Single-color, ratiometric biosensors for detecting signaling activities in live cells

Elife. 2018 Jul 3;7:e35458. doi: 10.7554/eLife.35458.


Genetically encoded fluorescent biosensors have revolutionized the study of signal transduction by enabling the real-time tracking of signaling activities in live cells. Investigating the interaction between signaling networks has become increasingly important to understanding complex cellular phenomena, necessitating an update of the biosensor toolkit to allow monitoring and perturbing multiple activities simultaneously in the same cell. We therefore developed a new class of fluorescent biosensors based on homo-FRET, deemed FLuorescence Anisotropy REporters (FLAREs), which combine the multiplexing ability of single-color sensors with a quantitative, ratiometric readout. Using an array of color variants, we were able to demonstrate multiplexed imaging of three activity reporters simultaneously in the same cell. We further demonstrate the compatibility of FLAREs for use with optogenetic tools as well as intravital two-photon imaging.

Keywords: anisotropy; biochemistry; cell biology; chemical biology; fluorescent proteins; kinases; mouse; polarization; second messengers; signal transduction.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Biosensing Techniques*
  • Cell Nucleus / metabolism
  • Cell Nucleus / ultrastructure
  • Color
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Cytosol / metabolism
  • Cytosol / ultrastructure
  • Fluorescence Polarization / methods*
  • Fluorescence Resonance Energy Transfer / instrumentation
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / metabolism*
  • Gene Expression Regulation
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Plasmids / chemistry
  • Plasmids / metabolism
  • Signal Transduction*
  • Single-Cell Analysis / methods*
  • Transfection
  • src-Family Kinases / genetics
  • src-Family Kinases / metabolism


  • Bacterial Proteins
  • Fluorescent Dyes
  • Luminescent Proteins
  • enhanced green fluorescent protein
  • red fluorescent protein
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins
  • lyn protein-tyrosine kinase
  • src-Family Kinases
  • Cyclic AMP-Dependent Protein Kinases