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
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Le résumé fourni par la source
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.
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Le contrôle bibliographique ouvert
DOI retrouvé dans Crossref DOI retrouvé ; titre concordant.
- Titre Crossref
- 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
- Date Crossref
- 14/04/2015
- Éditeur
- Public Library of Science (PLoS)
- Type
- journal-article
Ce recoupement confirme des métadonnées liées au DOI. Il ne confirme ni la méthode ni les conclusions de l’étude, et il ne compte pas comme une seconde source scientifique indépendante.
Où se fait cette recherche
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The Netherlands Cancer Institute Division of Cell Biology pays non établi dans la noticeÉtablissement de santé
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Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital pays non établi dans la noticeStructure de recherche
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Van Leeuwenhoek Centre for Advanced Microscopy pays non établi dans la noticeStructure de recherche
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Netherlands Institute for Neuroscience pays non établi dans la noticeStructure de recherche
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University of Amsterdam Swammerdam Institute for Life Sciences pays non établi dans la noticeUniversité ou école supérieure
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van Leeuwenhoek Centre of Advanced Microscopy pays non établi dans la noticeInstitution
Division of Cell Biology — The Netherlands Cancer Institute, Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital et Van Leeuwenhoek Centre for Advanced Microscopy, avec 3 autres affiliations.
Une affiliation ne permet pas de déduire la nationalité d’un auteur.