Expression of LINC complex proteins is increased in patient MD-1 (<i>SUN1</i> p.G68D/p.G338S) myoblasts.

<p>(<b>A</b>) Control and MD-1 myoblasts were fixed in methanol and analysed by immunofluorescence microscopy using SUN1, SUN2, emerin, nesprin-2G and lamin A/C antibodies, as indicated, together with DAPI staining of DNA. Scale bar, 22 µm. (<b>B</b>) Mean fluorescence intensity of SUN1, SUN2, emerin, nesprin 2 and lamin A/C was measured in individual DAPI-stained nuclei using an Olympus Scan∧R screening station and analysed using Scan∧R analysis software. The results are presented as mean ± S.E. of 1000 cells taken from at least 3 independent experiments. **<i>P</i>≤0.05. Significant <i>P</i>-value for SUN1 was <i>P</i> = 0.009. (<b>C</b>) Total protein extracts from control (C) and patient MD-1 myoblasts were Western blotted using antibodies against LINC complex-associated proteins, as indicated. (<b>D</b>) Samples prepared as in A were Western blotted using nesprin-2 (N2–N3) antibodies. (<b>E–F</b>) Protein expression was quantified by densitometric analysis of at least 3 independent experiments. The results are presented as mean ± S.E. *<i>P</i>≤0.05 and **<i>P</i>≤0.01. Each significant <i>P</i>- values are as follows: SUN1 <i>P</i> = 0.05, α-tubulin <i>P</i> = 0.003, nesprin-2 <i>P</i> = 0.002. <i>P</i>- value for emerin was <i>P</i> = 0.06.</p

SUN1 and SUN2 variants identified and associated family pedigrees.

<p>(<b>A</b>) Schematic diagram of the SUN1 and SUN2 protein domain organization and locations of disease-associated variants identified in our cohort. Mutation <i>SUN1</i> M50T, indicated in purple, did not disrupt LINC complex function in migration assays and thus may not be truly disease-causing. The mapped lamin A/C (green) and emerin (orange) binding sites, located in the nucleoplasmic N-terminal domain, are indicated. Regions of high hydrophobicity and the transmembrane domain are shown in grey and black, respectively. Coiled-coil domains responsible for oligomerization (blue) and the highly conserved SUN domain (red), found within the luminal C-terminal domain, are also indicated. (<b>B</b>) Pedigree with recessive inheritance of compound heterozygous <i>SUN1</i> variants. (<b>C</b>) Pedigrees where severely affected index cases carry <i>SUN1</i> and/or <i>SUN2</i> variants in combination with other gene mutations. Filled circles/squares indicate affected females/males. Circles containing a dot, in family 3, indicate unaffected female carriers of the X-linked <i>EMD</i> mutation. Arrows indicate index patients.</p

Schematic model of nuclear positioning and microtubule connections during differentiation of normal and SUN1/2 mutant myoblasts.

<p>(<b>A</b>) In myoblasts, the close positioning of centrosomes (red) to the outer nuclear surface is disrupted by SUN1/2 mutants, which is likely to be accompanied by impaired microtubule (green) association with the NE. (<b>B</b>) Upon commitment to differentiation in normal myoblasts, pericentrin and other centrosomal proteins redistribute from the centrosome to the nuclear surface, which becomes the major site of microtubule nucleation. In mutant committed myoblasts, pericentrin fails to associate with the NE and there is impairment of microtubule nucleation from the nuclear surface. (<b>C</b>) After cell fusion to form myotubes, the microtubules reorganize into overlapping parallel arrays along the long axis of the cell. The myonuclei become positioned evenly along the length of the cell in a microtubule-dependent manner with the involvement of dynein and kinesin motor proteins. In mutant myotubes, nuclei are clumped in a disorganized fashion and we propose that this is due to an inability to interact with the microtubule network.</p

Putative disease-causing variants in <i>SUN1</i> and <i>SUN2</i> in patients with EDMD-like phenotypes.

<p>Putative disease-causing variants in <i>SUN1</i> and <i>SUN2</i> in patients with EDMD-like phenotypes.</p

Impaired pericentrin localization and microtubule nucleation at the nuclear envelope in MD-1 myotubes.

<p>(<b>A</b>) Beta-tubulin (red) and pericentrin (green) double immunofluorescence staining of control and MD-1 myotubes. Chromatin was stained with DAPI (blue). Scale bar, 10 µm. Arrows indicate apparent pericentrin accumulation at the nuclear poles. (<b>B</b>) Control and MD-1 myotubes were treated with nocodazole followed by 5 min recovery in culture medium to allow microtubule regrowth. Samples were then fixed and stained as in panel A. Panels a and c show myotubes, whilst panels b and d show committed myoblasts from control and MD-1 cultures, respectively. Arrows in control cells indicate sites of microtubule regrowth at the nuclear poles, co-inciding with pericentrin localization. Arrowheads in patient cells indicate microtubule regrowth from cytoplasmic pericentrin foci. (<b>C</b>) Control and MD-1 myoblasts were fixed in methanol and subjected to immunofluorescence analysis using γ-tubulin antibodies to stain the centrosome and DAPI to stain the DNA. Distances between centrosomes and the nuclear periphery (µm) are indicated. Scale bar, 10 µm. (<b>D</b>) The number of microtubules nucleating from individual myotube nuclei prepared in B was counted and is presented as the mean ±S.D (n = 50 nuclei per sample). * p<0.05 as calculated using the Student's <i>t</i>-test. (<b>E</b>) Nucleus-centrosome distance was measured in 100 control and MD-1 myoblasts prepared in C, in two independent experiments, using Leica LAS AF Lite software and analysed using SPSS software. The median values (thick black lines) were 0.96 and 2.43 µm for control and MD-1 cells, respectively. <i>P</i> = 0.00012. ○ and * correspond to mild and extreme outliers, respectively.</p

Enhanced rate of differentiation, nuclear misalignment and clustering in MD-1 myotubes.

<p>(<b>A</b>) Immunofluorescence staining of MD-1 myotubes using SUN1 (green) and desmin antibodies (red). Chromatin was stained with DAPI (blue). Scale bar, 30 µm. (<b>B</b>) Graphical representation of the percentage of nuclei within myotubes counted in MD-1 and in five independent control cultures. (<b>C</b>) Phase contrast image of living control and MD-1 cultured myotubes (arrows) showing myonuclear clustering in patient cells. Scale bar, 10 µm. (<b>D</b>) Immunofluorescence staining of control and MD-1 myotubes with SUN2 (green) and caveolin 3 (red) antibodies. Arrows indicate SUN2 polarization at the nuclear poles in control cells. (<b>E–F</b>) Graphical representation of the percentage of myotubes with more than 10 nuclei and the percentage of myotubes with myonuclear clustering in control and MD-1 cultures. Data are presented as mean values ± S.D. of three independent experiments (50 myotubes per sample were counted). (<b>G</b>) Graphical representation of the percentage of committed myoblast and myotube nuclei with enrichment of SUN2 staining at the nuclear pole(s). Data are presented as mean values ±S.D. of 3 independent experiments (200 nuclei per sample). (<b>H</b>) Transmission electron microscopy analysis of control and MD-1 myotubes (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004605#s4" target="_blank">Materials and Methods</a> for details). Sarcomeric structures are evident in control myotubes (arrowheads), whereas they are absent from MD-1 myotubes showing myonuclear clustering. Arrows indicate myonuclei. Scale bar, 10 µm.</p

Emerin binding to p.G68D/p.G338S SUN1 is reduced <i>in vivo</i>.

<p>(<b>A</b>) SUN1 was immunoprecipitated from control (C) or MD-1 myoblast soluble lysates using 2383 SUN1 antibodies and samples Western blotted to detect co-immunopreciptated proteins. (<b>B</b>) Densitometric analysis of SUN1, lamin A/C and emerin bands from immunoprecipitated samples is plotted in arbitrary units (A.U.). (<b>C</b>) SUN1 was immunoprecipitated from control or MD-1 myoblasts and co-precipitated nesprin-2 was detected using N2–N3 antibody. Size markers (kDa) are indicated. Note that larger nesprin-2 isoforms are enriched in the immunoprecipitate compared to the lysate. (<b>D</b>) HEK293 cells were co-transfected with myc-SUN1 mutant and GFP-emerin plasmids and harvested after 48 hours. Co-precipitated GFP-emerin was detected by immunoblotting using GFP antibodies (myc-SUN1 IP). (<b>E</b>) Densitometric values of immunoblotted GFP-emerin bands are reported in arbitrary units (A.U.). (<b>F</b>) Human fibroblasts from an EDMD2 patient carrying the R401C <i>LMNA</i> mutation were transfected with SUN1-WT or SUN1-W377C cDNAs and fixed 48 hours after transfection. Lamin A/C was labelled using specific antibodies and revealed by FITC-conjugated secondary antibody (green). SUN1 was detected using Cy3-conjugated anti-myc antibody (red). Nuclei were counterstained with DAPI. Images show pairs of daughter cells from recent cell divisions. Increased dysmorphic nuclei with nuclear blebbing and honeycomb structures (arrows) are observed in double-mutant cells. The distance between daughter cells has been reduced using Photoshop 7 to allow magnification. (<b>G</b>) Percentage of dysmorphic nuclei in EDMD2 cells left untreated (untransfected), transfected with WT-SUN1 or transfected with W377C-SUN1 is reported in the graph as the mean of three independent experiments. Statistically significant differences (P<0.05) relative to controls or WT-transfected samples are indicated by asterisks.</p

Exogenously expressed SUN1 mutants impair pericentrin recruitment to the nuclear envelope.

<p>(<b>A</b>) Differentiated C2C12 myotubes transfected with wild-type (WT) SUN1, or the indicated mutants, were labelled with anti-myc (red) and anti-pericentrin antibodies (green). Desmin antibody (violet) was used as a muscle differentiation marker. Nuclei were counterstained using DAPI. Samples were observed using a Nikon laser confocal microscope. Bar, 10 µm. (<b>B–C</b>) Transfected myotubes prepared as in A were quantified for the absence of pericentrin staining at the nuclear envelope (B) and myonuclear clustering (C). Thirty myotubes per sample were counted in two independent experiments. Differences for all mutants were statistically significant with respect to wild-type-transfected myotubes (<i>P</i><0.01).</p

<i>SUN1</i> and <i>SUN2</i> variants with disease-modifying effects in patients with <i>MYBPC3</i>, <i>EMD</i> and <i>LMNA</i> mutations.

<p><i>SUN1</i> and <i>SUN2</i> variants with disease-modifying effects in patients with <i>MYBPC3</i>, <i>EMD</i> and <i>LMNA</i> mutations.</p