Atomic mechanisms of full-length ASC-mediated inflammasome assembly

ASC (Apoptosis-associated Speck-like protein containing a CARD) is a key adaptor protein that assembles inflammasomes by linking sensors such as NLRP3 to effectors like Caspase-1 via its PYD and CARD Death Domains. Due to ASC’s propensity to self-aggregate, most high-resolution structural studies focused on isolated PYD or CARD domains, leaving the atomic basis of full-length ASC assembly unknown. Here we determine atomic-resolution cryo-EM structures of PYD and CARD filaments from full-length ASC, revealing characteristic multitrack bundles composed of alternating ASCPYD and ASCCARD filaments that expose multiple interfaces for flexible assembly and efficient signaling. We further show that Caspase-1 filaments nucleate specifically from the B-end of ASCCARD filaments, and that the interdomain linker modulates bundle formation. The ASC isoform ASCb, with a four-residue linker, adopts a distinct architecture, correlating with reduced Caspase-1 activation efficiency. In ASC⁻/⁻ THP-1 cells, only wild-type ASC, not interface-disrupting mutants, restored ASC speck formation and Caspase-1 activation, underscoring the requirement for intact multitrack bundles. Cryo-electron tomography captures snapshots of higher-order inflammasome structures. These findings collectively define the structural and functional principles by which ASC organizes inflammasomes to amplify immune signaling. Inflammasomes are immune complexes built around the adaptor ASC (Apoptosis-associated Speck-like protein containing a CARD). Here, authors solve atomic structures of full-length ASC filaments, show that Caspase-1 starts at one filament end, and reveal a multitrack assembly model explaining rapid activation.