Detection of Initiator Caspase Induced Proximity in Single Cells by Caspase Bimolecular Fluorescence Complementation.
The caspase family of proteases includes key regulators of apoptosis and inflammation. The caspases can be divided into two groups, the initiator caspases and the executioner caspases. Initiator caspases include caspase-2, caspase-8, and caspase-9 and are activated by proximity-induced dimerization upon recruitment to large molecular weight protein complexes called activation platforms. This protocol describes an imaging-based technique called caspase Bimolecular Fluorescence Complementation (BiFC) that measures induced proximity of initiator caspases. This method uses nonfluorescent fragments of the fluorescent protein Venus fused to initiator caspase monomers. When the caspase is recruited to its activation platform, the resulting induced proximity of the caspase monomers facilitates refolding of the Venus fragments into the full molecule, reconstituting its fluorescence. Thus, the assembly of initiator caspase activation platforms can be followed in single cells in real time. Induced proximity is the most apical step in the activation of initiator caspases, and therefore, caspase BiFC is a robust and specific method to measure initiator caspase activation.
Department of Pediatrics-Hematology and Center for Cell and Gene Therapy, Baylor College of Medicine, 1102 Bates Avenue, Houston, TX, 77030, USA.
Melissa J
MJ
Parsons
Department of Pediatrics-Hematology and Center for Cell and Gene Therapy, Baylor College of Medicine, 1102 Bates Avenue, Houston, TX, 77030, USA.
Sara R
SR
Fassio
Department of Pediatrics-Hematology and Center for Cell and Gene Therapy, Baylor College of Medicine, 1102 Bates Avenue, Houston, TX, 77030, USA. bouchier@bcm.edu.
Lisa
L
Bouchier-Hayes
D017209:Apoptosis; D001426:Bacterial Proteins / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D010957:Plasmids; D059010:Single-Cell Analysis / Q000379:methods*; D059008:Time-Lapse Imaging / Q000379:methods
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D020169:Caspases
Apoptosis; BiFC; Caspases; Confocal; Fluorescence; Imaging; Microscopy; Venus