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A DNA nanodevice for mapping sodium at single-organelle resolution

Nature Biotechnology (2023)Cite this article

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Abstract

Cellular sodium ion (Na+) homeostasis is integral to organism physiology. Our current understanding of Na+ homeostasis is largely limited to Na+ transport at the plasma membrane. Organelles may also contribute to Na+ homeostasis; however, the direction of Na+ flow across organelle membranes is unknown because organellar Na+ cannot be imaged. Here we report a pH-independent, organelle-targetable, ratiometric probe that reports lumenal Na+. It is a DNA nanodevice containing a Na+-sensitive fluorophore, a reference dye and an organelle-targeting domain. By measuring Na+ at single endosome resolution in mammalian cells and Caenorhabditis elegans, we discovered that lumenal Na+ levels in each stage of the endolysosomal pathway exceed cytosolic levels and decrease as endosomes mature. Further, we find that lysosomal Na+ levels in nematodes are modulated by the Na+/H+ exchanger NHX-5 in response to salt stress. The ability to image subcellular Na+ will unveil mechanisms of Na+ homeostasis at an increased level of cellular detail.

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Fig. 1: RatiNa is a ratiometric, pH-independent and specific reporter of Na+.
Fig. 2: In-cell and in vivo calibration of RatiNa to measure lysosomal Na+.
Fig. 3: RatiNa captures physiological changes in organellar Na+.
Fig. 4: Lysosomal Na+ transport is vital for salt adaptation in C. elegans.

Data availability

The raw data supporting Figs. 1–4 are available for public access at Figshare: https://doi.org/10.6084/m9.figshare.23938503 (ref. 67). Source data are provided with this paper.

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Acknowledgements

We thank E. Perozo (University of Chicago), G. Ruvkun (Massachusetts General Hospital), A. Concepcion (University of Chicago) and A. L. Chun for valuable discussions and input on the manuscript. We thank C. Labno at the integrated light microscopy facilities at the University of Chicago for technical help and T. Wu for assistance with qRT–PCR. We thank K. Nehrke (University of Rochester Medical Center) for sharing NHX-5::GFP plasmid. Y.K. acknowledges funding from NIH grants 1DP1GM149751-01, 1R01NS112139-01A1, 1R21NS114428-01, 1R21HL161825-01A1 and 1R01GM147197-01 (to Y.K. and R.R.); FA9550-19-0003 from the AFOSR; HFSP grant RGP0032/2022 and the Ono Pharma Foundation.

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Authors and Affiliations

  1. Department of Chemistry, The University of Chicago, Chicago, IL, USA

    Junyi Zou, Koushambi Mitra, Palapuravan Anees, Daphne Oettinger, Joseph R. Ramirez, Aneesh Tazhe Veetil, Priyanka Dutta Gupta & Yamuna Krishnan

  2. Neuroscience Institute, The University of Chicago, Chicago, IL, USA

    Junyi Zou, Koushambi Mitra, Palapuravan Anees, Daphne Oettinger, Joseph R. Ramirez, Aneesh Tazhe Veetil, Priyanka Dutta Gupta, Jayson J. Smith, Paschalis Kratsios & Yamuna Krishnan

  3. Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Koushambi Mitra, Palapuravan Anees & Yamuna Krishnan

  4. Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Rajini Rao

  5. Department of Neurobiology, The University of Chicago, Chicago, IL, USA

    Jayson J. Smith & Paschalis Kratsios

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Contributions

J.Z. and Y.K. designed every aspect of the study. K.M., A.T.V. and J.R. designed and synthesized the CG dye. J.Z. made, characterized and validated RatiNa in vitro, in cells and in vivo. J.Z. and P.A. performed Na+ measurements in worms. D.O. performed all the brood size experiments. P.A. performed pH measurements in worms. J.J.S. and P.K. made the NHX-5::GFP worm. P.D.G. and R.R. provided NHE6 KO macrophages. J.Z. and Y.K. analyzed and interpreted all data. J.Z., K.M. and Y.K. wrote the paper. All authors provided input on the manuscript.

Corresponding author

Correspondence to Yamuna Krishnan.

