Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A DNA nanomachine chemically resolves lysosomes in live cells


Lysosomes are multifunctional, subcellular organelles with roles in plasma membrane repair, autophagy, pathogen degradation and nutrient sensing. Dysfunctional lysosomes underlie Alzheimer’s disease, Parkinson’s disease and rare lysosomal storage diseases, but their contributions to these pathophysiologies are unclear. Live imaging has revealed lysosome subpopulations with different physical characteristics including dynamics, morphology or cellular localization. Here, we chemically resolve lysosome subpopulations using a DNA-based combination reporter that quantitatively images pH and chloride simultaneously in the same lysosome while retaining single-lysosome information in live cells. We call this technology two-ion measurement or 2-IM. 2-IM of lysosomes in primary skin fibroblasts derived from healthy individuals shows two main lysosome populations, one of which is absent in primary cells derived from patients with Niemann–Pick disease. When patient cells are treated with relevant therapeutics, the second population re-emerges. Chemically resolving lysosomes by 2-IM could enable decoding the mechanistic underpinnings of lysosomal diseases, monitoring disease progression or evaluating therapeutic efficacy.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Design and characterization of ChloropHore.
Fig. 2: Trafficking pathway of ChloropHore in human dermal fibroblasts.
Fig. 3: Intracellular calibration of ChloropHore and ChloropHoreLy.
Fig. 4: 2-IM chemically resolves lysosome populations.

Data availability

The data that support the plots within this paper and the findings of this study are available from the corresponding author upon reasonable request.


  1. 1.

    Settembre, C., Fraldi, A., Medina, D. L. & Ballabio, A. Signals from the lysosome: a control centre for cellular clearance and energy metabolism. Nat. Rev. Mol. Cell Biol. 14, 283–296 (2013).

    CAS  Article  Google Scholar 

  2. 2.

    Fraldi, A., Klein, A. D., Medina, D. L. & Settembre, C. Brain disorders due to lysosomal dysfunction. Annu. Rev. Neurosci. 39, 277–295 (2016).

    CAS  Article  Google Scholar 

  3. 3.

    Pu, J., Guardia, C. M., Keren-Kaplan, T. & Bonifacino, J. S. Mechanisms and functions of lysosome positioning. J. Cell Sci. 129, 4329–4339 (2016).

    CAS  Article  Google Scholar 

  4. 4.

    Wasmeier, C., Hume, A. N., Bolasco, G. & Seabra, M. C. Melanosomes at a glance. J. Cell Sci. 121, 3995–3999 (2008).

    CAS  Article  Google Scholar 

  5. 5.

    Faurschou, M. & Borregaard, N. Neutrophil granules and secretory vesicles in inflammation. Microbes Infect. 5, 1317–1327 (2003).

    CAS  Article  Google Scholar 

  6. 6.

    Blott, E. J. & Griffiths, G. M. Secretory lysosomes. Nat. Rev. Mol. Cell Biol. 3, 122–131 (2002).

    CAS  Article  Google Scholar 

  7. 7.

    Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010).

    CAS  Article  Google Scholar 

  8. 8.

    Pertoft, H., Wärmegård, B. & Höök, M. Heterogeneity of lysosomes originating from rat liver parenchymal cells. Metabolic relationship of subpopulations separated by density-gradient centrifugation. Biochem. J. 174, 309–317 (1978).

    CAS  Article  Google Scholar 

  9. 9.

    Li, X. et al. A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation. Nat. Cell Biol. 18, 404–417 (2016).

    CAS  Article  Google Scholar 

  10. 10.

    Matteoni, R. & Kreis, T. E. Translocation and clustering of endosomes and lysosomes depends on microtubules. J. Cell Biol. 105, 1253–1265 (1987).

    CAS  Article  Google Scholar 

  11. 11.

    Pu, J. et al. BORC, a multisubunit complex that regulates lysosome positioning. Dev. Cell 33, 176–188 (2015).

    CAS  Article  Google Scholar 

  12. 12.

    Kornak, U. et al. Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104, 205–215 (2001).

    CAS  Article  Google Scholar 

  13. 13.

    Ba, Q., Raghavan, G., Kiselyov, K. & Yang, G. Whole-cell scale dynamic organization of lysosomes revealed by spatial statistical analysis. Cell Rep. 23, 3591–3606 (2018).

    CAS  Article  Google Scholar 

  14. 14.

    Seiji, M., Shimao, K., Birbeck, M. S. C. & Fitzpatrick, T. B. Subcellular localization of melanin biosynthests. Ann. NY Acad. Sci. 100, 497–533 (2006).

    Article  Google Scholar 

  15. 15.

    Raposo, G., Tenza, D., Murphy, D. M., Berson, J. F. & Marks, M. S. Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells. J. Cell Biol. 152, 809–824 (2001).

