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. 2022 Jul 22;11:e76281. doi: 10.7554/eLife.76281

Figure 3. Different ligands, agonists and inverse agonist, impact eGFP-CCR5 mobility differently.

eGFP-CCR5-WT expressing cells were treated or not with a saturating concentration of agonists (CCL4, 200 nM or PSC-RANTES, 20 nM) or inverse agonist (maraviroc, 10 μM) and single particle tracking analysis was performed. (A) Percentage of restricted tracklets after treatment over time (n=tracks from 10, 4, and 3 cells for PSC-RANTES, CCL4, and MVC conditions respectively, at least three independent experiments). (B) Distribution of tracklets motion after 10 min of treatment (mean ± SEM, n=40,564, 15,421, 11,213, 9828 tracks for each condition from 35, 12, 12, and 9 cells, respectively, at least three independent experiments). Unpaired t test on restricted motions only: ns, nonsignificant; **p≤0.01; ***p≤0.001. (C) Distribution of tracklets motion changes along tracks after 10 min of treatment (mean ± SEM, n=48,237, 8954, 16,668, 9828 tracks from 45, 9, 17, and 9 cells for each condition respectively, at least three experiments). Unpaired t test on all restricted motions only: ns, nonsignificant; ****p≤0.0001. (D) (Left) Single particle detection of eGFP-CCR5-WT after 3 min of stimulation with PSC-RANTES (20 nM) from frame 1 of live-imaging movie (one representative image). (Right) Mean of the sum of fluorescence intensity under large immobile spots and small mobile spots after 3–10 min of stimulation (mean ± SEM, n=at least 40 spots from 12 cells, three experiments). (E) Percentage of restricted tracklets after successive stimulation with maraviroc (10 μM, 5 min) and PSC-RANTES (20 nM, 5–12 min; one representative experiment). (F) Distribution of tracklets motions after successive stimulation with maraviroc (10 μM, during 5 min) and PSC-RANTES (20 nM, during 6 min) (mean ± SEM, n=14,467, 3601, 2075 tracks from 14, 2, and 2 cells respectively, one experiment).

Figure 3—source data 1. Source data for Figure 3.

Figure 3.

Figure 3—figure supplement 1. Effect of CCL4 on eGFP-CCR5 mobility.

Figure 3—figure supplement 1.

eGFP-CCR5-WT expressing cells were treated or not with a saturating concentration of CCL4 (200 nM) and single particle tracking analysis was performed. Percentage of restricted tracklets after treatment over time (left) and after 12–16 min of treatment (right) (mean ± SD, n=9951 and 4320 tracks for untreated and CCL4 conditions, from six and three cells, respectively). Unpaired t test: p value 0.0088**.
Figure 3—figure supplement 1—source data 1. Source data for Figure 3—figure supplements 1 and 2.
Figure 3—figure supplement 2. Different ligands, agonists and inverse agonist, impact FLAG-ST-CCR5 mobility differently.

Figure 3—figure supplement 2.

FLAG-ST-CCR5-WT expressing cells were treated or not with a saturating concentration of agonists (CCL4, 100 nM or PSC-RANTES, 100 nM) or inverse agonist (maraviroc, 1 μM) and single particle tracking analysis was performed. Distribution of tracklets motion after 10 min of treatment (mean +/-SEM, n=13,916, 8848, 17,160, 15,478 tracks for each condition from 24, 12, 16, and 22 cells, respectively). Unpaired t test on restricted motions only: **p<0.01, ****p<0.0001.