Journal: EMBO Reports

Article Title: Myddosome clustering in IL ‐1 receptor signaling regulates the formation of an NF‐kB activating signalosome

doi: 10.15252/embr.202357233

Figure Lengend Snippet: A Timelapse TIRF microscopy images showing an EL4‐MyD88‐GFP cell interacting with a IL‐1 functionalized SLB. MyD88‐GFP assembles into puncta that cluster and coalesce at the cell:SLB interface. Scale bar, 2 μm. B Schematic illustrating a working model for Myddosomes being tethered to the plasma membrane via interaction with IL‐1R bound to IL‐1. Based on this model, we predict physical barriers (on grid) would restrict the diffusion of IL‐1 on the SLB and limit Myddosome clustering. With no external barriers present (off grid), MyD88 puncta can merge to form larger multicomplex assemblies. C TIRF and bright‐field microscopy images of EL4‐MyD88‐GFP cells incubated for 30 min with IL‐1 functionalized SLBs formed off grid and on 1 and 2.5 μm grids. In the presence of a 1 and 2.5 μm grids, Myddosomes only coalesce within individual corrals and do not form multicomplex clusters. Scale bar, 5 μm. D Time series showing Myddosome formation in an EL4 cell interacting with both continuous and 2.5 μm gridded partitioned SLBs. t = 0 s denotes the start of cellular observation. Scale bar, 5 μm. E Kymographs from panel (D) showing the coalescence of MyD88‐GFP puncta off grid and the restricted movement of MyD88‐GFP puncta on 2.5 μm grids. Scale bar, 1 μm. F Quantification of MyD88‐GFP puncta maximum fluorescence intensity normalized to GFP from cells stimulated off and on 2.5 or 1 μm grids, at a ligand density of 10 IL‐1/μm 2 . Violin plots show the distribution of average max puncta intensities from individual cells across replicates. Data points superimposed on the violin plots are the averages from independent experimental replicates. The average max MyD88 puncta intensity (mean ± SEM): for off grid is 7.5 ± 1.8 GFPs, n = 8 biological replicates, with 88,304 MyD88‐GFP puncta from 161 cells measured in total across all replicates; for 2.5 μm grids is 5.2 ± 1.7 GFPs, n = 3 biological replicates, with 13,164 MyD88‐GFP puncta from 31 cells measured in total across all replicates; for 1 μm grids is 2.3 ± 0.1 GFPs, n = 8 biological replicates, with 126,600 MyD88‐GFP puncta from 254 cells measured in total across all replicates. Bars represent mean ± SEM. G Widefield images showing RelA localization in unstimulated EL4 cells and EL4 cells stimulated by SLB formed on and off grids. EL4 was fixed 30 min after addition to IL‐1‐functionalized SLBs and stained for RelA (magenta); DAPI‐stained nuclei (blue). Scale bar, 10 μm. H Quantification of RelA nucleus to cytoplasm ratio. Violin plots show the distribution of measurements from individual cells. Data points superimposed on the violin plots are the averages from independent experiments. The RelA nucleus‐to‐cytoplasm ratio of single cells marked with X in panel (G) is superimposed on the violin plot. RelA nucleus‐to‐cytoplasm ratio off grids, on 2.5 and 1 μm grids, and unstimulated conditions are 0.45 ± 0.01, 0.34 ± 0.02, 0.32 ± 0.02, 0.21 ± 0.03 (mean ± SEM), respectively. The P ‐value are * = 0.0133 and ** = 0.0027. Bars represent mean ± SEM ( n = 3–5 biological replicates, with a total of 18,370, 3,627, 2,988, and 965 cells measured off grids, on 2.5 and 1 μm grids, and unstimulated conditions, respectively). Statistical significance is determined using unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.

Article Snippet: We acquired 3D structured illumination microscopy of fixed EL4 cells on a Zeiss Elyra 7 microscope equipped with 405, 488, 561, and 642 nm laser lines for excitation.

