| Mechanotransduction is cellular signal transduction in response to mechanical stimuli. To determine if and how integrins are locally responsible for specific mechanotransduction pathways, we used magnetic methods (magnetic twisting cytometry and magnetic tweezers) to apply force to ligand-coated ferromagnetic particles bound specifically to integrins on the surface of adherent endothelial cells. Mechanical activation of the cAMP pathway required stress transmission to activated and clustered integrins. Additionally, these integrins (specifically the beta1 subunit) mediated such mechanosensitivity through the coordinated recruitment of the heterotrimeric G protein subunits Galphas and Gbeta, as detected by immunofluorescence staining. Importantly, subsequent mechanical stress induced activation of Gas, which was monitored with azido-anilido GTP binding assays. The same integrins, when clustered and activated, concurrently recruited the structural proteins paxillin, vinculin, and actin to mechanically stabilize newly forming focal adhesions. This stabilization was measured as local stiffness by the magnetic tweezer, which uncovered significantly higher cell-to-cell variability in mechanical properties compared to previous population-based techniques. The mechanical stress relayed through biochemically enriched and structurally fortified adhesions also elicited downstream expression of visualizable reporters of gene transcription in living cells (CRE-beta-lactamase and CRE-dsRed). Finally, we investigated the potential role of integrins in sensing the physiologically relevant mechanical stress imparted by fluid flow on endothelium and kidney epithelium. The cytoskeletal transmission of apically applied shear stress to basal integrin-mediated focal adhesions was required for some (NFkappaB in endothelium, Ca2+ in kidney epithelium), but not necessarily all (ERK in endothelium), signaling pathways induced by fluid flow. Overall, the work confirms that integrins represent points of mechanochemical signal convergence in many cell types under diverse mechanical loading conditions. |
