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  • intermediate filament  (15)
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  • 1
    Keywords: BIOLOGY ; ARCHITECTURE ; cytoskeleton ; PROTEIN ; CELL ; MICROSCOPY ; intermediate filament ; AMINO-ACID-SEQUENCE ; CROSS-LINKING ; review ; DESMIN ; ROD DOMAIN ; HUMAN VIMENTIN ; KERATIN FILAMENTS ; RAY SOLUTION SCATTERING ; SWITZERLAND ; COILED-COIL
    Type of Publication: Book chapter
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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: IN-VITRO ; VITRO ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; isolation ; intermediate filament
    Type of Publication: Journal article published
    PubMed ID: 15646613
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  • 4
    Keywords: EXPRESSION ; CELL ; Germany ; IN-VIVO ; MECHANISM ; mechanisms ; cytoskeleton ; MICROTUBULES ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; LIVING CELLS ; REVEALS ; ATOMIC-FORCE MICROSCOPY ; intermediate filament ; MOLECULAR-MECHANISM ; HUMAN HAIR KERATINS ; HUMAN VIMENTIN ; NUCLEAR LAMINA ; PROTEIN-FOLDING MOTIF ; TAIL DOMAIN
    Type of Publication: Journal article published
    PubMed ID: 15501448
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  • 5
    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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  • 6
    Keywords: CELLS ; IN-VITRO ; CELL ; human ; MICROSCOPY ; MODEL ; MODELS ; PATHWAY ; VITRO ; DISEASE ; DISTINCT ; PROTEIN ; PROTEINS ; mechanisms ; DYNAMICS ; polymorphism ; OLIGOMERS ; cytoskeleton ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; vimentin ; REVEALS ; DIMER ; electron microscopy ; intermediate filament ; ELECTRON-MICROSCOPY ; assembly ; PH ; HUMAN VIMENTIN ; analytical ultracentrifugation ; KERATIN FILAMENTS ; BIOLOGICAL MACROMOLECULES ; DIMERS ; AXES ; ADJACENT ; FILAMENTS ; 3D structure
    Abstract: intermediate filaments (IF's), along with microtubules, microfilaments, and associated cross-bridging proteins, constitute the cytoskeleton of metazoan cells. While crystallographic data on the dimer representing the elementary IF "building block" have recently become available, little structural detail is known about both the mature IF architecture and its assembly pathway. Here, we have applied solution small-angle x-ray scattering to investigate the in vitro assembly of a 53-kDa human IF protein vimentin at pH 8.4 by systematically varying the ionic strength conditions, and complemented these experiments by electron microscopy and analytical ultracentrifugation. While a vimentin solution in 5 mM Tris(.)HCl (pH 8.4) contains predominantly tetramers, addition of 20 mM NaCl induces further lateral assembly evidenced by the shift of the sedimentation coeficient and yields a distinct octameric intermediate. Four octamers eventually associate into unit-length filaments (ULFs) that anneal longitudinally. Based on the small-angle x-ray scattering experiments supplemented by crystallographic data and additional structural constraints, 3D molecular models of the vimentin tetramer, octamer, and ULF were constructed. Within each of the three oligomers, the adjacent dimers are aligned exclusively in an approximately half-staggered antiparallel A(11) mode with a distance of 3.2-3.4 nm between their axes. The ULF appears to be a dynamic and a relatively loosely packed structure with a roughly even mass distribution over its cross-section
    Type of Publication: Journal article published
    PubMed ID: 17050693
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  • 7
