Generation of one control and four iPSCs clones from patients with Emery-Dreifuss muscular dystrophy type 1.

Abstract Emery-Dreifuss muscular dystrophy type 1 (EDMD1) is a rare genetic disease caused by mutations in the EMD gene coding for a nuclear envelope protein emerin. We generated and characterized induced pluripotent stem cells (iPSCs) from two EDMD1 patients bearing a mutation c.del153C and from one healthy donor. That mutation leads to generation of premature STOP codon. Established iPSCs are very valuable tool for disease pathogenesis investigation and for the development of new therapeutic methods after differentiation to cardiac or muscle cells. Obtained iPSCs show the proper morphology, pluripotency markers expression, normal karyotype and potential to differentiate into three germ layers

The Different Function of Single Phosphorylation Sites of Drosophila melanogaster Lamin Dm and Lamin C

Lamins' functions are regulated by phosphorylation at specific sites but our understanding of the role of such modifications is practically limited to the function of cdc 2 (cdk1) kinase sites in depolymerization of the nuclear lamina during mitosis. In our study we used Drosophila lamin Dm (B-type) to examine the function of particular phosphorylation sites using pseudophosphorylated mutants mimicking single phosphorylation at experimentally confirmed in vivo phosphosites (S25E, S45E, T435E, S595E). We also analyzed lamin C (A-type) and its mutant S37E representing the N-terminal cdc2 (mitotic) site as well as lamin Dm R64H mutant as a control, non-polymerizing lamin. In the polymerization assay we could observe different effects of N-terminal cdc2 site pseudophosphorylation on A- and B-type lamins: lamin Dm S45E mutant was insoluble, in contrast to lamin C S37E. Lamin Dm T435E (C-terminal cdc2 site) and R64H were soluble in vitro. We also confirmed that none of the single phosphorylation site modifications affected the chromatin binding of lamin Dm, in contrast to the lamin C N-terminal cdc2 site. In vivo, all lamin Dm mutants were incorporated efficiently into the nuclear lamina in transfected Drosophila S2 and HeLa cells, although significant amounts of S45E and T435E were also located in cytoplasm. When farnesylation incompetent mutants were expressed in HeLa cells, lamin Dm T435E was cytoplasmic and showed higher mobility in FRAP assay

Hutchinson-Gilford Progeria Syndrome—Current Status and Prospects for Gene Therapy Treatment

Hutchinson-Gilford progeria syndrome (HGPS) is one of the most severe disorders among laminopathies—a heterogeneous group of genetic diseases with a molecular background based on mutations in the LMNA gene and genes coding for interacting proteins. HGPS is characterized by the presence of aging-associated symptoms, including lack of subcutaneous fat, alopecia, swollen veins, growth retardation, age spots, joint contractures, osteoporosis, cardiovascular pathology, and death due to heart attacks and strokes in childhood. LMNA codes for two major, alternatively spliced transcripts, give rise to lamin A and lamin C proteins. Mutations in the LMNA gene alone, depending on the nature and location, may result in the expression of abnormal protein or loss of protein expression and cause at least 11 disease phenotypes, differing in severity and affected tissue. LMNA gene-related HGPS is caused by a single mutation in the LMNA gene in exon 11. The mutation c.1824C > T results in activation of the cryptic donor splice site, which leads to the synthesis of progerin protein lacking 50 amino acids. The accumulation of progerin is the reason for appearance of the phenotype. In this review, we discuss current knowledge on the molecular mechanisms underlying the development of HGPS and provide a critical analysis of current research trends in this field. We also discuss the mouse models available so far, the current status of treatment of the disease, and future prospects for the development of efficient therapies, including gene therapy for HGPS

Emerin Is Required for Proper Nucleus Reassembly after Mitosis: Implications for New Pathogenetic Mechanisms for Laminopathies Detected in EDMD1 Patients

