title: Imaging-based methods for assessing caspase activity in single cells.
abstract: Caspases, a family of proteases that are essential mediators of apoptosis, are divided into two groups: initiator caspases and executioner caspases. Each initiator caspase is activated at the apex of its respective pathway, which generally leads to the cleavage and activation of executioner caspases. Executioner caspases in turn cleave numerous substrates in the cell, leading to its demise. Initiator caspases are activated when inactive monomers undergo induced proximity to form an active caspase. In contrast, executioner caspases are activated by cleavage. Based on this key difference, different imaging techniques have been developed to measure caspase activation and activity on a single-cell basis. Bimolecular fluorescence complementation (BiFC) is used to measure induced proximity of initiator caspases, whereas Förster resonance energy transfer (FRET) permits the investigation of caspase-mediated substrate cleavage in real time. Because many of the events in apoptosis, including caspase activation, are asynchronous in nature, these single-cell imaging techniques have proven to be immensely powerful in ordering and dissecting caspase pathways. When coupled with parallel detection of additional hallmark events of apoptosis, they provide detailed and quantitative kinetic and positional insights into the signal transduction pathways that regulate cell death. Here we provide a brief introduction into BiFC- and FRET-based imaging of caspase activation and activity in single cells.
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: Centre for Systems Medicine, Royal College of Surgeons in Ireland, Dublin 2, Ireland Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland.
authors.forename: Markus
authors.initials: M
authors.lastname: Rehm
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; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000494:pharmacology; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D010455:Peptides; C519706:TcapQ647 peptide; D020169:Caspases