Figure 4.
IRAK4 and IRAK1 are recruited to larger MyD88 oligomers. (A and B) Top: TIRF images of MyD88-GFP and IRAK4-mScarlet (A) or IRAK1-mScarlet (B). Region of interest (yellow box, merge image) shows an example of a MyD88-GFP spot colocalized with IRAK4-mScarlet (A) or IRAK1-mScarlet (B). Bottom: Time-series TIRF images from the region of interest (left) and fluorescence intensity time series (right) of MyD88-GFP and IRAK4-mScarlet (A) or IRAK1-mScarlet (B). (C and D) Quantification of the percentage of MyD88-GFP puncta per cell that colocalize with IRAK4 (C) or IRAK1 (D) for all puncta and puncta with lifetimes <50 s or ≥50 s. Violin plots show the distribution of individual cell measurements. Colored dots superimposed on violin plots correspond to the average value in the independent experiments (n = 3 for IRAK4/1; each replicate encompasses measurements from 16–34 cells). Bars represent mean ± SEM. (E) Density plot showing the distribution of MyD88 oligomer size (number of MyD88-GFP monomers is derived from the maximum intensity divided by the average intensity of GFP) for MyD88 puncta that are positive (+ve) or negative (-ve) for IRAK4 (top) or IRAK1 (bottom). Inset: Percentage of MyD88-GFP puncta per cell that colocalize with IRAK4 or IRAK1 with a maximum intensity of <4.5× GFP or ≥4.5× GFP. Violin plots show the distribution of individual cell measurements. Colored dots superimposed on the violin plots correspond to the mean value for the independent experiments (n = 3, IRAK4; n = 3, IRAK1). Bars represent mean ± SEM. (F) Analysis of IRAK4 and IRAK1 recruitment time during Myddosome assembly. Recruitment time was defined as the time interval from Myddosome nucleation (e.g., time = 0 s when MyD88-GFP puncta appears) to the appearance of IRAK4/1-mScarlet. Middle: Time series of TIRF images showing MyD88-GFP nucleation followed by IRAK4-mScarlet (top time series) or IRAK1-mScarlet (bottom time series) recruitment. Histogram of IRAK4 (n = 482 recruitment events, combined from 30 cells) and IRAK1 (n = 170 recruitment events, combined from 40 cells) recruitment times overlaid with the density plot of the distribution. Black horizontal lines on the histograms denote the average recruitment time (mean ± SD). Ave., average.

IRAK4 and IRAK1 are recruited to larger MyD88 oligomers. (A and B) Top: TIRF images of MyD88-GFP and IRAK4-mScarlet (A) or IRAK1-mScarlet (B). Region of interest (yellow box, merge image) shows an example of a MyD88-GFP spot colocalized with IRAK4-mScarlet (A) or IRAK1-mScarlet (B). Bottom: Time-series TIRF images from the region of interest (left) and fluorescence intensity time series (right) of MyD88-GFP and IRAK4-mScarlet (A) or IRAK1-mScarlet (B). (C and D) Quantification of the percentage of MyD88-GFP puncta per cell that colocalize with IRAK4 (C) or IRAK1 (D) for all puncta and puncta with lifetimes <50 s or ≥50 s. Violin plots show the distribution of individual cell measurements. Colored dots superimposed on violin plots correspond to the average value in the independent experiments (n = 3 for IRAK4/1; each replicate encompasses measurements from 16–34 cells). Bars represent mean ± SEM. (E) Density plot showing the distribution of MyD88 oligomer size (number of MyD88-GFP monomers is derived from the maximum intensity divided by the average intensity of GFP) for MyD88 puncta that are positive (+ve) or negative (-ve) for IRAK4 (top) or IRAK1 (bottom). Inset: Percentage of MyD88-GFP puncta per cell that colocalize with IRAK4 or IRAK1 with a maximum intensity of <4.5× GFP or ≥4.5× GFP. Violin plots show the distribution of individual cell measurements. Colored dots superimposed on the violin plots correspond to the mean value for the independent experiments (n = 3, IRAK4; n = 3, IRAK1). Bars represent mean ± SEM. (F) Analysis of IRAK4 and IRAK1 recruitment time during Myddosome assembly. Recruitment time was defined as the time interval from Myddosome nucleation (e.g., time = 0 s when MyD88-GFP puncta appears) to the appearance of IRAK4/1-mScarlet. Middle: Time series of TIRF images showing MyD88-GFP nucleation followed by IRAK4-mScarlet (top time series) or IRAK1-mScarlet (bottom time series) recruitment. Histogram of IRAK4 (n = 482 recruitment events, combined from 30 cells) and IRAK1 (n = 170 recruitment events, combined from 40 cells) recruitment times overlaid with the density plot of the distribution. Black horizontal lines on the histograms denote the average recruitment time (mean ± SD). Ave., average.

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