Emerin is an essential LEM (LAP2, Emerin, MAN1) domain protein in metazoans and an integral membrane protein associated with inner and outer nuclear membranes. Mutations in the human EMD gene coding for emerin result in the rare genetic disorder: Emery–Dreifuss muscular dystrophy type 1 (EDMD1). This disease belongs to a broader group called laminopathies—a heterogeneous group of rare genetic disorders affecting tissues of mesodermal origin. EDMD1 phenotype is characterized by progressive muscle wasting, contractures of the elbow and Achilles tendons, and cardiac conduction defects. Emerin is involved in many cellular and intranuclear processes through interactions with several partners: lamins; barrier-to-autointegration factor (BAF), β-catenin, actin, and tubulin. Our study demonstrates the presence of the emerin fraction which associates with mitotic spindle microtubules and centrosomes during mitosis and colocalizes during early mitosis with lamin A/C, BAF, and membranes at the mitotic spindle. Transfection studies with cells expressing EGFP-emerin protein demonstrate that the emerin fusion protein fraction also localizes to centrosomes and mitotic spindle microtubules during mitosis. Transient expression of emerin deletion mutants revealed that the resulting phenotypes vary and are mutant dependent. The most frequent phenotypes include aberrant nuclear shape, tubulin network mislocalization, aberrant mitosis, and mislocalization of centrosomes. Emerin deletion mutants demonstrated different chromatin binding capacities in an in vitro nuclear assembly assay and chromatin-binding properties correlated with the strength of phenotypic alteration in transfected cells. Aberrant tubulin staining and microtubule network phenotype appearance depended on the presence of the tubulin binding region in the expressed deletion mutants. We believe that the association with tubulin might help to “deliver” emerin and associated membranes to decondensing chromatin. Preliminary analyses of cells from Polish patients with EDMD1 revealed that for several mutations thought to be null for emerin protein, a truncated emerin protein was present. We infer that the EDMD1 phenotype may be strengthened by the toxicity of truncated emerin expressed in patients with certain nonsense mutations in EMD

Laminopathies: what can humans learn from fruit flies

Abstract Lamin proteins are type V intermediate filament proteins (IFs) located inside the cell nucleus. They are evolutionarily conserved and have similar domain organization and properties to cytoplasmic IFs. Lamins provide a skeletal network for chromatin, the nuclear envelope, nuclear pore complexes and the entire nucleus. They are also responsible for proper connections between the karyoskeleton and structural elements in the cytoplasm: actin and the microtubule and cytoplasmic IF networks. Lamins affect transcription and splicing either directly or indirectly. Translocation of active genes into the close proximity of nuclear lamina is thought to result in their transcriptional silencing. Mutations in genes coding for lamins and interacting proteins in humans result in various genetic disorders, called laminopathies. Human genes coding for A-type lamin (LMNA) are the most frequently mutated. The resulting phenotypes include muscle, cardiac, neuronal, lipodystrophic and metabolic pathologies, early aging phenotypes, and combined complex phenotypes. The Drosophila melanogaster genome codes for lamin B-type (lamin Dm), lamin A-type (lamin C), and for LEM-domain proteins, BAF, LINC-complex proteins and all typical nuclear proteins. The fruit fly system is simpler than the vertebrate one since in flies there is only single lamin B-type and single lamin A-type protein, as opposed to the complex system of B- and A-type lamins in Danio, Xenopus and Mus musculus. This offers a unique opportunity to study laminopathies. Applying genetic tools based on Gal4 and in vitro nuclear assembly system to the fruit fly model may successfully advance knowledge of laminopathies. Here, we review studies of the laminopathies in the fly model system

Neisseria gonorrhoeae FA1090 Carries Genes Encoding Two Classes of Vsr Endonucleases ▿

A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases V.NgoAXIII and V.NgoAXIV from Neisseria gonorrhoeae FA1090 based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli, the proteins were biochemically characterized and the endonucleolytic activities and specificities of V.NgoAXIII and V.NgoAXIV were determined. V.NgoAXIII was found to be multispecific and to recognize T:G mismatches in every nucleotide context tested, whereas V.NgoAXIV recognized T:G mismatches in the following sequences: GTGG, CTGG, GTGC, ATGC, and CTGC. Alanine mutagenesis of conserved residues showed that Asp50 and His68 of V.NgoAXIII and Asp51 and His69 of V.NgoAXIV are essential for hydrolytic activity. Glu25, His64, and Asp97 of V.NgoAXIV and Glu24, Asp63, and Asp97 of V.NgoAXIII are important but not crucial for the activity of V.NgoAXIII and V.NgoAXIV. However, Glu24 and Asp63 are also important for the specificity of V.NgoAXIII. On the basis of our results concerning features of Vsr endonucleases expressed by N. gonorrhoeae FA1090, we postulate that at least two types of Vsr endonucleases can be distinguished

All lamin Dm mutants localize to nuclear lamina in transfected <i>Drosophila</i> S2 cells but lamin Dm S⁴⁵E and T⁴³⁵E mutants show significantly different distribution.

