Cell confluence-dependent remodeling of endothelial membranes mediated by cholesterol.
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The plasma membranes of endothelial cells reaching confluence undergo profound structural and functional modifications, including the formation of adherens junctions, crucial for the regulation of vascular permeability and angiogenesis. Adherens junction formation is accompanied by the tyrosine dephosphorylation of adherens junctions proteins, which has been correlated with the strength and stability of adherens junctions. Here we show that cholesterol is a critical determinant of plasma membrane remodeling in cultures of growing cow pulmonary aortic endothelial cells. Membrane cholesterol increased dramatically at an early stage in the formation of confluent cow pulmonary aortic endothelial cell monolayers, prior to formation of intercellular junctions. This increase was accompanied by the redistribution of caveolin from a high density to a low density membrane compartment, previously shown to require cholesterol, and increased binding of the annexin II-p11 complex to membranes, consistent with other studies indicating cholesterol-dependent binding of annexin II to membranes. Furthermore, partial depletion of cholesterol from confluent cells with methyl-beta-cyclodextrin both induced tyrosine phosphorylation of multiple membrane proteins, including adherens junctions proteins, and disrupted adherens junctions. Both effects were dramatically reduced by prior complexing of methyl-beta-cyclodextrin with cholesterol. Our results reveal a novel physiological role for cholesterol regulating the formation of adherens junctions and other plasma membrane remodeling events as endothelial cells reach confluence. (+info)
Cell volume-dependent phosphorylation of proteins of the cortical cytoskeleton and cell-cell contact sites. The role of Fyn and FER kinases.
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Cell volume affects diverse functions including cytoskeletal organization, but the underlying signaling pathways remained undefined. We have shown previously that shrinkage induces Fyn-dependent tyrosine phosphorylation of the cortical actin-binding protein, cortactin. Because FER kinase was implicated in the direct phosphorylation of cortactin, we investigated the osmotic responsiveness of FER and its relationship to Fyn and cortactin. Shrinkage increased FER activity and tyrosine phosphorylation. These effects were abolished by the Src family inhibitor PP2 and strongly mitigated in Fyn-deficient but not in Src-deficient cells. FER overexpression caused cortactin phosphorylation that was further enhanced by hypertonicity. Exchange of tyrosine residues 421, 466, and 482 for phenylalanine prevented cortactin phosphorylation by hypertonicity and strongly decreased it upon FER overexpression, suggesting that FER targets primarily the same osmo-sensitive tyrosines. Because constituents of the cell-cell contacts are substrates of Fyn and FER, we investigated the effect of shrinkage on the adherens junctions. Hypertonicity provoked Fyn-dependent tyrosine phosphorylation in beta-catenin, alpha-catenin, and p120(Cas) and caused the dissociation of beta-catenin from the contacts. This process was delayed in Fyn-deficient or PP2-treated cells. Thus, FER is a volume-sensitive kinase downstream from Fyn, and the Fyn/FER pathway may contribute to the cell size-dependent reorganization of the cytoskeleton and the cell-cell contacts. (+info)
Myosin light chain kinase plays an essential role in S. flexneri dissemination.
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Shigella flexneri, the causitive agent of bacillary dysentery, has been shown to disseminate in colonic epithelial cells via protrusions that extend from infected cells and are endocytosed by adjacent cells. This phenomenon occurs in the region of the eukaryotic cell's adherens junctions and is inhibited by pharmacological reagents or host cell mutations that completely disrupt the junctional complex. In this study, inhibitors of the myosin light chain kinase (MLCK) were shown to dramatically decrease intercellular spread of S. flexneri but to have no inhibitory effect on bacterial entry, multiplication or actin-based motility within the host cell. Furthermore, cell-to-cell spread of Listeria monocytogenes, another bacterial pathogen that uses an actin-based mechanism to move within the eukaryotic cytoplasm and to spread from cell to cell, was not affected by the MLCK inhibitors, indicating that (1) the inhibition of S. flexneri cell-to-cell spread in treated cells is not due to a complete break down of cell-cell contacts, which was subsequently confirmed by confocal microscopy, and (2) MLCK plays a role in a S. flexneri-specific mechanism of dissemination. Myosin has been shown to play a role in a variety of membrane-based phenomena. The work presented here suggests that activation of this molecule via phosphorylation by MLCK, at the very least participates in the formation of the bacteria-containing protrusion, and could also contribute to the endocytosis of this structure by neighboring cells. (+info)
The molecular organization of endothelial junctions and their functional role in vascular morphogenesis and permeability.
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We review here our work on the molecular and functional organization of endothelial cell-to-cell junctions. The first part of the review is dedicated to VE-cadherin, characterized by our group few years ago. This protein is a member of the large family of transmembrane adhesion proteins called cadherins. It is endothelial cell specific and plays a major role in the organization of adherens junctions. Inactivation of VE-cadherin gene or in vivo truncation of its cytoplasmic tail leads to a lethal phenotype due to the lack of correct organization of the vasculature in the embryo. We found that the defect was due to apoptosis of endothelial cells, which became unresponsive to the survival signal induced by vascular endothelial cell growth factor. Our data indicate that VE-cadherin may act as a scaffolding protein able to associate vascular endothelial cell growth factor receptor and to promote its signaling. In the second part of the review we consider another protein more recently discovered by us and called junctional adhesion molecule (JAM). This protein is a small immunoglobulin which is located at tight junctions in the endothelium and in epithelial cells. Evidence is discussed indicating that JAM takes part in the organization of tight junctions and modulates leukocyte extravasation through endothelial intercellular junctions in vitro and in vivo. The general role of tight junctions in endothelial cells is also discussed. (+info)
Reversibility of increased microvessel permeability in response to VE-cadherin disassembly.
