Quantitative single-molecule imaging of TLR4 reveals ligand-specific receptor dimerization

Sci Signal. 2017 Oct 31;10(503):eaan1308. doi: 10.1126/scisignal.aan1308.


In humans, invading pathogens are recognized by Toll-like receptors (TLRs). Upon recognition of lipopolysaccharide (LPS) derived from the cell wall of Gram-negative bacteria, TLR4 dimerizes and can stimulate two different signaling pathways, the proinflammatory, MyD88-dependent pathway and the antiviral, MyD88-independent pathway. The balance between these two pathways is ligand-dependent, and ligand composition determines whether the invading pathogen activates or evades the host immune response. We investigated the dimerization behavior of TLR4 in intact cells in response to different LPS chemotypes through quantitative single-molecule localization microscopy. Quantitative superresolved data showed that TLR4 was monomeric in the absence of its co-receptors MD2 and CD14 in transfected HEK 293 cells. When TLR4 was present together with MD2 and CD14 but in the absence of LPS, 52% of the receptors were monomeric and 48% were dimeric. LPS from Escherichia coli or Salmonella minnesota caused the formation of dimeric TLR4 complexes, whereas the antagonistic LPS chemotype from Rhodobacter sphaeroides maintained TLR4 in monomeric form at the cell surface. Furthermore, we showed that LPS-dependent dimerization was required for the activation of NF-κB signaling. Together, these data demonstrate ligand-dependent dimerization of TLR4 in the cellular environment, which could pave the way for a molecular understanding of biased signaling downstream of the receptor.

MeSH terms

  • Escherichia coli / immunology
  • HEK293 Cells
  • Humans
  • Ligands
  • Lipopolysaccharide Receptors / genetics
  • Lipopolysaccharide Receptors / metabolism
  • Lipopolysaccharides / immunology*
  • Lymphocyte Antigen 96 / genetics
  • Lymphocyte Antigen 96 / metabolism
  • Myeloid Differentiation Factor 88 / metabolism
  • NF-kappa B p50 Subunit / genetics
  • NF-kappa B p50 Subunit / metabolism
  • Protein Multimerization*
  • Salmonella / immunology
  • Single Molecule Imaging / methods*
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / metabolism*
  • Transfection


  • LY96 protein, human
  • Ligands
  • Lipopolysaccharide Receptors
  • Lipopolysaccharides
  • Lymphocyte Antigen 96
  • MYD88 protein, human
  • Myeloid Differentiation Factor 88
  • NF-kappa B p50 Subunit
  • NFKB1 protein, human
  • TLR4 protein, human
  • Toll-Like Receptor 4