METHODS

Cell Culture and Materials

HEK293T cells (CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063) were maintained at 37°C/5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich, USA) supplemented with 10% Fetal Calf Serum (FCS; Sigma-Aldrich, USA). For a consistent cell background with functional studies performed using a reporter gene assay, all HEK293T cells also expressed a Firefly luciferase reporter gene (RE-Luc2P) that was inserted downstream of the NFAT promoter. Control experiments confirmed that HEK293T-NFAT-ReLuc2P cells did not emit luminescence in response to furimazine alone that interfered with NanoBiT or NanoBRET assays. Cells were passaged at 70-80% confluency using phosphate buffered saline (PBS; Lonza, Switzerland) and trypsin (0.25% w/v in versene; Lonza). Fluorescent VEGF165a and VEGF165b were labelled at a single N-terminal cysteine residue with TMR using the HaloTag mammalian protein detection and purification system (G6795; Promega Corporation, USA) as described previously (Kilpatrick et al., 2017; Peach et al., 2018a). Fluorescent ligands were characterised in terms of labelling efficiency, dimerisation and function as described in Kilpatrick et al. (2017) and Peach et al. (2018a). Ligands were stored at -20°C in 2.5mg/ml protease-free bovine serum albumin (BSA; Millipore, USA). Unlabelled recombinant human VEGF isoforms were purchased from R&D Systems (Abingdon, UK). Furimazine and purified NanoBiT fragments were purchased from Promega Corporation (Madison, USA).

Generating Constructs

N-terminal NanoLuc-tagged VEGFR2 (NM_002253) and NRP1 (NM_003873.5) were cloned in a pFN31K vector encoding the secretory IL‐6 signal peptide fused to the N‐terminus of NanoLuc, followed by a GSSGAIA linker before the receptor. HaloTag-VEGFR2 and HaloTag-NRP1 were cloned in a pFN21A vector with the IL‐6 signal peptide followed by a sequence encoding HaloTag and an EPTTEDLYFQSDNAIA linker at the receptor N‐terminus. SnapTag-NRP1 was cloned into a pcDNA3.1 vector encoding a murine 5HT3A signal sequence followed by the SnapTag and a STSPVWWNSADIQHSGGRSSGAIA linker. The receptor-encoded sequence from NanoLuc-NRP1 vector was used to generate SnapTag-NRP1 using the XhoI and XbaI restriction sites. N-terminal LgBiT-VEGFR2 and LgBiT-NRP1 were cloned in the pFN21A vector with the IL-6 signal peptide, LgBiT sequence and a flexible GSSGGGGSGGGGSSGGAIA linker. The LgBiT tag sequence from N198A pBiT1.1-N, available from the NanoBiT Multiple Cloning Site Starter System (N2014, Promega Corporation), was cut using SacII and SgfI. HiBiT-NRP1 (WT), HiBiT-NRP1 (Y297A), HiBiT-VEGFR2, SmBiT-NRP1 (WT), SmBiT-NRP1 (Y297A) and SmBiT-VEGFR2 were also cloned in a pFN21A vector with the IL-6 signal peptide, 11 amino acid sequence and a GSSGGSSGAIA linker. The VEGF-A binding-dead mutant of NRP1 (Y297A) was described previously (Peach et al., 2018a). The 11 amino acid NanoBiT tags (HiBiT: VSGWRLFKKIS; SmBIT VTGYRLFEEIL) were obtained as custom oligonucleotide sequences from Sigma-Aldrich, annealed into double stranded DNA and phosphorylated with T4 PNK (New England Biolabs) and inserted using SacII and SgfI sites.

NFAT Luciferase Reporter Gene Assay

HEK293T-NFAT-ReLuc2P cells stably expressed LgBiT-VEGFR2, HiBiT-VEGFR2 or SmBiT-VEGFR2. Cells were seeded at 25,000 cells/well in white 96-well plates pre-coated with poly-D-lysine in DMEM containing 10% FBS. Following incubation for 24 hours at 37%/5% CO2, medium was replaced with serum-free DMEM and cells were incubated for a further 24 hours. On the day of experimentation, medium was replaced with serum-free DMEM containing 0.1% BSA. Cells were stimulated with increasing concentrations of VEGF165a (R&D Systems) for 5 hours at 37%/5% CO2. Medium was replaced with 50 μl/well serum-free DMEM/0.1% BSA and 50 μl/well ONE-Glo Luciferase reagent. Following a 5 minute delay to allow reagent to react with luciferase and background luminescence to subside, luminescence emissions were measured using a TopCount platereader (Perkin Elmer, UK).

