TNF-R1 signaling pathway
Tumor necrosis factor-alpha (TNFα) is one of the best-characterized mediator of inflammation and binds to the transmembrane receptor TNF receptor 1 (TNF-R1). Evidence exists that TNF-R1 assembles in trimers upon ligand binding, followed by the formation of ligand-mediated clusters. But pre- and post-ligand assembly of TNF-R1 are still under debate (Chan 2007). Through recruitment of adapter proteins, activated TNF-R1 can either induce apoptosis by activation of Caspase-8, or survival via nuclear factor-kappa B (NF-kB) (Aggarwal 2003).
We applied single-molecule localization microscopy to obtain quantitative information on receptor cluster sizes and copy numbers. We investigated lateral dynamics of TNF-R1 before and after ligand binding using high-density single-particle tracking in combination with photoactivated localization microscopy (sptPALM). We established a method to measure ligand affinities directly on cells and determined the binding constant of TNF-R1 to its ligand TNFα (Dietz et al. 2014). We confirmed these binding data with microscale thermophoresis (Fricke et al. 2014).
Colocalization of TNF-R1 withTNFα
Figure 1: Two-colour localization microscopy of TNF-R1 and TNFα. (a) Two-colour image of TNFα-ATTO647N (red) and TNF-R1-tdEos (green). (b) Zoom of TNF-R1-tdEos PALM-image. The colour-code is based on coordinate-based-colocalization (CBC) analysis (Malkusch et al. 2012) ranging from +1 (perfect colocalization) to -1 (anti-correlation).
Figure 2: TNF-R1 Cluster analysis at the cell membrane. Clusters of TNF-R1 were sorted into ligand-induced (colocalization with TNFα) and non-induced types (no colocalization) with CBC analysis. Sorted clusters were then analyzed for radius and number of localizations. The cluster radius distribution shows a shift of the maximum and a slight increase of the mean from 33 nm (uninduced) to 39 nm for the induced receptor. The mean number of localizations almost doubles for ligand-bound receptor clusters. For cluster analysis, localization data of 9 cells were combined.
Lateral diffusion of TNF-R1 in living cells
Figure 3: Single-particle tracking of TNF-R1 in living cells. Relative frequency distributions of TNF-R1 diffusion coefficients D on HeLa cells either not induced or induced with xxx mM TNFα. TNF-R1 exhibits a fraction of fast diffusing particles and a small fraction of slowly diffusing receptors. Right: The fraction of slowly diffusion receptors decreases upon activation with TNFα (Heidbreder et al. 2012).
Table 1: Diffusion coefficients of TNF-R1 studied by FCS and sptPALM. Untreated cells and cells pre-incubated with TNFα or membrane nanodomain disrupting drugs as methyl-β-cyclodextrin (MCD) or nystatin were observered with sptPALM and compared to previously published data measured with fluorescence correlation spectroscopy (FCS) (Gerken et al. 2010).
D from FCS (µm2/s ± SD)
D from sptPALM (µm2/s ± SD)
|nativ||0.12 ± 0.09||0.14 ± 0.02|
|+ TNFα||0.11 ± 0.08||0.16 ± 0.03|
|+ MCD||0.16 ± n/a||0.20 ± 0.04|
|+ Nystatin||--||0.16 ± 0.05|
|+ TNFα + MCD||0.16 ± n/a||0.24 ± 0.03|
Binding studies of TNF-R1 to TNFα
Figure 4: Single-molecule binding study of TNFα to TNF-R1 in cells. Least-square fitting of the InlB binding curve with a Langmuir-binding model reveals a dissociations constant of KD = 15.7 nM (Dietz et al. 2014). On the right representative localization images at two different ligand concentrations are shown. The single-molecule images correspond to the respective data points with light and dark blue circles in the binding curve. The dissociation was confirmed in vitro by microscale thermophoresis to KD = 16.6 nM (Fricke et al. 2014)
Darius Widera lab @ University of Reading
Christian Kaltschmidt lab @ Bielefeld University
Jean-Baptiste Sibarita lab @ IINS Bordeaux
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Dietz MS, Fricke F, Krüger CL, Niemann HH & Heilemann M (2014) Receptor-ligand interactions. Binding affinities studied by single-molecule and super-resolution microscopy on intact cells. ChemPhysChem 15, 671.
Fricke F, Malkusch S, Wangorsch G, Greiner JF, Kaltschmidt B, Kaltschmidt C, Widera D, Dandekar T & Heilemann M (2014) Quantitative single-molecule localization microscopy combined with rule-based modeling reveals ligand-induced TNF-R1 reorganization toward higher-order oligomers. Histochemistry and Cell Biology 142, 91.
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Malkusch S, Endesfelder U, Mondry J, Gelléri M, Verveer PJ & Heilemann M (2012) Coordinate-based colocalization analysis of single-molecule localization microscopy data. Histochemistry and cell biology 137, 1.