Spectrin is a mechanoresponsive protein shaping fusogenic synapse architecture during myoblast fusion

Abstract

Spectrin is a membrane skeletal protein best known for its structural role in maintaining cell shape and protecting cells from mechanical damage. Here, we report that α/βH-spectrin (βH is also called karst) dynamically accumulates and dissolves at the fusogenic synapse between fusing Drosophila muscle cells, where an attacking fusion partner invades its receiving partner with actin-propelled protrusions to promote cell fusion. Using genetics, cell biology, biophysics and mathematical modelling, we demonstrate that spectrin exhibits a mechanosensitive accumulation in response to shear deformation, which is highly elevated at the fusogenic synapse. The transiently accumulated spectrin network functions as a cellular fence to restrict the diffusion of cell-adhesion molecules and a cellular sieve to constrict the invasive protrusions, thereby increasing the mechanical tension of the fusogenic synapse to promote cell membrane fusion. Our study reveals a function of spectrin as a mechanoresponsive protein and has general implications for understanding spectrin function in dynamic cellular processes.

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Fig. 1: α/βH-Spectrin is required for myoblast fusion and is enriched at the fusogenic synapse in founder cells.
Fig. 2: α/βH-Spectrin dynamically accumulates at the fusogenic synapse in response to PLS invasion.
Fig. 3: α/βH-Spectrin accumulates at the fusogenic synapse in the absence of chemical signalling from CAMs.
Fig. 4: α/βH-Spectrin exhibits mechanosensitive accumulation to shear deformation.
Fig. 5: α/βH-Spectrin restricts CAMs at the fusogenic synapse.
Fig. 6: The α/βH-spectrin network restricts the CAM Duf and constricts actin-propelled invasive protrusions.
Fig. 7: βV-spectrin, the mammalian homologue of Drosophila βH-spectrin, is required for C2C12 myoblast fusion.

References

  1. 1.

    Bennett, V. & Lorenzo, D. N. Spectrin- and ankyrin-based membrane domains and the evolution of vertebrates. Curr. Top. Membr. 72, 1–37 (2013).

    Article  PubMed  CAS  Google Scholar 

  2. 2.

    Bennett, V. & Lorenzo, D. N. An adaptable spectrin/ankyrin-based mechanism for long-range organization of plasma membranes in vertebrate tissues. Curr. Top. Membr. 77, 143–184 (2016).

    Article  PubMed  Google Scholar 

  3. 3.

    Machnicka, B. et al. Spectrins: a structural platform for stabilization and activation of membrane channels, receptors and transporters. Biochim. Biophys. Acta 1838, 620–634 (2014).

    Article  PubMed  CAS  Google Scholar 

  4. 4.

    Liu, S. C., Derick, L. H. & Palek, J. Visualization of the hexagonal lattice in the erythrocyte membrane skeleton. J. Cell Biol. 104, 527–536 (1987).

    Article  PubMed  CAS  Google Scholar 

  5. 5.

    Pielage, J. et al. A presynaptic giant ankyrin stabilizes the NMJ through regulation of presynaptic microtubules and transsynaptic cell adhesion. Neuron 58, 195–209 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. 6.

    Byers, T. J. & Branton, D. Visualization of the protein associations in the erythrocyte membrane skeleton. Proc. Natl Acad. Sci. USA 82, 6153–6157 (1985).

    Article  PubMed  CAS  Google Scholar 

  7. 7.

    Xu, K., Zhong, G. & Zhuang, X. Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science 339, 452–456 (2013).

    Article  PubMed  CAS  Google Scholar 

  8. 8.

    Hammarlund, M., Jorgensen, E. M. & Bastiani, M. J. Axons break in animals lacking β-spectrin. J. Cell Biol. 176, 269–275 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. 9.

    Krieg, M., Dunn, A. R. & Goodman, M. B. Mechanical control of the sense of touch by β-spectrin. Nat. Cell Biol. 16, 224–233 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. 10.

    Chen, E. H. & Olson, E. N. Unveiling the mechanisms of cell–cell fusion. Science 308, 369–373 (2005).

