ASC, a 22kDa adaptor protein consisting of a PYD and CARD domain, coordinates the function of apoptosis and inflammasomes in innate immunity.  Studies have shown that activation of the inflammasome sensors triggers ASC polymerization and formation of cellular aggregates/specks. This leads to recruitment and activation of pro-caspase-1 which promote maturation of pro- interleukin 1β (IL-1β) and pyroptotic cell death.

Here, we utilised a eukaryotic cell-free translational system and single molecule fluorescent spectroscopy to characterize the formation of the ASC aggregates. We observed that ASC self-associates spontaneously above a critical concentration to form distinctive massive aggregates (>1000 proteins) without forming of smaller oligomers. As this is indicative of fibrillation, we tested the presence of metastable species by seeding different concentrations of ASC-GFP with preformed ASC-Cherry seeds and looked for the recruitment of monomeric ASC to the seed using dual colour fluorescent spectroscopy. This analysis revealed the presence of a metastable zone.

While formation of ASC aggregates is driven by the PYD domain, it is surprising that the fibrils of ASC and its PYD domain are different. Both have distinct fluorescence spectroscopy signatures that are confirmed by negative staining electron microscopy images. ASC forms large clusters of star-shaped branching fibrils whereas fibrils of ASC-PYD lack the branching phenotype. Furthermore, they do not act as seeds for each other.

Additionally, data from single molecule FRET analysis of dual labelled ASC reveals that it undergoes conformational change during formation of the aggregates. In conclusion, we propose a new model for the formation of ASC aggregate. We have also established a platform to expand our studies to screen for proteins in innate immunity with prion-like behaviour.