Research ArticleCELL BIOLOGY

VPS4 triggers constriction and cleavage of ESCRT-III helical filaments

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Science Advances  10 Apr 2019:
Vol. 5, no. 4, eaau7198
DOI: 10.1126/sciadv.aau7198
  • Fig. 1 Local deformation of VPS4B-treated CHMP2A-CHMP3 tubes and disassembly upon ATP hydrolysis.

    (A) Clips of HS-AFM images of CHMP2A-CHMP3 tubes captured at 0.5 frame/s in the absence of VPS4B, showing no significant change in roughness of the tube surface. The inset represents the cross section (axial or radial) along the corresponding lines. (B) Clips of HS-AFM images captured at 1 frame/s, showing that the 10 μM VPS4-treated CHMP2A-CHMP3 tubes undergo discrete reductions of the tube radius (indicated with white arrows) without further structural changes in the absence of ATP Mg2+. The axial cross section at 231 s shows a clear reduction of at least 3 nm in the tube radius. Because of tip convolution, the exact value is likely higher than the observed result. (C) Clips of HS-AFM images captured at 1 frame/s from 10 μM VPS4-treated CHMP2A-CHMP3 tubes in presence of 200 μM ATP Mg2+. Upon ATP hydrolysis, CHMP2A-CHMP3 tubes disassembled within less than 1 min. White arrows show disassembly sites. Average tube height is 48 ± 3 nm (n = 30). Scale bars, 100 nm.

  • Fig. 2 In presence of ATP Mg2+, VPS4B mediates local constriction (asymmetric) of the CHMP2A-CHMP3 tubes before disassembly.

    (A) Clips of HS-AFM images captured at 0.5 frame/s, showing that the 5 μM VPS4B-treated CHMP2A-CHMP3 tubes undergo local constriction before disassembly. The constriction normally appears asymmetric (white arrows), i.e., the deformation starts from one side of the tube. Numbers in circles identify three constriction sites in the two tubes. Scale bar, 100 nm. (B) Kymographs of both tubes [green lines in (A)]. The progress of the constrictions (numbers in circles) is identifiable from the kymograph. (C) The height profiles of the tubes in (A) at nonconstricted site (in black curves) and at the constriction site over time derived from (A) and (B). Each color code is similar to the constriction sites (numbers in circles) in (A) and (B).

  • Fig. 3 Effect of VPS4 concentration on CHMP2A-CHMP3 tube remodeling in presence of ATP Mg2+.

    (A) Clips of HS-AFM images captured at 1 frame/s, showing a rapid disassembly of tube, treated with 10 μM VPS4B. (B) Kymograph taken from (A), showing the time and position of first constriction and/or disassembly (marked by red arrow). (C) Same as (A) but for 5 μM VPS4B-treated tubes. Frame rate, 1 frame/s. (D) Same as (B) but derived from (C). (E and F) Same as (C) and (D), respectively, but for 3 μM VPS4B-treated tubes. One can see that, at this concentration, the tube goes through constriction but not disassembly. (G and H) Same as (C) and (D), respectively, but for 1 μM VPS4B-treated tubes. Images in (G) show that at, 1 μM VPS4B concentration (with ATP Mg2+), the tube undergoes a partial constriction after a significant amount of time. Frame rate, 0.33 frame/s. (I) Effect of VPS4B concentration on the cleavage of CHMP2A-CHMP3 tubes. The cleavage time is considered from the moment of addition of 200 μM ATP Mg2+ until the first cleavage occurs (red arrows in the kymograph). For four different concentrations of VPS4B, more than 20 tubes were investigated. Scale bars, 100 nm.

  • Fig. 4 EM imaging of CHMP2A-CHMP3 tube remodeling by VPS4B.

    (A and B) Negative-stain images of CHMP2A-CHMP3 tubes incubated with 5 μM VPS4B and 200 μM ATP Mg2+, indicating (A) start sites of cleavage and (B) the generation of dome-like end caps. Scale bar, 100 nm. (C to E) Cryo-EM images of CHMP2A-CHMP3 tubes incubated with 5 μM VPS4B and 200 μM ATP Mg2+. (C) Images of early stages of constriction sites, (D) asymmetric constriction start sites, and (E) the generation of dome-like end caps. Scale bar, 100 nm.

  • Fig. 5 Model of membrane constriction induced by remodeling of CHMP2A-CHMP3 filaments.