    Article  PubMed  CAS  Google Scholar 

  11. 11.

    Aguilar, P. S. et al. Genetic basis of cell–cell fusion mechanisms. Trends Genet. 29, 427–437 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. 12.

    Sens, K. L. et al. An invasive podosome-like structure promotes fusion pore formation during myoblast fusion. J. Cell Biol. 191, 1013–1027 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. 13.

    Shilagardi, K. et al. Actin-propelled invasive membrane protrusions promote fusogenic protein engagement during cell–cell fusion. Science 340, 359–363 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. 14.

    Kim, J. H. et al. Mechanical tension drives cell membrane fusion. Dev. Cell 32, 561–573 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. 15.

    Kim, J. H., Jin, P., Duan, R. & Chen, E. H. Mechanisms of myoblast fusion during muscle development. Curr. Opin. Genet. Dev. 32, 162–170 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. 16.

    Shin, N. Y. et al. Dynamin and endocytosis are required for the fusion of osteoclasts and myoblasts. J. Cell Biol. 207, 73–89 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. 17.

    Haralalka, S. et al. Asymmetric Mbc, active Rac1 and F-actin foci in the fusion-competent myoblasts during myoblast fusion in Drosophila. Development 138, 1551–1562 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. 18.

    Jin, P. et al. Competition between Blown Fuse and WASP for WIP binding regulates the dynamics of WASP-dependent actin polymerization in vivo. Dev. Cell 20, 623–638 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. 19.

    Duan, R. et al. Group I PAKs function downstream of Rac to promote podosome invasion during myoblast fusion in vivo. J. Cell Biol. 199, 169–185 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. 20.

    Dubreuil, R. R., Byers, T. J., Stewart, C. T. & Kiehart, D. P. A β-spectrin isoform from Drosophila (βH) is similar in size to vertebrate dystrophin. J. Cell Biol. 111, 1849–1858 (1990).

    Article  PubMed  CAS  Google Scholar 

  21. 21.

    Thomas, G. H. & Kiehart, D. P. β Heavy-spectrin has a restricted tissue and subcellular distribution during Drosophila embryogenesis. Development 120, 2039–2050 (1994).

    PubMed  CAS  Google Scholar 

  22. 22.

    Tjota, M. et al. Annexin B9 binds to βH-spectrin and is required for multivesicular body function in Drosophila. J. Cell Sci. 124, 2914–2926 (2011).

    Article  PubMed  CAS  Google Scholar 

  23. 23.

    Mazock, G. H., Das, A., Base, C. & Dubreuil, R. R. Transgene rescue identifies an essential function for Drosophila β spectrin in the nervous system and a selective requirement for ankyrin-2-binding activity. Mol. Biol. Cell 21, 2860–2868 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. 24.

    Ruiz-Gomez, M., Coutts, N., Price, A., Taylor, M. V. & Bate, M. Drosophila Dumbfounded: a myoblast attractant essential for fusion. Cell 102, 189–198 (2000).

    Article  PubMed  CAS  Google Scholar 

  25. 25.

    Gardner, K. & Bennett, V. Modulation of spectrin–actin assembly by erythrocyte adducin. Nature 328, 359–362 (1987).

    Article  PubMed  CAS  Google Scholar 

  26. 26.

    Ungewickell, E., Bennett, P. M., Calvert, R., Ohanian, V. & Gratzer, W. B. In vitro formation of a complex between cytoskeletal proteins of the human erythrocyte. Nature 280, 811–814 (1979).

    Article  PubMed  CAS  Google Scholar 

  27. 27.

    Bennett, V. & Baines, A. J. Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol. Rev. 81, 1353–1392 (2001).

    Article  PubMed  CAS  Google Scholar 

  28. 28.

    Richardson, B. E., Beckett, K., Nowak, S. J. & Baylies, M. K. SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodeling at the site of myoblast fusion. Development 134, 4357–4367 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. 29.

