Figure 6.
Figure 6. HIV-1 and Vpr perturb the microtubule dynamics and localization of EB1 and p150Glued. (A) Primary human macrophages were noninfected (top), infected with HIV-1YU-2WT (middle), or infected with HIV-1YU-2ΔVpr (bottom) for 8 d. Cells were treated or not (basal) with nocodazole at 10 µM for 1 h at 37°C. After washing, cells were fixed (time 0) or incubated at 37°C without nocodazole for the indicated times before fixation. Macrophages were then labeled with an anti-p24 antibody, followed by Cy2-labeled anti–mouse IgG and a recombinant anti-tubulin, and then followed by Cy3-labeled anti–human IgG. Images were acquired under a spinning disk microscope. One optical section for tubulin and Z projections of sections for p24 staining, from stacks of representative cells, are shown. Bar, 10 µm. (B) Primary human macrophages were noninfected (top), infected with HIV-1YU-2WT (middle), or infected with HIV-1YU-2ΔVpr (bottom) for 8 d. The cells were then fixed and stained with an anti-p24, followed by Cy2-labeled anti–goat IgG (left) and an anti-EB1, followed by Cy3-labeled anti–mouse IgG (middle). Stacks of images were acquired with a wide-field microscope and analyzed to define (right) and quantify (C) the comet-shaped ellipsoid objects. Bar, 5 µm; magnification in insets is 3.3×. (C and D) Macrophages were treated as in B and the number of EB1-positive comets (C; n > 2000 comets per condition) or the total intensity of EB1 staining (D) was calculated in 12 noninfected (black bars), 12 HIV-1YU-2WT–infected (red bars), and 12 HIV-1YU-2ΔVpr (blue bars) cells. Results are expressed as a percentage of control noninfected cells. The means ± SEM of three independent experiments (donors) are plotted. (E) Primary human macrophages were noninfected (top), infected with HIV-1YU-2WT (middle), or infected HIV-1YU-2ΔVpr (bottom) for 8 d. The cells were then fixed and stained with an anti-p24 antibody, followed by Cy2-labeled anti–goat IgG (not depicted) and an anti-p150Glued, followed by Cy3-labeled anti–mouse IgG. Stack of images were acquired, and Z projection of images is shown after TopHatFilter treatment. Bar, 10 µm; magnification in insets is 2.2×. *, P < 0.05; ***, P < 0.0005.

HIV-1 and Vpr perturb the microtubule dynamics and localization of EB1 and p150Glued. (A) Primary human macrophages were noninfected (top), infected with HIV-1YU-2WT (middle), or infected with HIV-1YU-2ΔVpr (bottom) for 8 d. Cells were treated or not (basal) with nocodazole at 10 µM for 1 h at 37°C. After washing, cells were fixed (time 0) or incubated at 37°C without nocodazole for the indicated times before fixation. Macrophages were then labeled with an anti-p24 antibody, followed by Cy2-labeled anti–mouse IgG and a recombinant anti-tubulin, and then followed by Cy3-labeled anti–human IgG. Images were acquired under a spinning disk microscope. One optical section for tubulin and Z projections of sections for p24 staining, from stacks of representative cells, are shown. Bar, 10 µm. (B) Primary human macrophages were noninfected (top), infected with HIV-1YU-2WT (middle), or infected with HIV-1YU-2ΔVpr (bottom) for 8 d. The cells were then fixed and stained with an anti-p24, followed by Cy2-labeled anti–goat IgG (left) and an anti-EB1, followed by Cy3-labeled anti–mouse IgG (middle). Stacks of images were acquired with a wide-field microscope and analyzed to define (right) and quantify (C) the comet-shaped ellipsoid objects. Bar, 5 µm; magnification in insets is 3.3×. (C and D) Macrophages were treated as in B and the number of EB1-positive comets (C; n > 2000 comets per condition) or the total intensity of EB1 staining (D) was calculated in 12 noninfected (black bars), 12 HIV-1YU-2WT–infected (red bars), and 12 HIV-1YU-2ΔVpr (blue bars) cells. Results are expressed as a percentage of control noninfected cells. The means ± SEM of three independent experiments (donors) are plotted. (E) Primary human macrophages were noninfected (top), infected with HIV-1YU-2WT (middle), or infected HIV-1YU-2ΔVpr (bottom) for 8 d. The cells were then fixed and stained with an anti-p24 antibody, followed by Cy2-labeled anti–goat IgG (not depicted) and an anti-p150Glued, followed by Cy3-labeled anti–mouse IgG. Stack of images were acquired, and Z projection of images is shown after TopHatFilter treatment. Bar, 10 µm; magnification in insets is 2.2×. *, P < 0.05; ***, P < 0.0005.

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