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Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange

Nature Methods volume 12, pages 195198 (2015) | Download Citation

Abstract

We have developed a versatile new class of genetically encoded fluorescent biosensor based on reversible exchange of the heterodimeric partners of green and red dimerization-dependent fluorescent proteins. We demonstrate the use of this strategy to construct both intermolecular and intramolecular ratiometric biosensors for qualitative imaging of caspase activity, Ca2+ concentration dynamics and other second-messenger signaling activities.

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Acknowledgements

We thank the University of Alberta Molecular Biology Service Unit, C.W. Cairo and R. Derda for technical assistance, and T. Meyer (Stanford) for the PLCδ-encoding gene. Funding support was provided by Canada Research Chairs (R.E.C.), the Alberta Glycomics Centre (R.E.C.), the Canadian Institutes of Health Research (NHG 99085 and MOP 123514 to R.E.C. and MOP 119425 to D.M.), the Natural Sciences and Engineering Research Council of Canada (Discovery grant to R.E.C. and a CGSD3 Scholarship to S.C.A.), Alberta Ingenuity PhD Scholarships (S.C.A. and Y.S.), a National Science Foundation of China Major Research Grant (91132718 to Y.Z.), the Beijing Natural Science Foundation (7142085 to Y.Z.), US National Institutes of Health DP1 CA174423 (to J.Z.) and 5R44NS082222 (to A.M.Q. and T.E.H.), and US National Science Foundation Small Business Innovation Research (SBIR) 1248138 and Montana SBIR matching funds #13-50 RCSBIR-003 (A.M.Q.).

Author information

Affiliations

  1. Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.

    • Yidan Ding
    • , Jhon Ralph Enterina
    • , Yi Shen
    • , Spencer C Alford
    •  & Robert E Campbell
  2. State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, PKU (Peking University)-IDG (International Data Group)/McGovern Institute for Brain Research, Peking University, Beijing, China.

    • Jiao Li
    •  & Yan Zhang
  3. Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada.

    • Issan Zhang
    •  & Dusica Maysinger
  4. Montana Molecular, Bozeman, Montana, USA.

    • Paul H Tewson
    • , Anne Marie Quinn
    •  & Thomas E Hughes
  5. Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Gary C H Mo
    •  & Jin Zhang
  6. Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana, USA.

    • Thomas E Hughes
  7. Department of Bioengineering, Stanford University, Stanford, California, USA.

    • Spencer C Alford

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Contributions

Y.D., J.Z., P.H.T., A.M.Q., T.E.H., D.M., S.C.A., Y.Z. and R.E.C. conceived of and designed experiments. Y.D. assembled all constructs except for the PIP2, PKA and ERK biosensors and performed imaging of all caspase-3, caspase-8 and caspase-9 biosensors and the three-polypeptide Ca2+ biosensor. J.L. performed imaging with the caspase-3 biosensor in neurons. J.R.E. determined heterodimer affinities in vitro. Y.S. performed imaging of the single-polypeptide Ca2+ biosensor. I.Z. performed imaging of the caspase-1 biosensor. P.H.T. assembled and performed imaging of the PIP2 and PKA biosensors. G.C.H.M. assembled and performed imaging of the ERK biosensor. All authors were involved in data analysis, and Y.D., J.Z., T.E.H., D.M., S.C.A., Y.Z. and R.E.C. wrote the manuscript.

Competing interests

R.E.C., in partnership with the Alberta Glycomics Centre and the University of Alberta, has filed Canadian and US patent applications that describe the research in this manuscript. P.H.T., A.M.Q. and T.E.H. are employed by Montana Molecular, a for-profit company that develops genetically encoded fluorescent protein sensors. This does not alter the authors' adherence to all of the Nature Methods policies on sharing data and materials presented in this manuscript.

Corresponding author

Correspondence to Robert E Campbell.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15 and Supplementary Tables 1 and 2

Videos

  1. 1.

    Intermolecular green-to-red FPX-based biosensor for caspase-3 activity.

    Time-lapse fluorescence imaging of caspase-3 activity with GA-DEVD-B and RA. HeLa cells were treated with staurosporine to initiate apoptosis.

  2. 2.

    Translocating red-to-green FPX-based biosensor for caspase-3 activity.

    Time-lapse fluorescence imaging of caspase-3 activity with NESRA-DEVD-BNLS and GANLS. HeLa cells were treated with staurosporine to initiate apoptosis.

  3. 3.

    Translocating green-to-red FPX-based biosensor for caspase-3 activity.

    Time-lapse fluorescence imaging of caspase-3 activity with NESGA-DEVD-BNLS and RANLS. HeLa cells were treated with staurosporine to initiate apoptosis.

  4. 4.

    FPX biosensor for caspase-3 activity based on exchange of GA.

    Time-lapse fluorescence imaging of caspase-3 activity with GANLS-DEVD-BNES and BNLS. HeLa cells were treated with staurosporine to initiate apoptosis.

  5. 5.

    FPX biosensor for caspase-3 activity based on exchange of RA.

    Time-lapse fluorescence imaging of caspase-3 activity with RANLS-DEVD-BNES and BNLS. HeLa cells were treated with staurosporine to initiate apoptosis.

  6. 6.

    Two-color imaging of caspase-3 and caspase-9 activity by A copy exchange.

    Time-lapse fluorescence imaging of caspase-3 and caspase-9 activity with GANLS-DEVD-BNES, RANLS-LEHD-BNES, and BNLS. HeLa cells were treated with staurosporine to initiate apoptosis.

  7. 7.

    Intermolecular FPX-based biosensor for Ca2+.

    Time-lapse fluorescence imaging of histamine treated HeLa cells that are co-expressing RA-CaM, B-M13, and GA. The green and red fluorescence channels have been overlaid in this movie.

  8. 8.

    Intermolecular FPX-based biosensor for PIP2.

    Time-lapse fluorescence imaging of PIP2 concentration in the membrane by co-expression of RA and B fused to PH domains, and free GA. HEK293 cells have been treated with carbachol. The change occurs within one imaging interval (7 s) and thus appears quite sudden in this movie.

  9. 9.

    Intramolecular FPX-based biosensor for Ca2+.

    Time-lapse fluorescence imaging of histamine treated HeLa cells expressing the single polypeptide construct RA-CaM-B-M13-GA. The ratio of the green and red fluorescence intensities has been pseudocolored according to the look-up-table shown.

  10. 10.

    Intramolecular FPX-based biosensor for caspase-3.

    Time-lapse fluorescence imaging of caspase-3 activity in cells expressing the single polypeptide construct RA-linker-B-DEVD-GANES. HeLa cells were treated with staurosporine to initiate apoptosis.

  11. 11.

    Intramolecular FPX-based biosensor for caspase-8.

    Time-lapse fluorescence imaging of caspase-8 activity in cells expressing the single polypeptide construct RA-IETD-B-linker-GANES. HeLa cells were treated with staurosporine to initiate apoptosis.

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DOI

https://doi.org/10.1038/nmeth.3261