HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane
- Chemokine Signaling group, Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Spain
- X-ray Crystallography Unit, Department of Macromolecules Structure, Centro Nacional de Biotecnología/CSIC, Spain
- Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología/CSIC, Spain
- IrsiCaixa, Spain
- 12 de Octubre Health Research Institute (imas12), Spain
- Department of Public Health School of Medicine, School of Medicine Universidad Complutense de Madrid, Spain
- CIBERINFEC, Spain
- Germans Trias i Pujol Research Institute (IGTP), Spain
- University of Vic-Central University of Catalonia, Spain
- Catalan Institution for Research and Advanced Studies (ICREA), Spain
Figures
Figure 1 with 8 supplements
X4-gp120 modulates CXCR4 dynamics and nanoclustering.
Single-particle tracking analysis of JKCD4+X4- cells transiently transfected with CXCR4-AcGFP on fibronectin (FN)-, FN +CXCL12-, or FN +X4-gp120-coated coverslips (828 particles in 96 cells on FN; 2997 in 95 cells on FN +CXCL12 and 1547 in 91 cells on FN +X4-gp120) n=3. (A) Percentage of mobile and immobile CXCR4-AcGFP particles at the membrane of cells treated as indicated. (B) Diffusion coefficients (D1–4) of mobile particles at the membrane of cells treated as indicated with the median value of each experiment (black circles) and the median of all trajectories (dotted black lines; ****p≤0.0001). (C) Frequency of CXCR4-AcGFP particles containing monomers and dimers (≤2) or nanoclusters (≥3), mean ± SD calculated from mean spot intensity (MSI) values of each particle as compared with the value of monomeric CD86-AcGFP (980±86 a.u., **p≤0.01, ***p≤0.001). (D) Intensity distribution of individual CXCR4-AcGFP trajectories on unstimulated and CXCL12 or X4-gp120-stimulated cells. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=3; ****p≤0.0001). Statistical significance was determined by two-way ANOVA in panels A and C and by non-parametric Kruskal-Wallis tests followed by Dunn’s test for panels B and D.
Figure 1—figure supplement 1
Generation of functional recombinant X4-gp120.
(A) Representative Coomassie blue-stained polyacrylamide gel of 5 μg of purified recombinant X4-gp120 and commercial gp120 (gp120), used as a control (n=3). (B) Representative western blot of different amounts (μg indicated) of recombinant X4-gp120 and commercial gp120 (5 μg), used as control, analyzed with an anti-gp120 mAb (n=3). (C) Flow cytometry of X4-gp120 binding to JKCD4+CXCR4+ (JKCD4+X4+) and JKCD4+CXCR4- (JKCD4+X4-) cells analyzed with an anti-Histidine mAb. Mean MFI values (and SD) are shown (n=3; n.s.=not significant). (D) Flow cytometry of X4-gp120 binding on Daudi cells (CD4-X4+), pretreated or not with soluble recombinant human CD4 (sCD4), analyzed with an anti-His mAb. As control, the binding of sCD4 in the absence of X4-gp120 is also shown. Mean MFI values (and SD) are shown (n=3; **p≤0.01). Statistical significance was determined by paired t-test for panel C, and by two-way ANOVA in panel B.
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Figure 1—figure supplement 1—source data 1
Original files for Coomassie blue-stained polyacrylamide gel for Figure 1—figure supplement 1A.
- https://cdn.elifesciences.org/articles/110354/elife-110354-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
PDF file containing original Coomassie blue-stained polyacrylamide gel for Figure 1—figure supplement 1A.
Generation of functional recombinant X4-gp120. Coomassie blue-stained polyacrylamide gel of 5 μg of purified recombinant X4-gp120 and commercial gp120 (gp120), used as a control. Original files for Coomassie blue-stained polyacrylamide gel displayed in Figure 1—figure supplement 1—source data 1.
- https://cdn.elifesciences.org/articles/110354/elife-110354-fig1-figsupp1-data2-v1.zip
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Figure 1—figure supplement 1—source data 3
Original files for western blot analysis for Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/110354/elife-110354-fig1-figsupp1-data3-v1.zip
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Figure 1—figure supplement 1—source data 4
PDF file containing original western blot for Figure 1—figure supplement 1B.
Original membrane corresponding to Figure 1—figure supplement 1, panel B. Western blot of different amounts (μg indicated) of different batches of recombinant X4-gp120 and commercial gp120 (5 μg), used as control, analyzed with an anti-gp120 mAb. Batch#3 was selected for further assays. Original files for western blot analysis displayed in Figure 1—figure supplement 1—source data 3.
