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Bright Bioluminescent BRET Sensor Proteins for Measuring Intracellular Caspase Activity
Anniek den Hamer1, Pieterjan Dierickx2,3, Remco Arts1
1Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Den Dolech 2, 5612 AZ Eindhoven, The Netherlands.
ACS Sensors
|July 4, 2017
Summary
Researchers developed novel Bioluminescence Resonance Energy Transfer (BRET) caspase sensors. These genetically encoded probes bypass external light needs, overcoming limitations of FRET sensors for apoptosis research.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Fluorescence Resonance Energy Transfer (FRET) probes are vital for monitoring apoptosis.
- FRET probe limitations include dependence on external light, autofluorescence, and light scattering.
- These issues hinder applications in light-sensitive cells and certain assay formats.
Purpose of the Study:
- Develop novel caspase activity sensors using Bioluminescence Resonance Energy Transfer (BRET).
- Create genetically encoded, ratiometric probes that do not require external illumination.
- Overcome FRET limitations for improved intracellular caspase activity detection.
Main Methods:
- Constructed BRET sensor proteins fusing NanoLuc luciferase and mNeonGreen fluorescent protein.
- Engineered linkers containing recognition sites for caspase-3, -8, or -9.
- Performed in vitro characterization and high-throughput plate-reader assays, alongside long-term single-cell imaging.
Main Results:
- Developed three BRET caspase sensors with a robust 10-fold decrease in BRET ratio upon linker cleavage.
- Demonstrated successful high-throughput measurement of intracellular caspase activity, overcoming FRET-based limitations.
- Enabled long-term single-cell imaging to record cell heterogeneity in caspase activity.
Conclusions:
- BRET caspase sensors offer a powerful, illumination-independent alternative to FRET probes.
- These sensors facilitate high-throughput screening and detailed cellular analysis of apoptotic signaling.
- The technology allows for monitoring caspase activity in diverse cellular contexts and populations.
