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mCerulean3-Based Cameleon Sensor to Explore Mitochondrial Ca2+ Dynamics In Vivo
- Source :
- iScience, iScience, Vol 16, Iss, Pp 340-355 (2019)
- Publication Year :
- 2019
- Publisher :
- Elsevier Inc., 2019.
-
Abstract
- 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.<br />Graphical Abstract<br />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<br />Biological Sciences Tools; Cell Biology; Optical Imaging
- Subjects :
- 0301 basic medicine
Fluorescence-lifetime imaging microscopy
02 engineering and technology
Article
Biological Sciences Tool
03 medical and health sciences
0302 clinical medicine
In vivo
Organelle
Fluorescence microscope
lcsh:Science
030304 developmental biology
0303 health sciences
Multidisciplinary
biology
Chemistry
Optical Imaging
Correction
Cameleon (protein)
Cell Biology
021001 nanoscience & nanotechnology
Fluorescence
Cytosol
Förster resonance energy transfer
030104 developmental biology
biology.protein
Biophysics
lcsh:Q
Biological Sciences Tools
0210 nano-technology
030217 neurology & neurosurgery
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- iScience, iScience, Vol 16, Iss, Pp 340-355 (2019)
- Accession number :
- edsair.doi.dedup.....056a14ec189d5c031b403e8a322240e8