Techniques: Microscopy, Clinical Proteomics, Membrane, Diffusion-based Assay, Incubation, Fluorescence, Staining, Two Tailed Test

Journal: EMBO Reports

Article Title: Myddosome clustering in IL ‐1 receptor signaling regulates the formation of an NF‐kB activating signalosome

doi: 10.15252/embr.202357233

Figure Lengend Snippet: TIRF images of fixed EL4‐MyD88‐GFP cells off grids and stained with antibodies against K63‐Ub, M1‐Ub, pIKK, and pp65. Cells were activated on IL‐1 functionalized SLBs for 30 min before fixation. Scale bar, 5 μm. A magnified view of the large patch‐like Myddosome clusters from the highlighted region of interest in panel (A) (yellow box on merge images). Scale bar, 1 μm. Structured illumination microscopy images of Myddosome clusters stained with anti‐pIKK. Top row right, insets show the detail of Myddosome staining with anti‐pIKK. Inset taken from regions of interest overlaid the merge image (yellow boxes 1 and 2). Bottom row, x‐z view slice taken from yellow line overlaid on the merge image (top row). Myddosome and pIKK staining localize the cell–SLB interface. Blue dashed line defines the nucleus volume determined from the DAPI stain. Scale bar in main image and Z projection, 1 μm; scale bar inset, 0.5 μm. Schematic showing working model for how Myddosome clustering could enhance the generation of K63/M1‐Ub, pIKK, and pp65 and a NF‐kB signalosome. We hypothesize that the Myddosome clustering creates regions with a high density of complexes, and this will lead to enhanced production of signaling intermediates such as K63‐Ub and M1‐Ub chains, pIKK and pp65. Source data are available online for this figure.

Article Snippet: We acquired 3D structured illumination microscopy of fixed EL4 cells on a Zeiss Elyra 7 microscope equipped with 405, 488, 561, and 642 nm laser lines for excitation.

Techniques: Staining, Microscopy

Journal: EMBO Reports

Article Title: Myddosome clustering in IL ‐1 receptor signaling regulates the formation of an NF‐kB activating signalosome

doi: 10.15252/embr.202357233

Figure Lengend Snippet: A, B Top, TIRF images of fixed EL4‐MyD88‐GFP cells incubated with IL‐1 functionalized SLBs for 30 min and stained with antibodies against pp65 (A) or pIKK (B). Scale bar, 5 μm. Region of interest (red box, merge image) shows an example of MyD88‐GFP puncta that colocalizes with pp65 (A) or pIKK (B) puncta. Bottom, 2D histograms of the distribution of MyD88 puncta intensity and associated pp65 (A) or pIKK (B) staining intensity. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R , of linear fit labeled on 2D histograms). Blue‐shaded regions on scatter plot high MyD88 puncta classified as clustered Myddosomes. Bottom right, zoomed images of the region of interest (red box overlaid merge image, top) show MyD88‐GFP channel and associated pp65 (A) and pIKK (B) channel (pp65/pIKK images are displayed with Fire LUT). Red data points on the 2D histogram are from indicated puncta in the MyD88‐GFP image (numbered red arrows). Scale bar, 1 μm. C, D TIRF images of fixed EL4‐MyD88‐GFP cells incubated with partitioned IL‐1 functionalized SLBs (2.5 μm top row and 1 μm bottom row) and stained with anti‐pp65 (C) or anti‐pIKK (D). Region of interest (red box overlaid merge image) shows examples of MyD88‐GFP puncta that colocalize with pp65 (A) or pIKK (B) puncta. Scale bar, 5 μm. Far right, zoomed image of pp65 (C) or pIKK (D) puncta (from region of interest overlaid merge image) displayed with Fire LUT. Scale bar, 1 μm. E, F 2D histogram of MyD88‐GFP puncta intensity and associated pp65 (E) or pIKK (F) staining intensity on 2.5 and 1 μm grids. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R , of linear fit labeled on 2D histograms). G, H Quantification of mean pp65 (G) or pIKK (H) staining intensity for puncta classifieds as single or clustered Myddosomes, and MyD88 puncta formed on 2.5 and 1 μm grids. The normalized mean intensity for clusters, single Myddosomes MyD88 puncta on 2.5 and 1 μm grids are the following: for pp65 0.318 ± 0.044, 0.163 ± 0.009, 0.059 ± 0.005 and 0.057 ± 0.008; for pIKK 0.393 ± 0.051, 0.130 ± 0.004, 0.037 ± 0.006 and 0.035 ± 0.007 (a.u., mean ± SEM, mean value states in the order they appear on plot, left to right). Violin plots show the distribution of all segmented MyD88 puncta. Data points superimposed on the violin plots are the averages from independent experiments. P ‐values are * < 0.05, *** < 0.001, **** < 0.0001. Bars represent mean ± SEM ( n = 3–4 biological replicates for pp65, with 10,273, 14,009 and 2,675 puncta off grid, on 2.5 μm and 1 μm grid measured in total across all replicates; n = 4–5 biological replicates for pIKK, with 2,375, 35,496 and 59,593 puncta off grid, on 2.5 μm and 1 μm grid measured in total across all replicates). Statistical significance is determined using unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.

