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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.

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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).

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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.

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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.

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The authors declare no competing financial interests.

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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

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