Regulation of the association of alpha 6 beta 4 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 alpha 6 beta 4 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 alpha 6 beta 4-vimentin IF association. We first tested the requirement for alpha 6 beta 4-laminin interactions and actin microfilament assembly. We found that alpha 6 beta 4 associated with vimentin IFs when cells were adherent to either laminin 5 or fibronectin, indicating that this association can occur independent of alpha 6 beta 4-ligand interactions. Additionally, we found that alpha 6 beta 4 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 alpha 6 beta 4 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 alpha 6 beta 4-mediated adhesion. Finally, we tested the role of plectin in the association of alpha 6 beta 4 with vimentin IFs. Plectin is known to bind in vitro to both IFs and the beta 4 cytoplasmic domain (beta 4 tail), suggesting that it may be important for this linkage. Therefore, we generated deletion mutants of the beta 4 tail and compared the ability of alpha 6 beta 4 containing these deletions to associate with vimentin IFs. We targeted the two regions of the beta 4 tail known to bind to plectin
the N-terminal and C-terminal plectin binding sites. We found that deletion of the N-terminal binding site inhibited the association of alpha 6 beta 4 with vimentin IFs. Thus, plectin-beta 4 tail interactions may play an important role in connecting alpha 6 beta 4 with vimentin IFs and may prove to be important targets in the regulation of this association in endothelial cells.
Homan
Suzanne M
SM
Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208, USA.
Martinez
Robert
R
Benware
Angela
A
LaFlamme
Susan E
SE
D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D066298:In Vitro Techniques; D039161:Integrin alpha6beta4 / Q000235:genetics / Q000378:metabolism*; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007382:Intermediate Filaments / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism; D008024:Ligands; D008858:Microscopy, Phase-Contrast; D051190:Plectin; D011485:Protein Binding; D051381:Rats; D014162:Transfection; D014746:Vimentin / Q000378:metabolism*; D016596:Vinculin / Q000378:metabolism
D017931:DNA Primers; D039161:Integrin alpha6beta4; D007381:Intermediate Filament Proteins; D007797:Laminin; D008024:Ligands; C495332:PLEC protein, human; C495334:Plec protein, rat; D051190:Plectin; D014746:Vimentin; D016596:Vinculin