Dynamics of ASC speck formation during skin inflammatory responses in vivo

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signalling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested this speck-containing pyroptotic debris. A 3D ultrastructural reconstruction based on CLEM of in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas PYD or CARD alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis, but after substantial delay. Therefore, formation of a single compact speck and rapid cell death induction in vivo requires full-length ASC.