Strategies for imaging mitophagy in high-resolution and high-throughput

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Mitophagy, the selective autophagic removal of mitochondria, is crucial for cellular health and preventing disease.
• Defective mitophagy is linked to various pathologies, highlighting its importance as a therapeutic target.
• Accurate, real-time measurement of mitophagy is essential for identifying disease regulators and drug candidates.

Purpose of the Study:

• To develop and validate novel live-cell imaging assays for quantifying mitophagy in mammalian cells.
• To enable high-throughput screening for mitophagy regulators and inducers.
• To provide a sensitive and reliable method for studying mitophagy dynamics.

Main Methods:

• Utilized stable mammalian cell lines co-expressing fluorescent protein tags for mitochondria and autophagosomes/lysosomes (EGFP-LC3-Mito-DsRed and Mitochondria-EGFP-LAMP1-RFP).
• Employed wide-field, confocal, and super-resolution microscopy for live-cell imaging.
• Validated assays for spatial and temporal resolution and high-throughput adaptability.

Main Results:

• Developed two distinct mitophagy assays with high spatial and temporal resolution.
• Demonstrated the capability for high-throughput screening.
• Identified novel mitophagy inducers through limited compound screening.
• The assays enable real-time monitoring of mitochondrial delivery to lysosomes.

Conclusions:

• The developed live-cell imaging assays offer a sensitive, quantitative, and real-time approach to measure mitophagy.
• This platform is suitable for high-throughput screening of siRNA or compounds to identify mitophagy regulators.
• These assays advance the study of mitophagy dynamics and its role in disease, aiding drug discovery efforts.