title: Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes.
abstract: The human inflammatory caspases, including caspase-1, -4, -5 and -12, are considered as key regulators of innate immunity protecting from sepsis and numerous inflammatory diseases. Caspase-1 is activated by proximity-induced dimerization following recruitment to inflammasomes but the roles of the remaining inflammatory caspases in inflammasome assembly are unclear. Here, we use caspase bimolecular fluorescence complementation to visualize the assembly of inflammasomes and dimerization of inflammatory caspases in single cells. We observed caspase-1 dimerization induced by the coexpression of a range of inflammasome proteins and by lipospolysaccharide (LPS) treatment in primary macrophages. Caspase-4 and -5 were only dimerized by select inflammasome proteins, whereas caspase-12 dimerization was not detected by any investigated treatment. Strikingly, we determined that certain inflammasome proteins could induce heterodimerization of caspase-1 with caspase-4 or -5. Caspase-5 homodimerization and caspase-1/-5 heterodimerization was also detected in LPS-primed primary macrophages in response to cholera toxin subunit B. The subcellular localization and organization of the inflammasome complexes varied markedly depending on the upstream trigger and on which caspase or combination of caspases were recruited. Three-dimensional imaging of the ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)/caspase-1 complexes revealed a large spherical complex of ASC with caspase-1 dimerized on the outer surface. In contrast, NALP1 (NACHT leucine-rich repeat protein 1)/caspase-1 complexes formed large filamentous structures. These results argue that caspase-1, -4 or -5 can be recruited to inflammasomes under specific circumstances, often leading to distinctly organized and localized complexes that may impact the functions of these proteases.
authors.affiliation: 1] Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, TX, USA [2] Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA [3] Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA.
authors.forename: M G
authors.initials: MG
authors.lastname: Sanders
authors.affiliation: 1] Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, TX, USA [2] Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Parsons
authors.affiliation: 1] Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, TX, USA [2] Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA.
authors.forename: A G A
authors.initials: AG
authors.lastname: Howard
authors.affiliation: 1] Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, TX, USA [2] Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA [3] Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA.
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.affiliation: 1] Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, TX, USA [2] Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA.
authors.forename: S R
authors.initials: SR
authors.lastname: Fassio
authors.affiliation: Department of Immunity, Inflammation and Disease, National Institute of Environmental Health Sciences, 111 TW Alexander Drive, Research Triangle Park, NC, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Martinez
authors.affiliation: 1] Department of Pediatrics-Hematology, Baylor College of Medicine, Houston, TX, USA [2] Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA [3] Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: L
authors.initials: L
authors.lastname: Bouchier-Hayes
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D017209:Apoptosis / Q000276:immunology; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D053468:CARD Signaling Adaptor Proteins; D020170:Caspase 1 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D053454:Caspase 12 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020169:Caspases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D053471:Caspases, Initiator; D002772:Cholera Toxin / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate / Q000235:genetics; D058847:Inflammasomes / Q000737:chemistry / Q000378:metabolism; D007249:Inflammation / Q000201:enzymology* / Q000473:pathology; D008264:Macrophages / Q000201:enzymology; D057054:Molecular Imaging / Q000379:methods; D000070576:NLR Proteins; D055503:Protein Multimerization; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; D003598:Cytoskeletal Proteins; D058847:Inflammasomes; D000070576:NLR Proteins; C427903:NLRP1 protein, human; C402202:PYCARD protein, human; D002772:Cholera Toxin; C101136:Casp4 protein, mouse; D053454:Caspase 12; D020169:Caspases; D053471:Caspases, Initiator; D020170:Caspase 1