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Dynamin regulates the dynamics and mechanical strength of the actin cytoskeleton as a multifilament actin-bundling protein

Nature Cell Biology volume 22, pages 674–688 (2020)Cite this article

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Abstract

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12–16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.

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Fig. 1: Shi promotes myoblast fusion by facilitating PLS invasion at the fusogenic synapse.
Fig. 2: Shi bundles actin filaments.
Fig. 3: hDyn1 and hDyn2 bundle actin filaments.
Fig. 4: The PRD of Shi mediates Shi–actin interaction.
Fig. 5: Dynamin forms a helix that captures multiple actin filaments at the outer rim.
Fig. 6: GTP hydrolysis disassembles Shi helices to allow branched actin polymerization and cell–cell fusion.
Fig. 7: Actin bundling stimulates the GTP hydrolysis rate of Shi.
Fig. 8: Dynamin–actin interaction in vivo.

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Data availability

All data that support the findings of this study are available from the corresponding author upon reasonable request. Source data for Figs. 1–7 and Extended Data Figs. 1, 3–5 and 7–9 are presented with the paper.

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Acknowledgements

We thank the Bloomington Drosophila Stock Center for the fly stocks, B. Paterson for the myosin heavy chain antibody, K. Reed and A. Mohanakrishnan for help with protein purification and GTPase activity measurement, M. Mikolaj for help with generating the model in IMOD, H. He and H. Wang for help with generating the cryo-tomograms using SerialEM, K. Chinthalapudi for providing the actin filament for modelling in Chimera, and the Cryo-Electron Microscopy Core, NIDDK, NIH for Cryo-ET analyses. This work was supported by: NIH grants (R01 AR053173 and R01 GM098816), an American Heart Association Established Investigator Award and a HHMI Faculty Scholar Award to E.H.C.; an NIH grant (R01 GM095977) to T.M.S.; an NIH grant (R01 GM42455) and Welch Foundation grant (I-1823) to S.L.S.; an NIH grant (R01 GM104032) to M.G.; an NIH grant (R01 AI083359), HHMI and Simons Foundation grant and Welch Foundation grant (I-1704) to N.M.A.; and an NIH grant (R01 GM127673), Chan Zuckerberg Biohub Investigator Award and HHMI Faculty Scholar Award to A.F. M.K.R. is an HHMI investigator. R.Z. was supported by an American Heart Association postdoctoral fellowship. D.M.L. was supported by a Canadian Institute of Health Research postdoctoral fellowship. N.G. was supported by an American Heart Association predoctoral fellowship. J.A.D. was supported by a National Research Service Award from NIDDK (F32 DK101188). The electron microscopy imaging experiments at UT Southwestern were supported in part by an NIH grant (1S10OD021685-01A1).

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Author notes

  1. These authors contributed equally: Ruihui Zhang, Donghoon M. Lee, John R. Jimah.

Authors and Affiliations

  1. Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX, USA

    Ruihui Zhang, Donghoon M. Lee, Peter Keene, Pratima Pandey, Benjamin Ravaux & Elizabeth H. Chen

  2. Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, USA

    John R. Jimah & Jenny E. Hinshaw

  3. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Nathalie Gerassimov, Sangjoon Kim, Delgermaa Luvsanjav, Ji Hoon Kim & Elizabeth H. Chen

  4. Department of Biology, University of Pennsylvania, Philadelphia, PA, USA

    Changsong Yang & Tatyana M. Svitkina

  5. Department of Physics and Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA

    Jonathan Winkelman & Margaret Gardel

  6. Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA

    Marcel Mettlen, Sandra L. Schmid & Elizabeth H. Chen

  7. Department of Microbiology, UT Southwestern Medical Center, Dallas, TX, USA

    Michael E. Abrams & Neal M. Alto

  8. Department of Physiology, University of California, San Francisco, San Francisco, CA, USA

    Raghav Kalia

  9. Department of Biophysics and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX, USA

    Jonathon A. Ditlev & Michael K. Rosen

  10. Trans-NIH Shared Resource on Biomedical Engineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, Bethesda, MD, USA

    Guofeng Zhang

  11. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA

    Adam Frost

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  1. Ruihui Zhang
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Contributions

