Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution

  • Nature Methods volume 14, pages 10031009 (2017)
  • doi:10.1038/nmeth.4404
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GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.

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This work was supported by NIH grants CA151128 and GM118933 (R.S.); CA197996 (D.J.S.); CA120244, CA193981 and CA190533 (M.A.N.); Ruth L. Kirschstein National Research Service Award F32CA189622 (A.B.-S.); NIH grant 1F99CA21245501 (H.C.A.); Empire State Development Corporation Krabbe Disease Research Working Capital X561 and Krabbe Disease Research Capital Equipment U446 (M.L.F.); and the Jennifer Linscott Tietgen Foundation (M.A.N.). The pLV-SV4-puro lentiviral vector was obtained from P. Chumakov (Cleveland Clinic).

Author information

Author notes

    • Mitra S Rana
    •  & Archis Bagati

    Present addresses: NICHD, NIH, Bethesda, Maryland, USA (M.S.R.); Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA (A.B.).

    • Anna Bianchi-Smiraglia
    •  & Mitra S Rana

    These authors contributed equally to this work.


  1. Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York, USA.

    • Anna Bianchi-Smiraglia
    • , Colleen E Foley
    • , Leslie M Paul
    • , Brittany C Lipchick
    • , Sudha Moparthy
    • , Kalyana Moparthy
    • , Emily E Fink
    • , Archis Bagati
    • , Eugene S Kandel
    •  & Mikhail A Nikiforov
  2. Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.

    • Mitra S Rana
    •  & Rui Sousa
  3. Department of Biochemistry and Neurology, Hunter James Kelly Research Institute, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York, USA.

    • Edward Hurley
    •  & Maria Laura Feltri
  4. Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York, USA.

    • Hayley C Affronti
    • , Andrei V Bakin
    •  & Dominic J Smiraglia


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A.B.-S., M.S.R., R.S. and M.A.N. designed the experiments and wrote the manuscript; A.B.-S. and M.S.R. performed most of the experiments and analyzed the data; C.E.F., B.C.L., L.M.P., S.M., K.M., E.E.F. and A.B. performed some of the experiments; H.C.A. performed HPLC analysis; E.H. assisted with the microscopy acquisition and analysis; D.J.S., A.V.B., E.S.K. and M.L.F. supervised part of the study; R.S. and M.A.N. conceived the initial hypothesis and supervised the study. A.B.-S. and M.S.R. contributed equally to this study. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Rui Sousa or Mikhail A Nikiforov.

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