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

Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macromolecules. 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.


Tables, Figures & Movies:
Movie S-2: Elevated cytosolic Ca 2+ levels due to addition of an IP 3 -generating agonist in pH-Lemon -GPI expressing HeLa cells.
Movie S-3: Effects of extracellular, non-permeabilizing buffer change on the vesicular pH of GPI vesicles.

S -2 -
Movie S-4: Addition of sodium azide and ammonium chloride to neutralize acidic vesicles in HeLa cells.

Cloning
Constructs for recombinant expression of pH-Lemon and SypHer in E.coli were purchased from Gene Universal Inc. (Newark, USA) using pET-28a(+) vector and the flanking restriction enzyme sites XhoI and HindIII, with an internal EcoRI site separating the FPs. Primers for cloning were purchased ThermoFisher Scientific (Vienna, Austria). PCR reactions were performed using Herculase II fusion DNA polymerase (Agilent, Santa Clara, USA). Following the manufacturer´s guidelines for cloning, different pH-Lemon constructs for mammalian expression were generated using pcDNA3.1(-) vector (ThermoFisher Scientific). The correct sequence of respective constructs was confirmed by sequencing (Microsynth, Vienna, Austria). Mitochondrial targeting of pH-Lemon was achieved using an N-terminal tandem dimeric repeat of the COX8 targeting sequence. Cytosolic targeting of pH-Lemon was observed after addition of the lysine rich motif LPPLERLTL derived from GP41 human immunodeficiency virus, using HindIII-Stop-NES-EYFP_rev and EcoRI-EYFP_for Primers. Targeting pH-Lemon to the endoplasmic reticulum was achieved using the N-terminal targeting sequence of calreticulin (CaR) and the ER-retention sequence KDEL at the C-terminal end. Primers used for generation of ER targeted pH-Lemon comprised NheI-CaR-mT2_for and HindIII-Stop-KDEL_rev. Localization of pH-Lemon at the outer mitochondrial membrane was achieved by attaching pH-Lemon to the N-terminal end of OMP25. pH-Lemon was localized to autophago(lyso-)somes using LC3B sequence at the C-terminus of the sensor. Therefore, LC3B was cloned from HeLa cell cDNA using EcoRI-Lc3B_for and XhoI-Stop-Lc3B_rev primers and cloned into pcDNA3.1(+) vector (ThermoFisher). Subsequently, mTurquoise2 and EYFP were cloned into the vector using the primers NheI-mT2_for, BamHI-Linker-mT2_rev, BamHI-EYFP_for and EcoRI-EYFP_rev. Finally, pH-Lemon GPI was generated using the membrane leading sequence and the glycosylation sequence of cadherin 13 at the N-and C-terminus, respectively, using sequentially the primers MLS1-XhoI-mT2_for and NheI-MLS2_for, both in combination with EcoRI-Linker-mT2_rev. EYFP of pH-Lemon GPI was amplified using GPI1-KpnI-EYFP_rev and HindIII-Stop-GPI2_rev, both in combination with EcoRI-EYFP followed by ligation of MLS-mTurquoise2 PCR product into pcDNA3.1(-) vector. A detailed list of primer sequences used for cloning is presented in Supplementary Table 1.

Calibration of pH-Lemon -GPI
After perfusion of HeLa cells witth extracellular buffers with pH 10.0 and pH 4.0, which were nonpermeabilizing, the fluorescence intensities of mTurquoise2 and EYFP on the cell surface were measured. By using the formula below, the pH could be calculated.
Formula 1: Calculation of pH values using ratio values.    S -8 - S -9 - Cl (middle image) or after wash-out (right image). Scale bar in the right image represents 10 µm.

Table S-1: Primers used to generate pH-Lemon constructs
S -10 - (a) Schemtaic illustration of the effect of sodium azide and ammonium chloride, as well as bafilomycin-A on vesicular pH. Vacuolar-type H + -ATPases (V-ATPases) located in the vesicular membrane generates an acidic vesicular pH by pumping protons into the vesicular lumen upon ATP consumption. Neutralizing agents like NaN 3 and NH 4 Cl diffuse into the vesicular lumen, buffering the high H + concentration by generating HN 3 and NH 4 + , thus leading to neutralization of the vesicle. Treatment of cells with bafilomycin-A inhibits the V-ATPases, preventing H + import. (b) HeLa cells treated with 0,5 µM bafilomycin-A for 70 minutes at 37°C (Baf-A) showed an increased average pH in the vesicular region compared to untreated DMSO cells (control). Data represents average ± SD, n=8 for both conditions, ***p<0.0001 using unpaired t-test. (c) pH over-time course of HeLa cells expressing pH-Lemon -GPI, either incubated with 0,5µM Baf-A (blue line) or with the same concentration of DMSO (control, grey line) for 70 minutes at 37°C. Buffers were exchanged using a gravity-based perfusion system. Vesicles were neutralized using a "2CA"-buffer containing NaN 3 and NH 4 Cl, pH=9.0. Data shows representative single cell responses. (d) Representative pseudocolored ratio-images of HeLa cells expressing pH-Lemon -GPI after incubation with DMSO or Baf-A using the same protocol as shown in panel c. Region of interest (ROI) is indicated as a dashed white line. Scale bar represents 10 µm. (e) Average pH values measured in the ROIs of DMSO treated control HeLa cells expressing pH-Lemon -GPI under basal and neutralizing (using NaN 3 and NH 4 Cl) conditions, and after wash-out as demonstrated in (c) and (d). Data represent average ± SD, n=8, **p<0.005, ***p≤0.0005 using one-way ANOVA following Tukey's post-hoc test. (f) Average pH measured in the ROIs of Baf-A treated pH-Lemon -GPI expressing HeLa cells at basal and neutralized conditions and after wash-out, according to the protocol demonstrated in (c) and (d). Data represent average ± SD, n=8, *p<0.05, ***p<0.0001 using one-way ANOVA with Tukey's post-hoc test.