The CARD plays a critical role in ASC foci formation and inflammasome signalling

The ASC (apoptosis speck-like protein) is a key component of multimeric protein complexes that mediate inflammation and host defence. Comprising a PYD (Pyrin) domain and a CARD (caspase activation and recruitment domain), ASC functions downstream of NLRs (nucleotide-binding domain, leucine-rich repeat-containing receptors) and AIM2 (absent in melanoma 2) through the formation of supramolecular structures termed inflammasomes. However, the mechanism underlying ASC signalling and its dependency on oligomeric arrangements in inflammasome formation remain poorly understood. When expressed in cells, ASC forms discrete foci (called 'specks') typically with one speck per cell. We employed a BiFC (bimolecular fluorescence complementation) system to investigate and visualize ASC foci formation in living cells. We demonstrated that the CARD of ASC plays a central role in ASC inflammasome assembly, representing the minimal unit capable of forming foci in conjunction with the caspase 1 CARD. Mutational studies point to multiple surfaces on the ASC CARD and two predominant areas on the caspase 1 CARD mediating the formation of ASC/caspase 1 foci. The lack of foci formation for ASC CARD mutants correlates with a loss of IL-1β (interleukin 1β) processing in response to NLRP (NLR family, PYD domaincontaining) 3 or AIM2 agonists in RAW264.7 cell reconstitution assays. Analogously, we show that productive formation of the Salmonella typhimurium-induced NLRC4 (NLR family CARD domain-containing protein 4) inflammasome is dependent on ASC-CARD-mediated platform formation. Thus the results of the present study depict a central role of CARDs in the formation of ASC signalling platforms and provide an important tool for investigation of CARD-dependent networks.