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Updated: Sep 18, 2025
Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation
Published on: March 5, 2018
Designing an apoptosis reporter by mutagenesis-based insertion of caspase-3 cleavage motif into green fluorescence
Dong Gil Lee1, Hae-Jun Yang1, Unbin Chae1
1Futuristic Animal Resource & Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju-si 28116, Republic of Korea.
Introduction:
Apoptosis is an essential process for organisms and animal development, and its dysregulation is related to the progression of several diseases. Therefore, methods to detect apoptosis are necessary for mechanistic research and drug development. To overcome the disadvantages of traditional methods for detecting apoptosis, a variety of imaging strategies based on enzyme-mediated fluorescence activation have been developed.
Objectives:
This study aims to design a novel strategy for apoptosis fluorescent reporters which are inactivated by caspase-3.
Methods:
Four candidate EGFP mutations containing DEVD-similar sequences were selected by their structural positions and hydrophilicities, and an EGFP mutant was chosen by investigating fluorescence expression. This EGFP mutant was stably expressed in various cells using the safe harbor locus. To verify our apoptosis reporter system, protein levels, colocalization, and intensity were investigated in apoptosis-induced EGFP mutant-expressing cells.
Results:
The fluorescence intensity of the mutant EGFP was decreased in a time- and concentration-dependent manner by staurosporine and H2O2, which induce apoptosis. Furthermore, compared to a dark-to-bright reporter of apoptosis (caspase-activatable GFP), our bright-to-dark system showed greater sensitivity for apoptosis. Our system is useful in various models, including other species.
Conclusion:
Our method does not require additional peptides, which makes it easily adaptable to other systems. And, our apoptosis reporter may be useful in a variety of research fields as well as drug screening.
Insights
Researchers developed a novel bright-to-dark apoptosis reporter system. This system, inactivated by caspase-3, offers enhanced sensitivity for detecting apoptosis compared to existing methods, aiding disease research and drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Apoptosis is crucial for development and health; its dysregulation is linked to diseases.
- Accurate apoptosis detection methods are vital for research and drug discovery.
- Enzyme-mediated fluorescence imaging offers advantages over traditional apoptosis detection.
Purpose of the Study:
- To design a novel fluorescent reporter for apoptosis, specifically inactivated by caspase-3.
- To create a sensitive and adaptable tool for apoptosis detection.
Main Methods:
- Engineered enhanced green fluorescent protein (EGFP) mutants with caspase-3 cleavage sites.
- Selected an optimal EGFP mutant based on fluorescence expression and structural properties.
- Validated the reporter system in cells expressing the EGFP mutant, assessing protein levels, colocalization, and fluorescence intensity changes upon apoptosis induction.
Main Results:
- Apoptosis induction via staurosporine and H2O2 led to a time- and concentration-dependent decrease in mutant EGFP fluorescence.
- The developed bright-to-dark system demonstrated superior sensitivity in detecting apoptosis compared to a dark-to-bright reporter.
- The reporter system proved effective across various cell types and species.
Conclusions:
- The novel apoptosis reporter system is easily adaptable due to its self-contained nature, requiring no additional peptides.
- This reporter holds significant potential for diverse research applications, including high-throughput drug screening for apoptosis-related conditions.
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