It is now widely appreciated that cancer cells and stem cells can change their cell fate (differentiation, metastasis, etc.) depending on their mechanical environment. Mechanical sensing is likely to be initiated by individual membrane receptor proteins that are in direct contact with the mechanical environment. In order to understand how molecular level mechanical events trigger a cellular response, we need to examine the forces applied across individual cellular proteins during mechanical signaling.To define the single molecular forces required to activate signaling through a ligand‐receptor bond, we developed the tension gauge tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor‐ligand tension. Using a range of tethers with tunable tension tolerances, we showed that cells apply a peak tension of about 40 picoNewtons (pN) to single integrin‐ligand bonds during initial adhesion. This tension value was consistent across different cell types. We then presented a mixture of weak and strong TGTs (12 pN and 54 pN nominal tension tolerance) to the cells in order to better mimic native cellular environments that are likely heterogeneous. Surprisingly, just one or two strong TGTs were sufficient to allow the cells to adhere as long as there is a high density of weak tethers. Therefore, mechanical tugging through one or two integrins is enough to convince the cell that the substrate is stiff enough for adhesion. Even more surprisingly, when we presented a mixture of two different weak TGTs (12 pN and 23 pN), cells adhered to the surface despite the fact that individually neither of the two tethers could support cell adhesion. Furthermore, we found that the 23 pN tether can exert its influence even at very high dilution, down to as few as two tethers per cell. This phenomenon of ‘ultrasensitivity in differential force sensing through integrin’ can be explained by a model where simultaneous rupture of weaker bonds exerts a transient high tension across a single integrin to activate that integrin.Notch signaling, involved in development and tissue homeostasis, is activated at the cell‐cell interface through ligand‐receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. In our original TGT paper, we showed that Notch signaling is activated even for the weakest TGT (12 pN), suggesting that if Notch activation requires force, it must be less than 12 pN. Subsequently, the Blacklow lab show that the exposure of a Notch cleavage site required for its activation occurs at a tension of ~5 pN39, consistent with our estimate of a 12 pN upper limit. We recently developed LTGT (a low tension gauge tether) utilizing the low unbinding force of ~ 4 pN between single‐stranded (ss)DNA and E. coli ssDNA binding protein (SSB) and showed that Notch activation requires forces between 4 and 12 pN.
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