    CAS  Article  Google Scholar 

  16. 16.

    Chakraborty, K., Leung, K. & Krishnan, Y. High lumenal chloride in the lysosome is critical for lysosome function. eLife 6, e28862 (2017).

    Article  Google Scholar 

  17. 17.

    Saha, S., Prakash, V., Halder, S., Chakraborty, K. & Krishnan, Y. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells. Nat. Nanotech. 10, 645–651 (2015).

    CAS  Article  Google Scholar 

  18. 18.

    Van Dyke, R. W. Proton pump-generated electrochemical gradients in rat liver multivesicular bodies. Quantitation and effects of chloride. J. Biol. Chem. 263, 2603–2611 (1988).

    Google Scholar 

  19. 19.

    Luzio, J. P., Pryor, P. R. & Bright, N. A. Lysosomes: fusion and function. Nat. Rev. Mol. Cell Biol. 8, 622–632 (2007).

    CAS  Article  Google Scholar 

  20. 20.

    Weinert, S. et al. Lysosomal pathology and osteopetrosis upon loss of H+-driven lysosomal Cl accumulation. Science 328, 1401–1403 (2010).

    CAS  Article  Google Scholar 

  21. 21.

    Guzman, R. E., Grieschat, M., Fahlke, C. & Alekov, A. K. ClC-3 is an intracellular chloride/proton exchanger with large voltage-dependent nonlinear capacitance. ACS Chem. Neurosci. 4, 994–1003 (2013).

    CAS  Article  Google Scholar 

  22. 22.

    Biwersi, J., Tulk, B. & Verkman, A. S. Long-wavelength chloride-sensitive fluorescent indicators. Anal. Biochem. 219, 139–143 (1994).

    CAS  Article  Google Scholar 

  23. 23.

    Arosio, D. et al. Simultaneous intracellular chloride and pH measurements using a GFP-based sensor. Nat. Methods 7, 516–518 (2010).

    CAS  Article  Google Scholar 

  24. 24.

    Kuner, T. & Augustine, G. J. A genetically encoded ratiometric indicator for chloride. Neuron 27, 447–459 (2000).

    CAS  Article  Google Scholar 

  25. 25.

    Surana, S., Shenoy, A. R. & Krishnan, Y. Designing DNA nanodevices for compatibility with the immune system of higher organisms. Nat. Nanotech. 10, 741–747 (2015).

    CAS  Article  Google Scholar 

  26. 26.

    Modi, S., Nizak, C., Surana, S., Halder, S. & Krishnan, Y. Two DNA nanomachines map pH changes along intersecting endocytic pathways inside the same cell. Nat. Nanotech. 8, 459–467 (2013).

    CAS  Article  Google Scholar 

  27. 27.

    Chu, T. C. et al. Aptamer:toxin conjugates that specifically target prostate tumor cells. Cancer Res. 66, 5989–5992 (2006).

    CAS  Article  Google Scholar 

  28. 28.

    Pinheiro, A. V., Han, D., Shih, W. M. & Yan, H. Challenges and opportunities for structural DNA nanotechnology. Nat. Nanotech. 6, 763–772 (2011).

    CAS  Article  Google Scholar 

  29. 29.

    Bhatia, D. et al. Quantum dot-loaded monofunctionalized DNA icosahedra for single-particle tracking of endocytic pathways. Nat. Nanotech. 11, 1112–1119 (2016).

    CAS  Article  Google Scholar 

  30. 30.

    Prakash, V., Saha, S., Chakraborty, K. & Krishnan, Y. Rational design of a quantitative, pH-insensitive, nucleic acid based fluorescent chloride reporter. Chem. Sci. 7, 1946–1953 (2016).

    CAS  Article  Google Scholar 

  31. 31.

    Modi, S., Halder, S., Nizak, C. & Krishnan, Y. Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways. Nanoscale 6, 1144–1152 (2014).

    CAS  Article  Google Scholar 

  32. 32.

    Modi, S. et al. A DNA nanomachine that maps spatial and temporal pH changes inside living cells. Nat. Nanotech. 4, 325–330 (2009).

    CAS  Article  Google Scholar 

  33. 33.

    Surana, S., Bhat, J. M., Koushika, S. P. & Krishnan, Y. An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism. Nat. Commun. 2, 340 (2011).

    Article  Google Scholar 

  34. 34.

    Halder, S. & Krishnan, Y. Design of ultrasensitive DNA-based fluorescent pH sensitive nanodevices. Nanoscale 7, 10008–10012 (2015).

    CAS  Article  Google Scholar 

  35. 35.

    Gough, P. J. & Gordon, S. The role of scavenger receptors in the innate immune system. Microbes Infect. 2, 305–311 (2000).

    CAS  Article  Google Scholar 

  36. 36.

