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Structure and membrane remodeling activity of ESCRT-III helical polymers

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Science  18 Dec 2015:
Vol. 350, Issue 6267, pp. 1548-1551
DOI: 10.1126/science.aad8305
  • Fig. 1 IST1NTD and CHMP1B copolymerized into helical tubes comprising polar, double-stranded helical filaments.

    (A) Electron cryomicrograph showing IST1NTD-CHMP1B tubes (white arrows) assembled by incubating equimolar IST1NTD and CHMP1B in the presence of polymer-nucleating small acidic unilamellar vesicles (SUVs; yellow arrows). (Inset) End-on view of a short IST1NTD-CHMP1B tube. Scale bars, 40 nm (A) and 20 nm (inset). (B) End-on view of the reconstructed IST1NTD-CHMP1B tube highlighting single subunits of IST1NTD (light green, outer strand) and CHMP1B (dark green, inner strand). (C) External view of the reconstructed helix with a highlighted IST1NTD subunit. (D) Internal cutaway view of the reconstructed helix, with a highlighted CHMP1B subunit. (E) Ribbon diagram of the modeled IST1NTD subunit (closed conformation). (F) Ribbon diagram of the modeled CHMP1B subunit (open conformation). (G) Secondary structure diagrams for closed IST1NTD (top), open CHMP1B (middle), and closed CHMP1B (bottom).

  • Fig. 2 CHMP1B opening, strand structure, and electrostatic surface potentials of the IST1NTD-CHMP1B assembly.

    (A) Superposition of the open and closed CHMP1B conformations. (B) Five interlocked CHMP1B molecules from the inner strand of the filament. (C) “Top-end,” electrostatic surface view of the IST1NTD-CHMP1B tube, highlighting the acidity of the CHMP1B inner strand (including Glu130, Asp131, Asp147, Glu152, Asp155, Glu156, and Asp160) and the IST1NTD outer strand (including Asp49, Glu50, Glu57, Glu163, Glu168, Glu178, Asp180, and Glu186). (D) “Bottom-end,” electrostatic surface view of the IST1NTD-CHMP1B tube, highlighting the strongly basic characters of the CHMP1B inner strand (including Lys3, Lys87, Lys94, Lys101, Lys107, and Lys114) and the IST1NTD outer strand (including Lys7, Arg10, Lys90, Arg109, Lys118, Lys127, Lys130, Lys134, and Arg137). (E) Exterior, electrostatic surface view of the IST1NTD-CHMP1B tube, revealing the modestly basic character of the IST1NTD outer strand. (F) Internal cutaway electrostatic surface view of the IST1NTD-CHMP1B tube, revealing the strongly basic character of the lumenal surface, contributed primarily by basic residues in CHMP1B helix 1 (arrows), including Lys3, Lys13, Arg17, Lys20, Lys21, Lys24, Lys32, and Lys35.

  • Fig. 3 CHMP1B and IST1 tubulated cellular membranes.

    (A) Survey view of the cytoplasmic surface of the plasma membrane in an unroofed COS-7 cell expressing FLAG-CHMP1B. Tubular invaginations extending into the cell interior are apparent along the exposed plasma membrane and as stabilized openings at the edges of the cell. Use view glasses for 3D viewing of anaglyphs (left eye, red). (B) Higher-magnification view of tubular invaginations induced and coated by FLAG-CHMP1B filaments. (C) Immunodecoration confirmed the presence of CHMP1B around and along a tubule in a cell expressing untagged CHMP1B. Antibody detected with 12 nm gold is white in these contrast-reversed EM images. (D) Higher-magnification view of FLAG-CHMP1B filament spirals on exposed plasma membrane. (E) CHMP1B-immunoreactive organelle in an unroofed COS-7 cell expressing untagged CHMP1B. Antibody detected with 12 nm gold is white in these contrast reversed EM images; a representative gold particle is circled in blue. (F to I) Representative internal tubules from cells coexpressing untagged CHMP1B (12 nm gold; example circled in blue) and IST1-myc (18 nm gold; examples circled in red). (J and K) Clathrin-coated bud capping the end of (J) CHMP1B and (K) IST1-myc immunolabeled tubules from cotransfected cells. Measurements of filament diameter (and interstrand spacing) showed that when apparently unitary filaments were resolvable, their diameter varied from 5 to 10 nm, including platinum. These measurements are generally consistent with the dimensions of IST1-CHMP1B and CHMP1B filaments formed in vitro. Scale bars, 500 nm (A) and 100 nm (B) to (K).

