RT Journal Article SR Electronic T1 Dynamics of ASC speck formation during skin inflammatory responses in vivo JF bioRxiv FD Cold Spring Harbor Laboratory SP 111542 DO 10.1101/111542 A1 Paola Kuri A1 Nicole L. Schieber A1 Thomas Thumberger A1 Joachim Wittbrodt A1 Yannick Schwab A1 Maria Leptin YR 2017 UL http://biorxiv.org/content/early/2017/03/03/111542.abstract AB 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.One Sentence Summary With a new endogenous ASC real-time reporter we characterize speck dynamics in vivo as well as the concomitant pyroptosis speck formation causes in keratinocytes.