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

Abstract

Therapeutic ultrasound or shockwave has shown its great potential to stimulate neural and muscle tissue, where cavitation microbubble induced Ca 2+ 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. Ca 2+ 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 Ca 2+ signaling was assessed. Results: Ca 2+ 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 Ca 2+ response: increased integrin ligation by substrate ECM triggered ATP release and activation of P2X—but not Piezo1—ion channels. The resultant Ca 2+ influx caused dynamic changes in cell spread area. Conclusion: This approach to safely eliciting a Ca 2+ response with cavitation microbubbles and the uncovered mechanism by which increased integrin-ligation mediates ATP release and Ca 2+ signaling will inform new strategies to stimulate tissues with ultrasound and shockwaves.

Low-intensity pulsed ultrasound/nanomechanical force generators enhance osteogenesis of BMSCs through microfilaments and TRPM7

“…After washing with PBS, Opti-α-MEM was added and cells were observed under a laser scanning confocal microscope. The total observation and baseline times were 12 min and 30 s, respectively, whereas the LIPUS exposure period was 0.5–2.5 min; the treatment parameters were the same as those described in the “ LIPUS treatment ” subsection, and the interval between image captures was 4 s. The calcium response of each frame of image was quantified as Δ F / F 0 , where F 0 is the average baseline fluorescence intensity before LIPUS processing, Δ F = F P − F 0 , and F P is the real-time fluorescence intensity [ 34 ]. We compared the highest F P values of each group during and after LIPUS treatment.…”

Ultrasound-Responsive Systems as Components for Smart Materials

“…313−326 Besides studying behavioral changes in living organisms, 307,312,320,322−324 sonogenetics has also been used to trigger a cellular response against tumor cells. 318,319,322,326 For example, when T cells were engineered to express the MS channel Piezo1, ultrasound excitation in the presence of microbubbles triggered the expression of the chimeric antigen receptor (CAR), which could detect specific tumor-associated antigens. 319 Despite MS channels being widely expressed in cells, only a few types are useful to sonogenetics.…”

“…Subsequent works have extended the use of ultrasound-stimulated mechanosensitive channels to other research areas. − Besides studying behavioral changes in living organisms, ,,,− sonogenetics has also been used to trigger a cellular response against tumor cells. ,,, For example, when T cells were engineered to express the MS channel Piezo1, ultrasound excitation in the presence of microbubbles triggered the expression of the chimeric antigen receptor (CAR), which could detect specific tumor-associated antigens …”

“…Channel stimulation can be achieved either by locally induced thermal changes , (thermosonogenetics) or by ultrasound-induced shear stresses (mechanosonogenetics), although it is not clear which one is more important. The shear stresses can be amplified by microbubble oscillations during cavitation ,, or by fluid streaming. ,, A major difference between the thermal and mechanical mechanisms is their activation time scale. While thermal activation takes place over seconds, mechanical activation can trigger responses within milliseconds …”

Acoustofluidic Stimulation of Functional Immune Cells in a Microreactor

“…Mechanical stress has been shown to increase the concentration of intracellular Ca 2+ by affecting mechanosensitive ion channels (e.g., TRPC and Piezo1). [39][40][41][42] Importantly, acoustic pressure has been shown to be able to affect Ca 2+ influx into neuronal, [43] endothelial, [44] epithelial, [45] osteoblastic, [46] and cancer cells. [47,48] Acoustical streaming has also been reported to elicit Piezo1 activation and intracellular Ca 2+ response, [36,37] consistent with our results.…”