Abstract 305: Integrin β 3 Mediates Interaction of Vimentin with Focal Contacts in Endothelial Cells: Implications for Mechanical Stability of the Vascular Endothelium
Our previous studies have demonstrated the novel finding that vimentin-type intermediate filaments (IFs), in addition to microfilaments, associate with αvβ3 integrin-positive focal contacts (FCs) in endothelial cells and regulate FC dynamics. Here we investigated the role of integrins in mediating vimentin IF-focal contact interaction. Using siRNA to knock down β3 integrin in endothelial cells, we observed up to a 70% decrease in the numbers of FCs showing association with IFs. To provide further support for vimentin-FC interaction we transfected GFP-β3 integrin into CHO cells. These cells lack β3 integrin but contain vimentin. In “parental” CHO cells vimentin IFs are collapsed around the nucleus. In sharp contrast, in CHO cells transfected with full-length β3 integrin (CHOwtβ3) vimentin fibers extend to FCs at the cell periphery. To define which residue(s) in the β3 integrin cytoplasmic tail mediate vimentin-FC association, we tested the ability of mutated β3 integrin proteins to recruit vimentin to FCs in CHO cells. Deleting 9 residues at the β3 integrin C-terminus did not impact FC targeting of mutated β3 protein or its ability to mediate vimentin-FC association. Remarkably, in CHO cells (CHOβ3Y759F) expressing a mutated β3 integrin cytoplasmic tail, in which a tyrosine residue at position 759 has been replaced by phenylalanine (a non-phosphorylatable mimetic), we observe no significant association of vimentin with FCs. Nonetheless, β3Y759F integrin targets correctly to FCs and its expression has no impact on αvβ3 integrin-ligand binding or actin cytoskeleton organization. We assessed the functional consequences of inducing IF-focal contact interactions in CHO cells by using trypsinization assays to test the strength of attachment of CHO, CHOwtβ3 and CHOβ3Y759F cells. CHOwtβ3 cells exhibit significantly greater adhesive strength compared to parental CHO and CHOβ3Y759F cells. In summary, we have provided evidence that β3 integrin mediates vimentin association with FCs. We have also shown that differential phosphorylation of residue 759 in the β3 integrin cytoplasmic tail is a key, specific regulator of vimentin-FC association and we show that vimentin-FC interactions play an important role in regulating the adhesive strength of FCs.