Biochemical Isolation of the Myddosome from Murine Macrophages

  • Yunhao Tan
  • Jonathan C. KaganEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1714)


Ligand-induced macromolecular protein complex formation has emerged as a common means by which the innate immune system activates signal transduction pathways essential for host defense. Despite their structural divergence, key signaling molecules in diverse innate immune pathways mediate signal transduction by assembling higher-order protein complexes at specific subcellular locations in a stimulus-dependent manner. These protein complexes are collectively known as the supramolecular organizing centers (SMOCs), which link active receptors to a variety of downstream cellular responses. In the Toll-like receptor (TLR) pathway, the signaling adaptor MyD88 is the core of a SMOC called the myddosome, which is composed of the sorting adaptor TIRAP and the IRAK family kinases. Depending on the microbial ligands encountered, the myddosome can be assembled at the plasma membrane or endosomes, thereby leading to NF-ĸB and AP-1 activation, and the subsequent expression of pro-inflammatory cytokines. Herein, we provide a detailed protocol for studying myddosome assembly in murine bone marrow-derived macrophages (BMDMs).


TLR SMOCs MyD88 Myddosome Immunoprecipitation 



This work was supported by NIH grants AI093589, AI116550, and P30 DK34854 to J.C.K. J.C.K. holds an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. Y.T. is supported by a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research (the Merck Fellow).


  1. 1.
    Brubaker SW et al (2015) Innate immune pattern recognition: a cell biological perspective. Annu Rev Immunol 33:257–290CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Chu H, Mazmanian SK (2013) Innate immune recognition of the microbiota promotes host-microbial symbiosis. Nat Immunol 14(7):668–675CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140(6):805–820CrossRefPubMedGoogle Scholar
  4. 4.
    Vance RE, Isberg RR, Portnoy DA (2009) Patterns of pathogenesis: discrimination of pathogenic and nonpathogenic microbes by the innate immune system. Cell Host Microbe 6(1):10–21CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 11(5):373–384CrossRefPubMedGoogle Scholar
  6. 6.
    Horng T et al (2002) The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature 420(6913):329–333CrossRefPubMedGoogle Scholar
  7. 7.
    Yamamoto M et al (2003) Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301(5633):640–643CrossRefPubMedGoogle Scholar
  8. 8.
    Yamamoto M et al (2002) Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4. Nature 420(6913):324–329CrossRefPubMedGoogle Scholar
  9. 9.
    Yamamoto M et al (2003) TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway. Nat Immunol 4(11):1144–1150CrossRefPubMedGoogle Scholar
  10. 10.
    Medzhitov R et al (1998) MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways. Mol Cell 2(2):253–258CrossRefPubMedGoogle Scholar
  11. 11.
    Kagan JC, Medzhitov R (2006) Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling. Cell 125(5):943–955CrossRefPubMedGoogle Scholar
  12. 12.
    Fitzgerald KA et al (2003) IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol 4(5):491–496CrossRefPubMedGoogle Scholar
  13. 13.
    Lord KA, Hoffman-Liebermann B, Liebermann DA (1990) Nucleotide sequence and expression of a cDNA encoding MyD88, a novel myeloid differentiation primary response gene induced by IL6. Oncogene 5(7):1095–1097PubMedGoogle Scholar
  14. 14.
    Bonnert TP et al (1997) The cloning and characterization of human MyD88: a member of an IL-1 receptor related family. FEBS Lett 402(1):81–84CrossRefPubMedGoogle Scholar
  15. 15.
    Wesche H et al (1997) MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 7(6):837–847CrossRefPubMedGoogle Scholar
  16. 16.
    Muzio M et al (1997) IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science 278(5343):1612–1615CrossRefPubMedGoogle Scholar
  17. 17.
    Ngo VN et al (2011) Oncogenically active MYD88 mutations in human lymphoma. Nature 470(7332):115–119CrossRefPubMedGoogle Scholar
  18. 18.
    von Bernuth H et al (2008) Pyogenic bacterial infections in humans with MyD88 deficiency. Science 321(5889):691–696CrossRefGoogle Scholar
  19. 19.
    Lin SC, Lo YC, Wu H (2010) Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling. Nature 465(7300):885–890CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    George J et al (2011) Two human MYD88 variants, S34Y and R98C, interfere with MyD88-IRAK4-myddosome assembly. J Biol Chem 286(2):1341–1353CrossRefPubMedGoogle Scholar
  21. 21.
    Ferrao R et al (2014) IRAK4 dimerization and trans-autophosphorylation are induced by Myddosome assembly. Mol Cell 55(6):891–903CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kagan JC, Magupalli VG, Wu H (2014) SMOCs: supramolecular organizing centres that control innate immunity. Nat Rev Immunol 14(12):821–826CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Gay NJ, Gangloff M, O’Neill LA (2011) What the Myddosome structure tells us about the initiation of innate immunity. Trends Immunol 32(3):104–109CrossRefPubMedGoogle Scholar
  24. 24.
    Bonham KS et al (2014) A promiscuous lipid-binding protein diversifies the subcellular sites of toll-like receptor signal transduction. Cell 156(4):705–716CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Holler N et al (2003) Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex. Mol Cell Biol 23(4):1428–1440CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Micheau O, Tschopp J (2003) Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114(2):181–190CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Division of Gastroenterology, Harvard Medical SchoolBoston Children’s HospitalBostonUSA

Personalised recommendations