mCerulean3-Based Cameleon Sensor to Explore Mitochondrial Ca2+ Dynamics In Vivo

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.
Graphical Abstract
Highlights • Donor substitution in a mitochondrial Ca2+ sensor improves photo-physical properties • Mitochondria-targeting sequence amelioration enhances the sensor localization • Donor substitution allows FLIM-FRET analysis, with a compensation for pH bias • The performance of the sensor is improved in situ, ex vivo, and in vivo
Biological Sciences Tools; Cell Biology; Optical Imaging