title: Measuring initiator caspase activation by bimolecular fluorescence complementation.
abstract: Initiator caspases, including caspase-2, -8, and -9, are activated by the proximity-driven dimerization that occurs after their recruitment to activation platforms. Here we describe the use of caspase bimolecular fluorescence complementation (caspase BiFC) to measure this induced proximity. BiFC assays rely on the use of a split fluorescent protein to identify protein-protein interactions in cells. When fused to interacting proteins, the fragments of the split fluorescent protein (which do not fluoresce on their own) can associate and fluoresce. In this protocol, we use the fluorescent protein Venus, a brighter and more photostable variant of yellow fluorescent protein (YFP), to detect the induced proximity of caspase-2. Plasmids encoding two fusion products (caspase-2 fused to either the amino- or carboxy-terminal halves of Venus) are transfected into cells. The cells are then treated with an activating (death) stimulus. The induced proximity (and subsequent activation) of caspase-2 in the cells is visualized as Venus fluorescence. The proportion of Venus-positive cells at a single time point can be determined using fluorescence microscopy. Alternatively, the increase in fluorescence intensity over time can be evaluated by time-lapse confocal microscopy. The caspase BiFC strategy described here should also work for other initiator caspases, such as caspase-8 or -9, as long as the correct controls are used.
authors.affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030 Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Parsons
authors.affiliation: Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030 Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: Lisa
authors.initials: L
authors.lastname: Bouchier-Hayes
mesh_terms: D000818:Animals; D020169:Caspases / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D006358:Hot Temperature; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D013997:Time Factors; D014162:Transfection; D050257:Tubulin Modulators / Q000494:pharmacology; D014750:Vincristine / Q000494:pharmacology
chemical_list: D008164:Luminescent Proteins; D050257:Tubulin Modulators; D014750:Vincristine; D020169:Caspases