Confocal Imaging of HaloTag-VEGFR2 and SnapTag-NRP1

HEK293T-NFAT-ReLuc2P cells were plated in 8-well plates (Nunc Lab-Tek, Thermo Fisher Scientific) pre-coated with poly-D-lysine (0.01mg/ml in PBS) at 30,000 cells per well in DMEM/10% FBS. Following incubation for 24 hours, cells were transfected with a mixture of HaloTag-VEGFR2 and SnapTag-NRP1. Control wells were also transfected with a single construct and empty vector, such as SnapTag-NRP1 and pcDNA3.1/Neo. Transient transfections used FuGENE® HD at a 3:1 ratio of reagent to cDNA with a total 100 ng cDNA/well, with receptors transfected at equal amounts of 50 ng cDNA/well. Transfection solutions were made up in serum-free DMEM and added as 11 μl/well. Cells were incubated for a further 24 hours at 37°C/5% CO2. Receptors were then labelled with a solution of serum-free DMEM/0.1% BSA containing both 0.5 μM membrane impermeant HaloTag-AlexaFluor488 substrate (G1002; Promega Corporation, USA) and 0.5 μM membrane impermeant SNAP-Surface AlexaFluor647 (S9136S; New England BioLabs). These were incubated for 30 minutes (37°C/5% CO2). Cells were washed twice with 200 μl/well HBSS/0.1% BSA, then replaced with a final volume of 225 μl/well. Cells were incubated with vehicle, 10 nM unlabelled VEGF165b or 10 nM unlabelled VEGF165a for 60 minutes at 37°C, adding 25 μl to a total volume of 250 μl. Cells were imaged live using a temperature-controlled LSM710 confocal microscope fit with a 40x water objective (Pan Apochromat objective, NA 1.2). Wavelengths were imaged simultaneously using the 488/561/633 beamsplitter. HaloTag-VEGFR2 AlexaFluor488 was imaged using an Argon 488 nm laser (493-628 nm bandpass; 2.5% power); SnapTag-NRP1 AlexaFluor647 was imaged with a HeNe633 nm laser (638-747 nm; 2.5% power). All images were taken as 12 bit images with 1024x1024 pixels per frame with 4 averages and similar gains per replicate.

Bioluminescence Imaging of NanoBiT Complexes

HEK293T-NFAT-ReLuc2P cells were plated in 6-well plates at 400,000 cells/well in DMEM/10% FBS. On day 2, cells were transfected using FuGENE® HD at a 3:1 ratio of reagent to cDNA with a total 1500 ng cDNA/well made up in serum-free DMEM. Cells were transfected with equal amounts of LgBiT-VEGFR2 (750 ng cDNA/well) and HiBiT-NRP1 WT (750 ng cDNA/well). Alternatively, NanoLuc-VEGFR2 or NanoLuc-NRP1 were transfected at 750 ng cDNA/well with an equal amount of pcDNA3.1/Zeo (750 ng cDNA/well). On day 3, transfected cells were transferred to a 4- compartment 35/10 mm glass bottomed dish (CELLview, Greiner Bio-One). Dishes were pre-coated with poly-D-lysine (0.01mg/ml in PBS) and cells were plated at 75,000 cells/well in DMEM containing 0.1% FBS. On the day of experimentation (day 4), medium was replaced with HBSS/0.1% BSA. For cells expressing full-length NanoLuc, furimazine was added at 26 μM. In contrast, cells expressing the NanoBiT complex were incubated with a higher furimazine concentration (104 μM) for optimal imaging. Following incubation for 10 minutes to allow for substrate oxidation, cells were imaged live at 37°C using the inverted Olympus LV200 Bioluminescence Imaging System, fitted with a 60x oil immersion objective (super Apochromat UPLSAPO 60xO objective; NA 1.35) with a 0.5x tube lens to focus the image, therefore images had a final magnification of 30x. Luminescence was collected using a Hammamatsu Image EMx2 Electron Multiplying Charge Coupled Device (EMCCD) camera. Transmitted light images were collected using the camera in conventional CCD mode with a 200 ms exposure time. Luminescence emissions from the full-length NanoLuc or the NanoBiT complex were measured for 5 second exposure with a gain of 200. Images were taken as 8 bit images with 512x512 pixels per frame.

