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.