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

Abstract

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.

PIEZO1 regulates leader cell formation and cellular coordination during collective keratinocyte migration

“…work has linked PIEZO1-mediated Ca 2+ influx to impacting both focal adhesion dynamics [64][65][66] as well as Rac1 and RhoA levels. PIEZO1's effect on small GTPases has been shown to affect migration in both neural crest cells [67] and cancer cells [68], Cadherin remodeling in lymphatic endothelial cells [69], and macrophage mechanotransduction in iron metabolism [70]. We also observed that total levels of Rac1 and RhoA in healing monolayers are reduced in Yoda1-treated compared to DMSO-treated samples (S12 Fig) . While the downregulation of Rho GTPases provides an initial insight into the mechanism underlying PIEZO1-mediated inhibition of leader cells in collective migration, a detailed characterization of this relationship surpasses the scope of work covered within this paper.…”

“…Previous work has linked PIEZO1-mediated Ca 2+ influx to impacting both focal adhesion dynamics ( McHugh et al (2012) ; Li et al (2014) ; Yao et al (2022) ) as well as Rac1 and RhoA levels. PIEZO1’s effect on small GTPases has been shown to affect migration in both neural crest cells ( Canales Coutiño and Mayor (2021) ) and cancer cells ( Kim et al (2022) ), Cadherin remodeling in lymphatic endothelial cells ( Miroshnikova et al (2021) ), and macrophage mechanotransduction in iron metabolism ( Ma et al (2021) ). We also observed thattotal levels ofRac1 and RhoAin healing monolayers are reduced in Yoda1-treated compared to DMSO-treated samples( Figure 7—figure Supplement 1 ).…”

PIEZO1 regulates leader cell formation and cellular coordination during collective keratinocyte migration

“…Previous work has linked PIEZO1-mediated Ca 2+ influx to impacting both focal adhesion dynamics [64][65][66] as well as Rac1 and RhoA levels. PIEZO1's effect on small GTPases has been shown to affect migration in both neural crest cells [67] and cancer cells [68], Cadherin remodeling in lymphatic endothelial cells [69], and macrophage mechanotransduction in iron metabolism [70]. We also observed that total levels of Rac1 and RhoA in healing monolayers are reduced in Yoda1-treated compared to DMSOtreated samples (S12 Fig) . While the downregulation of Rho GTPases provides an initial insight into the mechanism underlying PIEZO1-mediated inhibition of leader cells in collective migration, a detailed characterization of this relationship surpasses the scope of work covered within this paper.…”

“…Previous work has linked PIEZO1-mediated Ca 2+ influx to impacting both focal adhesion dynamics [ 64 – 66 ] as well as Rac1 and RhoA levels. PIEZO1’s effect on small GTPases has been shown to affect migration in both neural crest cells [ 67 ] and cancer cells [ 68 ], Cadherin remodeling in lymphatic endothelial cells [ 69 ], and macrophage mechanotransduction in iron metabolism [ 70 ]. We also observed that total levels of Rac1 and RhoA in healing monolayers are reduced in Yoda1-treated compared to DMSO-treated samples ( S12 Fig ).…”

Pleiotropic physiological functions of Piezo1 in human body and its effect on malignant behavior of tumors

“…Both mesenchymal and amoeboid migration are closely related to the actin cytoskeleton ( Ruiz-Lafuente et al, 2021 ). Yoda1-induced Piezo1 activation increases intracellular calcium levels through extracellular calcium inward flow and release of calcium from endoplasmic reticulum storage, and upregulates the activity of a series of downstream effectors including PKA, ERK, Rac1 and ROCK, leading to cytosolic ruffling and thus promoting cancer cell migration ( Kim HS. et al, 2022 ).…”