Journal: bioRxiv

Article Title: Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain

doi: 10.1101/2021.05.01.442235

Figure Lengend Snippet: a, Schematic depiction of cotransfected plasmids, one plasmid carrying four orthogonal tRNA repeats (4x 7SK PylT, Simon Elsässer, Addgene number: 140008) and amber-mutated FZD 5 or FZD 6 carrying an 5-HT 3 receptor signal peptide, an N-terminal Nluc and a C-terminal 1D4 epitope tag, and the other plasmid carrying four orthogonal tRNA repeats and the tRNA synthetase (Addgene number: 140023). b, Schematic depiction of the CRD sensor design with the N-terminally Nluc-tagged FZD. Fluorescence labeling of residues in the linker (pink) or ECL3 (blue) region of the receptor with Tet-Cy3. c-d, BRET responses of FZD 6 linker (c) and ECL3 (d) amber mutant CRD sensors upon 3 µg/mL WNT-5A treatment or vehicle control. The arrow indicates the time point of WNT or vehicle application. e, BRET responses of FZD 5 ECL3 amber mutant CRD sensors upon 3 µg/mL WNT-5A treatment or vehicle control. The arrow indicates the time point of WNT/vehicle addition. f, Concentration-response curves of WNT-5A on HEK293T cells expressing FZD 5 -Q493Amb or FZD 6 -K466Amb. Concentrations-response curves are normalized to vehicle control. All experiments were performed in HEK293T cells cotransfected with the indicated FZD 5 or FZD 6 sensors and the orthogonal tRNA/synthetase pair. SS , signal sequence, Nluc , NanoLuciferase, Amb , amber mutant, Tet , tetrazine, ECL3 , extracellular loop 3.

Article Snippet: In addition, we defined a transfection ratio for the tRNA/FZD expression plasmid and the corresponding suppressor tRNA/aminoacyl tRNA synthetase plasmid (Addgene number: 140023) of 9:1 which results in an efficient uaa incorporation ( ).

Techniques: Plasmid Preparation, Fluorescence, Labeling, Mutagenesis, Control, Concentration Assay, Expressing, Sequencing

Journal: bioRxiv

Article Title: Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain

doi: 10.1101/2021.05.01.442235

Figure Lengend Snippet: a, Schematic depiction of the CRD sensor design with the N-terminally Nluc-tagged FZD. Fluorescence labeling of residues in the ECL3 (blue) region of the receptor with Tet-Cy3. b-c, BRET responses of FZD 6 (b) or FZD 5 (c) ECL3 amber mutant CRD sensors upon 3 µg/mL WNT-3A treatment or vehicle control. The arrow indicates the time point of WNT/vehicle addition. d, Concentration-response curves of WNT-3A on HEK293T cells expressing FZD 5 -Q493Amb or FZD 6 -K466Amb. Concentrations-response curves are normalized to vehicle control. All experiments were performed in HEK293T cells cotransfected with the indicated FZD 5 or FZD 6 sensors and the orthogonal tRNA/synthetase pair. Nluc , NanoLuciferase, Amb , amber mutant, Tet , tetrazine, ECL3 , extracellular loop 3.

Article Snippet: In addition, we defined a transfection ratio for the tRNA/FZD expression plasmid and the corresponding suppressor tRNA/aminoacyl tRNA synthetase plasmid (Addgene number: 140023) of 9:1 which results in an efficient uaa incorporation ( ).

Techniques: Fluorescence, Labeling, Mutagenesis, Control, Concentration Assay, Expressing

Journal: bioRxiv

Article Title: Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain

doi: 10.1101/2021.05.01.442235

Figure Lengend Snippet: a, Schematic depiction of the initiation of WNT-induced β-catenin signaling. b, TOPFlash reporter gene response induced by 1 μg/mL WNT-3A in absence or presence of 1 μg/mL DKK1 in ΔFZD 1-10 HEK293 cells transiently transfected with FZD 5 -Q493Amb. Data show mean ± s.e.m. of four independent experiments. c, Maximal BRET response induced by 3 μg/mL WNT-3A in absence or presence of 3 μg/mL DKK1 in ΔFZD 1-10 HEK293 cells transiently transfected with FZD 5 -Q493Amb. Results in (b) and (c) were analyzed with one-way ANOVA followed by Tukey post-hoc test. Significance levels are given as ** (p < 0.01), **** (p < 0.0001), and ns (not significant). d, TOPFlash reporter gene response induced by increasing concentration of WNT-surrogate in ΔFZD 1-10 HEK293 cells transiently transfected with FZD 5 -Q493Amb. The concentration-response curve of WNT-surrogate is normalized to vehicle control. e, BRET responses of the FZD 5 – Q493Amb sensor upon 3 µg/mL WNT-3A, 500 pM WNT-surrogate treatment or vehicle control four individual experiments. The arrow indicates the time point of WNT/WNT-surrogate or vehicle application. Differences between WNT-surrogate vehicle control and WNT-surrogate or WNT-3A vehicle control and WNT-3A-induced BRET responses were analyzed with multiple t-test followed by Holm-Sidak multiple comparison. Significance levels are given as * (p < 0.05), and ns (not significant). All experiments were performed in ΔFZD 1-10 HEK293 cells or HEK293T cells cotransfected with FZD 5 -Q493Amb and the orthogonal tRNA/synthetase pair. Amb , amber mutant.

Article Snippet: In addition, we defined a transfection ratio for the tRNA/FZD expression plasmid and the corresponding suppressor tRNA/aminoacyl tRNA synthetase plasmid (Addgene number: 140023) of 9:1 which results in an efficient uaa incorporation ( ).

Techniques: Transfection, Concentration Assay, Control, Comparison, Mutagenesis