S.K. initiated the project. R.Z., D.M.L., N.G., S.K. and E.H.C. designed the project, performed the experiments and discussed the data. R.Z. carried out the biochemistry, negative-stain electron microscopy and in vitro imaging experiments in Figs. 2 (except for Fig. 2e–g), 3, 4a–f, 5a,b, 6a–i and 7, Extended Data Figs. 3–5 (except for Extended Data Figs. 3e and 5b,c), 8 and 10a and Supplementary Videos 2 and 10–15. D.M.L. carried out the fly genetics, cell culture and in vivo super-resolution and electron microscopy imaging experiments in Figs. 1d–g, 4g–i, 6j,k and 8 and Extended Data Figs. 1e, 2g–j, 5b,c, 6, 9 and 10b. N.G. carried out the fly genetics and cell culture experiments in Figs. 1a,b,h,f and 6j,k and Extended Data Figs. 1a–d, 2f and 3e. S.K. performed the live imaging and immunostaining experiments in Fig. 1c, Extended Data Fig. 2a–e and Supplementary Video 1. R.Z. and E.H.C. collaborated with: J.R.J. and J.E.H. on the cryo-ET experiments in Fig. 5, Extended Data Figs. 7 and 10a and Supplementary Videos 5–9; J.W. and M.G. on the TIRF imaging experiment in Fig. 2f,g and Supplementary Video 4; R.K. and A.F. on the experiments with human Drp1 in Extended Data Fig. 5a; and J.A.D. and M.K.R. on the time-lapse imaging of actin bundling in Fig. 2e and Supplementary Video 3. D.M.L. and E.H.C. collaborated with C.Y. and T.M.S. on the PREM experiments in Fig. 8c–e and Extended Data Fig. 9b; M.M. and S.L.S. on the endocytosis experiments in Fig. 4i and Extended Data Fig. 6b; and M.E.A. and N.M.A. on the actin comet tail experiments in Extended Data Fig. 9e,f. D.M.L., N.G., D.L. and E.H.C. collaborated with G.Z. on the electron microscopy experiments in Fig. 1i. P.K. worked with D.M.L. on generating the constructs and performing the cell culture experiments in Figs. 1d–f and 6j,k and Extended Data Figs. 2h–j, 5b,c and 6. P.P. worked with R.Z. on the negative-stain electron microscopy experiments in Figs. 2–6 and Extended Data Figs. 3–5. B.R. worked with R.Z. on the lipid nanotube experiments in Figs. 2j, 3e and 5b and Extended Data Fig. 4c. J.H.K. worked with N.G. on the actin foci invasion measurements in Fig. 1h. R.Z., D.M.L. and E.H.C. made the figures and wrote the paper. All authors commented on the manuscript.

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Correspondence to Elizabeth H. Chen.

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Extended data

Extended Data Fig. 1 Shi is required for myoblast fusion.

a, Myoblast fusion defect in shits2 mutant embryo. The somatic muscles in stage 15 wild-type (WT) and shits2 mutant embryos were labeled by anti-muscle myosin heavy chain (MHC) antibody. Note the presence of unfused mononucleated myoblasts in shits2 mutant embryo at the restrictive temperature (several selected unfused FCMs indicated by arrowheads). n = 11, 8, 40 and 74 (top left, bottom left, top right, bottom right) embryos were imaged, with similar results. See quantification in Fig. 1b. b, Myoblast fusion defects in shits1 mutant embryo. The somatic muscles in stage 15 wild-type and shits1 mutant embryos were labeled by anti-Tropomyosin antibody. Several selected unfused FCMs in shits1 mutant embryo at the restrictive temperature are indicated by arrowheads. n = 8 and 24 (left to right) embryos were imaged, with similar results. See quantification in (c). c, Quantification of the fusion index of genotypes shown in (b). n = 40 DA1 muscles analyzed for wild type and shits1, respectively. Mean ± s.d. values are shown in the dot plot, and significance was determined by the two-tailed Student’s t-test. d, Phenotypic rescue of myoblast fusion in shits2 mutant embryos. Note that expression of Shi-GFP in all muscles (twi-GAL4) or FCMs (sns-GAL4), but not in founder cells (rP298-GAL4), restored myoblast fusion in shits2 embryos at the restrictive temperature. Several selected unfused FCMs indicated by arrowheads. n = 22, 7 and 8 (left to right) embryos were imaged, with similar results. See quantification in Fig. 1b. e, Myoblast fusion is normal in CortactinM/Z mutant embryos. The somatic muscles in late stage 15 wild type and CortactinM/Z mutant embryos were labeled by anti-MHC antibody. n = 20 and 15 (top to bottom) embryos were imaged, with similar results. Scale bars: 20 μm (a, b, d and e). Numerical source data are provided with the paper.

