Quantitative single-molecule localization microscopy combined with
rule-based modeling reveals ligand-induced TNF-R1 reorganization
towards higher order oligomers
Franziska Fricke1, #, Sebastian Malkusch1, #, Gaby Wangorsch2, Johannes F. Greiner3, Barbara
Kaltschmidt4, Christian Kaltschmidt 3, Darius Widera3, Thomas Dandekar2 , Mike Heilemann1, 5, *
1
Institute for Physical and Theoretical Chemistry, Johann-Wolfgang-Goethe-University Frankfurt,
Frankfurt am Main, Germany;
2
Bioinformatics, Biocenter, Julius-Maximilians-University Wuerzburg, Wuerzburg, Germany;
3
Department of Cell Biology, Faculty of Biology, University of Bielefeld, Bielefeld, Germany;
4
Molecular Neurobiology, Faculty of Biology, University of Bielefeld, Bielefeld, Germany;
5
BIOQUANT Centre, University of Heidelberg, Heidelberg, Germany;
#
*
F. Fricke and S. Malkusch contributed equally.
Corresponding author: heilemann@chemie.uni-frankfurt.de, phone +49 69 798 29736, fax +49
69 798 29560
Supplemental Material
NF-κB-reporter gene assay
NF-κB-reporter gene assay was performed as described in (Widera et al. 2006) and (Greiner et
al. 2013). Briefly, U251 cells cultivated as described above were transiently co-transfected with
TK(NF-κB)6LUC vector (1 μg, (Bachelerie et al. 1991) and pRL-CMV vector (2 μg, Promega
Corporation) (Fig. S1 a) by applying the Cell Line Nucleofector Kit (Lonza Group) as well as the
Nucleofector II device (Lonza Group) according to manufacturer's guidelines. TNFα-treatment
was performed 24 h after transient transfection by addition of uncoupled TNFα, Cy5-coupled
TNFα or ATTO647N-coupled TNFα (each 10 ng/ml). 24 h after respective TNFα-treatments,
reporter gene activity was assessed by harvesting of the cells using Cell scrapper S (TPP) and
subsequent lysis in 1 x Passive Lysis buffer (Promega Corporation) followed by measuring of
luciferase activity using Dual-Luciferase® Reporter Assay System (Promega Corporation, cat.
no. E1960) according to manufacturer's guidelines. Luciferase signals were normalized as a
ratio of firefly luciferase activity to Renilla luciferase activity. Graph Pad Prism software
(GraphPad Software, La Jolla, CA, USA) was applied for subsequent statistical analysis (P ≤
0.05 was considered significant, one way Anova test with bonferroni correction).
Biological activity of dye-coupled TNFα
In order to assure their biological activity, Cy5- and ATTO647N-coupled TNFα were applied to a
NF-κB-reporter gene assay. Being a well-described activator of the canonical NF-κB-pathway,
stimulation of cells with TNFα results in nuclear translocation of NF-κB, followed by subsequent
DNA-binding and increased NF-κB-driven transcriptional activity (Kaltschmidt et al. 2005).
Considering this enhanced NF-κB DNA-binding activity after TNFα-dependent stimulation, we
applied a dual luciferase-driven NF-κB reporter gene assay to determine the biological activity of
the dye-coupled TNFα (Fig. S1 a). In comparison to the untreated control approach, treatment
with Cy5- and ATTO647N-coupled TNFα resulted in significantly increased NF-κB-activity in
U251 cells, a well-established model to assess NF-κB-activity. Notably, application of Cy5coupled TNFα and ATTO647N-labeled TNFα led to NF-κB-activity levels similar to those after
treatment with native TNFα, indicating their unchanged biological activity (Fig. S1 b).
Simulation reaction volume
The reaction volume Vr was defined as the cleft between the lower membrane of the cell and the
glass surface of the microscope slide for reasons of comparison with imaging data. We
approximated the reaction volume by a cylinder. Hereby the base area is limited by the
dimension of the basal cell membrane. Cells were approximated as spherical areas with a
diameter of 10 µm (Winkel et al. 2012), leading to a basal surface area of FM =
0.5 ∙ 3.14 ∙ 10−8 dm2. The height hC of the cylinder is given by the mean distance between the
microscope glass slide and the basal membrane, assumed to be 100 nm.