<p>Localization of fusion GFP-lamin Dm and mutant proteins after 48 h (C) post-transfection into <i>Drosophila</i> S2 cells visualized under a confocal microscope and quantitative analyses of appearance of the particular phenotypes (A and B). Cells were stained for DNA with DAPI, for endogenous lamin Dm with mouse monoclonal antibodies ADL67. Exogenous lamin Dm proteins were visualized by eGFP fluorescence. Panel A demonstrates statistical analyses of distribution of lamin fusion proteins in nucleus only in nucleus and cytoplasm and in cytoplasm only. Panel B demonstrates statistical analyses of fusion proteins' localization to nuclear envelope and nuclear lamina (membrane), diffused phenotype, inclusion bodies phenotype and mixed phenotype respectively. Single confocal sections through the center of nuclei are shown. 200 cells were analyzed.</p

Comparison of the conserved amino acid sequences located in the N-terminal and C-terminal fragment of lamins containing the cdc2 kinase site, using Clustal W.

<p>A schematic view of the lamin monomer with <i>in vivo</i> identified embryonic phosphorylation sites as well as NLS, Ig-fold and CaaX motif are shown. Identical amino acids are marked by black boxes, similar by shadowed boxes. Four conserved regions were identified in vertebrate lamins and three in <i>Drosophila</i> lamins. The first region in all lamins also contains the N-terminal cdc2 site (SPTR motif). The second region located at the very beginning of the tail domain is present in vertebrates only and contains their C-terminal cdc2 site (SPXXR motif). The <i>Drosophila</i> C-terminal cdc2 site is located partially in the third conservative region (T/SRAT/S sequences) – TPSR motif for lamin Dm and TPSGR motif in lamin C. There is also an alternative C-terminal cdc2 site in lamin C (S⁴⁰⁵ in SPGR motif ). LDm-D <i>Drosophila melanogaster</i> lamin Dm, LC-D lamin C from fruit fly, LA/C-H human lamin A/C, LB1-H human lamin B1, LB-M mouse lamin B, LB2-G chicken lamin B2, LB1-X <i>Xenopus</i> lamin B1, LB3-X <i>Xenopus</i> lamin B3, LA-X <i>Xenopus</i> lamin A.</p

Graphical presentation of the data collected during FRAP experiments with farnesylation incompetent lamin Dm mutants in HeLa cells.

<p>Lamin Dm and all pseudophosphorylated mutants except lamin Dm T⁴³⁵E show the same low diffusion rate. Lack of recovery after photobleaching was observed for control human lamins (A, C and B1) (data not shown) as well as <i>D. melanogaster</i> wild type lamin Dm and the majority of lamin Dm mutants (S²⁵E, S45E, S⁵⁹⁵E), excluding lamin Dm T⁴³⁵E. They showed no measurable recovery after several minutes. In contrast, lamin Dm mutant with threonine 435 substituted by glutamic acid to mimic stable, permanent phosphorylation displayed increased dynamics (D = 2.7 µm²/s; Mf = 20%). The presented fluorescence recovery curve shows that <i>Drosophila</i> lamins show similar mobility as human lamins in HeLa cells. Only specific mutation (T⁴³⁵E) can increase protein mobility, indicating lower polymerization ability <i>in vivo</i>. For control experiments we used HeLa cells expressing free EGFP. EGFP expression was seen to be evenly distributed within the nucleus and cytoplasm. Cytoplasmic fraction of EGFP shows a slower recovery (t1/2 = 2.05 seconds) versus that observed in the nucleus (t1/2 = 1.2 seconds).</p