Mechanically induced integrin ligation mediates intracellular calcium signaling with single pulsating cavitation...

Therapeutic ultrasound or shockwave has shown its great potential to stimulate neural and muscle tissue, where cavitation microbubble induced Ca²⁺ signaling is believed to play an important role. However, the pertinent mechanisms are unknown, especially at the single-cell level. Particularly, it is still a major challenge to get a comprehensive understanding of the effect of potential mechanosensitive molecular players on the cellular responses, including mechanosensitive ion channels, purinergic signaling and integrin ligation by extracellular matrix. Methods: Here, laser-induced cavitation microbubble was used to stimulate individual HEK293T cells either genetically knocked out or expressing Piezo1 ion channels with different normalized bubble-cell distance. Ca²⁺ signaling and potential membrane poration were evaluated with a real-time fluorescence imaging system. Integrin-binding microbeads were attached to the apical surface of the cells at mild cavitation conditions, where the effect of Piezo1, P2X receptors and integrin ligation on single cell intracellular Ca²⁺ signaling was assessed. Results: Ca²⁺ responses were rare at normalized cell-bubble distances that avoided membrane poration, even with overexpression of Piezo1, but could be increased in frequency to 42% of cells by attaching integrin-binding beads. We identified key molecular players in the bead-enhanced Ca²⁺ response: increased integrin ligation by substrate ECM triggered ATP release and activation of P2X-but not Piezo1-ion channels. The resultant Ca²⁺ influx caused dynamic changes in cell spread area. Conclusion: This approach to safely eliciting a Ca²⁺ response with cavitation microbubbles and the uncovered mechanism by which increased integrin-ligation mediates ATP release and Ca²⁺ signaling will inform new strategies to stimulate tissues with ultrasound and shockwaves.