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Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis

Nature Cell Biology volume 19, pages 787–798 (2017)Cite this article

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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.

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Figure 1: ESCRT-III assemblies at the midbody dynamically turn over subunits in early and late abscission stages.
Figure 2: VPS4 is required for ESCRT-III accumulation and turnover.
Figure 3: VPS4 is required for constriction of the intercellular bridge.
Figure 4: Vps2 and Vps24 cooperatively bind Snf7 patches and inhibit ESCRT-III polymerization.
Figure 5: Vps2 and Vps24 polymerize side-by-side with Snf7 to form filament bundles.
Figure 6: Kinetics of ESCRT-III patch disassembly by Vps4.
Figure 7: Vps4 induces subunit turnover and net growth of ESCRT-III assemblies.
Figure 8: Vps4 induces dynamic reorganization of ESCRT-III assemblies.

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References

  1. Hurley, J. H. ESCRTs are everywhere. EMBO J. 34, 2398–2407 (2015).

    Article  CAS  Google Scholar 

  2. Hanson, P. I. & Cashikar, A. Multivesicular body morphogenesis. Annu. Rev. Cell Dev. Biol. 28, 337–362 (2012).

    Article  CAS  Google Scholar 

  3. Carlton, J. G. & Martin-Serrano, J. Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery. Science 316, 1908–1912 (2007).

    Article  CAS  Google Scholar 

  4. Guizetti, J. et al. Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments. Science 331, 1616–1620 (2011).

    Article  CAS  Google Scholar 

  5. Elia, N., Sougrat, R., Spurlin, T. A., Hurley, J. H. & Lippincott-Schwartz, J. Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission. Proc. Natl Acad. Sci. USA 108, 4846–4851 (2011).

    Article  CAS  Google Scholar 

  6. Lafaurie-Janvore, J. et al. ESCRT-III assembly and cytokinetic abscission are induced by tension release in the intercellular bridge. Science 339, 1625–1629 (2013).

    Article  CAS  Google Scholar 

  7. Mierzwa, B. & Gerlich, D. W. Cytokinetic abscission: molecular mechanisms and temporal control. Dev. Cell 31, 525–538 (2014).

    Article  CAS  Google Scholar 

  8. Vietri, M. et al. Spastin and ESCRT-III coordinate mitotic spindle disassembly and nuclear envelope sealing. Nature 522, 231–235 (2015).

    Article  CAS  Google Scholar 

  9. Olmos, Y., Hodgson, L., Mantell, J., Verkade, P. & Carlton, J. G. ESCRT-III controls nuclear envelope reformation. Nature 522, 236–239 (2015).

    Article  CAS  Google Scholar 

  10. Raab, M. et al. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death. Science 352, 359–362 (2016).

    Article  CAS  Google Scholar 

  11. Denais, C. M. et al. Nuclear envelope rupture and repair during cancer cell migration. Science 352, 353–358 (2016).

    Article  CAS  Google Scholar 

  12. Jimenez, A. J. et al. ESCRT machinery is required for plasma membrane repair. Science 343, 1247136 (2014).

    Article  Google Scholar 

  13. von Schwedler, U. K. et al. The protein network of HIV budding. Cell 114, 701–713 (2003).

    Article  CAS  Google Scholar 

  14. Bleck, M. et al. Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding. Proc. Natl Acad. Sci. USA 111, 12211–12216 (2014).

    Article  CAS  Google Scholar 

  15. Baietti, M. F. et al. Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat. Cell Biol. 14, 677–685 (2012).

    Article  CAS  Google Scholar 

  16. Choudhuri, K. et al. Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse. Nature 507, 118–123 (2014).

    Article  CAS  Google Scholar 

  17. Matusek, T. et al. The ESCRT machinery regulates the secretion and long-range activity of Hedgehog. Nature 516, 99–103 (2014).

    Article  CAS  Google Scholar 

  18. Henne, W. M., Stenmark, H. & Emr, S. D. Molecular mechanisms of the membrane sculpting ESCRT pathway. Cold Spring Harb. Perspect. Biol. 5, a016766 (2013).

    Article  Google Scholar 

  19. McCullough, J., Colf, L. A. & Sundquist, W. I. Membrane fission reactions of the mammalian ESCRT pathway. Annu. Rev. Biochem. 82, 663–692 (2013).

