title: A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells.
abstract: G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation.
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE, Amsterdam, The Netherlands.
authors.forename: Jakobus
authors.initials: J
authors.lastname: van Unen
authors.affiliation: Bio-Imaging-Center/Rudolf-Virchow-Zentrum and Department of Pharmacology and Toxicology, University of Wuerzburg, Versbacher Strasse 9, 97078, Wuerzburg, Germany.
authors.forename: Anette D
authors.initials: AD
authors.lastname: Stumpf
authors.affiliation: Bio-Imaging-Center/Rudolf-Virchow-Zentrum and Department of Pharmacology and Toxicology, University of Wuerzburg, Versbacher Strasse 9, 97078, Wuerzburg, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Schmid
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE, Amsterdam, The Netherlands.|Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, NL-1066 CX, Amsterdam, the Netherlands.
authors.forename: Nathalie R
authors.initials: NR
authors.lastname: Reinhard
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE, Amsterdam, The Netherlands.|Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, NL-1066 CX, Amsterdam, the Netherlands.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Hordijk
authors.affiliation: Bio-Imaging-Center/Rudolf-Virchow-Zentrum and Department of Pharmacology and Toxicology, University of Wuerzburg, Versbacher Strasse 9, 97078, Wuerzburg, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Hoffmann
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE, Amsterdam, The Netherlands.
authors.forename: Theodorus W J
authors.initials: TW
authors.lastname: Gadella
authors.affiliation: Swammerdam Institute for Life Sciences, Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, P.O. Box 94215, NL-1090 GE, Amsterdam, The Netherlands.
authors.forename: Joachim
authors.initials: J
authors.lastname: Goedhart
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D037342:Pertussis Toxin / Q000494:pharmacology; D043587:Receptor, Muscarinic M3 / Q000378:metabolism; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism; D049368:Receptors, Lysophosphatidic Acid / Q000378:metabolism
chemical_list: C547768:ADRA2A protein, human; D044387:GTP-Binding Protein beta Subunits; D043587:Receptor, Muscarinic M3; D018341:Receptors, Adrenergic, alpha-2; D049368:Receptors, Lysophosphatidic Acid; D037342:Pertussis Toxin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go