- https://cdn.elifesciences.org/articles/110354/elife-110354-fig1-figsupp1-data4-v1.zip
Figure 1—figure supplement 2
X4-gp120 activates CD4- and CXCR4-related signaling pathways in target cells.
(A) Western blot of lysed Jurkat cells activated with CXCL12 (50 nM) or X4-gp120 at the indicated time points and analyzed with anti-pAkt and pERK1/2 antibodies. A representative membrane is shown (n=3). (B) Western blot of primary CD4+ T blast lysates activated with X4-gp120 at the indicated time points and analyzed using anti-pLck and -pERK1/2 antibodies. A representative membrane is shown (n=3). In both cases, membranes were reblotted with an anti-tubulin mAb as a loading control. Molecular markers (kDa) are also shown.
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Figure 1—figure supplement 2—source data 1
Original files for western blot analysis for Figure 1—figure supplement 2A.
- https://cdn.elifesciences.org/articles/110354/elife-110354-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
PDF file containing original western blot for Figure 1—figure supplement 2A.
Original membrane corresponding to Figure 1—figure supplement 2, panel A. Western blot of lysed Jurkat cells activated with CXCL12 (50 nM) or X4-gp120 Batch#3 (0.05; 0.1; 0.3 μg/ml) at the indicated time points and analyzed with anti-pAkt and pERK1/2 antibodies. Membranes were reblotted with an anti-tubulin mAb as a loading control. Molecular markers (kDa) are also shown. Figure 1—figure supplement 2, panel A shows western blot of lysed Jurkat cells activated with CXCL12 (50 nM) and X4-gp120 Batch#3 0.3 μg/ml. Original files for western blot analysis displayed in Figure 1—figure supplement 2—source data 1.
- https://cdn.elifesciences.org/articles/110354/elife-110354-fig1-figsupp2-data2-v1.zip
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Figure 1—figure supplement 2—source data 3
Original files for western blot analysis for Figure 1—figure supplement 2B.
- https://cdn.elifesciences.org/articles/110354/elife-110354-fig1-figsupp2-data3-v1.zip
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Figure 1—figure supplement 2—source data 4
PDF file containing original western blot for Figure 1—figure supplement 2B.
Original membrane corresponding to Figure 1—figure supplement 2, panel B. Western blot of primary CD4+ T blast lysates activated with CXCL12 (50 nM) and X4-gp120 Batch#3 (0.3 μg/ml) at the indicated time points and analyzed using anti-pLck and pERK1/2 antibodies. Membranes were reblotted with an anti-tubulin mAb as a loading control. Molecular markers (kDa) are also shown. Figure 1—figure supplement 2, panel B shows western blot of lysed Jurkat cells activated with X4-gp120 Batch#3 0.3 μg/ml. Original files for western blot analysis displayed in Figure 1—figure supplement 2—source data 3.
- https://cdn.elifesciences.org/articles/110354/elife-110354-fig1-figsupp2-data4-v1.zip
Figure 1—figure supplement 3
X4-gp120 induces cell polarization.
(A) CD4+T cell blasts were perfused in flow chambers coated with ICAM-1-containing lipid bilayers, alone or with CXCL12- or X4-gp120. Polarized cells were determined from differential interference contrast (DIC) images using ImageJ2 and Imaris 7.0 and expressed as a percentage of the total number of cells per image (mean ± SD with the mean value of each experiment (black circles) indicated; n=6; n.s. not significant; *p≤0.05; ***p≤0.001). (B) Cells were perfused as in (A) and the percentage of adhered cells was determined using the interference reflection microscopy (IRM) signal. The frequency of adhesion (IRM+ cells) per image field was estimated as [n° of cells showing IRM contact/total n° of cells (estimated by DIC)]×100. More than 150 cells were analyzed of each condition. Data are shown as percentage of adhered cells. (Mean ± SD with the mean value of each experiment (black circles) indicated; n=6; *p≤0.05; ***p≤0.001). (C) Cells in B were analyzed for cell migration. We calculated the frequency of migration (cells showing an IRM+ contact and moving over time). Data are presented as percentage of migrating cells (mean ± SD with the mean value of each experiment (black circles) indicated; n=6; n.s.=not significant; **p≤0.01; *** p≤0.001). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test for all panels.
Figure 1—figure supplement 4
Classification of mobile receptor trajectories.
