The Molecular Organization of Endothelial Cell to Cell Junctions: Differential Association of Plakoglobin,/3-catenin, and ot-catenin with Vascular Endothelial Cadherin (VE-cadherin)

In this paper we report that the assembly of interendothelial junctions containing the cell type- specific vascular endothelial cadherin (VE-cadherin or cadherin-5) is a dynamic process which is affected by the functional state of the cells. Immunofluorescence double labeling of endothelial cells (EC) cultures indicated that VE-cadherin, a,-catenin, and fl-catenin colocalized in areas of cell to cell contact both in sparse and confluent EC monolayers. In contrast, plakoglobin became as- sociated with cell-cell junctions only in tightly confluent cells concomitantly with an increase in its protein and mRNA levels. Furthermore, the amount of plakoglobin coimmunoprecipitated with VE-cadherin increased in closely packed monolayers. Artificial wounding of confluent EC monolayers resulted in a major reorganization of VE-cadherin, ot-catenin, ~-catenin, and plakoglobin. All these pro- teins decreased in intensity at the boundaries of EC migrating into the lesion. In contrast, EC located immediately behind the migrating front retained junc- tional VE-cadherin, ot-catenin, and/3-catenin while plakoglobin was absent from these sites. In line with this observation, the amount of plakoglobin co- immunoprecipitated with VE-cadherin decreased in migrating EC. These data suggest that VE-cadherin, ot-catenin, and /3-catenin are already associated with each other at early stages of intercellular adhesion and become readily organized at nascent cell contacts. Plakoglobin, on the other hand, associates with junctions only when cells approach confluence. When cells migrate, this order is reversed, namely, plakoglobin dissociates first and, then, VE-cadherin, a-catenin, and/3-catenin disassemble from the junctions. The late association of plakoglobin with junctions suggests that while VE- cadherin/a-catenin//J-catenin complex can function as an early recognition mechanism between EC, the for- mation of mature, cytoskeleton-bound junctions re- quires plakoglobin synthesis and organization.