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Competing interests

The authors declare no competing interests. Y.K. is a cofounder of Esya Inc and MacroLogic Inc, which use DNA nanodevices to develop diagnostics and therapeutics, respectively.

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Nature Biotechnology thanks Haoxing Xu for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Chicago green (CG) is a pH insensitive and Na+ selective fluorophore.

a, Na+ sensing mechanism of CG b, Excitation (black) and emission (green) spectra of 100 nM CG increases with increasing Na+. c, Dissociation constant (Kd) of CG for Na+ does not vary with pH from pH 4.5–7.4. d, Individual in vitro calibration profiles of RatiNa at different pH in Fig. 1d. e, Kd of RatiNa for Na+ at different pH values as calculated from d. Kd of RatiNa is invariant from pH 4.5–7.4. f, RatiNa Na+ calibration profile from 1 mM to 200 mM Na+ in linear scale. g, RatiNa yields a Kd of 4.5 M for K+ and is 27-times more selective for Na+. Data in c-g are presented as mean values ± s.d. from n = 3 independent experiments.

Source data

Extended Data Fig. 2 Calibration of RatiNa and its stability in lysosomes of RAW 264.7 macrophages.

a, Lysosomes in RAW 264.7 macrophages prelabeled with TMR-dextran (cyan) and imaged at different chase times of RatiNaAT (magenta). Images are representative from n = 2 independent experiments. b, Histogram of intensity ratios of RatiNaAT/TMR-dextran (R/G) in single lysosomes at different chase times. Decreasing R/G indicates more DNA degradation. c, DNA degradation as a function of time quantified from b. Data are presented as mean values ± s.e.m. from n = 2 independent experiments. d, Normalized RatiNa signal (G/R) of n = 127 single lysosome in RAW macrophages versus the normalizing dye signal (R). No correlation between G/R and R, indicates RatiNa output depends only on Na+ level and independent of probe concentration. e, Fluorescence images of RatiNaAT labeled RAW 264.7 macrophages in CG (G) and ATTO (R) channels. Representative images from n = 3 independent experiments. f, Images of RatiNa-labeled lysosomes clamped at high and low Na+ of 145 mM and 5 mM in native lysosomes. G/R heat maps show RatiNa response in native lysosomes. Representative images from n = 3 independent experiments. g, Histogram of G/R values of RatiNa-labeled single lysosomes. Despite autofluorescence in each system, the fold change in G/R signal of RatiNa in lysosomes of RAW 264.7 macrophages is comparable to that in C. elegans and on beads. h, Schematic of workflow from raw images to Na+ heat maps of single organelles. Fluorescent images in the CG (G) and ATTO (R) channels are used to construct the G/R image. Then the Na+ heatmap was generated from the calibration curve of G/R to [Na+].

Source data

Extended Data Fig. 3 RatiNa is targeted specifically to each organelle on the endolysosomal pathway.

a, Representative images of C. elegans coelomocytes reveal negligible off-target labeling between RatiNaAT and indicated endocytic markers and chase times. b, RatiNa is targeted to a specific endocytic organelle at fixed chase time. Colocalization is calculated as percentage of organelles having both lumenal RatiNaAT and membrane marker fluorescence over all RatiNaAT containing organelles. Data are presented as mean values ± s.d. from n = 3 independent experiments. (n = 9 coelomocytes, 6 worms for 5 min chase of RAB-5::GFP; n = 7 coelomocytes, 6 worms for 17 min chase of RAB-5::GFP; n = 4 coelomocytes, 4 worms for 5 min chase of RAB-7::GFP; n = 6 coelomocytes, 5 worms for 17 min chase of RAB-7::GFP; n = 7 coelomocytes, 6 worms for 17 min chase of LMP-1::GFP; n = 9 coelomocytes, 6 worms for 60 min chase of LMP-1::GFP).