    Article  CAS  Google Scholar 

  20. Peel, S., Macheboeuf, P., Martinelli, N. & Weissenhorn, W. Divergent pathways lead to ESCRT-III-catalyzed membrane fission. Trends Biochem Sci. 36, 199–210 (2011).

    Article  CAS  Google Scholar 

  21. Hurley, J. H. & Hanson, P. I. Membrane budding and scission by the ESCRT machinery: it’s all in the neck. Nat. Rev. Mol. Cell Biol. 11, 556–566 (2010).

    Article  CAS  Google Scholar 

  22. Guizetti, J. & Gerlich, D. W. ESCRT-III polymers in membrane neck constriction. Trends Cell Biol. 22, 133–140 (2012).

    Article  CAS  Google Scholar 

  23. Schmidt, O. & Teis, D. The ESCRT machinery. Curr. Biol. 22, R116–R120 (2012).

    Article  CAS  Google Scholar 

  24. Schoneberg, J., Lee, I. H., Iwasa, J. H. & Hurley, J. H. Reverse-topology membrane scission by the ESCRT proteins. Nat. Rev. Mol. Cell Biol. 18, 5–17 (2017).

    Article  CAS  Google Scholar 

  25. Christ, L., Raiborg, C., Wenzel, E. M., Campsteijn, C. & Stenmark, H. Cellular functions and molecular mechanisms of the ESCRT membrane-scission machinery. Trends Biochem. Sci. 42, 42–56 (2017).

    Article  CAS  Google Scholar 

  26. Babst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T. & Emr, S. D. Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Dev. Cell 3, 271–282 (2002).

    Article  CAS  Google Scholar 

  27. Samson, R. Y., Obita, T., Freund, S. M., Williams, R. L. & Bell, S. D. A role for the ESCRT system in cell division in archaea. Science 322, 1710–1713 (2008).

    Article  CAS  Google Scholar 

  28. Teis, D., Saksena, S. & Emr, S. D. Ordered assembly of the ESCRT-III complex on endosomes is required to sequester cargo during MVB formation. Dev. Cell 15, 578–589 (2008).

    Article  CAS  Google Scholar 

  29. Teis, D., Saksena, S., Judson, B. L. & Emr, S. D. ESCRT-II coordinates the assembly of ESCRT-III filaments for cargo sorting and multivesicular body vesicle formation. EMBO J. 29, 871–883 (2010).

    Article  CAS  Google Scholar 

  30. Saksena, S., Wahlman, J., Teis, D., Johnson, A. E. & Emr, S. D. Functional reconstitution of ESCRT-III assembly and disassembly. Cell 136, 97–109 (2009).

    Article  CAS  Google Scholar 

  31. Wollert, T., Wunder, C., Lippincott-Schwartz, J. & Hurley, J. H. Membrane scission by the ESCRT-III complex. Nature 458, 172–177 (2009).

    Article  CAS  Google Scholar 

  32. Carlson, L. A. & Hurley, J. H. In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters. Proc. Natl Acad. Sci. USA 109, 16928–16933 (2012).

    Article  CAS  Google Scholar 

  33. Babst, M., Wendland, B., Estepa, E. J. & Emr, S. D. The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J. 17, 2982–2993 (1998).

    Article  CAS  Google Scholar 

  34. Obita, T. et al. Structural basis for selective recognition of ESCRT-III by the AAA ATPase Vps4. Nature 449, 735–739 (2007).

    Article  CAS  Google Scholar 

  35. Stuchell-Brereton, M. D. et al. ESCRT-III recognition by VPS4 ATPases. Nature 449, 740–744 (2007).

    Article  CAS  Google Scholar 

  36. Lata, S. et al. Helical structures of ESCRT-III are disassembled by VPS4. Science 321, 1354–1357 (2008).

    Article  CAS  Google Scholar 

  37. Adell, M. A. et al. Coordinated binding of Vps4 to ESCRT-III drives membrane neck constriction during MVB vesicle formation. J. Cell Biol. 205, 33–49 (2014).

    Article  CAS  Google Scholar 

  38. Yang, B., Stjepanovic, G., Shen, Q., Martin, A. & Hurley, J. H. Vps4 disassembles an ESCRT-III filament by global unfolding and processive translocation. Nat. Struct. Mol. Biol. 22, 492–498 (2015).

