Förster Resonance Energy Transfer-Based Single-Cell Imaging Reveals Piezo1-Induced Ca2+ Flux Mediates Membrane Ruffling and Cell Survival.

A mechanosensitive ion channel, Piezo1 induces non-selective cation flux in response to various mechanical stresses. However, the biological interpretation and underlying mechanisms of cells resulting from Piezo1 activation remain elusive. This study elucidates Piezo1-mediated Ca2+ influx driven by channel activation and cellular behavior using novel Förster Resonance Energy Transfer (FRET)-based biosensors and single-cell imaging analysis. Results reveal that extracellular Ca2+ influx via Piezo1 requires intact caveolin, cholesterol, and cytoskeletal support. Increased cytoplasmic Ca2+ levels enhance PKA, ERK, Rac1, and ROCK activity, which have the potential to promote cancer cell survival and migration. Furthermore, we demonstrate that Piezo1-mediated Ca2+ influx upregulates membrane ruffling, a characteristic feature of cancer cell metastasis, using spatiotemporal image correlation spectroscopy. Thus, our findings provide new insights into the function of Piezo1, suggesting that Piezo1 plays a significant role in the behavior of cancer cells.

Distinct calcium regulation of TRPM7 mechanosensitive channels at plasma membrane microdomains visualized by FRET-based single cell imaging.

Transient receptor potential subfamily M member 7 (TRPM7), a mechanosensitive Ca2+ channel, plays a crucial role in intracellular Ca2+ homeostasis. However, it is currently unclear how cell mechanical cues control TRPM7 activity and its associated Ca2+ influx at plasma membrane microdomains. Using two different types of Ca2+ biosensors (Lyn-D3cpv and Kras-D3cpv) based on fluorescence resonance energy transfer, we investigate how Ca2+ influx generated by the TRPM7-specific agonist naltriben is mediated at the detergent-resistant membrane (DRM) and non-DRM regions. This study reveals that TRPM7-induced Ca2+ influx mainly occurs at the DRM, and chemically induced mechanical perturbations in the cell mechanosensitive apparatus substantially reduce Ca2+ influx through TRPM7, preferably located at the DRM. Such perturbations include the disintegration of lipid rafts, microtubules, or actomyosin filaments; the alteration of actomyosin contractility; and the inhibition of focal adhesion and Src kinases. These results suggest that the mechanical membrane environment contributes to the TRPM7 function and activity. Thus, this study provides a fundamental understanding of how the mechanical aspects of the cell membrane regulate the function of mechanosensitive channels.