Source data

Extended Data Fig. 4 Na+ transporter mutants are less resistant to high salt stress and show elevated lysosomal Na+.

a, Brood sizes of Na+ transporter mutants upon high salt stress. Na+ transporter deletion mutants cannot adapt to 400 mM NaCl unlike WT worms. Arrowheads indicate points of lysosomal Na+ measurement that is, worms acutely stressed (Ac) with 200 mM. Data are presented from n = 3 independent experiments. b, Representative Na+ heatmaps of Na+ transporter deletion mutant worms. c, qRT-PCR shows that mRNA expression level of Na+ transporters do not change appreciably upon salt stress in N2 worms. Fold change in mRNA levels of Na+ transporters between Ac and Ch conditions is shown. act-1 was used as reference gene. Data are from n = 2–3 independent experiments. d. Lysosomal Na+ levels of Na+ transporter mutant worms under unstressed (Ns) and acutely stressed (Ac) condition. High lysosomal Na+ is observed in all investigated Na+ transporter mutants: 9 mM in Ns and 63 mM in Ac for nhx-5(−) worms. 24 mM in Ns and 51 mM in Ac for nhx-7(−) worms. 34 mM in Ns and 55 mM in Ac for nhx-8(−) worms. 25 mM in Ns and 36 mM in Ac for ncx-2(−) worms. Two sample two-tailed t test was used for statistical analysis assuming equal variance. ***P = 2.5 × 10−13, ***P = 1.7 × 10−10, ***P = 2.2 × 10−5, *P = 0.016 for nhx-5(−), nhx-7(−), nhx-8(−), ncx-2(−). All error bars are presented as mean values ± s.d.

Source data

Extended Data Fig. 5 Lysosomal pH of salt stressed worms.

a. PAGE analysis of the I-switch-based pH reporter module denoted Br-I-switch19. DD strand has Alexa488 as a donor, DA strand has Alexa647 as acceptor. b. pH calibration curve of Br-I-switch shows ~20 fold change of D/A signal from pH 5.0 to 6.0, with highest sensitivity near pH 5.5, the pH of coelomocyte lysosomes35. Data are presented as mean values ± s.d. from n = 3 independent experiments c. pH measurement of single lysosome of N2 and nhx-5(−) worms in unstressed (Ns) and acutely salt stressed (Ac) conditions. Ns and Ac nhx-5(−) worms show lower pH. Error bar represents ± s.d. Two sample two-tailed t-test was used for statistical analysis assuming equal variance. ***P = 1.4 × 10-120, ***P = 7.4 × 10-88, P = 0.25, *P = 0.042 for N2 Ns to N2 Ac, nhx-5(−) Ns to nhx-5(−) Ac, N2 Ns to nhx-5(−) Ns, N2 Ac to nhx-5(−) Ac. d. Representative images of Br-I-switch in Donor (D), Acceptor (A) FRET (D/A) channels and respective pH heatmaps.

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Extended Data Fig. 6 Mutants lacking key lysosomal genes adapt to high salt stress differently.

a, Brood sizes of deletion mutants of lysosomal storage disorder genes. Data are presented as mean values from n = 3 independent experiments. gba-3(−) and ctns-1(−), clh-6(−) and XT7 (cln-3.2(−);cln3.3(−); cln-3.1(−)) are worm models Gaucher’s disease, Cystinosis, osteopetrosis and Batten’s disease respectively, and more susceptible to chronic stress than N2 worms. b, A deletion mutant of F13H10.3, the C. elegans homolog of human SLC38A9, denoted (slc38a9(−)) is more susceptible to Ac salt stress. Brood sizes are presented as mean values from n = 3 independent experiments. Arrows indicate points of lysosomal Na+ measurement that is, worms acutely stressed (Ac) with 200 mM. c, Representative Na+ heatmaps of slc38a9(−) worms in Ns and Ac. d, Lysosomal Na+ measurements in the indicated slc38a9(−) worms with and without salt stress. Higher lysosomal Na+ level of 52 mM in Ac worms compared to 39 mM in Ns worms, Error bar represents ± s.d. Two sample two-tailed t-test was used for statistical analysis assuming equal variance. P = 0.4, ***P = 6.8 × 10−4 for N2 Ns to slc38a9(−) Ns, slc38a9(−) Ns to slc38a9(−) Ac.

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Zou, J., Mitra, K., Anees, P. et al. A DNA nanodevice for mapping sodium at single-organelle resolution. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01950-1

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