scholarly journals Cadherin Cytoplasmic Domains Inhibit the Cell Surface Localization of Endogenous E-Cadherin, Blocking Desmosome and Tight Junction Formation and Inducing Cell Dissociation

PLoS ONE ◽  
2014 ◽  
Vol 9 (8) ◽  
pp. e105313 ◽  
Author(s):  
Masayuki Ozawa ◽  
Wakako Kobayashi
Keyword(s):  
Cell Surface ◽  
Tight Junction ◽  
Cytoplasmic Domains ◽  
Cell Dissociation ◽  
Tight Junction Formation ◽  
Junction Formation ◽  
Surface Localization ◽  
Cell Surface Localization ◽  
E Cadherin

scholarly journals PALS1 Regulates E-Cadherin Trafficking in Mammalian Epithelial Cells

2007 ◽  
Vol 18 (3) ◽  
pp. 874-885 ◽  
Author(s):  
Qian Wang ◽  
Xiao-Wei Chen ◽  
Ben Margolis
Keyword(s):  
Tight Junction ◽  
Adherens Junction ◽  
Cell Periphery ◽  
Madin Darby Canine Kidney ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Evolutionarily Conserved ◽  
Junction Formation ◽  
Darby Canine Kidney ◽  
E Cadherin

Protein Associated with Lin Seven 1 (PALS1) is an evolutionarily conserved scaffold protein that targets to the tight junction in mammalian epithelia. Prior work in our laboratory demonstrated that the knockdown of PALS1 in Madin Darby canine kidney cells leads to tight junction and polarity defects. We have created new PALS1 stable knockdown cell lines with more profound reduction of PALS1 expression, and a more severe defect in tight junction formation was observed. Unexpectedly, we also observed a severe adherens junction defect, and both defects were corrected when PALS1 wild type and certain PALS1 mutants were expressed in the knockdown cells. We found that the adherens junction structural component E-cadherin was not effectively delivered to the cell surface in the PALS1 knockdown cells, and E-cadherin puncta accumulated in the cell periphery. The exocyst complex was also found to be mislocalized in PALS1 knockdown cells, potentially explaining why E-cadherin trafficking is disrupted. Our results suggest a broad and evolutionarily conserved role for the tight junction protein PALS1 in the biogenesis of adherens junction.

scholarly journals E-Cadherin Inhibits Cell Surface Localization of the Pro-Migratory 5T4 Oncofetal Antigen in Mouse Embryonic Stem Cells

2007 ◽  
Vol 18 (8) ◽  
pp. 2838-2851 ◽  
Author(s):  
Helen L. Spencer ◽  
Angela M. Eastham ◽  
Catherine L.R. Merry ◽  
Thomas D. Southgate ◽  
Flor Perez-Campo ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Cell Surface ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Contact ◽  
Es Cell ◽  
Surface Localization ◽  
Cell Surface Localization ◽  
E Cadherin ◽  
Cell Cell

Epithelial-mesenchymal transition (EMT) events occur during embryonic development and are important for the metastatic spread of epithelial tumors. We show here that spontaneous differentiation of mouse embryonic stem (ES) cells is associated with an E- to N-cadherin switch, up-regulation of E-cadherin repressor molecules (Snail and Slug proteins), gelatinase activity (matrix metalloproteinase [MMP]-2 and -9), and increased cellular motility, all characteristic EMT events. The 5T4 oncofetal antigen, previously shown to be associated with very early ES cell differentiation and altered motility, is also a part of this coordinated process. E- and N-cadherin and 5T4 proteins are independently regulated during ES cell differentiation and are not required for induction of EMT-associated transcripts and proteins, as judged from the study of the respective knockout ES cells. Further, abrogation of E-cadherin–mediated cell–cell contact in undifferentiated ES cells using neutralizing antibody results in a reversible mesenchymal phenotype and actin cytoskeleton rearrangement that is concomitant with translocation of the 5T4 antigen from the cytoplasm to the cell surface in an energy-dependent manner. E-cadherin null ES cells are constitutively cell surface 5T4 positive, and although forced expression of E-cadherin cDNA in these cells is sufficient to restore cell–cell contact, cell surface expression of 5T4 antigen is unchanged. 5T4 and N-cadherin knockout ES cells exhibit significantly decreased motility during EMT, demonstrating a functional role for these proteins in this process. We conclude that E-cadherin protein stabilizes cortical actin cytoskeletal arrangement in ES cells, and this can prevent cell surface localization of the promigratory 5T4 antigen.

scholarly journals Participation of plasma membrane proteins in the formation of tight junction by cultured epithelial cells

