Sue Shackleton

LMNA, encoding lamin A/C, is mutated in partial lipodystrophy

The lipodystrophies are a group of disorders characterized by the absence or reduction of subcutaneous adipose tissue. Partial lipodystrophy (PLD; MIM 151660) is an inherited condition in which a regional (trunk and limbs) loss of fat occurs during the peri-pubertal phase. Additionally, variable degrees of resistance to insulin action, together with a hyperlipidaemic state, may occur and simulate the metabolic features commonly associated with predisposition to atherosclerotic disease. The PLD locus has been mapped to chromosome 1q with no evidence of genetic heterogeneity. We, and others, have refined the location to a 5.3-cM interval between markers D1S305 and D1S1600 (refs 5 , 6). Through a positional cloning approach we have identified five different missense mutations in LMNA among ten kindreds and three individuals with PLD. The protein product of LMNA is lamin A/C, which is a component of the nuclear envelope. Heterozygous mutations in LMNA have recently been identified in kindreds with the variant form of muscular dystrophy (MD) known as autosomal dominant Emery-Dreifuss MD (EDMD–AD; ref. 7) and dilated cardiomyopathy and conduction-system disease (CMD1A). As LMNA is ubiquitously expressed, the finding of site-specific amino acid substitutions in PLD, EDMD–AD and CMD1A reveals distinct functional domains of the lamin A/C protein required for the maintenance and integrity of different cell types.

SUN1 Interacts with Nuclear Lamin A and Cytoplasmic Nesprins To Provide a Physical Connection between the Nuclear Lamina and the Cytoskeleton

ABSTRACT Nuclear migration and positioning within cells are critical for many developmental processes and are governed by the cytoskeletal network. Although mechanisms of nuclear-cytoskeletal attachment are unclear, growing evidence links a novel family of nuclear envelope (NE) proteins that share a conserved C-terminal SUN (Sad1/UNC-84 homology) domain. Analysis of Caenorhabditis elegans mutants has implicated UNC-84 in actin-mediated nuclear positioning by regulating NE anchoring of a giant actin-binding protein, ANC-1. Here, we report the identification of SUN1 as a lamin A-binding protein in a yeast two-hybrid screen. We demonstrate that SUN1 is an integral membrane protein located at the inner nuclear membrane. While the N-terminal domain of SUN1 is responsible for detergent-resistant association with the nuclear lamina and lamin A binding, lamin A/C expression is not required for SUN1 NE localization. Furthermore, SUN1 does not interact with type B lamins, suggesting that NE localization is ensured by binding to an additional nuclear component(s), most likely chromatin. Importantly, we find that the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain. Our data provide evidence of a physical nuclear-cytoskeletal connection that is likely to be a key mechanism in nuclear-cytoplasmic communication and regulation of nuclear position.

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.

Compound heterozygous ZMPSTE24 mutations reduce prelamin A processing and result in a severe progeroid phenotype

Hutchinson–Gilford progeria syndrome (HGPS; OMIM 176670) is an extremely rare but devastating disorder that mimics premature aging.1–3 Affected children appear normal at birth but typically develop failure to thrive in the first two years. Other features include alopecia, micrognathia, loss of subcutaneous fat with prominent veins, abnormal dentition, sclerodermatous skin changes, and osteolysis of the clavicles and distal phalanges. The mean age of death is at age 13 years, most commonly due to atherosclerosis. HGPS is mainly sporadic in occurrence, but a genetic cause has now been implicated following the identification of de novo heterozygous mutations in the LMNA gene in the majority of HGPS patients.4,5 A single family showing autosomal recessive inheritance of homozygous LMNA mutations has also been reported.6 LMNA encodes lamins A and C, components of the nuclear lamina, a meshwork underlying the nuclear envelope that serves as a structural support and is also thought to contribute to chromatin organisation and the regulation of gene expression.7,8 Interestingly, mutations in LMNA have recently been associated with at least eight inherited disorders, known as laminopathies, with differential dystrophic effects on a variety of tissues including muscle, neurones, skin, bone, and adipose tissue (reviewed in Mounkes et al 9). However, the realisation that these disorders share common genetic defects has led to clinical re-evaluation, with emerging evidence of significant phenotypic overlap.10 Hence the laminopathies might reasonably be considered as a spectrum of related diseases. HGPS has phenotypic similarities to several other laminopathies, in particular the atypical Werner’s syndrome11 and mandibuloacral dysplasia (MAD; OMIM 248370 and 608612).12 These diseases are associated with lipodystrophy,3,13 which is the most prominent feature of another laminopathy, familial partial lipodystrophy of the Dunnigan variety (OMIM 151660).14 MAD has been further classified as two …

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.

Hutchinson-Gilford progeria syndrome: clinical findings in three patients carrying the G608G mutation in LMNA and review of the literature.

