Quantitative measurement of pH dynamics in living cells using the mCherry‐TYG red fluorescent protein as a lifetime sensor

Abstract

pH regulation plays a crucial role in mammalian and bacterial cell metabolism, protein function, apoptotic signaling, and a wide range of other physiological processes. Monitoring intracellular pH after environmental perturbations would further knowledge about cellular activities in both healthy and disease states in mammalian cells, as well as mechanism of infection and antibiotic resistance in bacterial cells. Non‐optical techniques such as x‐ray crystallography and NMR cannot resolve live‐cell dynamics, and many fluorescent dye or protein‐based sensors are susceptible to concentration and intensity dependence of the fluorophore, sample thickness, and photo‐bleaching, making lab‐to‐lab translation difficult without extensive calibration. Therefore, there remains a need for options to quantitatively measure pH dynamics in live specimens. As an intrinsic property of fluorophores, fluorescence lifetime does not rely on protein concentration, method of measurement or fluorescence intensity. The utilization of FRET pairs in fluorescence lifetime imaging microscopy (FLIM), has shown applicability of lifetime measurement for dynamic quantitation, however dynamic range can be limited. Here we present in vitro and live‐cell characterization of a red monomeric pH sensitive mCherry mutant mCherry‐TYG for robust measurement of live bacterial and mammalian pH in real‐time. We were successfully able to translate lifetime measurements with ease, showing pH calibration of both isolated protein and bacterial suspensions overlaying with a 2 ns dynamic range between pH 5.5 and 9.0. We further tested applicability in monitoring environmental perturbations by quantifying changes in pH as a function of energy source in e. coli. Additionally, we are currently exploring application for monitoring receptor trafficking, endocytosis and degradation in mammalian cells. Our results demonstrate the possibility of red shifted protein‐based lifetime sensors as a robust tool for pH calibration in live cells.Support or Funding InformationNIH/NINDS R21 NS092010, R21 NS106319NIH/NEI R21 EY026425This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.