Letter | Published:

MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function

Nature volume 484, pages 120124 (05 April 2012) | Download Citation

Abstract

The basic unit of skeletal muscle in all metazoans is the multinucleate myofibre, within which individual nuclei are regularly positioned1. The molecular machinery responsible for myonuclear positioning is not known. Improperly positioned nuclei are a hallmark of numerous diseases of muscle2, including centronuclear myopathies3, but it is unclear whether correct nuclear positioning is necessary for muscle function. Here we identify the microtubule-associated protein ensconsin (Ens)/microtubule-associated protein 7 (MAP7) and kinesin heavy chain (Khc)/Kif5b as essential, evolutionarily conserved regulators of myonuclear positioning in Drosophila and cultured mammalian myotubes. We find that these proteins interact physically and that expression of the Kif5b motor domain fused to the MAP7 microtubule-binding domain rescues nuclear positioning defects in MAP7-depleted cells. This suggests that MAP7 links Kif5b to the microtubule cytoskeleton to promote nuclear positioning. Finally, we show that myonuclear positioning is physiologically important. Drosophila ens mutant larvae have decreased locomotion and incorrect myonuclear positioning, and these phenotypes are rescued by muscle-specific expression of Ens. We conclude that improper nuclear positioning contributes to muscle dysfunction in a cell-autonomous fashion.

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References

  1. 1.

    , , , & Number and spatial distribution of nuclei in the muscle fibres of normal mice studied in vivo. J. Physiol. (Lond.) 551, 467–478 (2003)

  2. 2.

    & Molecular basis of muscular dystrophies. Muscle Nerve 23, 1456–1471 (2000)

  3. 3.

    , & Centronuclear (myotubular) myopathy. Orphanet J. Rare Dis. 3, 26 (2008)

  4. 4.

    , , & SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodeling at the site of myoblast fusion. Development 134, 4357–4367 (2007)

  5. 5.

    & The development of the Drosophila larval body wall muscles. Int. Rev. Neurobiol. 75, 55–70 (2006)

  6. 6.

    , , & Absence of PS integrins or laminin A affects extracellular adhesion, but not intracellular assembly, of hemiadherens and neuromuscular junctions in Drosophila embryos. Dev. Biol. 196, 58–76 (1998)

  7. 7.

    Singling out Drosophila tendon cells: a dialogue between two distinct cell types. Trends Genet. 15, 448–453 (1999)

  8. 8.

    et al. Drosophila ensconsin promotes productive recruitment of Kinesin-1 to microtubules. Dev. Cell 15, 866–876 (2008)

  9. 9.

    & Marbles mutants: uncoupling cell determination and nuclear migration in the developing Drosophila eye. Development 120, 2609–2618 (1994)

  10. 10.

    , , & Lethal kinesin mutations reveal amino acids important for ATPase activation and structural coupling. J. Biol. Chem. 274, 31506–31514 (1999)

  11. 11.

    , & Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility. Cell 42, 39–50 (1985)

  12. 12.

    et al. Direct observation of single kinesin molecules moving along microtubules. Nature 380, 451–453 (1996)

  13. 13.

    & Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells. J. Cell Biol. 123, 357–371 (1993)

  14. 14.

    , , , & Bilateral olfactory sensory input enhances chemotaxis behavior. Nature Neurosci. 11, 187–199 (2008)

  15. 15.

    , & SALS, a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth. Dev. Cell 13, 828–842 (2007)

  16. 16.

    et al. Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila. Genes Dev. 15, 2967–2979 (2001)

  17. 17.

    et al. The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction. Neuron 39, 241–254 (2003)

  18. 18.

    , & Fate of microtubule-organizing centers during myogenesis in vitro. J. Cell Biol. 100, 35–46 (1985)

  19. 19.

    & RacGAP50C directs perinuclear γ-tubulin localization to organize the uniform microtubule array required for Drosophila myotube extension. Development 136, 1411–1421 (2009)

  20. 20.

    , & Reorganization of microtubule nucleation during muscle differentiation. Cell Motil. Cytoskeleton 60, 1–13 (2005)

  21. 21.

    The 3Ms of central spindle assembly: microtubules, motors and MAPs. Nature Rev. Mol. Cell Biol. 10, 9–20 (2009)

  22. 22.

    , , , & UNC-83 is a nuclear-specific cargo adaptor for kinesin-1-mediated nuclear migration. Development 136, 2725–2733 (2009)

  23. 23.

    , , , & Syne proteins anchor muscle nuclei at the neuromuscular junction. Proc. Natl Acad. Sci. USA 102, 4359–4364 (2005)

  24. 24.

    et al. SUN1 and SUN2 play critical but partially redundant roles in anchoring nuclei in skeletal muscle cells in mice. Proc. Natl Acad. Sci. USA 106, 10207–10212 (2009)

  25. 25.

    , , & Localization of muscle gene products in nuclear domains. Nature 337, 570–573 (1989)

  26. 26.

    , , , & Cytogenetic and molecular localization of tipE: a gene affecting sodium channels in Drosophila melanogaster. Genetics 139, 1679–1688 (1995)

  27. 27.

