FIGURE LEGENDS

Figure 1. Localisation of VEGFR2 and NRP1 co-expressed in living HEK293T cells . (a) HEK293T cells expressing HaloTag-VEGFR2 and SnapTag-NRP1 were simultaneously labelled with membrane-impermeant 0.5 μM HaloTag-AlexaFluor488 and 0.5 μM SnapTag-AlexaFluor647 for 30 minutes (37°C). Cells were washed twice in HEPES Buffered Saline Solution (HBSS) containing 0.1% Bovine Serum Albumin (BSA) and incubated at 37°C. Cells were imaged on the LSM710 Confocal Microscope (40X objective). The same cell population were imaged in the presence of vehicle or following treatment with 10 nM unlabelled VEGF165b or VEGF165a for 60 minutes (37°C). Images show HaloTag-VEGFR2 (green) and SnapTag-NRP1 (magenta), showing regions of spatial overlay in white. Images are representative from 4 independent experiments. (b,c) ImageJ/Fiji software was used to analyse images with channels corresponding to HaloTag-VEGFR2 or SnapTag-NRP1. Co-localisation was quantified based on regions of interest drawn around cells co-expressing both receptors. Mander’s Overlap Coefficients represent the proportion of SnapTag-NRP1 in HaloTag-VEGFR2+ regions (b), whereas Pearson’s Correlation Coefficients compare the relationship between the intensity of VEGFR2 and NRP1 pixels (c). All coefficient values were pooled from 4 independent experiments, with a total of 97 cells (vehicle), 68 cells (VEGF165b) or 54 cells (VEGF165a). Coefficients were compared between conditions using a Kruskal-Wallis test and post-Hoc Dunn’s multiple comparisons test between vehicle, VEGF165b or VEGF165a stimulation (* P< 0.05).
Figure 2. Oligomer formation between VEGFR2 and NRP1. (a,b) HEK293T cells were transiently transfected with a fixed concentration of NanoLuc-VEGFR2 (25 ng cDNA/well) and increasing concentrations of fluorescent acceptor (HaloTag-NRP1 or SnapTag-NRP1, 0-100 ng cDNA/well). All wells were transfected with 125 ng cDNA/well total with empty pcDNA3.1/Zeo vector. NRP1 was labelled with 0.2 µM HaloTag-AlexaFluor488 substrate or 0.2 μM SNAP-Surface AlexaFluor488 substrate for 30 minutes (37°C). Cells were washed twice with HBSS/0.1% BSA then incubated in 10 µM furimazine for 5 minutes (37°C). Emissions from the luminescent donor and fluorescent acceptor receptor were simultaneously monitored by the PHERAstar FS platereader. Data are expressed as (a) mean ± S.E.M. from 5 independent experiments with duplicate wells or (b) individual data points from a representative experiment plotting BRET ratio values against fluorescence emissions (485-520 nm).
Figure 3. Complementation of a VEGFR2/NRP1 NanoBiT complex . (a) To determine the optimal orientation of labelling with NanoLuc Binary Technology (NanoBiT) fragments, each receptor was tagged with the 18 kDa fragment (LgBiT) and a smaller 11 amino acid fragment. HiBiT has a higher intrinsic affinity to complement with LgBiT compared to SmBiT (Dixon et al., 2016). HEK293T cells were transiently transfected in 96-well plates with equal amounts of LgBiT-tagged receptor (50 ng cDNA/well) and HiBiT- or SmBiT-tagged receptor (50 ng cDNA/well). Cells were incubated with 10 µM furimazine in HBSS/0.1% BSA for 10 minutes (37°C). Data were normalised to un-transfected cells (0%) and HiBiT-NRP1/LgBiT-VEGFR2 (100%) per experiment. Data are expressed as mean ± S.E.M. from 5 independent experiments (LgBiT-VEGFR2) or 3 independent experiments (LgBiT-NRP1), each with triplicate wells. (b) To compare emissions from individual NanoBiT-tagged receptors relative to a complemented NanoBiT complex, HEK293T cells were transiently transfected in 96-well plates with LgBiT-VEGFR2, HiBiT-NRP1 or SmBiT-NRP1 (50 ng cDNA/well). Dual expression cells expressed a complemented NanoBiT complex (filled bars) whereas single constructs (empty bars) were transfected with 50 ng cDNA/well empty pcDNA3.1/Zeo vector for 100 ng total cDNA/well. Experiments were repeated in 5 independent experiments. Raw emissions were plotted from a representative experiment as mean ± S.E.M. from triplicate wells. (c) Cells expressed a single NanoBiT-tagged construct, in the absence (open bars) or presence (filled bars) of 20 nM purified HiBiT or LgBiT. Data are representative from 5 independent experiments from the same experiment as (b). Raw emissions were plotted as mean ± S.E.M. from triplicate wells. (d) Prevention of NanoBiT complex formation by co-expression of increasing amounts of competing VEGFR2 or NRP1. HEK293T cells were transfected with equal amounts of LgBiT-VEGFR2 (50 ng cDNA/well) and either HiBiT-NRP1 (lined bars) or SmBiT-NRP1 (solid bars) at 50 ng cDNA/well. Cells were also transfected with increasing amounts of HaloTag-NRP1 (0-200 ng cDNA/well), as well as with pcDNA3.1/Zeo empty vector (for 300 ng total cDNA/well). Data were normalised to un-transfected cells (0%) and the complemented NanoBiT complex in the absence of competing receptor (100%) per experiment. Data are expressed as mean ± S.E.M. from 3 independent experiments, each with triplicate wells. (a-d) Cells were incubated with furimazine (10 µM) in HBSS/0.1% BSA for 10 minutes (37°C). Luminescence emissions (475-505 nm) were measured by the PHERAstar FS platereader.
