Quantify Integrin Molecular Tensions using Tandem Tension Sensor

Introduction: Cell mechanotransduction, through which cells actively probe and react to the mechanical properties of local matrix, involves integrin tensions at the cell-matrix interfaces as a key biochemical signal. Previously, molecular tension sensors based on double-stranded DNAs (dsDNA), hairpin DNAs or shearing DNAs have substantially advanced the visualization and calibration of integrin tensions by converting force signal to fluorescence. However, these tension sensors mainly function as binary reporters, only indicating if a molecular force exceeds one predefined threshold. To achieve molecular force quantification in real time, here we developed Tandem Tension Sensor (TTS) consisting of multiple linked tension sensors, which individually tests a molecular force with different force thresholds and in separate fluorescence channels.

Materials and

Methods: We constructed TTS to quantify integrin tensions in a broad force range. TTS513 was designed and synthesized by linking two hairpin DNAs with 5 pN and 13 pN critical forces, respectively. TTS1320 was synthesized by linking a hairpin DNA and a shearing DNA with 13 and 20 pN critical forces, respectively (Fig. 1A). Each TTS construct contains an integrin peptide ligand, a Cy3 dye, an Atto 647N dye, two blackhole quenchers (BHQ2) and a biotin tag, which helps to immobilize the TTS on surfaces. The functionality of the sensors have been validated by visualizing integrin tensions in focal adhesions (HeLa cell) and platelets.

Results, Conclusions, and Discussions: By applying TTS513 and TTS1320 to wildtype HeLa cells and vinculin knockout cells, we showed that vinculin is not required for cells to generate low integrin tensions (5-13 pN; Figure 1B), but is essential for cells to generate integrin tensions beyond 20 pN (Figure 1B and 1C) in focal adhesions. Inhibiting myosin II by Y27632 led to a significant decrease of the molecular force ratio (the ratio of integrin tensions higher than 20 pN to those higher than 13 pN) reported by TTS1320 (Fig. 1D and 1E). TTS1320 also revealed two distinct force active regions in the platelets, with integrin tensions in the central region and in the peripheral region, respectively (Fig. 1F). Remarkably, integrin tensions in the central region appeared in all >5 pN, >13 pN and >20 pN force channels, but integrin tensions in the peripheral region only appeared in >5 pN and >13 pN channels. These results suggest that integrin tensions in the central region readily exceed 20 pN while the integrin tensions in the peripheral region is lower than 20 pN.

Conclusion: Using HeLa cells and platelets as cell models, we demonstrated that TTS, particularly the hairpin and shearing DNA assembled construct (TTS1320), can report integrin tensions at different force levels in single cells. TTS stands as a valuable tool for the quantification of receptor-transmitted molecular forces within living cells.
Reference: This work has been submitted for journal review.

Acknowledgements (Optional): This research was sponsored by National Institute of General Medical Sciences (1R35GM128747).