    Strunkelnberg, M. et al. rst and its paralogue kirre act redundantly during embryonic muscle development in Drosophila. Development 128, 4229–4239 (2001).

    PubMed  CAS  Google Scholar 

  30. 30.

    Bour, B. A., Chakravarti, M., West, J. M. & Abmayr, S. M. Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion. Genes Dev. 14, 1498–1511 (2000).

    PubMed  PubMed Central  CAS  Google Scholar 

  31. 31.

    Galletta, B. J., Chakravarti, M., Banerjee, R. & Abmayr, S. M. SNS: adhesive properties, localization requirements and ectodomain dependence in S2 cells and embryonic myoblasts. Mech. Dev. 121, 1455–1468 (2004).

    Article  PubMed  CAS  Google Scholar 

  32. 32.

    Shelton, C., Kocherlakota, K. S., Zhuang, S. & Abmayr, S. M. The immunoglobulin superfamily member Hbs functions redundantly with Sns in interactions between founder and fusion-competent myoblasts. Development 136, 1159–1168 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. 33.

    Bulchand, S., Menon, S. D., George, S. E. & Chia, W. The intracellular domain of Dumbfounded affects myoblast fusion efficiency and interacts with Rolling pebbles and Loner. PLoS ONE 5, e9374 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. 34.

    Chen, E. H. & Olson, E. N. Antisocial, an intracellular adaptor protein, is required for myoblast fusion in Drosophila. Dev. Cell 1, 705–715 (2001).

    Article  PubMed  CAS  Google Scholar 

  35. 35.

    Menon, S. D. & Chia, W. Drosophila Rolling pebbles: a multidomain protein required for myoblast fusion that recruits D-Titin in response to the myoblast attractant Dumbfounded. Dev. Cell 1, 691–703 (2001).

    Article  PubMed  CAS  Google Scholar 

  36. 36.

    Rau, A. et al. rolling pebbles (rols) is required in Drosophila muscle precursors for recruitment of myoblasts for fusion. Development 128, 5061–5073 (2001).

    PubMed  CAS  Google Scholar 

  37. 37.

    Menon, S. D., Osman, Z., Chenchill, K. & Chia, W. A positive feedback loop between Dumbfounded and Rolling pebbles leads to myotube enlargement in Drosophila. J. Cell Biol. 169, 909–920 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. 38.

    Mohler, W. A. et al. The type I membrane protein EFF-1 is essential for developmental cell fusion. Dev. Cell 2, 355–362 (2002).

    Article  PubMed  CAS  Google Scholar 

  39. 39.

    Stauffer, T. P., Ahn, S. & Meyer, T. Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells. Curr. Biol. 8, 343–346 (1998).

    Article  PubMed  CAS  Google Scholar 

  40. 40.

    Bennett, V. & Healy, J. Membrane domains based on ankyrin and spectrin associated with cell–cell interactions. Cold Spring Harb. Perspect. Biol. 1, a003012 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Graveley, B. R. et al. The developmental transcriptome of Drosophila melanogaster. Nature 471, 473–479 (2011).

    Article  PubMed  CAS  Google Scholar 

  42. 42.

    Luo, T., Mohan, K., Iglesias, P. A. & Robinson, D. N. Molecular mechanisms of cellular mechanosensing. Nat. Mater. 12, 1064–1071 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. 43.

    Kim, S. et al. A critical function for the actin cytoskeleton in targeted exocytosis of prefusion vesicles during myoblast fusion. Dev. Cell 12, 571–586 (2007).

    Article  PubMed  CAS  Google Scholar 

  44. 44.

    Massarwa, R., Carmon, S., Shilo, B. Z. & Schejter, E. D. WIP/WASp-based actin-polymerization machinery is essential for myoblast fusion in Drosophila. Dev. Cell 12, 557–569 (2007).

    Article  PubMed  CAS  Google Scholar 

  45. 45.

    Abmayr, S. M. & Pavlath, G. K. Myoblast fusion: lessons from flies and mice. Development 139, 641–656 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. 46.

    Schejter, E. D. Myoblast fusion: experimental systems and cellular mechanisms. Semin. Cell Dev. Biol. 60, 112–120 (2016).