Source Data Extended Data Fig. 1

Extended Data Fig. 2 Localization of Shibire at the fusogenic synapse in Drosophila embryos and S2R+ cells.

a–e, The recruitment of Shi to the fusogenic synapse depends on the FCM-specific cell adhesion molecule (CAM) Sns, but not on actin polymerization regulators. Stage 14 embryos of various genotypes were labeled with anti-Shi (endogenous Shi; green), phalloidin (F-actin; red), and anti-Duf (founder cell-specific CAM enriched at the fusogenic synapse; blue). Shi and F-actin were enriched at the fusogenic synapse in wild type (a), waspm/z (c), sltr (d), and kette (e) mutant embryos, but not in the sns mutant embryo (b). n = 72, 60, 92, 96, 65 fusogenic synapses (left to right) were imaged, with similar results. f–g, Two endocytosis-related proteins, Clc and AP-2α, are not enriched at the fusogenic synapse. Stage 14 embryos expressing Clc-GFP (f) or AP-2α-GFP (g) were labeled with phalloidin (red) and anti-Duf (blue). The FCM is outlined. n = 55 and 54 fusogenic synapses (left to right) were imaged, with similar results. h, Eff-1-mCherry localization in wild type and Shi knockdown S2R+ cells. Cells expressing Eff-1-mCherry (green) and Sns-GFP (blue) were labeled with phalloidin (red). Note the indistinguishable Eff-1-mCherry enrichment (arrowheads) at the fusogenic synapse in wild type and Shi knockdown cells. n = 65 (wild type) and 70 (Shi knockdown) fusogenic synapses were imaged, with similar results. i,j, Shi colocalizes with F-actin-enriched structures in S2R+ cells. Cells expressing Shi-3xHA (green) and Sns-GFP (blue) with (i) or without (j) Eff-1 were labeled with phalloidin (red). Note that Shi-3xHA colocalized with the F-actin and Sns enriched areas (arrowheads) regardless of Eff-1 expression. n = 92 (i) or 65 (j) fusogenic synapses were imaged, with similar results. Scale bars: 5 μm (a–e and h), 2 μm (f and g), 10 μm (i and j).

Extended Data Fig. 3 Shibire and ShibireAKE mutant bundle actin filaments.

a, Shi bundles actin filaments at low salt condition. Low speed co-sedimentation assays were performed and quantified as in Fig. 2a. 3 μM G-actin was incubated alone or with 0.5 μM Shi at different salt (KCl) concentrations as indicated. Mean ± s.d. values are shown in the dot plot, and significance was determined by the two-tailed Student’s t-test (as in b, f and h). n = 3 independent experiments. b, Shi binds actin filaments at low salt condition. High speed co-sedimentation assays were performed and quantified as in Fig. 3f. 3 μM G-actin or 0.5 μM Shi was incubated alone or together at different salt (KCl) concentrations as indicated. n = 3 independent experiments. c, High speed co-sedimentation assays were performed with 3 μM G-actin and increasing concentrations of Shi. Supernatants (S) and pellets (P) were monitored by SDS-PAGE after centrifugation at 50,000g. n = 3 independent experiments with similar results. See quantification in (d). d, Analysis of Shi-actin binding shown in (c). The concentration of bound Shi was plotted against that of free Shi and fitted to a hyperbolic function (one site binding). Scatchard analysis of Shi-actin binding was performed to calculate the stoichiometry of binding and dissociation equilibrium constant. n = 3 independent experiments. e, ShiAKE is less stable than Shi. Shi-3xHA and ShiAKE-3xHA were transfected into S2R+ cells co-expressing GFP and the cell lysates were subjected to western blot analysis. α-Tubulin and GFP were used as loading and transfection controls, respectively. n = 3 independent experiments with similar results. See quantification in (f). f, Quantification of western blot analyses, one of which is shown in (e). n = 3 independent experiments. g, Electron micrograph of negatively stained actin filaments in the presence of purified ShiAKE. n = 22 actin bundles were imaged, with similar results. Scale bar: 30 nm. h, Shi bundles actin filaments in the presence of GTP. 3 μM G-actin was incubated alone or with 0.5 μM Shi in the absence or presence of 1 mM GTP, and subjected to low speed co-sedimentation assays. n = 3 independent experiments. Numerical source data and unprocessed blots are provided with the paper.

