Fig. 2.

Dynamic recruitment of Gag puncta to the synapse and viral transfer into a target cell compartment revealed with rapid spinning-disc 3D confocal fluorescence microscopy. (A) Formation of a buttonlike accumulation of Gag at the site of adhesion, z projection at time = 0 (left), selected 3D reconstructions of contact site (arrows) over time (right four images). (B) A zone of Gag depletion, 2 to 3 μm wide, surrounds the synaptic button (dotted yellow line). (C) Patches of synapse-proximal Gag merge into the synapse. (D) A Gag-iGFP puncta moves out of and into the synapse. (E) During a transfer event, Gag puncta emerge from the synapse, separate, and then move to the distal pole. In (C) to (E), (a) selected frames highlight movement of Gag-iGFP puncta (yellow). (b) Object path is overlayed on the initial image. (c) Object distance to the synapse center and relative velocity are graphed over time. (d) Histogram distribution of the tracked objects velocities. (F and G) Immunostaining of Gag puncta requires membrane permeabilization. (F) Nonpermeabilized, anti-Env immunostain (red) does not stain the Gag-iGFP+ puncta (green) within the CD4 target cell (CellTracker Blue CMF2HC, Invitrogen), whereas surface Env-staining at synapse is observed. Three-color intensity profile along the 12-μm line (right). (G) Permeabilization of fixed cells reveals anti-Env immunostain (red) at the GFP puncta (green) within the CD4 target cell (blue). (H) Transmission electron micrograph of vesicles containing corelike structures in a CD4 cell engaged in synapse with an HIV-infected Jurkat cell. Low (left) and high (right) magnification of 70-nm sections.