Percentage of mobile receptor trajectories classified as confined, free, or directed for JK CD4+X4- cells transiently transfected with CXCR4-AcGFP. Statistical significance was determined by two-way ANOVA test for all panels.
Figure 1—figure supplement 5
Characterization and calculation of reference parameters for particles intensity, related to TIRF images.
(A) Fluorescence intensity histogram from monomeric CD86-AcGFP single particles, detected by one photobleaching step evaluation (data from 118 trajectories in 21 cells in 3 separate experiments). The reference fluorescence intensity value for monomer (980±86 a.u.) was obtained from the Gaussian fit (red line) of the histogram. Representative graphics of one-step (B), two-step (C), and three-step photobleaching (D) of the monomeric CXCR4-AcGFP particle.
Figure 1—video 1
Representative video of CXCR4-AcGFP on live JKCD4+X4- cells treated with DMSO and captured by SPT- TIRF, showing the diffusion of CXCR4 particles (monomers, dimers, and nanoclusters) at steady state (FN).
The video was acquired and displayed at 10.2 frames/s.
Figure 1—video 2
Representative video of CXCR4-AcGFP on live JKCD4+X4- cells treated with DMSO and captured by SPT-TIRF, showing the diffusion of CXCR4 particles (monomers, dimers, and nanoclusters) in response to CXCL12.
The video was acquired and displayed at 10.2 frames/s.
Figure 1—video 3
Representative video of CXCR4-AcGFP on live JKCD4+X4- cells treated with DMSO and captured by SPT-TIRF, showing the diffusion of CXCR4 particles (monomers, dimers, and nanoclusters) in response to X4-gp120.
The video was acquired and displayed at 10.2 frames/s.
Figure 2 with 1 supplement
gp120-VLPs are mature particles that express a low number of Env trimers.
(A) Representative images of clarified VLPs visualized by STED microscopy. Upper panels show images of the indicated VLPs stained for Gag p24 (blue) and gp120 (red). Lower panels show ×10 magnification of equivalent images. White arrows indicate mature VLPs (p24 condensation). (B) Percentage of mature VLPs, analyzed from the images in (A) using TrackAnalyzer in ImageJ, based on p24 intensity and aggregation level (mean ± SD; n=2; ****p≤0.0001; the significance indicated on immature VLPs bar shows the difference with all other conditions). (C) Percentage of VLPs expressing gp120 on their surface, as analyzed in ImageJ (mean ± SD; n=2; ***p≤0.001). (D) Distribution of gp120 mean fluorescence intensity. Each spot corresponds to the mean fluorescence intensity for each analyzed VLP in a.u. The black line represents the mean of all values (****p≤0.0001). (E) Frequency of gp120 intensity/particle. Statistical significance was determined by one-way-ANOVA followed by Tukey’s multiple comparisons test in panels B and C and by Mann-Whitney analysis for panel D.
Figure 2—figure supplement 1
Characterization of gp120-VLPs and LVPs.
(A) Representative transmission electron micrographs of gp120-VLP particles. Original scale bar 100 nm. (B) Culture media of transfected HEK-293T cells (cm) with different constructs, as indicated, and the corresponding clarified samples (cs) containing the VLPs or the LVPs generated, were analyzed by western blot with anti-p24, and -gp120 mAbs. The anti-p24 mAb also recognizes Pr55Gag, a precursor of p24 presents in immature particles. Molecular weight markers are indicated (kDa). (C) Flow cytometry analysis of VLPs (Env(-) and expressing x4-gp120) bound to latex beads in the presence of soluble human CD4, using an anti-Histidine mAb. A representative experiment is shown of 3 performed. (D) Representative transduction experiments using the indicated LVPs, where the reporter expression was captured using the Tecan SparkCyto reader. Upper panels show bright-field images of target HEK-293 CD4 cells. Lower panels show fluorescence signal from GFP expression in transduced cells. Images were captured with a 4× objective, using an exposure of 200ms in all cases and 80ms for control VSVG-VLPs (positive control). Env(-) LVPs were used as negative control (n=3). (E) Quantification of the Mean Fluorescence Intensity (MFI) of images obtained in transduction experiments.
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Figure 2—figure supplement 1—source data 1
Original files for western blot analysis for Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/110354/elife-110354-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
PDF file containing original western blots for Figure 2—figure supplement 1B.
