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

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Science Signaling  31 Oct 2017:
Vol. 10, Issue 503, eaan1308
DOI: 10.1126/scisignal.aan1308

Figures

  • Fig. 1 Single-molecule, superresolution imaging of membrane protein clusters and determination of protein stoichiometry in protein clusters.

    (A) A superresolved SMLM image is generated from a stack of images by determining the position of each molecule with nanometer precision. From the superresolved image, single clusters are selected, and the number of fluorescence bursts within each cluster is determined. The number of blinking events, which is the number of fluorescence bursts minus one, is plotted as a histogram and provides information on the average molecule count within the protein clusters. The selection of clusters for quantitative analysis is based on the superresolved image and removes overlapping clusters, clusters with irregular shape or size, or clusters that are composed of only very few points (pink boxes). Selected clusters (cyan boxes) were further analyzed. Illustration of these selection criteria is demonstrated for dimeric CTLA-4_mEos2. (B and C) PALM image (B) of HEK 293 cells expressing the monomeric protein CD86_mEos2 [inset, Protein Data Bank (PDB): 1NCN] in the plasma membrane and the blinking distribution (C) with fit function. (D and E) PALM image (D) of HEK 293 cells expressing the dimeric protein CTLA-4_mEos2 (inset, PDB: 3OSK) in the plasma membrane and the blinking distribution (E) with fit function. Scale bars, 200 nm. Data are from at least 500 clusters from at least nine cells recorded in at least three independent experiments.

  • Fig. 2 Stoichiometry analysis of TLR4_mEos2 in HEK 293 cells in situ.

    (A) Model-derived fit functions describing the blinking histogram for a protein monomer (purple), dimer (violet), and a weighted average of monomer and dimer in equal parts (blue). See Materials and Methods for further details. (B) Analysis of the distribution of blinking events in HEK 293 cells transiently transfected with plasmid encoding TLR4_mEos2 but lacking the co-receptors CD14 and MD2. We calculated the parameter p, which reports on the fraction of molecules that did not blink, as p = 0.31. (C) Analysis of blinking events in HEK293_CD14MD2 cells that were transfected with plasmid encoding TLR4_mEos2 but were not treated with LPS. TLR4 was found in a mixed population of monomers (52%) and dimers (48%). p = 0.32, q = 0.29. (D to F) HEK293_CD14MD2 cells transfected with plasmid encoding TLR4_mEos2 were treated with LPSEC (D), LPSSM (E), or LPSRS (F), and then, the distribution of blinking events was determined. (D) Treatment with LPSEC induced a weighted average of monomeric (26%) and dimeric (74%) TLR4 (used values: p = 0.32, q = 0.29). (E) For treatment with LPSSM, 27% monomers and 73% dimers were detected (used values: p = 0.32, q = 0.29). (F) Treatment with LPSRS led to monomeric TLR4 only (p = 0.33). Data are from at least 500 clusters from at least nine cells recorded in at least three independent experiments.

  • Fig. 3 Measurement of NF-κB– and IRF3-dependent luciferase activities to determine the potencies of different LPS chemotypes and the functionality of TLR4_mEos2.

    (A to C) HEK293_CD14MD2 cells were transiently transfected with (A) plasmids encoding EGFP, TLR4_GFP, or TLR4_mEos2 and an NF-κB–dependent luciferase reporter plasmid, (B) plasmid encoding TLR4_mEos2 together with the NF-κB–dependent luciferase reporter plasmid, or (C) plasmid encoding TLR4_mEos2 together with the IRF3-dependent luciferase reporter plasmid. Luciferase activity was detected in (A) unstimulated and LPSEC-stimulated cells; (B) untreated cells (control) and in cells stimulated with LPSEC or LPSSM in the absence or presence of LPSRS, as indicated; or (C) treated with LPSEC or LPSSM or left untreated (control). Intensity values were normalized to (A) untreated EGFP-expressing cells or (B and C) control cells. Data are means ± SEM of at least three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way analysis of variance (ANOVA) followed by Bonferroni correction.

Tables

  • Table 1 Values for the calibration proteins used for quantitative SMLM.

    Determination of the parameters p and q derived from the quantitative analysis of the blinking number distributions for the calibration proteins CD86_mEos2 and CTLA-4_mEos2. The average localization precision calculated with a NeNA is shown. Data are means ± SEM.

    pqNcellsNanalyzed clustersAverage localization precision (nm)
    CD86_mEos20.32950415.1 ± 0.4
    CTLA-4_mEos20.291084416.1 ± 1.0

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