Inflammation-induced subcellular redistribution of VE-cadherin, actin, andgamma-catenin in cultured human lung microvessel endothelial cells

The inflammation-induced subcellular redistribution of key cytoskeletal and junctional proteins in cultured human lung microvessel endothelial cells i s investigated as part of a study on the posttranslational regulation of pa racellular permeability. Inflammatory agonist-stimulated cells are detergen t fractionated into three subcellular compartments followed by quantitative immunoblot analysis. Actin, gamma -catenin, and VE-cadherin increasingly a ssociate with the cytoskeletal fraction upon thrombin stimulation. Concomit antly, actin is reduced in the cytosol fraction, whereas gamma -catenin and VE-cadherin are reduced in the membrane fraction. alpha- and beta -catenin show baseline distributions similar to those of VE-cadherin and gamma -cat enin, but do not significantly redistribute. Additionally, vimentin is foun d exclusively in the cytoskeletal fraction and also does not significantly redistribute following thrombin treatment. The VE-cadherin response is inde pendent of the presence of F-actin or actin redistribution. Immunofluoresce nce microscopy reveals that membrane and cytoskeletal VE-cadherin is presen t in alternating patches along the cell junctions. Furthermore, VE-cadherin is lost from zones of interendothelial cell pore formation. A model is for mulated describing these membrane-associated VE-cadherin patches as predete rmined zones of potential intercellular gap formation. During inflammation, VE-cadherin is lost from these zones and sequestered at the remaining cell -cell contact sites, anchored to the cytoskeleton in an actin-independent f ashion. (C) 2001 Academic Press.