Original membranes corresponding to Figure 2—figure supplement 1, panel B. Culture media of transfected HEK-293T cells (cm) with different constructs, as indicated, and the corresponding clarified samples (cs) containing the VLPs or the LVPs generated, were analyzed by western blot with anti-p24, and -gp120 mAbs. The anti-p24 mAb also recognizes Pr55Gag, a precursor of p24 presents in immature particles. Molecular weight markers are indicated (kDa). Figure 2—figure supplement 1, panel B shows the last six lanes of these membranes reorganized to separate LVPs from VLPs. Original files for western blot analysis displayed in Figure 2—figure supplement 1—source data 1.
- https://cdn.elifesciences.org/articles/110354/elife-110354-fig2-figsupp1-data2-v1.zip
Figure 3 with 4 supplements
gp120 VLPs modulate CXCR4 dynamics and nanoclustering.
Single-particle tracking analysis of JKCD4+X4- cells transiently transfected with CXCR4-AcGFP, on fibronectin (FN)-, FN +VLPs-, or FN +gp120 VLPs-coated coverslips (1087 particles in 159 cells on FN; 1400 in 153 cells on FN +VLPs and 1061 in 160 cells on FN +gp120 VLPs) n=6. (A) Diffusion coefficients (D1–4) of mobile particles at the membrane of cells treated as indicated. Figure shows the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=6; ****p≤0.0001). (B) Frequency of CXCR4-AcGFP particles containing monomers and dimers (≤2) or nanoclusters (≥3) in cells treated as indicated. Mean ± SD calculated from mean spot intensity (MSI) values of each particle as compared with the value of monomeric CD86-AcGFP (980±86 a.u., **p≤0.05, **p≤0.01, ****p≤0.0001). (C) Intensity distribution (arbitrary units, a.u.) from individual CXCR4-AcGFP trajectories on cells treated as indicated. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=6; ****p≤0.0001). Statistical significance was determined by non-parametric Kruskal-Wallis tests followed by Dunn’s test for panels A and C, and by two-way ANOVA in panel B.
Figure 3—figure supplement 1
Classification of mobile receptor trajectories.
Percentage of mobile receptor trajectories classified as confined, free, or directed for JK CD4+X4- cells transiently transfected with CXCR4-AcGFP in steady state or after Env(-) VLPs or gp120 VLPs stimulation. Statistical significance was determined by two-way ANOVA test.
Figure 3—video 1
Representative video of CXCR4-AcGFP on live JKCD4+X4- cells treated with DMSO and captured by SPT-TIRF, showing the diffusion of CXCR4 particles (monomers, dimers, and nanoclusters) at steady state (FN).
The video was acquired and displayed at 10.2 frames/s.
Figure 3—video 2
Representative video of CXCR4-AcGFP on live JKCD4+X4- cells treated with Env(-) VLPs and captured by SPT-TIRF, showing the diffusion of CXCR4 particles (monomers, dimers, and nanoclusters) at steady state (FN).
The video was acquired and displayed at 10.2 frames/s.
Figure 3—video 3
Representative video of CXCR4-AcGFP on live JKCD4+X4- cells treated with gp120 VLPs and captured by SPT-TIRF, showing the diffusion of CXCR4 particles (monomers, dimers, and nanoclusters) at steady state (FN).
The video was acquired and displayed at 10.2 frames/s.
Figure 4 with 4 supplements
gp120 VLPs modulate CXCR4R334X dynamics and nanoclustering.
Single-particle tracking analysis of JKCD4+X4- cells transiently transfected with CXCR4R334X-AcGFP, on fibronectin (FN)-, FN +VLPs-, or FN +gp120 VLPs-coated coverslips (341 particles in 63 cells on FN; 610 in 54 cells on FN +VLPs and 707 in 63 cells on FN +gp120 VLPs) n=2. (A) Intensity distribution (arbitrary units, a.u.) from individual CXCR4R334X-AcGFP trajectories on cells treated as indicated. Graph shows the distribution of all trajectories, with the mean value of each experiment (black circles) and the median of all trajectories ± SD (dotted black lines; n=2; ****p≤0.0001). (B) Diffusion coefficients (D1–4) of mobile single particle trajectories at the membrane of cells treated as indicated. Figure shows the mean value of each experiment (black circles) and the median of all trajectories (dotted black lines; n=2; n.s. not significant, *p≤0.05, ****p≤0.0001). (C) Frequency of CXCR4R334X-AcGFP particles containing monomers plus dimers (≤2) or nanoclusters (≥3), ± SD calculated from mean spot intensity values of each particle as compared with the value of monomeric CD86-AcGFP (*p≤0.05, ***p≤0.001). Statistical significance was determined by non-parametric Kruskal-Wallis tests followed by Dunn’s test for panels A and C, and by two-way ANOVA in panel B.
