Cytoskeletal activation of NHE1 regulates mechanosensitive cell volume adaptation and proliferation

Abstract

The regulation of mammalian cell volume is crucial for maintaining key cellular processes. Cells can rapidly respond to osmotic and hydrostatic pressure imbalances during environmental challenges, generating fluxes of water and ions that alter volume within minutes. While the role of ion pump and leak in cell volume regulation has been well-established, the role of the actomyosin cytoskeleton and its substantial interplay with ion transporters are still unclear. In this work, we discover a system of cell volume regulation controlled by cytoskeletal activation of ion transporters. Under hypotonic shock, NIH-3T3 and MCF-10A display a 20% secondary volume increase (SVI) following the initial regulatory volume decrease. We show that SVI is initiated by Ca2+influx through stretch activated channel Piezo1 and subsequent actomyosin remodeling. Rather than contracting cells, actomyosin triggers cell swelling by activating Na+-H+exchanger 1 (NHE1) through their co-binding partner ezrin. Cytoskeletal activation of NHE1 can be similarly triggered by mechanical stretch and attenuated by soft substrates. This mechanism is absent in certain cancer cell lines such as HT1080 and MDA-MB-231, where volume regulation is dominated by intrinsic response of ion transporters. Moreover, cytoskeletal activation of NHE1 during SVI induces nuclear deformation, leading to DNA demethylation and a significant, immediate transcriptomic response in 3T3 cells, a phenomenon that is absent in HT1080 cells. Overall, our findings reveal the central role of Ca2+and actomyosin-mediated mechanosensation in the regulation of ion transport, cell volume, DNA methylation, and transcriptomics.

Pump up the volume

“…Conversely, the intracellular ionic environment can influence cytoskeletal activity and force generation ( de Boer et al, 2023 ; Webb et al, 2011 ). Previous studies have shown that molecular interactions between actin, NHE1 and Akt (which is a target of PI3K) regulate actin organization, intracellular pH and cell migration, while recent work suggests that non-cancerous cells often use actin-NHE1 crosstalk to mediate mechanosensitive adaptations to environmental stimuli ( Denker et al, 2000 ; Denker and Barber, 2002 ; Meima et al, 2009 ; Ni et al, 2024 ).…”