Activated EGFR-αVβ3 interactions directly drive integrin-mediated cell mechanics.

Abstract

Cells sense and respond to physical forces, and aberrations in this exchange are implicated in disease and dysfunction. Integrins and EGFR have robust synergy, with integrins establishing the mechanical link between cytoskeleton and microenvironment and EGFR regulating growth and survival. Previously, we established ligand-activated EGFR tunes integrin mechanics and facilitates cell spreading and focal adhesion (FA) maturation. Sialylation enhanced this mechanical crosstalk by increasing retention of activated EGFR on the plasma membrane. However, the underlying mechanisms orchestrating EGFR's influence on cell mechanics remain unknown. To address if there was a direct interaction between EGFR and integrin, we used a proximity ligation assay. In Cos-7 cells, EGF stimulation increased EGFR-αVβ3 interactions at 1 hour post-plating over untreated controls. The number of interactions were reduced at 24 hours, indicating that they are transient and support initial cell attachment and FA formation. Sites of EGFR-αVβ3 interactions colocalize with paxillin and pFAK with higher efficiency at 1 hour than 24 hours, supporting their role in FA formation but not maintenance. EGFR-αVβ3integrin interactions were confirmed by co-immunoprecipitation with immunoblotting analysis. Applying high-resolution TIRF microscopy, we associate these interactions to lipid raft microdomains which indicate formation of EGFR-integrin based signaling hubs at the membrane to support downstream mechanical outcomes like FA formation. Finally, employing time-lapse live cell TIRF imaging of Cos-7 cells overexpressing tagged EGFR and paxillin we characterize the dynamic nature of EGFR-αVβ3 interactions and associate them with FAs. Activated EGFR accumulates at sites for FA formation before paxillin starts clustering and persists during FA maturation. Our results implicate direct EGFR-integrin interaction as the mechanism driving cellular outcomes including FA formation, maturation and cell spreading during initial cell adhesion. Overall, EGFR-integrin interplay performs a vital synergistic function as a joint-signaling rheostat orchestrating cell mechanics.