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

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Genetically Encoded Ca2+ Indicators (GECIs) are crucial for studying organelle Ca2+ homeostasis.
• Existing GECIs face challenges like low fluorescence, mistargeting, and pH sensitivity.
• Mitochondrial FRET-based GECIs often use ECFP, complicating fluorescence lifetime analysis due to its double exponential lifetime.

Purpose of the Study:

• To develop improved cytosolic and mitochondria-targeted Cameleon GECIs.
• To enhance probe brightness, stability, targeting efficiency, and Ca2+ sensitivity.
• To enable in vivo expression using adeno-associated viral vectors.

Main Methods:

• Substitution of the ECFP donor with mCerulean3, a brighter protein with single exponential lifetime.
• Extensive modification of GECI constructs for improved targeting and Ca2+ binding response.
• In situ characterization using fluorescence microscopy and Fluorescence Lifetime Imaging Microscopy (FLIM).
• Cloning into adeno-associated viral vectors for in vivo applications.

Main Results:

• Developed modified Cameleon GECIs with mCerulean3 offering improved fluorescence properties.
• Enhanced targeting efficiency and Ca2+ binding-induced fluorescence changes were achieved.
• Successful in situ characterization and demonstrated ex vivo and in vivo applications.

Conclusions:

• The modified Cameleon GECIs represent a significant advancement over existing probes.
• These improved GECIs are suitable for studying organelle Ca2+ dynamics in various biological contexts.
• The developed viral vectors facilitate in vivo applications of these advanced calcium indicators.