Email updates

Keep up to date with the latest news and content from BMC Biology and BioMed Central.

Journal App

google play app store
Open Access Research article

Organization and regulation of intracellular plasma membrane-connected HIV-1 assembly compartments in macrophages

Petra Mlcochova, Annegret Pelchen-Matthews and Mark Marsh*

Author Affiliations

Medical Research Council Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK

For all author emails, please log on.

BMC Biology 2013, 11:89  doi:10.1186/1741-7007-11-89

Published: 2 August 2013

Additional files

Additional file 1: Movie 1:

3D reconstruction of an uninfected MDM labeled with FM 4-64FX. Uninfected MDMs were labeled with 5 μg/ml FM 4-64FX, fixed and analyzed by confocal microscopy. Fiji software was used to assemble a 3D reconstruction from 131 optical z-slices (step size of 0.04 μm). Cell as shown in Figure 1A-C.

Format: MOV Size: 12.8MB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 2: Figure S1:

Cell-to-cell variability in the size and morphology of the IPMCs. (A) MDMs were nucleofected to express Gag-GFP and stained with FM 4-64FX 24 hours later. Confocal sections show co-localization of Gag-GFP and the FM 4–64 dye in the IPMC. (B) MDMs were labeled with the membrane-impermeable CellMask for 5 minutes at 37°C (or 30 minutes at 4°C; bottom row in panel B) and fixed. Confocal sections show labeled cell surface and IPMCs in uninfected and HIV-infected MDMs from the same donor. (C) MDMs were nucleofected with PH-GFP. Confocal sections show labeled cell surface and IPMCs in uninfected and HIV-infected MDMs from the same donor. Infected MDMs were detected by staining for the HIV matrix protein p17 (bottom panels). All scale bars: 10 μm.

Format: TIFF Size: 962KB Download file

Open Data

Additional file 3: Movie 2:

3D reconstruction of an uninfected MDM labeled with CellMask. Uninfected MDMs were labeled with CellMask for 5 minutes at 37°C. Confocal sections were recorded using an UltraVIEW Vox spinning disc confocal system (PerkinElmer, Cambridge, UK). Fiji software was used to build this 3D reconstruction assembled from 165 optical z-slices (step size of 0.1 μm). Cell as shown in Figure 1D-F.

Format: MOV Size: 9.2MB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 4: Movie 3:

3D reconstruction of an uninfected MDM expressing PH-GFP. Uninfected MDMs were nucleofected to express PH-GFP for 24 hours, fixed and imaged by confocal microscopy. Fiji software was used to assemble a 3D reconstruction from 230 optical z-slices (step size of 0.04 μm). Cell as shown in Figure 1I-K.

Format: MOV Size: 11.3MB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 5: Figure S2:

Immunostaining for PI(4,5)P2 in MDMs. MDMs were either (A) fixed with 4% paraformaldehyde/2% glutaraldehyde and permeabilized with 0.5% saponin or (B) fixed with 4% paraformaldehyde and permeabilized in 0.2% Triton X-100. Cells were labeled with a mouse monoclonal anti-PI(4,5)P2 antibody 2C11 and co-stained for CD81. Scale bars: 10 μm.

Format: TIFF Size: 917KB Download file

Open Data

Additional file 6: Movie 4:

Live cell imaging of an uninfected MDM nucleofected with PH-GFP. MDMs were nucleofected with PH-GFP and imaged after 24 hours using an UltraVIEW Vox spinning disc confocal system fitted on a Nikon ECLIPSE Ti microscope equipped with a temperature and CO2-controllable environment chamber. The movie was assembled from images taken every 10 seconds. Cell as shown in Figure 3A.

Format: MOV Size: 1.2MB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 7: Movie 5:

Live cell imaging of an uninfected MDM expressing PH-GFP. MDMs were nucleofected to express PH-GFP and imaged after 24 hours using an UltraVIEW Vox spinning disc confocal system fitted on a Nikon ECLIPSE Ti microscope equipped with a temperature and CO2-controllable environment chamber. The movie was assembled from images taken every 10 seconds. Cell as shown in Figure 3B.

Format: MOV Size: 2.7MB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 8: Figure S3:

Latrunculin A induces the translocation of actin into nuclei. MDMs were treated with 2 μM latrunculin A or DMSO (control) for 2 hours. Cells were stained with Alexa Fluor 594-conjugated phalloidin to label actin and 4′,6-diamidino-2-phenylindole to label nuclei. The images show single optical sections acquired with a Leica SPE confocal microscope. Scale bars: 10 μm.

