scholarly journals Occludin 1B, a Variant of the Tight Junction Protein Occludin

2000 ◽  
Vol 11 (2) ◽  
pp. 627-634 ◽  
Author(s):  
Zoia Muresan ◽  
David L. Paul ◽  
Daniel A. Goodenough
Keyword(s):  
Tight Junction ◽  
Base Pair ◽  
Mdck Cells ◽  
Human Colon ◽  
Reverse Transcription Pcr ◽  
Mouse Tissues ◽  
Junction Protein ◽  
Alternatively Spliced ◽  
Membrane Components ◽  
And Function

Occludin and claudin are the major integral membrane components of the mammalian tight junction. Although more than 11 distinct claudins have been identified, only 1 occludin transcript has been reported thus far. Therefore, we searched by reverse transcription–PCR for occludin-related sequences in Madin–Darby canine kidney (MDCK) mRNA and identified a transcript encoding an alternatively spliced form of occludin, designated occludin 1B. The occludin 1B transcript contained a 193-base pair insertion encoding a longer form of occludin with a unique N-terminal sequence of 56 amino acids. Analysis of the MDCK occludin gene revealed an exon containing the 193-base pair sequence between the exons encoding the original N terminus and the distal sequence, suggesting that occludin and occludin 1B arise from alternative splicing of one transcript. To assess the expression and distribution of occludin 1B, an antibody was raised against its unique N-terminal domain. Immunolabeling of occludin 1B in MDCK cells revealed a distribution indistinguishable from that of occludin. Furthermore, occludin 1B staining at cell-to-cell contacts was also found in cultured T84 human colon carcinoma cells and in frozen sections of mouse intestine. Immunoblots of various mouse tissues revealed broad coexpression of occludin 1B with occludin. The wide epithelial distribution and the conservation across species suggests a potentially important role for occludin 1B in the structure and function of the tight junction.

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.

scholarly journals Correlation of occludin protein mobility with paracellular leak pathway permeability in renal epithelia

2018 ◽  
Author(s):  
Josephine Axis ◽  
Alexander L. Kolb ◽  
Robert L. Bacallao ◽  
Kurt Amsler
Keyword(s):  
Tight Junction ◽  
Ischemia Reperfusion Injury ◽  
Mdck Cells ◽  
Ischemia Reperfusion ◽  
Paracellular Permeability ◽  
Tight Junction Protein ◽  
Protein Mobility ◽  
Junction Protein ◽  
Leak Pathway

ABSTRACTStudies have demonstrated regulation of the epithelial paracellular permeability barrier, the tight junction, by a variety of stimuli. Recent studies have reported a correlation between changes in paracellular permeability, particularly paracellular permeability to large solutes (leak pathway), and mobility of the tight junction protein, occludin, in the plane of the plasma membrane. This had led to the hypothesis that changes in occludin protein mobility are causative for changes in paracellular permeability. Using a renal epithelial cell model system, MDCK, we examined the effect of various manipulations on both leak pathway permeability, monitored as the paracellular movement of a fluorescent molecule (calcein), and occludin protein mobility, monitored through fluorescence recovery after photobleaching. Our results indicate that knockdown of the associated tight junction protein, ZO-1, increases baseline leak pathway permeability, whereas, knockdown of the related tight junction protein, ZO-2, does not alter baseline leak pathway permeability. Knockdown of either ZO-1 or ZO-2 decreases the rate of movement of occludin protein but only knockdown of ZO-2 protein alters the percent of occludin protein that is mobile. Further, treatment with hydrogen peroxide increases leak pathway permeability in wild type MDCK cells and in ZO-2 knockdown MDCK cells but not in ZO-1 knockdown MDCK cells. This treatment decreases the rate of occludin movement in all three cell lines but only alters the mobile fraction of occludin protein in ZO-1 knockdown MDCK cells. Finally, we examined the effect of renal ischemia/reperfusion injury on occludin protein mobility in vivo.Ischemia/reperfusion injury both increased the rate of occludin mobility and increased the fraction of occludin protein that is mobile. These results indicate that, at least in our cell culture and in vivo model systems, there is no consistent correlation between paracellular leak pathway permeability and occludin protein mobility.

Occludin TM4-: an isoform of the tight junction protein present in primates lacking the fourth transmembrane domain

2002 ◽  
Vol 115 (15) ◽  
pp. 3171-3180
Author(s):  
M. Reza Ghassemifar ◽  
Bhavwanti Sheth ◽  
Tom Papenbrock ◽  
Henry J. Leese ◽  
Franchesca D. Houghton ◽  
...  
Keyword(s):  
Tight Junction ◽  
Transmembrane Domain ◽  
Tight Junction Protein ◽  
Pcr Analysis ◽  
Weak Band ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
C Terminus ◽  
Junction Protein ◽  
Exon 4

The tight junction protein occludin possesses four transmembrane domains,two extracellular loops, and cytoplasmic N- and C-termini. Reverse transcription-PCR analysis of human tissues, embryos and cells using primers spanning the fourth transmembrane domain (TM4) and adjacent C-terminal region revealed two products. The larger and predominant product corresponded in sequence to canonical occludin (TM4+), while the smaller product exhibited a 162 bp deletion encoding the entire TM4 and immediate C-terminal flanking region (TM4-). Examination of the genomic occludin sequence identified that the 162 bp sequence deleted in TM4-coincided precisely with occludin exon 4, strongly suggesting that TM4- is an alternative splice isoform generated by skipping of exon 4. Indeed, the reading frame of downstream exons is not affected by exclusion of exon 4. The presence of both TM4+ and TM4- occludin isoforms was also identified in monkey epithelial cells but TM4-was undetected in murine and canine tissue and cells, indicating a late evolutionary origin for this alternative splicing event. Conceptual translation of TM4- isoform predicts extracellular localisation of the C-terminus. Immunocytochemical processing of living human Caco-2 cells using a C-terminal occludin antibody revealed weak, discontinuous staining restricted to the periphery of subconfluent islands of cells, or islands generated by wounding confluent layers. In occludin immunoblots, a weak band at ∼58 kDa, smaller than the predominant band at 65 kDa and corresponding to the predicted mass of TM4- isoform, is evident and upregulated in subconfluent cells. These data suggest that the TM4- isoform may be translated at low levels in specific conditions and may contribute to regulation of occludin function.

Phosphorylation of occludin correlates with occludin localization and function at the tight junction

1997 ◽  
Vol 273 (6) ◽  
pp. C1859-C1867 ◽  
Author(s):  
Vivian Wong
Keyword(s):  
Molecular Weight ◽  
Tight Junction ◽  
Tight Junctions ◽  
Growth Medium ◽  
Mdck Cells ◽  
Low Calcium ◽  
Phosphatase Treatment ◽  
And Function ◽  
Triton X ◽  
Mdck I

Multiple forms of occludin were found in Madin-Darby canine kidney (MDCK) cells. In the absence of cell-to-cell contacts, achieved by incubating cells in low-calcium growth medium, a cluster of lower-molecular-weight (LMW) occludin bands (∼65,000–68,000) was present in both MDCK I and II cells. On formation of tight junctions, achieved by changing the low-calcium growth medium to normal-calcium growth medium, a cluster of higher-molecular-weight (HMW) bands (∼72,000–75,000 for MDCK I cells and ∼70,000–73,000 for MDCK II cells) was also expressed. The HMW occludin bands could be eliminated by phosphatase treatment. Therefore, the HMW forms of occludin appeared to be the hyperphosphorylated product of the LMW forms. These HMW forms were Triton X-100 insoluble, which correlated with their localization at the tight junctions. Furthermore, depletion of tight junction-localized occludin by an occludin extracellular domian peptide (20) correlated with a decrease in the HMW forms of occludin. In conclusion, phosphorylation of occludin may be a mechanism by which occludin localization and function are regulated.

