LMNA variants cause cytoplasmic distribution of nuclear pore proteins in Drosophila and human muscle

Abstract

Mutations in the human LMNA gene, encoding A-type lamins, give rise to laminopathies, which include several types of muscular dystrophy. Here, heterozygous sequence variants in LMNA, which result in single amino-acid substitutions, were identified in patients exhibiting muscle weakness. To assess whether the substitutions altered lamin function, we performed in vivo analyses using a Drosophila model. Stocks were generated that expressed mutant forms of the Drosophila A-type lamin modeled after each variant. Larvae were used for motility assays and histochemical staining of the body-wall muscle. In parallel, immunohistochemical analyses were performed on human muscle biopsy samples from the patients. In control flies, muscle-specific expression of the wild-type A-type lamin had no apparent affect. In contrast, expression of the mutant A-type lamins caused dominant larval muscle defects and semi-lethality at the pupal stage. Histochemical staining of larval body wall muscle revealed that the mutant A-type lamin, B-type lamins, the Sad1p, UNC-84 domain protein Klaroid and nuclear pore complex proteins were mislocalized to the cytoplasm. In addition, cytoplasmic actin filaments were disorganized, suggesting links between the nuclear lamina and the cytoskeleton were disrupted. Muscle biopsies from the patients showed dystrophic histopathology and architectural abnormalities similar to the Drosophila larvae, including cytoplasmic distribution of nuclear envelope proteins. These data provide evidence that the Drosophila model can be used to assess the function of novel LMNA mutations and support the idea that loss of cellular compartmentalization of nuclear proteins contributes to muscle disease pathogenesis.

Figure 1.

Ig-fold domain of human and Drosophila A-type lamins. (A) Alignment of the Ig-fold domain of human Lamin A/C (Hs) and Drosophila Lamin C (Dm), using BLAST (bl2seq, NIH). One of the amino acids affected in the patient cases (G449) is identical between the two species; the second amino acid is similar (L489) and the third amino acid is not conserved (W514) but is flanked by conserved residues. Amino acid N456 is known to cause AD-EDMD when substituted with I. (+) Conserved residue based on similar amino-acid structure; (−) Gap in amino-acid sequence. (B) Ribbon diagram of the human Lamin A/C Ig-like fold (PDB: 1IFR) showing the relative position of the amino acids affected in patient cases studied here.

Figure 2.

Mutant forms of Lamin C cause locomotion defects. The results from locomotion assays of larvae expressing Lamin C transgenes by the C57 Gal4/UAS driver are shown. Three independent assays were performed using eight similarly staged larvae each. Box plots display 25–75 percentile range of the data and the thick black line indicates the median value. The Y-axis represents zones outlined in a Petri dish containing agarose. The X-axis shows the mutant form of Lamin C expressed in larvae.

Figure 3.

Expression of mutant forms of Lamin C in larval muscle causes mislocalization of A- and B-type lamins, nuclear and cytoskeletal defects. Staining of third instar larval body wall muscle fillets with anti-Lamin C (A, green), anti-lamin Dm₀ (B, green), phalloidin (A and B, purple) and DAPI (A and B/merge, blue) from larvae expressing wild-type Lamin C (wild-type) or mutant Lamin C transgenes by the muscle-specific driver C57. Drosophila amino-acid numbers are shown with human amino acid numbering in parentheses. Bar, 10 μm.

Figure 4.

Expression of mutant forms of Lamin C in larval muscle causes mislocalization of Klaroid. Staining of third instar larval body wall muscle fillets with anti-Klaroid (green), phalloidin (purple) and DAPI (merge, blue) from larvae expressing wild-type Lamin C (wild-type) or mutant Lamin C transgenes by the muscle-specific driver C57. Drosophila amino-acid numbers are shown with human numbers in parentheses. Bar, 10 μm.

Figure 5.

Expression of mutant forms of Lamin C in larval muscle causes mislocalization of nuclear pore proteins. Staining of third instar larval body wall muscle fillets with an antibody that recognizes nuclear pores possessing an FG-repeat (A, green), and the nuclear pore protein GP210 (B, green), phalloidin (A and B, purple) and DAPI (A and B/merge, blue) from larvae expressing wild-type Lamin C (wild-type) or mutant Lamin C transgenes by the muscle-specific driver C57. Drosophila amino-acid numbers are shown with human numbering in parentheses. Bar, 10 μm.

Figure 6.

Dystrophic features in human patient skeletal muscle biopsies. Staining of human muscle biopsies from healthy individuals (control) or novel LMNA mutation patients (G449V, L489P, W514R) with anti-Lamin A/C (A, green), anti-Lamin B1 (B, green), anti-dystrophin (A and B, purple) and DAPI (A and B, blue). Bar, 10 μm.

Figure 7.

Mislocalization of SUN2 and nuclear pore proteins in human patient skeletal muscle biopsies. Staining of human muscle biopsies from healthy individuals (control) or novel LMNA mutation patients (G449V, L489P, W514R) with anti-SUN2 (A, green), antibodies to nuclear pore proteins possessing FG repeats (B, green), phalloidin (A and B, purple) and DAPI (A and B, blue). Areas boxed in yellow are shown as enlargements below. Arrowheads indicate cytoplasmic localization of SUN2 and FG-repeat containing proteins in skeletal muscle fibers. Bar, 10 μm.

Figure 8.

Intact myofibers showing mislocalization of nuclear pore complexes in human patient skeletal muscle biopsies. Staining of human muscle biopsies from healthy individuals (control) or novel LMNA mutation patients (G449V, L489P, W514R) with antibodies that recognize nuclear pore proteins possessing FG repeats (green), anti-dystrophin (purple) and DAPI (blue). Areas boxed in yellow are enlarged below. Asterisks indicate cytoplasmic localization of NPCs in skeletal muscle fibers. Bar, 10 μm.

Figure 9.

Mislocalization of nuclear pore proteins in AD-EDMD, but not other types of MD patients. Staining of human muscle biopsies from healthy individuals (pediatric and adult control) or patients with LMNA mutations causing AD-EDMD (E358K/rod, R541P/Ig-fold), LGMD (R453W/Ig-fold), DMD or FSHD with antibodies that recognize nuclear pores possessing FG repeats (green), phalloidin (purple) and DAPI (blue). Area boxed in yellow shows cytoplasmic distribution of nuclear pore proteins. Bar, 10 μm.