    Article  CAS  Google Scholar 

  39. Ghazi-Tabatabai, S. et al. Structure and disassembly of filaments formed by the ESCRT-III subunit Vps24. Structure 16, 1345–1356 (2008).

    Article  CAS  Google Scholar 

  40. Pires, R. et al. A crescent-shaped ALIX dimer targets ESCRT-III CHMP4 filaments. Structure 17, 843–856 (2009).

    Article  CAS  Google Scholar 

  41. Henne, W. M., Buchkovich, N. J., Zhao, Y. & Emr, S. D. The endosomal sorting complex ESCRT-II mediates the assembly and architecture of ESCRT-III helices. Cell 151, 356–371 (2012).

    Article  CAS  Google Scholar 

  42. Shen, Q. T. et al. Structural analysis and modeling reveals new mechanisms governing ESCRT-III spiral filament assembly. J. Cell Biol. 206, 763–777 (2014).

    Article  CAS  Google Scholar 

  43. Chiaruttini, N. et al. Relaxation of loaded ESCRT-III spiral springs drives membrane deformation. Cell 163, 866–879 (2015).

    Article  CAS  Google Scholar 

  44. McCullough, J. et al. Structure and membrane remodeling activity of ESCRT-III helical polymers. Science 350, 1548–1551 (2015).

    Article  CAS  Google Scholar 

  45. McMillan, B. J. et al. Electrostatic interactions between elongated monomers drive filamentation of Drosophila shrub, a metazoan ESCRT-III protein. Cell Rep. 16, 1211–1217 (2016).

    Article  CAS  Google Scholar 

  46. Hanson, P. I., Roth, R., Lin, Y. & Heuser, J. E. Plasma membrane deformation by circular arrays of ESCRT-III protein filaments. J. Cell Biol. 180, 389–402 (2008).

    Article  CAS  Google Scholar 

  47. Cashikar, A. G. et al. Structure of cellular ESCRT-III spirals and their relationship to HIV budding. Elife 3, e02184 (2014).

    Article  Google Scholar 

  48. Babst, M., Sato, T. K., Banta, L. M. & Emr, S. D. Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p. EMBO J. 16, 1820–1831 (1997).

    Article  CAS  Google Scholar 

  49. Lee, I. H., Kai, H., Carlson, L. A., Groves, J. T. & Hurley, J. H. Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly. Proc. Natl Acad. Sci. USA 112, 15892–15897 (2015).

    Article  CAS  Google Scholar 

  50. Roll-Mecak, A. & Vale, R. D. Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature 451, 363–367 (2008).

    Article  CAS  Google Scholar 

  51. Elia, N., Fabrikant, G., Kozlov, M. M. & Lippincott-Schwartz, J. Computational model of cytokinetic abscission driven by ESCRT-III polymerization and remodeling. Biophys J. 102, 2309–2320 (2012).

    Article  CAS  Google Scholar 

  52. Kline-Smith, S. L. & Walczak, C. E. Mitotic spindle assembly and chromosome segregation: refocusing on microtubule dynamics. Mol. Cell 15, 317–327 (2004).

    Article  CAS  Google Scholar 

  53. Pollard, T. D., Blanchoin, L. & Mullins, R. D. Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu. Rev. Biophys. Biomol. Struct. 29, 545–576 (2000).

    Article  CAS  Google Scholar 

  54. Zemp, I. et al. Distinct cytoplasmic maturation steps of 40S ribosomal subunit precursors require hRio2. J. Cell Biol. 185, 1167–1180 (2009).

    Article  CAS  Google Scholar 

  55. Poser, I. et al. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat. Methods 5, 409–415 (2008).

    Article  CAS  Google Scholar 

  56. Hein, M. Y. et al. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 163, 712–723 (2015).

    Article  CAS  Google Scholar 

  57. Schmitz, M. H. & Gerlich, D. W. Automated live microscopy to study mitotic gene function in fluorescent reporter cell lines. Methods Mol. Biol. 545, 113–134 (2009).

    Article  CAS  Google Scholar 

  58. Lukinavicius, G. et al. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat. Methods 11, 731–733 (2014).

    Article  CAS  Google Scholar 

  59. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

  60. Morita, E. et al. Human ESCRT-III and VPS4 proteins are required for centrosome and spindle maintenance. Proc. Natl Acad. Sci. USA 107, 12889–12894 (2010).

