Presence of F-actin at phagocytic sites in HIV-1–infected macrophages. (A) Primary human macrophages were infected with HIV-1ADAWT or HIV-1ADAΔNef or noninfected for 8 days. The cells were incubated for 5 minutes with IgG-RBCs, then fixed and stained with AMCA-anti–rabbit IgG to reveal external RBCs. The cells were then permeabilized and labeled with anti-p24, followed by Cy2-anti–goat IgG and Alexa633-phalloidin to stain F-actin (iv-vi). Merged images show p24 in green and RBCs in red (i-iii). Arrowheads indicate examples of F-actin cups. Cells were analyzed by wide-field fluorescence microscopy and phase contrast (vii-ix). Bar represents 10 μm. (B) Noninfected cells were treated as described in panel A. (C) The profile of F-actin fluorescence intensities along the lines drawn at the phagocytic site (line 1) and in the cell body (line 2) are shown. (D) Noninfected cells and infected cells were treated as described in panel A. The fluorescence intensities measured in the phagocytic cups were background subtracted and divided by the fluorescence intensities measured for cortical actin in the cell body after background substration. This ratio defined the index of recruitment. The means ± SEM of 3 independent experiments are plotted (n = 78 actin cups per condition).