Homology model for TNF-R1-bound TNFα
The trivalent nature of TNFα was postulated by crystallography (PDB: 1TNF) (Eck and Sprang
1989), but no structural evidence for the interaction between TNF-R1 and TNFα was found in
the common data bases. However, the crystal structure for TNF-R1 bound to TNFβ exists (PBD:
1TNR) (Banner et al. 1993). We computed a homology model of TNF-R1 bound to TNFα by
multiple alignment of the two TNF template structures (PDB: 1TNR and 1TNF) using Modeller
(Sali and Blundell 1993). As the structure of TNFβ is very similar to the structure of TNFα, we
superimposed our homology model onto the known structure of TNFα (PDB: 1TNF). We used
the information about the receptor ligand interaction domains provided to visualize the possible
binding of three TNFR1 molecules to the TNFα trimer (Fig. S1 c) (Banner et al. 1993).
Model evaluation using receptor dimerization
The evaluation of our model was performed by a test model that describes the initial state of
TNF-R1 molecules at the cell membrane before TNFα application. As each TNF-R1 molecule R
exhibits a single PLAD, we chose the reversible dimerization of R to the dimer R-R as our test
model. In the BioNetGen language, rate constants represent the probability of a specific reaction
taking place. Therefore, multiplicative factors for multiple binding sites have to be considered (a
factor of 0.5 for pre-ligand dimerization).
0.5·𝑘𝑃𝐿𝐴𝐷,𝑜𝑛
𝑅+𝑅
→
←
𝑅−𝑅
𝑘𝑃𝐿𝐴𝐷,𝑜𝑓𝑓
Here, the rate constants are 𝑘𝑃𝐿𝐴𝐷,𝑜𝑓𝑓 and 𝑘𝑃𝐿𝐴𝐷,𝑜𝑛 . Assuming the system in a chemical
equilibrium, the concentrations of TNF-R1 in the monomeric and dimeric form are given by
𝑘𝑃𝐿𝐴𝐷,𝑜𝑓𝑓 [𝑅 − 𝑅]∞ − 0.5 ∙ 𝑘𝑃𝐿𝐴𝐷,𝑜𝑛 [𝑅]2∞ = 0 .
With the initial monomeric TNF-R1 concentration [𝑅]0 , the dissociation constant 𝐾𝑃𝐿𝐴𝐷 =
2 ∙ 𝑘𝑃𝐿𝐴𝐷,𝑜𝑓𝑓 /𝑘𝑃𝐿𝐴𝐷,𝑜𝑛
and [𝑅 − 𝑅] = 0.5 ∙ ([𝑅]0 − [𝑅]) we obtained the equilibrium receptor
concentration
[𝑅]∞ = √
𝐾𝑃𝐿𝐴𝐷 ∙ [𝑅]0
2
+
𝐾2𝑃𝐿𝐴𝐷
16
−
𝐾𝑃𝐿𝐴𝐷
4
.
Results from analytical derivation and simulation results at equilibrium were found to be similar.
Figure 5 (main text) shows the simulation results.
Supplemental Figures
Figure S1 A dual NF-κB-reporter system revealed biological activity of Cy5- and ATTO647N-coupled
TNFα a Schematic view of the dual NF-κB-reporter system. Constitutively expressed Renilla luciferase
served for normalization of NF-κB-driven firefly luciferase activity. b Stimulation with Cy5-coupled TNFα
and ATTO647N-coupled TNFα led to significantly increased levels of luciferase activity compared to
untreated control. Increased luciferase activity did not significantly differ from stimulation levels after
treatment with native TNFα. c Molecular structure of the TNF-R1-TNFα-complex with 3:3 binding
stoichiometry. The structure was obtained from homology modeling showing TNFα (blue), TNF-R1 (grey)
and the respective binding sites (green). By fluorescent coupling with NHS-ester, dyes are predominantly
linked to amino groups (purple). Most amino groups are freely accessible to the dye, but few lie in the
vicinity of binding sites (#). Hidden amino groups are marked with an asterisk *.
Figure S2 Morphological cluster analysis of TNF-R1-tdEOS images of unstimulated Hela cells (free TNFR1, unstimulated) and ligand-stimulated cells showing clusters not colocalizing with TNFα (free TNF-R1,
stimulated). Cluster radius (a) and localizations per cluster distributions (b) show no significant difference
Figure S3 Comparison of VSVG to TNF-R1 for protein calibration. Histograms of localizations per cluster
were obtained from morphological cluster analysis and show distinct peaks for all evaluated proteins
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