Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes.

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.

Purification of catalytically active caspase-12 and its biochemical characterization.

Caspase-12, mainly detected in endoplasmic reticulum (ER), has been suggested to play a role in ER-mediated apoptosis and inflammatory caspase activation pathway. Cleavage of the prodomain by caspase-3/-7 at the carboxyl terminus of Asp94 or m-calpain at the carboxyl terminus of Lys158 was reported to be a part of caspase-12-involved apoptosis. We biochemically characterized the prodomain-free forms of caspase-12 and the equivalent enzymes; Deltapro1(G95-D419), rev-Deltapro1[(T319-N419)-(G95-D318), a reverse form of Deltapro1] and rev-Deltapro2[(T319-N419)-(T159-D318)]. The three variants showed comparable activities which were dependent on salt concentration and pH. Auto-proteolytic cleavage was observed at two sites (carboxyl termini of Asp318 and Asp320) in Deltapro1. Constitutively active forms of caspase-12 (rev-Deltapro1 and rev-Deltapro2) could induce cell death in cells transfected with the corresponding expression vectors, but no cleavage of caspase-3, DFF45 or Bid was observed, indicating caspase-12 may mediate a distinct apoptotic pathway rather than caspase-8 or -9-mediated cell death.