  • Fig. 4 Topology of ESCRT-III membrane deformation in cells and in vitro.

    (A) Series of filament spirals on the plasma membrane of COS-7 cells expressing CHMP4A1-164 show development of the outwardly directed protrusions previously associated with ESCRT-III filaments (15, 16). Drawing highlights relationship between a CHMP4A conical spiral and a negatively curved plasma membrane tubule. (B) Series of filament spirals on the plasma membrane of COS-7 cells expressing FLAG-CHMP1B show development of invaginations directed into the cell. Drawing highlights relationship between a CHMP1B conical spiral and a positively curved plasma membrane tubule. (C) Negative stain electron micrograph showing that CHMP1B tubulates liposomes and forms a filamentous coat on the outside of the tubule. White arrows highlight regions coated by the CHMP1B helices, and the yellow arrow highlights a break in the coat where the internal lipid is visible. (D) Negative stain electron micrograph showing that the IST1NTD-CHMP1B copolymer forms on the outside of membrane tubules. White arrows highlight regions coated by the IST1NTD-CHMP1B copolymer, and the yellow arrows highlight breaks in the helical coat or uncoated regions of the liposome where the internal membrane is visible. Scale bars, 100 nm (A) and (B), 50 nm (C) and (D).

Supplementary Materials

  • Structure and membrane remodeling activity of ESCRT-III helical polymers

    John McCullough, Amy K. Clippinger, Nathaniel Talledge, Michael L. Skowyra, Marissa G. Saunders, Teresa V. Naismith, Leremy A. Colf, Pavel Afonine, Christopher Arthur, Wesley I. Sundquist, Phyllis I. Hanson, Adam Frost

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S12
    • Tables S1 and S2
    • Full Reference List

    Images, Video, and Other Other Media

    Movie S1
    Two and a half complete turns of the IST1NTD-CHMP1B reconstruction density, highlighting an outer strand subunit (light green, IST1NTD) and an inner strand subunit (dark green, CHMP1B).
    Movie S2
    Docking of the IST1NTD crystal structure (3FRR), and the final refined atomic model for the IST1NTD subunit into a segmented subunit from the outer strand cryo-EM density. The movie initially shows unsharpened EM density, then sharpened EM density, and then zooms in to show the density and model for IST1NTD helix 2, including side chains. The initial docking is with the IST1NTD crystal structure, and then shows the final refined structure. The major difference between the two structures involves remodeling of helix A to obtain a better fit to the density. This helix may be mobile because it makes a lattice contact in the 3D crystals (4), and is not well resolved in the EM density, as compared to the other helices.
    Movie S3
    Docking of the final refined de novo atomic model for the CHMP1B subunit into a segmented subunit from the inner strand cryo-EM density. The movie initially shows unsharpened EM density, then sharpened EM density, then the docking of the final refined model, and then zooms in to show the density for CHMP1B helix 2, including side chains.
    Movie S4
    Morph animation between the "closed" homology model of CHMP1B and the "open" structure from the reconstructed IST1NTD-CHMP1B copolymer.
    Movie S5
    Filtered electron cryotomogram of IST-CHMP1B conical assemblies imaged under frozen-hydrated conditions. The conical assembly resolved in the bottom half of the field of view was used as a starting model for the single particle reconstruction shown in fig. S12C-D.
    Movie S6
    Single particle reconstruction of a continuous conical spiral of the IST1-CHMP1B copolymer, low-pass filtered to 25 Å. The conical spiral comprises the same doublestranded filaments reconstructed to high resolution because: 1) the spiraling filaments are right handed, single start, and double-stranded; 2) each turn is separated by 5.1 ± 0.1 nm; 3) the narrow ends of the spiraling filaments taper to 24 nm, which matches the diameter of the IST1NTD-CHMP1B helices; and 4) subunits at the tapered ends of the cones are related by ~21° rotation angles, which again matches the IST1NTD-CHMP1B helices (fig S1)."

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