Article Snippet: We acquired 3D structured illumination microscopy of fixed EL4 cells on a Zeiss Elyra 7 microscope equipped with 405, 488, 561, and 642 nm laser lines for excitation.

Techniques: Incubation, Staining, Labeling, Two Tailed Test

Journal: EMBO Reports

Article Title: Myddosome clustering in IL ‐1 receptor signaling regulates the formation of an NF‐kB activating signalosome

doi: 10.15252/embr.202357233

Figure Lengend Snippet: A, B Top, TIRF images of fixed EL4‐MyD88‐GFP cells incubated with IL‐1 functionalized SLBs for 30 min and stained with anti‐K63‐Ub (A) or anti‐M1‐Ub (B). Scale bar, 5 μm. Region of interest (red box, merge image) shows an example of MyD88‐GFP puncta that is colocalized with K63‐Ub (A) or M1‐Ub (B) puncta. Bottom, 2D histograms of the distribution of MyD88 puncta intensity and associated K63‐Ub (A) or M1‐Ub (B) staining intensity. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient, R , of linear fit labeled on 2D histograms). Blue‐shaded region on scatter plot high MyD88 puncta classified as clustered Myddosomes. Bottom right, zoomed images of the region of interest (red box overlaid merge image, top) show MyD88‐GFP channel and associated K63‐Ub (A) and M1‐Ub (B) channel (K63/M1‐Ub images are displayed with Fire LUT). Red data points on the 2D histogram are from indicated puncta in the MyD88‐GFP image (numbered red arrows). Scale bar, 1 μm. C, D TIRF images of fixed EL4‐MyD88‐GFP cells incubated with partitioned IL‐1 functionalized SLBs (2.5 μm top row and 1 μm bottom row grids) and stained with anti‐K63‐Ub (C) or anti‐M1‐Ub (D). Region of interest (red box overlaid merge image) shows an example of MyD88‐GFP puncta that colocalize with K63‐Ub (A) or M1‐Ub (B) puncta. Scale bar, 5 μm. Far right, zoomed image of K63‐Ub (C) or M1‐Ub (D) puncta (from region of interest overlaid merge image) displayed with Fire LUT. Scale bar, 1 μm. E, F 2D histograms of the distribution of MyD88 puncta intensity and associated K63‐Ub (E) or M1‐Ub (F) staining intensity on 2.5 and 1 μm grids. Linear fit is shown as a blue line superimposed on 2D histograms (Pearson correlation coefficient of linear fit labeled on 2D histograms). G, H Quantification of mean K63‐Ub (G) or M1‐Ub (H) staining intensity for puncta classifieds as single or clustered Myddosomes, and MyD88 puncta formed on 2.5 and 1 μm grids. The normalized mean intensity for clustered, single Myddosomes, and MyD88 puncta on 2.5 and 1 μm grids are the following: for K63‐Ub 0.444 ± 0.030, 0.257 ± 0.025, 0.113 ± 0.015 and 0.104 ± 0.008; for M1‐Ub 0.520 ± 0.020, 0.183 ± 0.008, 0.174 ± 0.022 and 0.153 ± 0.016 (a.u., mean ± SEM, mean value states in the order they appear on plot, left to right). Violin plots show the distribution of all segmented MyD88 puncta. Data points superimposed on the violin plots are the averages from independent experiments. P ‐values are ** < 0.01, *** < 0.001. Bars represent mean ± SEM ( n = 3–4 biological replicates for K63, with 14,571, 27,494 and 24,026 puncta off grid, on 2.5 and 1 μm grid measured in total across all replicates; n = 3–4 biological replicates for M1, with 3,114, 6,091 and 1,844 puncta off grid, on 2.5 and 1 μm grid measured in total across all replicates). Statistical significance is determined using unpaired two‐tailed Student's t ‐test. Source data are available online for this figure.

