Figure 6. ESCRT-III filaments surround Gag assemblies in cells depleted of Vps4.
(A) HEK293T cells treated with Vps4A & Vps4B siRNA accumulate unique filament-encircled Gag assemblies. Inset is pseudo-colored to show the central Gag assembly (blue), surrounding filament (red), and perpendicular ‘struts’ between them (green). Each field also shows the occasionally seen subplasmalemmal VLP bump to provide a sense of scale. (B) Individual views of Gag assemblies, (C) Gag-GFP assemblies, and (D) Gag assemblies immunodecorated with indicated gold conjugated antibodies. Scale bars represent 100 nm. Each box in B–D is a 320 nm square. Use view glasses for 3D structure (left eye = red).
Figure 6—figure supplement 1. VLP release is impaired by inactivating or depleting Vps4.
(A) Confocal image of HEK293T cells expressing Vps4B-E228Q or Vps4A & Vps4B siRNA and transfected with Gag-GFP. Note that Gag accumulates on the plasma membrane instead of in released VLPs (compare with Figure 5—figure supplement 1A). (B) Deep-etch EM of an unbroken Vps4-depleted cell expressing Gag showing unbudded VLPs accumulating on the cell surface.