Molecular Characterization of the Tight Junction Protein ZO-1 in MDCK Cells

1999 ◽  
Vol 248 (1) ◽  
pp. 97-109 ◽  
Author(s):  
L. González-Mariscal ◽  
S. Islas ◽  
R.G. Contreras ◽  
M.R. Garcı́a-Villegas ◽  
A. Betanzos ◽  
...  
Keyword(s):  
Tight Junction ◽  
Molecular Characterization ◽  
Mdck Cells ◽  
Tight Junction Protein ◽  
Junction Protein

scholarly journals Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells.

1988 ◽  
Vol 106 (4) ◽  
pp. 1141-1149 ◽  
Author(s):  
J M Anderson ◽  
B R Stevenson ◽  
L A Jesaitis ◽  
D A Goodenough ◽  
M S Mooseker
Keyword(s):  
Tight Junction ◽  
Mdck Cell ◽  
Mouse Liver ◽  
Mdck Cells ◽  
Cell Protein ◽  
Scatchard Analysis ◽  
Madin Darby Canine Kidney ◽  
Mouse Tissues ◽  
Darby Canine Kidney ◽  
Canine Kidney

ZO-1, originally identified by mAb techniques, is the first protein shown to be specifically associated with the tight junction. Here we describe and compare the physical characteristics of ZO-1 from mouse liver and the Madin-Darby canine kidney (MDCK) epithelial cell line. The ZO-1 polypeptide has an apparent size of 225 kD in mouse tissues and 210 kD in canine-derived MDCK cells as determined by SDS-PAGE/immunoblot analysis. ZO-1 from both sources is optimally solubilized from isolated plasma membranes by either 6 M urea or high pH conditions; partial solubilization occurs with 0.3 M KCl. The nonionic detergents, Triton X-100 and octyl-beta-D-glucopyranoside, do not solubilize ZO-1. These solubility properties indicate that ZO-1 is a peripherally associated membrane protein. ZO-1 was purified to electrophoretic homogeneity from [35S]methionine metabolically labeled MDCK cells by a combination of gel filtration and immunoaffinity chromatography. Purified ZO-1 has an s20,w of 5.3 and Stokes radius of 8.6 nm. These values suggest that purified ZO-1 is an asymmetric monomeric molecule. Corresponding values for mouse liver ZO-1, characterized in impure protein extracts, were 6 s20,w and 9 nm. ZO-1 was shown to be a phosphoprotein in MDCK cells metabolically labeled with [32P]orthophosphate; analysis of phosphoamino acids from purified ZO-1 revealed only phosphoserine. ZO-1 epitope number was determined by Scatchard analysis of competitive and saturable binding of two different 125I-mAbs to SDS-solubilized proteins from liver and MDCK cells immobilized on nitrocellulose. Saturation binding occurs at 26 ng mAb/mg liver and 63 ng/mg of MDCK cell protein. This is equivalent to 30,000 ZO-1 molecules per MDCK cell assuming a single epitope/ZO-1 molecule.

Expression of alternatively spliced isoforms of tight junction protein ZO-1 in gastrointestinal tumors

2003 ◽  
Vol 124 (4) ◽  
pp. A135
Author(s):  
Shigeru Kanaoka ◽  
Ken-Ichi Yoshida ◽  
Mutsuhiro Ikuma ◽  
Naoyuki Miura ◽  
Haruhiko Sugimura ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junction Protein ◽  
Gastrointestinal Tumors ◽  
Junction Protein ◽  
Alternatively Spliced

scholarly journals The Bile Acids Specifically Modulate Colonic MUC2 and Tight Junction Protein Expression in the Human Colon Cancer Cell Line

