Emery-Dreifuss muscular dystrophy (EDMD) is a heterogeneous late-onset disease involving skeletal muscle wasting and heart defects caused, in a minority of cases, by mutations in either of two genes encoding the inner nuclear membrane (INM) proteins, emerin and lamins A/C. Nesprin-1 and -2 are multi-isomeric, spectrin-repeat proteins that bind both emerin and lamins A/C and form a network in muscle linking the nucleoskeleton to the INM, the outer nuclear membrane, membraneous organelles, the sarcomere and the actin cytoskeleton. Thus, disruptions in nesprin/lamin/emerin interactions might play a role in the muscle-specific pathogenesis of EDMD. Screening for DNA variations in the genes encoding nesprin-1 (SYNE1) and nesprin-2 (SYNE2) in 190 probands with EDMD or EDMD-like phenotypes identified four heterozygous missense mutations. Fibroblasts from these patients exhibited nuclear morphology defects and specific patterns of emerin and SUN2 mislocalization. In addition, diminished nuclear envelope localization of nesprins and impaired nesprin/emerin/lamin binding interactions were common features of all EDMD patient fibroblasts. siRNA knockdown of nesprin-1 or -2 in normal fibroblasts reproduced the nuclear morphological changes and mislocalization of emerin and SUN2 observed in patient fibroblasts. Taken together, these data suggest that EDMD may be caused, in part, by uncoupling of the nucleoskeleton and cytoskeleton because of perturbed nesprin/emerin/lamin interactions.

Background-Hutchinson-Gilford progeria syndrome is a rare inherited disorder of premature aging caused by mutations in LMNA or Zmpste24 that disrupt nuclear lamin A processing, leading to the accumulation of prelamin A. Patients develop severe premature arteriosclerosis characterized by vascular smooth muscle cell (VSMC) calcification and attrition. Methods and Results-To determine whether defective lamin A processing is associated with vascular aging in the normal population, we examined the profile of lamin A expression in normal and aged VSMCs. In vitro, aged VSMCs rapidly accumulated prelamin A coincidently with nuclear morphology defects, and these defects were reversible by treatment with farnesylation inhibitors and statins. In human arteries, prelamin A accumulation was not observed in young healthy vessels but was prevalent in medial VSMCs from aged individuals and in atherosclerotic lesions, where it often colocalized with senescent and degenerate VSMCs. Prelamin A accumulation correlated with downregulation of the lamin A processing enzyme Zmpste24/FACE1, and FACE1 mRNA and protein levels were reduced in response to oxidative stress. Small interfering RNA knockdown of FACE1 reiterated the prelamin A-induced nuclear morphology defects characteristic of aged VSMCs, and overexpression of prelamin A accelerated VSMC senescence. We show that prelamin A acts to disrupt mitosis and induce DNA damage in VSMCs, leading to mitotic failure, genomic instability, and premature senescence. Conclusions-This study shows that prelamin A is a novel biomarker of VSMC aging and disease that acts to accelerate senescence. It therefore represents a novel target to ameliorate the effects of age-induced vascular dysfunction. (Circulation.

Nesprin-2 is a multi-isomeric, modular protein composed of variable numbers of spectrin-repeats linked to a C-terminal transmembrane domain and/or to N-terminal paired calponin homology (CH) domains. The smaller isoforms of nesprin-2 co-localize with and bind lamin A and emerin at the inner nuclear envelope (NE). In SW-13 cells, which lack lamin A/C, nesprin-2 epitopes and emerin were both mislocalized and formed aggregates in the endoplasmic reticulum (ER). The larger isoforms and other CH-domain-containing isoforms co-localize with heterochromatin within the nucleus and are also present at the outer NE and in multiple cytoplasmic compartments. Nesprin-2 isoforms relocalize during in vitro muscle differentiation of C2C12 myoblasts to the sarcomere of myotubes. Immunogold electron microscopy using antibodies specific for three different epitopes detected nesprin-2 isoforms at multiple locations including intranuclear foci, both membranes of the NE, mitochondria, sarcomeric structures and plasma membrane foci. In adult skeletal muscle, confocal immunolocalization studies demonstrated that nesprin-2 epitopes were present at the Z-line and were also associated with the sarcoplasmic reticulum (SR) in close apposition to SERCA2. These data suggest that nesprin-2 isoforms form a linking network between organelles and the actin cytoskeleton and thus may be important for maintaining sub-cellular spatial organisation. Moreover, its association at the NE with lamin and emerin, the genes mutated in Emery-Dreifuss muscular dystrophy, suggests a mechanism to explain how disruption of the NE leads to muscle dysfunction.

