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  • MUTATIONS  (17)
  • 1
    Keywords: CELLS ; EXPRESSION ; IN-VITRO ; CELL ; human ; IN-VIVO ; VITRO ; VIVO ; NETWORK ; GENE ; PROTEIN ; PROTEINS ; TIME ; INFECTION ; RAT ; CONTRAST ; STAGE ; DISRUPTION ; MUTATION ; MUTATIONS ; MUSCLE ; ASSEMBLY PROPERTIES ; SERIES ; GREEN FLUORESCENT PROTEIN ; ORGANIZATION ; ADENOVIRUS ; ALPHA-B-CRYSTALLIN ; DILATED CARDIOMYOPATHY ; EPIDERMOLYSIS-BULLOSA SIMPLEX ; INTERMEDIATE-FILAMENT PROTEINS ; MICE LACKING DESMIN ; MUSCULAR-DYSTROPHY ; SKELETAL MYOPATHY ; SMOOTH-MUSCLE ; Z-DISCS
    Abstract: Mutations in desmin have been associated with a subset of human myopathies. Symptoms typically appear in the second to third decades of life, but in the most severe cases can manifest themselves earlier. How desmin mutations lead to aberrant muscle function, however, remains poorly defined. We created a series of four mutations in rat desmin and tested their in vitro filament assembly properties. RDM-G, a chimera between desmin and green fluorescent protein, formed protofilament-like structures in vitro. RDM-1 and RDM-2 blocked in vitro assembly at the unit-length filament stage, while RDM-3 had more subtle effects on assembly. When expressed in cultured rat neonatal cardiac myocytes via adenovirus infection, these mutant proteins disrupted the endogenous desmin filament to an extent that correlated with their defects in in vitro assembly properties. Disruption of the desmin network by RDM-1 was also associated with disruption of plectin, myosin, and a-actinin organization in a significant percentage of infected cells. In contrast, expression of RDM-2, which is similar to previously characterized human mutant desmins, took longer to disrupt desmin and plectin organization and had no significant effect on myosin or alpha-actinin organization over the 5-day time course of our studies. RDM-3 had the mildest effect on in vitro assembly and no discernable effect on either desmin, plectin, myosin, or a-actinin organization in vivo. These results indicate that mutations in desmin have both direct and indirect effects on the cytoarchitecture of cardiac myocytes
    Type of Publication: Journal article published
    PubMed ID: 12529857
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  • 2
    Keywords: CELL ; Germany ; human ; DISEASE ; DISEASES ; DISTINCT ; GENE ; PROTEIN ; PROTEINS ; INTERMEDIATE-SIZED FILAMENTS ; IMPACT ; BIOLOGY ; NUMBER ; MUTATION ; MUTATIONS ; US ; cytoskeleton ; intermediate filaments ; vimentin ; DE-NOVO ; ALPHA-B-CRYSTALLIN ; MICE LACKING DESMIN ; HUMAN-DISEASE ; ARCHITECTURE ; intermediate filament ; CHICKEN SKELETAL-MUSCLE ; desmin-related myopathy ; desminopathy ; filament assembly ; HEAD DOMAIN ; IMMUNOCYTOCHEMICAL ANALYSIS ; MYOFIBRILLAR MYOPATHY ; RESTRICTIVE CARDIOMYOPATHY ; VERTEBRATE SMOOTH-MUSCLE
    Abstract: Desmin, the major intermediate filament (IF) protein of muscle, is evolutionarily highly conserved from shark to man. Recently, an increasing number of mutations of the desmin gene has been described to be associated with human diseases such as certain skeletal and cardiac myopathies. These diseases are histologically characterised by intracellular aggregates containing desmin and various associated proteins. Although there is progress regarding our knowledge on the cellular function of desmin within the cytoskeleton, the impact of each distinct mutation is currently not understood at all. In order to get insight into how such mutations affect filament assembly and their integration into the cytoskeleton we need to establish IF structure at atomic detail. Recent progress in determining the dimer structure of the desmin-related IF-protein vimentin allows us to assess how such mutations may affect desmin filament architecture. (C) 2004 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15477095
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  • 3
    Keywords: brain ; ENVIRONMENT ; CELLS ; IN-VITRO ; CELL ; human ; IN-VIVO ; MICROSCOPY ; MODEL ; VITRO ; VIVO ; NETWORKS ; PROTEIN ; PROTEINS ; TISSUE ; murine ; REDUCTION ; RAT ; SKIN ; IDENTIFICATION ; MUTATION ; FIBER ; MUTATIONS ; LENGTH ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; keratin ; vimentin ; LIVING CELLS ; ATOMIC-FORCE MICROSCOPY ; BEHAVIOR ; INSIGHTS ; intermediate filament ; SINGLE ; RECOMBINANT ; PROTOCOL ; DESMIN ; SWITZERLAND ; LEVEL ; INTERMEDIATE ; PERSISTENCE ; function ; AFM ; ALPHA-KERATIN FIBERS ; HAGFISH SLIME THREADS ; MECHANICAL-BEHAVIOR ; neurofilament ; stretching ; EUKARYOTIC CELLS ; NEUROFILAMENTS
