PROJECT SUMMARY AND ABSTRACT Macromolecular signaling complexes play fundamental roles in biology, ranging from the control of development to regulation of the immune system. The inflammasome is an example of a macromolecular signaling platform that activates the innate immune system in response to invading pathogens or endogenous host-derived danger signals. As the innate immune system regulates inflammation and has crosstalk with the adaptive immune system, dysregulation of inflammasome signaling is associated with autoinflammatory and autoimmune disorders. Activation of the inflammasome involves the recognition of intracellular danger signals by germline- encoded pattern recognition receptors that then recruit an adaptor protein known as ASC (although in some instances ASC is dispensable) to form the inflammasome. ASC then recruits a cysteine protease known as caspase-1 to the inflammasome where it undergoes autoproteolytic maturation. Active caspase-1 then cleaves substrates that are important for restoring cellular homeostasis, including the interleukin family of cytokines, IL- 1 and IL-18, and the pore forming protein GSDMD to induce a lytic type of cell death known as pyroptosis. The mechanism of how inflammasomes are assembled, in particular, how the different proteins are organized in space and time to ensure proper function, is not well understood. Of note, the incorporation of ASC on the inflammasome is required for cytokine processing, but the mechanism of how ASC induces cytokine activation has remained unclear for over a decade. Additionally, some inflammasomes are ASC-independent but can form ASC-containing inflammasomes. How these inflammasomes decide when to form an ASC-independent or ASC containing inflammasome is a mystery. The goal of this proposal is to address these fundamental questions in innate immunity. We will elucidate the mechanism of cytokine activation and delineate the spatiotemporal regulation of inflammasomes. To accomplish this, we have developed new tools that will allow us to build a spatiotemporal map of inflammasome formation and signaling, which will provide key mechanistic insights to aid therapeutic development for immune related disorders. Furthermore, we anticipate these tools will become important biomedical research tools.