It is extremely challenging to quantitate lumenal Ca2+ in acidic Ca2+ stores of the cell because all Ca2+ indicators are pH sensitive, and Ca2+ transport is coupled to pH in acidic organelles. We have developed a fluorescent DNA-based reporter, CalipHluor, that is targetable to specific organelles. By ratiometrically reporting lumenal pH and Ca2+ simultaneously, CalipHluor functions as a pH-correctable Ca2+ reporter. By targeting CalipHluor to the endolysosomal pathway, we mapped lumenal Ca2+ changes during endosomal maturation and found a surge in lumenal Ca2+ specifically in lysosomes. Using lysosomal proteomics and genetic analysis, we found that catp-6, a Caenorhabditis elegans homolog of ATP13A2, was responsible for lysosomal Ca2+ accumulation—an example of a lysosome-specific Ca2+ importer in animals. By enabling the facile quantification of compartmentalized Ca2+, CalipHluor can expand the understanding of subcellular Ca2+ importers.
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The data that support the plots within this paper and the findings of this study are available from the corresponding author upon reasonable request.
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We thank J. Kuriyan and M. Zajac for valuable comments. We thank the Integrated Light Microscopy facility at the University of Chicago, the Caenorhabditis Genetic Center for strains, and Ausubel Lab for Arhinger Library RNAi clones. We thank the Krainc lab at Northwestern University (Chicago, IL, USA) for the fibroblast cells harboring mutations in ATP13A2 (L6025). HDF cells were a kind gift from the lab of J. Rowley (University of Chicago, Chicago, IL, USA). This work was supported by the University of Chicago Women’s Board; a Pilot and Feasibility award from an NIDDK center grant no. P30DK42086 to the University of Chicago Digestive Diseases Research Core Center; MRSEC grant no. DMR-1420709; the National Center for Advancing Translational Sciences of the National Institutes of Health through grant no. 1UL1TR002389-01 that funds the Institute for Translational Medicine, Chicago Biomedical Consortium, with support from the Searle Funds at The Chicago Community Trust, C-084; and University of Chicago start-up funds to Y.K. Y.K. is a Brain Research Foundation Fellow.
The authors declare no competing interests.
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(a) Chemical structure of Rhod-5F. (b) Fluorescence emission spectra of Rhod-5F(green) and Alexa Fluor 647 (red) with increasing [Ca2+] upon exciting Rhod-5F and Alexa Fluor 647 at 560 nm and 650 nm, respectively. (c) Normalized O/R ratio of Rhod-5F/Alexa Fluor 647 with increasing [Ca2+] at pH 7.2 and 5.5. Error bar represents mean + s.e.m. of three independent experiments.
(a) Gel showing the conjugation of Rhod-5F to D2-DBCO strand. Gels were visualized in EtBr and TMR channels. (b) Native PAGE showing formation of CalipHluorLy. Gels were visualized in Alexa Fluor 488, TMR and Alexa Fluor 647 channels. (c) Schematic of working principle of CalipHluor. pH-induced FRET changes between Alexa Fluor 488 (donor, green sphere) and Alexa Fluor 647 (acceptor, red star) is used to report pH ratiometrically. A Ca2+ sensitive fluorophore (Rhod-5F, yellow diamond) and Alexa Fluor 647 report Ca2+ (at a given pH) ratiometrically by direct excitation of each dye. (d) Gel showing the conjugation of Rhod-5F to O3-DBCO strand. Gels were visualized in EtBr and TMR channels. (e) Native PAGE showing formation of CalipHluor. Gels were visualized in Alexa Fluor 488, TMR and Alexa Fluor 647 channels. (f) Emission spectra of CalipHluor at pH values ranging from 7.5 to 5.0 upon excitation at 488 nm. (g) Normalized ratio of fluorescence intensity of donor to that of acceptor (D/A) of CalipHluor as a function of pH. (D λex = 495 nm, λem = 520 nm; A λex = 495 nm, λem = 665 nm). Gels were performed twice independently. Error bar represents mean + S.E.M of three independent experiments.
Representative pseudo color images of coelomocytes labeled with CalipHluorLy and clamped at the indicated (a) pH and (b) free [Ca2+] at pH 5.5. Scale bar 5 μm. Experiments were repeated three times independently with similar results.
Supplementary Figure 4 Comparison of in vitro and in vivo pH and Ca2+ calibration profile of CalipHluorLy.
(a-d) D/A ratios of CalipHluorLy as a function of pH clamped at different amounts of added [Ca2+]. (e-h) Normalized O/R ratios of CalipHluorLy as a function of free [Ca2+] clamped at different pH points. For in vivo n = 10 worms; 15 cells and 50 endosomes were quantified; in vitro n = 2. Error bar represents mean + s.e.m.
(a-c) Representative confocal images taken 5 min, 17 min, and 60 min following injection of CalipHluorA647 in worms expressing GFP::RAB-5, GFP::RAB-7 and LMP-1::GFP. Scale bar 5 μm. Experiment was performed once. n = 10 worms.
a) catp-6 rescues lethality of cup-5 +/−. Representative images showing the number of progeny of cup-5 +/− worms in plates containing RNAi bacteria of mrp-4 (positive control), clh-6, catp-6, catp-5 and e.v. (control). Experiments were repeated twice independently with similar results. b) RT-PCR analysis of total RNA isolated from C. elegans pre- and post-RNAi. Lanes correspond to PCR-amplified cDNA of the indicated gene product isolated from wild type without RNAi treatment (denoted by gene name) and the corresponding dsRNA-fed worms (denoted as. ‘— gene name) c) Representative images of worms expressing LMP-1::GFP (green) in the background of various indicated RNAi, which were injected with CalipHluorLyA647 (red) and imaged 60 mins post-injection. Scale bar: 5 μm. d) Quantification of colocalization between the CalipHluorLyA647 and GFP in LMP-1::GFP worms. n = 10 cells; error bars represent mean + s.e.m.
a) Schematic of the working principle of CalipHluormLy. An Oregon Green based pH sensor (green sphere), an ion insensitive Alexa Fluor 647 (red star) and a Ca2+ sensitive fluorophore (Rhod-5F, yellow diamond). Calibration curves comparing in vitro (red) and on beads (orange) calibration at; b) pH 4.6 and c) pH 5.1. Comparison of d) Kd and e) Fold change (FC) in O/R of CalipHluorLy (pink) and CalipHluormLy (black). f) Representative images Comparison of g) Fold change (FC) in O/R and h) Kd of CalipHluormLy in vitro (pink), on beads (orange) and in cellulo (gray). (n = 5 cells; 30 endosomes; n = 60 beads). Experiments were performed three times independently. *Error is obtained from Hill equation fit. Error bars represent mean + s.e.m. Scale bar: 10 µm.
a)-b) CalipHluormLy internalization by primary human skin fibroblasts is competed out by excess maleylated BSA (mBSA, 10 μM), revealing that uptake is mediated by scavenger receptors. Cells are imaged in Alexa Fluor 647 channel. AF: autofluorescence. Scale bar: 10 µm. Experiments were performed in triplicate. Error bars indicate the mean of three independent experiments +/− s.e.m. (n = 25 cells).
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Narayanaswamy, N., Chakraborty, K., Saminathan, A. et al. A pH-correctable, DNA-based fluorescent reporter for organellar calcium. Nat Methods 16, 95–102 (2019). https://doi.org/10.1038/s41592-018-0232-7
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