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

Jeffrey Klarenbeek; Joachim Goedhart; Aernoud van Batenburg; Daniella Groenewald; Kees Jalink

doi:10.1371/journal.pone.0122513

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.

Authors

Jeffrey Klarenbeek¹, Joachim Goedhart², Aernoud van Batenburg¹, Daniella Groenewald¹, Kees Jalink¹

Affiliations

¹ Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands; van Leeuwenhoek Centre of Advanced Microscopy, Amsterdam, The Netherlands.
² Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands; van Leeuwenhoek Centre of Advanced Microscopy, Amsterdam, The Netherlands.

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

Grants and funding

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.