Source Data Extended Data Fig. 3

Extended Data Fig. 4 The PRD domain of Shibire mediates Shibire-actin interaction.

a, The PRD domain is required for Shi-actin binding. 0.5 μM Shi (or a Shi deletion mutant as indicated) was incubated alone or with 3 μM G-actin. The wild-type Shi and Shi mutant bands are marked by arrowheads. Mean ± s.d. values are shown in the dot plot, and significance was determined by the two-tailed Student’s t-test (as in b, e and f). n = 3 independent experiments. b, The PRD and G domains, but not the PH domain, are involved in actin bundling. 3 μM G-actin was incubated alone or with 0.5 μM Shi (or a Shi deletion mutant as indicated), and subjected to low speed co-sedimentation assays. The wild-type Shi and Shi mutant bands are marked by arrowheads. n = 3 independent experiments. c, Electron micrographs of negatively stained lipid nanotubes that were incubated with Shi (or a Shi deletion mutant). n = 63, 76, 75, 94 (top to bottom) nanotubes were imaged, with similar results. Scale bar: 30 nm. d, SH3 domains interfere with, but do not block, Shi-actin interaction. 6 μM G-actin was incubated with 0.5 μM Shi in the absence or presence of a SH3 domain as indicated, and subjected to high speed co-sedimentation assays. n = 2 independent experiments (one is shown) with similar results. e, Actin-bundling activity of tandem PRD. 3 μM G-actin was incubated alone or with 0.5 μM Shi, 1.5 μM ShiPRD or 1 μM Shi2xPRD, respectively, and subjected to low speed co-sedimentation assays. n = 3, 3, 5, 5 (left to right) independent experiments. f, Actin-binding activity of single and tandem PRD. 0.5 μM Shi, 1.5 μM ShiPRD or 1 μM Shi2xPRD was incubated alone or with 3 μM G-actin, and subjected to high speed co-sedimentation assays. n = 3, 3, 4, 4, 4, 4 (left to right) independent experiments. Numerical source data and unprocessed blots are provided with the paper.

Source Data Extended Data Fig. 4

Extended Data Fig. 5 Critical role for the PRD in actin bundling.

a, Electron micrographs of negatively stained actin filaments mediated by Drosophila Shi, Drp1-ShiPRD, Drp1, and human Drp1 (hDrp1). Different ratios of actin to Shi/Drp1-ShiPRD/Drp1/hDrp1 are indicated on the left. Note that Shi formed rings along actin bundles when actin:Shi = 8:1, whereas Drp1-ShiPRD started to form rings along actin bundles when actin:Drp1-ShiPRD = 1:1. Neither Drosophila or hDrp1 formed rings along actin bundles. n = 12, 11, 24, 10, 11, 7, 10, 12, 30, 59, 6, 5, 5, 16 (top to bottom and left to right) actin bundles imaged with similar results. b, Quantification of the fusion indices of S2R+ cells expressing Shi, Drp1-ShiPRD or Drp1. Note that Drp1-ShiPRD, but not Drp1, partially rescued the cell fusion defect in Sns-Eff-1-expressing Shi knockdown cells. Overexpression (OE) of Drp1-ShiPRD, but not Drp1, caused a dominant negative effect on the fusion of Sns-Eff-1-expressing cells. All Shi and mutant constructs were tagged with 3xHA. n = 7, 6, 9, 9, 9, 6, 4, 5 and 4 (left to right) independent experiments. Mean ± s.d. values are shown in the dot plot, and significance was determined by the two-tailed Student’s t-test (as in d and e). c, Drp1-ShiPRD, but not Drp1, is enriched at the fusogenic synapse of S2R+ cells. Sns-Eff-1-expressing S2R+ cells were labeled by anti-HA (green), phalloidin (red) and Sns-iBlueberry (blue). The fusogenic synapses were marked by F-actin and Sns enrichment. n = 30, 90 and 102 (top to bottom) fusogenic synapses were imaged, with similar results. d, Actin-binding activity of Shi and its PRD domain mutants. 0.5 μM Shi (or a PRD mutant as indicated) was incubated alone or with 3 μM G-actin, and subjected to high speed co-sedimentation assays. n = 3 independent experiments. e, Actin-bundling activity of Shi and its PRD domain mutants. 3 μM G-actin was incubated alone or with 0.5 μM Shi (or a PRD mutant as indicated), and subjected to low speed co-sedimentation assays. n = 4 independent experiments. Scale bars: 30 nm (a), 5 μm (c). Numerical source data and unprocessed blots are provided with the paper.

