ASC speck formation leads to pyroptosis via activation of Caspa through PYD–PYD domain interaction. 3-dpf Tg(HSE:asc-mKate2) larvae treated with the pan-caspase inhibitor Q-VD-Oph (100 µM) after or without heat shock were stained with acridine orange at 17 hphs. Acridine orange (AO) spots (A) and specks (B) were quantified. Treatment with Q-VD-Oph significantly diminished cell death caused by speck formation compared with nontreated controls (one-way ANOVA; ****, P < 0.0001) but did not affect speck formation. (C) Live imaging of transient expression of HSE:caspa-EGFP, HSE:caspb-EGFP, or HSE:casp3a-EGFP with HSE:asc-mKate2. Recruitment to the ASC–mKate2 specks only occurs in the case of Caspa–GFP coexpression. Enlarged view of single muscle cells for each case (yellow boxes) are shown below their corresponding row. Live imaging of heat-shock–induced transient expression of HSE:PYD_A-mKate2 or HSE:CARD_A-mKate2 with HSE:caspa-EGFP in 3-dpf larvae at 19 hphs (D) with enlarged view of single cells (D′ and D″). (E) PYD_A, but not CARD_A, aggregates recruit Caspa-GFP. Live imaging of transient expression of HSE:caspa-EGFP, HSE:caspb-EGFP, or HSE:casp3a-EGFP between 9 and 17 hphs. Vast amounts of epidermal cellular debris are seen only when Caspa–GFP is overexpressed. (F) Single plane of HSE:caspa-EGFP transient expression at 17 hphs in muscle cells showing morphological changes upon Caspa–GFP overexpression. caspa antisense wish in 3-dpf larvae (G). Enlarged view shows expression in skin (G′) and ventral fin (G″). (H) Time-lapse imaging of caspa mutants transiently expressing HSE:asc-mKate2 with GFP at 3 hphs. Cell death response is severely affected in caspa^−/− keratinocytes, with cells dying an apoptotic-like death >7 h after speck formation. Full time-lapses are included in Video 9. Bars: (full larvae) 300 µm; (all others) 40 µm.