Optical tweezers combined with FRET tension sensor reveal force-dependent vinculin dynamics
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Methods to visualize and quantify the molecular responses of cells to local forces exerted at adhesions are crucial to elucidate how physical forces control cellular behavior. Of the many proteins involved in focal adhesions, vinculin plays a key role in mediating force-sensitive processes. Here, we combine optical tweezers and Förster resonance energy transfer (FRET) microscopy to measure the intensity and FRET efficiency of the vinculin tension sensor, VinTS, in response to a force. Fibroblasts expressing VinTS formed adhesions on fibronectin-coated, 3μm-diameter, polystyrene beads, which were subjected to an optical trap with adjustable stiffness up to 0.26 pN/nm. After 5 min, the mean bead displacement was ∼200nm in all trapping conditions inducing counteracting forces in the 10-100pN range. In response to the optical trap, vinculin recruitment and tension increased as a function of time. The mean increase in vinculin tension (1-2% decrease in absolute FRET efficiency) attained after 5 min was not significantly different at the different trapping conditions. However, vinculin recruitment was significantly higher at the higher trap stiffness with up to 35% increase in intensity at 0.26 pN/nm compared with <10% increase at 0.13 pN/nm, and an average 3% decrease in intensity for untrapped beads. The increase in vinculin intensity was correlated with the decrease in FRET efficiency at 0.26 pN/nm but not at lower stiffness. Thus, the presence of the high stiffness optical trap over 5 min appears to induce a positive correlation between vinculin recruitment and vinculin tension. In a few instances, vinculin puncta migrated a few microns away from the bead exceeding the bead’s movement speed while experiencing an increase in both vinculin intensity and tension. Taken together, the results suggest that combining an optical trap with vinculin tension measurements uncovers novel vinculin dynamics in the presence of a force.
Statement of significance
A fully calibrated system combining optical tweezers with FRET microscopy is developed and used to measure vinculin tension and recruitment in response to a force. The results demonstrate a correlation between vinculin recruitment and vinculin tension when the cell’s adhesion force is counteracted for 5 min by an optical trap stiffness of 0.26 pN/nm. This response was absent when a lower trap stiffness (0.13 pN/nm) was used or in the absence of the optical trap. Such dynamic measurements, enabled by the techniques presented in this paper, can help elucidate the mechanisms by which cells sense physical forces and the properties of proteins, such as vinculin, which play a fundamental role in cellular behaviors involving tissue growth and repair.