Source Data Extended Data Fig. 5

Extended Data Fig. 6 Localization and endocytic function of Shi PRD domain mutants.

a, Shi PRD domain mutants (ShiR804D, ShiR829D, ShiR846/853D and Shi4RD) are enriched at the fusogenic synapse. Sns-Eff-1-expressing S2R+ cells were labeled by anti-HA (green), phalloidin (red) and Sns-iBlueberry (blue). The fusogenic synapses were marked by F-actin and Sns enrichment. n = 52, 65, 56, 77 and 73 (top to bottom) fusogenic synapses were imaged, with similar results. b, Pulse-chase transferrin (Tf) uptake assays were performed with Shi mutants in Shi knockdown S2R+ cells. Cells were labeled with Tf-Alexa488 (green) and anti-human Tf receptor (hTfR) (red). Note that Shi PRD domain mutants (ShiR804D, ShiR829D, and ShiR846/853D) rescued the endocytosis defect in Shi knockdown cells as did wild type Shi. Shi4RD partially rescued endocytosis, whereas the GTPase domain mutant ShiQ35E did not rescue the endocytosis defect. n = 25, 28, 27, 22, 35, 20, 20 and 20 (left to right) cells were imaged, with similar results. See quantification in Fig. 4i. Scale bars: 5 μm (a), 20 μm (b).

Extended Data Fig. 7 Cryo-ET of hDyn1- and Shi-mediated actin bundles.

a, Sections through the cryo-tomogram with hDyn1 (light blue) and associated actin filaments (rainbow colored) (area corresponding to that in Fig. 5e left panel). The top section reveals a right-handed tilt of hDyn1, the middle shows the T-shaped structures, and the bottom shows a left-handed tilt, consistent with a helical polymer. n = 3 tomograms were collected, with similar results. b, Tomogram-derived model of actin filaments bundled by a hDyn1 helix showing the side, front, and back views. Colors are the same as in (a). Figure is representative from three experiments of tomograms showing hDyn1-actin interaction. c, Cryo-ET of three parallel Shi-mediated actin bundles. Boxed area of the tomogram enlarged in (d). n = 13 tomograms were collected, with similar results. d, Shi assembles as helices surrounded by actin filaments. Top, middle, and bottom views of the boxed region in (c) are consistent with a Shi helical polymer (light blue). The top panels are the boxed area binned for ease of visualization. The middle panels have the modelled Shi helix docked onto the unbinned tomogram. The bottom panel is a view of the unbinned tomogram. Figure is representative of 13 experiments of tomograms showing Shi-actin interaction. Scale bar: 20 nm (a, c and d). Numerical source data are provided with the paper.

Source Data Extended Data Fig. 7

Extended Data Fig. 8 GTP addition triggers increased Arp2/3-mediated branched actin polymerization on dynamin-actin bundles.

