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 positioned. The molecular machinery responsible for myonuclear positioning is not known. Improperly positioned nuclei are a hallmark of numerous diseases of muscle, including centronuclear myopathies, 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.

Figure 1. Myonuclear positioning requires Ensconsin/MAP7

a. Hemi-segments from control and ens^swo embryos at beginning (stage 14) and end (stage 17) of nuclear migration. Muscles, tropomyosin (green), Nuclei, dsRed (red). Bar, 10 µm. b. Timelapse of nuclear migration in control and ens^swo hemisegment. Bar, 10 µm. c. Timelapse of nuclear migration in control and Map7 depleted C2C12-H1B-GFP myotubes. Nuclei, red; Bar, 15 um. d. Nuclear distribution in C2C12 myotubes that were untreated (control) or treated with indicated siRNA and Map7 depleted cells expressing full length MAP7 (#118 + FL-MAP7). Error bars, s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05 (scrambled vs. experimental condition).

Figure 2. Kinesin is required for myonuclear positioning

a. Single hemi-segments from stage 17 (22h AEL) embryos of indicated genotypes; Bar, 10 µm. b. Representative immunofluorescence images of control and Kif5b depleted C2C12 myotubes differentiated for 4 days and immunostained for MHC (green) and DAPI (red). Bars, 15 µm. c. Histogram of nuclear distribution in C2C12 myotubes that were untreated (control) or treated with the indicated siRNA or Kif5b depleted cells expressing full length Kif5b (#781 + FL-Kif5b). Error bars, s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05 (scrambled vs. experimental condition).

Figure 3. Kinesin and Ensconsin/MAP7 interact to regulate nuclear position

a. Kif5b, MAP7 and chimera constructs N-terminally tagged with GFP (MAP7/chimeras) or c-Myc (Kif5b). Numbers indicate amino acids. b. Western blot with indicated antibodies (right) of GFP-MAP7 (top) immunoprecipitations. c. Anti-GFP Western blot of Kif5b immunoprecipitations from C2C12s expressing indicated constructs (top). (*) anti-Kif5b IgG. d. Anti-c-Myc Western blot from C2C12s expressing Kif5b constructs. e. Anti-c-Myc Western blot from GFP immunoprecipitations using C2C12s expressing indicated constructs. f. Nuclear distribution in Map7 depleted C2C12s expressing indicated chimeras. Error bars s.e.m. **p < 0.01, *p < 0.05 g. Map7 depleted C2C12s expressing indicated chimeras. Anti-GFP (green), Nuclei, DAPI (red). Bar, 15 µm.

Figure 4. Ensconsin/MAP7 is required for intracellular muscle organization and efficient larval locomotion

a. Maximum intensity XY projections of muscle VL3 from segment A3 of L3 larvae from the indicated genotypes stained for actin (red), nuclei (white), and Z-bands (green). Bar, 20 µm. b. Average velocity of migration for L3 larvae of the indicated genotypes. Each of the indicated Gal4 drivers is expressing UAS-ensHA in a homozygous ens^swo mutant background. Error bars, s.e.m. ***p < 0.001, **p < 0.01. c. Nearest neighbor analysis of nuclei within muscle VL3 from segment A3 from L3 larvae of the indicated genotype. Error bars, s.e.m. ***p < 0.001, **p < 0.01.