Mammalian SUN Protein Interaction Networks at the Inner Nuclear Membrane and Their Role in Laminopathy Disease Processes

The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have overlapping binding sites distinct from the lamin A binding site. However, we demonstrate that tight association of SUN1 with the nuclear lamina depends upon a short motif within residues 209–228, a region that does not interact significantly with known SUN1 binding partners. Moreover, SUN1 localizes correctly in cells lacking emerin. Importantly then, the major determinant of SUN1 NE localization has yet to be identified. We further find that a subset of lamin A mutations, associated with laminopathies Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS), disrupt lamin A interaction with SUN1 and SUN2. Despite this, NE localization of SUN1 and SUN2 is not impaired in cell lines from either class of patients. Intriguingly, SUN1 expression at the NE is instead enhanced in a significant proportion of HGPS but not EDMD cells and strongly correlates with pre-lamin A accumulation due to preferential interaction of SUN1 with pre-lamin A. We propose that these different perturbations in lamin A-SUN protein interactions may underlie the opposing effects of EDMD and HGPS mutations on nuclear and cellular mechanics.     

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.     

LINC complex alterations in DMD and EDMD/CMT fibroblasts

Emery-Dreifuss muscular dystrophy (EDMD) is a late onset-disease characterized by skeletal muscle wasting and heart defects with associated risk of sudden death. The autosomal dominant form of the disease is caused by mutations in the LMNA gene encoding LaminA and C, the X-linked form results from mutations in the gene encoding the inner nuclear membrane protein Emerin (STA). Both Emerin and LaminA/C interact with the nuclear envelope proteins Nesprin-1 and -2 and mutations in genes encoding C-terminal isoforms of Nesprin-1 and -2 have also been implicated in EDMD. Here we analyse primary fibroblasts from patients affected by either Duchenne muscular dystrophy (DMD) or Emery-Dreifuss muscular dystrophy/Charcot-Marie-Tooth syndrome (EDMD/CMT) that in addition to the disease causing mutations harbour mutations in the Nesprin-1 gene and in the SUN1 and SUN2 gene, respectively. SUN proteins together with the Nesprins form the core of the LINC complex which connects the nucleus with the cytoskeleton. The mutations are accompanied by changes in cell adhesion, cell migration, senescence, and stress response, as well as in nuclear shape and nuclear envelope composition which are changes characteristic for laminopathies. Our results point to a potential influence of mutations in components of the LINC complex on the clinical outcome and the molecular pathology in the patients.     

Increased solubility of lamins and redistribution of lamin C in X-linked Emery-Dreifuss muscular dystrophy fibroblasts

Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the gene encoding the nuclear membrane protein emerin (X-linked EDMD) or in the gene encoding lamins A/C (autosomal dominant EDMD). One hypothesis explaining the disease suggests that the mutations lead to weakness of the nuclear lamina. To test this hypothesis we investigated lamin solubility and distribution in skin fibroblasts from X-EDMD patients. Using in situ extraction of cells and immunofluorescence microscopy or biochemical fractionation and immunoblotting, we found that all lamin subtypes displayed increased solubility properties in fibroblasts from X-EDMD patients compared to normal individuals. Lamin and emerin solubility was mildly increased in fibroblasts from an X-EDMD carrier. Biochemical fractionation and immunoblotting also indicated that lamin C but no other lamin became redistributed from the nuclear lamina to the nucleoplasm in X-EDMD fibroblasts. Indirect immunofluorescence and confocal microscopy studies using lamin A- and lamin C-specific antibodies confirmed that lamin C but not lamin A became redistributed to the nucleoplasm. Interestingly, the lamin A/C binding protein LAP2alpha was also mislocalized in X-EDMD fibroblasts.     

