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. 2017 Jul;19(7):787-798.
doi: 10.1038/ncb3559. Epub 2017 Jun 12.

Dynamic Subunit Turnover in ESCRT-III Assemblies Is Regulated by Vps4 to Mediate Membrane Remodelling During Cytokinesis

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Free PMC article

Dynamic Subunit Turnover in ESCRT-III Assemblies Is Regulated by Vps4 to Mediate Membrane Remodelling During Cytokinesis

Beata E Mierzwa et al. Nat Cell Biol. .
Free PMC article

Abstract

The endosomal sorting complex required for transport (ESCRT)-III mediates membrane fission in fundamental cellular processes, including cytokinesis. ESCRT-III is thought to form persistent filaments that over time increase their curvature to constrict membranes. Unexpectedly, we found that ESCRT-III at the midbody of human cells rapidly turns over subunits with cytoplasmic pools while gradually forming larger assemblies. ESCRT-III turnover depended on the ATPase VPS4, which accumulated at the midbody simultaneously with ESCRT-III subunits, and was required for assembly of functional ESCRT-III structures. In vitro, the Vps2/Vps24 subunits of ESCRT-III formed side-by-side filaments with Snf7 and inhibited further polymerization, but the growth inhibition was alleviated by the addition of Vps4 and ATP. High-speed atomic force microscopy further revealed highly dynamic arrays of growing and shrinking ESCRT-III spirals in the presence of Vps4. Continuous ESCRT-III remodelling by subunit turnover might facilitate shape adaptions to variable membrane geometries, with broad implications for diverse cellular processes.

