Skip to main content

Thank you for visiting nature.com. 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 pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells

Abstract

The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5–130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Design and characterization of Clensor and ClensorTf.
Figure 2: Programming Clensor delivery to specific endocytic organelles.
Figure 3: Quantitative performance of Clensor within subcellular organelles.
Figure 4: Spatiotemporal mapping of [Cl] along the endolysosomal pathway using Clensor in living cells.
Figure 5: ClensorTf maps [Cl] within recycling endosomes (REs).

References

  1. Sheppard, D. N. & Welsh, M. J. Structure and function of the CFTR chloride channel. Physiol. Rev. 79, S23–S45 (1999).

    CAS  Article  Google Scholar 

  2. Stauber, T., Weinert, S. & Jentsch, T. J. Cell biology and physiology of CLC chloride channels and transporters. Compr. Physiol. 2, 1701–1744 (2012).

    Google Scholar 

  3. Stauber, T. & Jentsch, T. J. Chloride in vesicular trafficking and function. Annu. Rev. Physiol. 75, 453–477 (2013).

    CAS  Article  Google Scholar 

  4. Kuner, T. & Augustine, G. J. A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 27, 447–459 (2000).

    CAS  Article  Google Scholar 

  5. Markova, O., Mukhtarov, M., Real, E., Jacob, Y. & Bregestovski, P. Genetically encoded chloride indicator with improved sensitivity. J. Neurosci. Methods 170, 67–76 (2008).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  7. Casey, J. R., Grinstein, S. & Orlowski, J. Sensors and regulators of intracellular pH. Nature Rev. Mol. Cell Biol. 11, 50–61 (2010).

    CAS  Article  Google Scholar 

  8. Sonawane, N. D., Thiagarajah, J. R. & Verkman, A. S. Chloride concentration in endosomes measured using a ratioable fluorescent Cl indicator: evidence for chloride accumulation during acidification. J. Biol. Chem. 277, 5506–5513 (2002).

    CAS  Article  Google Scholar 

  9. Krapf, R., Illsley, N. P., Tseng, H. C. & Verkman, A. S. Structure–activity relationships of chloride-sensitive fluorescent indicators for biological application. Anal. Biochem. 169, 142–150 (1988).

    CAS  Article  Google Scholar 

  10. Verkman, A. S. Development and biological applications of chloride-sensitive fluorescent indicators. Am. J. Physiol. 259, C375–C388 (1990).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  12. Geddes, C. D. Optical halide sensing using fluorescence quenching: theory, simulations and applications—a review. Meas. Sci. Technol. 12, R53 (2001).

    CAS  Article  Google Scholar 

  13. Sonawane, N. D. & Verkman, A. S. Determinants of [Cl] in recycling and late endosomes and Golgi complex measured using fluorescent ligands. J. Cell Biol. 160, 1129–1138 (2003).

    CAS  Article  Google Scholar 

  14. Bhatia, D., Sharma, S. & Krishnan, Y. Synthetic, biofunctional nucleic acid-based molecular devices. Curr. Opin. Biotechnol. 22, 475–484 (2011).

    CAS  Article  Google Scholar 

  15. Krishnan, Y. & Bathe, M. Designer nucleic acids to probe and program the cell. Trends Cell Biol. 22, 624–633 (2012).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  18. Bhatia, D. et al. Icosahedral DNA nanocapsules by modular assembly. Angew. Chem. Int. Ed. 48, 4134–4137 (2009).

    CAS  Article  Google Scholar 

  19. Lee, H. et al. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nature Nanotech. 7, 389–393 (2012).

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  21. 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 

  22. Wilner, S. E. et al. An RNA alternative to human transferrin: a new tool for targeting human cells. Mol. Ther. Nucleic Acids 1, e21 (2012).

    Article  Google Scholar 

  23. Guha, A., Sriram, V., Krishnan, K. S. & Mayor, S. Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes. J. Cell Sci. 116, 3373–3386 (2003).