    Article  PubMed  CAS  Google Scholar 

  47. 47.

    Deng, S., Azevedo, M. & Baylies, M. Acting on identity: myoblast fusion and the formation of the syncytial muscle fiber. Semin. Cell Dev. Biol. 72, 45–55 (2017).

    Article  PubMed  CAS  Google Scholar 

  48. 48.

    Johnson, C. P., Tang, H.-Y., Carag, C., Speicher, D. W. & Discher, D. E. Forced unfolding of proteins within cells. Science 317, 663–666 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. 49.

    Popowicz, G. M., Schleicher, M., Noegel, A. A. & Holak, T. A. Filamins: promiscuous organizers of the cytoskeleton. Trends Biochem. Sci. 31, 411–419 (2006).

    Article  PubMed  CAS  Google Scholar 

  50. 50.

    Schiffhauer, E. S. et al. Mechanoaccumulative elements of the mammalian actin cytoskeleton. Curr. Biol. 26, 1473–1479 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. 51.

    Sheetz, M. P., Schindler, M. & Koppel, D. E. Lateral mobility of integral membrane proteins is increased in spherocytic erythrocytes. Nature 285, 510–511 (1980).

    Article  PubMed  CAS  Google Scholar 

  52. 52.

    Tsuji, A. & Ohnishi, S. Restriction of the lateral motion of band 3 in the erythrocyte membrane by the cytoskeletal network: dependence on spectrin association state. Biochemistry 25, 6133–6139 (1986).

    Article  PubMed  CAS  Google Scholar 

  53. 53.

    Thomas, G. H. et al. Drosophila βHeavy-spectrin is essential for development and contributes to specific cell fates in the eye. Development 125, 2125–2134 (1998).

    PubMed  CAS  Google Scholar 

  54. 54.

    Lee, S. K. & Thomas, G. H. Rac1 modulation of the apical domain is negatively regulated by βHeavy-spectrin. Mech. Dev. 128, 116–128 (2011).

    Article  PubMed  CAS  Google Scholar 

  55. 55.

    Kocherlakota, K. S., Wu, J. M., McDermott, J. & Abmayr, S. M. Analysis of the cell adhesion molecule sticks-and-stones reveals multiple redundant functional domains, protein-interaction motifs and phosphorylated tyrosines that direct myoblast fusion in Drosophila melanogaster. Genetics 178, 1371–1383 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. 56.

    Wei, Q., Rong, Y. & Paterson, B. M. Stereotypic founder cell patterning and embryonic muscle formation in Drosophila require nautilus (MyoD) gene function. Proc. Natl Acad. Sci. USA 104, 5461–5466 (2007).

    Article  PubMed  CAS  Google Scholar 

  57. 57.

    Byers, T. J., Brandin, E., Lue, R. A., Winograd, E. & Branton, D. The complete sequence of Drosophila β-spectrin reveals supra-motifs comprising eight 106-residue segments. Proc. Natl Acad. Sci. USA 89, 6187–6191 (1992).

    Article  PubMed  CAS  Google Scholar 

  58. 58.

    Zhang, S. & Chen, E. H. in Cell Fusion: Overviews and Methods (ed. Chen, E. H.) 275–297 (Humana Press, Totowa, NJ, 2008).

  59. 59.

    Sato, T. A modified method for lead staining of thin sections. J. Electron Microsc. (Tokyo) 17, 158–159 (1968).

    CAS  Google Scholar 

  60. 60.

    Kee, Y. S. & Robinson, D. N. Micropipette aspiration for studying cellular mechanosensory responses and mechanics. Methods Mol. Biol. 983, 367–382 (2013).

    Article  PubMed  CAS  Google Scholar 

  61. 61.

    Discher, D. E., Boal, D. H. & Boey, S. K. Simulations of the erythrocyte cytoskeleton at large deformation. II. Micropipette aspiration. Biophys. J. 75, 1584–1597 (1998).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. 62.