Distinct functional domains in nesprin-1alpha and nesprin-2beta bind directly to emerin and both interactions are disrupted in X-linked Emery-Dreifuss muscular dystrophy

Emerin and specific isoforms of nesprin-1 and -2 are nuclear membrane proteins which are binding partners in multi-protein complexes spanning the nuclear envelope. We report here the characterisation of the residues both in emerin and in nesprin-1alpha and -2beta which are involved in their interaction and show that emerin requires nesprin-1 or -2 to retain it at the nuclear membrane. Using several protein-protein interaction methods, we show that residues 368 to 627 of nesprin-1alpha and residues 126 to 219 of nesprin-2beta, which show high homology to one another, both mediate binding to emerin residues 140-176. This region has previously been implicated in binding to F-actin, beta-catenin and lamin A/C suggesting that it is critical for emerin function. Confirmation that these protein domains interact in vivo was shown using GFP-dominant negative assays. Exogenous expression of either of these nesprin fragments in mouse myoblast C2C12 cells displaced endogenous emerin from the nuclear envelope and reduced the targeting of newly synthesised emerin. Furthermore, we are the first to report that emerin mutations which give rise to X-linked Emery-Dreifuss muscular dystrophy, disrupt binding to both nesprin-1alpha and -2beta isoforms, further indicating a role of nesprins in the pathology of Emery-Dreifuss muscular dystrophy.     

Increased solubility of lamins and redistribution of lamin C in X-linked Emery–Dreifuss muscular dystrophy fibroblasts

Emery–Dreifuss muscular dystrophy (EDMD) is caused by mutations in the gene encoding the nuclear membrane protein emerin (X-linked EDMD) or in the gene encoding lamins A/C (autosomal dominant EDMD). One hypothesis explaining the disease suggests that the mutations lead to weakness of the nuclear lamina. To test this hypothesis we investigated lamin solubility and distribution in skin fibroblasts from X-EDMD patients. Using in situ extraction of cells and immunofluorescence microscopy or biochemical fractionation and immunoblotting, we found that all lamin subtypes displayed increased solubility properties in fibroblasts from X-EDMD patients compared to normal individuals. Lamin and emerin solubility was mildly increased in fibroblasts from an X-EDMD carrier. Biochemical fractionation and immunoblotting also indicated that lamin C but no other lamin became redistributed from the nuclear lamina to the nucleoplasm in X-EDMD fibroblasts. Indirect immunofluorescence and confocal microscopy studies using lamin A- and lamin C-specific antibodies confirmed that lamin C but not lamin A became redistributed to the nucleoplasm. Interestingly, the lamin A/C binding protein LAP2α was also mislocalized in X-EDMD fibroblasts.     

Nuclear envelope alterations in fibroblasts from LGMD1B patients carrying nonsense Y259X heterozygous or homozygous mutation in lamin A/C gene

Mutations in the LMNA gene encoding nuclear lamins A and C are responsible for seven inherited disorders affecting specific tissues. We have analyzed skin fibroblasts from a patient with type 1B limb-girdle muscular dystrophy and from her deceased newborn grandchild carrying, respectively, a heterozygous (+/mut) and a homozygous (mut/mut) nonsense Y259X mutation. In fibroblasts(+/mut), the presence of only 50% lamins A and C promotes no detectable abnormality, whereas in fibroblasts(mut/mut) the complete absence of lamins A and C leads to abnormally shaped nuclei with lobules in which none of the analyzed nuclear proteins were detected, i.e., B-type lamins, emerin, nesprin-1alpha, LAP2beta, and Nup153. These lobules perturb cell division as fibroblast(mut/mut) cultures with large proportions of cells with dysmorphic nuclei grow more slowly than controls and the cell proliferation normalizes when the number of these abnormally shaped nuclei declines. In all fibroblasts(mut/mut), nesprin-1alpha-like emerin exhibited aberrant localization in the endoplasmic reticulum. Transfection of wild-type lamin A or C cDNAs restored the correct localization of both emerin and nesprin-1alpha. These data demonstrate that lamin C, like lamin A, interacts in vivo directly with nesprin-1alpha and with emerin and that lamin A or C is sufficient for the correct anchorage of emerin and nesprin-1alpha at the nuclear envelope in human cells.     

