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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{"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", "type"=>"journal", "authors"=>[{"first_name"=>"Jeffrey", "last_name"=>"Klarenbeek", "scopus_author_id"=>"15831559300"}, {"first_name"=>"Joachim", "last_name"=>"Goedhart", "scopus_author_id"=>"6602537873"}, {"first_name"=>"Aernoud", "last_name"=>"Van Batenburg", "scopus_author_id"=>"56648148300"}, {"first_name"=>"Daniella", "last_name"=>"Groenewald", "scopus_author_id"=>"56647993200"}, {"first_name"=>"Kees", "last_name"=>"Jalink", "scopus_author_id"=>"7003869556"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"doi"=>"10.1371/journal.pone.0122513", "sgr"=>"84929486051", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"25875503", "issn"=>"19326203", "scopus"=>"2-s2.0-84929486051", "pui"=>"604107445"}, "id"=>"e8bf1b83-d152-3079-80c3-901dd7b5011a", "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.", "link"=>"http://www.mendeley.com/research/fourthgeneration-epacbased-fret-sensors-camp-feature-exceptional-brightness-photostability-dynamic-r", "reader_count"=>110, "reader_count_by_academic_status"=>{"Unspecified"=>8, "Professor > Associate Professor"=>1, "Researcher"=>27, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>37, "Student > Postgraduate"=>7, "Student > Master"=>13, "Other"=>4, "Student > Bachelor"=>7, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>8, "Professor > Associate Professor"=>1, "Researcher"=>27, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>37, "Student > Postgraduate"=>7, "Student > Master"=>13, "Other"=>4, "Student > Bachelor"=>7, "Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>8, "Engineering"=>2, "Biochemistry, Genetics and Molecular Biology"=>20, "Agricultural and Biological Sciences"=>46, "Medicine and Dentistry"=>8, "Neuroscience"=>9, "Pharmacology, Toxicology and Pharmaceutical Science"=>5, "Business, Management and Accounting"=>1, "Physics and Astronomy"=>1, "Chemistry"=>9, "Energy"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>2}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>8}, "Neuroscience"=>{"Neuroscience"=>9}, "Chemistry"=>{"Chemistry"=>9}, "Energy"=>{"Energy"=>1}, "Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>46}, "Business, Management and Accounting"=>{"Business, Management and Accounting"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>20}, "Unspecified"=>{"Unspecified"=>8}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>5}}, "reader_count_by_country"=>{"Netherlands"=>2, "Mexico"=>1, "Germany"=>1}, "group_count"=>5}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/2020524"], "description"=>"<div><p>Mentioned are construct details (column 1–3), unique Lab ID (column 4), % ratio change (expressed as cAMP-induced change from an initial baseline ratio of 1, column 5) with its Standard Error of Mean, FRET efficiency E in rest (column 6) with SEM, E after saturation of the sensor with IBMX and Forskolin with SEM (column 7), absolute change of lifetime in ns with SEM (column 8) and % change in lifetime with SEM (column 9). Column 10 contains the fully descriptive name. As mentioned in the main text, we will from now on for brevity name the sensors \"Epac-S<sup>LabID</sup>\", for example, Epac-S<sup>H96</sup> for mECFPΔ_Epac(CD, ΔDEP)_cp<sup>173</sup>Ven_Ven.</p>\n<p>In gray is the reference construct Epac-S<sup>H74</sup>. Abbreviations: nd, not determined; mCer3, monomeric Cerulean 3; mECFP, monomeric enhanced CFP; mTurq, monomeric Turquoise fluorescent protein; Hi-aff, High-affinity (Q270E mutant); <sup>cp173</sup>V, circular permutation of the fluorescent protein Venus(etc); td <sup>cp173</sup>V (etc), tandem of two <sup>cp173</sup>V fluorophores; <sup>cp173</sup>VV, tandem of cpVenus and Venus; CD, catalytically dead mutant (T781A & F782A amino acids of the Epac1 wild type protein); ΔDEP, deletion of the DEP domain to prevent membrane localization. Note that unlike Venus, Citrine contained the A206K monomerizing mutation. FRET efficiency E was calculated as E = 1-τ<sub>DonorAcceptor</sub>/τ<sub>Donor</sub> using τ<sub>Donor</sub> = 4.10 ns for mTurquoise2 and 3.70 ns for mTurquoise1.</p></div>", "links"=>[], "tags"=>["flim", "report variants", "Dynamic Range", "sensors favourite", "Q 270E", "cAMP sensors", "future studies", "Point mutation", "Epac moiety", "Dedicated Sensors", "cAMP Feature Exceptional Brightness", "fluorescence lifetime", "cAMP levels", "fret", "acceptor cassette", "cell biologists", "cp 173Venus fluorophores", "cAMP concentrations"], "article_id"=>1379480, "categories"=>["Uncategorised"], "users"=>["Jeffrey Klarenbeek", "Joachim Goedhart", "Aernoud van Batenburg", "Daniella Groenewald", "Kees Jalink"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0122513.g002", "stats"=>{"downloads"=>0, "page_views"=>24, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Summary_of_FRET_changes_of_the_constructs_mentioned_in_this_study_/1379480", "title"=>"Summary of FRET changes of the constructs mentioned in this study.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-04-14 03:15:12"}
