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Bright Bioluminescent BRET Sensor Proteins for Measuring Intracellular Caspase Activity.
den Hamer, Anniek; Dierickx, Pieterjan; Arts, Remco; de Vries, Joost S P M; Brunsveld, Luc; Merkx, Maarten.
Afiliación
  • den Hamer A; Laboratory 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.
  • Dierickx P; Hubrecht Institute-KNAW and University Medical Center Utrecht, 3584 CT Utrecht, The Netherlands.
  • Arts R; Division of Heart and Lungs, Department of Cardiology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.
  • de Vries JSPM; Laboratory 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.
  • Brunsveld L; Laboratory 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.
  • Merkx M; Laboratory 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 Sens ; 2(6): 729-734, 2017 Jun 23.
Article en En | MEDLINE | ID: mdl-28670623
FRET-based caspase activity probes have become important tools to monitor apoptotic cell signaling. However, their dependence on external illumination is incompatible with light sensitive cells and hampers applications that suffer from autofluorescence and light scattering. Here we report the development of three caspase sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) that retain the advantages of genetically encoded, ratiometric optical probes but do not require external illumination. These sensors consist of the bright and stable luciferase NanoLuc and the fluorescent protein mNeonGreen, fused together via a linker containing a recognition site for caspase-3, -8, or -9. In vitro characterization showed that each caspase sensor displayed a robust 10-fold decrease in BRET ratio upon linker cleavage, with modest caspase specificity. Importantly, whereas scattering and background fluorescence precluded FRET-based detection of intracellular caspase activity in plate-reader assays, such measurements could be easily performed using our caspase BRET sensors in a high throughput format. The brightness of the BRET sensors also enabled long-term single-cell imaging, allowing BRET-based recording of cell heterogeneity in caspase activity in a heterogenic cell population.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: ACS Sens Año: 2017 Tipo del documento: Article País de afiliación: Países Bajos Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: ACS Sens Año: 2017 Tipo del documento: Article País de afiliación: Países Bajos Pais de publicación: Estados Unidos