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 specific time points and subjected to photobleaching analysis. (A) Photobleaching analysis with stills from cells stimulated with LPS (10 nM) for 5 min. If the labeled TLR4 is a monomer then photobleaching occurs in a single step. If the labeled TLR4 is a dimer then two photobleaching steps occur. Since it was not possible to independently determine the efficiency of labeling of TLR4, this analysis allows the relative number of TLR4 monomers and dimers on the cell surface to be determined. (B) (i) TLR4 expression in unstimulated cells: monomeric (78 ± 3%); dimeric (22 ± 3%) (ii) increased number of detected TLR4 dimers after LPS stimulation (10 nM) for 5 min (p<0.05) (iii) the number of detected TLR4 dimers is unchanged by CRX555 (iv) less TLR4 dimers were detected with the antagonist Rhodobacter sphaeroides Lipid A (RSLA) compared to unstimulated cells (p<0.05) and (v) LPS-stimulation of TLR4-Pro712His-Halo shows reduced number of dimers compared to wild-type TLR4 (p<0.05). At least 16 cells were analyzed at each time point in three independent repeat experiments, data are expressed as mean ± SEM, and data were analyzed by a two-tailed unpaired Student’s t-test. (C) A two-step model for TLR4 signaling: ligand induced dimer stabilization followed by apposition of the TIRs. (D) Porcupine plots from molecular dynamics simulations of TLR4/MD2, with magnitudes of atomic motion indicated by length and color of associated arrows: minor rotational motions of the ECDs with lipid A brings the C-termini of the TLR4 ECD into close apposition.