Article Snippet: We acquired 3D structured illumination microscopy of fixed EL4 cells on a Zeiss Elyra 7 microscope equipped with 405, 488, 561, and 642 nm laser lines for excitation.

Techniques: Incubation, Staining, Labeling, Two Tailed Test

Journal: EMBO Reports

Article Title: Myddosome clustering in IL ‐1 receptor signaling regulates the formation of an NF‐kB activating signalosome

doi: 10.15252/embr.202357233

Figure Lengend Snippet: TIRF images of an EL4 cell expressing MyD88‐GFP and mScarlet‐TRAF6. Scale bar, 2 μm. Time‐series images indicate MyD88 and TRAF6 puncta from the yellow boxed area. Scale bar in time‐series images, 1 μm. TRAF6 is transiently recruited to MyD88 until TRAF6 becomes stable and nucleates on MyD88 puncta. Fluorescence intensities of MyD88 and TRAF6 overtime are shown at top right. Density plot of single molecules of GFP (green, n = 40,229 GFP particles) and estimated intensity distribution of a 6× GFP multimer (blue) and a 12× GFP multimer (purple). Shaded light blue and dark blue regions designate intensity values ≥ 4.5× GFP and ≥ 9× GFP, respectively, which were used to categorize MyD88 puncta as containing ≥ 1 or ≥ 2 Myddosome complexes. 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 TRAF6. The average size for puncta positive or negative for TRAF6 recruitment is 10.4 versus 3.9 MyD88s, measured from 13,526 positive MyD88 puncta versus 83,462 negative MyD88 puncta measured in 191 cells and combined from six biological replicates. Quantification of the percentage of MyD88‐GFP puncta per cell that colocalizes with TRAF6 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 = 6 biological replicates, with 17–48 cells measured per replicate). Bars represent mean ± SEM. 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 HOIL1. The average size for puncta positive or negative for HOIL1 recruitment is 47.4 versus 11.6 MyD88s, mean calculated from 10,300 HOIL1‐positive MyD88 puncta versus 108,054 negative MyD88 puncta measured from 230 cells and combined across nine biological replicates. Quantification of the percentage of MyD88‐GFP puncta per cell that colocalizes with HOIL1 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 = 9 biological replicates, with 9–46 cells measured per replicate). Bars represent mean ± SEM.

Article Snippet: We acquired 3D structured illumination microscopy of fixed EL4 cells on a Zeiss Elyra 7 microscope equipped with 405, 488, 561, and 642 nm laser lines for excitation.

Techniques: Expressing, Fluorescence, Derivative Assay

Journal: EMBO Reports

Article Title: Myddosome clustering in IL ‐1 receptor signaling regulates the formation of an NF‐kB activating signalosome

doi: 10.15252/embr.202357233

Figure Lengend Snippet: A, B TIRF images of EL4 cells expressing MyD88‐GFP and mScarlet‐TRAF6 stimulated on IL‐1 functionalized SLBs at a ligand density of 1 IL‐1/μm 2 off grids (A) or on 1 μm grids (B). Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bars, 5 μm. C Quantification 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/μm 2 , 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, E TIRF 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 μm 2 off grids (D) or on 1 μm grids (E). Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bars, 5 μm. F Quantification 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/μm 2 , 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, H TIRF images of EL4 cells expressing MyD88‐GFP and mScarlet‐HOIL1 stimulated on IL‐1 functionalized SLBs at a ligand density of 10 IL‐1/μm 2 off grids (G) or on 1 μm grids (H). Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bars, 5 μm. I Quantification 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/μm 2 , 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, K TIRF images of EL4 cells expressing MyD88‐GFP and mScarlet‐HOIL1 stimulated on IL‐1 functionalized SLBs at a ligand density of 32 IL‐1/μm 2 off grids (J) or on 1 μm grids (K). Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bars, 5 μm. L Quantification 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/μm 2 , 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.