    Keywords: CELLS ; IN-VITRO ; CELL ; COMBINATION ; PROTEIN ; PROTEINS ; DIFFERENTIATION ; TISSUE ; COMPLEX ; COMPLEXES ; FAMILY ; primary ; DOMAIN ; TISSUES ; BIOLOGY ; MEMBER ; MEMBERS ; polymorphism ; ELEMENT ; PATTERNS ; ELEMENTS ; NUCLEUS ; CRYSTAL-STRUCTURE ; MUSCLE ; STABILITY ; tomography ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; Jun ; keratin ; vimentin ; epidermis ; DIMER ; ATOMIC-STRUCTURE ; intermediate filament ; molecular ; ONCOLOGY ; PROGRAM ; review ; RE ; FAMILIES ; PATTERN ; ELECTRON-MICROSCOPY ; assembly ; COILED-COIL ; EPIDERMAL KERATIN FILAMENTS ; ROD DOMAIN ; analysis ; methods ; LONG ; USA ; FILAMENTS ; STEM ; modeling ; X-RAY ; SHAPE ; cryo-electron tomography ; FIBROUS PROTEINS ; HELICAL COILED COILS ; IV ALPHA-INTERNEXIN ; PLASTICITY ; small-angle X-ray scattering ; TRICHOCYTE KERATIN
    Abstract: Intermediate filaments (IFs) represent one of the prominent cytoskeletal elements of metazoan cells. Their constituent proteins are coded by a multigene family, whose members are expressed in complex patterns that are controlled by developmental programs of differentiation. Hence, IF proteins found in epidermis differ significantly from those in muscle or neuronal tissues. Due to their fibrous nature, which stems from a fairly conserved central alpha-helical coiled-coil rod domain, IF proteins have long resisted crystallization and thus determination of their atomic structure. Since they represent the primary structural elements that determine the shape of the nucleus and the cell more generally, a major challenge is to arrive at a more rational understanding of how their nanomechanical properties effect the stability and plasticity of cells and tissues. Here, we review recent structural results of the coiled-coil dimer, assembly intermediates and growing filaments that have been obtained by a hybrid methods approach involving a rigorous combination of X-ray crystallography, small angle X-ray scattering, cryo-electron tomography, computational analysis and molecular modeling. (c) 2007 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17521629
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  • 8
    Keywords: DISEASE ; MECHANISM ; INTERMEDIATE-FILAMENTS ; vimentin ; ATOMIC-FORCE MICROSCOPY ; DIMER ; electron microscopy ; INVITRO ; intermediate filament ; analytical ultracentrifugation ; DESMIN ; SCAFFOLDS ; UNITS ; Assembly kinetics ; Recombinant keratins
    Abstract: We have generated human recombinant keratins K8 and K18 and describe conditions to quantitatively follow their assembly into filaments. When renatured individually from 8M urea into a low ionic strength/high pH-buffer, K8 was present in a dimeric to tetrameric form as revealed by analytical ultracentrifugation. In contrast, K18 sedimented as a monomer. When mixed in 8M urea and renatured together, K8 and K18 exhibited s-value profiles compatible with homogeneous tetrameric complexes. This finding was confirmed by sedimentation equilibrium centrifugation. Subsequently, these tetrameric starter units were subjected to assembly experiments at various protein concentrations. At low values such as 0.0025g/l, unit-length filaments were abundantly present after 2s of assembly. During the following 5min, filaments grew rapidly and by measuring the length of individual filaments we were able to generate time-dependent length profiles. These data revealed that keratins K8/K18 assemble several times faster than vimentin and desmin. In addition, we determined the persistence length l(p) of K8/K18 filaments to be in the range of 300nm. Addition of 1mM MgCl(2) increases l(p) to 480nm indicating that magnesium ions affect the interaction of keratin subunits within the filament during assembly to some extent.
