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Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis

Nature Methods volume 6, pages 737–740 (2009)Cite this article

Abstract

We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn2+ binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn2+ was buffered at 0.4 nM in pancreatic β cells, and we found substantially higher Zn2+ concentrations in insulin-containing secretory vesicles.

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Figure 1: Design and properties of eCALWY sensors.
Figure 2: Determination of cytosolic free Zn2+ concentration in INS-1(832/13) cells using eCALWY variants.
Figure 3: Subcellular targeting of Zn2+ probes to insulin-storing granules.

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Acknowledgements

We thank S.M.J. van Duijnhoven for expressing and characterizing eZinCh, A. McDonald for assisting in the spinning disc confocal microscopy experiments, A. Tarasov for setting up the α-toxin incubation, H. Bayley (University of Oxford) for providing the α-toxin, C. Newgard (Duke University) for providing INS-1(832/13) cells, and L. Klomp and P. van den Berghe (University Medical Center Utrecht) and E.W. Meijer for their support at various stages of this research. M.M. and M.S.K. acknowledge support by the Human Frontier of Science Program (Young Investigator grant, (RGY)0068-2006). G.A.R. thanks the US National Institutes of Health for project grant RO1 DK071962-01, the Wellcome Trust for programme grants 067081/Z/02/Z and 081958/Z/07/Z, Medical Research Council (UK) for research grant G0401641, and the EU FP6 (“SaveBeta” consortium grant). T.J.N. and E.A.B. were supported by Imperial College divisional studentships.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Laboratory of Chemical Biology, Eindhoven University of Technology, Eindhoven, The Netherlands

    Jan L Vinkenborg, Melissa S Koay & Maarten Merkx

  2. Division of Medicine, Section of Cell Biology, Imperial College London, London, UK

    Tamara J Nicolson, Elisa A Bellomo & Guy A Rutter

Authors
  1. Jan L Vinkenborg
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  2. Tamara J Nicolson
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  3. Elisa A Bellomo
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  4. Melissa S Koay
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  5. Guy A Rutter
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  6. Maarten Merkx
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Contributions

J.L.V., G.A.R. and M.M. designed research; J.L.V., T.J.N., E.A.B. and M.S.K. conducted experiments, J.L.V., T.J.R., E.A.B., M.S.K., G.A.R. and M.M. analyzed data and J.L.V., G.A.R. and M.M. wrote the paper.

Corresponding authors

Correspondence to Guy A Rutter or Maarten Merkx.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14 and Supplementary Tables 1–5 (PDF 887 kb)

Supplementary Video 1

Ratiometric response of changes in cytosolic zinc for HEK293 expressing eCALWY-5. (AVI 6765 kb)

Supplementary Video 2

Ratiometric response of changes in vesicular zinc levels upon treatment of INS-1(832/13) cells expressing VAMP2-eZinCh with 10 μM monensin. (AVI 3797 kb)

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Vinkenborg, J., Nicolson, T., Bellomo, E. et al. Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis. Nat Methods 6, 737–740 (2009). https://doi.org/10.1038/nmeth.1368

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