BRET Between NanoLuc-VEGFR2 and Fluorescent NRP1

HEK293T-NFAT-ReLuc2P cells were plated in white 96-well plates pre-coated with poly-D-lysine (0.01mg/ml in PBS) at 25,000 cells per well in DMEM containing 10% FBS. Following 24 hours, cells were transiently transfected with a total 125 ng cDNA/well using FuGENE® HD at a 3:1 ratio of reagent to cDNA. Cells were transfected with a constant amount of NanoLuc-VEGFR2 (25 ng cDNA/well). Cells were simultaneously transfected with increasing concentrations of HaloTag-NRP1 or SnapTag-NRP1 (2.5-100 ng cDNA/well). Additional wells only contained NanoLuc-VEGFR2. These transfection solutions were made up to equivalent to 125 ng per well using empty pcDNA3.1/Zeo vector in serum-free DMEM. Cells were incubated for another 24 hours at 37°C/5% CO2. On the day of the experiment, cells were treated with 0.2 μM membrane impermeant HaloTag-AlexaFluor488 substrate or 0.2 μM SNAP-Surface AlexaFluor488 substrate in serum-free-DMEM/0.1% BSA. Cells were incubated for 30 minutes at 37°C/5% CO2. They were then washed twice with 100 μl/well HBSS/0.1% BSA and replaced with a final volume of 50 μl/well HBSS/0.1% BSA. At this stage, fluorescence emissions were quantified using the PHERAstar FS platereader using filters for excitation at 485 nm and emission at 520 nm. Cells were then incubated with the NanoLuc substrate furimazine (10 μM) for 5 minutes. Emissions were recorded using the PHERAstar FS platereader using filters simultaneously measuring NanoLuc emissions at 475 nm (30 nm bandpass) and AlexaFluor488 emissions at 535 nm (30 nm bandpass). BRET ratios were calculated as fluorescence over luminescence emissions from the second of three cycles.

