Abstract
Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling.
Highlights
During infections and tissue injury, large oligomeric complexes of proteins are assembled
In contrast to stimulatory LPS, structurally related antagonists such as Eritoran or Rhodobacter sphaeroides Lipid A (RSLA) bind to TLR4/MD2 co-receptors but do not induce the formation of hetero-tetrameric complexes in vitro. Consistent with this we find that the number of TLR4 dimers on the surface of macrophages treated with RSLA is significantly lower than that seen in unstimulated cells (Figure 2B(iv))
We show that in the absence of ligand, TLR4/MD2 on the surface of living immune system cells is in a dynamic equilibrium with populations of heterodimers and heterotetramers
Summary
During infections and tissue injury, large oligomeric complexes of proteins are assembled. The complexes represent supramolecular organizing centers (SMOCs), which serve as the principal subcellular source of signals that promote inflammation (Kagan et al, 2014). In the Toll-like receptor (TLR) pathways, the most commonly discussed organizing center is the MyDDosome, which induces NF-k B and AP-1 activation to drive inflammatory transcriptional responses to infection (Motshwene et al, 2009; Lin et al, 2010; Gay et al, 2014). A common feature of these organizing centers is their ability to be assembled inducibly during infection or other stressful experiences. It is believed that the purpose of assembling these complexes is to create an activation threshold in the innate immune system, such that all-or-none
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