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-kB signalling.
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Funding Info:
This work was supported by grants from the Medical Research Council (G1000133) to NJG and CEB. and a Wellcome Trust Investigator award to NJG. (WT100321/z/12/Z) and to CEB (WT108045AIA). We would like to thank GSK for the gift of CRX555, Iain Fraiser (NIH) for the reporter macrophages, Doug Golenbock and Kate Fitzgerald (University of Massachusetts Medical School) for a gift of the immortalized wild type, MyD88-/- and TLR4-/- macrophage cell lines (commercially available from BEI; see Materials and methods). Data from this paper are archived at http://dx.doi.org/10.17863/ CAM.6018.Funding Funder Grant reference number Author Medical Research Council G1000133 Nicholas J Gay Clare E Bryant
Wellcome Trust WT100321/z/12/Z Nicholas J Gay
Wellcome Trust WT108045AIA Clare E Bryant