Abstract

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with cellular compartmentalization. Here, we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to threefold better than the diffraction limit. These 'FLINC' biosensors use binding-induced changes in protein fluorescence dynamics to translate kinase activities or protein–protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A protein kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membranes of living cells and to uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture whose structural and functional characteristics can be revealed by these new biosensors.

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Acknowledgements

The authors acknowledge Q. Ni, L.M. Amzel, S. Mehta and P.A. Iglesias for critical reading of the manuscript, and X. Zhou for aid in protein purification. This work was supported by NIH DP1 CA174423, R35 CA197622 and R01 DK073368 (to J.Z.); R01 GM079440, T32 GM008403 and NSF MCB 141210B (to K.G.F.); R01CA74305 (to P.A.C.); NCI1K22CA154600 and 1R03A188439 (to E.J.K.); a Research-Foundation Flanders (FWO-Vlaanderen) postdoctoral fellowship, KU Leuven Research Professorship and European Research Council ERC Starting Grant 714688 (to P.D.); and a Graduate Research Fellowship from the NSF DGE-1232825 (to A.M.P.). J.Z. and G.M. acknowledge the UCSD Specialized Cancer Center Support Grant P30 2P30CA023100-28. R.J. acknowledges NIST and the NSF Physics Frontier Center at JILA for support. R.J. is a staff member in the Quantum Physics Division of the National Institute of Standards and Technology (NIST). Certain commercial equipment, instruments or materials are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the NIST, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.

Author information

Affiliations

  1. Department of Pharmacology, University of California San Diego, La Jolla, California, USA.

    • Gary C H Mo
    • , Brian Ross
    • , Fabian Hertel
    • , Eric Greenwald
    • , Chris Booth
    •  & Jin Zhang
  2. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA.

    • Brian Ross
  3. JILA, University of Colorado and NIST, Boulder, Colorado, USA.

    • Premashis Manna
    •  & Ralph Jimenez
  4. Department of Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, Colorado, USA.

    • Premashis Manna
    • , Amy Palmer
    •  & Ralph Jimenez
  5. Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Xinxing Yang
    • , Brian Tenner
    •  & Jie Xiao
  6. T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, USA.

    • Ashlee M Plummer
    •  & Karen G Fleming
  7. Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Zan Chen
    • , Philip A Cole
    •  & Jin Zhang
  8. Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, USA.

    • Yuxiao Wang
    •  & Eileen J Kennedy
  9. BioFrontiers Institute, University of Colorado, Boulder, Boulder, Colorado, USA.

    • Amy Palmer
  10. Department of Chemistry, KU Leuven, Heverlee, Belgium.

    • Peter Dedecker

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Contributions

G.C.H.M. and J.Z. made the initial discovery and designed all experiments. P.D. was also involved in the initial proposal of the research direction. G.C.H.M., B.R., F.H., P.M., E.G., X.Y., C.B., B.T., A.M.P., Z.C. and K.G.F. performed experiments and analyzed data with input from J.Z., J.X., R.J., A.P., K.G.F. and P.A.C.; Y.W. and E.J.K. provided reagents. G.C.H.M., B.R., E.G. and P.D. developed and validated all postprocessing algorithms; P.D. formalized the normalization approach. G.C.H.M. and J.Z. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jin Zhang.

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    Fluorescence fluctuations from FLINC and control constructs

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DOI

https://doi.org/10.1038/nmeth.4221