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

Anna Bianchi-Smiraglia, Mitra S. Rana, Colleen E. Foley, Leslie M. Paul, Brittany C. Lipchick, Sudha Moparthy, Kalyana Moparthy, Emily E. Fink, Archis Bagati, Edward Hurley, Hayley C. Affronti, Andrei V. Bakin, Eugene S. Kandel, Dominic J. Smiraglia, Maria Laura Feltri, Rui Sousa, Mikhail A. Nikiforov

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1003-1009
Number of pages7
JournalNature Methods
Volume14
Issue number10
DOIs
StatePublished - Oct 1 2017

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution'. Together they form a unique fingerprint.

  • Cite this

    Bianchi-Smiraglia, A., Rana, M. S., Foley, C. E., Paul, L. M., Lipchick, B. C., Moparthy, S., Moparthy, K., Fink, E. E., Bagati, A., Hurley, E., Affronti, H. C., Bakin, A. V., Kandel, E. S., Smiraglia, D. J., Feltri, M. L., Sousa, R., & Nikiforov, M. A. (2017). Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution. Nature Methods, 14(10), 1003-1009. https://doi.org/10.1038/nmeth.4404