Keeping the LINC: the importance of nucleocytoskeletal coupling in intracellular force transmission and cellular function

Providing a stable physical connection between the nucleus and the cytoskeleton is essential for a wide range of cellular functions and it could also participate in mechanosensing by transmitting intra- and extra-cellular mechanical stimuli via the cytoskeleton to the nucleus. Nesprins and SUN proteins, located at the nuclear envelope, form the LINC (linker of nucleoskeleton and cytoskeleton) complex that connects the nucleus to the cytoskeleton; underlying nuclear lamins contribute to anchoring LINC complex components at the nuclear envelope. Disruption of the LINC complex or loss of lamins can result in disturbed perinuclear actin and intermediate filament networks and causes severe functional defects, including impaired nuclear positioning, cell polarization and cell motility. Recent studies have identified the LINC complex as the major force-transmitting element at the nuclear envelope and suggest that many of the aforementioned defects can be attributed to disturbed force transmission between the nucleus and the cytoskeleton. Thus mutations in nesprins, SUN proteins or lamins, which have been linked to muscular dystrophies and cardiomyopathies, may weaken or completely eliminate LINC complex function at the nuclear envelope and result in impaired intracellular force transmission, thereby disrupting critical cellular functions.     

Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy

The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.     

Structural analysis of emerin, an inner nuclear membrane protein mutated in X-linked Emery-Dreifuss muscular dystrophy

Like Duchenne and Becker muscular dystrophies, Emery-Dreifuss muscular dystrophy (EDMD) is characterized by myopathic and cardiomyopathic abnormalities. EDMD has the particularity of being linked to mutations in nuclear proteins. The X-linked form of EDMD is caused by mutations in the emerin gene, whereas autosomal dominant EDMD is caused by mutations in the lamin A/C gene. Emerin colocalizes with lamin A/C in interphase cells, and binds in vitro to lamin A/C. Recent work suggests that lamin A/C might serve as a receptor for emerin. We have undertaken a structural analysis of emerin, and in particular of its N-terminal domain, which is comprised in the emerin segment critical for binding to lamin A/C. We show that region 2-54 of emerin adopts the LEM fold. This fold was originally described in the two N-terminal domains of another inner nuclear membrane protein called lamina-associated protein 2 (LAP2). The existence of a conserved solvent-exposed surface on the LEM domains of LAP2 and emerin is discussed, as well as the nature of a possible common target.     

Expression of lamin A mutated in the carboxyl-terminal tail generates an aberrant nuclear phenotype similar to that observed in cells from patients with Dunnigan-type partial lipodystrophy and Emery-Dreifuss muscular dystrophy

Autosomal dominantly inherited missense mutations in lamins A and C cause familial partial lipodystrophy of the Dunnigan-type (FPLD), and myopathies including Emery-Dreifuss muscular dystrophy (EDMD). While mutations responsible for FPLD are restricted to the carboxyl-terminal tails, those responsible for EDMD are spread throughout the molecules. We observed here the same structural abnormalities in the nuclear envelope and chromatin of fibroblasts from patients with FPLD and EDMD, harboring missense mutations at codons 482 and 453, respectively. Similar nuclear alterations were generated in fibroblasts, myoblasts, and preadipocytes mouse cell lines overexpressing lamin A harboring either of these two mutations. A large variation in sensitivity to lamin A overexpression was observed among the three cell lines, which was correlated with their variable endogenous content in A-type lamins and emerin. The occurrence of nuclear abnormalities was reduced when lamin B1 was coexpressed with mutant lamin A, emphasizing the functional interaction of the two types of lamins. Transfected cells therefore develop similar phenotypes when expressing lamins mutated in the carboxyl-terminal tail at sites responsible for FPLD or EDMD.     

The role of the nuclear envelope in Emery–Dreifuss muscular dystrophy

The X-linked form of Emery-Dreifuss muscular dystrophy (X-EDMD) is caused by absence, or greatly reduced amounts, of the inner nuclear-membrane protein, emerin. The autosomal dominant form (AD-EDMD) is caused by missense mutations in lamins A and C, two components of the nuclear lamina that interact directly with emerin. Lamin A/C mutations also cause one form of dilated cardiomyopathy (CMD1A) and one form of limb-girdle muscular dystrophy (LGMD1B), both of which have clinical features in common with EDMD, as well as a rare, unrelated form of lipodystrophy (FPLD). Evidence is now emerging that defective assembly of the nuclear lamina is a feature of all these diseases, although not necessarily the direct cause. Why only heart and skeletal muscle, and possibly connective tissue, are affected in EDMD and why expression of the disease is so extremely variable between individuals remains to be explained.     