Format: TIFF Size: 1.4MB Download file

Open Data

Additional file 9: Figure S4:

Latrunculin A, cytochalasin E or cytochalasin D alter IPMC morphology and enhance HIV-1 release from MDMs. HIV-infected MDMs were treated with 2 μM latrunculin A (Lat), 1 μM cytochalasin E (CCE), 5 μM cytochalasin D (CCD) or DMSO (control) 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. The images show single optical sections acquired with a Leica SPE confocal microscope. The cells marked by white squares are enlarged in the bottom row. Scale bars: 10 μm. (B) Single optical sections showing examples of compact, dispersed or both (mixed) compartments. Cells were stained with antibodies against CD81 and p17. (C) MDMs were analyzed according to the morphology of the IPMCs. Ten single optical sections through the cells were acquired, inspected for the presence of IPMCs, and cells containing either compact or dispersed IPMCs or both (mixed) were counted. (D) The amount of virus released during treatment of MDMs with the actin polymerization inhibitors was analyzed by p24 ELISA assay (AIDS and Cancer Virus Program NCI-Frederick, MD, USA). Results are shown relative to the control untreated MDMs (DMSO).

Format: TIFF Size: 1.1MB Download file

Open Data

Additional file 10: Figure S5:

HIV particles still assemble in IPMCs after treatment with latrunculin A. HIV-infected MDMs were treated with DMSO or 2 μM Latrunculin A for 2 hours and processed for cryosectioning. Ultrathin cryosections from (A, B) infected control or (C, D, E) latrunculin A-treated macrophages were immunolabeled with anti-p24 antibodies, a rabbit anti-mouse bridging antibody and protein A-gold (5 nm in A, B and D, or 10 nm in C and E). EM analysis showed that HIV particles accumulated in complex IPMCs in both control and latrunculin A-treated cells. Arrows in B and D show representative immature virus particles and budding profiles, indicating virus assembly within IPMCs. Arrowheads in E mark an example of the electron-dense coats containing β2-integrins and focal adhesion proteins [14]. Images were taken on an FEI Tecnai G2 Spirit transmission EM with a Morada 11 MegaPixel TEM camera and AnalySIS software. Scale bars: 1 μm in A and C, and 200 nm in B, D, E.

Format: TIFF Size: 1.2MB Download file

Open Data

Additional file 11: Movie 6:

3D reconstruction of an infected MDM labeled with CellMask and treated with latrunculin A. MDMs infected for 7 days with HIV-1BaL were treated with DMSO (control) or latrunculin A for 2 hours and labeled with CellMask. Fiji software was used to build a 3D reconstruction of an IPMC from 200 optical z-slices (step size of 0.04 μm). The 3D reconstructions were cut out from whole cells to display the individual compartments. The control cell is shown on the left, and the reconstruction from a latrunculin-treated cell on the right. Note multiple connections to the cell surface.

Format: MOV Size: 7MB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 12: Figure S6:

Effect of latrunculin A on PH-GFP expressing MDMs. MDMs were nucleofected to express PH-GFP for 24 hours and then treated with DMSO (control) or latrunculin A for 2 hours. (A) The images show single optical sections acquired with a Leica SPE confocal microscope. (B) Both 3D reconstructions of IPMCs were built from 142 optical z-slices (step size of 0.04 μm). The 3D reconstructions were cut out from whole cells to display the individual compartments. Scale bars: 10 μm.

Format: TIFF Size: 220KB Download file

Open Data

Additional file 13: Movie 7:

3D reconstruction of uninfected MDMs expressing PH-GFP and treated with latrunculin A. MDMs were nucleofected to express PH-GFP for 24 hours and then treated with DMSO (control) or latrunculin A for 2 hours. Fiji software was used to build 3D reconstructions of IPMCs from 142 optical z-slices (step size of 0.04 μm). The 3D reconstructions were cut out from the whole cell to better display the individual compartments. The control cell is shown on the left, and the reconstruction from a latrunculin-treated cell on the right. Note multiple connections to the cell surface. Cells as shown in Figure S6 in Additional file 12.

Format: MOV Size: 6MB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 14: Movie 8:

Live cell imaging of MDMs expressing Gag-GFP. Cells were imaged with the UltraVIEW Vox spinning disc confocal system. Images were recorded at 6 frames/minute. Cell as shown in Figure 6D.

Format: MOV Size: 880KB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 15: Movie 9:

Live cell imaging of Gag-GFP in an IPMC. MDMs were nucleofected to express Gag-GFP and imaged with the UltraVIEW Vox spinning disc confocal system. Images were recorded at 6 frames/minute. Cell as shown in Figure 6E.

Format: MOV Size: 861KB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 16: Movie 10:

Live cell imaging of MDMs expressing Gag-GFP and LifeAct-Ruby: Effect of latrunculin treatment. MDMs were nucleofected to express Gag-GFP and LifeAct-Ruby for 24 hours then treated with 2 μM latrunculin A and imaged for 1.5 hours with the UltraVIEW Vox spinning disc confocal system. Images were recorded at 6 frames/minute. Cell as shown in Figure 6F.

Format: MOV Size: 10.8MB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data