The talin–vinculin interaction is required for efficient force transduction. (A) Live-cell imaging of talinKO cells coexpressing either GFP-paxillin or GFP-talinFL with RFP-LifeAct (top panel). The cell edge was traced over time using the RFP-LifeAct signal. Black outline indicates the cell position at the first frame; yellow indicates the position in the last frame. Temporal color maps of adhesion movement obtained from the GFP signal of the same cell (lower panel) show that cells without talin after Mn²⁺ treatment are highly dynamic compared with talin-expressing Mn²⁺-treated cells. Images were acquired every 2 min for 2 h. (B) Representative force maps of talinKO cells expressing either GFP-paxillin or GFP-talinFL spread on a PAA hydrogel containing fluorescent beads for traction force microscopy. White line indicates the outline of the cell. Blue color indicates regions of low force exertion; red indicates regions of high force exertion. Scale bars in A–D indicate 10 µm. (C) Quantification of the total force exerted per cell from traction force microscopy experiments. Graphs show the mean and SEM; n = 13 cells, results are representative of three independent experiments. **, P < 0.01, unpaired two-tailed t test. (D) Schematic showing the role of talin in force transduction. In wild-type cells (+talin), vinculin (purple) reinforces the link between talin (gray) and actin, engaging the molecular clutch for efficient force transduction, stabilizing adhesion turnover. Without talin (−talin), vinculin is weakly anchored to additional adhesion proteins and is only able to transduce low forces. These adhesions are unstable and rapidly turned over. (E) Tables summarizing the binding between indicated constructs.