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Spontaneous assembly and active disassembly balance adherens junction homeostasis.

ABSTRACT: The homeostasis of adherens junctions was studied using E-cadherin and its two mutants tagged by the photoconvertible protein Dendra2 in epithelial A-431 cells and in CHO cells lacking endogenous cadherin. The first mutant contained point mutations of two elements, Lys738 and the dileucine motif that suppressed cadherin endocytosis. The second mutant contained, in addition, an extensive truncation that uncoupled the mutant from beta-catenin and p120. Surprisingly, the intact cadherin and its truncated mutant were recruited into the junctions with identical kinetics. The full-size cadherin was actively removed from the junctions by a process that was unaffected by the inactivation of its endocytic elements. The cadherin's apparent half-residence time in the junction was about 2 min. Cadherin clusters made of the truncated mutant exhibited much slower but ATP-independent junctional turnover. Taken together, our experiments showed that adherens junction homeostasis consists of three distinctive steps: cadherin spontaneous recruitment, its lateral catenin-dependent association, and its active release from the resulting clusters. The latter process, whose mechanism is not clear, may play an important role in various kinds of normal and abnormal morphogenesis.

SUBMITTER: Hong S

PROVIDER: S-EPMC2840517 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Spontaneous assembly and active disassembly balance adherens junction homeostasis.

Hong Soonjin S Troyanovsky Regina B RB Troyanovsky Sergey M SM

Proceedings of the National Academy of Sciences of the United States of America 20100202 8

The homeostasis of adherens junctions was studied using E-cadherin and its two mutants tagged by the photoconvertible protein Dendra2 in epithelial A-431 cells and in CHO cells lacking endogenous cadherin. The first mutant contained point mutations of two elements, Lys738 and the dileucine motif that suppressed cadherin endocytosis. The second mutant contained, in addition, an extensive truncation that uncoupled the mutant from beta-catenin and p120. Surprisingly, the intact cadherin and its tru ...[more]

PMID: 20133579

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Project description:Background: Negative-pressure wound therapy has become widely available in modern chronic wound cares. Accelerated keratinocyte (HaCaT cell) movements and decreased E-cadherin expressions induced by the applied negative pressure gradient of 125 mmHg (NP) have been reported in previous studies. However, the molecular mechanism for E-cadherin regulations under NP remains unexplored. We highlighted the signal transduction involved in NP-induced E-cadherin regulation for keratinocytes in the study. Results: Microarray results showed that catenin 1 (CTNND1) gene, the gene encoding the p120-catenin (p120ctn) in cell-cell junctions, was significantly (p = 0.0005) upregulated in HaCaT cells under NP for 12 hours in comparison with those at ambient pressure (AP). Cell fractionations and immunoblotting data showed that NP increased p120ctn phosphorylation, and resulted in p120ctn translocation from the plasma membrane to the cytoplasm. Fluorescent stains suggested that NP diminished the co-localization of p120ctn and E-cadherin on the plasma membrane. Similar phenomenon was also observed in the cells with overexpressed p120ctn at NP. Interestingly, overexpression of dN- p120ctn, a deletion mutant of p120ctn without the N-terminal phosphorylation sites, restored the adherens junctions (AJ) at NP. Knockdown of p120ctn with lentiviral small hairpin RNA not only diminished E-cadherin expressions but also accelerated cell movement at AP. Conclusions: Phosphorylation of p120ctn is endowed to respond rapidly to NP and contributes to the AJ disassembly. This NP-induced E-cadherin downregulation can possibly accelerate wound-healing process. Conclusions: Phosphorylation of p120ctn is endowed to respond rapidly to NP and contributes to the AJ disassembly. This NP-induced E-cadherin downregulation can possibly accelerate wound-healing process. HaCaT cells under negative pressure (NP) gradient of 125 mmHg for 12 hours in comparison with those at ambient pressure (AP) were tested for RNA extraction and hybridization on Affymetrix microarrays. The experiments have been biological replicated three times. The differential expression gene between NP and AP were selected and validated with qPCR method.

Project description:AimsMicrovesicles (MVs) conduct intercellular communication and impact diverse biological processes by transferring bioactive cargos to other cells. We investigated whether and how endothelial production of MVs contribute to vascular dysfunction during inflammation.Methods and resultsWe measured the levels and molecular properties of endothelial-derived MVs (EC-MVs) from mouse plasma following a septic injury elicited by cecal ligation and puncture, as well as those from supernatants of cultured endothelial cells stimulated by inflammatory agents including cytokines, thrombin, and complement 5a. The mouse studies showed that sepsis caused a significant increase in total plasma vesicles and VE-cadherin+ EC-MVs compared to sham control. In cultured ECs, different inflammatory agents caused diverse patterns of EC-MV production and cargo contents. When topically applied to endothelial cells, EC-MVs induced a cytoskeleton-junction response characterized by myosin light chain phosphorylation, contractile fibre reorganization, VE-cadherin phosphorylation, and adherens junction dissociation, functionally measured as increased albumin transendothelial flux and decreased barrier resistance. The endothelial response was coupled with protein tyrosine phosphorylation promoted by MV cargo containing c-Src kinase, whereas MVs produced from c-Src deficient cells did not exert barrier-disrupting effects. Additionally, EC-MVs contribute to endothelial inflammatory injury by promoting neutrophil-endothelium adhesion and release of neutrophil extracellular traps containing citrullinated histones and myeloperoxidase, a response unaltered by c-Src knockdown.ConclusionEndothelial-derived microparticles cause endothelial barrier dysfunction by impairing adherens junctions and activating neutrophils. The signalling mechanisms underlying the endothelial cytoskeleton-junction response to EC-MVs involve protein phosphorylation promoted by MV cargo carrying c-Src. However, EC-MV-induced neutrophil activation was not dependent on c-Src.

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Spontaneous assembly and active disassembly balance adherens junction homeostasis.

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Spontaneous assembly and active disassembly balance adherens junction homeostasis.

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