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We determined the role of vascular endothelial (VE)-cadherin complex in regulating the permeability of pulmonary microvessels. Studies were made in mouse lungs perfused with albumin-Krebs containing EDTA, a Ca(2+) chelator, added to study the VE-cadherin junctional disassembly. We then repleted the perfusate with Ca(2+) to restore VE-cadherin integrity. Confocal microscopy showed a disappearance of VE-cadherin immunostaining in a time- and dose-dependent manner after Ca(2+) chelation and reassembly of the VE-cadherin complex within 5 min after Ca(2+) repletion. We determined the (125)I-labeled albumin permeability-surface area product and capillary filtration coefficient (K(fc)) to quantify alterations in the pulmonary microvessel barrier. The addition of EDTA increased (125)I-albumin permeability-surface area product and K(fc) in a concentration-dependent manner within 5 min. The permeability response was reversed within 5 min after repletion of Ca(2+). An anti-VE-cadherin monoclonal antibody against epitopes responsible for homotypic adhesion augmented the increase in K(fc) induced by Ca(2+) chelation and prevented reversal of the response. We conclude that the disassembled VE-cadherins in endothelial cells are mobilized at the junctional plasmalemmal membrane such that VE-cadherins can rapidly form adhesive contact and restore microvessel permeability by reannealing the adherens junctions. (+info)
Actin-dependent membrane association of a Drosophila epithelial APC protein and its effect on junctional Armadillo.
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BACKGROUND: The adenomatous polyposis coli (APC) protein is an important tumour suppressor in the colon. It promotes the destabilisation of free cytoplasmic beta-catenin (the vertebrate homologue of the Drosophila protein Armadillo), a critical effector of the Wnt signalling pathway. The beta-catenin protein is also a component of adherens junctions, linking these to the actin cytoskeleton. In Drosophila epithelial cells, the ubiquitous form of APC, known as E-APC, is associated with adherens junctions. This association appears to be necessary for E-APC to function in destabilising Armadillo. RESULTS: Using actin-depolymerising drugs, we established that an intact actin cytoskeleton is required for the association of E-APC with adherens junctions in the Drosophila embryo. From an analysis of profilin mutants, whose actin cytoskeleton is disrupted, we found that E-APC also requires actin filaments to associate with adhesive cell membranes in the ovary. Notably, conditions that delocalised E-APC from membranes, including a mutation in E-APC itself, caused partial detachment of Armadillo from adhesive membranes. CONCLUSIONS: Actin filaments are continuously required for E-APC to be associated with junctional membranes. These filaments may serve as tracks for E-APC to reach the adherens junctions. The failure of E-APC to do so appears to affect the integrity of junctional complexes. (+info)
Reversal of the Ras-induced transformed phenotype by HR12, a novel ras farnesylation inhibitor, is mediated by the Mek/Erk pathway.
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We have used the selective farnesylation inhibitor HR12 [cysteine-N(methyl)valine-N(cyclohexyl) glycine-methionine-O-methyl-ester] to study the role of oncogenic Ras in cytoskeletal reorganization in Ha-ras(V12)-transformed Rat1 cells (Rat1/ras). Application of HR12 resulted in complete restoration of the cytoskeleton and associated cell adhesions disrupted by oncogenic Ras. This included an increase in the number and size of focal adhesions, accompanied by massive stress fiber formation and enhanced tyrosine phosphorylation. Furthermore, HR12 induced assembly of adherens junctions and dramatically elevated the level of the junctional components, cadherin and beta-catenin. HR12 was unable to restore the nontransformed phenotype in cells expressing farnesylation-independent, myristylated Ras. Examination of the main Ras-regulated signaling pathways revealed that HR12 induced a dose- and time-dependent decline in Erk1&2 activation (t(1/2) approximately 6 h), which correlated with the accumulation of nonfarnesylated oncogenic-Ras. Inhibition of the Mek/Erk pathway in Rat1/ras cells, using the Mek inhibitor, PD98059, resulted in complete cytoskeletal recovery, indistinguishable from that induced by HR12. Moreover, a constitutively active Mek mimicked the effect of ras transformation in Rat1 cells, and prevented HR12-induced cytoskeletal effects in Rat1/ras cells. No such effects were observed after treatment of Rat1/ras cells with the phosphatidylinositol 3-kinase inhibitor LY294002. These findings establish the Mek/Erk pathway as the dominant pathway involved in conferring the cytoskeletal and junctional manifestations of the Ras-induced transformed phenotype. (+info)
Endothelial adherens junctions.
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The principle of the molecular organization of adherens junctions follows a uniform pattern, which is found in epithelial, muscular, neuroneal as well as in endothelial cells and is highly conserved among species. Transmembrane molecules of the cadherin family link to catenins, which anchor the adhesion plaque to the cytoskeleton. The kind of cadherin used in adherens junctions is cell-type specific, vascular endothelial (VE)-cadherin is specific for endothelial cells. The assembly and disassembly of the cadherin/catenin complex is dynamic and regulated by growth factors. The functional status of adherens junctions controls endothelial cell-to-cell adhesion, cell scattering, vessel morphogenesis and has intracellular signaling properties, thereby playing an important role in vasculogenesis and angiogenesis. (+info)