Conflict of interest statement

Competing Financial Interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. ESCRT-III assemblies at the midbody dynamically turn over subunits in early and late abscission stages.
(a) Validation of mmCHMP4B-LAP functionality by RNAi phenotype rescue. Cumulative histograms indicate duration from complete cleavage furrow ingression until abscission for wildtype HeLa cells and for HeLa cells expressing mmCHMP4B-LAP at 55-80 h after siRNA transfection (3 independent experiments with combined sample numbers of n = 48 cells for wildtype+siControl, n = 38 cells for wildtype+siCHMP4B, n = 60 cells for mmCHMP4B-LAP+siControl, and n = 46 cells for mmCHMP4B-LAP+siCHMP4B). siCHMP4B (hs) targets only endogenous human CHMP4B but not mmCHMP4B-LAP. (b) FRAP of mmCHMP4B-LAP at a HeLa cell midbody at early and late abscission stages, stained with SiR-tubulin. Dashed circles indicate photobleaching region; time 0 indicates first image after photobleaching. High-resolution example of experiment in c-e. (c-d) Fluorescence recovery curves for (c) early abscission (n = 18 cells from 4 independent experiments) or (d) late abscission stages (n = 17 cells from 4 independent experiments). Single exponential function f(t)=1-e^(-k*t), or double exponential function f(t)=A1*(1-e^(-k1*t))+(1-A1*)(1-e^(-k2*t)) were fitted to the data. Points and shaded areas indicate mean ± SEM of fluorescence; dashed lines indicate fits of exponential functions. (e) Quantification of highly mobile fractions by fitting double exponential functions to data from c, d. Dots represent individual cells. (f) 3D live-cell confocal microscopy of the intercellular bridge during telophase, in HeLa cells expressing hsCHMP2B-LAP or hsCHMP3-LAP, respectively. Arrowheads indicate abscission. High-resolution example of experiment in g. (g) Quantification of hsCHMP2B-LAP (n = 17 cells from 4 independent experiments), hsCHMP3-LAP (n = 13 cells from 3 independent experiments), and hsCHMP4B-LAP (n = 17 cells from 3 independent experiments) midbody accumulation. Points and shaded areas indicate mean ± SEM; normalized to intercellular bridge fluorescence after cleavage furrow ingression, and temporally aligned to abscission (time point 0). (h) Highly mobile fractions of LAP-tagged ESCRT-III subunits derived from double exponential fits to FRAP curves. Each dot represents a single FRAP experiment acquired in 3 independent experiments; bars indicate medians. (i) Residence times of highly mobile fractions for cells shown in h. Scale bars, 1 μm in b, f.
Figure 2
Figure 2. VPS4 is required for ESCRT-III accumulation and turnover.
(a) Confocal microscopy of the intercellular bridge in HeLa cells expressing mmCHMP4B-LAP or mmVPS4B-LAP, respectively. Arrowheads indicate abscission. High-resolution example of experiment in b. (b) Midbody accumulation of mmCHMP4B-LAP (n = 15 cells from 3 independent experiments) or mmVPS4B-LAP (n = 16 cells from 3 independent experiments) relative to abscission (time point 0). Points and shaded areas indicate mean and SEM. (c) Live-cell images of telophase cells expressing mmCHMP4B-LAP after transfection of a non-targeting control siRNA, or siRNAs targeting hsVPSP4A/B after 20 h or 48 h. Insets show enlarged midbody regions. The same contrast settings were used for all panels. High-resolution example of experiment in Supplementary Fig. 3c. (d) Quantification of cytoplasmic mmCHMP4B-LAP levels from data in e, f. Dots represent individual cells from 3 independent experiments; bars indicate medians. (e) FRAP curves and double exponential fits for mmCHMP4B-LAP at pre-constriction stages transfected with control siRNAs (n = 18 cells from 3 independent experiments) or siRNAs targeting VPS4A/B (n = 18 cells for siVPS4A/B 20h, and n = 17 cells for siVPS4A/B 48h from 3 independent experiments). Points and shaded areas indicate mean ± SEM. (f) Highly mobile fractions of mmCHMP4B-LAP determined by double exponential fits to FRAP curves shown in e (3 independent experiments with combined sample numbers of n = 9 cells for siControl 48h, n = 18 cells for siVPS4A/B 20h, and n = 13 cells for siVPS4A/B 48h). Statistical test using the two-sided Kolmogorov–Smirnov test yielded P = 6.562e-3 for siControl 48h relative to siVPS4A/B 20h, and P = 4.021e-6 for siControl 48h relative to siVPS4A/B 48h. Dots represent individual cells; bars indicate medians. Scale bars, 1 μm in a; 5 µm or 1 µm (inset) in c.
Figure 3
Figure 3. VPS4 is required for constriction of the intercellular bridge.
