Mechanosensitive regulation of cell volume and its implications in cell functions

Introduction: Cell volume is an essential cell biophysical property that responds sensitively to both intra- and extracellular physical and physiological changes. Mammalian cells can rapidly adjust to osmotic and hydrostatic pressure imbalances during an environmental change, triggering water and ion fluxes that can alter cell volume within minutes. The regulation of cell volume through ion fluxes has been well established. However, whether actomyosin-mediated cell mechanics and mechanosensation control cell volume is still unclear. In this work, we combine microfluidics, physical stimulations, and cell biology techniques to demonstrate how the interplay between actomyosin and Sodium-Hydrogen exchanger 1 (NHE1) plays a key role in cell volume regulation. We demonstrate that physical and physiological stimulations, such as hypotonic shock, mechanical stretching, and hydrostatic pressure, activate a mechanosensitive pathway in normal-like cells through actomyosin-NHE1 interplay, modulating cell volume and intracellular pH. Importantly, this active and adaptive cell volume regulatory system is lacking in many cancer cell lines. Moreover, we found that such cytoskeletal activation of NHE1 is associated with important cellular functions, including proliferation and migration. Overall, our study provides novel insights into the role of cytoskeletal mechanosensation in cell volume regulation, and underscores its significance in fluid-related physiological and pathological processes.

Materials and

Methods: In this study, we combined precision microfluidics with biomechanical tools to explore how cells actively and adaptively respond to various fluidic-based stimuli. We developed a microfluidic assay utilizing the fluorescence exclusion method, allowing us to quantitatively assess cell volume with high-throughput efficiency and single-cell resolution. Additionally, we engineered an advanced air-pressure system, suitable for both live-cell imaging and long-term cell culture, to examine the effects of hydrostatic pressure on cell dynamics and functions. We augmented these techniques with various of advanced indicators and fluorescent dyes to monitor the dynamics of intracellular calcium, potassium, and pH in response to fluidic or mechanical stimulations. Furthermore, we constructed mathematical models to elucidate the principles by which cell mechanics and electrophysiology coordinate during mechanosensitive cell volume regulation.

Results, Conclusions, and Discussions: In this work, we identified a previously unestablished role of actomyosin in cell volume regulation. Instead of generating contractile force that directly controls cell volume, fluidic and mechanical stimuli trigger actomyosin-mediated mechanosensation that activates NHE1. In normal-like cells, such as NIH-3T3 fibroblasts and MCF-10A breast epithelial cells, hypotonic shock, mechanical stretching, or reductions in hydrostatic pressure initiate calcium influx through the stretch-activated channel Piezo1, which in turn induces actomyosin remodeling. This remodeling leads to the activation of NHE1 via its interaction with the actin-binding partner ezrin, resulting in increased cell volume and proton efflux. Additionally, we demonstrate that such mechanosensitive activity induces substantial nuclear deformation and triggers a cascade of epigenetic remodeling, evidenced by significant changes in DNA methylation patterns and gene expression profiles. These changes subsequently influence cell proliferation through the ERK/MAPK pathway and cell migration via the "Osmotic Engine Model". Notably, this mechanosensitive modulation of cell volume is markedly absent in cancer cell lines, such as HT1080 fibrosarcoma and MDA-MB-231 breast cancer cells, or in 3T3 cells with Piezo1 knockout. Cancer cells and Piezo1 knockout cells also exhibit distinct responses in gene expression profiles, proliferation control, and migration behaviors, suggesting a significant mechanistic divergence from normal cells.

Overall, our study highlights the critical role of actomyosin and Piezo1 in the mechanosensitive regulation of NHE1 in regulating cell volume and intracellular pH. The absence of such regulation in cancer cells offers a novel insight into cancer cell biology and opens avenues for exploring mechanotransduction as a therapeutic target.

Acknowledgements (Optional):