Separating Actin-Dependent Chemokine Receptor ...€¦ · intensity profile of the monomeric...
Transcript of Separating Actin-Dependent Chemokine Receptor ...€¦ · intensity profile of the monomeric...
Molecular Cell, Volume 70
Supplemental Information
Separating Actin-Dependent Chemokine Receptor
Nanoclustering from Dimerization Indicates a Role
for Clustering in CXCR4 Signaling and Function
Laura Martínez-Muñoz, José Miguel Rodríguez-Frade, Rubén Barroso, Carlos Óscar S.Sorzano, Juan A. Torreño-Pina, César A. Santiago, Carlo Manzo, Pilar Lucas, Eva M.García-Cuesta, Enric Gutierrez, Laura Barrio, Javier Vargas, Graciela Cascio, Yolanda R.Carrasco, Francisco Sánchez-Madrid, María F. García-Parajo, and Mario Mellado
A B
0.000
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Figure S1. Characterization and calculation of reference parameters for particles diffusion and intensity, related to Figure 1.
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PitStop2 FN+CXCL12PitStopNeg FN+CXCL12
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Fig. 2C
Jurkat CD4+ + isotypeJurkat CD4+ + anti−CXCR4Jurkat CD4+ siRNA CXCR4 + anti−CXCR4Jurkat CD4+ siRNA control + anti−CXCR4
Rel
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ll nu
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anti-CXCR4100 101 102 103 104
A
JK-CD4
JKCD4 JKCD4
Figure S2. Effects of endogenous CXCR4 on CXCR4-AcGFP lateral difussion and nanoclustering, related to Figures 1 and 2.
0 2 4 6 80.0
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0.8CD4-CFP/CXCR4-YFPCD4-CFP/5HT2B-YFP
A
0 2 4 60.0
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0.8B CXCR4-CFP/CD4-YFP CHeterodimers FRET50 FRETmax
CD4-CFP/CXCR4-YFP 1.57 ± 0.20 0.56 ± 0.02CD4-CFP/5HT2B-YFP NDND CXCR4-CFP/CD4-YFP 3.71 ± 0.76 0.92 ± 0.11
Ratio YFP/CFPRatio YFP/CFP
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Fig. 2C
Figure S3. CD4 forms heterodimers with CXCR4 and their coexpression reduces preformed CXCR4 nanoclus-ters, related to Figure 3.
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Figure S4. Effects of actin cytoskeleton and tubulin network blockade on CXCR4 dimerization and on the spatio-temporal distribution of CXCR4, related to Figure 4.
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Ratio YFP/Rluc Ratio YFP/Rluc Ratio YFP/Rluc
Gαi-Rluc/CXCR4-YFP Gαi-Rluc/CXCR4mut-YFP Gαi-Rluc/5HT2B-YFP
C D E
FGαi-Rluc/CXCR4-YFP
Gαi-Rluc/CXCR4mut-YFP
BRETmax BRET50
34.94 ± 3.073
36.48 ± 4.079
1.737 ± 0.489
1.285 ± 0.377
Figure S5. Effects of the blockade of actin polymerization and CD4 co-expression in CXCL12-mediated responses, related to Figure 5.
DMSO221TMV
239TMVI
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CCL21
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***
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Streptavidin-PE100 101 102 103
Jurkat CD4+ + 239TMVIJurkat CD4+ + 221TMV
Jurkat CD4+ + DMSO
0 20 40 60 80 100
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ut ConfinedFreeDirected
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anti-CXCR4100 101 102 103 104
Rel
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ell N
umbe
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IsotypeJK-CD4 wtJK-CD4 mut
Figure S6. TMVI region is essential for CXCR4 clustering, related to Figure 6.