(a-b) Transfection of siRNAs targeting hsVPS4A/B causes abscission failure in wild-type HeLa cells, but not in HeLa cells stably expressing mmVPS4B-LAP. (a) Progression from cleavage furrow ingression (time point 0) until abscission in wildtype HeLa cells at 30-50 h after transfection of indicated siRNAs (n = 84 cells for wildtype+siControl, and n = 80 cells for wildtype+siVPS4A/B for 3 fields of view from 2 independent experiments). (b) Rescue of abscission failure in HeLa cells stably expressing mmVPS4B-LAP (data from a, n = 54 cells for mmVPS4B-LAP+siControl, and n = 45 cells for mmVPS4B-LAP+siVPS4A/B for 3 fields of view from 2 independent experiments). Bars and error bars indicate mean ± SEM. (c) Representative electron micrograph of an intercellular bridge of a control cell (n = 10 cells, out of which 3 cells had filaments without constriction, and 4 showed filaments with constriction). Arrowheads indicate 17 nm diameter filaments. (d) Intercellular bridge of a cell 26 h after transfection of VPS4A/B siRNA (n = 26 cells, out of which 4 cells showed filaments without constriction). Arrowheads indicate 17 nm diameter filaments. Scale bars, 200 nm in c, d.
Figure 4
Figure 4. Vps2 and Vps24 cooperatively bind Snf7 patches and inhibit ESCRT-III polymerization.
(a) Time-lapse microscopy of ESCRT-III polymerization on supported lipid membranes in a microfluidic flow chamber. Recombinant Snf7-AlexaFluor-488 was injected at t = 0 min; Vps2-Atto-565 and Vps24 were added at t = 22 min. (b) Kymograph of a single ESCRT-III patch from a. (c-d) Quantification of (c) mean fluorescence and (d) patch diameters from 24 patches as in a-b (quantified from 4 fields of view within a representative experiment, and consistent results in 3 independent experiments using differently labeled proteins, e.g. Supplementary Fig. 6a-c). Curves and shaded areas represent mean ± SEM. (e) Kymograph of an experiment where Snf7-AlexaFluor-647N was added at t = 0 min, followed by sequential addition of Vps24-AlexaFluor-488 and Vps2-Atto-565 (representative image from 24 patches within the shown experiment, and 1 additional independent experiment). (f) Kymograph of an ESCRT-III patch, where Snf7-AlexaFluor-488 was added at t = 0 min, then washed out during 28-32 min (shaded area), followed by addition of Vps2-Atto-565 and Vps24 at t = 47 min. The transient increase of Vps2 signal during washout resulted from background ambient light. (g) Fluorescence quantification of 37 patches as in f (analyzed from 4 fields of view within the shown experiment, and 3 additional independent experiments). Curves and shaded areas represent mean ± SEM. Scale bars, 5 μm in a; 5 µm (vertical) and 5 min (horizontal) in b, e, f.
Figure 5
Figure 5. Vps2 and Vps24 polymerize side-by-side with Snf7 to form filament bundles.
(a) Transmission electron microscopy of Snf7 spirals polymerized on liposomes. Colored overlays indicate the number of parallel filament strands. (b) Distribution of filament bundle lengths quantified in 11 spirals from 3 independent experiments as in a. (c) Snf7 was polymerized on liposomes, followed by Vps2 and Vps24 addition. Colored overlays indicate the number of parallel filament strands. (d) Quantification of 17 spirals from 2 independent experiments as in c. (e) Examples of filament morphologies with different strand numbers, corresponding to colored overlays used in a-d. (f) Averaged line profiles across ESCRT-III filament bundles from a-d (n = 3 filaments for 1 strand, n = 8 filament bundles for 2 strands, n = 3 filament bundles for 3 strands, n = 8 filament bundles for 4 strands; and n = 3 filament bundles for 6 strands). Curves and shaded areas indicate mean ± SEM. (g-i) HS-AFM imaging of ESCRT-III polymers on supported lipid membranes. Snf7 was polymerized on lipid membranes, followed by addition of Vps2 and Vps24 at t = 0. (g) Spiral morphology before and after addition of Vps2 and Vps24. Green and magenta lines indicate line profiles used to (h) measure height variability. (i) Height variability was measured as coefficient of variation along radial line profiles within spirals before and after addition of Vps2 and Vps24, respectively, as shown in g, h (n = 28 spirals for Snf7, and n= 26 spirals for Snf7+Vps2+Vps24 from 2 independent experiments). Statistical test using the two-sided Kolmogorov–Smirnov test yielded P = 2.875e-14 for Snf7 relative to Snf7+Vps2+Vps24. Dots represent individual line profile measurements; bars indicate medians. Scale bars, 50 nm in a, c, e; 200 nm in g.
Figure 6
Figure 6. Kinetics of ESCRT-III patch disassembly by Vps4.
