Quantifying Acute Fuel and Respiration Dependent pH Homeostasis in Live Cells Using the mCherryTYG Mutant as a Fluorescence Lifetime Sensor.

Intracellular pH plays a key role in physiology, and its measurement in living specimens remains a crucial task in biology. Fluorescent protein-based pH sensors have gained widespread use, but there is limited spectral diversity for multicolor detection, and it remains a challenge to measure absolute pH values. Here we demonstrate that mCherryTYG is an excellent fluorescence lifetime pH sensor that significantly expands the modalities available for pH quantification in live cells. We first report the 1.09 Å X-ray crystal structure of mCherryTYG, exhibiting a fully matured chromophore. We next determine that it has an extraordinarily large dynamic range with a 2 ns lifetime change from pH 5.5 to 9.0. Critically, we find that the sensor maintains a p Ka of 6.8 independent of environment, whether as the purified protein in solution or expressed in live cells. Furthermore, the lifetime measurements are robustly independent of total fluorescence intensity and scatter. We demonstrate that mCherryTYG is a highly effective sensor using time-resolved fluorescence spectroscopy on live-cell suspensions, which has been previously overlooked as an easily accessible approach for quantifying intracellular pH. As a red fluorescent sensor, we also demonstrate that mCherryTYG is spectrally compatible with the ATeam sensor and EGFP for simultaneous dual-color measurements of intracellular pH, ATP, and extracellular pH. In a proof-of-concept, we quantify acute respiration-dependent pH homeostasis that exhibits a stoichiometric relationship with the ATP-generating capacity of the carbon fuel choice in E. coli. Broadly speaking, our work presents a previously unemployed methodology that will greatly facilitate continuous pH quantification.

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

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 pKa 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.