Fig 2: Clustering increases TRAF6 and HOIL1 lifetime at Myddosomes A, BHistogram showing the lifetime of TRAF6 and HOIL1 recruitment to single Myddosomes, and clusters containing 2–4 and 5–7 Myddosome complexes, off (blue) and on (red) 1 μm grids.CModel showing how a single Myddosome has a digital signaling output. However, the amplitude of this output increases proportionally as the density of Myddosome complexes increases within clusters. The increased amplitude of the M1/K63‐Ub, pIKK, and pp65 signaling output is likely due to the increase in stability of LUBAC and TRAF6 at larger clusters. As Myddosomes are biochemically coupled to extracellular IL‐1, this mechanism examines how IL‐1 signaling can generate both digital and analog signaling responses that are proportional to the stimulating dose of IL‐1. Source data are available online for this figure.

Fig 3: Characterization of dynamics of MyD88‐GFP puncta size in MyD88‐GFP/mScarlet‐TRAF6 cells off grids and on 1 μm grids A, BScaled density distribution of MyD88 max normalized intensity off grids and on 1 μm grids at a ligand density of 1 (A) or 10 (B) IL1/μm2. The average MyD88 max normalized intensity (dashed line) at 1 IL1/μm2 off grids versus on 1 μm grids are 2.2 versus 1.7, and at 10 IL1/μm2 are 4.4 versus 2.6. Insets are quantifications of the percentages of Myddosome clusters. A Myddosome cluster is defined as a MyD88‐GFP puncta containing equal to or greater than two Myddosomes. At 1 IL‐1/μm2, the percentages of Myddosome clusters off grids versus on 1 μm grids are 2.7 ± 1.4% versus 1.2 ± 0.7% and at 10 IL1/μm2 are 19.7 ± 2.0% versus 4.7 ± 0.8%. Bars represent mean ± SEM. At 1 IL1/μm2, data are measured from 24,315 MyD88 puncta off grids from 91 cells and four replicates, and 23,161 MyD88 puncta on 1 μm grids from 70 cells and three replicates. At 10 IL1/μm2, data are measured from 34,452 MyD88 puncta off grids from 87 cells and four biological replicates, and 71,525 MyD88 puncta on 1 μm grids from 100 cells and four biological replicates.

Fig 4: E3 ligases TRAF6 and HOIL1 are recruited to Myddosomes A, BTop: TIRF images of MyD88‐GFP and mScarlet‐TRAF6 (A) or mScarlet‐HOIL1 (B). Region of interest (yellow box, merge image) shows an example of a MyD88‐GFP puncta colocalized with mScarlet‐TRAF6 (A) or mScalet‐HOIL1 (B). Bottom: Time‐series TIRF images from the region of interest (left) and fluorescence intensity times series (right) of MyD88 and TRAF6 (A) or HOIL1 (B).C, DQuantifications of percentage of MyD88‐GFP puncta that colocalized with TRAF6 (C) or HOIL1 (D) grouped for all puncta, puncta containing ≤ 1×, > 1×, and ≥ 2× Myddosome complexes. The percentages for TRAF6 (C) in these groups are 15.4 ± 2.4%, 6.1 ± 1.8%, 36.2 ± 5.2%, and 58.4 ± 5.9%, respectively (mean ± SEM). The percentages for HOIL1 (D) in these groups are 8.6 ± 0.5%, 0.4 ± 0.2%, 14.9 ± 1.9%, and 25.9 ± 5.0%, respectively (mean ± SEM). Violin plots indicate the distribution of individual cell measurements. Colored dots superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 6 biological replicates for TRAF6, with 191 cells measured in total across all replicates, see also Appendix Fig S4A; n = 9 biological replicates for HOIL1, with 230 cells measured in total across all replicates, see also Appendix Fig S4B).EQuantifications of the percentage of TRAF6‐ or HOIL1‐positive MyD88 puncta on single Myddosomes and clusters containing 2–4, 5–7, 8–10, 11–13, and 14–16 Myddosome complexes. With greater Myddosome numbers per puncta, the percentage of MyD88 colocalized with TRAF6 or HOIL1 increases. The data points represent the average across replicates and bars represent mean ± SEM (n = 6 biological replicates for TRAF6, with 191 cells measured in total across all replicates, see also Appendix Fig S4A; n = 9 biological replicates for HOIL1, with 230 cells measured in total across all replicates, see also Appendix Fig S4B).FAnalysis of TRAF6 and HOIL1 landing size and recruitment time to MyD88 puncta. Left, time‐series TIRF images showing MyD88‐GFP puncta nucleation and the appearance of TRAF6. Right, the associated fluorescence intensity time trace for the time series shown. Recruitment time is defined as the time interval from Myddosome nucleation (e.g., time = 0 s when MyD88‐GFP puncta appears) to the appearance of a TRAF6 or HOIL1 puncta. Landing size is defined as the fluorescent intensity of the MyD88 puncta at the time when TRAF6 or HOIL1 appears (indicated on the fluorescent intensity trace with arrow).GHistogram of landing size of MyD88 puncta, expressed as number of Myddosome complexes per puncta, for TRAF6 (top, n = 6,015 recruitment events, technical replicates, from 183 cells pooled from six biological replicates) and HOIL1 (bottom, n = 5,562 recruitment events, technical replicates, from 212 cells pooled from nine biological replicates) recruitment. Histogram is overlaid with a density plot of the distribution. Black horizontal lines on the histograms denote the average landing size (mean ± SEM).HHistogram for the recruitment time of TRAF6 (top, n = 94 recruitment events, technical replicates, from four cells pooled from three biological replicates) and HOIL1 (bottom, n = 69 recruitment events, technical replicates, from four cells pooled from four biological replicates) overlaid with the density plot of the distribution. Black horizontal lines on the histograms denote the average recruitment time (mean ± SD). Source data are available online for this figure.