1983 ◽  
Vol 96 (3) ◽  
pp. 693-702 ◽  
Author(s):  
EB Griepp ◽  
WJ Dolan ◽  
ES Robbins ◽  
DD Sabatini
Keyword(s):  
Protein Synthesis ◽  
Steady State ◽  
Tight Junction ◽  
Messenger Rna ◽  
Freeze Fracture ◽  
Epithelial Cell Line ◽  
Experimental Conditions ◽  
Tight Junction Formation ◽  
Junction Formation ◽  
Spinner Culture

Measurements of the transepithelial electrical resistance correlated with freeze-fracture observations have been used to study the process of tight junction formation under various experimental conditions in monolayers of the canine kidney epithelial cell line MDCK. Cells derived from previously confluent cultures and plated immediately after trypsin- EDTA dissociation develop a resistance that reaches its maximum value of several hundred ohms-cm(2) after approximately 24 h and falls to a steady-state value of 80-150 ohms- cm(2) by 48 h. The rise in resistance and the development of tight junctions can be completely and reversibly prevented by the addition of 10 μg/ml cycloheximide at the time of plating, but not when this inhibitor is added more than 10 h after planting. Thus tight junction formation consists of separable synthetic and assembly phases. These two phases can also be dissociated and the requirement for protein synthesis after plating eliminated if, following trypsinization, the cells are maintained in spinner culture for 24 h before plating. The requirement for protein synthesis is restored, however, if cells maintained in spinner culture are treated with trypsin before plating. Actinomycin D prevents development of resistance only in monolayers formed from cells derived from sparse rather than confluent cultures, but new mRNA synthesis is not required if cells obtained from sparse cultures are maintained for 24 h in spinner culture before plating. Once a steady-state resistance has been reached, its maintenance does not require either mRNA or protein synthesis; in fact, inhibition of protein synthesis causes a rise in the resistance over a 30-h period. Following treatments that disrupt the junctions in steady- state monolayers recovery of resistance also does not require protein synthesis. These observations suggest that proteins are involved in tight junction formation. Such proteins, which do not turn over rapidly under steady-state conditions, are destroyed by trypsinization and can be resynthesized in the absence of stable cell-cell or cell-substratum contact. Messenger RNA coding for proteins involved in tight junction formation is stable except when cells are sparsely plated, and can also be synthesized without intercellular contacts or cell-substratum attachment.

Tight junction protein cingulin is expressed by maternal and embryonic genomes during early mouse development

Development ◽  
1993 ◽  
Vol 117 (3) ◽  
pp. 1145-1151 ◽  
Author(s):  
Q. Javed ◽  
T.P. Fleming ◽  
M. Hay ◽  
S. Citi
Keyword(s):  
Tight Junction ◽  
Cell Mass ◽  
Preimplantation Embryos ◽  
Cell Contact ◽  
Mouse Development ◽  
Early Cleavage ◽  
Cell Stage ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Junction Formation

The expression of the tight junction peripheral membrane protein, cingulin (140 × 10(3) M(r), was investigated in mouse eggs and staged preimplantation embryos by immunoblotting and immunoprecipitation. Polyclonal antibody to chicken brush cingulin detected a single 140 × 10(3) M(r) protein in immunoblots of unfertilised eggs and all preimplantation stages. Relative protein levels were high in eggs and early cleavage stages, declined during later cleavage and increased again in expanding blastocysts. Quantitative immunoprecipitation of metabolically labelled eggs and staged embryos also revealed a biphasic pattern for cingulin synthesis with relative net levels being high in unfertilised eggs, minimal during early cleavage, rising 2.3-fold specifically at the onset of compaction (8-cell stage, when tight junction formation begins), and increasing further at a linear rate during morula and blastocyst stages. Cingulin synthesis in eggs is not influenced by fertilisation (or aging, if unfertilised), but this level declines sharply after first cleavage. These results indicate that cingulin is expressed by both maternal and embryonic genomes. The turnover of maternal cingulin (unfertilised eggs) and embryonic cingulin at a stage before tight junction formation begins (4-cell stage) is higher (t1/2 approximately 4 hours) than cingulin synthesised after tight junction formation (blastocysts; t1/2 approximately 10 hours). This increase in cingulin stability is reversed in the absence of extracellular calcium. Cingulin synthesis is also tissue-specific in blastocysts, being up-regulated in trophectoderm and down-regulated in the inner cell mass. Taken together, the results suggest that (i) cingulin may have a role during oogenesis and (ii) cell-cell contact patterns regulate cingulin biosynthesis during early morphogenesis, contributing to lineage-specific epithelial maturation.

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