Nuclear envelope protein Lem2 is required for mouse development and regulates MAP and AKT kinases

The nuclear lamina, along with associated nuclear membrane proteins, is a nexus for regulating signaling in the nucleus. Numerous human diseases arise from mutations in lamina proteins, and experimental models for these disorders have revealed aberrant regulation of various signaling pathways. Previously, we reported that the inner nuclear membrane protein Lem2, which is expressed at high levels in muscle, promotes the differentiation of cultured myoblasts by attenuating ERK signaling. Here, we have analyzed mice harboring a disrupted allele for the Lem2 gene (Lemd2). No gross phenotypic defects were seen in heterozygotes, although muscle regeneration induced by cardiotoxin was delayed. By contrast, homozygous Lemd2 knockout mice died by E11.5. Although many normal morphogenetic hallmarks were observed in E10.5 knockout embryos, most tissues were substantially reduced in size. This was accompanied by activation of multiple MAP kinases (ERK1/2, JNK, p38) and AKT. Knockdown of Lem2 expression in C2C12 myoblasts also led to activation of MAP kinases and AKT. These findings indicate that Lemd2 plays an essential role in mouse embryonic development and that it is involved in regulating several signaling pathways. Since increased MAP kinase and AKT/mTORC signaling is found in other animal models for diseases linked to nuclear lamina proteins, LEMD2 should be considered to be another candidate gene for human disease

Uncoordinated Transcription and Compromised Muscle Function in the Lmna-Null Mouse Model of Emery-Dreifuss Muscular Dystrophy

LMNA encodes both lamin A and C: major components of the nuclear lamina. Mutations in LMNA underlie a range of tissue-specific degenerative diseases, including those that affect skeletal muscle, such as autosomal-Emery-Dreifuss muscular dystrophy (A-EDMD) and limb girdle muscular dystrophy 1B. Here, we examine the morphology and transcriptional activity of myonuclei, the structure of the myotendinous junction and the muscle contraction dynamics in the lmna-null mouse model of A-EDMD. We found that there were fewer myonuclei in lmna-null mice, of which ∼50% had morphological abnormalities. Assaying transcriptional activity by examining acetylated histone H3 and PABPN1 levels indicated that there was a lack of coordinated transcription between myonuclei lacking lamin A/C. Myonuclei with abnormal morphology and transcriptional activity were distributed along the length of the myofibre, but accumulated at the myotendinous junction. Indeed, in addition to the presence of abnormal myonuclei, the structure of the myotendinous junction was perturbed, with disorganised sarcomeres and reduced interdigitation with the tendon, together with lipid and collagen deposition. Functionally, muscle contraction became severely affected within weeks of birth, with specific force generation dropping as low as ∼65% and ∼27% of control values in the extensor digitorum longus and soleus muscles respectively. These observations illustrate the importance of lamin A/C for correct myonuclear function, which likely acts synergistically with myotendinous junction disorganisation in the development of A-EDMD, and the consequential reduction in force generation and muscle wasting

Lamin A/C truncation in dilated cardiomyopathy with conduction disease

BACKGROUND: Mutations in the gene encoding the nuclear membrane protein lamin A/C have been associated with at least 7 distinct diseases including autosomal dominant dilated cardiomyopathy with conduction system disease, autosomal dominant and recessive Emery Dreifuss Muscular Dystrophy, limb girdle muscular dystrophy type 1B, autosomal recessive type 2 Charcot Marie Tooth, mandibuloacral dysplasia, familial partial lipodystrophy and Hutchinson-Gilford progeria. METHODS: We used mutation detection to evaluate the lamin A/C gene in a 45 year-old woman with familial dilated cardiomyopathy and conduction system disease whose family has been well characterized for this phenotype [1]. RESULTS: DNA from the proband was analyzed, and a novel 2 base-pair deletion c.908_909delCT in LMNA was identified. CONCLUSIONS: Mutations in the gene encoding lamin A/C can lead to significant cardiac conduction system disease that can be successfully treated with pacemakers and/or defibrillators. Genetic screening can help assess risk for arrhythmia and need for device implantation

Specific Loss of Histone H3 Lysine 9 Trimethylation and HP1γ/Cohesin Binding at D4Z4 Repeats Is Associated with Facioscapulohumeral Dystrophy (FSHD)

Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy in which no mutation of pathogenic gene(s) has been identified. Instead, the disease is, in most cases, genetically linked to a contraction in the number of 3.3 kb D4Z4 repeats on chromosome 4q. How contraction of the 4qter D4Z4 repeats causes muscular dystrophy is not understood. In addition, a smaller group of FSHD cases are not associated with D4Z4 repeat contraction (termed “phenotypic” FSHD), and their etiology remains undefined. We carried out chromatin immunoprecipitation analysis using D4Z4–specific PCR primers to examine the D4Z4 chromatin structure in normal and patient cells as well as in small interfering RNA (siRNA)–treated cells. We found that SUV39H1–mediated H3K9 trimethylation at D4Z4 seen in normal cells is lost in FSHD. Furthermore, the loss of this histone modification occurs not only at the contracted 4q D4Z4 allele, but also at the genetically intact D4Z4 alleles on both chromosomes 4q and 10q, providing the first evidence that the genetic change (contraction) of one 4qD4Z4 allele spreads its effect to other genomic regions. Importantly, this epigenetic change was also observed in the phenotypic FSHD cases with no D4Z4 contraction, but not in other types of muscular dystrophies tested. We found that HP1γ and cohesin are co-recruited to D4Z4 in an H3K9me3–dependent and cell type–specific manner, which is disrupted in FSHD. The results indicate that cohesin plays an active role in HP1 recruitment and is involved in cell type–specific D4Z4 chromatin regulation. Taken together, we identified the loss of both histone H3K9 trimethylation and HP1γ/cohesin binding at D4Z4 to be a faithful marker for the FSHD phenotype. Based on these results, we propose a new model in which the epigenetic change initiated at 4q D4Z4 spreads its effect to other genomic regions, which compromises muscle-specific gene regulation leading to FSHD pathogenesis

Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately reactivated it could affect muscle differentiation and contribute to disease pathology

Lamin A Rod Domain Mutants Target Heterochromatin Protein 1α and β for Proteasomal Degradation by Activation of F-Box Protein, FBXW10

Lamins are major structural proteins of the nucleus and contribute to the organization of various nuclear functions. Mutations in the human lamin A gene cause a number of highly degenerative diseases, collectively termed as laminopathies. Cells expressing lamin mutations exhibit abnormal nuclear morphology and altered heterochromatin organization; however, the mechanisms responsible for these defects are not well understood.The lamin A rod domain mutants G232E, Q294P and R386K are either diffusely distributed or form large aggregates in the nucleoplasm, resulting in aberrant nuclear morphology in various cell types. We examined the effects of these lamin mutants on the distribution of heterochromatin protein 1 (HP1) isoforms. HeLa cells expressing these mutants showed a heterogeneous pattern of HP1alpha and beta depletion but without altering HP1gamma levels. Changes in HP1alpha and beta were not observed in cells expressing wild-type lamin A or mutant R482L, which assembled normally at the nuclear rim. Treatment with proteasomal inhibitors led to restoration of levels of HP1 isoforms and also resulted in stable association of lamin mutants with the nuclear periphery, rim localization of the inner nuclear membrane lamin-binding protein emerin and partial improvement of nuclear morphology. A comparison of the stability of HP1 isoforms indicated that HP1alpha and beta displayed increased turnover and higher basal levels of ubiquitination than HP1gamma. Transcript analysis of components of the ubiquitination pathway showed that a specific F-box protein, FBXW10 was induced several-fold in cells expressing lamin mutants. Importantly, ectopic expression of FBXW10 in HeLa cells led to depletion of HP1alpha and beta without alteration of HP1gamma levels.Mislocalized lamins can induce ubiquitin-mediated proteasomal degradation of certain HP1 isoforms by activation of FBXW10, a member of the F-box family of proteins that is involved in E3 ubiquitin ligase activity

Integrated high-content quantification of intracellular ROS levels and mitochondrial morphofunction

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-contentmicroscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells

TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation

<div><p>Recent work indicates that the nuclear envelope is a major signaling node for the cell that can influence tissue differentiation processes. Here we present two nuclear envelope trans-membrane proteins TMEM120A and TMEM120B that are paralogs encoded by the <i>Tmem120A</i> and <i>Tmem120B</i> genes. The TMEM120 proteins are expressed preferentially in fat and both are induced during 3T3-L1 adipocyte differentiation. Knockdown of one or the other protein altered expression of several genes required for adipocyte differentiation, <i>Gata3</i>, <i>Fasn</i>, <i>Glut4</i>, while knockdown of both together additionally affected <i>Pparg</i> and <i>Adipoq</i>. The double knockdown also increased the strength of effects, reducing for example <i>Glut4</i> levels by 95% compared to control 3T3-L1 cells upon pharmacologically induced differentiation. Accordingly, TMEM120A and B knockdown individually and together impacted on adipocyte differentiation/metabolism as measured by lipid accumulation through binding of Oil Red O and coherent anti-Stokes Raman scattering microscopy (CARS). The nuclear envelope is linked to several lipodystrophies through mutations in lamin A; however, lamin A is widely expressed. Thus it is possible that the TMEM120A and B fat-specific nuclear envelope transmembrane proteins may play a contributory role in the tissue-specific pathology of this disorder or in the wider problem of obesity.</p></div