A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor
Fig 2
Vpu-APEX2 localizes to enlarged juxtanuclear endosomes and the limiting membranes of multi-vesicular bodies.
(A) Schematic depicting APEX2 staining protocol for visualization by electron microscopy. (B) HeLa P4.R5 cells were transfected with the codon-optimized Vpu constructs bearing a C-terminal APEX2 tag. 24 hr later the cells were fixed before APEX2-dependent polymerization of DAB in the presence of hydrogen peroxide. The cells were then stained with OsO4, processed, and imaged by transmission electron microscopy (TEM). Left panel: Cells expressing Vpu-APEX2 contain juxtanuclear accumulations of osmium-highlighted enlarged vesicles (EV) likely derived from the Golgi and endosomes. Right panel: a higher magnification image of the juxtanuclear region of the cell shown at left. The limiting membranes of vesicles resembling multivesicular bodies (MVB) are highlighted by osmium. (C) Cells expressing Vpu (without an APEX2 tag) also contain enlarged juxtanuclear vesicles. (D) A control image showing Golgi (G) stacks in non-transfected HeLa P4.R5 cells.