File:Human-T-Lymphotropic-Virus-1-Visualized-at-the-Virological-Synapse-by-Electron-Tomography-pone.0002251.s003.ogv

English: Tomographic reconstruction showing the virological synapse between a naturally HTLV-1-infected CD4+ T-cell (PBMC) (right) and a target cell (autologous uninfected CD4+ T-cell) (left). Virus transmission can be seen in two separate synaptic clefts. The HTLV-1 particles are in simultaneous contact with the two cell membranes and are extremely electron-dense due to the labelling of the viral Gag p19 matrix protein (see also Movies S4 and S5). Parts of the two plasma membranes are also darkly stained, indicating the presence of viral Gag protein. The presence of two centrioles (long white arrows), two Golgi apparatuses (short, thick white arrows) and several mitochondria close to the VS reflects the polarization of the microtubule organizing centre (MTOC) of the HTLV-1-infected CD4+ T cell towards the cell-cell contact. Parts of the cell nucleus can be seen in both cells. Tomogram slices through the 3D reconstruction can be seen in Figure 4A,B and a surface representation of the synapse is shown in Figure 4C. The sample was fixed with PFA/glutaraldehyde, labelled against viral Gag p19 matrix protein, embedded in Agar 100 resin and post-fixed with OsO4. The reconstructed 3D volume is composed of tomograms obtained from five serial sections. The tomograms were calculated from tilt series of 131 images (tilt range −65° to +65°, 1° increment). The images were recorded with a 2k×2k Gatan CCD camera (pixel size 1.29 nm) at an under focus of −3.0 µm with an FEI Tecnai F30 FEG-TEM (300 kV) using the FEI software. The reconstruction was calculated using the software IMOD. The tomogram represents a volume with an area of 1.55×2.66 µm2 and a thickness of 343 nm. The scale bar at the end of the movie corresponds to 500 nm.