Regulation of the association of α6β4 with vimentin intermediate filaments in endothelial cells

The adhesion of microvascular endothelial cells to their underlying basement membrane is important for the maintenance of vascular integrity. Most integrins function in endothelial cell adhesion by forming a transmembrane link between their basement membrane ligand and the actin microfilament cytoskeleton. The alpha6beta4 laminin-binding integrin, however, associates with vimentin intermediate filaments (IFs) in microvascular endothelial cells and therefore is likely to uniquely contribute to the barrier function of the endothelium. In this study, we examined the regulation of alpha6beta4-vimentin IF association. We first tested the requirement for alpha6beta4-laminin interactions and actin microfilament assembly. We found that alpha6beta4 associated with vimentin IFs when cells were adherent to either laminin 5 or fibronectin, indicating that this association can occur independent of alpha6beta4-ligand interactions. Additionally, we found that alpha6beta4 was associated with vimentin IFs prior to cell spreading, indicating that changes in the microfilament cytoskeleton associated with changes in cell shape are also not required. Thus, although the association of alpha6beta4 with vimentin IFs may strengthen cell adhesion by providing endothelial cells with an additional transmembrane linkage between the basement membrane and the cytoskeleton, this association is not itself regulated by alpha6beta4-mediated adhesion. Finally, we tested the role of plectin in the association of alpha6beta4 with vimentin IFs. Plectin is known to bind in vitro to both IFs and the beta4 cytoplasmic domain (beta4 tail), suggesting that it may be important for this linkage. Therefore, we generated deletion mutants of the beta4 tail and compared the ability of alpha6beta4 containing these deletions to associate with vimentin IFs. We targeted the two regions of the beta4 tail known to bind to plectin in vitro. the N-terminal and C-terminal plectin binding sites. We found that deletion of the N-terminal binding site inhibited the association of alpha6beta4 with vimentin IFs. Thus, plectin-beta4 tail interactions may play an important role in connecting alpha6beta4 with vimentin IFs and may prove to be important targets in the regulation of this association in endothelial cells. (C) 2002 Elsevier Science (USA).