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Single-cell intracellular pH dynamics regulate the cell cycle by timing G1 exit and the G2 transition

Transient changes in intracellular pH (pHi) regulate normal cell behaviors like migration and proliferation, while pHi dynamics are dysregulated in cancer. However, it is unclear how spatiotemporal pHi dynamics influence single-cell behaviors. We performed single-cell pHi measurements in normal and cancer cell lines and measured spatiotemporal pHi dynamics during cell cycle progression. Single-cell pHi is dynamic (0.16±0.07 pH units) throughout the cell cycle, with decreased pHi at G1/S, late S, and mitosis and increased pHi in mid-S and G2/M. We next experimentally manipulated pHi and measured effects on cell cycle progression. We show that decreased pHi is a permissive cue for G1 exit, with decreased pHi shortening G1 and increased pHi elongating G1. We also show that dynamic pHi is required for S phase timing, as high pHi elongates S phase and low pHi inhibits S/G2 transition. This work shows dynamic pHi is necessary for cell cycle progression in single cells. SUMMARY Spear et al. characterize an approach for measuring single-cell intracellular pH (pHi) and monitoring single-cell pHi dynamics during cell cycle progression. By tracking pHi in single cells, Spear et al. confirm prior work at the population level showing an increase in pHi at the G2/M transition. However, single-cell pHi measurements reveal new features of pHi dynamics during cell cycle progression, including significantly decreased pHi at the G1/S boundary, S/G2 boundary, and just prior to division, and increases during mid-S phase and G2. Using pHi manipulation, Spear et al. determine that decreased pHi shortens G1 phase and increased pHi elongates G1 phase, indicating low pHi is a cue for G1 exit. S phase progression was also reliant on temporal pHi changes, suggesting pHi dynamics are necessary for cell cycle progression.