Video 2. T cell en face visualization of F-actin and multivesicular bodies (MVB) at the immune synapse (IS) interface.
Untransfected C3 cells (Control YFP-) (first column) or C3 cells transfected with either the bi-cistronic, YFP expression plasmid interfering all FMNL1 isoforms (shFMNL1-HA-YFP) (second column), or FMNL1 interfering and expressing interference-resistant YFP-FMNL1βWT (shFMNL1-HA-YFP-FMNL1βWT) (third column), YFP-FMNL1βS1086A (fourth column), or YFP-FMNL1βS1086D (fifth column) constructs, were mixed with CMAC-labeled, SEE-pulsed Raji cells (blue) attached to slides as described in Materials and methods. After 1 hr of culture, synaptic conjugates were fixed and labeled for F-actin (acquired in magenta, changed to blue) and for CD63 (red). Fixed whole cells (top row) and synapse contact areas (bottom row) were imaged with confocal microscopy and Z stacks were processed to generate en face, zx views of the synaptic interface, as indicated in Materials and methods. The video (10 fps) shows the generation via a 90° turn of the en face view of the Jurkat cell where the MVB are accumulated at the central region of the immune synapse (cIS) if they are polarized. When MVB are not polarized, some MVB can still be observed at cIS because they are scattered throughout the cell. This video is related to Figure 4—figure supplement 4.