Myddosome clustering in IL‐1 receptor signaling regulates the formation of an NF‐kB activating signalosome

IL‐1 receptor (IL‐1R) signaling can activate thresholded invariant outputs and proportional outputs that scale with the amount of stimulation. Both responses require the Myddosome, a multiprotein complex. The Myddosome is required for polyubiquitin chain formation and NF‐kB signaling. However, how these signals are spatially and temporally regulated to drive switch‐like and proportional outcomes is not understood. During IL‐1R signaling, Myddosomes dynamically reorganize into multi‐Myddosome clusters at the cell membrane. Blockade of clustering using nanoscale extracellular barriers reduces NF‐kB activation. Myddosomes function as scaffolds that assemble an NF‐kB signalosome consisting of E3‐ubiquitin ligases TRAF6 and LUBAC, K63/M1‐linked polyubiquitin chains, phospho‐IKK, and phospho‐p65. This signalosome preferentially assembles at regions of high Myddosome density, which enhances the recruitment of TRAF6 and LUBAC. Extracellular barriers that restrict Myddosome clustering perturbed the recruitment of both ligases. We find that LUBAC was especially sensitive to clustering with 10‐fold lower recruitment to single Myddosomes than clustered Myddosomes. These data reveal that the clustering behavior of Myddosomes provides a basis for digital and analog IL‐1R signaling.