    Abstract: Intermediate filaments (IFs) are structural elements of eukaryotic cells with distinct mechanical properties. Tissue integrity is severely impaired, in particular in skin and muscle, when IFs are either absent or malfunctioning due to mutations. Our knowledge on the mechanical properties of IFs is mainly based on tensile testing of macroscopic fibers and on the rheology of IF networks. At the single filament level, the only piece of data available is a measure of the persistence length of vimentin IFs. Here, we have employed an atomic force microscopy (AFM) based protocol to directly probe the mechanical properties of single cytoplasmic IFs when adsorbed to a solid support in physiological buffer environment. Three IF types were studied in vitro: recombinant murine desmin, recombinant human keratin K5/K14 and neurofilaments isolated from rat brains, which are composed of the neurofilament triplet proteins NF-L, NF-M and NF-H. Depending on the experimental conditions, the AFM tip was used to laterally displace or to stretch single IFs on the support they had been adsorbed to. Upon applying force, IFs were stretched on average 2.6-fold. The maximum stretching that we encountered was 3.6-fold. A large reduction of the apparent filament diameter was observed concomitantly. The observed mechanical properties therefore suggest that IFs may indeed function as mechanical shock absorbers in vivo.
    Type of Publication: Journal article published
    PubMed ID: 16257415
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  • 4
    Keywords: APOPTOSIS ; CELLS ; IN-VITRO ; Germany ; human ; IN-VIVO ; DISEASE ; GENE ; GENES ; PROTEIN ; DOMAIN ; CLEAVAGE ; resistance ; MUTATIONS ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; vimentin ; ALPHA-B-CRYSTALLIN ; EPIDERMOLYSIS-BULLOSA SIMPLEX ; MICE LACKING VIMENTIN ; CYTOTOXICITY ; assembly ; keratins ; aggregates
    Abstract: To get new insights into the function of the intermediate filament (IF) protein vimentin in cell physiology, we generated two mutant cDNAs, one with a point mutation in the consensus motif in coillA (R113C) and one with the complete deletion of coil 2B of the rod domain. In keratins and glia filament protein (GFAP). analogous mutations cause keratinopathies and Alexander disease, respectively. Both mutants prevented filament assembly in vitro and inhibited assembly of wild-type vimentin when present in equal amounts. In stably transfected preadipocytes, these mutants caused the complete disruption of the endogenous vimentin network, demonstrating their dominant-negative behaviour. Cytoplasmic vimentin aggregates colocalised with the chaperones alpha B-crystallin and HSP40. Moreover, vimR(113)C mutant cells were more resistant against staurosporine-induced apoptosis compared to controls. We hypothesise that mutations in the vimentin gene, like in most classes of IF genes, may contribute to distinct human diseases. (c) 2005 Elsevier GmbH. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16373170
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  • 5
    Keywords: DYNAMICS ; MUTATIONS ; vimentin ; lamin ; ARCHITECTURE ; keratins ; STIFFNESS ; Phylogeny ; VISCOELASTIC PROPERTIES ; TAIL DOMAINS
    Abstract: Intermediate filament proteins form filaments, fibers and networks both in the cytoplasm and the nucleus of metazoan cells. Their general structural building plan accommodates highly varying amino acid sequences to yield extended dimeric alpha-helical coiled coils of highly conserved design. These 'rod' particles are the basic building blocks of intrinsically flexible, filamentous structures that are able to resist high mechanical stresses, that is, bending and stretching to a considerable degree, both in vitro and in the cell. Biophysical and computer modeling studies are beginning to unfold detailed structural and mechanical insights into these major supramolecular assemblies of cell architecture, not only in the 'test tube' but also in the cellular and tissue context.