Nuclear envelope dynamics during mammalian spermatogenesis: new insights on male fertility

The production of highly specialized spermatozoa from undifferentiated spermatogonia is a strictly organized and programmed process requiring extensive restructuring of the entire cell. One of the most remarkable cellular transformations accompanying the various phases of spermatogenesis is the profound remodelling of the nuclear architecture, in which the nuclear envelope (NE) seems to be crucially involved. In recent years, several proteins from the distinct layers forming the NE (i.e. the inner and outer nuclear membranes as well as the nuclear lamina) have been associated with meiosis and/or spermiogenesis in different mammalian species. Among these are A‐ and B‐type lamins, Dpy‐19‐like protein 2 (DPY19L2), lamin B receptor (LBR), lamina‐associated polypeptide 1 (LAP1), LAP2/emerin/MAN1 (LEM) domain‐containing proteins, spermatogenesis‐associated 46 (SPATA46) and diverse elements of the linker of nucleoskeleton and cytoskeleton (LINC) complex, namely Sad‐1/UNC‐84 homology (SUN) and Klarsicht/ANC‐1/Syne‐1 homology (KASH) domain‐containing proteins. Herein, we summarize the current state of the art on the cellular and subcellular distribution of NE proteins expressed during mammalian spermatogenesis, and discuss the latest research developments regarding their testis‐specific functions. This review provides a comprehensive and innovative overview of the NE network as a regulatory platform and as an essential determinant of efficient meiotic chromosome recombination as well as spermiogenesis‐associated nuclear remodelling and differentiation in mammalian male germline cells. Thus, this review provides important novel insights on the biological relevance of NE proteins for male fertility.     

Structure of Sad1-UNC84 homology (SUN) domain defines features of molecular bridge in nuclear envelope

The SUN (Sad1-UNC-84 homology) domain is conserved in a number of nuclear envelope proteins involved in nuclear migration, meiotic telomere tethering, and antiviral responses. The LINC (linker of nucleoskeleton and cytoskeleton) complex, formed by the SUN and the nesprin proteins at the nuclear envelope, serves as a mechanical linkage across the nuclear envelope. Here we report the crystal structure of the SUN2 protein SUN domain, which reveals a homotrimer. The SUN domain is sufficient to mediate binding to the KASH (Klarsicht, ANC-1, and Syne homology) domain of nesprin 2, and the regions involved in the interaction have been identified. Binding of the SUN domain to the KASH domain is abolished by deletion of a region important for trimerization or by point mutations associated with nuclear migration failure. We propose a model of the LINC complex, where the SUN and the KASH domains form a higher ordered oligomeric network in the nuclear envelope. These findings provide the structural basis for understanding the function and the regulation of the LINC complex.     

Emerin organizes actin flow for nuclear movement and centrosome orientation in migrating fibroblasts

In migrating fibroblasts, rearward movement of the nucleus orients the centrosome toward the leading edge. Nuclear movement results from coupling rearward-moving, dorsal actin cables to the nucleus by linear arrays of nesprin-2G and SUN2, termed transmembrane actin-associated nuclear (TAN) lines. A-type lamins anchor TAN lines, prompting us to test whether emerin, a nuclear membrane protein that interacts with lamins and TAN line proteins, contributes to nuclear movement. In fibroblasts depleted of emerin, nuclei moved nondirectionally or completely failed to move. Consistent with these nuclear movement defects, dorsal actin cable flow was nondirectional in cells lacking emerin. TAN lines formed normally in cells lacking emerin and were coordinated with the erratic nuclear movements, although in 20% of the cases, TAN lines slipped over immobile nuclei. Myosin II drives actin flow, and depletion of myosin IIB, but not myosin IIA, showed similar nondirectional nuclear movement and actin flow as in emerin-depleted cells. Myosin IIB specifically coimmunoprecipitated with emerin, and emerin depletion prevented myosin IIB localization near nuclei. These results show that emerin functions with myosin IIB to polarize actin flow and nuclear movement in fibroblasts, suggesting a novel function for the nuclear envelope in organizing directional actin flow and cytoplasmic polarity.     