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 33-33
Author(s):  
Yafang Ma ◽  
Zixin Huang ◽  
Chunbao Li
Keyword(s):  
Bile Acid ◽  
Protein Expression ◽  
Tight Junction ◽  
Bile Acids ◽  
Human Colon ◽  
Tight Junction Protein ◽  
Human Colon Cancer ◽  
Tight Junction Protein Expression ◽  
Junction Protein ◽  
Muc2 Expression

Abstract Objectives Alterations to mucin secretion and epithelial tight junctions can compromise the ability of the epithelium to act as a barrier for the host to prevent pathogenic attack. Bile acids are synthesized in hepatocytes and released into the intestine, further modified by gut bacteria. Although many studies have investigated the changes of intestinal bile acids in the pathogenesis of various immune disorders, there are few reports about its function in preventing or interventing the dysfunction of the intestinal barrier. In this study, we sought to investigate the effects of the colonic bile acids on MUC2 and tight junction protein expression, which are crucial to colonic barrier. Methods Regulation of MUC2 and tight junction protein expression was assayed in the human colon cancer LS174T and T84 cells. The cells were treated with deoxycholic acid (DCA), lithocholic acid (LCA), 3-oxo-DCA, 3-oxo-LCA, isoDCA and isoLCA (100 μM or 200 μM), respectively. Proliferation of the cells was investigated with the MTT assay. mRNA expression of MUC2, ZO-1, occludin, claudin1 were measured by RT-PCR. Nuclear bile acid receptor FXR and TGR5, toll-like receptors and TLR adaptor MyD88, and genes (CDX2, AGR2, MyD88) related to mucin synthesis and secretion were also measured. Results In comparison with the untreated control, DCA, 3-oxo-DCA, isoDCA and isoLCA (100 μM) significantly upregulated the ZO-1, occludin and bile acid receptor FXR gene expression in the T84 cell. LCA, 3-oxo-LCA and isoLCA upregulated MUC2 expression at 200 μM, but showed no significant effect at 100 μM. DCA only significantly upregulated MUC2 expression at 200 μM, but isoDCA upregulated MUC2 expression independent of concentration in the LS174T cell. The expression of CDX2, AGR2, MyD88 was consistent with MUC2. Conclusions Bile acids at various concentrations specifically modulate MUC2 and tight junction protein expression, and thereby alter the colonic barrier function. This regulatory effect of bile acids could be mediated by activating bile acid receptors FXR. Funding Sources This work was financially supported by Ministry of Science and Technology of China (10000 Talent Project).

scholarly journals Force-dependent remodeling of a tight junction protein ZO-1 is regulated by phase separation

2020 ◽  
Author(s):  
Noriyuki Kinoshita ◽  
Takamasa S. Yamamoto ◽  
Naoko Yasue ◽  
Toshihiko Fujimori ◽  
Naoto Ueno
Keyword(s):  
Phase Separation ◽  
Tight Junction ◽  
Inner Cell Mass ◽  
Mdck Cells ◽  
Cell Mass ◽  
Tight Junction Protein ◽  
Cell Junction ◽  
Physiological Importance ◽  
Junction Protein ◽  
Tensile Forces

SummaryAlthough the physiological importance of biomolecular condensates is widely recognized, how it is controlled in time and space during development is largely unknown. Here we show that a tight junction protein ZO-1 forms cytoplasmic condensates in the trophectoderm (TE) of the mouse embryo before E4.0. These disappear via dissolution, and ZO-1 accumulates at the cell junction as the blastocyst cavity grows, and internal pressure on TE cells increases. In contrast, the dissolution is less evident in TE cells attached to the inner cell mass, as they receive weaker tensile forces. Furthermore, analyses using MDCK cells have shown that the ZO-1 condensates are generated and maintained by liquid-liquid phase separation. Our study also highlights that the dynamics of these condensates depends on the physical environment via the interaction between ZO-1 and F-actin. We propose that the force-dependent regulation of ZO-1 condensation contributes to establishing robust cell-cell adhesion during early development.

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