Supplemental Figure legends
Figure S1. Characterization and calculation of reference parameters for particles
diffusion and intensity, related to Figure 1. (A) AcGFP monomeric protein
immobilized on glass was used to calculate the smallest detectable diffusion coefficient;
∼95% of immobile AcGFP monomeric protein had a diffusion coefficient ≤0.0015
µm2/s. Thus, we considered this as the threshold for discriminating immobile vs. mobile
trajectories. Median indicated (red). Similar results were obtained on fixed Jurkat CD4+
cells transfected with CXCR4-AcGFP. (B-C) CXCR4-AcGFP confined particles show
restricted mobility within ∼200 nm regions. Jurkat CD4+ cells were electroporated with
CXCR4-AcGFP (JKCD4) and assessed by SPT-TIRF. (B) Three representative 2D
trajectories of CXCR4-AcGFP on steady state cells. Boxes around the trajectories are
340 x 340 nm and show CXCR4wt diffusion in regions of ∼200 nm. Trajectories were
acquired at 10 frames/s. (C) Confinement size (L) in µm is shown; each dot represents
mean L/cell. We evaluated L for CXCR4 confined trajectories in 12 cells. Data shown
as mean ± SEM. (D-F) Detection of monomeric CD86-AcGFP expressed on Jurkat
CD4+ cells by SPT-TIRF. (D) Representative frame showing different CD86-AcGFP
fluorescent spots. The position of each particle was detected automatically (in the
example, 8 particles were detected, red circles). Scale bar, 1 µm. (E) Representative
intensity profile of the monomeric CD86-AcGFP particle, marked as 8 in D). A single
photobleaching step was detected, as expected for single molecules. The inset shows the
fluorescence intensity of particle 8 in frame 10 (1s, i) and in frame 100 (10 s, ii). (F)
Fluorescence intensity histogram from monomeric CD86-AcGFP single particles,
detected by one photobleaching step evaluation (data from 161 trajectories in 25 cells in
3-5 separate experiments). The reference fluorescence intensity value for monomer
(1336 ± 156 a.u.) was obtained from the Gaussian fit (red line) of the histogram. (G)
Representative multi-step photobleaching of CXCR4-AcGFP trajectories. Histograms
of the number of steps detected from 151 trajectories over eight different cells are
shown in Fig. 1J of the main manuscript.
Figure S2. Effects of endogenous CXCR4 on CXCR4-AcGFP lateral diffusion and
nanoclustering, related to Figures 1 and 2. (A) Representative flow cytometry
analysis using the specific anti-CXCR4 antibody (clone 44717) in Jurkat CD4+, Jurkat
CD4+ transfected with specific siRNA CXCR4 3´UTR and Jurkat CD4+ transfected with
non-targeting siRNA control cells (n = 7). (B-D) SPT-TIRF analysis of CXCR4-AcGFP
in Jurkat CD4+ transfected with siRNA CXCR4 3´UTR and 24 h post-transfection, cells
were electroporated with CXCR4-AcGFP (JK-CD4 cells), on fibronectin (FN)- or
fibronectin+CXCL12 (FN+CXCL12)-coated coverslips. (B) Diffusion coefficient (D1-4)
of single CXCR4-AcGFP particles in steady-state (FN) or CXCL12-stimulated
(CXCL12) JK-CD4 cells, with median indicated (red bar) (≥200 trajectories (n = 3; not
significant, p >0.05, two-tailed Mann-Whitney nonparametric t test). (C) Percentage of
the number of CXCR4 receptors/particle as determined from the trajectories intensities.
397 trajectories from 23 unstimulated cells (FN, gray) and 497 trajectories from 24
CXCL12-stimulated cells (FN+CXCL12, blue), n = 3). Inset, Fig. 2C of the main
manuscript is reproduced here to facilitate comparison with data for JKCD4 cells. (D)
D1-4 of individual CXCR4-AcGFP trajectories exhibiting confinement (left) or with
Brownian motion (right) associated with particle size for monomer/dimer or
nanoclusters (≥3 receptors/particle) in unstimulated (FN, black boxes) or CXCL12-
stimulated (FN+CXCL12, blue boxes) cells. One-way ANOVA followed by Tukey’s
multiple comparison test were used (not significant, p >0.05). (E-F) CXCL12-mediated
CXCR4 nanoclusters do not correspond to internalization vesicles, related to Figure 2.
