Figure 3. Biogenesis of TCR-enriched microvesicles is mediated by ESCRT proteins and antagonized by HIV GAG.

a. Optical-EM correlation of TCR and and MCC/I-E^k distributions of a representative AND T cell following siRNA-mediated TSG101 suppression. Scale bar 3 μm. b. Electron micrographs of en face sections of human CD4+ T cells forming IS in response to TCR engagement. Cells were transfected with a construct encoding dominant negative VPS4 fused to GFP (VPS4dn-GFP) or GFP only. Red boxes and arrowheads indicate regions imaged at higher magnification in right panels. Arrowheads in c. indicate nascent vesicles tethered to the plasma membrane. d. Quantitation of limiting membrane-tethered microvesicles expressed as a percentage of total microvesicles within central compartments of the IS in cells expressing indicated constructs. Means and s.d. are shown, P value is for Student’s t-test. N is indicated above data bars. Results are pooled from two independent experiments. e–g. Representative optical-EM correlation of the IS in human CD4+ T cells transfected with a construct encoding HIV GAG fused to GFP (GAG-GFP, green). Red, TCR; scale bar, 3 μm. e. T cell without central GAG-GFP accumulation resuming migration and releasing TCR-enriched microvesicles (arrowhead). f. T cell forming an IS with centrally accumulated GAG-GFP. g. T cell with centrally accumulated GAG-GFP resuming motility and releasing GAG-GFP-containing microvesicles (arrowheads). h. Higher magnification image of boxed region in g. showing internal juxta-membrane density in GAG-containing microvesicles. Arrowhead, plasma membrane; scale bar, 500 nm.