    Type of Publication: Journal article published
    PubMed ID: 22085677
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  • 9
    Keywords: CELLS ; CELL ; Germany ; MICROSCOPY ; SYSTEM ; SITE ; SITES ; PROTEINS ; COMPLEX ; COMPLEXES ; CONTRAST ; DYNAMICS ; BIOLOGY ; polymorphism ; DIVERSITY ; MICROTUBULES ; tomography ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; vimentin ; ORGANIZATION ; DIMER ; RECONSTRUCTION ; CRYOELECTRON MICROSCOPY ; ATOMIC-STRUCTURE ; intermediate filament ; MOLECULAR-BASIS ; ELECTRON-MICROSCOPY ; COILED-COIL ; MOLECULAR ARCHITECTURE ; KERATIN FILAMENTS ; microtubule ; USA ; DIMERS ; UNIT ; SPECIMENS ; FILAMENTS ; 3D structure ; cryo-electron tomography ; protofibrils
    Abstract: Vimentin polymerizes via complex lateral interactions of coiled-coil dimers into long, flexible filaments referred to as intermediate filaments (IFs). Intermediate in diameter between microtubules and microfilaments, IFs constitute the third cytoskeletal filament system of metazoan cells. Here we investigated the molecular basis of the 3-D architecture of vimentin IFs by cryo-electron microscopy (cryo-EM) as well as cryo-electron tomography (Cryo-ET) 3-D reconstruction. We demonstrate that vimentin filaments in cross-section exhibit predominantly a four-stranded protofibrilar organization with a right-handed supertwist with a helical pitch of about 96 nm. Compact filaments imaged by cryo-EM appear surprisingly straight and hence appear very stiff. In addition, IFs exhibited an increased flexibility at sites of partial unraveling. This is in strong contrast to chemically fixed, negatively stained preparations of vimentin filaments that generally exhibit smooth bending without untwisting. At some point along the filament unraveling may be triggered and propagates in a cooperative manner so that long stretches of filaments appear to have unraveled rapidly in a coordinated fashion. (c) 2006 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17289402
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  • 10
    Keywords: PEPTIDE ; CELLS ; IN-VITRO ; CELL ; human ; VITRO ; PROTEIN ; DOMAIN ; BIOLOGY ; SPECTROSCOPY ; FORM ; SUBUNIT ; MUTATION ; CRYSTAL-STRUCTURE ; STABILITY ; INTERMEDIATE-FILAMENTS ; vimentin ; DIMER ; lamin ; DOMAINS ; ATOMIC-STRUCTURE ; intermediate filament ; SINGLE ; molecular biology ; assembly ; REARRANGEMENT ; coiled coil ; COILED-COIL ; nuclear lamins ; TEMPERATURE ; AMINO-ACID SUBSTITUTIONS ; circular dichroism ; POSITION ; STATE ; biophysical analysis ; CONSENSUS MOTIF ; GCN4 LEUCINE-ZIPPER ; HYDROPHOBIC CORE ; Oligomerisation ; OLIGOMERIZATION STATE ; PROTEIN STRUCTURES
    Abstract: Interestingly, our previously published structure of the coil 1A fragment of the human intermediate filament protein vimentin turned out to be a monomeric alpha-helical coil instead of the expected dimeric coiled coil. However, the 39-amino-acid-long helix had an intrinsic curvature compatible with a coiled coil. We have now designed four mutants of vimentin coil 1A, modifying key a and d positions in the heptad repeat pattern, with the aim of investigating the molecular criteria that are needed to stabilize a dimeric coiled-coil structure. We have analysed the biophysical properties of the mutants by circular dichroism spectroscopy, analytical ultracentrifugation and X-ray crystallography. All four mutants exhibited an increased stability over the wild type as indicated by a rise in the melting temperature (T-m). At a concentration of 0.1 mg/ml, the T-m of the peptide with the single point mutation Y117L increased dramatically by 46 degrees C compared with the wild-type peptide. In general, the introduction of a single stabilizing point mutation at an a or a d position did induce the formation of a stable dimer as demonstrated by sedimentation equilibrium experiments. The dimeric oligomerisation state of the Y117L peptide was furthermore confirmed by Xray crystallography, which yielded a structure with a genuine coiled-coil geometry. Most notably, when this mutation was introduced into full-length vimentin, filament assembly was completely arrested at the unit-length filament (ULF) level, both in vitro and in cDNA-transfected cultured cells. Therefore, the low propensity of the wild-type coil 1A to form a stable two-stranded coiled coil is most likely a prerequisite for the end-to-end annealing of ULFs into filaments. Accordingly, the coil 1A domains might "switch" from a dimeric alpha-helical coiled coil into a more open structure, thus mediating, within the ULFs, the conformational rearrangements of the tetrameric subunits that are needed for the intermediate filament elongation reaction. (C) 2009 Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 19422834
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