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The regulation of pH is essential for proper organelle function, and organelle-specific changes in pH often reflect the dynamics of physiological signaling and metabolism. For example, mitochondrial energy production depends on the proton gradient maintained between the alkaline mitochondrial matrix and neutral cytosol. However, we still lack a quantitative understanding of how pH dynamics are coupled between compartments and how pH gradients are regulated at organelle boundaries. Genetically encoded pH sensors are well suited to address this problem because they can be targeted to specific subcellular locations and they facilitate live, single-cell analysis. However, most of these pH sensors are derivatives of green and yellow fluorescent proteins that are not spectrally compatible for dual-compartment imaging. Therefore, there is a need for ratiometric red fluorescent protein pH sensors that enable quantitative multicolor imaging of spatially resolved pH dynamics. In this work, we demonstrate that the I158E/Q160A mutant of the red fluorescent protein mCherry is an effective ratiometric pH sensor. It has a p K  a of 7.3 and a greater than 3-fold change in ratio signal. To demonstrate its utility in cells, we measured activity and metabolism-dependent pH dynamics in cultured primary neurons and neuroblastoma cells. Furthermore, we were able to image pH changes simultaneously in the cytosol and mitochondria by using the mCherryEA mutant together with the green fluorescent pH sensor, ratiometric-pHluorin. Our results demonstrate the feasibility of studying interorganelle pH dynamics in live cells over time and the broad applicability of these sensors in studying the role of pH regulation in metabolism and signaling.

Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry