pH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments

Abstract

Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macro-molecules. Here, we engineered a novel genetically encoded pH probe by fusing the pH-stable cyan fluorescent protein (FP) variant, mTurquoise2, to the highly pH-sensitive enhanced yellow fluorescent protein, EYFP. This approach yielded a ratiometric biosensor—referred to as pH-Lemon—optimized for live imaging of distinct pH conditions within acidic cellular compartments. Protonation of pH-Lemon under acidic conditions significantly decreases the yellow fluorescence while the cyan fluorescence increases due to reduced Förster resonance energy transfer (FRET) efficiency. Because of its freely reversible and ratiometric responses, pH-Lemon represents a fluorescent biosensor for pH dynamics. pH-Lemon also shows a sizable pH-dependent fluorescence lifetime change that can be used in fluorescence lifetime imaging microscopy as an alternative observation method for the study of pH in acidic cellular compartments. Fusion of pH-Lemon to the protein microtubule-associated protein 1A/1B-light chain 3B (LC3B), a specific marker of autophagic membranes, resulted in its targeting within autolysosomes of HeLa cells. Moreover, fusion of pH-Lemon to a glycophosphatidylinositol (GPI) anchor allowed us to monitor the entire luminal space of the secretory pathway and the exoplasmic leaflet of the plasma membrane. Utilizing this new pH probe, we revealed neutral and acidic vesicles and substructures inside cells, highlighting compartments of distinct pH throughout the endomembrane system. These data demonstrate, that this novel pH sensor, pH-Lemon, is very suitable for the study of local pH dynamics of subcellular microstructures in living cells.

Time Evolution of Local pH Around a Photo‐Acid in Water and a Polymer Hydrogel: Time Resolved Fluorescence Spectroscopy of Pyranine

“…Acid‐base reaction is one of the fundamental chemical reactions in nature and has implications in many natural and biological processes . The dissociation equilibrium of an acid HA into proton and its deprotonated species (A − ) is, …”

“…The ratio of the signal (i. e. fluorescence intensity) from HA* and A − * in the excited state has been used for measuring intra‐cellualr pH, apoptosis, and intracellular hypoxia …”

“…The ratio of the signal (i. e. fluorescence intensity) from HA* and A À * in the excited state has been used for measuring intracellualr pH, [1][2] apoptosis, [3] and intracellular hypoxia. [4] Acidity of a photo-acid (e. g. pyranine, HPTS, Scheme 1), is low in the ground state and extremely high in the electronically excited state (because of redistribution of the electronic charges).…”

Synthesis and Supramolecular Functional Assemblies of Ratiometric pH Probes

“…[169] Recent examples demonstrated that genetically encoded protein pH probes couldb ee mployed to image cellular compartments. [170] The balance betweenc ovalent and non-covalent forces in protein-dye hybridsw as used to develop fluorescent ratiometric probes for intracellular pH. Interactions between serum albumina nd squaraine dye nanoparticles were employed to control the degree of assembly of the hybrid nanosized system.…”

Purification and Application of Genetically Encoded Potassium Ion Indicators for Quantification of Potassium Ion Concentrations within Biological Samples

“…Today, a wide variety of these FRET-based indicators is available, including sensors for various metal ions, pH, cell metabolites, or even small molecules, often having short half-lives (Bischof et al, 2019; Burgstaller et al, 2019; Imamura et al, 2009; Tanimura, 2011). All of these probes are based on diverse FP variants, enabling a FRET-based read-out, fused to a specific analyte-binding domain, undergoing a conformational rearrangement upon analyte binding.…”