    , , , & Kinesin light chains are essential for axonal transport in Drosophila. J. Cell Biol. 141, 443–454 (1998)

  28. 28.

    et al. Drosophila fragile X-related gene regulates the MAP1B homolog Futsch to control synaptic structure and function. Cell 107, 591–603 (2001)

  29. 29.

    & The β subunit of the Drosophila melanogaster ATP synthase: cDNA cloning, amino acid analysis and identification of the protein in adult flies. Biochem. Biophys. Res. Commun. 195, 785–791 (1993)

  30. 30.

    et al. Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development. Nature Cell Biol. 12, 257–266 (2010)

  31. 31.

    et al. New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes. Genesis 34, 135–138 (2002)

  32. 32.

    Method of Feeding Ethane Methylsulfonate (EMS) to Drosophila Males. (Drosophila Information Service, 1968)

  33. 33.

    et al. Skeletal muscle phenotypically converts and selectively inhibits metastatic cells in mice. PLoS ONE 5, e9299 (2010)

  34. 34.

    et al. Nitric oxide inhibition of Drp1-mediated mitochondrial fission is critical for myogenic differentiation. Cell Death Differ. 17, 1684–1696 (2010)

  35. 35.

    et al. A perinuclear actin cap regulates nuclear shape. Proc. Natl Acad. Sci. USA 106, 19017–19022 (2009)

  36. 36.

    , & Drosophila larval NMJ dissection. J. Vis. Exp. 10.3791/1107. (2009)

  37. 37.

    , & Drosophila larval NMJ immunohistochemistry. J. Vis. Exp. (25). e1108 10.3791/1108 (2009)

  38. 38.

    , & Live imaging of Drosophila myoblast fusion. Methods Mol. Biol. 475, 263–274 (2008)

  39. 39.

    , & High-resolution measurement of odor-driven behavior in Drosophila larvae. J. Vis. Exp. (11). e638 10.3791/638 (2008)

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Acknowledgements

We thank K. Anderson, K. Hadjantonakis, A. Hall and D. Sassoon for comments on the manuscript. We thank the Baylies and Gomes Laboratories for discussions, and R. Fernandez-Gonzalez for his assistance in computational analysis. The initial screens in Drosophila were supported by National Institutes of Health (NIH) grants GM056989 and GM0781318 to M.B.; the Drosophila nuclear positioning analysis was supported by a Muscular Dystrophy Association (MDA) grant to M.B. T.M. was supported initially by NIH Training Grant T32 BM008539. B.C. was supported initially by a Fondation pour la Recherche Médicale (FRM) fellowship. V.G. was supported initially by a Région Île-de-France fellowship. Mammalian work was supported by Muscular Dystrophy Association (MDA), INSERM Avenir programme and Agence Nationale de la Recherche (ANR) grants to E.R.G.

Author information

Author notes

    • Thomas Metzger
    • , Vincent Gache
    • , Edgar R. Gomes
    •  & Mary K. Baylies

    These authors contributed equally to this work.

Affiliations

  1. Program in Developmental Biology, Sloan-Kettering Institute, New York, New York 10065, USA

    • Thomas Metzger
    • , Mu Xu
    • , Eric S. Folker
    • , Brian E. Richardson
    •  & Mary K. Baylies
  2. Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10065, USA

    • Thomas Metzger
    •  & Mary K. Baylies
  3. UMR S 787 INSERM, Université Pierre et Marie Curie Paris 6, 75634 Paris, France

    • Vincent Gache
    • , Bruno Cadot
    •  & Edgar R. Gomes
  4. Groupe Hospitalier Pitié-Salpêtrière, Institut de Myologie, 75013 Paris, France

    • Edgar R. Gomes

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Contributions

T.M. and V.G. are joint first authors. E.G. and M.B. are joint senior authors. M.B., M.X., T.M., E.G., V.G. and B.C. conceived, designed and analysed the experiments. T.M., M.X., E.S. and B.R. conducted the Drosophila experimental work. V.G. and B.C. conducted the mouse primary cultures and C2C12 experimental work. The manuscript was written by T.M., E.S., V.G., E.G. and M.B. with assistance from other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Edgar R. Gomes or Mary K. Baylies.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figures 1-10 and legends for Supplementary Movies 1-8.

Videos

  1. 1.

    Supplementary Movie 1

    Nuclear migration in the Lateral Transverse muscles of a wildtype Drosophila embryo - see Supplementary Information file for full legend.

  2. 2.

    Supplementary Movie 2

    Nuclear migration in the Lateral Transverse muscles of an ensswo mutant embryo - see Supplementary Information file for full legend.

  3. 3.

    Supplementary Movie 3

    Control primary myotubes - see Supplementary Information file for full legend.

  4. 4.

    Supplementary Movie 4

    Map7 siRNA depleted primary myotubes - see Supplementary Information file for full legend.

  5. 5.

    Supplementary Movie 5

    Control C2C12-H1B-GFP myotubes - see Supplementary Information file for full legend.

  6. 6.

    Supplementary Movie 6

    Map 7 siRNA C2C12-H1B-GFP myotubes - see Supplementary Information file for full legend.

  7. 7.

    Supplementary Movie 7

    Control primary myotubes - see Supplementary Information file for full legend.

  8. 8.

    Supplementary Movie 8

    Kif5b siRNA depleted primary myotubes - see Supplementary Information file for full legend.

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DOI

https://doi.org/10.1038/nature10914

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