mCerulean3-Based Cameleon Sensor to Explore Mitochondrial Ca2+ Dynamics In Vivo
Elisa Greotti,
Ilaria Fortunati,
Diana Pendin,
Camilla Ferrante,
Luisa Galla,
Lorena Zentilin,
Mauro Giacca,
Nina Kaludercic,
Moises Di Sante,
Letizia Mariotti,
Annamaria Lia,
Marta Gómez-Gonzalo,
Michele Sessolo,
Giorgio Carmignoto,
Renato Bozio,
Tullio Pozzan
Affiliations
Elisa Greotti
Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy; Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
Ilaria Fortunati
Department of Chemical Sciences and INSTM, University of Padua, 35131 Padua, Italy
Diana Pendin
Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy; Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
Camilla Ferrante
Department of Chemical Sciences and INSTM, University of Padua, 35131 Padua, Italy
Luisa Galla
Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy; Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
Lorena Zentilin
Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), 34149 Trieste, Italy
Mauro Giacca
Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), 34149 Trieste, Italy
Nina Kaludercic
Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy; Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
Moises Di Sante
Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
Letizia Mariotti
Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy; Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
Annamaria Lia
Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
Marta Gómez-Gonzalo
Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy; Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
Michele Sessolo
Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy; Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
Giorgio Carmignoto
Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy; Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy
Renato Bozio
Department of Chemical Sciences and INSTM, University of Padua, 35131 Padua, Italy
Tullio Pozzan
Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy; Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy; Venetian Institute of Molecular Medicine (VIMM), 35131 Padua, Italy; Corresponding author
Summary: Genetically Encoded Ca2+ Indicators (GECIs) are extensively used to study organelle Ca2+ homeostasis, although some available probes are still plagued by a number of problems, e.g., low fluorescence intensity, partial mistargeting, and pH sensitivity. Furthermore, in the most commonly used mitochondrial Förster Resonance Energy Transfer based-GECIs, the donor protein ECFP is characterized by a double exponential lifetime that complicates the fluorescence lifetime analysis. We have modified the cytosolic and mitochondria-targeted Cameleon GECIs by (1) substituting the donor ECFP with mCerulean3, a brighter and more stable fluorescent protein with a single exponential lifetime; (2) extensively modifying the constructs to improve targeting efficiency and fluorescence changes caused by Ca2+ binding; and (3) inserting the cDNAs into adeno-associated viral vectors for in vivo expression. The probes have been thoroughly characterized in situ by fluorescence microscopy and Fluorescence Lifetime Imaging Microscopy, and examples of their ex vivo and in vivo applications are described. : Biological Sciences Tools; Cell Biology; Optical Imaging Subject Areas: Biological Sciences Tools, Cell Biology, Optical Imaging
