Genetically Encoded Cell Fate Reporter System for Multiplex Single-Cell Detection of DNA and Mitochondrial Damage.

Area of Science:

• Cell biology
• Toxicology
• Drug discovery

Background:

• Predicting compound mechanisms of action is crucial for drug discovery and toxicology.
• Reporter cell lines are essential for phenotypic screening of large compound libraries.
• Developing cell lines with predictive biomarkers is a key requirement for phenotypic screening.

Purpose of the Study:

• To develop a stable cell line for real-time, single-cell level monitoring of DNA damage and mitochondrial permeabilization.
• To engineer cells to simultaneously report on DNA damage, mitochondrial permeabilization, and autophagy for comprehensive cell fate analysis.
• To utilize this multi-phenotypic reporter system for profiling toxicants and understanding their mechanisms of action.

Main Methods:

• Developed a stable cell line expressing TagBFP2-53BP1 as a real-time DNA damage sensor (53BP1 foci formation).
• Engineered cells to report mitochondrial permeabilization using Smac-RFP.
• Further expressed cells with EGFP-LC3 to visualize autophagy, enabling simultaneous imaging of distinct cell fates.

Main Results:

• Demonstrated that telomerase inhibitor-induced cell death involves double-strand breaks and mitochondrial permeabilization.
• Successfully profiled several toxicants based on distinct, observable cell fates using the developed system.
• Showcased the ability to extract heterogeneous cell responses over time for deeper mechanistic insights.

Conclusions:

• The developed multi-phenotypic live single-cell sensor system offers significant advantages over single-parameter assays.
• This approach provides enhanced insights into toxicant mechanisms of action and facilitates predictive toxicology.
• The system enables comprehensive analysis of distinct cell fates, improving the efficiency of drug discovery and safety testing.