Fig. 1.

Nuclear import and export sequence combinations enhance myonuclear GFP trafficking in myotubes and myofibers. (A) HCR-FISH against GFP mRNA (magenta) in a tibialis anterior (TA) myofiber of a mouse treated with an AAV encoding GFP. (Scale bar: 10 µm.) (B) Model for why protein cargoes with nuclear localization sequences accumulate in certain nuclei and not others. Peptide tags that facilitate both nuclear import and export may improve trafficking of protein cargoes to nontransduced nuclei. (C) Schematic of the C2C12 fusion experiment to identify NLS/NES combinations that improve propagation of GFP across multiple myonuclei. (D) Representative images of GFP fluorescence (green) in chimeric C2C12 myotubes expressing NLS/NES combinations. (Scale bar: 40 µm.) (E) Cumulative distribution functions of nuclear GFP signal for NLS/NES combinations in chimeric C2C12 myotubes. Significance by Kolmogorov–Smirnov test. (F) Schematic of the experiment to assess nuclear propagation of GFP in 4-wk-old WT mice treated systemically with 5E+13vg/kg 2xSV40 NLS GFP AAV or Myospreader GFP AAV. (G) GFP signal (green) in representative TA myofibers isolated from treated mice. Myonuclei borders are indicated with dashed lines. (Scale bar: 1 mm.) (H) Density plot of nuclear GFP signal in TA myofibers of treated mice. Significance by Kolmogorov–Smirnov test (ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001) (error bars = 95% CI).