A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals

Area of Science:

• Cellular Biology
• Mitochondrial Physiology
• Genetics and Genomics

Background:

• Mitochondria are crucial for cellular calcium (Ca²⁺) homeostasis, and their dysfunction is implicated in diseases like neurodegeneration and cancer.
• Studying mitochondrial Ca²⁺ dynamics in living cells is vital, but existing genetically encoded Ca²⁺ indicators (GECIs) for mitochondria lack adequate spatial resolution in mouse models.
• There is a need for advanced tools to investigate mitochondrial Ca²⁺ handling in various tissues without invasive procedures.

Purpose of the Study:

• To generate and characterize a novel transgenic mouse line for controlled, mitochondria-targeted expression of a Förster resonance energy transfer (FRET)-based Ca²⁺ indicator.
• To enable tissue-specific and non-invasive studies of mitochondrial Ca²⁺ dynamics in vivo.
• To provide a tool for investigating the role of mitochondrial Ca²⁺ in health and disease across different tissues and developmental stages.

Main Methods:

• Engineered the mouse ROSA26 locus to insert an optimized sequence for a mitochondria-targeted Cameleon (4mtD3cpv) probe, preceded by a loxP-STOP-loxP cassette.
• Achieved tissue-specific expression of the probe via Cre recombinase-mediated excision, allowing controlled mitochondrial Ca²⁺ indicator expression.
• Validated probe localization in the mitochondrial matrix and functionality using Ca²⁺ imaging in various tissues and brain slices from the generated mouse line.

Main Results:

• Successfully generated the mt-Cam transgenic mouse line enabling controlled expression of the 4mtD3cpv probe specifically within the mitochondrial matrix.
• Demonstrated ubiquitous and specific mitochondrial localization of the probe across diverse tissues and ages, from embryonic to aged animals.
• Confirmed probe functionality through in vitro and ex vivo Ca²⁺ imaging experiments, showing accurate detection of mitochondrial Ca²⁺ dynamics.

Conclusions:

• The mt-Cam mouse line provides a powerful, non-invasive tool for studying mitochondrial Ca²⁺ dynamics in a tissue-specific manner.
• This resource facilitates research into the role of mitochondrial Ca²⁺ in various physiological and pathological conditions without requiring invasive interventions.
• The developed probe allows for potential calibration of fluorescent signals to mitochondrial Ca²⁺ concentration ([Ca²⁺]), enhancing quantitative analysis.