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Nesprins are a recently discovered family of ubiquitously expressed intracellular proteins. Through alternative transcriptional initiation, termination and splicing, two genes - nesprin-1 and nesprin-2 (also known as syne-1 and syne-2) - give rise to many protein isoforms that vary markedly in size. The largest of these isoforms comprise a C-terminal transmembrane domain (the KLS domain) linked by a spectrin-repeat rod domain to an N-terminal paired, actin-binding, calponin-homology domain. This structure suggests that they are well suited to orchestrate signalling between cell membranes and the cytoskeleton. Other isoforms have variable lengths of this rod domain linked to either end of the protein. Smaller isoforms with the KLS domain are localised at the inner nuclear membrane, where they bind lamin A/C and emerin. Larger nesprin isoforms link the outer nuclear membrane with intracellular organelles and the actin cytoskeleton and are thought to regulate nuclear anchorage and organelle migration. Thus, nesprins might have a variety of fundamental roles in cells, particularly muscle cells where they are highly expressed. We speculate that nesprin mutations might contribute to a broad range of human disease syndromes, including laminopathies.

Nesprins-1 and -2 are highly expressed in skeletal and cardiac muscle and together with SUN (Sad1p/UNC84)-domain containing proteins and lamin A/C form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) bridging complex at the nuclear envelope (NE). Mutations in nesprin-1/2 have previously been found in patients with autosomal dominant Emery–Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). In this study, three novel rare variants (R8272Q, S8381C and N8406K) in the C-terminus of the SYNE1 gene (nesprin-1) were identified in seven DCM patients by mutation screening. Expression of these mutants caused nuclear morphology defects and reduced lamin A/C and SUN2 staining at the NE. GST pull-down indicated that nesprin-1/lamin/SUN interactions were disrupted. Nesprin-1 mutations were also associated with augmented activation of the ERK pathway in vitro and in hearts in vivo. During C2C12 muscle cell differentiation, nesprin-1 levels are increased concomitantly with kinesin light chain (KLC-1/2) and immunoprecipitation and GST pull-down showed that these proteins interacted via a recently identified LEWD domain in the C-terminus of nesprin-1. Expression of nesprin-1 mutants in C2C12 cells caused defects in myoblast differentiation and fusion associated with dysregulation of myogenic transcription factors and disruption of the nesprin-1 and KLC-1/2 interaction at the outer nuclear membrane. Expression of nesprin-1α2 WT and mutants in zebrafish embryos caused heart developmental defects that varied in severity. These findings support a role for nesprin-1 in myogenesis and muscle disease, and uncover a novel mechanism whereby disruption of the LINC complex may contribute to the pathogenesis of DCM.

Objective-Reactive oxygen species-generating nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase proteins (Noxs) are involved in cell differentiation, migration, and apoptosis. Nox4 is unique among Noxs in being constitutively active, and its subcellular localization may therefore be particularly important. In this study, we identified and characterized a novel nuclear-localized 28-kDa splice variant of Nox4 in vascular cells. Approach and Results-Nox4 immunoreactivity was noted in the nucleus and nucleolus of vascular smooth muscle cells and multiple other cell types by confocal microscopy. Cell fractionation, sequence analyses, and siRNA studies indicated that the nuclear-localized Nox4 is a 28-kDa splice variant, Nox4D, which lacks putative transmembrane domains. Nox4D overexpression resulted in significant NADPH-dependent reactive oxygen species production as detected by several different methods and caused increased phosphorylation of extracellular-signal-regulated kinase1/2 and the nuclear transcription factor Elk-1. Overexpression of Nox4D could also induce DNA damage as assessed by γ-H2AX phosphorylation. These effects were inhibited by a single amino acid substitution in the Nox4D NADPH-binding region. Conclusions-Nox4D is a nuclear-localized and functionally active splice variant of Nox4 that may have important pathophysiologic effects through modulation of nuclear signaling and DNA damage. Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results Nuclear-Localized Nox4 ImmunoreactivityWe used a well-validated polyclonal antibody directed against the C-terminal region of Nox4 11,20,21 to investigate the localization of endogenous Nox4 by immunofluorescence in several cell types, including VSMC, endothelial cells, fibroblasts, and cardiomyocytes. A diffuse reticular staining pattern through the cell that colocalized with an ER marker, protein disulfide isomerase, was evident in most cell types ( Figures 1A and 2) as reported previously. 15,16 In addition, however, there was strong focal and granular Nox4 immunostaining within the nucleus in all cell types. Electron microscopy indicated that the focal Nox4 staining was in the nucleolus ( Figure 1C). We confirmed that the focal intranuclear staining was nucleolar by costaining with 2 different nucleolar markers, nucleophosmin and fibrillarin ( Figure 1B). Preincubation of the Nox4 antibody with the antigenic peptide against which it was raised completely abolished both the perinuclear and nucleolar staining, verifying the specificity of antigen-antibody interaction ( Figure 1D). Furthermore, treatment with a prevalidated Nox4 siRNA to knock down Nox4 protein expression also abolished staining as compared with a scrambled siRNA ( Figure 3A). Nuclear-Localized Nox4 Is a 28-kDa Nox4 Splice VariantTo confirm nuclear and nucleolar localization, VSMCs were fractionated into membrane and nuclear fractions. Immunoblotting for Nox4 demonstrated an ≈65-kDa band corresponding to the expected molecular size of Nox4 but ...

The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.