Figure 4.
Characterization of the fusogenic contact and role of actin cytoskeleton in fusion of macrophages with infected Jurkat T cells. (A–F) Characterization of the fusogenic contact between infected Jurkat T cells and MDMs. (A) Time-lapse images of MDM infection by fusion: Jurkat cells were infected with a GFP-expressing viral strain (ADA-GFP-VSVG), infected cells were sorted by flow cytometry and co-cultured with MDMs 30 min before starting live-microscopy (see also Video 4). HIV-GFP in green, dashed white lines delineate the MDM. Scale bars, 10 µm. Representative images of more than 10 cell interactions leading to fusion. (B) Representative scanning electron microscopy (SEM) image of MDMs (blue) in co-culture for 30 min with Jurkat cells uninfected (NI, yellow) or infected with HIV-GFP (green). Scale bars, 5 µm. (C) Left: Representative confocal microscopy image of MDMs in co-culture for 30 min with a mix of uninfected (NI) and infected (HIV) Jurkat cells. F-actin (phalloidin, blue), HIV-GFP (green), and CD18 (yellow). Right: Twofold magnification of F-actin and CD18 fluorescence intensity (false colors). Scale bars, 20 and 10 µm. (D) Quantification of normalized F-actin and CD18 signal intensity at cell contacts between MDMs and Jurkat cells infected (HIV) or not (NI), from images corresponding to Fig. 4 C (for actin, n = 23–33 images and for CD18, n = 8–11 images, from three donors, median ± interquartile range). (E and F) Quantification by flow cytometry of infection of MDMs incubated with isotype control (Control) or antibody targeting CD18 (anti-CD18) and co-cultured during 24 h with infected Jurkat cells (C, n = 11 donors) or autologous primary T cells (D, n = 7 donors), normalized to the isotype control condition. Mean ± SD. (G and H) MDMs were pre-treated with different inhibitors (Jasplakinolide, Cytochalasin D, CK666, Blebbistatin, Y27632, TAT-C3, or Calyculin A) or DMSO (Control) and co-cultured during 24 h with infected Jurkat cells. (G) Representative images of co-cultures treated with Jasplakinolide, Blebbistatin, Y27632, or not (Control). F-actin (phalloidin, blue), HIV-p24 (yellow) and nuclei (DAPI, gray). Scale bar, 200 and 50 µm. (H) Quantification by microscopy analysis of MDM infection, normalized to the control condition (n = 6 to 8 donors, median ± interquartile range). (I and J) Analysis of infection of MDMs transfected with non-targeting siRNA (siCTRL) or targeting myosin IIA (siMYO) before co-culture with infected Jurkat cells. (I) Representative microscopy images of MDM infection. F-actin (phalloidin, blue), HIV-p24 (yellow), and nuclei (DAPI, gray). Scale bars, 200 and 50 µm. (J) Quantification of MDM infection by flow cytometry, normalized to the siCTRL condition (n = 8 donors). Mean ± SD. Statistical analyses: (D) Mann-Whitney (F-actin) or unpaired t test (CD18) test, (E, F, and J) Paired t test, (H) Wilcoxon test or Paired t test. * P ≤ 0.05; ** P ≤ 0.01.

Characterization of the fusogenic contact and role of actin cytoskeleton in fusion of macrophages with infected Jurkat T cells. (A–F) Characterization of the fusogenic contact between infected Jurkat T cells and MDMs. (A) Time-lapse images of MDM infection by fusion: Jurkat cells were infected with a GFP-expressing viral strain (ADA-GFP-VSVG), infected cells were sorted by flow cytometry and co-cultured with MDMs 30 min before starting live-microscopy (see also Video 4). HIV-GFP in green, dashed white lines delineate the MDM. Scale bars, 10 µm. Representative images of more than 10 cell interactions leading to fusion. (B) Representative scanning electron microscopy (SEM) image of MDMs (blue) in co-culture for 30 min with Jurkat cells uninfected (NI, yellow) or infected with HIV-GFP (green). Scale bars, 5 µm. (C) Left: Representative confocal microscopy image of MDMs in co-culture for 30 min with a mix of uninfected (NI) and infected (HIV) Jurkat cells. F-actin (phalloidin, blue), HIV-GFP (green), and CD18 (yellow). Right: Twofold magnification of F-actin and CD18 fluorescence intensity (false colors). Scale bars, 20 and 10 µm. (D) Quantification of normalized F-actin and CD18 signal intensity at cell contacts between MDMs and Jurkat cells infected (HIV) or not (NI), from images corresponding to Fig. 4 C (for actin, n = 23–33 images and for CD18, n = 8–11 images, from three donors, median ± interquartile range). (E and F) Quantification by flow cytometry of infection of MDMs incubated with isotype control (Control) or antibody targeting CD18 (anti-CD18) and co-cultured during 24 h with infected Jurkat cells (C, n = 11 donors) or autologous primary T cells (D, n = 7 donors), normalized to the isotype control condition. Mean ± SD. (G and H) MDMs were pre-treated with different inhibitors (Jasplakinolide, Cytochalasin D, CK666, Blebbistatin, Y27632, TAT-C3, or Calyculin A) or DMSO (Control) and co-cultured during 24 h with infected Jurkat cells. (G) Representative images of co-cultures treated with Jasplakinolide, Blebbistatin, Y27632, or not (Control). F-actin (phalloidin, blue), HIV-p24 (yellow) and nuclei (DAPI, gray). Scale bar, 200 and 50 µm. (H) Quantification by microscopy analysis of MDM infection, normalized to the control condition (n = 6 to 8 donors, median ± interquartile range). (I and J) Analysis of infection of MDMs transfected with non-targeting siRNA (siCTRL) or targeting myosin IIA (siMYO) before co-culture with infected Jurkat cells. (I) Representative microscopy images of MDM infection. F-actin (phalloidin, blue), HIV-p24 (yellow), and nuclei (DAPI, gray). Scale bars, 200 and 50 µm. (J) Quantification of MDM infection by flow cytometry, normalized to the siCTRL condition (n = 8 donors). Mean ± SD. Statistical analyses: (D) Mann-Whitney (F-actin) or unpaired t test (CD18) test, (E, F, and J) Paired t test, (H) Wilcoxon test or Paired t test. * P ≤ 0.05; ** P ≤ 0.01.

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