title: 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.
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.
authors: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands; van Leeuwenhoek Centre of Advanced Microscopy, Amsterdam, The Netherlands.
authors: Jeffrey
authors: J
authors: Klarenbeek
authors: Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands; van Leeuwenhoek Centre of Advanced Microscopy, Amsterdam, The Netherlands.
authors: Joachim
authors: J
authors: Goedhart
authors: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands; van Leeuwenhoek Centre of Advanced Microscopy, Amsterdam, The Netherlands.
authors: Aernoud
authors: A
authors: van Batenburg
authors: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands; van Leeuwenhoek Centre of Advanced Microscopy, Amsterdam, The Netherlands.
authors: Daniella
authors: D
authors: Groenewald
authors: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands; van Leeuwenhoek Centre of Advanced Microscopy, Amsterdam, The Netherlands.
authors: Kees
authors: K
authors: Jalink
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000032:analysis* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D061848:Optical Imaging / Q000295:instrumentation / Q000379:methods*; D010006:Osteoblasts / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; D011993:Recombinant Fusion Proteins; C503160:VC6.1 cameleon protein, recombinant; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP