N-terminal tags impair the ability of lamin A to provide structural support to the nucleus.

Lamins are intermediate filament proteins that contribute to numerous cellular functions, including nuclear morphology and mechanical stability. The N-terminal head domain of lamin is crucial for higher order filament assembly and function, yet the effects of commonly used N-terminal tags on lamin function remain largely unexplored. Here, we systematically studied the effect of two differently sized tags on lamin A (LaA) function in a mammalian cell model engineered to allow for precise control of expression of tagged lamin proteins. Untagged, FLAG-tagged and GFP-tagged LaA completely rescued nuclear shape defects when expressed at similar levels in lamin A/C-deficient (Lmna-/-) MEFs, and all LaA constructs prevented increased nuclear envelope ruptures in these cells. N-terminal tags, however, altered the nuclear localization of LaA and impaired the ability of LaA to restore nuclear deformability and to recruit emerin to the nuclear membrane in Lmna-/- MEFs. Our finding that tags impede some LaA functions but not others might explain the partial loss of function phenotypes when tagged lamins are expressed in model organisms and should caution researchers using tagged lamins to study the nucleus.

Maintenance of germline stem cell homeostasis despite severe nuclear distortion.

Stem cell loss in aging and disease is associated with nuclear deformation. Yet, how nuclear shape influences stem cell homeostasis is poorly understood. We investigated this connection using Drosophila germline stem cells, as survival of these stem cells is compromised by dysfunction of the nuclear lamina, the extensive protein network that lines the inner nuclear membrane and gives shape to the nucleus. To induce nuclear distortion in germline stem cells, we used the GAL4-UAS system to increase expression of the permanently farnesylated nuclear lamina protein, Kugelkern, a rate limiting factor for nuclear growth. We show that elevated Kugelkern levels cause severe nuclear distortion in germline stem cells, including extensive thickening and lobulation of the nuclear envelope and nuclear lamina, as well as alteration of internal nuclear compartments. Despite these changes, germline stem cell number, proliferation, and female fertility are preserved, even as females age. Collectively, these data demonstrate that disruption of nuclear architecture does not cause a failure of germline stem cell survival or homeostasis, revealing that nuclear deformation does not invariably promote stem cell loss.

Altered expression and localization of nuclear envelope proteins in a prostate cancer cell system.

BACKGROUND: The nuclear envelope (NE), which is composed of the outer and inner nuclear membranes, the nuclear pore complex and the nuclear lamina, regulates a plethora of cellular processes, including those that restrict cancer development (genomic stability, cell cycle regulation, and cell migration). Thus, impaired NE is functionally related to tumorigenesis, and monitoring of NE alterations is used to diagnose cancer. However, the chronology of NE changes occurring during cancer evolution and the connection between them remained to be precisely defined, due to the lack of appropriate cell models. METHODS: The expression and subcellular localization of NE proteins (lamins A/C and B1 and the inner nuclear membrane proteins emerin and ß-dystroglycan [ß-DG]) during prostate cancer progression were analyzed, using confocal microscopy and western blot assays, and a prostate cancer cell system comprising RWPE-1 epithelial prostate cells and several prostate cancer cell lines with different invasiveness. RESULTS: Deformed nuclei and the mislocalization and low expression of lamin A/C, lamin B1, and emerin became more prominent as the invasiveness of the prostate cancer lines increased. Suppression of lamin A/C expression was an early event during prostate cancer evolution, while a more extensive deregulation of NE proteins, including ß-DG, occurred in metastatic prostate cells. CONCLUSIONS: The RWPE-1 cell line-based system was found to be suitable for the correlation of NE impairment with prostate cancer invasiveness and determination of the chronology of NE alterations during prostate carcinogenesis. Further study of this cell system would help to identify biomarkers for prostate cancer prognosis and diagnosis.

Emerin deficiency drives MCF7 cells to an invasive phenotype.

During metastasis, cancer cells traverse the vasculature by squeezing through very small gaps in the endothelium. Thus, nuclei in metastatic cancer cells must become more malleable to move through these gaps. Our lab showed invasive breast cancer cells have 50% less emerin protein resulting in smaller, misshapen nuclei, and higher metastasis rates than non-cancerous controls. Thus, emerin deficiency was predicted to cause increased nuclear compliance, cell migration, and metastasis. We tested this hypothesis by downregulating emerin in noninvasive MCF7 cells and found emerin knockdown causes smaller, dysmorphic nuclei, resulting in increased impeded cell migration. Emerin reduction in invasive breast cancer cells showed similar results. Supporting the clinical relevance of emerin reduction in cancer progression, our analysis of 192 breast cancer patient samples showed emerin expression inversely correlates with cancer invasiveness. We conclude emerin loss is an important driver of invasive transformation and has utility as a biomarker for tumor progression.

Natural History of Dilated Cardiomyopathy Due to c.77T>C (p.Val26Ala) in Emerin Protein.

(1) Introduction: Dilated cardiomyopathy (DCM) mainly affects young individuals and is the main indication of heart transplantation. The variant c.77T>C (p.Val26Ala) of the gene coding for emerin (EMD) in chromosome Xq28 has been catalogued as a pathogenic variant for the development of DCM, exhibiting an X-linked inheritance pattern. (2) Methods: A retrospective study was conducted covering the period 2015-2023 in patients with DCM of genetic origin. The primary endpoint was patient age at onset of the first composite major cardiac event, in the form of a first episode of heart failure, malignant ventricular arrhythmia, or end-stage heart failure, according to the presence of truncating variant in titin gene (TTNtv) versus the p.Val26Ala mutation in the EMD protein. (3) Results: A total of 31 and 22 patients were included in the EMD group and TTNtv group, respectively. The primary endpoint was significantly higher in the EMD group, with a hazard ratio of 4.16 (95% confidence interval: 1.83-9.46; p = 0.001). At 55 years of age, all the patients in the EMD group had already presented heart failure, nine presented malignant ventricular arrhythmia (29%), and 13 required heart transplantation (42%). (4) Conclusions: DCM secondary to the c.77T>C (p.Val26Ala) mutation in the EMD gene is associated to an increased risk of major cardiac events compared to patients with DCM due to TTNtv, with a large proportion of transplanted patients in the fifth decade of life.

Developmental changes in nuclear lamina components during germ cell differentiation.

The nuclear lamina (NL) changes composition for regulation of nuclear events. We investigated changes that occur in Drosophila oogenesis, revealing switches in NL composition during germ cell differentiation. Germline stem cells (GSCs) express only LamB and predominantly emerin, whereas differentiating nurse cells predominantly express LamC and emerin2. A change in LamC-specific localization also occurs, wherein phosphorylated LamC redistributes to the nuclear interior only in the oocyte, prior to transcriptional reactivation of the meiotic genome. These changes support existing concepts that LamC promotes differentiation, a premise that was tested. Remarkably ectopic LamC production in GSCs did not promote premature differentiation. Increased LamC levels in differentiating germ cells altered internal nuclear structure, increased RNA production, and reduced female fertility due to defects in eggshell formation. These studies suggest differences between Drosophila lamins are regulatory, not functional, and reveal an unexpected robustness to level changes of a major scaffolding component of the NL.

Proteomic characterization of human LMNA-related congenital muscular dystrophy muscle cells.

LMNA-related congenital muscular dystrophy (L-CMD) is caused by mutations in the LMNA gene, encoding lamin A/C. To further understand the molecular mechanisms of L-CMD, proteomic profiling using DIA mass spectrometry was conducted on immortalized myoblasts and myotubes from controls and L-CMD donors each harbouring a different LMNA mutation (R249W, del.32 K and L380S). Compared to controls, 124 and 228 differentially abundant proteins were detected in L-CMD myoblasts and myotubes, respectively, and were associated with enriched canonical pathways including synaptogenesis and necroptosis in myoblasts, and Huntington's disease and insulin secretion in myotubes. Abnormal nuclear morphology and reduced lamin A/C and emerin abundance was evident in all L-CMD cell lines compared to controls, while nucleoplasmic aggregation of lamin A/C was restricted to del.32 K cells, and mislocalization of emerin was restricted to R249W cells. Abnormal nuclear morphology indicates loss of nuclear lamina integrity as a common feature of L-CMD, likely rendering muscle cells vulnerable to mechanically induced stress, while differences between L-CMD cell lines in emerin and lamin A localization suggests that some molecular alterations in L-CMD are mutation specific. Nonetheless, identifying common proteomic alterations and molecular pathways across all three L-CMD lines has highlighted potential targets for the development of non-mutation specific therapies.

Clinical and Genetic Heterogeneity of Nuclear Envelopathy Related Muscular Dystrophies in an Indian Cohort.

Introduction: Nuclear envelopathies occur due to structural and/or functional defects in various nuclear envelope proteins such as lamin A/C and lamin related proteins. This study is the first report on the phenotype-genotype patterns of nuclear envelopathy-related muscular dystrophies from India. Methods: In this retrospective study, we have described patients with genetically confirmed muscular dystrophy associated with nuclear envelopathy. Data on clinical, laboratory findings and muscle MRI were collected. Results: Sixteen patients were included with median age at onset of 3 years (range: 1 month - 17 years). Three genes were involved: LMNA (11, 68.75%), EMD (4, 25%) and SYNE1 (1, 6.25%). The 11 patients with LMNA variants were Congenital muscular dystrophy (MDCL)=4, Limb Girdle Muscular Dystrophy (LGMD1B)=4 and Emery-Dreifuss Muscular Dystrophy (EDMD2)=3. On muscle biopsy, one patient from each laminopathy phenotype (n = 3) revealed focal perivascular inflammatory infiltrate. Other notable features were ophthalmoparesis in one and facial weakness in one. None had cardiac involvement. Patients with EDMD1 had both upper (UL) and lower limb (LL) proximo-distal weakness. Cardiac rhythm disturbances such as sick sinus syndrome and atrial arrhythmias were noted in two patients with EDMD1. Only one patient with variant c.654_658dup (EMD) lost ambulation in the 3rd decade, 18 years after disease onset. Two had finger contractures with EMD and SYNE1 variants respectively. All patients with LMNA and SYNE1 variants were ambulant at the time of evaluation. Mean duration of illness (years) was 11.6±13 (MDCL), 3.2±1.0 (EDMD2), 10.4±12.8 (LGMD1B), 11.8±8.4 (EDMD1) and 3 (EDMD4). One patient had a novel SYNE1 mutation (c.22472dupA, exon 123) and presented with UL phenotype and prominent finger and wrist contractures. Conclusion: The salient features included ophthalmoparesis and facial weakness in LMNA, prominent finger contractures in EMD and SYNE1 and upper limb phenotype with the novel pathogenic variant in SYNE1.

FAK regulates tension transmission to the nucleus and endothelial transcriptome independent of kinase activity.

The mechanical environment generated through the adhesive interaction of endothelial cells (ECs) with the matrix controls nuclear tension, preventing aberrant gene synthesis and the transition from restrictive to leaky endothelium, a hallmark of acute lung injury (ALI). However, the mechanisms controlling tension transmission to the nucleus and EC-restrictive fate remain elusive. Here, we demonstrate that, in a kinase-independent manner, focal adhesion kinase (FAK) safeguards tension transmission to the nucleus to maintain EC-restrictive fate. In FAK-depleted ECs, robust activation of the RhoA-Rho-kinase pathway increased EC tension and phosphorylation of the nuclear envelope protein, emerin, activating DNMT3a. Activated DNMT3a methylates the KLF2 promoter, impairing the synthesis of KLF2 and its target S1PR1 to induce the leaky EC transcriptome. Repleting FAK (wild type or kinase dead) or inhibiting RhoA-emerin-DNMT3a activities in damaged lung ECs restored KLF2 transcription of the restrictive EC transcriptome. Thus, FAK sensing and control of tension transmission to the nucleus govern restrictive endothelium to maintain lung homeostasis.

Clinical features and identification of LEMD3 gene mutation in 4 osteopoikilosis patients / 中华内分泌代谢杂志

Objective To analyze the clinical features and the mutation of LEMD3 gene in four osteopoikilosis patients.Methods Clinical data of 4 patients were collected, peripheral blood samples were obtained for DNA extract, and LEMD3 gene mutation was analyzed by direct DNA sequencing.Results 4 patients with osteopoikilosis included a male aged 44, a female aged 42, a 26-year-old male, a 21-year-old female.All these patients were from families of non-consanguineous marriage.The main complaint of these patients was pain on arthrosis.Abnormal X-ray radiography was found in medical examination, while markers of bone metabolism were normal.The results of X-ray examination showed that numerous, discrete round or ovoid calcification were scattered throughout the wrist, pelvis and scapula.A de novo mutation c.595delG(NM_014319.4) localized in exon1 of the LEMD3 gene resulting in p.Ala199ProfsX46 of Case 3, while the mutation is not found in his parents and the remaining 3 patients.Conclusions A de novol LEMD3 mutation led to osteopoikilosis was found, and the pathogenesis of molecular mechanism in Chinese remained further exploration.

Cardiac Dysrhythmias, Cardiomyopathy and Muscular Dystrophy in Patients with Emery-Dreifuss Muscular Dystrophy and Limb-Girdle Muscular Dystrophy Type 1B

Emery-Dreifuss muscular dystrophy (EDMD) and limb-girdle muscular dystrophy type 1B (LGMD1B) are characterized by cardiac dysrhythmias, late-onset cardiomyopathy, slowly progressive skeletal myopathy and contractures of the neck, elbows and ankles. The causative mutation is either in the emerin gene (X-linked recessive EDMD) or lamin A/C gene (autosomal dominant EDMD2 or LGMD1B). We report three cases of EDMD, EDMD2 and LGMD1B. A 14-yr-old boy showed limitation of cervical flexion and contractures of both elbows and ankles. Sinus arrest with junctional escape beats was noted. He was diagnosed as X-linked recessive EDMD (MIM 310300). A 28-yr-old female showed severe wasting and weakness of humeroperoneal muscles. Marked limitation of cervical flexion and contractures of both elbows and ankles were noted. Varying degrees of AV block were noted. She was diagnosed as autosomal dominant EDMD2 (MIM 181350). A 41-yr-old female had contractures of both ankles and limb-girdle type muscular dystrophy. ECG revealed atrial tachycardia with high grade AV block. She was diagnosed as autosomal dominant LGMD1B (MIM 159001). Cardiac dysrhythmias in EDMD and LGMD1B include AV block, bradycardia, atrial tachycardia, atrial fibrillation, and atrial standstill, causing sudden death necessitating pacemaker implantation. Cardiologists should know about these unusual genetic diseases with conduction defects, especially in young adults.

Emery Dreifuss muscular Dystrophy: A case report

Emery-Dreifuss muscular dystrophy(EDMD) is a very rare, has never reported in Korea, relatively benign muscle disorder caused by defects of emerin. The clinical triad include 1) early contracture of the elbows, Achilles tendons, and postcervical muscles, 2) progressive weakness and atrophy in a humeroperoneal distribution, and 3) cardiomyopathy characterized by conduction defect. Heart block is a frequent cause of death. The detection of this disorder is important because insertion of a cardiac pacemaker can be life saving. As emerin was not found in biopsies from patients affected by EDMD and most mutations in EDMD are null, the immunohistochemical diagnosis can be easily performed by detection the absence of emerin. We report a 14-year-old boy with slowly progressive scapuloperoneal muscle weakness and atrophy, and contracture of the Achilles tendons, elbows and postcervical muscles. Muscle biopsy showed marked atrophy of myofiber and increased intermysial fibrosis and immunohistochemical study showed emerin deficiency.

Characters of clinical and pathological and the expressions of emerin protein and STA gene on Emery-Dreifuss muscular dystrophy:report of one case / 临床神经病学杂志

Objective To investigate clinical manifestation and pathological changes and the expressions of Emerin protein and STA gene of Emery-Dreifuss muscular dystrophy (EDMD).Methods The clinical features and STA gene detection from one patient with EDMD were analyzed retrospectively. Results The onset age of this patient was in early childhood. The four limbs were progressive muscle weakness and muscular atrophy. There were joint contractures and cardiac involvement in the early stage. The serum muscle enzymes increased slightly. The pathological changes in muscles showed that the sizes of muscle fibers were different, the fibers became spherical and some fibers were replaced by fat. Because of normal spinal anterior horn cells and sural nerves, neurogenic muscular atrophy might be ruled out. Emerin protein could not be tested in striated muscle and cardiac muscle. No mutation of STA gene was found in this case. Conclusions Emery-Dreifuss muscular dystrophy is one particular type of muscular dystrophy. It is characterized with joint contractures and cardiac involvement in the early stage. Emerin protein is deficient in EDMD. There is no mutation of STA gene in sporadic EDMD.