Figure 4. Bioluminescence imaging of NanoLuc-VEGFR2, NanoLuc-NRP1 or NanoBiT-complemented VEGFR2-NRP1 complexes. HEK293T cells were transfected with LgBiT-VEGFR2 (750 ng cDNA/well) and HiBiT-NRP1 (750 ng cDNA/well). Following 24 hours, transfected cells were seeded into 35 mm2 glass-bottomed dishes. Cells were incubated with furimazine for 10 minutes at 37°C (26 μM for full-length NanoLuc; 104 μM for NanoBiT complex). Cells were imaged live using a widefield Olympus LV200 Bioluminescence Imaging System as described under Methods. Images are representative from 3 independent experiments.
Figure 5. Characterisation of NFAT signalling from VEGFR2 tagged with LgBiT, HiBiT or SmBiT moieties. HEK293T cells stably expressed both NFAT-ReLuc2P and either LgBiT-VEGFR2, HiBiT-VEGFR2 or SmBiT-VEGFR2. (a) Cells were serum-starved for 24 hours. On the day of experimentation, cells were stimulated with increasing concentrations of VEGF165a for 5 hours and 37°C/5% CO2. Data were normalised to mean vehicle (0%) or 10 nM unlabelled VEGF165a (100%) per experiment. Data are expressed as mean ± S.E.M. from 5 independent experiments with duplicate wells per experiment.
Figure 6. Saturation binding of VEGF165b-TMR and VEGF165a-TMR at a HiBiT complex of VEGFR2 and NRP1 . (a) Fluorescent VEGF-A ligand binding was monitored at a defined complex of LgBiT-VEGFR2 and HiBiT-NRP1. In the presence of furimazine, individual receptors do not emit luminescence in isolation. Upon NanoBiT complementation, luminescence emissions can excite the tetramethylrhodamine (TMR) in close proximity. NanoBiT therefore only acts as a luminescent donor when VEGFR2 and NRP1 are in complex. (b,c) HEK293T cells were transfected in 6-well plates with equal amounts of LgBiT-VEGFR2 (750 ng cDNA/well) and HiBiT-NRP1 (750 ng cDNA/well). Following 24 hours, transfected cells were seeded in 96-well plates. On the day of experimentation, cells were incubated with increasing concentrations of VEGF165b-TMR (b) or VEGF165a-TMR (c). This was performed in the presence or absence of 100 nM VEGF165b (b) or VEGF165a (c) to determine non-specific binding. Following 60 minutes at 37°C, 10 μM furimazine was added for 10 minutes (37°C). Emissions were measured on the PHERAstar platereader. BRET ratios are expressed as mean ± S.E.M. from 3 independent experiments with duplicate wells.
Figure 7. Real-time binding of fluorescent VEGF-A isoforms at the NanoBiT complex compared to isolated receptors. (a) HEK293T cells were transfected in 6-well plates with equal amounts of LgBiT-VEGFR2 (750 ng cDNA/well) and HiBiT-NRP1 (750 ng cDNA/well). Alternatively, cells were transfected with equal amounts of NanoLuc-VEGFR2 or NanoLuc-NRP1 (750 ng cDNA/well) and empty pcDNA3.1/Zeo vector (750 ng cDNA/well). Following 24 hours, transfected cells were seeded in 96-well plates. On the day of experimentation, cells were pre-treated with furimazine (10 µM) and left to equilibrate at 37°C for 10 minutes. (a) Cells expressing the NanoBiT complex (LgBiT-VEGFR2/HiBiT-NRP1) were stimulated with 4 concentrations of VEGF165b-TMR added at x=0. Kinetic data were fitted to a global association model with an unconstrained kon from the 90 minute time course. (b) On the same plate, the real-time binding profile of 20 nM VEGF165b-TMR was monitored in cells only expressing either NanoLuc-VEGFR2 or NanoLuc-NRP1 (left y axis, grey symbols). This was directly compared to binding of the same concentration of VEGF165b-TMR at the LgBiT-VEGFR2/HiBiT-NRP1 NanoBiT Complex (right y axis, red circular symbols). (c) Cells expressing the HiBiT complex were stimulated with 4 concentrations of VEGF165a-TMR. Kinetic data were fitted to a global association model without a constrained kon from the initial 20 minutes due to the latter decline in BRET ratio. (d) The real-time binding profile of a saturating concentration of VEGF165a-TMR (10 nM) was compared between cells expressing LgBiT-VEGFR2/HiBiT-NRP1 (right y axis, blue circular symbols) to cells only expressing NanoLuc-VEGFR2 or NanoLuc-NRP1 (left y axis, open symbols). For each experiment, emissions were simultaneously measured on the PHERAstar FS platereader every 30 seconds for 90 minutes at 37°C. BRET ratios were baseline-corrected to vehicle at each time point per experimental replicate. In (b) and (d), the x axis was split to highlight the initial association (20 minutes) and long-term BRET signal (90 minutes). Data represent mean ± S.E.M. from 5 independent experiments with duplicate wells. Derived kon, koff and kinetic pKd parameters are in Table 1.
