Figure 2. ESCRT-III filaments form conical spirals on the plasma membrane of cells depleted of Vps4A & Vps4B.
(A) Survey view of the cytoplasmic surface of the plasma membrane from a HeLa cell unroofed ∼60 hr after transfection with siRNA targeting Vps4A & Vps4B. Pseudocoloring shows clathrin (orange), caveolae (green), and ESCRT-III spirals (yellow). Similar filament spirals were seen in siRNA treated HEK293T, U2OS, and MCF-7 cells (not shown). (B) Immunodecoration of ESCRT-III proteins in spirals on Vps4-depleted HeLa cell plasma membranes. Antibodies recognizing CHMP6 (left), CHMP2B (middle), and CHMP1B (right) detected with 18 nm gold that appears white in these contrast reversed EM images. Use view glasses for 3D structure in both panels (left eye = red). Scale bars represent 100 nm.
Figure 2—figure supplement 1. Immunodecoration of ESCRT-III proteins beneath protrusions from the cell surface.
Fortuitous breaks in HeLa cells depleted of Vps4A & B subjected to immunogold labeling allowed antibody staining inside broken cells. (A) Immunodecoration of CHMP1B, (B) immunodecoration of CHMP2B. 18 nm gold particles appear white in these contrast reversed deep-etch EM images. Insets show enlarged images (blue edges) with gold particles marked yellow. Use view glasses for 3D structure. Scale bars represent 100 nm for main panels and 50 nm for insets.