A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells

Journal name:
Nature Nanotechnology
Volume:
10,
Pages:
645–651
Year published:
DOI:
doi:10.1038/nnano.2015.130
Received
Accepted
Published online

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.

At a glance

Figures

  1. Design and characterization of Clensor and ClensorTf.
    Figure 1: Design and characterization of Clensor and ClensorTf.

    a, Structure and working principle of Clensor. P, sensing module (pink line) containing a Cl-sensitive fluorophore, BAC (green filled circle); D2, normalizing module (brown line) containing a Cl-insensitive fluorophore, Alexa 647 (red filled circle); D1, targeting module (orange line). In the presence of Cl, BAC undergoes collisional quenching, whereas fluorescence of Alexa 647 is Cl-independent. b, Modified sensor design for targeting to the recycling pathway (ClensorTf). D1Tfapt, targeting module modified with an RNA aptamer (Tfapt) against the human transferrin receptor (cyan line). c, Fluorescence emission spectra of Clensor at the indicated [Cl] obtained using λExBAC = 435 nm (green) and λExAlexa 647 = 650 nm (red). d, In vitro Cl calibration profile of Clensor showing normalized Alexa 647 and BAC fluorescence intensity ratio (R/G) versus [Cl]. R/G values at different chloride concentrations were normalized to the value at 5 mM chloride. e, Plot of KSV for Clensor versus pH. Error bars indicate the mean of three independent experiments ± s.e.m.

  2. Programming Clensor delivery to specific endocytic organelles.
    Figure 2: Programming Clensor delivery to specific endocytic organelles.

    a, ClensorA647 internalization by haemocytes in the presence (+mBSA) and absence (−mBSA) of excess competitor ligand maleylated BSA (mBSA, 10 μM) and autofluorescence (AF) in Drosophila haemocytes. Error bars indicate the mean of three independent experiments ± s.e.m. (n = 20 cells). b, Trafficking of Clensor (ClensorA647) in haemocytes isolated from flies expressing YFP-Rab5 (upper row), YFP-Rab7 (middle row) and GFP-LAMP (lower row) at the indicated chase times. ClensorA647-positive vesicles are shown in red and YFP-Rab5/YFP-Rab7/GFP-LAMP vesicles are shown in green. Scale bars, 10 μm. c, Competition experiments with ClensorTfA647 and excess unlabelled transferrin (Tf, 25 μM). Normalized intensities of cells pulsed with ClensorTfA647 in the presence (+Tf) and absence (–Tf) of Tf and ClensorA647 are shown. Error bars indicate the mean of three independent experiments ± s.e.m. (n = 15 cells). d, Co-localization of ClensorTfA647 with transferrin (TfA568) in S2R+ cells. Scale bars, 10 μm.

  3. Quantitative performance of Clensor within subcellular organelles.
    Figure 3: Quantitative performance of Clensor within subcellular organelles.

    a, Alexa 647 channel and respective pseudocolour R/G map of Drosophila haemocytes pulsed with Clensor and clamped at 5, 40 and 80 mM Cl. Scale bars, 10 μm. b, Histograms showing typical spread of R/G ratios of vesicles clamped at 5, 40 and 80 mM Cl (n ≈ 10 cells, ≥50 endosomes). c, In vitro and intracellular fold change in R/G ratios of Clensor at 5 and 120 mM Cl. d, Normalized R/G intensity (Alexa 647/BAC) ratios inside the endosomes, plotted as a function of [Cl], yield the intracellular calibration profile (red), which is overlaid on the in vitro chloride calibration profile (black). Error bars indicate the mean of three independent experiments ± s.e.m. (n ≈ 10 cells, ≥50 endosomes).

  4. Spatiotemporal mapping of [Cl−] along the endolysosomal pathway using Clensor in living cells.
    Figure 4: Spatiotemporal mapping of [Cl] along the endolysosomal pathway using Clensor in living cells.

    a, Representative pseudocolour R/G map of live haemocytes isolated from wild-type Drosophila and labelled with Clensor. Scale bar, 10 μm. b, Histograms of R/G ratios of EE at 5 min (green), LE at 60 min (orange) and LY at 120 min (red) (n ≈ 20 cells, ∼100 endosomes/lysosomes). c,d, RT-PCR analysis of total RNA isolated from 3rd instar larvae of UAS-DmClC-b RNAi/Coll GAL4 larvae (DmClC-b RNAi) (c) and UAS-DmClC-c RNAi/Coll GAL4 larvae (DmClC-c RNAi) (d). Lanes marked as CS correspond to PCR-amplified cDNA of the indicated gene products (DmClC-b and DmClC-c) from wild-type Drosophila, and lanes marked as RNAi are for the corresponding gene products isolated from 3rd instar larvae of UASDmClC-b RNAi/Coll GAL4 (c) and UAS-DmClC-c RNAi/CollGAL4 (d) flies. RpL32 was used as a loading control. eg, Box plots showing [Cl¯] distributions for EE (e), LE (f) and LY (g) in haemocytes of the indicated genetic background (n ∼ 15 cells, ∼75 endosomes/lysosomes). h,i, Box plots showing pH distributions in EE (h) and LE (i) in haemocytes of the indicated genetic background using the FD10 dual excitation method. j, Box plots showing pH distribution in LY in haemocytes of the indicated genetic background using I4LY A488/A647 (n > 15 cells, >50 endosomes/lysosomes. In ej: CS, wild-type; b RNAi, RNAi against DmClC-b; b mutant, DmClC-b mutant; c RNAi, DmClC-c RNAi. Boxes represent 25–75% of the population. Horizontal lines within boxes represent the median. Filled circles represent mean of the data obtained for each indicated genotype. Error bars on filled circles represent s.e.m.

  5. ClensorTf maps [Cl−] within recycling endosomes (REs).
    Figure 5: ClensorTf maps [Cl] within recycling endosomes (REs).

    a,b, RNAi knockdown of DmClC-c and DmClC-b in Drosophila S2R+ cells. PCR-amplified cDNA of the indicated gene products (DmClC-b and DmClC-c) isolated from untreated and RNAi treated Drosophila S2R+ cells for RNAi against DmClC-c (a) and RNAi against DmClC-b (b). RpL32 was used as loading control.

References

  1. Sheppard, D. N. & Welsh, M. J. Structure and function of the CFTR chloride channel. Physiol. Rev. 79, S23S45 (1999).
  2. Stauber, T., Weinert, S. & Jentsch, T. J. Cell biology and physiology of CLC chloride channels and transporters. Compr. Physiol. 2, 17011744 (2012).
  3. Stauber, T. & Jentsch, T. J. Chloride in vesicular trafficking and function. Annu. Rev. Physiol. 75, 453477 (2013).
  4. Kuner, T. & Augustine, G. J. A genetically encoded ratiometric indicator for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 27, 447459 (2000).
  5. Markova, O., Mukhtarov, M., Real, E., Jacob, Y. & Bregestovski, P. Genetically encoded chloride indicator with improved sensitivity. J. Neurosci. Methods 170, 6776 (2008).
  6. Arosio, D. et al. Simultaneous intracellular chloride and pH measurements using a GFP-based sensor. Nature Methods 7, 516518 (2010).
  7. Casey, J. R., Grinstein, S. & Orlowski, J. Sensors and regulators of intracellular pH. Nature Rev. Mol. Cell Biol. 11, 5061 (2010).
  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, 55065513 (2002).
  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, 142150 (1988).
  10. Verkman, A. S. Development and biological applications of chloride-sensitive fluorescent indicators. Am. J. Physiol. 259, C375C388 (1990).
  11. Biwersi, J., Tulk, B. & Verkman, A. S. Long-wavelength chloride-sensitive fluorescent indicators. Anal. Biochem. 219, 139143 (1994).
  12. Geddes, C. D. Optical halide sensing using fluorescence quenching: theory, simulations and applications—a review. Meas. Sci. Technol. 12, R53 (2001).
  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, 11291138 (2003).
  14. Bhatia, D., Sharma, S. & Krishnan, Y. Synthetic, biofunctional nucleic acid-based molecular devices. Curr. Opin. Biotechnol. 22, 475484 (2011).
  15. Krishnan, Y. & Bathe, M. Designer nucleic acids to probe and program the cell. Trends Cell Biol. 22, 624633 (2012).
  16. Modi, S. et al. A DNA nanomachine that maps spatial and temporal pH changes inside living cells. Nature Nanotech. 4, 325330 (2009).
  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, 459467 (2013).
  18. Bhatia, D. et al. Icosahedral DNA nanocapsules by modular assembly. Angew. Chem. Int. Ed. 48, 41344137 (2009).
  19. Lee, H. et al. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nature Nanotech. 7, 389393 (2012).
  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).
  21. Modi, S., Halder, S., Nizak, C. & Krishnan, Y. Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways. Nanoscale 6, 11441152 (2014).
  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).
  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, 33733386 (2003).
  24. Mayle, K. M., Le, A. M. & Kamei, D. T. The intracellular trafficking pathway of transferrin. Biochim. Biophys. Acta 1820, 264281 (2012).
  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).
  26. Huotari, J. & Helenius, A. Endosome maturation. EMBO J. 30, 34813500 (2011).
  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).
  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, 955962 (1988).
  29. Weinert, S. et al. Lysosomal pathology and osteopetrosis upon loss of H+-driven lysosomal Cl accumulation. Science 328, 14011403 (2010).
  30. Hara-Chikuma, M. et al. ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation. J. Biol. Chem. 280, 12411247 (2005).
  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, 941946 (2005).
  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, 12291237 (2009).
  33. Poët, M. et al. Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6. Proc. Natl Acad. Sci. USA 103, 1385413859 (2006).
  34. Kasper, D. et al. Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J. 24, 10791091 (2005).
  35. Mindell, J. A. Lysosomal acidification mechanisms. Annu. Rev. Physiol. 74, 6986 (2012).
  36. DiCiccio, J. E. & Steinberg, B. E. Lysosomal pH and analysis of the counter ion pathways that support acidification. J. Gen. Physiol. 137, 385390 (2011).
  37. Kornak, U. et al. Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104, 205215 (2001).
  38. Scott, C. C. & Gruenberg, J. Ion flux and the function of endosomes and lysosomes: pH is just the start. BioEssays 33, 103110 (2011).
  39. Weisz, O. A. Acidification and protein traffic. Int. Rev. Cytol. 226, 259319 (2003).
  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, 593607 (2003).
  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, 64996503 (2000).

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Author information

Affiliations

  1. National Centre for Biological Sciences, TIFR, GKVK, Bellary Road, Bangalore 560065, India

    • Sonali Saha,
    • Saheli Halder &
    • Yamuna Krishnan
  2. Department of Chemistry, University of Chicago, 929E, 57th Street, E305A, GCIS, Chicago, Illinois 60637, USA

    • Ved Prakash,
    • Kasturi Chakraborty &
    • Yamuna Krishnan

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.

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