Changes at P183 of emerin weaken its protein-protein interactions resulting in X-linked Emery-Dreifuss muscular dystrophy

Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked recessive muscular dystrophy characterized by early contractures of the elbows, Achilles tendons and spine, slowly progressive muscle wasting and weakness, and cardiomyopathy associated with cardiac conduction defects. The emerin gene has been mapped to Xq28 and encodes a 34-kDa serine-rich protein, emerin, which has been localized to the nuclear envelope in a wide variety of tissues, including skeletal and cardiac muscle. Mutations spanning the emerin gene have been identified in patients with EDMD. We present here the effect, on emerin protein expression, of two missense mutations identified in unrelated EDMD patients. These alterations predict the replacement of a proline residue at position 183 with either a histidine or a threonine. Biochemical analysis has demonstrated that the mobility and expression levels of the mutant forms of emerin are indistinguishable from that of wild-type emerin, but that they have weakened interactions with nuclear lamina components. In comparison with the usual EDMD phenotype, patients with P183 missense mutations have a later age at onset of first symptoms, elbow contractures, ankle contractures, upper limb weakness and lower limb weakness, but there is no difference for the age at onset of cardiac involvement. This is the first report of protein studies on patients with missense mutations resulting in the clinical features of EDMD. These studies demonstrate the importance of proline 183 for the proper structure/function of emerin. Received: 29 September 1998 / Accepted: 14 January 1999     

Nesprin-3: a versatile connector between the nucleus and the cytoskeleton

The cytoskeleton is connected to the nuclear interior by LINC (linker of nucleoskeleton and cytoskeleton) complexes located in the nuclear envelope. These complexes consist of SUN proteins and nesprins present in the inner and outer nuclear membrane respectively. Whereas SUN proteins can bind the nuclear lamina, members of the nesprin protein family connect the nucleus to different components of the cytoskeleton. Nesprin-1 and -2 can establish a direct link with actin filaments, whereas nesprin-4 associates indirectly with microtubules through its interaction with kinesin-1. Nesprin-3 is the only family member known that can link the nuclear envelope to intermediate filaments. This indirect interaction is mediated by the binding of nesprin-3 to the cytoskeletal linker protein plectin. Furthermore, nesprin-3 can connect the nucleus to microtubules by its interactions with BPAG1 (bullous pemphigoid antigen 1) and MACF (microtubule-actin cross-linking factor). In contrast with the active roles that nesprin-1, -2 and -4 have in actin- and microtubule-dependent nuclear positioning, the role of nesprin-3 is likely to be more passive. We suggest that it helps to stabilize the anchorage of the nucleus within the cytoplasm and maintain the structural integrity and shape of the nucleus.     

A novel mutation in human EMD gene and mitochondrial dysfunction in emerin knockdown cardiomyocytes

Emerin is an inner nuclear envelope protein encoded by the EMD gene, mutations in which cause Emery–Dreifuss muscular dystrophy type 1 (EDMD1). Cardiac involvement has become a major threat to patients with EDMD1; however, the cardiovascular phenotype spectrums of emerinopathy and the mechanisms by which emerin regulates cardiac pathophysiology remain unclear. Here, we identified a novel nonsense mutation (c.C57G, p.Y19X) in the EMD gene in a Han Chinese family through high‐throughput sequencing. Two family members were found to have EDMD1 with muscle weakness and cardiac arrhythmia. Mechanistically, we first discovered that knockdown of emerin in HL‐1 or H9C2 cardiomyocytes lead to impaired mitochondrial oxidative phosphorylation capacity with downregulation of electron transport chain complex I and IV and upregulation of complex III and V. Moreover, loss of emerin in HL‐1 cells resulted in collapsed mitochondrial membrane potential, altered mitochondrial networks and downregulated multiple factors in RNA and protein level, such as PGC1α, DRP1, MFF, MFN2, which are involved in regulation of mitochondrial biogenesis, fission and fusion. Our findings suggest that targeting mitochondrial bioenergetics might be an effective strategy against cardiac disorders caused by EMD mutations.