Real-Time Measurement of Molecular Tension during Cell Adhesion and Migration Using Multiplexed Differential Analysis of Tension Gauge Tethers.

Cells must respond specifically and dynamically to mechanical cues from the extracellular environment and dysregulation of extracellular force sensing leads to a variety of diseases. Therefore, it is important to deconvolve the many inputs that transduce mechanical signals and understand how these signals are interpreted and responded to. DNA and peptide-based molecular force sensors have been previously developed to measure forces applied through single membrane receptors including integrins and Notch receptors. The tension gauge tether (TGT) exploits the physical rupture force of double-stranded DNA to measure and modulate the force applied through single receptor-ligand bonds and can cover a wide range of tension (10-60 pN). By exploiting a fluorescent dye-quencher pair and collecting differential fluorescence signals over time, we characterized the quenched tension gauge tether (qTGT) system and developed an image analysis protocol to measure molecular tension in quasi-real time. We show that this differential qTGT analysis method can simultaneously measure multiple levels of integrin-mediated molecular tension over a wide time scale during the onset of adhesion and cell migration.