    Article  CAS  Google Scholar 

  61. Wollert, T. & Hurley, J. H. Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature 464, 864–869 (2010).

    Article  CAS  Google Scholar 

  62. Aguet, F., Van De Ville, D. & Unser, M. Model-based 2.5-d deconvolution for extended depth of field in brightfield microscopy. IEEE Trans. Image Process 17, 1144–1153 (2008).

    Article  Google Scholar 

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Acknowledgements

D.W.G. has received financial support from the European Community’s Seventh Framework Programme FP7/2007-2013 under grant agreements no. 241548 (MitoSys) and no. 258068 (Systems Microscopy), from an ERC Starting Grant (agreement no. 281198), from the Wiener Wissenschafts-, Forschungs- und Technologiefonds (WWTF; project no. LS14-009), and from the Austrian Science Fund (FWF; project no. SFB F34-06). B.E.M. has received a PhD fellowship from the Boehringer Ingelheim Fonds. A.R. acknowledges funding from: Human Frontier Science Program (HFSP), Young Investigator Grant no. RGY0076-2008: the European Research Council (ERC), starting (consolidator) grant no. 311536-MEMFIS: the Swiss National Fund for Research, grants no. 131003A_130520 and no. 131003A_149975. N.C. acknowledges the European Commission for the Marie-Curie post-doctoral fellowship CYTOCUT no. 300532-2011. J.M.v.F. acknowledges funding by an EMBO long-term fellowship (ALTF 1065-2015). T.M.-R. has received funding from the Deutsche Forschungsgemeinschaft (DFG) grant MU1423/4-1. S.S. acknowledges funding by an ANR grant ANR-Nano (ANR-12-BS10-009-01) and a European Research Council (ERC) Starting Grant (no. 310080, MEM-STRUCT-AFM). The authors thank D. Teis, M. Alonso Y Adell, C. Campsteijn and J. Gruenberg for comments on the manuscript, the IMBA/IMP/GMI BioOptics core facility for technical support, the EM Facility of the Vienna Biocenter Core Facilities (VBCF), who performed parts of the sample preparation and electron microscopy, F. Humbert for protein purification, C. Sommer and R. Höfler for statistical advice, C. Blaukopf for technical support, W. Reiter for providing S. cerevisiae genomic DNA, and Life Science Editors for editing assistance.

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Author notes

  1. Julia König

    Present address: Electron Microscopy Unit, Francis Crick Institute, London, NW1 1AT, UK

  2. Simon Scheuring

    Present address: Departments of Anesthesiology and Physiology & Biophysics, Weill Cornell Medicine, New York, New York, 10065, USA

  3. Beata E. Mierzwa, Nicolas Chiaruttini and Lorena Redondo-Morata: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), AT-1030, Vienna, Austria

    Beata E. Mierzwa & Daniel W. Gerlich

  2. Department of Biochemistry, University of Geneva, CH-1211, Geneva, Switzerland

    Nicolas Chiaruttini, Joachim Moser von Filseck, Jorge Larios & Aurélien Roux

  3. U1006 INSERM, Aix-Marseille Université, 13009, Marseille, France

    Lorena Redondo-Morata & Simon Scheuring

  4. Experimental Center, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, D-01307, Dresden, Germany

    Julia König & Thomas Müller-Reichert

  5. Max Planck Institute of Molecular Cell Biology and Genetics, D-01307, Dresden, Germany

    Ina Poser

  6. Swiss National Centre for Competence in Research Programme Chemical Biology, CH-1211, Geneva, Switzerland

    Aurélien Roux

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Contributions

B.E.M. designed, conducted and analysed all cell biological experiments, and analysed part of the HS-AFM data. N.C. designed, conducted and analysed in vitro reconstitution experiments based on fluorescence microscopy. L.R.-M. designed, conducted and analysed HS-AFM experiments. J.M.v.F. and N.C. designed, conducted and analysed electron microscopy of in vitro-assembled ESCRT-III polymers. J.K. and T.M.-R. designed, conducted and analysed electron microscopy experiments of intercellular bridges. J.L. established the CHMP4B purification and produced labelled CHMP4B. I.P. generated HeLa cells stably expressing mmVPS4B-LAP. B.E.M., N.C., D.W.G., A.R. and S.S. conceived the project, analysed data and wrote the manuscript.

