Recovery after photobleaching of super MyD88 oligomers in IRAK4 KO cells is time-dependent. (A and B) TIRF images of MyD88-GFP in IRAK4 KO cells after 5 min (A) or 1 h incubation on IL1 SLBs. The white boxes indicate the position of photobleached Myddosomes. Middle: Cropped time series of the photobleached Myddosomes. Right: Schematics show possible explanation of fluorescence recovery due to continual MyD88 oligomerization at early time points (A). Conversely, MyD88 depletion after 1 h could explain loss of fluorescence recovery (B). (C) Quantification of super MyD88 oligomers recovery 2 min after photobleaching after 5 min or 1 h incubation. Bar represents mean ± SD; scatter plot symbols represent single MyD88-GFP oligomer photobleaching recovery measurements (n = 28 and n = 42 MyD88-GFP puncta for 5 min and 60 min, respectively). (D) Speculative model describing Myddosome assembly. MyD88 recruitment to the IL1-bound IL1R nucleates MyD88 oligomerization. Initially, the small oligomer of MyD88 is unstable and can disassemble. However, as MyD88 oligomer size increases, so does complex stability, and the formation of larger MyD88 complexes triggers downstream signaling in the form of the sequential recruitment of IRAK4 followed by IRAK1.