Research ArticleCell Biology

Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells

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Science Signaling  14 Jan 2020:
Vol. 13, Issue 614, eaax5647
DOI: 10.1126/scisignal.aax5647

Oligomerizing for optimal activity

Drugs that prevent the activation of the TNFα receptor TNFR1 are of great interest because of the many roles of this ligand/receptor pair in pathophysiological processes such as inflammation. Karathanasis et al. determined the oligomerization state of TNFR1 by quantitatively analyzing single-molecule superresolution microscopy data. TNFα stimulation triggered the clustering of TNFR1 monomers and dimers into trimers and nonamers. Forms of TNFR1 with mutations that impaired basal dimerization or that abolished ligand binding did not assemble into higher-order oligomers upon TNFα stimulation. These results provide insight into the oligomerization states of TNFR1 that must be targeted before and after TNFα stimulation.


Ligand-induced tumor necrosis factor receptor 1 (TNFR1) activation controls nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling, cell proliferation, programmed cell death, and survival and is crucially involved in inflammation, autoimmune disorders, and cancer progression. Despite the relevance of TNFR1 clustering for signaling, oligomerization of ligand-free and ligand-activated TNFR1 remains controversial. At present, models range from ligand-independent receptor predimerization to ligand-induced oligomerization. Here, we used quantitative, single-molecule superresolution microscopy to study TNFR1 assembly directly in native cellular settings and at physiological cell surface abundance. In the absence of its ligand TNFα, TNFR1 assembled into monomeric and dimeric receptor units. Upon binding of TNFα, TNFR1 clustered predominantly not only into trimers but also into higher-order oligomers. A functional mutation in the preligand assembly domain of TNFR1 resulted in only monomeric TNFR1, which exhibited impaired ligand binding. In contrast, a form of TNFR1 with a mutation in the ligand-binding CRD2 subdomain retained the monomer-to-dimer ratio of the unliganded wild-type TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-κB signaling.

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