Fluorescence Resonance Energy Transfer Optomechanics Approach To Construct Intermolecular Domain Tension Probes for...

Integrin-talin-cytoskeleton-mediated force transduction plays crucial roles in mechanobiology, especially in tumor invasion and metastasis and the regulation of neurite outgrowth and synaptic plasticity. However, gene cloning of mechanics-related protein probes remains challenging for protein gain-of-function and signal overactivation. Leveraging AlphaFold2 prediction of talin/actin-binding domains, we constructed intermolecular tension probes for talin-microfilament interactions. These probes comprised an EF domain binding on the C-terminus of talin and various actin-binding domains (SH3, PDZ, LIM, and FERM). The intermolecular domain tension probes were applied in the talin-microfilament force transduction system, which successfully avoided the adverse effects of gene cloning and talin-overactivation-induced cell invasion. We found that talin-microfilament force transduction depends on the specific actin-binding domain of talin, while the binding of microfilaments themselves is nonspecific. Microfilament depolymerization, however, can reverse the increase in tension induced by integrin activation. In this study, we developed intermolecular domain tension probes with an optimized structure and molecular weight, allowing real-time fluorescence monitoring of tension transduction and cellular distribution in live cells and force detection independent of the direct modulation of protein function.