    CAS  Article  Google Scholar 

  24. Mayle, K. M., Le, A. M. & Kamei, D. T. The intracellular trafficking pathway of transferrin. Biochim. Biophys. Acta 1820, 264–281 (2012).

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  26. Huotari, J. & Helenius, A. Endosome maturation. EMBO J. 30, 3481–3500 (2011).

    CAS  Article  Google Scholar 

  27. Gupta, G. D. et al. Analysis of endocytic pathways in Drosophila cells reveals a conserved role for GBF1 in internalization via GEECs. PLoS ONE 4, e6768 (2009).

    Article  Google Scholar 

  28. Krapf, R., Berry, C. A. & Verkman, A. S. Estimation of intracellular chloride activity in isolated perfused rabbit proximal convoluted tubules using a fluorescent indicator. Biophys J. 53, 955–962 (1988).

    CAS  Article  Google Scholar 

  29. 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 

  30. Hara-Chikuma, M. et al. ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation. J. Biol. Chem. 280, 1241–1247 (2005).

    CAS  Article  Google Scholar 

  31. Hara-Chikuma, M., Wang, Y., Guggino, S. E., Guggino, W. B. & Verkman, A. S. Impaired acidification in early endosomes of ClC-5 deficient proximal tubule. Biochem. Biochem. Biophys. Res. Commun. 329, 941–946 (2005).

    CAS  Article  Google Scholar 

  32. Mohammad-Panah, R. et al. An essential role for ClC-4 in transferrin receptor function revealed in studies of fibroblasts derived from Clcn4-null mice. J. Cell Sci. 122, 1229–1237 (2009).

    CAS  Article  Google Scholar 

  33. Poët, M. et al. Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6. Proc. Natl Acad. Sci. USA 103, 13854–13859 (2006).

    Article  Google Scholar 

  34. Kasper, D. et al. Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J. 24, 1079–1091 (2005).

    CAS  Article  Google Scholar 

  35. Mindell, J. A. Lysosomal acidification mechanisms. Annu. Rev. Physiol. 74, 69–86 (2012).

    CAS  Article  Google Scholar 

  36. DiCiccio, J. E. & Steinberg, B. E. Lysosomal pH and analysis of the counter ion pathways that support acidification. J. Gen. Physiol. 137, 385–390 (2011).

    CAS  Article  Google Scholar 

  37. 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 

  38. Scott, C. C. & Gruenberg, J. Ion flux and the function of endosomes and lysosomes: pH is just the start. BioEssays 33, 103–110 (2011).

    CAS  Article  Google Scholar 

  39. Weisz, O. A. Acidification and protein traffic. Int. Rev. Cytol. 226, 259–319 (2003).

    CAS  Article  Google Scholar 

  40. Sriram, V., Krishnan, K. S. & Mayor, S. Deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster. J. Cell Biol. 161, 593–607 (2003).

    CAS  Article  Google Scholar 

  41. Clemens, J. C. et al. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc. Natl Acad. Sci. USA 97, 6499–6503 (2000).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors thank the Central Imaging and Flow Facility at the National Centre for Biological Sciences (NCBS) for imaging. This work was funded by the Wellcome Trust Department of Biotechnology (DBT), the India Alliance and the University of Chicago. S.S., S.H. and V.P. acknowledge the Council of Scientific and Industrial Research (CSIR), Government of India, for fellowship. FLIM experiments were performed at the Northwestern University Center for Advanced Microscopy supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center.

Author information

Authors and Affiliations

Authors

Contributions

S.S., K.C., V.P. and Y.K. conceived and designed the experiments. S.S., V.P. and K.C. performed the experiments. S.H. designed the I4LYA488/A647 used herein. S.S., V.P., K.C. and Y.K. analysed the data. S.S. and Y.K. co-wrote the paper. All authors commented on the manuscript.

Corresponding author

Correspondence to Yamuna Krishnan.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary Information (PDF 1979 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Saha, S., Prakash, V., Halder, S. et al. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells. Nature Nanotech 10, 645–651 (2015). https://doi.org/10.1038/nnano.2015.130

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2015.130

Further reading

Search

Quick links

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