Actin assembly is required for the integrity of the IPMCs. (A-C) HIV-infected MDMs were treated with medium containing dimethyl sulfoxide (control) or 2 μM latrunculin A for 2 hours. (A) Cells were stained with an anti-p17 antibody that only recognizes mature virus particles and Alexa Fluor 594-conjugated phalloidin to label actin. (B) Enlargements of the marked areas in panel A. The images show single optical sections acquired with a Leica SPE confocal microscope as above. (C) HIV-infected macrophages were labeled with CellMask for 5 minutes at 37°C, fixed and imaged with an UltraVIEW Vox spinning disc confocal system as described in Figure 3 (see Additional file 11). (D,E) MDMs were nucleofected to express Gag-GFP and imaged after 24 hours using an UltraVIEW Vox spinning disc confocal system as described above (see Additional files 14 and 15). Images were recorded at 6 frames/minute with a 60× Nikon oil immersion objective (NA 1.4) using Volocity 5.3.2 (Perkin Elmer) and analyzed with Volocity, Adobe Photoshop, ImageJ and Fiji software. The cell periphery is indicated with the dashed white line. The white arrowheads in (E) show Gag-GFP in thin channels between the IPMC and the cell surface. The image E’ shows an enlargement of the area marked by the red box. (F) MDMs, co-transfected to express Gag-GFP and LifeAct-Ruby for 24 hours, were treated with 2 μM latrunculin A and imaged for 1.5 hours with the spinning disc confocal system (see also Additional file 16). All experiments were repeated with cells from at least two different donors. All scale bars = 10 μm.
Mlcochova et al. BMC Biology 2013 11:89 doi:10.1186/1741-7007-11-89