a, Quantification of Arp2/3-mediated branched actin filaments on Shi-actin bundles in the absence or presence of GTP shown in Fig. 6h. n = 34 actin bundles. Mean ± s.d. values are shown in the dot plot, and significance was determined by the two-tailed Student’s t-test (as in c and e). b, Time-lapse stills of TIRF images of Arp2/3-mediated branched actin polymerization on Shi-actin bundles in the absence or presence of GTP (Supplementary Video 13). 2 μM Shi was first incubated with 1 μM Alexa Fluor 488 phalloidin-labeled actin (green) to generate actin bundles. Subsequently, Arp2/3, VCA and rhodamine-G-actin (magenta) were added to the Shi-actin bundles to start branched actin polymerization without (top three panels) or with (bottom three panels) 1 mM GTP. n = 32 actin bundles in the absence or presence of GTP were imaged, with similar results. See quantification in (c). c, Quantification of Arp2/3-mediated branched actin filaments in (b). n = 32 actin bundles. d, Time-lapse stills of TIRF images of Arp2/3-mediated branched actin polymerization on hDyn1-actin bundles in the absence or presence of GTP (Supplementary Video 15). 2 μM hDyn1 was first incubated with 1 μM Alexa Fluor 488 phalloidin-labeled actin (green) to generate actin bundles, followed by a wash with TIRF buffer without (top three panels) or with (bottom three panels) 0.01 mM GTP. Subsequently, Arp2/3, VCA and rhodamine-G-actin (magenta) were added to start branched actin polymerization. n = 34 and 35 actin bundles in the absence or presence of GTP, respectively, imaged with similar results. See quantification in (e). e, Quantification of Arp2/3-mediated branched actin filaments in (d). n = 34 and 35 (left to right) actin bundles. Scale bar: 5 μm (b and d). Numerical source data are provided with the paper.

Source Data Extended Data Fig. 8

Extended Data Fig. 9 Localization of dynamin on actin-enriched structures in cells.

a, Shi localization on actin bundles at the fusogenic synapse revealed by STED microscopy. S2R+ cells co-expressing Shi-GFP, Sns-HA, and Eff-1-FLAG were labeled with anti-GFP (green), phalloidin (red), and anti-HA (blue). Boxed areas in the left panels are enlarged on the right. Note the Shi punctae along the actin bundles. n = 16 fusogenic synapses were imaged, with similar results. b, Specificity of antibodies in the immunogold PREM experiments. Boxed area in the left panel is enlarged on the right. Shi knockdown S2R+ cells co-expressing Sns, Eff-1 and Shits2-3xHA were stained with anti-HA for immunogold labeling. Note the absence of 18-nm colloidal gold particle labeling in these actin bundles, suggesting that this cell was untransfected and demonstrating the labeling specificity of the primary and secondary antibodies. n = 15 fusogenic synapses were imaged, with similar results. c, Quantification of gold particle density on actin bundles. The gold particle density was calculated as the number of gold particles divided by the area of the actin bundle examined. n = 14 and 21 actin bundles from 4 (Shi-GFP) and 7 (ShiQ35E-GFP) fusogenic synapses. Mean ± s.d. values are shown in the dot plot, and significance was determined by the two-tailed Student’s t-test (as in d). d, Quantification of Shi cluster occurrence in Shi knockdown S2R+ cells co-expressing Sns, Eff-1 and Shi-GFP (or ShiQ35E-GFP). Shi cluster occurrence was calculated as the percentage of gold particles in clusters (containing ≥3 gold particles in a 100 nm2 square box) vs. the total number of gold particles on an actin bundle. Note the increase occurrence of ShiQ35E-GFP clusters than that of Shi. n = 14 and 21 actin bundles from 4 (Shi-GFP) and 7 (ShiQ35E-GFP) fusogenic synapses. e,f, hDyn2 localization on actin comet tails in Listeria-infected HeLa cell revealed by SIM (e) and STED (f). Over two dozen Listeria (GFP-Lm; blue) were protruding from a host cell, each propelled by an actin comet tail (e). Note the hDyn2 punctae (green) along each comet tail labeled with phalloidin (red) (e and f). n = 57 (e) and 9 (f) actin comet tails (biologically independent samples) were imaged, with similar results. Scale bars: 2 μm (a, left panels, b, left panel, e and f), 500 nm (a, right panels), 100 nm (b, right panel). Numerical source data are provided with the paper.