Fig 5: Extracellular barriers that restrict Myddosome clustering reduce TRAF6 and HOIL1 recruitment A, BTIRF images of EL4 cells expressing MyD88‐GFP and mScarlet‐TRAF6 stimulated on IL‐1 functionalized SLBs at a ligand density of 1 IL‐1/μm2 off grids (A) or on 1 μm grids (B). Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bars, 5 μm.CQuantification of percentage of MyD88‐GFP puncta that colocalized with TRAF6 off grids and on 1 μm grids at a ligand density of 1 IL‐1/μm2, and the percentages are 16.2 ± 2.5% and 6.1 ± 1.5%, respectively (mean ± SEM). Violin plots indicate the distribution of individual cell measurements. Colored dots superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 4 experimental replicates off grids, with a total of 24,315 MyD88 puncta from 91 cells; n = 3 biological replicates on 1 μm grids, with a total of 23,161 MyD88 puncta from 70 cells). Statistical significance is determined using unpaired two‐tailed Student's t‐test.D, ETIRF images of EL4 cells expressing MyD88‐GFP and mScarlet‐TRAF6 stimulated on IL‐1 functionalized SLBs at a ligand density of 10 IL‐1 per μm2 off grids (D) or on 1 μm grids (E). Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bars, 5 μm.FQuantification of percentage of MyD88‐GFP puncta that colocalized with TRAF6 off grids and on 1 μm grids at a ligand density of 10 IL‐1/μm2, and the percentages are 25.0 ± 1.6% and 12.7 ± 1.8%, respectively (mean ± SEM). Violin plots indicate the distribution of individual cell measurements. Colored dots superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 4 biological replicates off grids, with a total of 34,452 MyD88 puncta from 87 cells; n = 4 biological replicates on 1 μm grids, with a total of 71,525 MyD88 puncta from 100 cells). Statistical significance is determined using unpaired two‐tailed Student's t‐test.G, HTIRF images of EL4 cells expressing MyD88‐GFP and mScarlet‐HOIL1 stimulated on IL‐1 functionalized SLBs at a ligand density of 10 IL‐1/μm2 off grids (G) or on 1 μm grids (H). Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bars, 5 μm.IQuantification of percentage of MyD88‐GFP puncta that colocalized with HOIL1 off grids and on 1 μm grids at a ligand density of 10 IL‐1/μm2, and the percentages are 7.0 ± 0.8% and 1.7 ± 0.7%, respectively (mean ± SEM). Violin plots indicate the distribution of individual cell measurements. Colored dots superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 4 biological replicates off grids, with a total of 53,852 MyD88 puncta from 74 cells; n = 4 biological replicates on 1 μm grids, with a total of 55,075 MyD88 puncta from 154 cells). Statistical significance is determined using unpaired two‐tailed Student's t‐test.J, KTIRF images of EL4 cells expressing MyD88‐GFP and mScarlet‐HOIL1 stimulated on IL‐1 functionalized SLBs at a ligand density of 32 IL‐1/μm2 off grids (J) or on 1 μm grids (K). Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bars, 5 μm.LQuantification of percentage of MyD88‐GFP puncta that colocalized with HOIL1 off grids and on 1 μm grids at a ligand density of 32 IL‐1/μm2, and the percentages are 8.6 ± 0.5% and 4.2 ± 0.5%, respectively (mean ± SEM). Violin plots indicate the distribution of individual cell measurements. Colored dots superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 9 biological replicates off grids, with a total of 118,354 MyD88 puncta off grids from 230 cells; n = 4 biological replicates on 1 μm grids, with a total of 68,819 MyD88 puncta from 138 cells). Statistical significance is determined using unpaired two‐tailed Student's t‐test. Source data are available online for this figure.