Optical tweezers combined with FRET tension sensor reveal force-dependent vinculin dynamics

Visualizing and quantifying molecular responses to local forces exerted at cell adhesions is crucial to elucidate how physical forces control cellular behavior. Here, we combine optical tweezers with Förster resonance energy transfer (FRET) microscopy of the vinculin tension sensor, VinTS, to measure the response of vinculin, a key mechanical load-bearing protein, to an applied force. Fibroblasts expressing VinTS formed adhesions on fibronectin-coated, 3-μm-diameter, polystyrene beads. As the beads were displaced by the cell, we applied an optical trap to counteract this movement and increase the traction force required by the cell to maintain the bead's displacement. The median bead displacement after 5 min was ∼200 nm in all trapping conditions tested, from zero (no laser) up to 0.26 pN/nm, inducing counteracting forces in the 10-100 pN range. To maintain this displacement, vinculin recruitment increased at high stiffness (up to 35% in relative intensity), while vinculin tension increased only moderately in all trapping conditions (1%-2% decrease in absolute FRET efficiency). Vinculin recruitment was governed by stiffness rather than the magnitude of the traction force and was correlated with vinculin tension at 0.26 pN/nm but not at lower stiffness. In rare instances, vinculin puncta migrated a few micrometers 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 in living cells uncovers novel vinculin dynamics in the presence of a force.