(a) Kymograph of a representative patch from time-lapse microscopy of Snf7 patches on supported lipid membranes in a flow chamber. A solution of Snf7-AlexaFluor-488 was injected into the flow chamber and incubated until patches polymerized on the membrane. Snf7 was washed out at t = 22 min, followed by addition of ATP and 8 µM Vps4 at t = 33 min. (b) Quantification of mean fluorescence of 13 patches as in a (4 different fields of view within the shown experiment, and 1 additional independent experiment). Curves and shaded areas indicate mean ± SEM. (c) A solution of Snf7-AlexaFluor-488 was injected into the flow chamber and incubated until patches polymerized. At t = 34 min, Snf7 was removed from the solution and Vps2-Atto-565, Vps24, Vps4, and ATP were injected. (d) Quantification of mean fluorescence of 33 patches as in c (4 fields of view within the shown experiment, and 1 additional independent experiment). Curves and shaded areas indicate mean ± SEM. (e-g) Vps4-induced dynamic turnover and lateral mobility of ESCRT-III filament spirals depends on ATP. (e) HS-AFM imaging of ESCRT-III spirals. Assemblies were generated by polymerization of Snf7 on supported lipid membranes, followed by addition of Vps2 and Vps24. After washout of all soluble components, Vps4 was injected. Then, ATP and Mg2+ were added, and imaging was started 22 s later (t = 0). Images represent averages of 2 consecutive time frames to improve signal-to-noise ratio. (f) Quantification of spiral diameters from e (119 spirals from a representative experiment, out of 6 independent experiments). Dots represent single spirals; bars indicate medians. (g) Snf7 was polymerized on supported lipid membranes, followed by addition of Vps2 and Vps24, and subsequent addition of Vps4, as in Fig. 8a but without ATP. Imaging was started 30 s after addition of Vps4 (t = 0). Images represent averages of 3 consecutive time frames. Corresponding spiral diameter quantification is shown in Fig. 8c. Scale bars, 5 µm (vertical) and 5 min (horizontal) in a, c; 100 nm in e; 200 nm in g.
Figure 7
Figure 7. Vps4 induces subunit turnover and net growth of ESCRT-III assemblies.
(a) Time-lapse microscopy of ESCRT-III polymerization on supported lipid membranes. Snf7-AlexaFluor-488 was injected at t = 0 min. Vps2-Atto-565 and Vps24 were added at t = 36 min while maintaining Snf7 in the solution. At t = 45 min, Snf7-AlexaFluor-488 was removed and a mix containing Snf7-Atto-647N, Vps2-Atto-565, Vps24 and Vps4 was added, followed by addition of ATP at t = 54 min. (b) Kymograph of a single patch from a. (c) Mean fluorescence quantification of 35 patches as in b (4 fields of view within the shown experiment, and consistent results in 2 additional independent experiments using differently labeled proteins). Curves and shaded areas represent mean ± SEM. (d-e) Time-lapse microscopy of in vitro polymerization as in a, but for a mixed solution containing Snf7-AlexaFluor-488, Vps2-Atto-565, Vps24, and ATP (d) in the presence of Vps4, or (e) without Vps4. Representative images of 2 independent experiments per condition are shown. Scale bars, 10 μm in a, d, e; 5 µm (vertical) and 5 min (horizontal) in b.
Figure 8
Figure 8. Vps4 induces dynamic reorganization of ESCRT-III assemblies.
HS-AFM imaging of ESCRT-III polymers on supported lipid membranes. (a) Snf7 was polymerized on supported lipid membranes, followed by addition of Vps2, Vps24, and Vps4. Then, Mg2+ and ATP were added and imaging was started 5.5 min later (t = 0). Overlays highlight pre-formed spirals (blue) or newly formed spirals (orange). Bottom panels show a close-up of the nucleation of a new spiral. Images represent averages of 3 consecutive time frames to improve the signal-to-noise ratio. Scale bars, 200 nm (top panel) or 5 nm (bottom panel). (b) Quantification of spiral diameters from a (274 spirals from a representative experiment, from a total of 3 independent experiments). Dots represent single spirals; bars indicate medians. (c) Quantification of spiral diameters from Fig. 6g (175 spirals from a representative experiment, from a total of 6 independent experiments). Dots represent single spirals; bars indicate medians. (d) Tracking of spiral centers from a. (e) Tracking as in d, but for an experiment without ATP and Mg2+ as shown in Fig. 6g. (f) Quantification of mean velocity of spiral centers from d, e (n = 34 spirals for with ATP, and n = 31 spirals for without ATP, for a representative experiment per condition, from a total of 3 or 6 independent experiments, respectively). Statistical test using the two-sided Kolmogorov–Smirnov test yielded P = 4.441e-16 for spiral velocities in the presence of ATP relative to velocities in the absence of ATP. Dots represent single tracks; bars indicate medians. (g) Model of dynamic ESCRT-III assembly and constriction. Vps4 mediates continuous subunit turnover in ESCRT-III assemblies during growth and constriction. (1) At the tip, dynamic turnover of growth-inhibitory Vps2 and Vps24 subunits could sustain extension of inward-curving filaments. (2) At the core of filament bundles, Vps4-mediated subunit turnover could facilitate sliding of neighboring helical turns to promote constriction.

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