    Bhatia, D., Surana, S., Chakraborty, S., Koushika, S. P. & Krishnan, Y. A synthetic icosahedral DNA-based host–cargo complex for functional in vivo imaging. Nat. Commun. 2, 339 (2011).

    Article  Google Scholar 

  37. 37.

    Chakraborty, K., Veetil, A. T., Jaffrey, S. R. & Krishnan, Y. Nucleic acid-based nanodevices in biological imaging. Annu. Rev. Biochem. 85, 349–373 (2016).

    CAS  Article  Google Scholar 

  38. 38.

    Veetil, A. T. et al. Cell-targetable DNA nanocapsules for spatiotemporal release of caged bioactive small molecules. Nat. Nanotech. 12, 1183–1189 (2017).

    CAS  Article  Google Scholar 

  39. 39.

    Surana, S., Bhatia, D. & Krishnan, Y. A method to study in vivo stability of DNA nanostructures. Methods 64, 94–100 (2013).

    CAS  Article  Google Scholar 

  40. 40.

    Schuchman, E. H. & Desnick, R. J. Types A and B Niemann–Pick disease. Mol. Genet. Metab. 120, 27–33 (2017).

  41. 41.

    Vanier, M. T. & Millat, G. Niemann–Pick disease type C. Clin. Genet. 64, 269–281 (2003).

    CAS  Article  Google Scholar 

  42. 42.

    Desnick, R. J. & Schuchman, E. H. Enzyme replacement and enhancement therapies: lessons from lysosomal disorders. Nat. Rev. Genet. 3, 954–966 (2002).

    CAS  Article  Google Scholar 

  43. 43.

    Ory, D. S. et al. Intrathecal 2-hydroxypropyl-β-cyclodextrin decreases neurological disease progression in Niemann-Pick disease, type C1: a non-randomised, open-label, phase 1–2 trial. Lancet 390, 1758–1768 (2017).

    CAS  Article  Google Scholar 

  44. 44.

    Johnson, D. E., Ostrowski, P., Jaumouillé, V. & Grinstein, S. The position of lysosomes within the cell determines their luminal pH. J. Cell Biol. 212, 677–692 (2016).

    CAS  Article  Google Scholar 

  45. 45.

    Kornhuber, J. et al. Identification of new functional inhibitors of acid sphingomyelinase using a structure–property–activity relation model. J. Med. Chem. 51, 219–237 (2008).

    CAS  Article  Google Scholar 

  46. 46.

    Cenedella, R. J. Cholesterol synthesis inhibitor U18666A and the role of sterol metabolism and trafficking in numerous pathophysiological processes. Lipids 44, 477–487 (2009).

    CAS  Article  Google Scholar 

  47. 47.

    He, X. et al. Characterization of human acid sphingomyelinase purified from the media of overexpressing Chinese hamster ovary cells. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1432, 251–264 (1999).

    CAS  Article  Google Scholar 

  48. 48.

    Devany, J., Chakraborty, K. & Krishnan, Y. Subcellular nanorheology reveals lysosomal viscosity as a reporter for lysosomal storage diseases. Nano Lett. 18, 1351–1359 (2018).

    CAS  Article  Google Scholar 

  49. 49.

    Aue, W. P., Bartholdi, E. & Ernst, R. R. Two‐dimensional spectroscopy. Application to nuclear magnetic resonance. J. Chem. Phys. 64, 2229–2246 (1976).

    CAS  Article  Google Scholar 

  50. 50.

    Boaz, H. & Rollefson, G. K. The quenching of fluorescence. Deviations from the Stern–Volmer law. J. Am. Chem. Soc. 72, 3435–3443 (1950).

    CAS  Article  Google Scholar 

Download references


This work was supported by the University of Chicago Women’s Board, Pilot and Feasibility award from an NIDDK center grant P30DK42086 to the University of Chicago Digestive Diseases Research Core Center, MRSEC grant no. DMR-1420709, Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust, C-084, ANL-UChicago collaborative grant and University of Chicago start-up funds to Y.K. Y.K. is a Brain Research Foundation Fellow.

Author information




K.L., K.C., A.S. and Y.K. designed the project. K.L., K.C. and A.S. performed experiments. K.L., K.C., A.S. and Y.K. analysed the data. K.L., K.C., A.S. and Y.K. wrote the paper. All authors discussed the results and gave input on the manuscript.

Corresponding author

Correspondence to Yamuna Krishnan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–13, Supplementary Tables 1–2 and Supplementary References

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Leung, K., Chakraborty, K., Saminathan, A. et al. A DNA nanomachine chemically resolves lysosomes in live cells. Nature Nanotech 14, 176–183 (2019).

Download citation

Further reading


Quick links

Find nanotechnology articles, nanomaterial data and patents all in one place. Visit Nano by Nature Research