(E) Jurkat CD4+ cells expressing CXCR4-AcGFP were untreated, pretreated with
PitStop2 (30 µM, red box), an inhibitor of clathrin-coated pits, or with PitStopNeg, a
chemical compound used as a PitStop2 control (30 µM, control, hatched blue box) (15
min, 37ºC). Cells were placed on FN or FN+CXCL12 (100 nM) coverslips (5 min,
37ºC, 5% CO2), fixed and evaluated by SPT. CXCL12-mediated nanoclustering of
CXCR4 particles, defined as increase of particle intensity, was similar in all
experimental conditions tested. One-way ANOVA followed by Tukey’s multiple
comparison test were used to determine significant differences between means (not
significant, p >0.05, ** p <0.001). (F) CXCL12-mediated CXCR4 nanoclusters size
(particle intensity, a.u.) was evaluated by SPT in CXCR4-AcGFP-expressing Jurkat
CD4+ cells pre-treated with brefeldin A, an inhibitor of intracellular protein transport
(10 µg/ml; 30 min, 37ºC, 5% CO2). *** p <0.0001; two-tailed Mann-Whitney
nonparametric t test.
Figure S3. CD4 forms heterodimers with CXCR4 and their coexpression reduces
preformed CXCR4 nanoclusters, related to Figure 3. (A-D) CD4/CXCR4
heterodimers at the cell membrane in live cells. (A-B) FRET saturation curves were
generated in 293T cells transiently cotransfected with a constant amount of CD4-CFP
(A) or CXCR4-CFP (B) and increasing amounts of CXCR4-YFP or CD4-YFP,
respectively. As negative control, we used 5HT2B-YFP. (C) FRET50 and FRETmax values
were calculated by nonlinear regression applied to a single binding-site model. The data
for the negative control fitted a linear regression equation (random interactions). (n ≥ 3)
(ND, not determined). (D) FRET analysis by acceptor photobleaching of CD4/CXCR4
and CXCR4/CD4 heterodimers. As negative controls we used CD4/5HT2B and
CXCR4/mGluR1α. Representative images of CFP and YFP staining before (CFP-Pre
and YFP-Pre) and after (CFP-Post and YFP-Post) photobleaching. Right, zoom of
FRET at the photobleached areas using a false-color scale. FRET efficiency is shown as
mean ± SE. Only areas with a ∼2:1 YFP/CFP ratio for CD4/CXCR4 and CD4/5HT2B,
and ∼3:1 YFP/CFP ratio for CXCR4/CD4 and CXCR4/mGluR1α were used for
bleaching analysis (outline). (E-G) CD4 reduces preformed CXCR4 nanoclusters on the
T cell membrane. (E) Jurkat CD4+ and Jurkat cells were electroporated with CXCR4-
AcGFP (JKCD4 and JK, respectively). Intensity distributions of CXCR4-AcGFP
particles as obtained from SPT trajectories on unstimulated cells. 595 trajectories in 23
JKCD4 cells, and 937 trajectories in 20 JK cells (n = 3-5 experiments); mean ± SEM
(red bar). *** p <0.0001; two-tailed Mann-Whitney nonparametric t test. (F) Percentage
of receptors/particle from unstimulated (FN, gray; 937 trajectories in 21 cells) and
CXCL12-stimulated JK cells (FN+CXCL12, black; 834 trajectories in 18 cells) (n = 3).
