Supporting Online Material


Quantitative 3D Video Microscopy of HIV Transfer Across T Cell Virological Synapses
Wolfgang Hübner, Gregory P. McNerney, Ping Chen, Benjamin M. Dale, Ronald E. Gordon, Frank Y. S. Chuang, Xiao-Dong Li, David M. Asmuth, Thomas Huser, Benjamin K. Chen

Supporting Online Material

This supplement contains:
Materials and Methods
Figs. S1 to S7
Tables S1 to S3
References

This file is in Adobe Acrobat PDF format.

Other Supporting Online Material for this manuscript includes the following: available at www.sciencemag.org/cgi/content/full/323/5922/1743/DC1
Movies S1 to S12

Movie s1
Synaptic buttons are intense accumulations of Gag at the virological synapse, visualized here in a three-dimensional representation of an HIV Gag-iGFPexpressing Jurkat cell attached to three CD4+ target cells (red). HIV Gag-iGFP (green) is focused at the sites of cell-cell contact.

Movie s2
Oligomerization of Gag at the virological synapse is detected by fluorescence resonance energy transfer between HIV Gag-iCerulean and HIV Gag-iVenus. Three-dimensional reconstruction with donor cerulean signal (blue) and FRET signal (green) shown in the HIV-expressing cell. CD4+ target cells are visualized with the fluorescent dye CMRA (red).

Movie s3
Formation of a synaptic button at a site where a target cell has recently attached to a donor cell. Brief animation illustrates the perspective of the 3D reconstruction, followed by the movie in 3D view. The new button forms by movement of nearby Gag-iGFP towards the site of cell-cell contact. Spinning disc confocal images were recorded at one stack per 1.4 sec. Elapsed time is depicted in minutes:seconds.

Movie s4
Overview spinning disc confocal movie of a synapse that displays the recruitment of Gag to a synaptic button, followed by the coordinated budding and endocytosis into the CD4+ target cell. Maximum intensity projection shown first, followed by a 3D reconstruction of the same images. Elapsed time is depicted in minutes:seconds.

Movie s5
Movement of a vesicular Gag puncta into the virological synapse. Same puncta shown first without, then with, the object highlighted in a 3D reconstruction. Elapsed time is depicted in minutes:seconds.

Movie s6
Coalescing of Gag puncta, and vectorial movement into the virological synapse. Same puncta is first shown first without, then with, the object highlighted in a 3D reconstruction. Elapsed time is depicted in minutes:seconds.

Movie s7
Rapid movement of a small puncta out of and back into the synaptic button. 3D reconstruction image is first shown without, then with, the object highlighted. Elapsed time is depicted in minutes:seconds.

Movie s8
Budding of large HIV Gag-iGFP-containing vesicles into CD4+ target cells is followed by vectorial movement of the HIV-laden compartment toward the distal pole of the cell. 3D reconstruction image shown first without, then with, the object highlighted. Elapsed time is depicted in minutes:seconds.

Movie s9
Transfer of an entire synaptic button to the CD4+ target cell. Image is an overlay of brightfield image and green fluorescence in a maximum intensity projection of the acquired stack. Elapsed time is depicted in minutes:seconds.

Movie s10
Accumulation of HIV Gag-iGFP (green) virus particles in target cells recorded with laser scanning confocal microscopy. 3D stack acquired every 5 minutes for 13 hours. Elapsed time is depicted in hours:minutes:seconds.

Movie s11
Productive infection of VS target cells is observed with long duration wide field microscopy, cell #1 from Fig. 4A. Images acquired every 10 minutes over 67 hours. The HIV green fluorescence signal alone is on the left while an overlay of green fluorescence and phase contrast is shown on the right. Elapsed time is depicted in hours:minutes.

Movie s12
After contact with HIV-infected cells, productive infection is observed in long duration imaging of cells #2 through #5 depicted in Fig. 4B. Images acquired with long duration wide field microscopy every 10 minutes over 67 hours. Elapsed time for each individual cells is depicted in hours:minutes.

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