Cytoskeletal activation of NHE1 regulates mechanosensitive cell volume adaptation and proliferation.

Mammalian cells can rapidly respond to osmotic and hydrostatic pressure imbalances during an environmental change, generating large fluxes of water and ions that alter cell volume within minutes. While the role of ion pump and leak in cell volume regulation has been well-established, the potential contribution of the actomyosin cytoskeleton and its interplay with ion transporters is unclear. We discovered a cell volume regulation system that is controlled by cytoskeletal activation of ion transporters. After a hypotonic shock, normal-like cells (NIH-3T3, MCF-10A, and others) display a slow secondary volume increase (SVI) following the immediate regulatory volume decrease. We show that SVI is initiated by hypotonic stress induced Ca influx through stretch activated channel Piezo1, which subsequently triggers actomyosin remodeling. The actomyosin network further activates NHE1 through their synergistic linker ezrin, inducing SVI after the initial volume recovery. We find that SVI is absent in cancer cell lines such as HT1080 and MDA-MB-231, where volume regulation is dominated by intrinsic response of ion transporters. A similar cytoskeletal activation of NHE1 can also be achieved by mechanical stretching. On compliant substrates where cytoskeletal contractility is attenuated, SVI generation is abolished. Moreover, cytoskeletal activation of NHE1 during SVI triggers nuclear deformation, leading to a significant, immediate transcriptomic change in 3T3 cells, a phenomenon that is again absent in HT1080 cells. While hypotonic shock hinders ERK-dependent cell growth, cells deficient in SVI are unresponsive to such inhibitory effects. Overall, our findings reveal the critical role of Ca and actomyosin-mediated mechanosensation in the regulation of ion transport, cell volume, transcriptomics, and cell proliferation.