Real time observation of chaperone-modulated talin mechanics with single molecule resolution

Recent single-molecule studies have recognized talin as a mechanosensitive hub in focal adhesion, where its function is strongly regulated by mechanical force. For instance, at low force (below 5 pN), folded talin binds RIAM for integrin activation; whereas at high force (above 5 pN), it unfolds to activate vinculin binding for focal adhesion stabilization. Being a cytoplasmic protein, talin might interact with several cytosolic chaperones: however, the role of chaperones in talin mechanics is unknown. To address this question, we investigated the force response of a mechanically stable talin domain with a set of well-known holdase (DnaJ, DnaK, Hsp70, and Hsp40) and foldase (DnaKJE, DsbA) chaperones, using single-molecule magnetic tweezers. Our findings demonstrate that chaperone could affect adhesion proteins stability by changing their folding mechanics; while holdase chaperones reduce their unfolding force to ∼6 pN, foldase chaperones shift it up to ∼15 pN. Since talin is mechanically synced within 2 pN force ranges, these changes are significant in cellular condition. Furthermore, we determined the fundamental mechanism of this altered mechanical stability, where chaperones directly reshape their energy landscape: unfoldase chaperone (DnaK) decreases the unfolding barrier height from 26.8 to 21.7 k B T, while foldase chaperone (DsbA) increases it to 33.5 k B T. We reconciled our observations with eukaryotic Hsp70 and Hsp40 chaperones and observed their similar function of decreasing the talin unfolding barrier to 23.1 k B T. The quantitative mapping of this chaperone-induced talin folding landscape directly illustrates that chaperones perturb the adhesion protein stability under physiological force, thereby influencing their force-dependent interactions and adhesion dynamics.