Figure 8. Fluorescent VEGF-A binding kinetics at a NanoBiT VEGFR2/NRP1 complex using split tags with lower intrinsic affinity . HEK293T cells were transfected in 6-well plates with equal amounts of LgBiT-VEGFR2 (750 ng cDNA/well) and SmBiT-NRP1 (750 ng cDNA/well). Following 24 hours, transfected cells were seeded in 96-well plates. Cells were pre-treated with furimazine (10 µM) and left to equilibrate at 37°C for 10 minutes. (a) Cells were stimulated with 4 concentrations of VEGF165b-TMR added at x=0. Kinetic data were fitted to a global association model without a constrained konfrom the 90 minute time course. For clarity, the 10 nM data set has not been included in the figure. (b) The derived rate constant, kobs, was obtained from exponential association curves fitted for each of the four fluorescent ligand concentrations. These were plotted against VEGF165b-TMR concentration and fitted against a linear regression (HiBiT Complex y = 0.0023x + 0.034, R2 = 0.46; SmBiT Complex y = 0.0026x + 0.01, R2 = 0.65). (c) Cells were stimulated with 4 concentrations of VEGF165a-TMR. Kinetic data were fitted to a global association model without a constrained konfrom the initial 20 minutes due to the latter decline in BRET ratio. For clarity, the 5 nM data set has not been included in the figure. (d) The derived kobs for each fluorescent ligand at all four concentrations were plotted against each VEGF165a-TMR concentration and fit with a linear regression (HiBiT Complex y = 0.0024x + 0.10, R2 = 0.72; SmBiT Complex y = 0.0025x + 0.09, R2 = 0.62). Emissions were simultaneously measured on the PHERAstar FS platereader every 30 seconds for 90 minutes at 37°C. BRET ratios were baseline-corrected to vehicle at each time point per replicate. Data represent mean ± S.E.M. from 5 independent experiments with duplicate wells in each independent experiment. Derived kon, koff and kinetic pKd parameters are in Table 1.
Figure 9. Ligand binding of VEGF165a-TMR at a NanoBiT complex with a binding-dead NRP1 mutant . (a) VEGF165a-TMR ligand binding was monitored at a defined NanoBiT complex between LgBiT-VEGFR2 and a HiBiT-NRP1 VEGF-A binding-dead mutant in the b1 domain. (b) HEK293T cells were transiently transfected in 96-well plates with LgBiT-VEGFR2, HiBiT-NRP1 Y297A or SmBiT-NRP1 Y297A (50 ng cDNA/well). Dual expression cells expressed a complemented NanoBiT complex with the HiBiT or SmBiT tag. Cells also expressed single constructs (empty bars) were transfected with 50 ng cDNA/well empty pcDNA3.1/Zeo vector. Cells were incubated with 10 µM furimazine in HBSS/0.1% BSA for 10 minutes (37°C). Luminescence emissions (475-505 nm) were measured by the PHERAstar FS platereader. Data were normalised to un-transfected cells (0%) and HiBiT-NRP1 Y297A/LgBiT-VEGFR2 (100%) per experiment. Data are expressed as mean ± S.E.M. from 5 independent experiments, each with triplicate wells. (c,d) HEK293T cells were transfected in 6-well plates with equal amounts of LgBiT-VEGFR2 (750 ng cDNA/well) and HiBiT-NRP1 Y297A (750 ng cDNA/well). Following 24 hours, transfected cells were seeded in 96-well plates. (c) On the day of experimentation, cells were incubated with increasing concentrations of VEGF165a-TMR in the presence or absence of 100 nM VEGF165a to determine non-specific binding. Following 60 minutes at 37°C, 10 μM furimazine was added for 10 minutes (37°C). Emissions were measured on the PHERAstar platereader (550-LP/460-480 nm). BRET ratios are expressed as mean ± S.E.M. from 3 independent experiments with duplicate wells. Derived equilibrium dissociation constants (pKd) are in the text. (d) Cells were pre-treated with furimazine (10 µM) and left to equilibrate at 37°C for 10 minutes. Cells were incubated with 4 concentrations of VEGF165a-TMR. Kinetic data were fitted to a global association model without a constrained kon from the initial 20 minutes. Emissions were simultaneously measured on the PHERAstar FS platereader every 30 seconds for 90 minutes at 37°C. BRET ratios were baseline-corrected to vehicle at each time point per experimental replicate. Data represent mean ± S.E.M. from 5 independent experiments with duplicate wells. Derived kon, koff and kinetic pKd parameters are noted in the text.