Luminescence from NanoBiT Complementation

To characterise luminescence emissions from a NanoBiT complex, HEK293T-NFAT-ReLuc2P cells were plated as 25,000 cells/well in white 96-well plates pre-coated with poly-D-lysine (0.01mg/ml in PBS) in DMEM containing 10% FBS. Following 24 hours, cells were transiently transfected using FuGENE® HD at a 3:1 ratio of reagent to cDNA with a total 100 ng cDNA/well. Cells were transfected with a combination of LgBiT-tagged (50 ng cDNA/well) and HiBiT-/SmBiT-tagged receptors (50 ng cDNA/well). Alternatively, cells were transfected with single constructs (50 ng cDNA/well) with empty pcDNA3.1/Zeo vector (50 ng cDNA/well). Transfection mixtures were made up in serum-free DMEM and added as 5 μl/well without replacing DMEM/10% FBS on cells. Cells were incubated at 37°C/5% CO2 for a further 24 hours. Medium was replaced with HBSS/0.1% BSA containing 10 μM furimazine, in the absence or presence of purified LgBiT protein (N401B, Promega Corporation) or HiBiT protein (N301A, Promega Corporation). Cells were incubated at 37°C for 10 minutes to allow NanoBiT complementation and the oxidation of furimazine. To prevent the loss of signal through the bottom of the plate, an adhesive plate BackSeal was added at this point. Luminescence emissions were measured on the PHERAstar platereader using the filter settings measuring emissions between 475-505 nm.
Additional experiments aimed to disrupt the recomplemented NanoBiT complex using increasing concentrations of competing receptor. HEK293T-NFAT-ReLuc2P cells were plated as 25,000 cells/well in white 96-well plates pre-coated with poly-D-lysine (0.01mg/ml in PBS) in DMEM containing 10% FBS. Following 24 hours, cells were transiently transfected using FuGENE® HD at a 3:1 ratio of reagent to cDNA. Cells were transfected with a constant amount of LgBiT-VEGFR2 (50 ng cDNA/well) and either HiBiT-NRP1 or SmBiT-NRP1 (50 ng cDNA/well). Cells were also transfected with increasing concentrations of HaloTag-NRP1 (25-200 ng cDNA/well). This was made up to 300 ng cDNA/well with empty pcDNA3.1/Zeo vector. Additional wells only contained the LgBiT-VEGFR2 and HiBiT/SmBiT-NRP1 complex. Cells were incubated with transfection solution for 24 hours at 37°C/5% CO2. On the day of the experiment, cells were treated with 0.2 μM membrane impermeant HaloTag-AlexaFluor488 substrate in serum-free-DMEM/0.1% BSA (30 minutes, 37°C/5% CO2). They were then washed twice with 100 μl/well HBSS/0.1% BSA and replaced with a final volume of 50 μl/well HBSS/0.1% BSA. Fluorescence emissions were quantified using the PHERAstar FS platereader using filters for excitation at 485 nm and emission at 520 nm. Cells were incubated with 10 μM furimazine for 10 minutes, then luminescence and fluorescence emissions were recorded using PHERAstar FS platereader. Emissions were simultaneously measured for NanoLuc at 475 nm (30 nm bandpass) and AlexaFluor488 at 535 nm (30 nm bandpass).

Fluorescent VEGF-A Binding at a VEGFR2/NRP1 NanoBiT Complex

HEK293T-NFAT-ReLuc2P cells were plated in 6-well plates at 400,000 cells/well in DMEM containing 10% FBS. On day 2, cells were transfected using FuGENE® HD at a 3:1 ratio of reagent to cDNA with a total 1500 ng cDNA/well made up in serum-free DMEM. Cells were transfected with equal amounts of LgBiT-VEGFR2 (750 ng cDNA/well) and HiBiT-NRP1 WT or Y297A (750 ng cDNA/well), or equal amounts of LgBiT-VEGFR2 (750 ng cDNA/well) with SmBiT-NRP1 WT (750 ng cDNA/well). For experiments monitoring kinetics at HiBiT complexes, matched controls were performed alongside in which cells were transfected with single receptors conjugated to full-length NanoLuc. NanoLuc-VEGFR2 or NanoLuc-NRP1 were transfected at 750 ng cDNA/well, made up to 1500 ng cDNA/well with empty pcDNA3.1/Zeo vector (750 ng cDNA/well). On day 3, cells were transferred from 6-well plates. Cells were washed with 1 ml/well PBS, detached with 500 μl/well trypsin and resuspended in 2 ml DMEM containing 10% FBS. Cells were seeded in white 96-well plates pre-coated with poly-D-lysine (0.01mg/ml in PBS) at 30,000 cells/well. On the day of experimentation (day 4), medium was replaced with HBSS/0.1% BSA.
For saturation experiments, increasing concentrations of VEGF165a–TMR or VEGF165b-TMR (0.5-20 nM) were added in the presence or absence of a high concentration of corresponding unlabelled ligand (100nM, ~100-fold greater than the estimated Kd value). Following incubation for 60 minutes in the dark at 37°C, the NanoLuc substrate furimazine (10 μM) was added to each well and equilibrated for 5 minutes to enable NanoLuc-mediated furimazine oxidation and resulting luminescence emissions. Emissions were recorded using the PHERAstar FS platereader (BMG Labtech) using a filter simultaneously measuring NanoLuc emissions at 450 nm (30 nm bandpass) and TMR emissions using a longpass filter at 550 nm. BRET ratios were calculated as fluorescence over luminescence emissions from the second of three cycles.
For kinetic experiments, cells were pre-treated with furimazine (10 μM) for 5 minutes to enable NanoLuc-mediated furimazine oxidation and resulting luminescence emissions. BRET ratios were then measured per well using the PHERAstar FS platereader using the filters above. Following 4 initial measurements, intact cells were stimulated with 0.5-20 nM VEGF165a-TMR or VEGF165b-TMR Emissions were recorded every 30 seconds for 20 minutes or 90 minutes, using the temperature control function of the PHERAstar FS platereader to maintain conditions at 37°C.