Source Data Extended Data Fig. 9

Extended Data Fig. 10 Models describing the mechanisms of dynamin-actin interaction.

a, A schematic diagram of the cross section view (left) of the dynamin helix with associated actin filaments. During dynamin’s assembly into a helical structure, actin filaments are bundled to the outer rim of the partially assembled dynamin helices via the PRD domains. Once a dynamin helix is fully assembled, the assembly-stimulated GTPase activity of dynamin triggers rapid GTP hydrolysis and helix disassembly, freeing dynamin dimers/tetramers to bundle new actin filaments. A schematic diagram of the side view of the dynamin helix with associated actin filaments (right). At its full capacity, a hDyn1 helix (illustrated here) can bundle 16 actin filaments, whereas Shi helix can bundle 12 filaments. b, Dynamin-mediated actin bundling enhances mechanical stiffness of the actin network and promotes membrane protrusions at the fusogenic synapse. Each actin super bundle is likely organized by multiple dynamin helices that are being assembled, the dynamic asynchronous GTP hydrolysis results in a “hybrid” actin bundle that contains stretches of unprotected filaments held together at random locations by partial dynamin helices. Arp2/3-mediated actin polymerization on the unprotected areas of the actin bundles generate additional branched actin filaments, which may join the partially occupied bundles via trans-bundle PRD–actin interactions and crosslink multiple bundles. Through dynamin’s cycling between helix assembly and disassembly as a function of GTP hydrolysis, the actin bundles grow thicker and mechanically stiffer. As a consequence, the podosome-like structure at the fusogenic synapse is able to propel membrane protrusions to promote cell-cell fusion.

Supplementary information

Reporting Summary

Supplementary Table 1

All molecular reagents used in this study.

Supplementary Video 1

Shi is dynamically associated with the F-actin focus at the fusogenic synapse. Time-lapse imaging of muscle cells expressing Shi–GFP (green) and Actin–mRFP (red) in a stage 14 wild-type embryo. The accumulation and dissolution of Shi coincided with that of the F-actin foci. n = 31 fusogenic synapses were imaged, with similar results.

Supplementary Video 2

Shi bundles Arp2/3-mediated branched actin filaments. Time-lapse TIRF images of Arp2/3-mediated branched actin polymerization (actin labelled with Alexa Fluor 568-phalloidin) and thick actin bundle formation at numerous locations upon the addition of Shi–SNAP-Surface 488. The TIRF assay was performed in NEM-myosin-coated flow chambers. n = 12 independent imaging experiments were performed, with similar results.

Supplementary Video 3

Shi bundles pre-assembled actin filaments. Time-lapse TIRF imaging of Shi-mediated bundling of pre-assembled actin filaments at the single-filament level. Left, actin alone; right, actin and Shi. The TIRF assay was performed on SLBs. Shi was added into the reaction mix of pre-assembled actin filaments. n = 10 independent imaging experiments were performed, with similar results.

Supplementary Video 4

Shi bundles actin filaments during filament growth. Time-lapse TIRF imaging of Shi-mediated actin bundling during actin polymerization. The TIRF assay was performed in mPEG-Silane-coated flow chambers. Shi and actin were mixed for imaging. n = 22 actin bundles were imaged, with similar results.

Supplementary Video 5

Cryo-ET of actin filaments incubated with hDyn1. The video shows z sections of cryo-ET generated from a tilt series collected on cryo grids containing actin filaments and hDyn1. n = 3 tomograms were collected, with similar results. Scale bar: 10 nm.

Supplementary Video 6

Modelling of the hDyn1–actin interaction based on cryo-ET (I). This video includes: (1) z sections of a cryo-ET; and (2) segmentation of actin filaments and hDyn1 by points in order to develop a model of the hDyn1–actin interaction. The hDyn1 helix is sky blue and actin filaments are rainbow coloured. The video is representative of three experiments of tomograms showing the hDyn1–actin interaction (as in Supplementary Videos 7 and 8). Scale bar: 10 nm.

Supplementary Video 7

Modelling of the hDyn1–actin interaction based on cryo-ET (II). This video includes: (1) z sections of a cryo-ET; (2) modelling of the hDyn1 helix; (3) modelling of the actin filaments; (4) modelling of the hDyn1 helix and actin filaments; (5) a model of turning the full 360° along the x axis; (6) a model of turning the full 360° along the y axis; and (7) a model of transitioning to the front view. The hDyn1 helix is light blue and the actin filaments are rainbow coloured. Scale bar: 10 nm.

Supplementary Video 8

Modelling of the hDyn1–actin interaction based on cryo-ET (III). This video includes: (1) z sections through the cryo-ET; and (2) modelling of the hDyn1 helix and actin filaments on the background of the tomogram.

