THE MOLECULAR-ORGANIZATION OF ENDOTHELIAL-CELL TO CELL-JUNCTIONS - DIFFERENTIAL ASSOCIATION OF PLAKOGLOBIN, BETA-CATENIN, AND ALPHA-CATENINWITH VASCULAR ENDOTHELIAL CADHERIN (VE-CADHERIN)

In this paper we report that the assembly of interendothelial junction s 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 e ndothelial cells (EC) cultures indicated that VE-cadherin, alpha-caten in, and beta-catenin colocalized in areas of cell to cell contact both in sparse and confluent EC monolayers. In contrast, plakoglobin becam e associated with cell-cell junctions only in tightly confluent cells concomitantly with an increase in its protein and mRNA levels. Further more, the amount of plakoglobin coimmunoprecipitated with VE-cadherin increased in closely packed monolayers. Artificial wounding of conflue nt EC monolayers resulted in a major reorganization of VE-cadherin, al pha-catenin, beta-catenin, and plakoglobin. All these proteins decreas ed in intensity at the boundaries of EC migrating into the lesion. In contrast, EC located immediately behind the migrating front retained j unctional VE-cadherin, alpha-catenin, and beta-catenin while plakoglob in was absent from these sites. In line with this observation, the amo unt of plakoglobin co-immunoprecipitated with VE-cadherin decreased in migrating EC. These data suggest that VE-cadherin, alpha-catenin, and beta-catenin are already associated with each other at early stages o f intercellular adhesion and become readily organized at nascent cell contacts. Plakoglobin, on the other hand, associates with junctions on ly when cells approach confluence. When cells migrate, this order is r eversed, namely, plakoglobin dissociates first and, then, VE-cadherin, alpha-catenin, and beta-catenin disassemble from the junctions. The l ate association of plakoglobin with junctions suggests that while VE-c adherin/alpha-catenin/beta-catenin complex can function as an early re cognition mechanism between EC, the formation of mature, cytoskeleton- bound junctions requires plakoglobin synthesis and organization.