Article Snippet: We acquired 3D structured illumination microscopy of fixed EL4 cells on a Zeiss Elyra 7 microscope equipped with 405, 488, 561, and 642 nm laser lines for excitation.

Techniques: Expressing, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: Myddosome clustering in IL ‐1 receptor signaling regulates the formation of an NF‐kB activating signalosome

doi: 10.15252/embr.202357233

Figure Lengend Snippet: A, B Scaled density distribution of MyD88 max normalized intensity off grids and on 1 μm grids at a ligand density of 10 (A) or 32 (B) IL1/μm 2 . The average MyD88 max normalized intensity (dashed line) at 10 IL1/μm 2 off grids versus on 1 μm grids is 10.6 versus 2.1 and at 32 IL1/μm 2 is 14.3 versus 3.8. Insets are quantifications of the percentage of Myddosome clusters. A Myddosome cluster is defined as a MyD88‐GFP puncta containing equal to or greater than 2 Myddosomes. At 10 IL1/μm 2 , the percentages of Myddosome clusters off grids versus on 1 μm grids are 43.0 ± 9.5% versus 2.9 ± 1.3%, and at 32 IL1/μm 2 are 49.8 ± 6.3% versus 13.1 ± 3.9%. Bars represent mean ± SEM. At 10 IL1/μm 2 , data are measured from 53,852 MyD88 puncta off grids from 74 cells and four biological replicates and 55,075 MyD88 puncta on 1 μm grids from 154 cells and four biological replicates. At 32 IL1/μm 2 , data are measured from 118,354 MyD88 puncta off grids from 230 cells and nine biological replicates and 68,819 MyD88 puncta on 1 μm grids from 138 cells and four biological replicates. C An example of TIRF images of HOIL1 recruitment in EL4 cells expressing MyD88‐GFP and mScarlet‐HOIL1 stimulated on IL1 functionalized SLBs on 1 μm grids at a ligand density of 10 IL1/μm 2 . Kymographs derived from dashed lines overlaid TIRF images (left panel). Scale bar, 5 μm. D, E Histogram of the average landing size for HOIL1 at 10 IL1/μm 2 (H) and 32 IL1/μm 2 (I) off and on 1 μm grids, overlaid with density plots of the distribution. The landing size of Myddosome is calculated with landing size of MyD88 puncta divided by the intensity of 4.5× GFP. The average landing size of Myddosome at 10 IL1/μm 2 off grids versus on 1 μm grids is 4.4 ± 1.2 versus 0.7 ± 0.2 Myddosomes (Mean ± SEM), measured from 1762 versus 691 MyD88‐GFP puncta from 66 versus 86 cells and 4 versus 4 replicates. The average landing size of Myddosome at 32 IL1/μm 2 off grids versus on 1 μm grids is 6.2 ± 1.1 versus 1.4 ± 0.2 Myddosomes (Mean ± SEM), measured from 5,562 versus 2,189 MyD88‐GFP puncta from 212 versus 124 cells and 9 versus 4 biological replicates.

Article Snippet: We acquired 3D structured illumination microscopy of fixed EL4 cells on a Zeiss Elyra 7 microscope equipped with 405, 488, 561, and 642 nm laser lines for excitation.

Techniques: Expressing, Derivative Assay

Journal: EMBO Reports

Article Title: Myddosome clustering in IL ‐1 receptor signaling regulates the formation of an NF‐kB activating signalosome

doi: 10.15252/embr.202357233

Figure Lengend Snippet:

Article Snippet: We acquired 3D structured illumination microscopy of fixed EL4 cells on a Zeiss Elyra 7 microscope equipped with 405, 488, 561, and 642 nm laser lines for excitation.

Techniques: Virus, Recombinant, DNA Extraction, Enzyme-linked Immunosorbent Assay, Knock-In, Plasmid Preparation, Software