals after a 20-h interruption of suckling were prepared and the width of the tight junction of the secretory epithelium was analyzed. At the ultrastructural level, it was shown that accumulation of milk caused a significant increase in the width of the tight junction between epithelial cells. In the control group, the width of this structure was 2.1 ± 0.1 µm; in the experimental group — 4.2 ± 0.1 μm. The marked increase in the width of tight junctions between epithelial cells is in accordance with an observed increase in the level of claudin-1 and -3 in the secretory epithelium and can be interpreted as adaptive changes aimed at maintaining the structure of the alveoli.

scholarly journals Distinct claudins and associated PDZ proteins form different autotypic tight junctions in myelinating Schwann cells

2002 ◽  
Vol 159 (2) ◽  
pp. 361-372 ◽  
Author(s):  
Sebastian Poliak ◽  
Sean Matlis ◽  
Christoph Ullmer ◽  
Steven S. Scherer ◽  
Elior Peles
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Schwann Cells ◽  
Pdz Domain ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Guanylate Kinase ◽  
Pdz Proteins ◽  
Junction Protein ◽  
Zona Occludens

The apposed membranes of myelinating Schwann cells are joined by several types of junctional specializations known as autotypic or reflexive junctions. These include tight, gap, and adherens junctions, all of which are found in regions of noncompact myelin: the paranodal loops, incisures of Schmidt-Lanterman, and mesaxons. The molecular components of autotypic tight junctions have not been established. Here we report that two homologues of Discs Lost–multi PDZ domain protein (MUPP)1, and Pals-associated tight junction protein (PATJ), are differentially localized in myelinating Schwann cells and associated with different claudins. PATJ is mainly found at the paranodal loops, where it colocalized with claudin-1. MUPP1 and claudin-5 colocalized in the incisures, and the COOH-terminal region of claudin-5 interacts with MUPP1 in a PSD-95/Disc Large/zona occludens (ZO)-1 (PDZ)-dependent manner. In developing nerves, claudin-5 and MUPP1 appear together in incisures during the first postnatal week, suggesting that they coassemble during myelination. Finally, we show that the incisures also contain four other PDZ proteins that are found in epithelial tight junctions, including three membrane-associated guanylate-kinase proteins (membrane-associated guanylate-kinase inverted-2, ZO-1, and ZO-2) and the adaptor protein Par-3. The presence of these different tight junction proteins in regions of noncompact myelin may be required to maintain the intricate cytoarchitecture of myelinating Schwann cells.

scholarly journals AMPK Activation Promotes Tight Junction Assembly in Intestinal Epithelial Caco-2 Cells

2019 ◽  
Vol 20 (20) ◽  
pp. 5171 ◽  
Author(s):  
Séverine Olivier ◽  
Jocelyne Leclerc ◽  
Adrien Grenier ◽  
Marc Foretz ◽  
Jérôme Tamburini ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Tight Junctions ◽  
Paracellular Permeability ◽  
Cell Junctions ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Ampk Activation ◽  
Energy Status ◽  
The Impact

The AMP-activated protein kinase (AMPK) is principally known as a major regulator of cellular energy status, but it has been recently shown to play a key structural role in cell-cell junctions. The aim of this study was to evaluate the impact of AMPK activation on the reassembly of tight junctions in intestinal epithelial Caco-2 cells. We generated Caco-2 cells invalidated for AMPK α1/α2 (AMPK dKO) by CRISPR/Cas9 technology and evaluated the effect of the direct AMPK activator 991 on the reassembly of tight junctions following a calcium switch assay. We analyzed the integrity of the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), the paracellular permeability, and quantification of zonula occludens 1 (ZO-1) deposit at plasma membrane by immunofluorescence. Here, we demonstrated that AMPK deletion induced a delay in tight junction reassembly and relocalization at the plasma membrane during calcium switch, leading to impairments in the establishment of TEER and paracellular permeability. We also showed that 991-induced AMPK activation accelerated the reassembly and reorganization of tight junctions, improved the development of TEER and paracellular permeability after calcium switch. Thus, our results show that AMPK activation ensures a better recovery of epithelial barrier function following injury.

