TNF-R1 signaling pathway

Tumor necrosis factor-alpha (TNFα) is a mediator of inflammation and binds to the TNF receptor 1 (TNF-R1). Evidence exists that TNF-R1 assembles in trimers upon ligand binding, followed by the formation of larger, ligand-mediated clusters.

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). How TNF-R1 assembles and initiates it diverse signaling responses is an exciting field or research. 


We apply single-molecule localization microscopy to obtain quantitative information about the molecular composition of TNF-R1 signaling clusters. We are interested to understand how the formation of specific signaling complexes ("hubs") initiate specific cellular responses. Using single-particle tracking, fluorescence correlation spectroscopy (FCS) and imagingFCS, we investigate the dynamics of TNF-R1 in the plasma membrane of live cells.


Assembly of TNF-R1 signaling complexes



Figure 1. (a) Two-colour single-molecule localization microscopy of TNF-R1_tdEos (green) and TNFα_ATTO647N (red) and (b) colocalization image.


Figure 2. Single-molecule localization microscopy provides quatitative information on the cluster size and intensity (number of localizations) for single TNF-R1_tdEos clusters. TNF-R1_tdEos receptor clusters that bind TNFα (top row) are larger and contain more single-molecule localizations than the resting receptor (bottom row). 



Figure 3. We aplpy single-molecule localization microscopy to measure binding constants of TNFα-TNF-R1-interactions directly at the plasma membrane of intact cells. For TNFα_ATTO647N, we determined a dissociation constant of KD = 15.7 nM (Dietz et al. 2014), which is in good accordance to the value determined by microscale thermophoresis, KD = 16.6 nM (Dietz et al., 2014).


Dynamics of TNF-R1 in the plasma membrane of live cells



Figure 4. Single-particle tracking of TNF-R1_tdEos in live cells report on the diffusion coefficient before (D = 0.14 µm²/s) and after binding TNFα (D=0.16 µm²/s) (left), with a decrease in the fraction of slow TNF-R1 molecules (right) (Heidbreder et al., 2012).




Dietz, M. S., Fricke, F., Krüger, C. L., Niemann, H. H. & Heilemann, M. (2014) Receptor-ligand interactions: Binding affinities studied by single-molecule and super-resolution microscopy on intact cells. ChemPhysChem. 15 (4), 671–676.

Fricke, F., Malkusch, S., Wangorsch, G., Greiner, J. F., 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 (1), 91–101.

Heidbreder, M., Zander, C., Malkusch, S., Widera, D., Kaltschmidt, B., Kaltschmidt, C., Nair, D., Choquet, D., Sibarita, J. B. & Heilemann, M. (2012) TNF-alpha influences the lateral dynamics of TNF receptor I in living cells. Biochimica et biophysica acta. 1823 (10), 1984–1989.

Malkusch, S., Endesfelder, U., Mondry, J., Gelléri, M., Verveer, P. J. & Heilemann, M (2012) Coordinate-based colocalization analysis of single-molecule localization microscopy data. Histochemistry and cell biology. 137 (1), 1–10.