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Recruitment of HIV and Its Receptors to Dendritic Cell�T Cell Junctions
David McDonald, Li Wu, Stacy M. Bohks, Vineet N. KewalRamani, Derya Unutmaz, Thomas J. Hope

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Materials and Methods
Figs. S1 and S2

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  • Movie S1
    Localization of HIV in DCs in suspension. MDDCs were incubated with GFP-Vpr�labeled HIVLAI (green) for 1 hour at 37°C, washed and stained for surface HLA class II (HLA-DR, -DP, -DQ) by indirect immunofluorescence (red). Cells were fixed, stained with Hoechst dye (blue), and resuspended in Gel Mount. Cells were imaged in z-section (0.2 Greek Letter Mum) and deconvolved. Movie shows entire z-stack from the bottom to the top of the cell.
  • Movie S2
    Recruitment of HIV to sites of cell contact. MDDCs were incubated with GFP-Vpr�labeled HIVLAI (green) for 1 hour at 37°C, washed and stained with a mitochondrial dye (red). The DCs were added to Hos/CD4 cells growing on a coverslip chamber premounted on a microscope and warmed to 37°C. Hos/CD4 are the reticular structures under the bright DCs. Starting at 1 min after the DCs contacted the target cells, images were collected in z-series every 2 min, deconvolved and reconstructed as 3-D volume projections and imported into QuickTime. Display is 2 frames/second.
  • Movie S3   Movie S4   Movie S5
    Recruitment of HIV in protealyzed DCs. DCs were incubated with GFP-Vpr�labeled HIVLAI (green) for 1 hour at 37°C, washed in PBS, resuspended in 0.05% Trypsin for 10 min at 37°C, then washed in complete medium. The cells were allowed to recover at 37°C for 1 hour and then plated onto Hos/CD4 cells as in Fig. 2. Cells were imaged every minute in z-section in the GFP channel and by differential interference contrast (DIC) (grey) and shown as 3-D volume reconstructions displayed as QuickTime movies. Movie S3. Full-frame display of the entire time-lapse film shows heavily labeled DC (top right) that rolls into frame along the Hos/CD4 cell process. Hos/CD4 cell body can be seen to the upper left. Movie S4. Magnification of volume projection of the DC over time, beginning at 28 min, when the DC rolled into position. Movie S5. Side view of DC over time. 3-D volume reconstruction is rotated so the left side of the cell faces the viewer. The signal tends to concentrate downward and right toward the Hos cell projection in this view. All movies are displayed at 6 frames/second.