VAP-33 localizes to both an intracellular vesicle population and with occludin at the tight junction

1999 ◽  
Vol 112 (21) ◽  
pp. 3723-3732 ◽  
Author(s):  
L.A. Lapierre ◽  
P.L. Tuma ◽  
J. Navarre ◽  
J.R. Goldenring ◽  
J.M. Anderson
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Tight Junctions ◽  
Growth Regulation ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Subcellular Fractionation ◽  
Tissue Culture Cell ◽  
Cellular Functions ◽  
Vesicle Docking

Tight junctions create a regulated intercellular seal between epithelial and endothelial cells and also establish polarity between plasma membrane domains within the cell. Tight junctions have also been implicated in many other cellular functions, including cell signaling and growth regulation, but they have yet to be directly implicated in vesicle movement. Occludin is a transmembrane protein located at tight junctions and is known to interact with other tight junction proteins, including ZO-1. To investigate occludin's role in other cellular functions we performed a yeast two-hybrid screen using the cytoplasmic C terminus of occludin and a human liver cDNA library. From this screen we identified VAP-33 which was initially cloned from Aplysia by its ability to interact with VAMP/synaptobrevin and thus was implicated in vesicle docking/fusion. Extraction characteristics indicated that VAP-33 was an integral membrane protein. Antibodies to the human VAP-33 co-localized with occludin at the tight junction in many tissues and tissue culture cell lines. Subcellular fractionation of liver demonstrated that 83% of VAP-33 co-isolated with occludin and DPPIV in a plasma membrane fraction and 14% fractionated in a vesicular pool. Thus, both immunofluorescence and fractionation data suggest that VAP-33 is present in two distinct pools in the cells. In further support of this conclusion, a GFP-VAP-33 chimera also distributed to two sites within MDCK cells and interestingly shifted occludin's localization basally. Since VAP-33 has previously been implicated in vesicle docking/fusion, our results suggest that tight junctions may participate in vesicle targeting at the plasma membrane or alternatively VAP-33 may regulate the localization of occludin.

scholarly journals Contribution of the Cytoplasmic Determinants of Vpu to the Expansion of Virus-Containing Compartments in HIV-1-Infected Macrophages

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Olivier Leymarie ◽  
Leslie Lepont ◽  
Margaux Versapuech ◽  
Delphine Judith ◽  
Sophie Abelanet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Viral Particle ◽  
Transmembrane Domain ◽  
Immune Surveillance ◽  
Restriction Factor ◽  
Viral Particles ◽  
Protein Functions ◽  
Specific Accumulation ◽  
Model Cell ◽  
Hiv 1

ABSTRACTHIV-1 infection of macrophages leads to the sequestration of newly formed viruses in intracellular plasma membrane-connected structures termed virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The cellular restriction factor bone marrow stromal cell antigen 2 (BST2), which prevents HIV-1 dissemination by tethering budding viral particles at the plasma membrane, can be found in VCCs. The HIV-1 accessory protein Vpu counteracts the restriction factor BST2 by downregulating its expression and removing it from viral budding sites. Numerous studies described these Vpu countermeasures in CD4+T cells or model cell lines, but the interplay between Vpu and BST2 in VCC formation and HIV-1 production in macrophages is less explored. Here, we show that Vpu expression in HIV-1-infected macrophages enhances viral release. This effect is related to Vpu’s ability to circumvent BST2 antiviral activity. We show that in absence of Vpu, BST2 is enriched in VCCs and colocalizes with capsid p24, whereas Vpu expression significantly reduces the presence of BST2 in these compartments. Furthermore, our data reveal that BST2 is dispensable for the formation of VCCs and that Vpu expression impacts the volume of these compartments. This Vpu activity partly depends on BST2 expression and requires the integrity of the Vpu transmembrane domain, the dileucine-like motif E59XXXLV64and phosphoserines 52 and 56 of Vpu. Altogether, these results highlight that Vpu controls the volume of VCCs and promotes HIV-1 release from infected macrophages.IMPORTANCEHIV-1 infection of macrophages leads to the sequestration of newly formed viruses in virus-containing compartments (VCCs), where virions remain infectious and hidden from immune surveillance. The restriction factor BST2, which prevents HIV-1 dissemination by tethering budding viral particles, can be found in VCCs. The HIV-1 Vpu protein counteracts BST2. This study explores the interplay between Vpu and BST2 in the viral protein functions on HIV-1 release and viral particle sequestration in VCCs in macrophages. The results show that Vpu controls the volume of VCCs and favors viral particle release. These Vpu functions partly depend on Vpu’s ability to antagonize BST2. This study highlights that the transmembrane domain of Vpu and two motifs of the Vpu cytoplasmic domain are required for these functions. These motifs were notably involved in the control of the volume of VCCs by Vpu but were dispensable for the prevention of the specific accumulation of BST2 in these structures.

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