Corresponding authors

Correspondence to Aurélien Roux or Daniel W. Gerlich.

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Integrated supplementary information

Supplementary Figure 1 Validation of CHMP4B depletion and FRAP fitting of a single exponential model with a variable mobile fraction.

(a) Western blot of whole cell lysates from HeLa cells stably expressing mmCHMP4B-LAP from an endogenous promoter, probed by anti-CHMP4B antibody, 48 h after transfection of an siRNA specifically targeting hsCHMP4B, or non-targeting negative control siRNA. Anti-α-actin antibodies were used as a loading control. Unprocessed scans are shown in Supplementary Fig. 8a, b. Western Blot was repeated in 2 independent experiments. (b,d) Comparison of a single exponential fit that allows an immobile fraction f(t) = A∗(1 − e(−k∗t)), and a double exponential fit f(t) = A1 ∗ (1 − e(−k1∗t)) + (1 − A1∗)(1 − e(−k2∗t)) to data in Fig. 1c (n = 18 cells from 4 independent experiments). (b) Overlay of the fitted curves. Points and shaded area indicate mean ± s.e.m. of fluorescence; dashed lines indicate fits of single or double exponential functions. (c) Residuals of the fits from b. (d) Residence times and mobile fractions derived from single exponential fits and highly mobile pools from double exponential fits. Dots represent individual cells.

Supplementary Figure 2 Different LAP-tagged ESCRT-III subunits do not perturb abscission and have similar dynamics.

(a–d) Abscission timing was measured by time-lapse microscopy and related to expression levels based on GFP fluorescence. Expression was induced in HeLa cell lines carrying stable integrations of (a) hsCHMP2B-LAP (n = 50 cells from 4 independent experiments), (b) hsCHMP3-LAP (n = 57 cells from 3 independent experiments), (c) hsCHMP4B-LAP (n = 21 cells from 3 independent experiments), or (d) mmCHMP4B-LAP (n = 25 cells from 3 independent experiments). Each dot represents a single cell. (e–g) Quantification of FRAP in telophase cells at pre-constriction stages for (e) hsCHMP2B-LAP (n = 19 cells from 3 independent experiments), (f) hsCHMP3-LAP (n = 16 cells from 3 independent experiments), or (g) hsCHMP4B-LAP (n = 24 cells from 3 independent experiments). Points and shaded areas indicate mean ± s.e.m.; dashed lines indicate optimal fits in the upper panels of single exponential functions f(t) = A∗(1 − e(−k∗t)) and double exponential functions f(t) = A1∗(1 − e(−k1∗t)) + (1 − A1∗)(1 − e(−k2∗t)) to the mean recovery curves, with the residuals plotted in the lower panels.

Supplementary Figure 3 LAP-tagged VPS4B is functional and VPS4A/B depletion perturbs ESCRT-III assembly at the midbody.

(a,b) Western blot of whole cell lysates from cells stably expressing mmVPS4B-LAP from an endogenous promoter, probed by (a) anti-GFP or (b) anti-VPS4B antibodies, 48 h after transfection of siRNAs specifically targeting hsVPSP4A and hsVPS4B, or non-targeting negative control siRNA. Anti-GAPDH antibodies were used as a loading control. The asterisk marks a non-specific band. Unprocessed scans are shown in Supplementary Fig. 8. (c–f) Western Blots were repeated in 2 independent experiments. (c) Quantification of mmCHMP4B-LAP accumulation at the midbody 48 h after transfection of siRNAs targeting hsVPSP4A/B (n = 19 cells from 3 independent experiments), or non-targeting negative control siRNAs (n = 15 cells from 3 independent experiments), normalized and temporally aligned to the time point of complete cleavage furrow ingression (time point 0). Curves and shaded areas represent mean ± s.e.m. (d,e) Cytoplasmic levels of (d) hsCHMP2B-LAP or (e) hsCHMP3-LAP 20 h after transfection of siRNAs targeting hsVPSP4A/B (n = 39 cells from 2 independent experiments, or n = 79 cells from 4 independent experiments, respectively), or non-targeting negative control siRNAs (n = 76 cells from 2 independent experiments, or n = 59 cells from 4 independent experiments, respectively). Dots represent individual cells; bars indicate medians.