Figure 4—figure supplement 1
Classification of mobile receptor trajectories.
Percentage of mobile receptor trajectories classified as confined, free, or directed for JK CD4+X4- cells transiently transfected with CXCR4R334X-AcGFP in steady state or after Env(-) VLPs or gp120 VLPs stimulation. Statistical significance was determined by two-way ANOVA test.
Figure 4—video 1
Representative video of CXCR4R334X-AcGFP on live JKCD4+X4- cells treated with DMSO and captured by SPT-TIRF, showing the diffusion of CXCR4 particles (monomers, dimers, and nanoclusters) at steady state (FN).
The video was acquired and displayed at 10.2 frames/s.
Figure 4—video 2
Representative video of CXCR4R334X-AcGFP on live JKCD4+X4- cells treated with Env(-) VLPs and captured by SPT-TIRF, showing the diffusion of CXCR4 particles (monomers, dimers, and nanoclusters) at steady state (FN).
The video was acquired and displayed at 10.2 frames/s.
Figure 4—video 3
Representative video of CXCR4R334X-AcGFP on live JKCD4+X4- cells treated with gp120 VLPs and captured by SPT-TIRF, showing the diffusion of CXCR4 particles (monomers, dimers, and nanoclusters) at steady state (FN).
The video was acquired and displayed at 10.2 frames/s.
Figure 5 with 1 supplement
CD4 forms heterodimers with CXCR4 and CXCR4R334X.
FRET saturation curves generated using HEK-293T cells transiently transfected with a constant amount of CD4-CFP DNA (2 μg) and increasing amounts of (A) CXCR4-YFP (0.5–8.0 μg), (B) CXCR4R334X-YFP (0.5–8.0 μg) or (C) 5HT2B DNA (0.5–12 μg). KD and FRETmax values were calculated using a nonlinear regression equation for a single binding-site model (n=2). (D) FRET efficiency in HEK-293 cells transiently transfected with CXCR4-YFP/ CD4-CFP (ratio 15:9), in the absence or presence of gp120 VLPs or Env(-) VLPs. Data shows FRET efficiency (arbitrary units, a.u.; mean ± SD; n=3; n.s. not significant, *p≤0.05, ***p≤0.001). (E) FRET efficiency in HEK-293 cells transiently transfected with CXCR4R334X-YFP/CD4-CFP (ratio 15:9), in the absence or presence of gp120-VLPs or Env(-) VLPs. Data shows FRET efficiency (a.u.; mean ± SD; n=3; n.s. not significant, *p≤0.05, ***p≤0.001, ****p≤0.0001). Statistical significance was determined by unpaired t-test in panels D and E.
Figure 5—figure supplement 1
Predicted interaction between CD4 and CXCR4 or CXCR4R334X by AlphaFold3.
Predicted interactions between CD4 transmembrane and cytoplasmic domain (residues 361–433) and CXCR4 (left panel) or CXCR4R334X (right panel) at 1:1 ratio.
Figure 6
X4-gp120 promote similar internalization patterns of CXCR4 and CXCR4R334X receptors.
(A) Surface receptor expression of CXCR4 (white dots) or CXCR4R334X (black dots), after stimulation with CXCL12 (blue lines) or X4-gp120 (red lines). Results show mean ± SD of the percentage of receptor expression at the cell surface (n=3). (B) Surface receptor expression of CD4 in JK CD4+ CXCR4+ (white dots) or JK CD4+ CXCR4R334X (black dots), after stimulation with CXCL12 (blue lines) or X4-gp120 (red lines). Results show mean ± SD of the percentage of receptor expression at the cell surface (n=3). Statistical significance was determined by one-way-ANOVA of AUC (*p≤0.05, **p≤0.01).
Figure 7 with 1 supplement
CD4/CXCR4 and CD4/CXCR4R334X complexes support similar HIV-1 infection.
