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Supplementary Information

Membrane curvature regulates ligand-specific membrane sorting of

GPCRs in living cells

Kadla R. Rosholm1,2,6, Natascha Leijnse2,4, Anna Mantsiou1,2, Vadym Tkach1,2, Søren L. Pedersen2,3,7, Volker F. Wirth1,2, Lene

B. Oddershede2,4, Knud J. Jensen2,3, Karen L. Martinez1,2, Nikos S. Hatzakis1,2, Poul Martin Bendix4, Andrew Callan-Jones5

and Dimitrios Stamou1,2,★

1Bio-Nanotechnology and Nanomedicine Laboratory, Nano-Science Center, Department of Chemistry, University of

Copenhagen, 2Lundbeck Foundation Center Biomembranes in Nanomedicine, 3Department of Chemistry, University of

Copenhagen, 4Niels Bohr Institute, University of Copenhagen, 5Laboratoire Matière et Systèmes Complexes, Université Paris-

Diderot

Present affiliations: 6The Victor Chang Cardiac Research Institute, Sydney, Australia, 7Gubra Aps, Hørsholm, Denmark,

★email: stamou@nano.ku.dk

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Supplementary Results

Supplementary Figure 1 Quantification of filopodium radius and normalized Y2R density in single

filopodia. PC12 or HEK293 cells were transfected to express the Y2-receptor (Y2R), which were N-

terminally fused to a water-soluble fluorophore (DY-647) using SNAP-tag labeling1. Subsequent labeling

of the cell membrane by a lipid dye, DiOC18 (DiO), allowed imaging by fluorescence microscopy. (a)

Left: Fluorescence micrographs of a HEK293 cell, focused on the filopodia and surface-adhered cell

membrane, and a zoom in on a single filopodium, in the DiO channel (top) and the Y2R channel

(bottom). Scale bars are 5 µm. Right: The corresponding intensity profiles quantified as an average of 50

pixels along the yellow line in the micrographs. The integrated intensities of the two dyes on a

filopodium, I(DiO)f and I(Y2R)f, were quantified by Gaussian fits to the intensity profiles (black lines).

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(b) Left: Fluorescence micrographs of the same cell as in (a), focused on the cell body plasma membrane

in the middle of the cell, in the DiO channel (top) and the Y2R channel (bottom). Scale bars are 5 µm.

Right: The corresponding intensity profiles taken as an average of 50 pixels along the yellow line in the

micrographs. The integrated membrane intensities of the two dyes, I(DiO)PM and I(Y2R)PM, were

quantified by Gaussian fits to the intensity profiles (black lines). The relative filopodium radius, I(DiO),

and normalized Y2R density were quantified as the intensity ratios: I(DiO) = I(DiO)f/I(DiO)PM and

normalized Y2R density = (I(Y2R)f/I(DiO)f)/(I(Y2R)PM/I(DiO)PM) (see online Methods). (c) Top:

Fluorescence micrograph of a tether pulled from a living DiO-labeled HEK293 cell, using an optically

trapped streptavidin-coated bead. The integrated intensity, I(DiO), was quantified by a Gaussian fit to the

intensity profile across the tether (yellow line). Bottom: Histogram of I(DiO) values (green) extracted

from 25 short (< 15 µm) tethers. The mean of the distribution was extracted by fitting a Gaussian function

to the data (black line) and related to the mean tether radius that was previously quantified by scanning

electron microscopy2 (see online Methods). (d) Live-cell micrographs of PC12 cell filopodia:

transmission (left), DiO fluorescence (middle) and protein/lipid dye fluorescence (right). From top to

bottom the micrographs are displayed for Y2R (top), DiDC18 (DiD) (middle) and Aquaporin 0 (AQP0)

(bottom). Scale bars are 5 µm. (e) Histograms of normalized densities and the corresponding errors

(propagated from the s.e.m. between three intensity measurements) displayed for Y2R (Nfilopodia = 459,

red, top), DiD (Nfilopodia = 442, blue, middle) and AQP0 (Nfilopodia = 475, green, bottom). The arithmetic

mean of the distributions was quantified by fitting a lognormal function (solid line). (f) Normalized

density as a function of radius quantified in PC12 cell filopodia, HEK293 cell filopodia and HEK293

pulled tethers plotted together for Y2R (red, top), DiD (blue, middle) and AQP0 (green, bottom). The

data from the three different systems collapsed to the same master trend, and could be fitted together to

either a power function (Y2R) or a straight line (DiD and AQP0) (black lines), demonstrating that they

exhibit the same sensitivity to membrane curvature. The error on the quantified values of fold-increase in

density, F, are for the filopodia experiments the s.e.m between two to five biological replicates (see Table

S2) and for the tether experiments the propagated s.d. from the extracted fitting parameters.

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Supplementary Figure 2 Verification that DiD is not sorted by membrane curvature and that Y2R

sorting is not influenced by the membrane dye or by GPCR density. We established that DiD was not

sorted by membrane curvature in vitro by employing a well-characterized technique for preparing

nanometer sized cylindrical membrane tethers, i.e. pulling tethers from micropipette aspirated giant

unilamellar vesicles (GUVs). (a) Micrographs of DiD labeled tethers of two different radii pulled from

GUVs. Scale bars are 5 µm. Using a setup analogous to the one described by Ramesh et al.3 with DiD-

labeled GUVs we simultaneously could assign tether radius, by regulating membrane tension, and extract

the integrated intensity of the pulled tether using the same quantification strategy as for the cell assays.

(b) Integrated intensity as a function of radius for five individual tethers. The intensity is quantified as the

average of three independent measurements (N = 3) on the same tether and the error bars are the s.e.m.

between the three measurements. The integrated intensity scales linearly with tether radius demonstrating

that DiD is not sorted by membrane curvature in vitro. (c) The normalized DiD density as a function of

relative filopodia radius (N = 3, Ncells = 29, Nfilopodia = 273) displayed a linear dependency with the data

points scattered around one, demonstrating that DiD is not sorted by membrane curvature in filopodia.

We verified that Y2R sorting was not influenced by the lipid dye, DiO, by reproducing the curvature-

dependent sorting of Y2R in pulled membrane tethers in which actin is temporarily depleted

(Supplementary Fig. 3f) using the intensity of a cytosolic dye, calcein-AM (calcein), for measuring tether

size (see online Methods). (d) Micrograph of the tether recorded in the calcein channel. The scale bar is 5

µm. (e) Integrated calcein intensity as a function of time upon tether elongation. The data points represent

the average of two tethers (N = 2). The calcein signal had fully equilibrated 10 min upon tether

elongation. (f) The normalized Y2R density as a function of relative tether radius, I(Cal) (se online

Methods), displaying an increased density of Y2R in the more highly curved tethers. The curvature-

dependent sorting of the Y2R was recurrent in calcein-labeled tethers, verifying that Y2R sorting is not

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caused by the lipid dye. Finally, we investigated whether GPCR density influenced the observed sorting.

We plotted the normalized Y2R density as a function of filopodium radius for two different ranges of

Y2R area fraction, ϕ: (g) 0.1% < ϕ < 0.4% and (h) 0.5% < ϕ < 2%. The fold increase in density, F,

between filopodia of 250 nm and 25 nm radius, was quantified by the error-weighted fit of a power

function, f(x) = f(0) + c . x-1 to the raw data (solid lines) and revealed that the sorting was identical within

error between the two density ranges (F = 3.4 ± 0.3 and F = 3.7 ± 0.3 for the low and high density,

respectively). The error on the F values is the propagated s.d. from the fitting parameters.

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Supplementary Figure 3 Verification that Y2R and DiO were fully equilibrated in pulled membrane

tethers on timescales over which tethers were devoid of the actin network. First we ensured that Y2R and

DiO were freely diffusing in the tether using fluorescence recovery after photobleaching (FRAP)

experiments. In both channels we bleached a part of the tether of length L close to the bead and measured

the fluorescence recovery as a function of time. (a) Fluorescence micrographs of Y2R in a pulled

membrane tether before bleaching (left), immediately after bleaching (middle) and 400 s after bleaching

(right). Scale bars are 5 µm. (b) Normalized intensity recovery curve of the Y2R. (c) Normalized

intensity recovery curve of DiO. The quantified half-life and minimum diffusion constants are averages ±

s.d. between three FRAP experiments (N = 3). The minimum diffusion constant of the Y2R (DT,min = 1.2 ⋅

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10-9 ± 0.6 ⋅ 10-9 cm2/s) was in good agreement with the previously measured diffusion of TMPs in red

blood cell tethers (DT,min = 1.5 ⋅ 10-9 ± 0.6 ⋅ 10-9 cm2/s)4. The half-life quantified for DiO in cell

membrane tethers (t1/2 = 22 ± 5 s) was in good agreement with the half-life previously quantified in vitro

for DiOC16 in tethers pulled from GUVs (t1/2 = 21.6 ± 3.8 s)5, suggesting that the behavior of the dye is

similar in cellular membranes and in vitro model membranes. Next, we quantified the equilibration time

of the Y2R and DiO after elongating a tether. For both of them we pulled a tether and imaged every 2 min

until the intensity was equilibrated. (d) Integrated Y2R intensity as a function of time. (e) Integrated DiO

intensity as a function of time. Each data point represents the average between two tethers (N = 2). Both

Y2R and DiO equilibrated within 2 min upon tether elongation. Finally, we verified that the actin network

did not extend significantly into the pulled membrane tethers during the time course of our experiments.

(f) Micrographs of a tether pulled from a HEK293 cell co-expressing fluorescently labeled Utrophin

(GFP-Utrophin), previously established as a reporter of F-Actin6, 7, and SNAP-tag fused Y2R, which was

subsequently labeled with DY-647. Scale bars are 5 µm. The micrographs are depicting the F-Actin

channel (left), Y2R channel (middle) and an overlay of the two channels (right) after 2 min equilibration,

demonstrating that actin is absent in the tether within the timescale of Y2R equilibration. Previous work

with HEK293 cells has revealed a typical time lag of ~100 s between the formation of short membrane

tethers and the polymerization of actin within the tether7. However, here we pulled significantly longer

tethers (20 – 100 µm) and we did not include regions near the cell for our sorting analysis.

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Supplementary Figure 4 Fits of the thermodynamic sorting model displayed together with the raw data.

Plots of normalized GPCR density versus filopodium curvature before (red) and after (black) activation

for (a) Y2R (Ninactive = 2572 and Nactive = 2012), (b) β2AR (Ninactive = 1272 and Nactive = 2240) and (c)

β1AR (Ninactive = 1041 and Nactive = 980). The receptors were activated by addition of saturation

concentration of agonist (100 nM PYY3-368 to the Y2R and 100 µM Isoproterenol9 (ISO) to the β2AR

and β1AR). Error-weighted fits of the thermodynamic sorting model (Supplementary Notes, Eq. S8) to the

raw data are displayed as solid lines. To visualize the trend in the data we included the binned data points

(N = 50 points per bin) in the graphs (dark red or black points for inactive and active receptor,

respectively). The error bars on the binned data are error-weighted s.d. on the x-axis and error-weighted

s.e.m. on the y-axis. (d) Crystal structure of the β2AR in the inactive (orange, PDB: 2RH110) and active

(cyan, PDB: 3P0G11) conformation, displaying the asymmetric shape of the receptor across the

membrane. (e) Schematic illustration of the asymmetric shape depicting how on average ligand binding

has been reported to induce a small contraction in the extracellular and a larger expansion in the

intracellular part of GPCRs. Adapted from Nygaard et al.12. (f) The change in protein rigidity, Δκp

(black), and intrinsic curvature, Δ(1/cp) (red), upon receptor activation as a function of Gibbs free energy

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of binding (ΔG) for the three ligand:GPCR couples, PYY3-36:Y2R8, ISO:β2AR9 and ISO:β1AR9. The

error bars are the propagated experimental errors on the x-axis and the extracted standard error of the

fitting parameters on the y-axis. Straight-line fits to the data (red and black line) are displayed to visualise

the correlation.

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Supplementary Figure 5 UPLC spectra (215 nm) of PYY3-36. The chromatogram demonstrates the

purity to be > 95%.

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Supplementary Table 1 Comparison of the quantified curvature-dependent sorting of Y2R, DiD and

AQP0 in all individual filopodia experiments. Each experiment represents a different transfection

(biological replicate). To compare the curvature-dependent sorting of Y2R and the negative controls we

quantified the fold increase in density, F, upon a tenfold decrease in filopodia radius (250 nm to 25 nm).

The table contains the cell line, the number of technical replicates within each experiment (the number of

filopodia, N(filopodia) and the number of cells, N(cells)) and the fold increase in density, F, for all

individual experiments as well as the average and s.e.m. between the individual experiments (bold) for

the Y2R (top), DiD (middle) and AQP0 (bottom).

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