Data Analysis

Data were analysed using GraphPad Prism 7.02 (GraphPad Software, La Jolla, CA, USA). Data are presented as mean ± S.E.M.. Statistical significance was defined as P< 0.05. Confocal images were collected using Zen 2010 software (Zeiss, Germany). Confocal images were processed and analysed using ImageJ Fiji 1.52 software (National Institutes of Health, US).
For colocalization analysis, confocal images were corrected to the background fluorescence intensity from each experimental replicate determined using un-transfected cells in each field of view (HaloTag-VEGFR2, 488 nm; SnapTag-NRP1, 647 nm). The mean background intensity was calculated for each experimental replicate (n=4) and subtracted from each image for manual thresholding. To quantify colocalization, regions of interest (ROIs) were drawn around each cell that co-expressed HaloTag-VEGFR2 and SnapTag-NRP1. Following subtraction of the region outside the ROI, colocalisation was determined using pixel-based measures between HaloTag-VEGFR2 and SnapTag-NRP1 using the ImageJ plugin Coloc 2. Mander’s Overlap Coefficients measure co-occurrence as the proportion of SnapTag-NRP1 pixels (red) overlapping with HaloTag-VEGFR2 (green). Pearson’s Correlation Coefficients measure whether there is a correlation between these channels. Colocalisation parameters were calculated on a per cell basis, with a total number of 97 cells (vehicle) and 54 cells (VEGF165a stimulation) and 68 cells (VEGF165b stimulation), pooled from 4 independent experiments.
Saturation binding curves were fitted simultaneously for total (VEGF165a-TMR or VEGF165b-TMR alone) and non‐specific binding (obtained in the presence of 100 nM of unlabelled VEGF‐A) using the equation:
\begin{equation} Total\ Binding=\ B_{\max}.\frac{\ [L]}{\left[L\right]+\ K_{d}}+M.\left[L\right]+C\nonumber \\ \end{equation}
describing the nanomolar fluorescent ligand concentration, [L]; maximal binding, Bmax; the equilibrium dissociation constant of the labelled ligand, Kd, in the same units as [L], the slope of the non-specific binding component, M; and the y axis intercept, c.
Kinetic studies of fluorescent ligand binding measured over time were fitted to a mono-exponential association function:
\begin{equation} Binding=y_{\max}\ .\ (1\ -e^{-k_{\text{obs}}\text{\ .\ t}})\nonumber \\ \end{equation}
describing time, t, plotted on the x axis; maximum response at infinite time, ymax; and the rate constant observed for association, kobs. Additionally, kon and koff values were determined by simultaneously fitting association curves at different fluorescent ligand concentrations ([L]). This utilised the following relationship with kobs:
\begin{equation} k_{\text{obs}}=k_{\text{on}}.\left[L\right]+\ k_{\text{off}}\nonumber \\ \end{equation}
further describing association rate, kon, in units of min-1 M-1; and dissociation rate, koff, in min-1. These kinetic data were also used to estimate the binding affinities, due to the relationship between dissociation and association rates within an equilibrium:
\begin{equation} K_{d}=\ \frac{k_{\text{off}}}{k_{\text{on}}}\nonumber \\ \end{equation}
Residence time was calculated as the reciprocal of koff. Additionally, assuming a first order reaction, half life (t1/2) was calculated for a given concentration.
\begin{equation} t_{1/2}=\ \frac{\ln 2}{k_{\text{on}}.\left[L\right]+\ k_{\text{off}}}\nonumber \\ \end{equation}\begin{equation} t_{1/2}=\ \frac{0.693}{k_{\text{obs}}}\nonumber \\ \end{equation}