Supplementary Video 9

Cryo-ET of Shi and actin. Z sections of cryo-ET were generated from a tilt series collected on cryo grids containing Shi and actin filaments. n = 13 tomograms were collected, with similar results. Scale bar: 10 nm.

Supplementary Video 10

GTP hydrolysis induces dynamic disassembly of Shi helices. FRAP of Shi–SNAP-Surface 488 along an actin bundle in the absence (left) or presence (right) of GTP. A segment of the Shi–actin bundle was photobleached and the fluorescence recovery was monitored live. Note that the fluorescence was recovered in the presence, but not absence, of GTP. n = 9 and 13 independent imaging experiments were performed, respectively, with similar results.

Supplementary Video 11

GTP hydrolysis triggers dynamic assembly/disassembly of Shi-mediated actin bundles. Time-lapse TIRF imaging of GTP hydrolysis-induced dynamic actin bundling and disassembly by Shi. Arp2/3-mediated branched actin polymerization with rhodamine-G-actin (red) was generated, followed by the addition of Shi–SNAP-Surface 488 (green) to bundle the actin filaments. Subsequent GTP addition triggered the dynamic disassembly and reassembly of Shi helices and actin bundles. n = 3 independent imaging experiments were performed, with similar results.

Supplementary Video 12

GTP hydrolysis promotes dynamic Arp2/3-mediated branched actin polymerization on Shi–actin bundles. Time-lapse TIRF imaging of Arp2/3-mediated branched actin polymerization on Shi–actin bundles in the absence (top) or presence (bottom) of GTP. Shi–actin bundles were generated by incubating unlabelled actin with Shi–SNAP-Surface 488 (green). Arp2/3, VCA and rhodamine-G-actin (magenta) were added to the Shi–actin bundles to start branched actin polymerization with or without GTP. Note the disassembly of Shi from the actin bundle upon GTP addition, and the numerous branched actin filaments emanating from the same actin bundle. n = 34 Shi–actin bundles with or without GTP were imaged, with similar results.

Supplementary Video 13

GTP addition leads to increased Arp2/3-mediated branched actin polymerization on Shi–actin bundles. Time-lapse TIRF imaging of Arp2/3-mediated branched actin polymerization on Shi–actin bundles in the absence (top) or presence (bottom) of GTP. Shi–actin bundles were generated by incubating unlabelled Shi with Alexa Fluor 488-phalloidin-actin (green). Subsequently, Arp2/3, VCA and rhodamine-G-actin (magenta) were added to the Shi–actin bundles to start branched actin polymerization with or without GTP. Note the enhanced number of branched actin filaments emanating from the actin bundles in the presence of GTP compared to in its absence. n = 32 Shi–actin bundles with or without GTP were imaged, with similar results.

Supplementary Video 14

Shi bundles newly polymerized branched actin filaments generated from the Shi–actin bundle. Time-lapse TIRF imaging of Shi-mediated bundling of new branched actin filaments. Arp2/3-mediated branched actin filaments on Shi–actin bundles were generated as in Supplementary Video 13. Note that the branched actin filaments were bundled rapidly upon the addition of Shi. n = 7 actin bundles were imaged, with similar results.

Supplementary Video 15

GTP hydrolysis promotes dynamic Arp2/3-mediated branched actin polymerization on hDyn1–actin bundles. Time-lapse TIRF imaging of Arp2/3-mediated branched actin polymerization on hDyn1–actin bundles in the absence (top) or presence (bottom) of GTP. hDyn1 was first incubated with Alexa Fluor 488-phalloidin-actin (green) to generate actin bundles, followed by a wash with TIRF buffer with or without 0.01 mM GTP. Subsequently, Arp2/3, VCA and rhodamine-G-actin (magenta) were added to start branched actin polymerization. Note the enhanced number of branched actin filaments emanating from the actin bundles in the presence of GTP compared to in its absence. n = 35 and 34 hDyn1-mediated actin bundles with and without GTP, respectively, were imaged, with similar results.

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Zhang, R., Lee, D.M., Jimah, J.R. et al. Dynamin regulates the dynamics and mechanical strength of the actin cytoskeleton as a multifilament actin-bundling protein. Nat Cell Biol 22, 674–688 (2020). https://doi.org/10.1038/s41556-020-0519-7

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