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

Abstract

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.

Visualizing the pH inEscherichia colicoloniesviathe sensor protein mCherryEA allows high-throughput screening of mutant libraries

“…The mCherry variant with the I158E and Q160A amino acid exchanges, originally engineered to support excited-state proton transfer for generating a long Stokes shift variant, exhibits at neutral pH two excitation peaks corresponding to the protonated and deprotonated chromophore with a single emission peak (Piatkevich et al , 2010). Based on this property, the mCherry variant named mCherryEA, was found to function as a ratiometric pH sensor protein, because the protonation state of Glu158 is sensitive to the pH of the surrounding solution, which results in pH-dependent protonation of the chromophore (Rajendran et al , 2018). To generate a sensor plasmid encoding the mCherryEA, the gene was synthesized and cloned into the backbone of the expression plasmid pEKEx2, resulting in the plasmid pEKEx2_mCherryEA.…”

“…Following the identification of a mutant strain possessing a pH-dependent growth phenotype, the cytoplasmic pH of the isolated mutant is measured via fluorescent dyes (e.g. BCECF and SNARF), radioactive probes (Kashket, 1985; Han and Burgess, 2010) or genetically encoded sensor proteins (Martynov et al , 2018; Rajendran et al , 2018). For this purpose, different ratiometric pH responsive RFPs such as pHluorin and pHred have been developed, both possessing a pK a of 6.9 but different intrinsic fluorescence properties (Miesenböck et al , 1998; Tantama et al , 2011; Martynov et al , 2018).…”

“…For this purpose, different ratiometric pH responsive RFPs such as pHluorin and pHred have been developed, both possessing a pK a of 6.9 but different intrinsic fluorescence properties (Miesenböck et al , 1998; Tantama et al , 2011; Martynov et al , 2018). Recently, the mCherry variant mCherryEA was shown to be an effective ratiometric red fluorescent protein pH biosensor possessing a pK a of 7.3 (Rajendran et al , 2018). This is close to the range of internal pH values reported for E. coli (7.4-7.9) (Slonczewski et al , 2009), making this sensor protein well suited for applications in E. coli .…”

Visual pH Sensors: From a Chemical Perspective to New Bioengineered Materials

“…Most of FPs used for living cell pH determination are variants of green and yellow fluorescent proteins, not spectrally compatible for dual-compartment imaging. Using a double mutant (I158E/Q160A) of the red fluorescent protein mCherry (pdb entry ID 3nt3), M. Tantama and coworkers developed an effective ratiometric pH sensor with a pKa of 7.3 [ 128 , 142 ]. This variant of mCherry protein shows an activity and metabolism-dependent pH dynamics in cultured primary neurons and neuroblastoma cells.…”

Cancer and pH Dynamics: Transcriptional Regulation, Proteostasis, and the Need for New Molecular Tools

“…To combat some of the issues with pHluorin, red-shifted fluorescent proteins (RFP) that function as pH biosensors have been recently developed, including pHred [ 105 ], pHuji [ 107 ], and more recently mCherry variants [ 108 , 119 ]. The mCherry EA mutant is a bright ratiometric pH sensor that has been used in combination with pHluorin to measure pH simultaneously in both the cytosol and mitochondria [ 108 ]. While red-shifted fluorescence does improve tissue and tumor imaging compatibility, RFP-based pHi sensors suffer from low brightness and significant aggregation, which make quantification more challenging than their GFP-based counterparts.…”