Supplementary Figure 4 VPS4 is required for rapid incorporation of microinjected recombinant CHMP4B into midbody-localized ESCRT-III.

(a) Recombinant human CHMP4B-Atto-565 was microinjected into telophase HeLa cells stably expressing mouse CHMP4B-LAP from an endogenous promoter, stained with SiR-tubulin. Time 0 indicates first image after injection. (b) As in a but 48 h after transfection of siRNAs specifically targeting hsVPSP4A/B. (c) Quantification of hsCHMP4B-Atto-565 midbody incorporation in untreated cells (n = 8 cells from 3 independent experiments), or 48 h after transfection of siRNAs targeting hsVPSP4A/B (n = 10 cells from 3 independent experiments). Curves and shaded areas indicate mean ± s.e.m. Fluorescence intensity of injected CHMP4B-Atto-565 was measured in midbody regions based on the CHMP4B-LAP signal. To correct for cytoplasmic background within the midbody region, the mean intensity of the surrounding area was subtracted from the region of interest. Scale bars, 5 μm, or 1 μm (inset) in a,b.

Supplementary Figure 5 Validation of in vitro ESCRT-III reconstitution assay.

(a) Localization of budding yeast Snf7-LAP in live HeLa cells during abscission. (b) FRAP of Snf7-LAP at a midbody at late abscission stages. (c) Fluorescence recovery and double exponential fit for early and late abscission as in b (n = 10 cells from 3 independent experiments). Points and shaded area indicate mean ± s.e.m. (d) Highly mobile fraction and residence time for data shown in c. Dots represent individual cells; bars indicate medians. (e–g) ESCRT-III patches form exclusively on lipid membranes. Lipid membranes were labelled with DOPE-Atto647N (left) in a flow chamber. Snf7-Alexa488 was then added, followed by addition of Vps2-Atto565 and Vps24. (e) Regions covered by burst GUVs are visible as dark regions in the Vps2 channel because they lack unspecific background fluorescence. Images show a representative experiment, with consistent observations in 2 additional independent experiments with differently labeled proteins. (f,g) Kymographs of single ESCRT-III patches as indicated by lines in in e. Patch 1 (f) localizes to central area on the membrane and shows unconstrained growth, whereas patch 2 (g) shows that growth stops when ESCRT-III patches reach the edge of the membrane. Vps2-Atto565 and Vps24 were injected together in two steps, first at 1 nM where they did not prevent Snf7 growth, second at 10 nM where they bind and prevent growth. Scale bars, 5 μm or 1 μm (inset) in a; 1 μm in b; 10 μm in e; 5 μ m (vertical) and 5 min (horizontal) in f,g.

Supplementary Figure 6 Vps2 and Vps24 cooperatively bind Snf7 patches and inhibit Snf7 polymerization.

(a) Long-term time-lapse microscopy of fluorescently labeled budding yeast ESCRT-III subunits during polymerization on supported lipid membranes in microfluidic chambers. A solution of Snf7-AlexaFluor-488 was injected into the flow chamber at t = 0 min; Vps2-Atto-565 and unlabeled Vps24 were added at t = 22 min. (b) Quantification of patch fluorescence as in a (n = 7 patches from 4 fields of view within the shown experiment, and additional independent experiment is shown in Fig. 4a–d). Curves and shaded areas indicate mean ± s.e.m. (c) The addition of Vps2 and Vps24 inhibits patch growth independently of their size. Kymograph of multiple patches of a representative experiment where Snf7-AlexaFluor-647N was added at t = 0 min, followed by addition of Vps2-Atto-565 and unlabeled Vps24 at t = 29 min. Similar experiments are shown in a, and Fig. 4a–d. (d) Snf7-AlexaFluor-647N was added at t = 0 min, followed by addition of Vps2-Atto-565 at t = 26 min, and Vps24-AlexaFluor-488 at t = 33 min. (e) Kymograph of an ESCRT-III patch from d (representative example from 24 patches in 4 different fields of view within the shown experiment, and 1 additional independent experiment with differently labelled proteins). Scale bars, 5 μm (vertical) or 5 min (horizontal) in a,c,e; 10 μm in d.

Supplementary Figure 7 ESCRT-III patch growth and disassembly kinetics under various conditions.

