Real-Time Monitoring Mitochondrial Viscosity during Mitophagy Using a Mitochondria-Immobilized Near-Infrared
Xiaodong Wang1, Li Fan1, Shuohang Wang2
1Institute of Environmental Science, College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, P. R. China.
Abstract:
Mitophagy plays a crucial role in maintaining intracellular homeostasis through the removal of dysfunctional mitochondria and recycling their constituents in a lysosome-degradative pathway, which leads to microenvironmental changes within mitochondria, such as the pH, viscosity, and polarity. However, most of the mitochondrial fluorescence viscosity probes only rely on electrostatic attraction and readily leak out from the mitochondria during mitophagy with a decreased membrane potential, thus easily leading to an inaccurate detection of viscosity changes. In this work, we report a mitochondria-immobilized NIR-emissive aggregation-induced emission (AIE) probe CS-Py-BC, which allows for an off-on fluorescence response to viscosity, thus enabling the real-time monitoring viscosity variation during mitophagy. This system consists of a cyanostilbene skeleton as the AIE active core and viscosity-sensitive unit, a pyridinium cation for the mitochondria-targeting group, and a benzyl chloride subunit that induces mitochondrial immobilization. As the viscosity increased from 0.903 cP (0% glycerol) to 965 cP (99% glycerol), CS-Py-BC exhibited an about 92-fold increase in fluorescence intensity at 650 nm, which might be attributed to the restriction of rotation and inhibition of twisted intramolecular charge transfer in a high viscosity system. We also revealed that CS-Py-BC could be well immobilized onto mitochondria, regardless of the mitochondrial membrane potential fluctuation. Most importantly, using CS-Py-BC, we have successfully visualized the increased mitochondrial viscosity during starvation or rapamycin-induced mitophagy in real time. All these features render CS-Py-BC a promising candidate to investigate mitophagy-associated dynamic physiological and pathological processes.
Insights
Researchers developed a novel probe, CS-Py-BC, for real-time monitoring of mitochondrial viscosity changes during mitophagy. This probe overcomes limitations of previous methods, enabling accurate detection of viscosity variations in mitophagy.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Imaging
Background:
- Mitophagy is essential for cellular homeostasis, involving lysosomal degradation of damaged mitochondria.
- Existing mitochondrial viscosity probes suffer from leakage and inaccurate readings due to membrane potential changes during mitophagy.
- Accurate monitoring of mitochondrial microenvironment changes during mitophagy is crucial for understanding cellular processes.
Purpose of the Study:
- To develop a novel, mitochondria-immobilized probe for real-time viscosity monitoring during mitophagy.
- To overcome the limitations of current probes that leak from mitochondria.
- To enable accurate visualization of viscosity variations within mitochondria during mitophagy.
Main Methods:
- Design and synthesis of a mitochondria-immobilized, near-infrared (NIR) emissive aggregation-induced emission (AIE) probe (CS-Py-BC).
- The probe incorporates a cyanostilbene core, a pyridinium cation for mitochondrial targeting, and a benzyl chloride subunit for immobilization.
- Evaluation of the probe's fluorescence response to viscosity changes and its immobilization efficiency under varying mitochondrial membrane potentials.
Main Results:
- CS-Py-BC exhibits an "off-on" fluorescence response to viscosity, with a 92-fold intensity increase in high viscosity environments.
- The probe demonstrates robust immobilization on mitochondria, independent of mitochondrial membrane potential.
- Real-time visualization of increased mitochondrial viscosity during starvation- or rapamycin-induced mitophagy was achieved.
Conclusions:
- CS-Py-BC is a reliable and effective tool for real-time monitoring of mitochondrial viscosity variations.
- The probe's ability to remain immobilized overcomes previous detection limitations.
- CS-Py-BC holds promise for investigating mitophagy-related physiological and pathological processes.