    (A) CHMP2A-CHMP3 helical filaments assemble within a membrane neck structure such as a vesicle or virus bud or at the midbody. Currently, we do not know how many turns assemble in vivo. (B) VPS4 forms an asymmetric hexamer structure in the presence of ATP and Mg2+. This structure needs to assemble on functional ESCRT-III filaments, and ATP-driven rotation threads the substrate via its central pore. Because CHMP2A-CHMP3 polymer assembly is directional, the assembly of VPS4 acting clockwise or anticlockwise is likely to be important. The assembly of one VPS4 complex might be sufficient to induce CHMP2A-CHMP3 constriction, which often starts asymmetrically (middle). This can lead to membrane constriction and cleavage of the CHMP2A-CHMP3 filament. (C) Alternatively, two adjacent VPS4B complexes remodel CHMP2A-CHMP3 filaments acting clockwise and anticlockwise, thereby leading to the generation of two dome-like end caps and cleavage of the CHMP2A-CHMP3 filament. In both scenarios, constriction of CHMP2A-CHMP3 could prime the site for fission, and cleavage of the filament might play an important role in tension release as proposed (53).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaau7198/DC1

    Fig. S1. Stability of VPS4B-treated CHMP2A-CHMP3 tubular structure over an extended period of time.

    Fig. S2. Constriction time for 5 μM VPS4B-treated CHMP2A-CHMP3 tube in the presence of ATP Mg2+.

    Fig. S3. Effect of VPS4B E235Q and I124E mutants on CHMP2A-CHMP3 tube remodeling.

    Fig. S4. Snapshots of constricted tubes.

    Fig. S5. Negative staining EM images of CHMP2A-CHMP3 tubes.

    Fig. S6. Cryo-EM images of CHMP2A-CHMP3 tubes incubated with 50 μM VPS4B in the presence of 50 μM adenylyl-imidodiphosphate (AMP-PNP) Mg2+.

    Fig. S7. Negative staining images of CHMP2A-CHMP3 tubes incubated with 5 μM VPS4B and 200 μM ATP Mg2+, indicating asymmetric and symmetric constriction sites, along with dome-like end cap formation at the constriction sites.

    Movie S1. CHMP2A-CHMP3 tube immobilized on a membrane bilayer without VPS4B.

    Movie S2. CHMP2A-CHMP3 tube immobilized on a membrane bilayer with 10 μM VPS4B.

    Movie S3. CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 10 μM VPS4B.

    Movie S4. CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 5 μM VPS4B.

    Movie S5. CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 10 μM VPS4B E235Q mutant and 200 μM ATP Mg2+.

    Movie S6. CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 10 μM VPS4B I124E and 200 μM ATP Mg2+.

    Movie S7. CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 10 μM VPS4B.

    Movie S8. CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 5 μM VPS4B.

    Movie S9. CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 3 μM VPS4B.

    Movie S10. CHMP2A-CHMP3 tubes immobilized on membrane bilayer, preincubated with 1 μM VPS4B.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Stability of VPS4B-treated CHMP2A-CHMP3 tubular structure over an extended period of time.
    • Fig. S2. Constriction time for 5 μM VPS4B-treated CHMP2A-CHMP3 tube in the presence of ATP Mg2+.
    • Fig. S3. Effect of VPS4B E235Q and I124E mutants on CHMP2A-CHMP3 tube remodeling.
    • Fig. S4. Snapshots of constricted tubes.
    • Fig. S5. Negative staining EM images of CHMP2A-CHMP3 tubes.
    • Fig. S6. Cryo-EM images of CHMP2A-CHMP3 tubes incubated with 50 μM VPS4B in the presence of 50 μM adenylyl-imidodiphosphate (AMP-PNP) Mg2+.
    • Fig. S7. Negative staining images of CHMP2A-CHMP3 tubes incubated with 5 μM VPS4B and 200 μM ATP Mg2+, indicating asymmetric and symmetric constriction sites, along with dome-like end cap formation at the constriction sites.
    • Legends for movies S1 to S10

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). CHMP2A-CHMP3 tube immobilized on a membrane bilayer without VPS4B.
    • Movie S2 (.avi format). CHMP2A-CHMP3 tube immobilized on a membrane bilayer with 10 μM VPS4B.
    • Movie S3 (.avi format). CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 10 μM VPS4B.
    • Movie S4 (.avi format). CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 5 μM VPS4B.
    • Movie S5 (.avi format). CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 10 μM VPS4B E235Q mutant and 200 μM ATP Mg2+.
    • Movie S6 (.avi format). CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 10 μM VPS4B I124E and 200 μM ATP Mg2+.
    • Movie S7 (.avi format). Movie S7. CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 10 μM VPS4B.
    • Movie S8 (.avi format). CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 5 μM VPS4B.
    • Movie S9 (.avi format). CHMP2A-CHMP3 tubes immobilized on a membrane bilayer, preincubated with 3 μM VPS4B.
    • Movie S10 (.avi format). CHMP2A-CHMP3 tubes immobilized on membrane bilayer, preincubated with 1 μM VPS4B.

    Files in this Data Supplement:

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