(a) Representative patches of three experiments with different Vps4 concentrations. For each experiment, Snf7-AlexaFluor-488 patches were polymerized, then Snf7 was washed out while Vps2-Atto-565 and Vps24 were added for ∼10 min. ATP and Vps4 (at 1, 2, or 4 μM, respectively) were then added to the solutions. Scale bars, 5 μm (vertical) or 5 min (horizontal). (b) Quantification of mean fluorescence of several patches shown in e (n = 16 patches for 1 μM Vps4, n = 14 patches for 2 or 4 μM Vps4, from 4 fields of view per condition from 1 independent experiment). Curves and shaded areas indicate mean ± s.e.m.; dotted lines indicate optimal fits to f(t) = (1 − a) ∗ e−(t/τ) + a. Fitted parameters for 1 μM Vps4: τ = 4.7 ± 1.0 min, a = 37 ± 3%; 2 μM Vps4: τ = 2.6 ± 1.5 min, a = 38 ± 2%; and for 4 μM Vps4: τ = 1.4 ± 0.5 min, a = 30 ± 3%. (c) Quantification of patch radial growth speed in presence of Snf7/Vps2/Vps24/Vps4 + ATP as a function of Vps4 concentration. Patches were pre-nucleated with 500 nM Snf7, followed by addition of Vps2/Vps24, ATP and different concentrations of Vps4 (0.5, 1, 2, or 4 μM). Dots represent individual patches from 4 independent experiments per condition; bars and error bars indicate mean ± SD.

Supplementary Figure 8 Unprocessed images of Western Blots.

Full scans of Western Blots shown in (a,b) Supplementary Fig. 1a, (c,d) Supplementary Fig. 3a, and (e–f) Supplementary Fig. 3b. Red rectangles indicate cropped regions shown in figures.

Supplementary information

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Dynamic subunit turnover in CHMP4B at the midbody before and during constriction.

FRAP of mmCHMP4B-LAP (green) at the midbody, stained with SiR-tubulin (magenta). Left panel shows early abscission (before constriction), and right panel shows late abscission (during constriction). Circles indicate photobleaching region; time point 0 is the first frame recorded immediately after photobleaching. Scale bars, 1 μm. (AVI 315 kb)

Accumulation of CHMP2B and CHMP3 at the midbody.

3-D live cell confocal microscopy of the intercellular bridge during telophase in HeLa cells expressing hsCHMP2B-LAP (left) or hsCHMP3-LAP (right), stained with SiR-tubulin. Time point 0 indicates complete disassembly of the midbody-associated microtubule bundle during abscission. Scale bars, 1 μm. (AVI 1238 kb)

Accumulation of CHMP4B and VPS4B at the midbody.

Telophase intercellular bridges of HeLa cells expressing mmCHMP4B-LAP (left) or mmVPS4B-LAP (right), stained with SiR-tubulin. Time point 0 indicates complete disassembly of the midbody-associated microtubule bundle during abscission. Scale bars, 1 μm. (AVI 1278 kb)

Vps2 and Vps24 inhibit Snf7 polymer growth.

Time-lapse microscopy of fluorescently labeled budding yeast ESCRT-III subunits during polymerization on supported lipid membranes in a microfluidic flow chamber. A solution of Snf7-AlexaFluor-488 (green) was injected into the flow chamber at t = 0 min. Vps2-Atto-565 (magenta) together with unlabeled Vps24 were added after 22 min while maintaining initial concentration of Snf7 in the solution, as depicted in the timeline above the movie. Insets show kymographs of a selected patch indicated by a yellow line. Dashed yellow line indicates time point of solution exchange in the flow chamber. Scale bar, 10 μm. (MOV 5347 kb)

Vps2 requires Vps24 to bind Snf7 polymers.

Time-lapse microscopy of fluorescently labeled budding yeast ESCRT-III subunits during polymerization on supported lipid membranes in a microfluidic flow chamber. A solution of Snf7-AlexaFluor-647N was injected into the flow chamber at t = 0 min. Vps2-Atto-565 was added after 25 min, and Vps24-AlexaFluor-488 was added after 32 min while maintaining the initial Snf7 concentration in the solution, as depicted in the timeline above the pictures. Insets show kymographs of a selected patch indicated by a yellow line. Dashed yellow lines indicate time points of solution exchange in the flow chamber. Scale bar, 10 μm. (MOV 2547 kb)

Vps24 requires Vps2 to bind Snf7 polymers.

