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

Abstract

Signaling pathways can produce digital outputs that are invariant and analogue outputs that scale with the amount of stimulation. In IL-1 receptor (IL-1R) signaling both types of outputs require the Myddosome, a multi-protein complex. The Myddosome is required for polyubiquitin chain formation and NF-kB signaling. However, the ways in which these signals are spatially and temporally regulated to drive switch-like and proportional outcomes is not understood. We find that during IL-1R signaling, Myddosomes dynamically re-organize into large, multi-Myddosome clusters at the cell membrane. Blockade of Myddosome clustering using nanoscale extracellular barriers reduces NF-kB activation. We find that Myddosomes function as a scaffold that assembles 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 found that LUBAC was especially sensitive to clustering, with a sevenfold lower recruitment to single Myddosomes than clustered Myddosomes. This data reveals that the clustering behavior of Myddosome provides the basis for digital and analogue IL-1R signaling.

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“…This IL-1 variant had been engineered to have a 10x His tag, and could therefore label membranes that contained DGS-NTA lipids. Full details on the design, expression and purification of this construct have been previously reported ( 21 , 28 ). Briefly, His10-Halo-IL-1β was expressed and purified as a spycatcher-spytag conjugation of two separate engineered proteins (IL-1β-Spytag and His10-Halo-Spycatcher).…”

“…Before each imaging experiment we acquired calibration images using recombinant mEGFP and mScarlet-i. To image single GFP/mScarlet-i fluorophores, recombinant purified mEGFP and mScarlet-i ( 28 ) was diluted in HBS and adsorbed to KOH cleaned glass. Single molecules of GFP or mScarlet-i were imaged using identical microscope acquisition settings to those used for cellular imaging.…”

“…We quantified spinning disk confocal images of RelA nuclear localization in an analysis pipeline implemented in FIJI and Cell Profiler, previously reported here ( 28 ). Briefly, first we performed background subtraction from the GFP and RelA (Cy5 channel) immunofluorescence staining micrographs in FIJI.…”

“…To estimate the number of CHARMS monomers and TRAF6 within tracked signalosomes, the fluorescent intensity was divided by the median intensity of single mEGFP or mScarlet-I fluorophores respectively, using a methodology previously described ( 28 , 29 ). Briefly, to compute the distribution of single GFP/mScarlet-i fluorophores intensities, we processed images of single mGFP or mScarlet fluorophores absorbed to glass identically to CHARMS and mScarlet-TRAF6 images.…”