title: A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change.
abstract: Genetically encoded calcium biosensors have become valuable tools in cell biology and neuroscience, but some aspects such as signal strength and response kinetics still need improvement. Here we report the generation of a FRET-based calcium biosensor employing troponin C as calcium-binding moiety that is fast, is stable in imaging experiments, and shows a significantly enhanced fluorescence change. These improvements were achieved by engineering magnesium and calcium-binding properties within the C-terminal lobe of troponin C and by the incorporation of circularly permuted variants of the green fluorescent protein. This sensor named TN-XL shows a maximum fractional fluorescence change of 400% in its emission ratio and linear response properties over an expanded calcium regime. When imaged in vivo at presynaptic motoneuron terminals of transgenic fruit flies, TN-XL exhibits highly reproducible fluorescence signals with the fastest rise and decay times of all calcium biosensors known so far.
authors.affiliation: AG Zelluläre Dynamik, Abteilung Neuronale Informationsverarbeitung, Max-Planck-Institut für Neurobiologie 82152 Martinsried, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Mank
authors.forename: Dierk F
authors.initials: DF
authors.lastname: Reiff
authors.forename: Nicola
authors.initials: N
authors.lastname: Heim
authors.forename: Michael W
authors.initials: MW
authors.lastname: Friedrich
authors.forename: Alexander
authors.initials: A
authors.lastname: Borst
authors.forename: Oliver
authors.initials: O
authors.lastname: Griesbeck
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000032:analysis* / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D004330:Drosophila; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006624:Hippocampus / Q000196:embryology / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D002118:Calcium