    Li, J., Dao, M., Lim, C. T. & Suresh, S. Spectrin-level modeling of the cytoskeleton and optical tweezers stretching of the erythrocyte. Biophys. J. 88, 3707–3719 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

We thank the Bloomington Stock Center for fly stocks, B. Paterson for the MHC antibody, F. Li for technical assistance, G. Zhang for help with the high-pressure freezing and freeze substitution method, J. Nathans for sharing confocal microscopes and D. Pan for critically reading the manuscript. This work was supported by the NIH grants (R01 AR053173 and R01 GM098816), the American Heart Association Established Investigator Award and the HHMI Faculty Scholar Award to E.H.C.; the NIH grants (R01 GM66817 and R01 GM109863) to D.N.R.; the NIH grants (R01 GM074751 and R01 GM114671) and the Chan Zucherberg Biohub Investigator Award to D.A.F.; the NSF grant MCB-1122013 to C.T.; the NSFC grant 11572316 to T.L.; and the NSFC grant 31771256 to R.D. R.D. was supported by an American Heart Association postdoctoral fellowship, K.S. by an American Heart Association Scientist Development Grant, D.M.L. by a Canadian Institute of Health Research postdoctoral fellowship and S.S. by a Life Sciences Research Foundation postdoctoral fellowship.

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R.D. initiated the project. R.D., J.H.K., K.S. and E.H.C. planned the project, performed the experiments in Figs. 1–3,5,7, Supplementary Figs. 1,2,4,6 and Supplementary Videos 1–4,6–9, and discussed the data. J.H.K. and E.H.C. collaborated with E.S. and D.N.R. on the MPA experiments in Fig. 4A–E and Supplementary Fig. 3, and with S.S. and D.A.F. on the AFM experiments in Fig. 4K–L and Supplementary Video 5. D.M.L. carried out the SIM experiments in Fig. 6 and Supplementary Video 10. S.L. carried out the electron microscopy experiments in Fig. 5H. T.L. developed the coarse-grained models in Fig. 4F–J and Supplementary Fig. 5. C.T. contributed spectrin constructs. R.D., J.K., K.S., D.M.L., E.S., T.L., D.N.R. and E.H.C. generated the figures. J.H.K. and E.H.C. wrote the paper. All authors commented on the manuscript.

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Correspondence to Elizabeth H. Chen.

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Integrated supplementary information

Supplementary Figure 1 β-spectrin cannot replace βH-spectrin in myoblast fusion.

(a) Myoblast fusion defects caused by β-spectrin expression in muscle cells. Stage 15 embryos labeled with anti-MHC. Ventral lateral muscles of three hemisegments are shown in each panel. Unfused myoblasts are indicated by arrowheads. Expression of a functional, amino-terminal-tagged full-length Myc-β-Spec in all muscle cells with twi-GAL4 exacerbated the fusion defect in the βH-spec-/- mutant (left panel) and caused a minor fusion defect in wild-type (right panel) embryos. For each genotype, 10 embryos were imaged with similar results. Scale bar, 20 μm. (bd) β-spectrin, adducin and protein 4.1 are not enriched at the fusogenic synapse. Fusogenic synapses (arrowheads) in stage 13–14 wild-type embryos marked by anti-Sltr. β-Spec (b), adducin (known as hu-li tai shao in Drosophila1) (c), and protein 4.1 (known as coracle in Drosophila2) (d) were visualized by immunostaining with respective antibodies. In each case, 10 embryos were imaged with similar results. Note that none of these proteins was enriched at the fusogenic synapse. Scale bar, 5 μm. (e) Ectopically expressed β-spectrin in muscle cells is not enriched at the fusogenic synapse. Myc-β-Spec was ectopically expressed with a muscle-specific mef2-GAL4 driver. βH-Spec and β-Spec were visualized by anti-βH-Spec and anti-Myc, respectively. The fusogenic synapse (arrowhead) was marked by phalloidin staining (F-actin) and the FCM was outlined with white dotted line. Note that βH-Spec was highly enriched at the fusogenic synapse, whereas β-Spec exhibited a general cortical localization pattern. Ten fusogenic synapses were imaged with similar results. Scale bar, 5 μm. (f) Mini-βH-spectrin is enriched at the fusogenic synapse. mCherry-βH-Spec was expressed in muscle cells with twi-Gal4 and visualized by the mCherry fluorescent signal. The fusogenic synapse (arrowhead) was marked by phalloidin staining (F-actin) and the FCM is outlined with white dotted line. Note that mini-βH-Spec is highly enriched at the fusogenic synapse. Immunostainings were repeated three times. Ten fusogenic synapses were imaged with similar results. Scale bar, 5 μm.

