Mitochondrial flashes: From indicator characterization to in vivo imaging.

Area of Science:

• Biochemistry
• Cell Biology
• Biomedical Engineering

Background:

• Mitochondria are vital for cellular energy and signaling; their dysfunction is linked to human diseases.
• Mitochondrial superoxide, a primary reactive oxygen species (ROS), is challenging to detect in vivo.
• Understanding mitochondrial dynamics is crucial for disease research.

Purpose of the Study:

• To characterize a circularly permuted yellow fluorescent protein (cpYFP) as a biosensor for mitochondrial superoxide and pH.
• To enable real-time detection of mitochondrial superoxide and pH dynamics in living systems.
• To investigate mitochondrial flash events and their triggers.

Main Methods:

• In vitro characterization of cpYFP for selectivity to superoxide and pH sensitivity.
• Confocal and two-photon imaging of transgenic cpYFP targeted to the mitochondrial matrix.
• Monitoring mitochondrial flashes in living cells, perfused hearts, and live animals.

Main Results:

• cpYFP exhibits high selectivity for superoxide over other ROS and dual sensitivity to pH.
• Mitochondrial flash events, triggered by transient mitochondrial permeability transition (tMPT), were detected in real-time.
• These flashes consist of superoxide bursts and transient alkalization signals.

Conclusions:

• cpYFP serves as a robust biosensor for mitochondrial superoxide and pH.
• Real-time monitoring of mitochondrial flashes provides a novel tool to study mitochondrial function.
• This technology can advance research in mitochondrial respiration, ROS production, pH regulation, and tMPT kinetics in health and disease.