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

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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Figure 1: Myonuclear positioning requires ensconsin/MAP7.
Figure 2: Kinesin is required for myonuclear positioning.
Figure 3: Kinesin and ensconsin/MAP7 interact to regulate nuclear position.
Figure 4: Ensconsin/MAP7 is required for intracellular muscle organization and efficient larval locomotion.

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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.

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Authors

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.

Corresponding authors

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

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10 and legends for Supplementary Movies 1-8. (PDF 18266 kb)

Supplementary Movie 1

Nuclear migration in the Lateral Transverse muscles of a wildtype Drosophila embryo - see Supplementary Information file for full legend. (MOV 1658 kb)

Supplementary Movie 2

Nuclear migration in the Lateral Transverse muscles of an ensswo mutant embryo - see Supplementary Information file for full legend. (MOV 1547 kb)

Supplementary Movie 3

Control primary myotubes - see Supplementary Information file for full legend. (MOV 422 kb)

Supplementary Movie 4

Map7 siRNA depleted primary myotubes - see Supplementary Information file for full legend. (MOV 315 kb)

Supplementary Movie 5

Control C2C12-H1B-GFP myotubes - see Supplementary Information file for full legend. (MOV 308 kb)

Supplementary Movie 6

Map 7 siRNA C2C12-H1B-GFP myotubes - see Supplementary Information file for full legend. (MOV 217 kb)

Supplementary Movie 7

Control primary myotubes - see Supplementary Information file for full legend. (MOV 419 kb)

Supplementary Movie 8

Kif5b siRNA depleted primary myotubes - see Supplementary Information file for full legend. (MOV 305 kb)

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Metzger, T., Gache, V., Xu, M. et al. MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function. Nature 484, 120–124 (2012). https://doi.org/10.1038/nature10914

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