Molecular Compressive Force Sensor for Mapping Forces at the Cell–Substrate Interface

Mechanical forces are crucial for biological processes such as T cell antigen recognition. A suite of molecular tension probes to measure pulling forces have been reported over the past decade; however, there are no reports of molecular probes for measuring compressive forces, representing a gap in the current mechanobiology toolbox. To address this gap, we report a molecular compression reporter using pseudostable hairpins (M-CRUSH). The design principle was based on a pseudostable DNA structure that folds in response to an external compressive force. We created a library of DNA stem-loop hairpins with varying thermodynamic stability, and then used Förster Resonance Energy Transfer (FRET) to quantify hairpin folding stability as a function of temperature and crowding. We identified an optimal pseudostable DNA hairpin highly sensitive to molecular crowding that displayed a shift in melting temperature (T_m) of 7 °C in response to a PEG crowding agent. When immobilized on surfaces, this optimized DNA hairpin showed a 29 ± 6% increase in FRET index in response to 25% w/w PEG 8K. As a proof-of-concept demonstration, we employed M-CRUSH to map the compressive forces generated by primary naïve T cells. We noted dynamic compressive forces that were highly sensitive to antigen presentation and coreceptor engagement. Importantly, mechanical forces are generated by cytoskeletal protrusions caused by acto-myosin activity. This was confirmed by treating cells with cytoskeletal inhibitors, which resulted in a lower FRET response when compared to untreated cells. Furthermore, we showed that M-CRUSH signal is dependent on probe density with greater density probes showing enhanced signal. Finally, we demonstrated that M-CRUSH probes are modular and can be applied to different cell types by displaying a compressive signal observed under human platelets. M-CRUSH offers a powerful tool to complement tension sensors and map out compressive forces in living systems.