Activation of Toll-like receptors nucleates assembly of the MyDDosome signaling hub
- University of Cambridge, United Kingdom
- University of Oxford, United Kingdom
- Bioinformatics Institute (A*STAR), Singapore
- National University of Singapore, Singapore
Figures
Figure 1 with 5 supplements
The kinetics and size of MyDDosome formation depend on the efficacy of TLR4 stimulation.
MyD88-/- iBMM were virally transduced with pHRMyD88-GFP and either left unstimulated or stimulated with LPS (10–500 nM) or CRX-555 (500 nM-1µM). (A) Stills showing MyDDosome formation in a live cell …
Figure 1—figure supplement 1
(i) To determine whether addition of a fluorescent tag to MyD88 effects its ability to signal HEK cells were transiently transfected with 10 ng MyD88-GFP, along with an 10 ng p-NFκΒ Luc reporter and 5 ng of phRG (constitutively active renilla control plasmid).
Cells were lysed 48 hr after transfection and NFκΒ activity measured normalised to renilla activity in response to MyD88 over expression (data are expressed as mean luciferase/renilla ± SEM; n = 3). …
Figure 1—figure supplement 2
An overview of the MyDDosome tracking process using a TrackMate plugin.
(A) shows an example of raw data collected over a 9 min period following cell stimulation with 500 nM LPS. In this case imaging commences 3 min post-stimulation and ends at 11.5 min …
Figure 1—figure supplement 3
Representative MyD88-GFP data for 500 nM LPS treated cells.
(A and B) represent 18 frames of acquired data for a single cell post-stimulation with 500 nM LPS. Acquisition commenced at 3 mi. $$BOX_TXT_END$$ n utes post-stimulation and continued for 9 min (one …
Figure 1—figure supplement 4
Representative MyD88-GFP data for 50 nM LPS treated cells.
(A and B) represent 18 frames of acquired data for a single cell post-stimulation with 50 nM LPS. Acquisition commenced at 6 min post-stimulation and continued for 9 min (one frame recorded every 30 …
Figure 1—figure supplement 5
Representative MyD88-GFP data for 1 μM CRX-555-treated cells.
(A and B) represent 18 frames of acquired data for a single cell post-stimulation with 1 uM CRX-555. Acquisition commenced at 4 min post-stimulation and continued for 9 min (one frame recorded every …
Figure 2 with 3 supplements
The effect of ligands upon TLR4 dimerization.
TLR4-/- iBMM virally transduced with pHR-TLR4-Halo were incubated with HaloTag R110Direct. Cells were either left unstimulated or stimulated with LPS (10–500 nM) or CRX-555 (500 nM-1µM), fixed at …
Figure 2—figure supplement 1
(i) To determine whether addition of a Halo tag to TLR4 effects its ability to signal HEK cells were transfected with 1 ng Wild-type (TLR4WT) or Halo-Tagged TLR4 (TLR4Ha), 1 ng each of CD14 and MD2, 10 ng p-NFκΒ Luc reporter and 5 ng of phRG (constitutively active renilla control plasmid).
After 48 h cells were stimulated with LPS (1 ng/ml or 10 ng/ml); data are expressed as mean luciferase/renilla ± SEM; n = 3). (ii) TLR4-/- iBMDMs were lentivirally transduced with TLR4-Halo. After …
Figure 2—figure supplement 2
Porcupine plots based on three independent replica simulations of apo, ligand-free TLR4/MD2, with magnitudes of atomic motion indicated by length and color of associated arrows, reveal large lateral fluctuations of C-termini, consistent across all replicas.
This suggests that ligand binding brings the C-termini of the TLR4 ECD into close apposition.
Figure 2—figure supplement 3
Dynamic motion of MD2 relative to TLR4.
Starting from the LPS bound X-ray structure (pdb ID 3FXI) (http://www.nature.com/nature/journal/v458/n7242/full/nature07830.html) of MD2 (transparent grey) bound to dimeric TLR4 (transparent pink), …
Figure 3 with 1 supplement
Macrophages stimulated with CRX555 show delayed NF-κB nuclear translocation in comparison to cells stimulated with LPS.
RAW264.7 macrophages stably expressing RelA-EGFP and a TNFα promoter-mCherry reporter were stimulated with LPS or CRX555. Confocal time-lapse images were captured every 3 min for 15 hr. (A) Stills …
Figure 3—figure supplement 1
MATLAB-based automated analysis of NF-κB translocation and mCherry fluorescence intensity.
segmentation to determine the area of a cell body was performed based on GFP pictures and bright field pictures. A nuclear region was determined as a circle with a diameter which was selected …
Additional files
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Supplementary file 1
Structural properties of TLR4/MD2 heterotetrameric complex observed during final 20 ns of molecular dynamics simulations
The modeled TLR4/MD-2 heterotetramer exhibited increased structural drift with respect to the LPS-bound X-ray structure in the absence of ligand (apo state), as reflected in the mean RMSD values. Measurement of the surface areas buried between protein chains reveals that the largest conformational changes are evident at the primary TLR4 dimerization interfaces (which govern the stability of the higher-order heterotetrameric complex) than at the secondary TLR4 dimerization interfaces, with shifts of up to ~ 60% versus~20%, respectively. This is consistent with the observed relative motion of up to ~ 10 Å of MD2 relative to its primary TLR4 partner in the apo state, as described in the main text.
- https://doi.org/10.7554/eLife.31377.015
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Transparent reporting form
- https://doi.org/10.7554/eLife.31377.016
