ASC specks are highly intercrossed, filamentous structures whose clustering is altered by point mutations. (A–C) CLEM of high-pressure frozen 3 dpf Tg(HSE:asc-mKate2) larvae at 18 hphs. Low magnification electron micrograph (A, left) and overlay with red channel (A, right) imaged with light microscope. Black arrowhead shows location of speck. (B) Area of interest (red box) imaged with electron microscope. TEM tomography slice of the speck (B, black arrowhead) and overlay with 3D reconstruction of speck after manual tracking of individual filaments (B′). (C) Zoom in of three-dimensional reconstruction model. Entire TEM tomography stack and three-dimensional model are found in Video 4. Bars, 10 µm, unless otherwise indicated. (D) Results from phosphorylation-site analysis using the online tool GPS version 2.1.1, depicting Syk and JNK-specific, predicted phosphorylation sites in zebrafish ASC. Full results are found in Table S1. (E) Live imaging of larvae transiently expressing HSE:asc(4xmut)-mKate2, containing four missense mutations (T38A, Y152F, T160A, and T170A). (F) Single muscle cell in larvae transiently expressing either HSE:asc-mKate2, or HSE:asc(4xmut)-mKate2, or HSE:asc(Y152F)-mKate2. Bars, 30 µm.