Roles for Spatiotemporal Intracellular pH Dynamics in Single-Cell Phenotypes and Behaviors

Transient changes in intracellular pH (pHi) regulate normal cell behaviors such as proliferation, differentiation, migration, and apoptosis. Most studies investigating pHi-dependent cell behaviors have used population measurements, pH-sensitive dyes, and non-physiological experimental conditions. Thus, the field lacks knowledge of accurate spatiotemporal pHi measurements at the single-cell level. Many pH-dependent cell behaviors are composed of highly-ordered processes that occur on the minutes to hours timescale within an organism, so understanding spatiotemporal pHi dynamics during these processes is crucial. In addition to pHi regulating normal cell biology, dysregulated pHi is an emerging hallmark of cancer. Cancer cells have a constitutively increased pHi compared to normal cells of the same tissue, and this is an early event in cancer development. Prior work has identified that genetic, phenotypic, and extracellular heterogeneity are drivers of cancer progression, but we lack knowledge of the pHi heterogeneity present within a tumor and whether pHi heterogeneity is biologically meaningful. Here, we measure spatiotemporal pHi in single cells to improve our understanding of pHi-driven cell behaviors in both normal and cancer cells. We characterize spatial pHi heterogeneity in 2D cell cultures and 3D spheroid models, showing that pHi heterogeneity increases in 3D spheroids compared to 2D cultures. We measure single-cell temporal pHi dynamics on a short timescale (minutes) during growth factor stimulation and on a longer timescale (hours) during cell cycle progression. We determine that single-cell pHi dynamics are correlated with cell cycle phases and additionally find that pHi dynamics are necessary for correct timing of cell cycle phase transitions and successful cell division. Finally, we show that increasing pHi in normal cells can induce cytokinesis defects and cancer-associated phenotypes such as multinucleation, micronuclei, and entosis. We demonstrate the significance of both spatial and temporal pHi dynamics in regulating single-cell biology and lay a framework for future studies to investigate the molecular mechanisms of how single-cell pHi drives single-cell behaviors, especially in cancer.