    Type of Publication: Journal article published
    PubMed ID: 25621895
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  • 6
    Keywords: RECEPTOR ; CELLS ; EXPRESSION ; BLOOD ; human ; MICROSCOPY ; MODEL ; DISTINCT ; GENE ; PROTEIN ; PROTEINS ; MICE ; BINDING ; chromosome ; MOUSE ; PHENOTYPES ; CHROMATIN ; MEMBRANE ; MUTATION ; PRODUCT ; MUTATIONS ; ANOMALIES ; ABNORMALITIES ; MUSCULAR-DYSTROPHY ; NUCLEAR-ENVELOPE ; A/C ; LBR ; LEMLI-OPITZ-SYNDROME ; REDUCTASE GENE
    Abstract: The nature of the wild-type gene product at the mouse ichthyosis (ic) locus has been of great interest because mutations at this locus cause marked abnormalities in nuclear heterochromatin, similar to those observed in Pelger-Huet anomaly (PHA). We recently found that human PHA is caused by mutations in the gene (LBR) encoding lamin B receptor, an evolutionarily conserved inner nuclear membrane protein involved in nuclear assembly and chromatin binding. Mice homozygous for deleterious alleles at the ichthyosis (ic) locus present with a blood phenotype similar to PHA, and develop other phenotypic abnormalities, including alopecia, variable expression of syndactyly and hydrocephalus. The ic locus on mouse chromosome 1 shares conserved synteny with the chromosomal location of the human LBR locus on human chromosome 1. In this study, we identified one nonsense (815ins) and two frameshift mutations (1088insCC and 1884insGGAA) within the Lbr gene of mice homozygous for either of three independent mutations (ic, ic and ic(4J), respectively) at the ichthyosis locus. These allelic mutations are predicted to result in truncated or severely impaired LBR protein. Our studies of mice homozygous for the ic mutation revealed a complete loss of LBR protein as shown by immunofluorescence microscopy and immunoblotting. The findings provide the molecular basis for the heterochromatin clumping and other distinct phenotypes caused by ic mutations. These spontaneous Lbr mutations confirm the molecular basis of human PHA and provide a small animal model for determination of the precise function of LBR in normal and pathological states
    Type of Publication: Journal article published
    PubMed ID: 12490533
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  • 7
    Keywords: CELLS ; ENDOTHELIAL-CELLS ; EXPRESSION ; CELL ; Germany ; human ; SYSTEM ; DISEASE ; PROTEIN ; PROTEINS ; TISSUE ; LINES ; MICE ; TIME ; DOMAIN ; TISSUES ; BIOLOGY ; DELETION ; MOUSE ; MUTANT ; NO ; TRANSGENIC MICE ; HUMANS ; MUTATION ; LINE ; MUTATIONS ; cytoskeleton ; intermediate filaments ; keratin ; PHENOTYPE ; vimentin ; pathology ; PROTEASOME ; ALPHA-B-CRYSTALLIN ; EPIDERMOLYSIS-BULLOSA SIMPLEX ; INTERMEDIATE-FILAMENT PROTEINS ; keratins ; INCREASE ; POINT MUTATIONS ; ENGLAND ; NOV ; HSP70 ; FOCAL ADHESIONS ; response ; proteasomes ; NO EVIDENCE ; CELL BIOLOGY ; Cataract formation ; CHAPERONE ACTIVITY ; Chaperones ; Dominant-negative mutation ; EYE LENS ; FIBER CELL-DIFFERENTIATION ; Mouse model systems ; Protein misfolding
    Abstract: Vimentin is the main intermediate filament ( IF) protein of mesenchymal cells and tissues. Unlike other IF-/- mice, vimentin(-/-) mice provided no evidence of an involvement of vimentin in the development of a specific disease. Therefore, we generated two transgenic mouse lines, one with a ( R113C) point mutation in the IF-consensus motif in coil1A and one with the complete deletion of coil 2B of the rod domain. In epidermal keratins and desmin, point mutations in these parts of the alpha-helical rod domain cause keratinopathies and desminopathies, respectively. Here, we demonstrate that substoichiometric amounts of vimentin carrying the R113C point mutation disrupted the endogenous vimentin network in all tissues examined but caused a disease phenotype only in the eye lens, leading to a posterior cataract that was paralleled by the formation of extensive protein aggregates in lens fibre cells. Unexpectedly, central, postmitotic fibres became depleted of aggregates, indicating that they were actively removed. In line with an increase in misfolded proteins, the amounts of Hsp70 and ubiquitylated vimentin were increased, and proteasome activity was raised. We demonstrate here for the first time that the expression of mutated vimentin induces a protein-stress response that contributes to disease pathology in mice, and hypothesise that vimentin mutations cause cataracts in humans
    Type of Publication: Journal article published
    PubMed ID: 18940912
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  • 8
    Keywords: APOPTOSIS ; EXPRESSION ; IN-VITRO ; human ; VITRO ; DISEASE ; PROTEIN ; PROTEINS ; transcription ; TISSUE ; DNA ; animals ; TISSUES ; DYNAMICS ; CELL-CYCLE ; CHROMATIN ; MUTATIONS ; CAENORHABDITIS-ELEGANS ; INTERMEDIATE-FILAMENTS ; DNA-REPLICATION ; ORGANIZATION ; ARCHITECTURE ; MITOSIS ; nuclear envelope ; filament assembly ; molecular ; MOLECULAR-BASIS ; C-ELEGANS ; NUCLEAR ; MUTANTS ; ENVELOPE ; USA ; animal ; laminopathic diseases
