Fourth-generation Epac-Based FRET Sensors for cAMP Feature Exceptional Brightness, Photostability and Dynamic Range: Characterization of Dedicated Sensors for FLIM, for Ratiometry and With High Affinity

PLoS One. 2015 Apr 14;10(4):e0122513. doi: 10.1371/journal.pone.0122513. eCollection 2015.

Abstract

Epac-based FRET sensors have been widely used for the detection of cAMP concentrations in living cells. Originally developed by us as well as others, we have since then reported several important optimizations that make these sensors favourite among many cell biologists. We here report cloning and characterization of our fourth generation of cAMP sensors, which feature outstanding photostability, dynamic range and signal-to-noise ratio. The design is based on mTurquoise2, currently the brightest and most bleaching-resistant donor, and a new acceptor cassette that consists of a tandem of two cp173Venus fluorophores. We also report variants with a single point mutation, Q270E, in the Epac moiety, which decreases the dissociation constant of cAMP from 9.5 to 4 μM, and thus increases the affinity ~ 2.5-fold. Finally, we also prepared and characterized dedicated variants with non-emitting (dark) acceptors for single-wavelength FLIM acquisition that display an exceptional near-doubling of fluorescence lifetime upon saturation of cAMP levels. We believe this generation of cAMP outperforms all other sensors and therefore recommend these sensors for all future studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biosensing Techniques / instrumentation
  • Biosensing Techniques / methods*
  • Biosensing Techniques / standards
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism
  • Cell Line, Tumor
  • Cyclic AMP / analysis*
  • Cyclic AMP / metabolism
  • Fluorescence Resonance Energy Transfer / instrumentation
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / chemistry
  • Gene Expression
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism
  • HEK293 Cells
  • Humans
  • Mice
  • Neurons / metabolism
  • Neurons / ultrastructure
  • Optical Imaging / instrumentation
  • Optical Imaging / methods*
  • Osteoblasts / metabolism
  • Osteoblasts / ultrastructure
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction

Substances

  • Calcium-Binding Proteins
  • Fluorescent Dyes
  • Guanine Nucleotide Exchange Factors
  • RAPGEF3 protein, human
  • Recombinant Fusion Proteins
  • VC6.1 cameleon protein, recombinant
  • Green Fluorescent Proteins
  • Cyclic AMP

Grant support

JG is supported by NanoNextNL. KJ is supported by grants from KWF (NKI 2010-4626) and the Stichting Technische Wetenschappen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.