Supplementary Figure 2 βH-spectrin is dynamic at the cortex of epithelial cells.

FRAP of βH-Spec in Drosophila embryonic epithelial cells. mCherry-βH-Spec was ectopically expressed in epithelial cells with the 69B-GAL4 driver. (a) Time-lapse stills of representative FRAP experiment in a stage 14 embryo. Arrowhead indicates the photo-bleached region. Scale bar, 5 μm. (b) Recovery kinetics of the mCherry fluorescence after photobleaching. The representative curve shows the fluorescence recovery of mCherry-βH-Spec from (a). The recovery half-time (t1/2) and percentage were quantified from multiple experiments. Each dot represents a fusogenic synapse; n = 31 fusogenic synapses were analyzed. The horizontal bars represent median value. The average t1/2 was 116 ± 38 sec (median: 112 sec) and percentage recovery was 64 ± 11% (median: 66%). Note that the half-time is longer than that at the fusogenic synapse (Fig. 2f).

Supplementary Figure 3 The mechanosensitivity of spectrin proteins revealed by MPA.

(a, b) Actin and β-spectrin do not show mechanosensitive accumulation. (a) Representative DIC and fluorescent images of aspirated cells. Arrowheads indicate the base areas of aspirated cells. (i) No mechanosensitive accumulation of mCherry-α-Spec and GFP-actin when co-expressed. (ii) mCherry-α-Spec accumulation at the base area when co-expressed with βH-Spec. (iii) mCherry-β-Spec did not show mechanosensitive accumulation as did βH-Spec when the two spectrin proteins were co-expressed. Sample sizes ranged from n = 8 to 10 as reflected in the dot plots in (b). Scale bars, 5 μm. (b) Quantification of protein accumulation at the base areas of aspirated cells. Each data point represents an independent MPA experiment; n = 8, 8, 10, 10, 9, 9 (from left to right). The horizontal bars represent the mean ratio. An ANOVA with Fisher’s least significant difference test was applied to determine statistical significance. (c, d) The mechanosensitive accumulation of βH-spectrin is time- and force-dependent. (c) βH-Spec accumulation over time shown by MPA experiments. The data points were collected at a fixed pressure of 0.4 nN/μm2, and plotted every 3s for a total of 2.5 min. Different color codes indicate traces of individual cells examined (n = 12). Each raw data point was normalized to the initial value to remove variability caused by differences in protein distribution at the start of the experiment. The average values of the 12 cells measured at each time point are shown as black dots with error bars (SEM). βH-Spec accumulation reached its peak at around 80–90s after the onset of aspiration. (d) βH-Spec accumulation depends on applied force. Each value is the peak accumulation reached during the experiment normalized to the initial Ib/Io value. Note that βH-Spec accumulation increases with increased pressure.

Supplementary Figure 4 The N-terminal CH domains of βH-spectrin bind F-actin.

(a) Domain structure of full length and mutant βH-Spec used in MPA and F-actin co-sedimentation assays. Actin-binding domains and the tetramerization site are indicated. Each distinct segment from the N-terminus to the C-terminus of βH-Spec is designated by a number. CH: calponin homology; SH3: Src homology 3; PH: pleckstrin homology; Spec: spectrin repeat. (b) F-actin co-sedimentation assay with purified βH-Spec fragments. The numbers (1, 29–31, 34) indicate βH-Spec fragments depicted in (a). S: supernatant; P: pellet. Note that βH-Spec fragment 1 precipitated with F-actin, whereas βH-Spec fragments 29–31 and 34 remained in the supernatant, confirming that the CH domains of βH-Spec bind F-actin. Experiments were repeated three times for each spectrin fragment with similar results.