Time-lapse microscopy of fluorescently labeled budding yeast ESCRT-III subunits during polymerization on supported lipid membranes in a microfluidic flow chamber. A solution of Snf7-AlexaFluor-647N was injected into the flow chamber at t = 0 min. Vps24-AlexaFluor-488 was added after 19 min, and Vps2-Atto-565 was added after 30 min while maintaining the initial Snf7 concentration in the solution, as depicted in the timeline above the pictures. Insets show kymographs of a selected patch indicated by a yellow line. Dashed yellow lines indicate time points of solution exchange in the flow chamber. Scale bar, 10 μm. (MOV 4310 kb)

Snf7 polymers have very low intrinsic subunit dissociation rates.

Time-lapse microscopy of Snf7 patch polymerization on supported lipid membranes in a microfluidic flow chamber. Snf7-AlexaFluor-488 was added, and then removed from the buffer solution at t = 34 min. Vps2-Atto-565 and unlabeled Vps24 were simultaneously added to the solution at t = 50 min, as depicted in the timeline above the pictures. Insets show kymographs of a selected patch indicated by a yellow line. Dashed yellow lines indicate time points of solution exchange in the flow chamber. Scale bar, 10 μm. (MOV 1186 kb)

Vps4 rapidly disassembles ESCRT-III patches in the absence of soluble Snf7.

Time-lapse microscopy of Snf7 polymer patches on supported lipid membranes in a microfluidic flow chamber. A solution of Snf7-AlexaFluor-488 was injected into the flow chamber and incubated until patches polymerized on the supported lipid membrane. At t = 34 min Snf7 was removed from the solution and Vps2, Vps24, Vps4, and ATP were injected, as depicted in the timeline above. Insets show kymographs of a selected patch indicated by a yellow line. The dashed yellow line indicates the time point of solution exchange in the flow chamber. Scale bar, 10 μm. (MOV 999 kb)

Vps4 induces shrinkage of ESCRT-III spirals in the presence of ATP.

HS-AFM movie showing disassembly of ESCRT-III spirals by Vps4. ESCRT-III patches were assembled by polymerization of Snf7, and subsequent 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). Scale bar, 100 nm. (AVI 9522 kb)

ESCRT-III spirals form an immobile and stable array in presence of Vps4 and absence of ATP.

Snf7 was polymerized into on supported lipid membranes, followed by addition of Vps2 and Vps24. Then, Vps4 but no ATP was added, and imaging was started 30 s later (t = 0). Scale bar, 200 nm. (AVI 5241 kb)

Vps4 counteracts growth-inhibition imposed by Vps2/Vps24 and promotes rapid turnover of Snf7 subunits.

Visualization of subunit exchange in ESCRT-III polymers by time-lapse microscopy using two distinctly labeled Snf7 pools. Snf7-AlexaFluor-488 was injected into the flow chamber until polymer patches formed on supported lipid membranes. Vps2-Atto-565 and Vps24 were added at t = 36 min while maintaining Snf7-AlexaFluor-488 in the solution. At t = 45 min, soluble Snf7-AlexaFluor-488 was removed from the solution, and a mix containing Snf7-Atto-647N, Vps2-Atto-565, Vps24 and Vps4 was injected. At t = 54 min, the same mix plus ATP was added to the solution. Insets show kymographs of a selected patch indicated by a yellow line. Dashed yellow lines indicate time points of exchanging the solution in the flow chamber. Scale bar, 10 μm. (MOV 5433 kb)

Vps4 induces dynamic reorganization of ESCRT-III assemblies in the presence of soluble ESCRT-III subunits.

HS-AFM movie showing dynamic reorganization of ESCRT-III spirals by Vps4. ESCRT-III patches were assembled by polymerization of Snf7, and subsequent addition of Vps2 and Vps24, Vps4, and ATP/Mg2 + . Imaging was started 5.5 min later (t = 0). Scale bar, 200 nm. (AVI 6661 kb)

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Mierzwa, B., Chiaruttini, N., Redondo-Morata, L. et al. Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis. Nat Cell Biol 19, 787–798 (2017). https://doi.org/10.1038/ncb3559

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