Background  Hutchinson–Gilford progeria syndrome (HGPS) is a rare premature ageing disorder that belongs to a group of conditions called laminopathies which affect nuclear lamins. Classical and atypical forms of HGPS have been reported and there are clinical overlaps with mandibulo‐acral dysplasia and restrictive dermopathy. To date, mutations in two genes, LMNA and ZMPSTE24, have been found in patients with HGPS. The p.G608G LMNA mutation is the most commonly reported mutation. Correlations between genotype and phenotype in children with progeroid syndromes are beginning to emerge.

A novel interaction between FRMD7 and CASK: evidence for a causal role in idiopathic infantile nystagmus

Idiopathic infantile nystagmus (IIN) is a genetically heterogeneous disorder of eye movement that can be caused by mutations in the FRMD7 gene that encodes a FERM domain protein. FRMD7 is expressed in the brain and knock-down studies suggest it plays a role in neurite extension through modulation of the actin cytoskeleton, yet little is known about its precise molecular function and the effects of IIN mutations. Here, we studied four IIN-associated missense mutants and found them to have diverse effects on FRMD7 expression and cytoplasmic localization. The C271Y mutant accumulates in the nucleus, possibly due to disruption of a nuclear export sequence located downstream of the FERM-adjacent domain. While overexpression of wild-type FRMD7 promotes neurite outgrowth, mutants reduce this effect to differing degrees and the nuclear localizing C271Y mutant acts in a dominant-negative manner to inhibit neurite formation. To gain insight into FRMD7 molecular function, we used an IP-MS approach and identified the multi-domain plasma membrane scaffolding protein, CASK, as a FRMD7 interactor. Importantly, CASK promotes FRMD7 co-localization at the plasma membrane, where it enhances CASK-induced neurite length, whereas IIN-associated FRMD7 mutations impair all of these features. Mutations in CASK cause X-linked mental retardation. Patients with C-terminal CASK mutations also present with nystagmus and, strikingly, we show that these mutations specifically disrupt interaction with FRMD7. Together, our data strongly support a model whereby CASK recruits FRMD7 to the plasma membrane to promote neurite outgrowth during development of the oculomotor neural network and that defects in this interaction result in nystagmus.

MicroRNA expression profiling in patients with lamin A/C-associated muscular dystrophy

Mutations in the lamin A/C gene (LMNA) cause several disorders referred to as laminopathies, which include premature aging syndromes, lipodystrophy, and striated muscle disorders. There is evidence that lamin A/C plays a role in gene expression. MicroRNAs (miRNAs) are short noncoding RNAs regulating mRNAs involved in various biological processes, including the pathophysiology of striated muscles. Here, we profiled the expression of the miRNA transcriptome in skeletal muscle from patients with LMNA‐related muscular dystrophy. Results show that control and patient groups can be distinguished based on their miRNA expression profile. Sixteen miRNAs are significantly dysregulated in patients compared with controls. Pathway enrichment analysis in the predicted targets of these miRNAs revealed pathways involved in muscle repair, such as MAPK, transforming growth factor‐β, and Wnt signaling. Interestingly, 9 of these miRNAs (hsa‐miR‐100, −127–3p, −148a, −136∗, −192, −335, −376c, −489, and −502–3p) are highly expressed in fetal muscle, suggesting that the fetal miRNA gene program mediates a regenerative process. Overexpression of these miRNAs in C2C12 mouse myoblasts revealed that 3 of them (miR‐100, −192, and −335) participate in muscle proliferation and differentiation. We identified target genes that likely mediate this effect, which include the calcineurin gene PPP3CA. Our findings are the first to demonstrate that miRNA expression is affected in laminopathies.—Sylvius, N., Bonne, G., Straatman, K., Reddy, T., Gant, T. W., Shackleton, S. MicroRNA expression profiling in patients with lamin A/C‐associated muscular dystrophy. FASEB J. 25, 3966–3978 (2011). www.fasebj.org

The Role of FRMD7 in Idiopathic Infantile Nystagmus

Idiopathic infantile nystagmus (IIN) is an inherited disorder in which the nystagmus arises independently of any other symptoms, leading to the speculation that the disorder represents a primary defect in the area of the brain responsible for ocular motor control. The inheritance patterns are heterogeneous, however the most common form is X-linked. FRMD7 resides at Xq26-27 and approximately 50% of X-linked IIN families map to this region. Currently 45 mutations within FRMD7 have been associated with IIN, confirming the importance of FRMD7 in the pathogenesis of the disease. Although mutations in FRMD7 are known to cause IIN, very little is known about the function of the protein. FRMD7 contains a conserved N-terminal FERM domain suggesting that it may provide a link between the plasma membrane and actin cytoskeleton. Limited studies together with the knowledge of the function of other FERM domain containing proteins, suggest that FRMD7 may play a role in membrane extension during neuronal development through remodeling of the actin cytoskeleton.

Mitotic phosphorylation of SUN1 loosens its connection with the nuclear lamina while the LINC complex remains intact.

At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD.