Muscular Dystrophy-Associated SUN1 and SUN2 Variants Disrupt Nuclear-Cytoskeletal Connections and Myonuclear Organization

Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning

Molecular Dissection of the Centrosome Overduplication Pathway in S-Phase-Arrested Cells ▿ †

Cancer cells frequently exhibit overduplicated centrosomes that lead to formation of multipolar spindles, chromosome missegregation, and aneuploidy. However, the molecular events involved in centrosome overduplication remain largely unknown. Experimentally, centrosome overduplication is observed in p53-deficient cells arrested in S phase with hydroxyurea. Using this assay, we have identified distinct roles for Cdk2, microtubules, dynein, and Hsp90 in the overduplication of functional centrosomes in mammalian cells and show that Cdk2 is also required for the generation of centriolar satellites. Moreover, we demonstrate that nuclear export is required for centriolar satellite formation and centrosome overduplication, with export inhibitors causing a Cdk-dependent accumulation of nuclear centrin granules. Hence, we propose that centrosome precursors may arise in the nucleus, providing a novel mechanistic explanation for how nuclear Cdk2 can promote centrosome overduplication in the cytoplasm. Furthermore, this study defines a molecular pathway that may be targeted to prevent centrosome overduplication in S-phase-arrested cancer cells

A two-step model of mast cell adhesion.

<p>A diagram illustrates cell membrane complexes of MC and ASMC at the top and bottom, respectively. In MCs, CADM1 dimer forms a complex with 4.1 proteins and CASK, bound to F-actin. In ASMCs, nectin-3 dimer forms a complex with afadin, connected to F-actin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone.0085980-Ogita1" target="_blank">[73]</a>. In an initial stage of MC adhesion to ASMCs, CADM1 dimers interact with nectin-3 dimers on the surface of ASMCs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone.0085980-Moiseeva2" target="_blank">[19]</a>. This places inactive integrins in close proximity to their ECM ligands on ASMC surface. Kit, stabilised by CADM1, becomes close to membranous CSF in ASMCs. During an advanced adhesion stage, active Kit dimers bind to mCSF and initiate signal transduction to integrins in MCs. Integrins and other ECM receptors on MCs find their ECM ligands and interact with them. Activated integrins bind talin, vinculin and other structural proteins, which link integrins to cortical F-actin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone.0085980-Morgan1" target="_blank">[57]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone.0085980-Humphries1" target="_blank">[74]</a>, organised by CADM1.</p

Downregulation of CADM1 in human lung mast cells reduced the assembly of filamentous actin and tyrosine phosphorylation.

<p><b>A.</b> HLMCs from various donors were transduced with SP4, control shRNA (LucSh) or CADM1 shRNA (Shm) viral particles, followed by fluorescent staining of surface CADM1 and F-actin. Surface CADM1 expression is shown in histograms. <b>B.</b> CADM1 in the SP4 and Shm groups was expressed as percentages of the control LucSh group and quantified as percentage and log-transformed percentage. (n = 5 for LucSh and SP4, n = 4 for Shm). These data were presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone-0085980-g001" target="_blank">Fig. 1</a> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone.0085980-Moiseeva2" target="_blank">[19]</a>. <b>C.</b> HLMCs with modulated CADM1 were also co-stained for F-actin. The data are shown in the graph. Correlation between surface CADM1 and F-actin in LucSh- or Shm-transduced HLMCs is shown in a scatter plot with correlation parameters. <b>E.</b> Western blotting of protein extracts from LucSh-, SP4- and Shm-transduced HLMCs from D682 and D674, developed with Abs shown on the right. The protein bands were quantified and the data are shown in the graph.</p

The effect of EDTA and CADM1 modulation on HMC-1 adhesion to airway smooth muscle cells and their extracellular matrices at 30 min and 60 min.

<p><b>A.</b> HMC-1 cells (n = 2 in quadruplicate) adhered to HASMCs (n = 3) or HASMC ECM for 30 or 60 minutes in the absence or presence of 5 mM EDTA. <b>B and C</b>. HMC-1 cells (n = 2 in quadruplicate), transduced with CADM1 SP4 or shRNA (Sh5) adenoviral particles, adhered to HASMCs (n = 3 in quadruplicate)(B) or their ECM (<b>C</b>). <b>D and E</b>. HMC-1 cells (n = 2 in quadruplicate), pre-incubated with inhibitory antibodies for CADM1 (9D2) and aplha5beta1 integrin, isotype controls IgY and IgG1 or 5 mM EDTA, adhered to fibronectin (D) or HASMC ECM (E). * P<0.05; ** P<0.01; *** P<0.001.</p

Modulated CADM1 levels in HMC-1 cells influenced Kit.

<p>LucSh-, SP4- and Shm-transduced HMC-1 cells from an experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone-0085980-g002" target="_blank">Fig. <b>2C</b></a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone-0085980-g003" target="_blank"><b>3A</b></a>, stained for surface Kit and CADM1, were examined by confocal laser scanning microscopy. The same optical section of the cell surface (equivalent to the side of a crumpet) is shown for each protein. Kit and CADM1 are shown in false colours. The right column shows light-transmission images. Bar is 5 µm. <b>B.</b> Graphs are shown for the thresholded Manders’s coefficients M1 for colocalisation of Kit with CADM1 and M2 for colocalisation of CADM1 with Kit in the SP4, LucSh and Shm-groups of cells (n = 7, n = 7 and n = 6 cells with two surfaces each, respectively). * P<0.05, *** P<0.001.<b>C.</b> The middle optical section is shown for the LucSh-transduced HMC-1 cell shown in A. The line through the dual stained cell was scanned and the intensity of staining is presented in a graph on the right.</p

CADM1 Downregulation in human lung mast cells increased the length of cortical actin filaments.

<p>LucSh-, SP4- and Shm-transduced HLMCs D682 from an experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone-0085980-g003" target="_blank">Fig. <b>3A</b> and <b>3E</b></a> were stained for F-actin and surface CADM1. Glass slides with HLMCs in suspension were examined by confocal laser scanning microscopy. The same optical section of the cell surface is shown for each protein. F-actin and CADM1 are shown in false colours. Bar is 10 µm. <b>B.</b> The maximal length of actin filaments was measured as the means of 4 longest filaments on the cell membrane for each examined cell, then the data for all groups of transduced cells were analysed (n = 10 cell for each group). **, P<0.01; ***, P<0.001. <b>C.</b> The maximal and minimal cross-sectional (X, Y) and Z axial distances were measured and analysed for LucSh- (n = 13), SP4- (n = 9) and Shm-transduced (n = 15) cells.</p

Modulated CADM1 levels in HMC-1 cells influenced surface Kit expression, an assembly of filamentous actin and tyrosine phosphorylation.

<p><b>A.</b> HMC-1 cells were transduced with SP4, SP1, control shRNA LucSh, CADM1 shRNA (Sh5 or Shm) viral particles. Then examined for expression of surface CADM1 (total n = 30 groups from 7 transductions), surface Kit (n = 27 from 7 transductions) and amounts of F-actin (n = 23 from 4 transductions) by FACS. The control group combines LucSh-transduced and non-transduced cells, CADM1 downregulated group combines Sh5- and Shm-transduced cells. All data were expressed as a percentage of the levels in the SP-overexpressing cell group. <b>B and C.</b> Scatter plots for the data presented in <b>A</b> with correlation or regression model parameters are shown for Kit (<b>B</b>) and F-actin (<b>C</b>) as a function of CADM1. Data for SP4- and SP1-expressing cells are shown in different colours. <b>D.</b> Western blotting of protein extracts from LucSh-, SP4- and Shm-transduced HMC-1 cells, with 3 independent transductions for each group, developed with Abs shown on the right of the blots. <b>E.</b> Protein bands, shown in <b>D</b> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone.0085980.s002" target="_blank">Fig. S<b>2C</b></a>, were quantified. Bands in SP4 (n = 5) and Shm (n = 4) groups were expressed as percentages of control LucSh/GFP group (n = 5) for each protein, except phosphotyrosine 58–63 kDa bands (n = 3). * P<0.05; ** P<0.01; *** P<0.001.</p

CADM1 downregulation in HMC-1 cells increased the length of cortical actin filaments.

<p>LucSh-, SP4- and Shm-transduced HMC-1 cells from an experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone-0085980-g002" target="_blank">Fig. <b>2C</b></a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085980#pone-0085980-g003" target="_blank"><b>3A</b></a>, stained for F-actin and CADM1, were examined by confocal laser scanning microscopy. The same optical section of the cell surface (equivalent to the side of a crumpet) is shown for each protein. F-actin and CADM1 are shown in false colours. The right column shows light-transmission images. Bar is 5 µm. <b>B.</b> The maximal length of actin filaments was calculated as the means of 4 longest distances between crossed filaments for each examined HMC-1 cell, then the data for n = (10–11) cells for each group were analysed. ** P<0.01; *** P<0.001. <b>C.</b> Graphs are shown for the thresholded Manders’s coefficients M1 for colocalisation of F-actin with CADM1 and M2 for colocalisation of CADM1 with F-actin in the SP4, LucSh and Shm-groups of cells (n = 7, n = 7 and n = 6 cells with two surfaces each, respectively). * P<0.05, ** P<0.01; *** P<0.001.</p