Mechanical Activity At Focal Adhesion Sites

Abstract

We investigated whether mechanical force applied to extracellular matrix proteins (ECM)-integrin focal adhesion sites would induce mechanical activity characteristic of specific ECM type. We used atomic force microscopy (AFM) to apply forces to ECM adhesion sites on vascular smooth muscle cells (VSMC) isolated from resistance arterioles. The tip of the AFM probes were fused with a borosilicate bead (5 um diameter) coated with fibronectin (FN), collagen type-I (CNI), collagen type-IV (CNIV), laminin (LN) or vitronectin (VN). ECM-coated beads induced clustering of a5 and b3 integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward (z-axis) pulling force (800∼1600 pN) applied to the bead-cell contact site for FN specific focal adhesions induced a force-generating response from the cells resulting in a downward pull by the cell. Depolymerization of the actin cytoskeleton with cytochalasin D blocked whilst stabilization of the actin cytoskeleton with jasplakinolide enhanced this micromechanical event. Myosin light chain kinase inhibition (ML7) and an inhibitor of cSrc tyrosine kinase (PP2) also blocked the response. Furthermore, inhibitory antibodies to a5 and b3 integrins blocked the micromechanical cell event in a concentration-dependent manner. Similar experiments with CNI, CNIV, VN, or LN failed to induce micromechanical events. Our results demonstrate that mechanical force applied through FN at single focal adhesion sites induces a micromechanical event that is actin, myosin light chain kinase and tyrosine kinase dependent. Importantly, the data illustrate that there are different mechanical characteristics for focal adhesions formed by different ECM proteins. FN appears of particular relevance in its ability to induce a force-generating reaction from sites of focal adhesion in VSMC in response to applied forces.