    Abstract: Lamins are nuclear intermediate filament proteins and the major building blocks of the nuclear lamina. Besides providing nuclear shape and mechanical stability, lamins are required for chromatin organization, transcription regulation, DNA replication, nuclear assembly, nuclear positioning, and apoptosis. Mutations in human lamins cause many different heritable diseases, affecting various tissues and causing early aging. Although many of these mutations result in nuclear deformation, their effects on lamin filament assembly are unknown. Caenorhabditis elegans has a single evolutionarily conserved lamin protein, which can form stable 10-nm-thick filaments in vitro. To gain insight into the molecular basis of lamin filament assembly and the effects of laminopathic mutations on this process, we investigated mutations in conserved residues of the rod and tail domains that are known to cause various laminopathies in human. We show that 8 of 14 mutant lamins present WT-like assembly into filaments or paracrystals, whereas 6 mutants show assembly defects. Correspondingly, expressing these mutants in transgenic animals shows abnormal distribution of Ce-lamin, abnormal nuclear shape or change in lamin mobility. These findings help in understanding the role of individual residues and domains in laminopathy pathology and, eventually, promote the development of therapeutic interventions
    Type of Publication: Journal article published
    PubMed ID: 18162544
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  • 9
    Keywords: CELLS ; IN-VITRO ; MUTATIONS ; cytoskeleton ; intermediate filaments ; vimentin ; ATOMIC-FORCE MICROSCOPY ; DESMIN ; STIFFNESS ; ACTIN ; Atomic force microscopy ; VISCOELASTIC PROPERTIES ; ARTICULAR-CARTILAGE ; DEFICIENT FIBROBLASTS ; Dynamic elastic modulus ; ELASTIC-MODULUS ; INDENTATION ; Nanomechanics
    Abstract: The contribution of the intermediate filament (IF) network to the mechanical response of cells has so far received little attention, possibly because the assembly and regulation of IFs are not as well understood as that of the actin cytoskeleton or of microtubules. The mechanical role of IFs has been mostly inferred from measurements performed on individual filaments or gels in vitro. In this study we employ atomic force microscopy (AFM) to examine the contribution of vimentin IFs to the nanomechanical properties of living cells under native conditions. To specifically target and modulate the vimentin network, Rat-2 fibroblasts were transfected with GFP-desmin variants. Cells expressing desmin variants were identified by the fluorescence microscopy extension of the AFM instrument. This allowed us to directly compare the nanomechanical response of transfected and untransfected cells at high spatial resolution by means of AFM. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. Expression of the non-filament forming GFP-DesL345P mutant led to a collapse of the endogenous vimentin network in the perinuclear region that was accompanied by localized stiffening. Correlative confocal microscopy indicates that the expression of desmin variants specifically targets the endogenous vimentin IF network without major rearrangements of other cytoskeletal components. By measuring functional changes caused by IF rearrangements in intact cells, we show that IFs play a crucial role in mechanical behavior not only at large deformations but also in the nanomechanical response of individual cells.
    Type of Publication: Journal article published
    PubMed ID: 21426942
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  • 10
    Keywords: CELLS ; CELL ; Germany ; human ; SYSTEM ; SYSTEMS ; DISEASE ; DISEASES ; DISTINCT ; PROTEIN ; PROTEINS ; BIOLOGY ; ELEMENT ; STRESS ; MUTATION ; LINE ; PATHOGENESIS ; MUTATIONS ; NUCLEUS ; MUSCLE ; cytoskeleton ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; ATOMIC-FORCE MICROSCOPY ; MUSCULAR-DYSTROPHY ; MICE LACKING VIMENTIN ; ARCHITECTURE ; intermediate filament ; DYSTROPHY ; muscular dystrophy ; review ; RE ; NUCLEAR LAMINA ; analysis ; function ; animal ; FILAMENTS ; physiological function ; CYTOPLASM ; DESMIN CYTOSKELETON ; FOCAL ADHESIONS ; MECHANOTRANSDUCTION
    Abstract: Intermediate filaments (IFs) constitute a major structural element of animal cells. They build two distinct systems, one in the nucleus and one in the cytoplasm. In both cases, their major function is assumed to be that of a mechanical stress absorber and an integrating device for the entire cytoskeleton. In line with this, recent disease mutations in human IF proteins indicate that the nanomechanical properties of cell-type-specific IFs are central to the pathogenesis of diseases as diverse as muscular dystrophy and premature ageing. However, the analysis of these various diseases suggests that IFs also have an important role in cell-type-specific physiological functions
    Type of Publication: Journal article published
    PubMed ID: 17551517
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