  • {"files"=>["https://ndownloader.figshare.com/files/2020522"], "description"=>"<p>A. Localization of the FRET-sensors: N1E-115 cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)-<sup>cp173</sup>Venus-Venus (Epac-S<sup>H126</sup>); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td<sup>cp173</sup>Venus (Epac-S<sup>H187</sup>); mTurquoise2Δ-Epac(CD, ΔDEP)-td<sup>cp173</sup>Dark Venus (Epac-S<sup>H159</sup>) and mTurquoise2Δ-Epac(CD, ΔDEP)-<sup>cp174</sup>Cit (Epac-S<sup>H147</sup>). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S<sup>H105</sup>); mTurquoise (Epac-S<sup>H74</sup>) or mTurquoise2 (Epac-S<sup>H126</sup>) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor <sup>cp173</sup>Venus-Venus (Epac-S<sup>H134</sup>); td<sup>cp173</sup>Venus (Epac-S<sup>H187</sup>) or td<sup>cp173</sup>Dark Venus (Epac-S<sup>H189</sup>), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of HEK293T cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S<sup>H126</sup> or Epac-S<sup>H134</sup>) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S<sup>H187</sup>. After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S<sup>H189</sup>. A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.</p>", "links"=>[], "tags"=>["flim", "report variants", "Dynamic Range", "sensors favourite", "Q 270E", "cAMP sensors", "future studies", "Point mutation", "Epac moiety", "Dedicated Sensors", "cAMP Feature Exceptional Brightness", "fluorescence lifetime", "cAMP levels", "fret", "acceptor cassette", "cell biologists", "cp 173Venus fluorophores", "cAMP concentrations"], "article_id"=>1379478, "categories"=>["Uncategorised"], "users"=>["Jeffrey Klarenbeek", "Joachim Goedhart", "Aernoud van Batenburg", "Daniella Groenewald", "Kees Jalink"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0122513.g001", "stats"=>{"downloads"=>3, "page_views"=>35, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Characterization_of_novel_FRET_sensors_/1379478", "title"=>"Characterization of novel FRET sensors.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-04-14 03:15:12"}
  • {"files"=>["https://ndownloader.figshare.com/files/2020530"], "description"=>"<div><p>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 cp<sup>173</sup>Venus 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.</p></div>", "links"=>[], "tags"=>["flim", "report variants", "Dynamic Range", "sensors favourite", "Q 270E", "cAMP sensors", "future studies", "Point mutation", "Epac moiety", "Dedicated Sensors", "cAMP Feature Exceptional Brightness", "fluorescence lifetime", "cAMP levels", "fret", "acceptor cassette", "cell biologists", "cp 173Venus fluorophores", "cAMP concentrations"], "article_id"=>1379486, "categories"=>["Uncategorised"], "users"=>["Jeffrey Klarenbeek", "Joachim Goedhart", "Aernoud van Batenburg", "Daniella Groenewald", "Kees Jalink"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0122513", "stats"=>{"downloads"=>8, "page_views"=>35, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_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_/1379486", "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", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2015-04-14 03:15:12"}
  • {"files"=>["https://ndownloader.figshare.com/files/2020526"], "description"=>"<p>Mentioned are construct details (column 1–3), Unique Lab ID (column 4), optimal application (Sensitized Emission, SE, or Fluorescence Lifetime IMaging, FLIM; column 5) and the fully descriptive name (column 6). Abreviations: GFP, Green Fluorescent Protein; mRFP, monomeric Red Fluorescent Protein; pAdeno, plasmid with Adenoviral backbone; pLVX, plasmid with LentiViral eXpression backbone; for other abbreviations see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122513#pone.0122513.g002\" target=\"_blank\">Fig 2</a>. The lentiviral construct, H183) was prepared by dr. J. Karczewski (Wageningen University, NL) and has not been separately evaluated on our equipment.</p><p>Recommended constructs for new experiments.</p>", "links"=>[], "tags"=>["flim", "report variants", "Dynamic Range", "sensors favourite", "Q 270E", "cAMP sensors", "future studies", "Point mutation", "Epac moiety", "Dedicated Sensors", "cAMP Feature Exceptional Brightness", "fluorescence lifetime", "cAMP levels", "fret", "acceptor cassette", "cell biologists", "cp 173Venus fluorophores", "cAMP concentrations"], "article_id"=>1379482, "categories"=>["Uncategorised"], "users"=>["Jeffrey Klarenbeek", "Joachim Goedhart", "Aernoud van Batenburg", "Daniella Groenewald", "Kees Jalink"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0122513.t001", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Recommended_constructs_for_new_experiments_/1379482", "title"=>"Recommended constructs for new experiments.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2015-04-14 03:15:12"}

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{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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