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A DNA-based fluorescent probe maps NOS3 activity with subcellular spatial resolution

Nature Chemical Biology volume 16, pages 660–666 (2020)Cite this article

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Abstract

Nitric oxide synthase 3 (NOS3) produces the gasotransmitter nitric oxide (NO), which drives critical cellular signaling pathways by S-nitrosylating target proteins. Endogenous NOS3 resides at two distinct subcellular locations: the plasma membrane and the trans-Golgi network (TGN). However, NO generation arising from the activities of both these pools of NOS3 and its relative contribution to physiology or disease is not yet resolvable. We describe a fluorescent DNA-based probe technology, NOckout, that can be targeted either to the plasma membrane or the TGN, where it can quantitatively map the activities of endogenous NOS3 at these locations in live cells. We found that, although NOS3 at the Golgi is tenfold less active than at the plasma membrane, its activity is essential for the structural integrity of the Golgi. The newfound ability to spatially map NOS3 activity provides a platform to discover selective regulators of the distinct pools of NOS3.

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Fig. 1: NOckout reporters image endogenous NO production at the plasma membrane and Golgi.
Fig. 2: Simultaneous, quantitative NO imaging at two distinct subcellular locations.
Fig. 3: Phosphorylated NOS3 is enriched at the plasma membrane.
Fig. 4: The Golgi is an S-nitrosylation hotspot in breast cancer cells.

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Data availability

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

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Acknowledgements

We thank J. Kuriyan and W.C. Sessa for valuable discussions. We thank Y. Fang for technical discussions and for the reagents used in biochemical studies of NOS3. We thank the Integrated Light Microscopy facility and BioPhysics core facility at the University of Chicago. We thank M. Zajac for a critical reading of the manuscript. This work was supported by a research grant from the University of Pennsylvania Orphan Disease Center in partnership with the Andrew Coppola Foundation, the University of Chicago Women’s Board; a Pilot and Feasibility award from an NIDDK center grant no. P30DK42086 to the University of Chicago Digestive Diseases Research Core Center; the Chicago Biomedical Consortium, with support from the Searle Funds at The Chicago Community Trust, C-084; a Scientific Innovation Award from the Brain Research Foundation and the Mergel Funsky award to Y.K.

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Author notes

  1. These authors contributed equally: Junyi Zou, Aneesh T. Veetil.

Authors and Affiliations

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

    Maulik S. Jani, Junyi Zou, Aneesh T. Veetil & Yamuna Krishnan

  2. Grossman Institute of Neuroscience, Quantitative Biology and Human Behavior, University of Chicago, Chicago, IL, USA

    Maulik S. Jani, Junyi Zou, Aneesh T. Veetil & Yamuna Krishnan

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Contributions

M.S.J., A.T.V. and Y.K. designed studies. M.S.J., A.T.V. and J.Z. characterized the sensor. M.S.J. and A.T.V. performed imaging experiments. M.S.J. and J.Z performed quantitation of enzymatic rate. M.S.J. performed immunostaining and western blot based biochemical/cell biology assays. M.S.J., A.T.V., J.Z and Y.K. analyzed data. M.S.J. and Y.K. wrote the manuscript with input from all authors.

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Correspondence to Yamuna Krishnan.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–23, synthetic procedures and Note

Reporting Summary

Supplementary Video 1

NOckoutPM reports real-time change in NO levels at the plasma membrane on treatment with ionomycin in combination with cholesterol (500 μM)

Supplementary Video 2

NOckoutPM reports real-time change in NO levels at the plasma membrane on treatment with thapsigargin (1 μM). NOckoutPM signal from T-47D cell pulsed with NOckoutPM and NOckoutTGN is represented as the ratio of DAR intensity to that of A488

Supplementary Video 3

NOckoutTGN reports real-time change in NO levels at the TGN on treatment with thapsigargin (1 μM). NOckoutTGN signal from T-47D cell pulsed with NOckoutPM and NOckoutTGN is represented as the ratio of DAR intensity to that of A647

Supplementary Video 4

Pharmacological NO scavenging in T-47D cells using Methylene blue (20 μM) leads to Golgi fragmentation

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Jani, M.S., Zou, J., Veetil, A.T. et al. A DNA-based fluorescent probe maps NOS3 activity with subcellular spatial resolution. Nat Chem Biol 16, 660–666 (2020). https://doi.org/10.1038/s41589-020-0491-3

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