Supplementary Figure 5

Unprocessed western blots for Fig. 5i (a) and Fig. 7d (b).

Supplementary Figure 6 Working models on the function of the mechanoresponsive protein α/βH-spectrin at the fusogenic synapse.

(a) The mechanosensitive accumulation of α/βH-spectrin. Based on coarse-grained modeling, an invasive membrane protrusion generate by a pushing force causes maximal shear deformation (shape change) of the actin network in the receiving cell at the base area of the protrusion. The extensibility and flexibility of α/βH-spectrin heterotetramers enable them to accommodate a range of angle/distance changes triggered by shear stress and stay bound to the shear-deformed actin network, resulting in their mechanosensitive accumulation. Actin polymers and spectrin heterotetramers are not drawn to scale. Actin-binding domains at the ends of the spectrin heterotetramer are colored in blue, and the PH domains in the middle of the heterotetramer are indicated as a short stub. (b) α/βH-spectrin functions as a cellular fence and a cellular sieve. The top diagram shows a pair of a founder cell (FC; now a binucleated myotube) and an FCM engaging at the fusogenic synapse marked by the actin focus (green). The relative localization domains of α/βH-spectrin (Spec; red), Duf (blue) and F-actin (green) are shown at the early and late stages of the fusogenic synapse from different viewing angles (bottom four square panels). The plasma membrane is shaded in gray, and F-actin underneath the plasma membrane in the top view panels is shaded in light green. At the early stage, α/βH-spectrin accumulates at the base of invasive protrusions and is not closely associated with Duf due to their different modes of recruitment. After multiple rounds of mechanoresponsive feedback between the two fusion partners, α/βH-spectrin accumulates in large areas of the fusogenic synapse to (1) restrict Duf diffusion via biochemical interactions and/or molecular collision, and (2) constrict the diameter of invasive protrusions with spectrin-free microdomains within the spectrin network. See text for details.

Supplementary Figure 7 The contour plots of the maxima of dilation and shear deformations induced by invasive protrusions.

The maxima of dilation (a) and shear (b) deformations induced by invasive protrusions with different lengths (vertical axis) and radii (horizontal axis) were plotted. The values of the physical parameters can be found in the description of the coarse-grained molecular mechanics modeling in the Methods section. Note that the dilation and shear deformations increase with the protrusion length and decrease with the protrusion radius.

Supplementary information

Supplementary Information

Supplementary Figures 1–7, Supplementary Video legends and Supplementary References.

Reporting Summary

Supplementary Video 1

Spectrin is dynamically associated with the F-actin focus at the fusogenic synapse

Supplementary Video 2

Spectrin accumulation correlates with the F-actin foci intensity

Supplementary Video 3

Dynamics of Spectrin at the fusogenic synapse

Supplementary Video 4

Dynamics of Spectrin at the cortex of epithelial cells

Supplementary Video 5

Mechanosensitive accumulation of Spectrin and MyoII induced by lateral indentation using AFM

Supplementary Video 6

Duf dispersal at the fusogenic synapse in α/βH-spectrin double-mutant embryos

Supplementary Video 7

Duf resides in a tight cluster at the fusogenic synapse in wild type embryos

Supplementary Video 8

F-actin dispersal in the absence of chemical signalling from Duf

Supplementary Video 9

βH-spectrin dispersal in duf,rst mutant embryos expressing DufΔintra

Supplementary Video 10

3D reconstruction of F-actin and βH-spectrin at the fusogenic synapse based on SIM imaging

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Duan, R., Kim, J.H., Shilagardi, K. et al. Spectrin is a mechanoresponsive protein shaping fusogenic synapse architecture during myoblast fusion. Nat Cell Biol 20, 688–698 (2018). https://doi.org/10.1038/s41556-018-0106-3

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