Inset, Fig. 2C of the main manuscript is reproduced here to facilitate comparison with
data for JKCD4 cells. (G) Jurkat cells were electroporated with siRNA specific for
CXCR4 or non-targeting control siRNA, and after 24 h, cells were electroporated with
CXCR4-AcGFP (JK- and JKc, respectively). Percentage of receptors/particle as obtained
from SPT trajectory intensities on unstimulated cells. 592 trajectories in 21 JK- cells and
397 trayectories in 20 JKc cells. (H-J) SPT of CXCR4-AcGFP in Jurkat cells with
endogenous CXCR4 downregulated with siCXCR4 (JK-), on FN- or FN+CXCL12-
coated coverslips. (H) D1-4 of single CXCR4-AcGFP particles in steady state (FN) or
CXCL12-stimulated (CXCL12) with median indicated in red (data from ≥343
trajectories in 24 cells, n = 3); not significant, p >0.05, two-tailed Mann-Whitney
nonparametric t test. (I-J) D1-4 of CXCR4-AcGFP single particles in confined regions
(I) or with Brownian motion (J) associated with particle size for monomer/dimer or
nanoclusters (≥3 receptors/particle) in unstimulated (FN, black boxes) or CXCL12-
stimulated cells (FN+CXCL12, hatched black boxes). One-way ANOVA followed by
Tukey’s multiple comparison test were used (not significant, p >0.05, ** p <0.01).
Figure S4. Effects of actin cytoskeleton and tubulin network blockade on CXCR4
dimerization and on the spatiotemporal distribution of CXCR4, related to Figure
4. (A) LatA treatment blocks CXCL12-promoted conformational change on CXCR4
homodimers. FRET saturation curves were generated in 293T cells expressing a
constant amount of CXCR4-CFP and increasing amounts of CXCR4-YFP. Cells were
pre-treated with LatA (1 µM, 15 min, 37ºC; red) and then stimulated with CXCL12
(100 nM, 30 min, 37ºC). Control experiments were done using ethanol (diluent for
LatA) (control, blue). Data were analyzed using a nonlinear regression for a single
binding site model. One representative experiment of seven is shown. (B-E) SPT was
used to assess the effect of nocodazole on CXCR4 dynamics and clustering. JKCD4
untreated or treated with the nocodazole diluent (DMSO) as control (control), or
nocodazole (1 µg/ml, 15 min, 37ºC). (B) Intensity distribution of CXCR4-AcGFP
particles (595 trajectories on steady-state untreated JKCD4 cells, 520 trajectories on
DMSO-treated JKCD4 cells; 641 trajectories on nocodazole-treated JKCD4 cells; 673
trajectories on CXCL12-stimulated untreated JKCD4 cells; 786 trajectories on DMSO-
treated JKCD4 cells and 842 trajectories on nocodazole-treated JKCD4 cells. 10-22
cells per condition; n ≥ 3); black bars indicate mean ± SEM. (not significant, p >0.05, *
p <0.05, ** p <0.01, *** p <0.0001; one-way ANOVA followed by Tukey’s multiple
comparison test). (C) Percentage of receptors/particle from unstimulated (FN, gray) or
CXCL12-stimulated (FN+CXCL12) cells. (D) D1-4 distribution of CXCR4-AcGFP
single particles in confined regions (left) or with Brownian motion (right) associated
with particle size for monomer/dimer or nanoclusters (≥3 receptors/particle) in
unstimulated (none) or CXCL12-stimulated (CXCL12) cells. One-way ANOVA
followed by Tukey’s multiple comparison test; not significant, p >0.05). (E) Effect of
nocodazole on the type of trajectory motion. Percentage of CXCR4-AcGFP single
particle trajectories classified as confined, Brownian or directed motion using the
moment scaling spectrum (MSS) analysis on unstimulated or CXCL12-stimulated cells
(236 trajectories on steady state control; 309 trajectories on steady state nocodazole;
177 trajectories on CXCL12-stimulated control and 179 trajectories on CXCL12-
stimulated nocodazole; in > 10 JKCD4 cells per condition, n = 3, not significant, p
>0.05, ** p <0.01; Chi-square test). Notice that CXCL12 stimulation leads to a slight
increase on the percentage of trajectories exhibiting directed transport, which are fully
abrogated by nocodazole. This indicates that although some vesicle trafficking might be
present, it constitutes only a very small fraction of the total trajectories analyzed.
