Role of PtdIns(4,5)P2 and the IL-7Rα cytoplasmic domain in the control of IL-7R dynamic structure. (A) The PtdIns(4,5)P2 signal was analyzed using flow cytometry and confocal microscopy in plasma membrane–permeabilized cells from fractions A and B (for flow cytometry) and in plasma membrane–permeabilized sorted bone marrow B220⁺CD43⁺ cells (for confocal microscopy) from control (WT, red areas and columns) and VAV-CRE (blue areas and columns) mice. Representative MFI histograms of PtdIns(4,5)P2 signal (left). Quantitative PtdIns(4,5)P2 signal after MFI normalization to WT with mean MFI (middle). Results represent individual mice (n = 9 mice per group) and means ± SEM. Representative confocal microscopy pictures (original magnification ×100) of bone marrow B220⁺CD43⁺ sorted cells from WT and VAV-CRE mice. The IL-7Rα (red) antibody was added to cells before plasma membrane–permeabilization, whereas the PtdIns(4,5)P2 (green) antibody was added after membrane permeabilization. White scale bars: 1 μm. (B) The positively charged polybasic 45 amino acid sequence in the juxtamembrane region of the mouse IL-7Rα cytoplasmic domain is presented. Box 1 and 2 (blue) represent the 2 JAK1 binding domains on the IL-7Rα chain. The transmembrane region (green), hydrophobic (red), positively (purple), and negatively (orange) charged amino acids are represented. (C) A tryptophan fluorescence emission spectrum assay was used to detect the binding of the 45 amino acids peptide 265 to 309 presented in Figure 4B to acidic POPG or zwitterionic POPC bicelles (left) as well as to 10% acidic PtdIns(4,5)P2/90% zwitterionic POPC bicelles at different lipid concentrations. One representative of 3 independent experiments is shown. (D) An indirect FRET strategy was used to analyze the proximity between the fluorescent plasma membrane DiO′ probe and p–IL-7Rα^Y449 located at the carboxyterminal end of the IL-7Rα cytoplasmic domain in bone marrow B220⁺CD43⁺ sorted cells from control (WT, red columns) and VAV-CRE (blue columns) mice, before and after the addition of IL-7 (2 ng/mL) for 2 minutes at 37°C. Representative confocal pictures (original magnification ×63) of energy transfer (FRET efficiency) between AF488 (DIO′)– and AF546 (p–IL-7Rα^Y449)–labeled secondary antibody. FRET efficiency was calculated according to the indirect FRET method validated by Guala et al (left).²³ The rainbow scale represents the energy transfer between the 2 fluorophores from 0.5 (blue) to 2 (white). The quantitative energy transfer between the 2 fluorophores-labeled DiO′ and p–IL-7Rα^Y449 (FRET efficiency) is presented (right). Results represent means ± SEM (n = 135-206 cells analyzed per group, from 6 mice). P values were calculated using unpaired nonparametric t test. NS: P > .05; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. NS, nonsignificant; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol; SEM, standard error of the mean; WT, wild type; MFI, mean fluorescence intensity; FRET, fluorescence resonance energy transfer.