The presence of CXCR4R334X on JKCD4+ cells does not alter gp120 binding and increases fusion events with target cells expressing HIV pHXB2 envelope. (A) Binding of X4-gp120 to target cells expressing CD4 and CXCR4 or CD4 and CXCR4R334X analyzed by flow cytometry. Cells were incubated with 0.3 mg/mL of X4-gp120 at 37 °C for 30 min. Data show MFI (arbitrary units, a.u.) mean ± SD; (n=2). Statistical significance was determined using Student’s t-test (n.s.=not significant). (B) Cell-cell fusion between JKHXBc2-expressing HIV-1 envelope and different target cells (JKCD4+CXCR4+, JKCD4+CXCR4- , and JKCD4+CXCR4R334X). Prior to co-culture, each cell type was loaded with the corresponding cell-tracker. Data show the percentage of fusion events ± SD (n=6). We used as reference the fusions events detected in JKCD4+CXCR4+ cells (100%). Statistical significance was determined by one-way-ANOVA (*p<0.05, ****p≤0.0001). (C) Representative biparametric histograms from cells in B showing CMAC versus orange fluorophores. (D) Human PBMCs isolated from a WHIM patient (WHIM) and three healthy donors (HD1-3) in two independent experiments were infected with X4-pseudotyped HIV-1NL4-3 (MOI: 0.001). At 2 hr post infection (p.i.), supernatant samples were obtained at different time points (days post-infection) and p24 levels (pg/mL) in each sample were determined using a commercial ELISA. Results show mean ± SD (n=2).
Figure 7—figure supplement 1
Characterization of CD4+T cell populations in blood from healthy controls and a WHIM patient.
(A) Gating strategy and (B) analysis of CD4 and CXCR4 expression on CD4+ T cells. (C) Mean fluorescence intensity (MFI) of CD4 and CXCR4 on CD4+ T cells from samples in A.
Author response image 1
Purified AcGFP monomeric protein was immobilized on glass at various concentrations.
Dependency of the distribution of particle components on particle density was calculated; >95% were monomeric single particles at 2.0-4.5 particles/µm2. This range of particle density was used to analyze the dynamics of CXCR4-AcGFP, or CXCR4R334X-AcGFP single particles on JKCD4 cells.
Author response image 2
Single molecule photobleaching times measured directly from single molecule trajectories of CD86-AcGFP, considering only traces that exhibit single molecule photobleaching steps.
The experimental data are shown in gray bars (n=273 trajectories over 3 independent experiments). The red line corresponds to a single exponential decay fitting of the experimental data, from where to has been extracted.
Author response image 3
Representative confocal images of microwell dishes coated with fibronectin ((left panel) or fibronectin + VLPs (right panel)) and stained with wheat germ agglutinin (WGA) coupled to Alexa647.
Bar scale 1µm.
Author response image 4
Representative MSD plots from individual trajectories of CXCR4AcGFP detected by SPT-TIRF in resting JKCD4 cells showing different types of motion: (A) confined, (B) Brownian/Free, (C) direct transport.
Author response image 5
CD4/CXCR4 complexes were superimposed with CD4/CXCR4 complexes (left panel) or CD4/CXCR4R334X complexes (right panels).
Arrows indicate the CD4 molecule used as reference for the superimposing.
Author response image 6
Comparison of the angle between the transmembrane domains of CD4 in CXCR4 WT and WHIM complexes.
The angle between residues N196 from one CXCR4 molecule and E416 from the two CD4 dimer molecules was calculated for the CXCR4 WT (12°) and WHIM (24°) complexes to demonstrate the difference in CD4 positioning.
Author response image 7
Interacting residues at the CD4/CXCR4 interface.
The panel displays the interface residues from the CXCR4 and CD4 oligomer. CD4 residues labeled with a red sphere show the interacting residues present in both CXCR4-WT and –WHIM hetero- oligomers. The continuous red lines represent a saline bridge, while the blue lines indicate a hydrogen bond and the dashed red lines represent non-bonded interactions. As illustrated in the figure, half of the interacting residues differ between the WT and WHIM models, indicating that the interacting surfaces are also distinct.
Author response image 8
VLPs treatment does not alter cell membrane fluidity.
Diffusion values obtained by RICS from JKCD4X4 cells. (n = 3, with at least 10 cells analyzed per experiment and condition; n.s., not significant).
Author response image 9
Flow cytometry analysis of gp120 binding to Jurkat cells expressing CD4/CXCR4 or CD4/CXCR4R334X in the presence of different concentrations of the neutralizing anti-gp120 antibodies b12 (left panel) and VRC01 (right panel).
AUC comparison by Welch’s t-test: pvalues 0.2950 and 0.2112 for b12 and VRC01 respectively (n = 2).
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HIV-1 envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane
eLife 15:RP110354.
https://doi.org/10.7554/eLife.110354.2
