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Updated: May 16, 2026
Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro
Published on: July 17, 2018
Real-time detection of cellular death receptor-4 activation by fluorescence resonance energy transfer
Zeynep Dereli-Korkut1, Harmeet Gandhok, Ling Ge Zeng
1Department of Biomedical Engineering, City University of New York/City College, 160 Convent Avenue, Steinman Hall, T-434, New York, NY 10031, USA.
Abstract:
Targeted therapy involving the activation of death receptors DR4 and/or DR5 by its ligand, TRAIL, can selectively induce apoptosis in certain tumor cells. In order to profile the dynamic activation or trimerization of TRAIL-DR4 in live cells in real-time, the development of an apoptosis reporter cell line is essential. Fluorescence resonance energy transfer (FRET) technology via a FRET pair, cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP), was used in this study. DR4-CFP and DR4-YFP were stably expressed in human lung cancer PC9 cells. Flow cytometer sorting and limited dilution coupled with fluorescence microscopy were used to select a monoclonal reporter cell line with high and compatible expression levels of DR4-CFP and DR4-YFP. FRET experiments were conducted and FRET efficiencies were monitored according to the Siegel's YFP photobleaching FRET protocol. Upon TRAIL induction a significant increase in FRET efficiencies from 5% to 9% demonstrated the ability of the DR4-CFP/YFP reporter cell line in monitoring the dynamic activation of TRAIL pathways. 3D reconstructed confocal images of DR4-CFP/YFP reporter cells exhibited a colocalized expression of DR4-CFP and DR4-YFP mainly on cell membranes. FRET results obtained during this study complements the use of epi-fluorescence microscopy for FRET analysis. The real-time FRET analysis allows the dynamic profiling of the activation of TRAIL pathways by using the time-lapse fluorescence microscopy. Therefore, DR4-CFP/YFP PC9 reporter cells along with FRET technology can be used as a tool for anti-cancer drug screening to identify compounds that are capable of activating TRAIL pathways.
Insights
Researchers developed a novel apoptosis reporter cell line to track TRAIL-DR4 activation in real-time. This tool enables dynamic profiling of TRAIL pathways for effective anti-cancer drug screening.
Area of Science:
- Molecular Biology
- Cell Biology
- Biophysics
Background:
- Targeted cancer therapy can utilize the Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) to selectively trigger apoptosis in tumor cells by activating death receptors DR4 and/or DR5.
- Real-time monitoring of TRAIL-DR4 activation and trimerization in live cells is crucial for understanding and developing these targeted therapies.
- The development of a specialized apoptosis reporter cell line is essential for this dynamic profiling.
Purpose of the Study:
- To create and validate a live-cell reporter system for real-time monitoring of TRAIL-DR4 activation.
- To utilize Fluorescence Resonance Energy Transfer (FRET) technology for quantifying receptor-ligand interactions.
- To establish a tool for screening anti-cancer drugs that activate the TRAIL pathway.
Main Methods:
- Engineered human lung cancer PC9 cells to stably express DR4 fused with cyan fluorescence protein (DR4-CFP) and yellow fluorescence protein (DR4-YFP).
- Utilized flow cytometry and fluorescence microscopy to select a monoclonal reporter cell line with high, compatible expression of DR4-CFP and DR4-YFP.
- Applied the Siegel's YFP photobleaching FRET protocol to measure FRET efficiencies and monitored dynamic changes using time-lapse fluorescence microscopy.
Main Results:
- A significant increase in FRET efficiency from 5% to 9% upon TRAIL induction confirmed the reporter cell line's ability to monitor dynamic TRAIL pathway activation.
- 3D confocal imaging showed colocalized expression of DR4-CFP and DR4-YFP predominantly on the cell membrane.
- FRET analysis using time-lapse microscopy provided real-time insights into TRAIL pathway dynamics, complementing epi-fluorescence microscopy.
Conclusions:
- The developed DR4-CFP/YFP PC9 reporter cell line effectively monitors TRAIL-DR4 activation in real-time.
- This FRET-based system serves as a valuable tool for anti-cancer drug discovery, identifying compounds that activate the TRAIL pathway.
- The study demonstrates the utility of FRET technology in live-cell assays for profiling dynamic biological processes relevant to cancer therapy.
