Labeling and measuring stressed mitochondria using a PINK1-based ratiometric fluorescent sensor.

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Mitochondria are vital for cellular homeostasis, and their dysfunction is linked to numerous diseases, including diabetes, cancer, and neurodegeneration.
• Understanding the triggers of mitochondrial stress is crucial for disease research, yet comprehensive analyses have been lacking.

Purpose of the Study:

• To develop and utilize a novel fluorescent tool, mito-Pain, for broad analysis of mitochondrial stress.
• To identify compounds that induce mitochondrial stress and characterize their effects on mitochondria.

Main Methods:

• Developed mito-Pain, a fluorescent probe utilizing PTEN-induced putative kinase 1 (PINK1) stabilization to label stressed mitochondria.
• Screened a library of 3,374 compounds using mito-Pain to identify mitochondrial stress inducers.
• Classified compounds based on their effects on mitochondrial depolarization, morphology, and Parkin recruitment.

Main Results:

• Identified 57 compounds that induce mitochondrial stress.
• Observed that Parkin recruitment is often associated with mitochondrial depolarization and aggregation, indicating recruitment to severely damaged mitochondria.
• Documented diverse mitochondrial morphological changes (fragmentation, aggregation, elongation, swelling) induced by compounds, with or without depolarization or Parkin recruitment.
• Discovered PINK1-dependent ectopic Parkin recruitment to cytosolic puncta.

Conclusions:

• Mito-Pain is an effective tool for detecting stressed mitochondria across various conditions.
• The study provides novel insights into mitochondrial quality control pathways, including PINK1/Parkin signaling.
• Identified new chemical compounds that modulate mitochondrial function and stress responses.