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J Vis Exp. 2018 Mar 5;(133). doi: 10.3791/57316.

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation.

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Department of Pediatrics, Division of Hematology-Oncology, Baylor College of Medicine.
Department of Pediatrics, Division of Hematology-Oncology and Department of Molecular and Cellular Biology, Baylor College of Medicine;


The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

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