A genetically encoded fluorescent protein sensor for mitochondrial membrane damage detection.

Area of Science:

• Cellular Biology
• Toxicology
• Biotechnology

Background:

• Mitochondria are vital organelles, and detecting mitochondrial damage is critical for cellular biology and toxicology.
• Existing chemical probes require exogenous reagents, limiting direct cellular event monitoring.
• Genetically encoded fluorescent protein sensors offer a reagent-free alternative for detecting molecular events.

Purpose of the Study:

• To introduce and validate the MMD-Sensor, a novel molecular sensor system for monitoring mitochondrial membrane damage.
• To demonstrate the sensor's ability to detect mitochondrial damage induced by various chemical agents.
• To provide a direct, visual method for assessing mitochondrial integrity in living cells.

Main Methods:

• The MMD-Sensor comprises two modules: Module I (MLS-CSS-NLS-mNeonGreen N-terminus-mCherry) and Module II (mNeonGreen C-terminus-NLS-mtagBFP2).
• Under normal conditions, Module I is in the mitochondrial inner membrane, and Module II is in the nucleus.
• Mitochondrial damage triggers CSS cleavage, releasing Module I to the nucleus, where mNeonGreen fragment complementation generates green fluorescence.

Main Results:

• The sensor system successfully detected mitochondrial membrane damage in HeLa and 293T cells.
• Dynamic red fluorescence migration from mitochondria to the nucleus and nucleus-localized green fluorescence indicated damage.
• The MMD-Sensor effectively visualized damage induced by uncoupling agents, ATP synthase inhibitors, cationic carriers, and reactive oxygen species (ROS).

Conclusions:

• The MMD-Sensor provides a direct and sensitive method for monitoring mitochondrial membrane damage.
• This genetically encoded system eliminates the need for exogenous reagents, simplifying cellular damage assessment.
• The MMD-Sensor is a valuable tool for research in cell biology, toxicology, and drug discovery.