Figure 3.
Analysis of MyD88-GFP puncta dynamics, lifetime, and size. (A) TIRF images of MyD88-GFP in an EL4 cell landing on an IL1β-functionalized SLB. Overlaid colored boxes highlight examples of individual MyD88-GFP puncta. Red and orange ROI show the fluorescence intensity time series (left) and TIRF images (right) from Myddosomes that are short-lived (<50 s) and dim (<3× GFP average intensity). Blue regions of interest show fluorescence intensity time series (bottom) and TIRF images (top) from two example MyD88-GFP puncta that grow in intensity and have a long lifetime (≥50 s). The dashed gray lines on intensity plots mark the quantal MyD88-GFP fluorescence intensities, estimated from single GFP fluorophores. (B) Density plot of the maximum fluorescent intensity of MyD88-GFP puncta (dark blue, n = 2,422 tracked MyD88-GFP particles from 14 cells) compared with single molecules of GFP (green, n = 397 GFP particles) and estimated intensity distribution of a 6× GFP multimer (light blue). Shaded blue region designates intensity values >4.5× GFP. (C) Quantification of the proportion (%) of MyD88-GFP puncta per cell that have a maximum fluorescence intensity <4.5× GFP or ≥4.5× GFP. Violin plots show the distribution of the cell data. Data points superimposed on violin plots are the averages of independent experiments. Bars represent mean ± SEM (n = 6 experimental replicates, with 6–24 cells measured per replicate). (D) Distribution of MyD88-GFP puncta lifetimes. Myddosomes were classified by maximum fluorescence intensity <4.5× or ≥4.5× GFP (n = 2,037 <4.5× GFP versus n = 385 ≥4.5× GFP tracked MyD88-GFP puncta combined from 14 cells). (E) Quantification of the proportion (%) per cell of MyD88-GFP puncta with an intensity maximum ≥4.5× GFP categorized by lifetimes <50 s or ≥50 s. Violin plots show the distribution of the cell data. Data points superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 6 experimental replicates, with 6–24 cells measured per replicate). (F) Correlation between growth in intensity and lifetime of MyD88-GFP puncta. Left: TIRF images and intensity trace of a representative MyD88-GFP puncta. The change in intensity calculated as maximum intensity subtracted by the initial intensity. Right: 2D histogram of MyD88-GFP puncta lifetime versus changed in fluorescent intensity. Linear fit is shown as a blue line with the 95% CI shown in gray (Spearman’s rank correlation coefficient R = 0.59, P < 0.001, n = 1,763 MyD88 tracks, combined from 14 cells). (G) Activation of phospho-p38 at different IL1 ligand densities on SLBs. Left: Phospho-p38 staining on SLB labeled with 0.1 and 300 IL1β per square micrometer. EL4 cell lines (30 min after addition to IL1β-labeled SLBs) were fixed and stained for phospho-p38 (magenta); DAPI stained nuclei (blue). Right: Quantification of phospho-p38 staining. Violin plots show the single-cell distribution of staining intensities. Data points superimposed on the violin plots are the averages from independent experiments (n = 3 experimental replicates, with >6,000 cells measured per replicate). Bars represent mean ± SEM. P values were calculated using a two-tailed unpaired Student's t test. (H) Quantification of the number of MyD88-GFP puncta per cell that assemble with intensity maxima ≥4.5× GFP on SLB labeled with high and low IL1 densities. Violin plots show the single-cell distribution of the total number of MyD88-GFP puncta per cell and those with lifetimes <50 s or ≥50 s. Data points superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 3 experimental replicates, with 6–24 cells measured per replicate). P values were calculated using an unpaired Student's t test. fluo., fluorescence; Max, maximum; Norm., normalized.

Analysis of MyD88-GFP puncta dynamics, lifetime, and size. (A) TIRF images of MyD88-GFP in an EL4 cell landing on an IL1β-functionalized SLB. Overlaid colored boxes highlight examples of individual MyD88-GFP puncta. Red and orange ROI show the fluorescence intensity time series (left) and TIRF images (right) from Myddosomes that are short-lived (<50 s) and dim (<3× GFP average intensity). Blue regions of interest show fluorescence intensity time series (bottom) and TIRF images (top) from two example MyD88-GFP puncta that grow in intensity and have a long lifetime (≥50 s). The dashed gray lines on intensity plots mark the quantal MyD88-GFP fluorescence intensities, estimated from single GFP fluorophores. (B) Density plot of the maximum fluorescent intensity of MyD88-GFP puncta (dark blue, n = 2,422 tracked MyD88-GFP particles from 14 cells) compared with single molecules of GFP (green, n = 397 GFP particles) and estimated intensity distribution of a 6× GFP multimer (light blue). Shaded blue region designates intensity values >4.5× GFP. (C) Quantification of the proportion (%) of MyD88-GFP puncta per cell that have a maximum fluorescence intensity <4.5× GFP or ≥4.5× GFP. Violin plots show the distribution of the cell data. Data points superimposed on violin plots are the averages of independent experiments. Bars represent mean ± SEM (n = 6 experimental replicates, with 6–24 cells measured per replicate). (D) Distribution of MyD88-GFP puncta lifetimes. Myddosomes were classified by maximum fluorescence intensity <4.5× or ≥4.5× GFP (n = 2,037 <4.5× GFP versus n = 385 ≥4.5× GFP tracked MyD88-GFP puncta combined from 14 cells). (E) Quantification of the proportion (%) per cell of MyD88-GFP puncta with an intensity maximum ≥4.5× GFP categorized by lifetimes <50 s or ≥50 s. Violin plots show the distribution of the cell data. Data points superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 6 experimental replicates, with 6–24 cells measured per replicate). (F) Correlation between growth in intensity and lifetime of MyD88-GFP puncta. Left: TIRF images and intensity trace of a representative MyD88-GFP puncta. The change in intensity calculated as maximum intensity subtracted by the initial intensity. Right: 2D histogram of MyD88-GFP puncta lifetime versus changed in fluorescent intensity. Linear fit is shown as a blue line with the 95% CI shown in gray (Spearman’s rank correlation coefficient R = 0.59, P < 0.001, n = 1,763 MyD88 tracks, combined from 14 cells). (G) Activation of phospho-p38 at different IL1 ligand densities on SLBs. Left: Phospho-p38 staining on SLB labeled with 0.1 and 300 IL1β per square micrometer. EL4 cell lines (30 min after addition to IL1β-labeled SLBs) were fixed and stained for phospho-p38 (magenta); DAPI stained nuclei (blue). Right: Quantification of phospho-p38 staining. Violin plots show the single-cell distribution of staining intensities. Data points superimposed on the violin plots are the averages from independent experiments (n = 3 experimental replicates, with >6,000 cells measured per replicate). Bars represent mean ± SEM. P values were calculated using a two-tailed unpaired Student's t test. (H) Quantification of the number of MyD88-GFP puncta per cell that assemble with intensity maxima ≥4.5× GFP on SLB labeled with high and low IL1 densities. Violin plots show the single-cell distribution of the total number of MyD88-GFP puncta per cell and those with lifetimes <50 s or ≥50 s. Data points superimposed on the violin plots are the averages from independent experiments. Bars represent mean ± SEM (n = 3 experimental replicates, with 6–24 cells measured per replicate). P values were calculated using an unpaired Student's t test. fluo., fluorescence; Max, maximum; Norm., normalized.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal