IL-4R dynamics 1

Dynamics and interaction of Interleukin-4 receptor subunits in

living cells

Hetvi Gandhi, Remigiusz Worch, Martin Hintersteiner, Kristina Kurgonaite, Petra Schwille,

Christian Bökel & Thomas Weidemann

Supporting Material

Figures S1-S8

Tables S1-S3

IL-4R dynamics 2

FIGURE S1 HEK293T as a model for IL-4R mediated JAK/STAT signaling

FIGURE S1 Endogenous type 2 IL-4R signaling in HEK293T cells transiently transfected

with human STAT6 and assayed by phospho-STAT6 immunoblotting. At identical ligand

concentrations, IL-4 causes stronger activation than IL-13.

IL-4R dynamics 3

FIGURE S2 trisNTA coupled fluorescent dyes bind reversibly to hexahistidine tags

FIGURE S2 (A) FRET experiment: Using non-fluorescent trisNTA-QSY7 as an acceptor,

eGFP-H6 donor fluorescence was reversibly quenched. (B) Kinetics of the same experimental

procedure. Binding is completed in less than 5 minutes. All measurements were done at room

temperature in PBS using a Fluorolog τ-3 (Jobin-Yvon).

IL-4R dynamics 4

FIGURE S3 Co-expression of full length IL-4Rα chain with the type 2 receptor IL-

13Rα1

FIGURE S3 (A) Transiently expressed IL-13Rα1-eGFP shows efficient transport to the

plasma membrane with a mild accumulation in perinuclear membrane compartments at higher

expression levels. Cell morphology is indistinguishable from non-transfected cells. The

endogenous IL-4Rα chains are expressed at such a low density that cannot be visualized by

labeled IL-4 ligand (B). Ectopically expressed IL-4Rα chain can be detected by labeled IL-4-

A647 at the cell surface. However, a large fraction of cells transform into a rounded shape

with strong ER retention of the second receptor chain IL-13Rα1-eGFP. The morphology of

those cells is not suitable for single-cell FCCS analysis.

IL-4R dynamics 5

FIGURE S4 FCCS quantification of IL-4R subunit interactions in the plasma

membrane

FIGURE S4 (A) Histograms of the average cross-correlation ratio (CCav) from individual

cells were fitted by Gaussian distributions, and (B) their overlay for the indicated experiments

(color code). A shift towards higher CCav values indicates stronger binding. (C) Correlation

between the Kd values derived for single cells using either the eGFP (index G) or the A647

(index R) channel for normalization of the cross-correlation amplitude. The slope should be

close to one. (for details see the main article's appendix: "Kd determination from FCCS data").

IL-4R dynamics 6

FIGURE S5 FCCS analysis of type 2 IL-4R employing a His-tagged full length IL-4Rα

chain.

FIGURE S5 (A) Example for transformed, rounded HEK293T cells showing insufficient co-

expression of the two receptor subunits at the plasma membrane. (B) Exceptional case of

stretched cells showing good surface expression of both receptor subunits which were

amenable for FCCS measurements. Note their co-localization within cortical endosomes

(CEs, inset, arrow heads). (C) Cross-correlation amplitudes show a significant, ligand induced

increase indicating receptor subunit dimerization of the type 2 IL-4R complex under

overexpression conditions. Maximum cross-correlation (CCmax) reflects the number of dimers

with respect to the less abundant receptor subunit expressed in that particular cell (each data

points represents an average of 1-4 15 second FCCS runs). Cross-correlation levels were

normalized with a positive control (PC, H6-IL4Ram266-eGFP, dashed red line) which was

measured for each Labtek slide to combine independent experiments (n = 5). Box-and-

whisker plots indicate 2nd and 3rd quartile (box), mean (square), median (horizontal line),

and 1.5x interquartile range (whiskers). *** indicates p < 0.01 (ANOVA).

IL-4R dynamics 7

FIGURE S6 IL-4R subunits accumulate in cortical endosomes

FIGURE S6 HEK293T cells were transiently transfected with a non-tagged IL-2Rγ and

JAK3-eGFP to label CEs. Confocal images show the bottom membrane of a cell expressing

rather low amounts of JAK3-eGFP, therefore the background from unbound cytoplasmic

JAK3-eGFP is low. Dots of high intensity represent IL-2Rγ/JAK3-eGFP complexes enriched

in CEs. Comparing the fluorescence pattern at 0 min and 31.1 min reveals that their relative

positions are largely maintained. Smaller endosomes can show jump distances of a few µm

per minute (open arrow head), whereas larger endosomes move on a submicrometer length

scale (filled arrow head). Imaging was done at 22 °C in air buffer. The positions of the arrow

heads are fixed relative to the frames.

IL-4R dynamics 8

FIGURE S7 Cortical endosomes decorate with markers for early sorting and recycling

FIGURE S7 (A) CEs do not carry markers for late degradatory compartments. (B) IL-13Rα1

co-localizes with CEs marked by JAK3-tagRFP and H6-IL-2Rγ labeled with trisNTA-A647

(arrows) suggesting that type 1 and type 2 receptor subunits engage the same endosomal

trafficking route.

IL-4R dynamics 9

FIGURE S8 Constitutive internalization concentrates ligands and receptors within

cortical endosomes

FIGURE S8 (A) The truncated IL-4Ram266 chain promotes similar redistribution kinetics of

fluorescently labeled IL-4-A647 into CEs as the full length receptors. (B) Examples showing

cells before and after loading of trisNTA-A647. The amount of trisNTA-A647 (red) loaded

per CE (insets) was used for quantifying the kinetics. (C) Schematic workflow: Cells were

stained at 4°C, excess trisNTA-A647 was removed from the bulk, and endocytosis was

released by transfer to 37°C. The amount of receptor mediated trisNTA-A647 in CEs was

determined for different time points after the washing step by APD imaging.

IL-4R dynamics 10

TABLE S1: Receptor mobility determined by FCCS. Mean values for the indicated color

channels and constructs measured in HEK293T stained with trisNTA-A647: diffusion

coefficient of the fast component (fraction in %) in the A647 channel accounting for unbound

trisNTA-A647 in bulk solution (DR1), diffusion coefficient of the slow component in the

A647 channel reflecting lateral diffusion of trisNTA-A647 bound to H6-IL-4Ram266 in the

membrane (DR2), a short correlation time (fraction in %) measured in the eGFP channel (τ1),

diffusion coefficient of the slow component in the eGFP channel reflecting lateral receptor

diffusion of eGFP-tagged receptors in the membrane (DG2), diffusion coefficient of the cross-

correlation time (DX), and the size of sample (n). "n.d." indicates not determined. Mean values

in the grey shaded column correspond to Figure 3 D.

Experiment

DR1 ± SD

A647

[µm-2/s]

DR2 ± SD

A647

[µm-2/s]

τ 1 ± SD

eGFP

[ms]

DG2 ± SD

eGFP

[µm-2/s]

DX ± SD

[µm-2/s]

n

cells

H6- IL-4Rαm266-

eGFP

+trisNTA-A647

380 ± 220

(23 ± 7 %) 0.23 ± 0.07

0.70 ± 0.34

(24 ± 5 %) 0.24 ± 0.09 0.19 ± 0.08 55

Fas-eGFP 340 ± 210

(25 ± 6 %) 0.25 ± 0.06

0.76 ± 0.27

(29 ± 5 %) 0.42 ± 0.12 n.d. 8

Fas-eGFP + IL-4 590 ± 250

(31 ± 6 %) 0.31 ± 0.06

0.54 ± 0.16

(29 ± 5 %) 0.51 ± 0.13 n.d. 8

IL-2Rγ-eGFP / JAK3 450 ± 320

(25 ± 6 %) 0.25 ± 0.06

0.44 ± 0.15

(34 ± 10 %) 0.16 ± 0.05 n.d. 13

IL-2Rγ-eGFP / JAK3

+IL-4

410 ± 290

(23 ± 8 %) 0.23 ± 0.08

0.46 ± 0.24

(36 ± 6 %) 0.17 ± 0.08 0.08 ± 0.08 30

IL-13Rα1-eGFP 420 ± 270

(26 ± 10 %) 0.26 ± 0.10

0.62 ± 0.32

(32 ± 7 %) 0.38 ± 0.17 n.d. 25

IL-13Rα1-eGFP

+ IL-4

320 ± 170

(24 ± 6 %) 0.24 ± 0.06

0.73 ± 0.26

(32 ± 7 %) 0.27 ± 0.09 0.17 ± 0.11 30

IL-13Rα1-eGFP

+ IL-13

320 ± 150

(29 ± 7 %) 0.29 ± 0.07

0.74 ± 0.34

(31 ± 4 %) 0.29 ± 0.10 0.28 ± 0.23 39

IL-4R dynamics 11

TABLE S2: Receptor densities and binding data determined by FCCS. Numerical values

for the lateral affinities as mentioned in the text (Kd,2D), normalized average cross-correlation

ratio (CCav,n), surface density in the A647 channel specifying the amount of truncated, His-

tagged H6-IL-4Rαm266 chain per µm2 (Nr), surface density in the eGFP channel specifying

the second, eGFP-tagged receptor subunit (Ng), ratio of receptor densities averaged across the

measured cell population (Nr/Ng), and the sample size (n). Mean values in the grey shaded

columns correspond to Figure 3 E and 3 F. Note comparable expression levels of all

constructs.

Experiment Kd,2D ± SEM

[µm-2]

CCav,n ± SD

[%]

Nr ± SD

[µm-2]

Ng ± SD

[µm-2] Nr/ Ng

n

(cells)

H6- IL-4Rαm266-

eGFP

+trisNTA-A647

n.d. 100 ± 25 200 ± 100 220 ± 130 1.0 62

Fas-eGFP n.d. 4 ± 2 560 ± 500 390 ± 290 1.4 8

Fas-eGFP + IL-4 n.d. 6 ± 3 370 ± 180 210 ± 90 1.8 10

IL-2Rγ-eGFP / JAK3 n.d. 13 ± 10 270 ± 140 150 ± 100 2.1 16

IL-2Rγ-eGFP / JAK3

+IL-4 1000 ± 150 19 ± 11 260 ± 170 140 ± 70 1.8 32

IL-13Rα1-eGFP n.d. 11 ± 10 340 ± 180 170 ± 150 2.5 20

IL-13Rα1-eGFP

+ IL-4 180 ± 40 51 ± 11 420 ± 290 250 ± 180 1.9 25

IL-13Rα1-eGFP

+ IL-13 480 ± 90 29 ± 11 240 ± 180 220 ± 140 1.1 38

IL-4R dynamics 12

TABLE S3: Co-localization of JAK3/IL-2Rγ positive CEs with endocytosis markers.

Values of the grey shaded column were plotted in Figure 5 B.

Marker [%]

co-localization ± SD

n

(endosomes)

n

(cells)

mRFP-Rab5 96.1 1.8 955 13

EEA1 83.8 7.1 1640 18

mRCherry-Rab11 91.3 1.9 442 10

mRFP-Rab7 15.4 2.4 991 15

Lamp1 12.4 8.3 1209 14

Lysotracker 17.1 9.5 1078 14