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)

 

Collaboration partners

Darius Widera lab @ University of Reading
Christian Kaltschmidt lab @ Bielefeld University
Jean-Baptiste Sibarita lab @ IINS Bordeaux
 

Publications

Aggarwal BB (2003) Signalling pathways of the TNF superfamily: a double-edged sword. Nature Reviews Immunology 3, 745.

Chan FK-M (2007) Three is better than one: pre-ligand receptor assembly in the regulation of TNF receptor signaling. Cytokine 37, 101.

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.

Gerken M, Krippner-Heidenreich A, Steinert S, Willi S, Neugart F, Zappe A, Wrachtrup J, Tietz C & Scheurich (2010) Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors. Biochimica et biophysica acta 1798, 1081.

Heidbreder M, Zander C, Malkusch S, Widera D, Kaltschmidt B, Kaltschmidt C, Nair D, Choquet D, Sibarita JB & Heilemann M (2012) TNF-alpha influences the lateral dynamics of TNF receptor I in living cells. Biochimica et biophysica acta 1823, 1984.

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.