Force-Induced Calcium Concentration Change and Focal Adhesion Translocation: Effects of Force Amplitude and Frequency

Vascular endothelial cells rapidly sense and transduce external forces into biological signals through a process known as mechanotransduction. Numerous biological processes are involved in mechanotransduction, including calcium signaling and activation of focal adhesion sites, but little is known about how cells initially sense changes in the external mechanical environment. In order to examine the rapid mechanosensing thresholds involved with mechanotransduction, calcium concentration changes and focal adhesion site translocations were observed while applying nN-level magnetic trap shear forces to the cell apex via integrin-linked magnetic beads. Both biological responses were monitored with fluorescent microscopy by labeling intracellular calcium with Fluo-3 calcium dye and by infecting cells with GFP-paxillin fusion proteins. Monitoring calcium concentration changes proved unreliable for determining mechanotransduction thresholds, while a non-graded, time dependent (minutes) steady load threshold for mechanotransduction was established between 0.90 and 1.45 nN for focal adhesion site activation. Activation was greatest near the point of forcing (< 7.5 pm), indicating that shear forces imposed on the apical cell membrane transmit nonuniformly to the basal cell surface and that focal adhesion sites may function as individual mechanosensors responding to local levels of force. Results from a continuum, viscoelastic finite element model of magnetocytometry that represented experimental focal adhesion attachments provided support for a non-uniform force transmission to basal surface focal adhesion sites. Frequency variation between 0.1 and 50 Hz altered focal adhesion translocation and resulted in a biphasic response minimized at 1.0 Hz. Furthermore, applying the tyrosine kinase inhibitors genistein and PP2, a specific Src family kinase inhibitor, resulted in differential effects on force-induced translocation. These results highlight the mutual importance of force transmission and biochemical signaling in focal adhesion mechanotransduction. Principal Thesis Supervisor: Roger D. Kamm Title: Professor of Mechanical Engineering Force-induced calcium concentration change and focal adhesion translocation: Effects of force amplitude and frequency Chapter