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Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells
Published on: March 16, 2017
A Novel "Double-Responsive" and "Dual-Targeted" Multifunctional Fluorescent Probe Monitors the Level Changes of ONOO-
Di Wei1, Yingshu Dai1, Xixian Yan1
1Ministry of Education Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, College of Chemistry, Fuzhou University, Fuzhou 350108, P. R. China.
Abstract:
Pyroptosis, often referred to as inflammatory necrosis, is a type of programmed cell death, characterized by the swelling of cells until the cell membranes rupture, resulting in the release of intracellular substances and a strong inflammatory response. Lipid droplets and mitochondria play important roles in cellular activities. A strong correlation exists between pyroptosis and mitochondrial dysfunction, which can be reflected through physiological functions and involves changes in the mitochondrial microenvironment and morphology. In this work, a "double-responsive" and "dual-targeted" fluorescent probe named WD-2 was constructed. It has excellent response performance to viscosity and ONOO-, and can simultaneously monitor the relevant levels in lipid droplets and mitochondria. Its remarkable targeting ability toward mitochondria and lipid droplets has been verified through colocalization experiments. In cell imaging experiments, the interaction between mitochondria and lipid droplets during nutritional stress was preliminarily studied. With the help of doxorubicin hydrochloride, the changes in the level of ONOO- in mitochondria during pyroptosis were explored, providing a new perspective for understanding the mechanism of this process.
Insights
Researchers developed a novel fluorescent probe, WD-2, to track cellular changes during pyroptosis (programmed cell death). This probe monitors viscosity and reactive oxygen species in lipid droplets and mitochondria, offering new insights into cell death mechanisms.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Medicine
Background:
- Pyroptosis, or inflammatory necrosis, is a programmed cell death pathway marked by cell swelling, membrane rupture, and inflammatory mediator release.
- Lipid droplets and mitochondria are crucial organelles involved in cellular functions and are implicated in pyroptosis.
- Mitochondrial dysfunction is strongly correlated with pyroptosis, involving alterations in mitochondrial microenvironment and morphology.
Purpose of the Study:
- To construct a novel fluorescent probe, WD-2, capable of dual targeting (mitochondria and lipid droplets) and double response (viscosity and peroxynitrite, ONOO⁻).
- To utilize WD-2 for simultaneous monitoring of viscosity and ONOO⁻ levels within lipid droplets and mitochondria.
- To investigate the interplay between mitochondria and lipid droplets under nutritional stress and explore ONOO⁻ dynamics during doxorubicin-induced pyroptosis.
Main Methods:
- Construction and characterization of a novel "double-responsive" and "dual-targeted" fluorescent probe, WD-2.
- Verification of the probe's targeting capabilities using colocalization experiments in cellular models.
- Application of WD-2 in cell imaging to study mitochondrial-lipid droplet interactions and ONOO⁻ levels during pyroptosis induced by doxorubicin hydrochloride.
Main Results:
- The WD-2 probe demonstrated excellent responsiveness to viscosity and ONOO⁻.
- Effective simultaneous monitoring of viscosity and ONOO⁻ in both lipid droplets and mitochondria was achieved.
- Colocalization experiments confirmed the probe's specific targeting of mitochondria and lipid droplets.
- Preliminary studies revealed interactions between mitochondria and lipid droplets under nutritional stress.
- Changes in mitochondrial ONOO⁻ levels during pyroptosis were successfully explored using WD-2.
Conclusions:
- The developed WD-2 fluorescent probe is a valuable tool for simultaneously monitoring viscosity and ONOO⁻ in mitochondria and lipid droplets.
- WD-2 facilitates the investigation of organelle interactions and reactive species dynamics during pyroptosis.
- This study provides a novel perspective for understanding the mechanisms underlying pyroptosis and related cellular processes.
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