Figure S5. Effects of the blockade of actin polymerization and CD4 co-expression
in CXCL12-mediated responses, related to Figure 5. (A) CD4 coexpression does not
modify cell surface CXCR4 levels. Jurkat cells were electroporated with CD4-YFP or
YFP vector (YFP). After 24 h, CXCR4 membrane expression was determined by flow
cytometry. (B) Purified human naïve CD4+ T lymphocytes were pretreated LatA (1 µm,
15 min, 37ºC) or with LatA diluent (ethanol, control). Ca2+ mobilization assays were
performed in response to CXCL12 (50 nM, 20 nM and 10 nM). Quantification of Ca2+
flux response (%) is shown. (C-F) BRET titration curves using 293T cells transiently
contransfected with a constant amount of Gαi-Rluc (0.5 µg) and increasing amounts of
CXCR4-YFP (0.15-3.5 µg, C), or CXCR4mut-YFP (0.15-3.5 µg, D), or 5HT2B-YFP as
a negative control (0.15-8.0 µg, E). (F) BRETmax and BRET50 values were calculated by
using a nonlinear regression equation for a single binding-site model and expressed as
mean ± SEM (n=4).
Figure S6. TMVI region is essential for CXCR4 clustering, related to Figure 6. (A)
239TMVI peptide specifically blocks CXCL12-mediated migration in KG1a cells. KG1a
cells were electroporated with CXCR4-YFP, CXCR5-YFP, or CCR7-YFP. Migration of
cells expressing CXCR4-YFP, CXCR5-YFP or CCR7-YFP in response to 20 nM
CXCL12, 300 nM CXCL13, or 20 nM CCL21, respectively. Data as mean ± SD (n=3,
*** p ≤ 0.0001; two-tailed Mann-Whitney nonparametric t test). (B) 221TMV and
239TMVI peptides are incorporated into the T cell membrane. Jurkat CD4+ cells were
treated with DMSO (peptides diluent, control), 221TMV- or 239TMVI-biotinylated
peptides (50 µg/ml; 30 min 37ºC, 5% CO2), followed by streptavidin-PE, and membrane
peptide levels analyzed by flow cytometry. (C) Representative flow cytometry analysis
of JK-CD4 transiently transfected with CXCR4wt-AcGFP or with CXCR4mut-AcGFP
(JK-CD4 wt and JK-CD4 mut cells, respectively). (D) CXCL12-induced internalization
of JK-CD4 wt and JK-CD4 mut cells. (E) Jurkat CD4+ cells were electroporated with
CXCR4wt-AcGFP (JKCD4wt) or CXCR4mut-AcGFP (JKCD4mut). SPT-TIRF was
used to analyze the percentage of CXCR4 single particle trajectories classified as
confined, Brownian or directed motion by MSS analysis, on unstimulated or CXCL12-
stimulated cells (steady-state JKCD4wt 192 trajectories from 26 cells; steady-state
JKCD4mut 113 from 14 cells; CXCL12-stimulated JKCD4wt 254 from 24 cells;
CXCL12-stimulated JKCD4mut 227 trajectories from 22 cells; n = 3-5) (not significant,
p >0.05; Chi-square test).
DIMERS FRET50 FRETmax
CXCR4wt-CFP/CXCR4wt-YFP 0.524 ± 0.019 0.739 ± 0.008CXCR4mut-CFP/CXCR4mut-YFPCXCR4wt-CFP/CXCR4mut-YFP 0.955 ± 0.096 0.724 ± 0.031
0.570 ± 0.045 0.672 ± 0.021
Table S1,related to Figure 6F. FRET50 and FRETmax values were calculated by nonlinear regression applied to a single binding site model (n>3).
CXCR4 CD4 siRNA CXCR4-AcGFPJurkat
JK
Jurkat CD4+
JKCD4
JK-
JK-CD4JKCCD4
+
+
+++ +
+
++
+
+
+
+
+
none
none
none
none
control
CXCR4
CXCR4
--
-
-- -
-
Table S2, related to STAR methods; experimental model and subject details, cell lines and primary cells.
Notation of the Jurkat cells used in the study and protein expression or lack of expression (+ or -, respectively) and the siRNA employed. When required, cells were transfected with wt or mutant CXCR4-AcGFP.