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  • 1
    Keywords: Germany ; MICROSCOPY ; IMAGES ; VISUALIZATION ; RNA ; transcription ; MOLECULES ; TIME ; DNA ; BINDING ; polymer ; NUMBER ; ELECTRON ; SURFACE ; LENGTH ; MICA ; MONTE-CARLO SIMULATIONS ; PARAMETERS ; SCATTERING ; STATISTICAL-ANALYSIS ; SUPERHELIX
    Abstract: The conformations of supercoiled (sc) DNA and linear DNA bound to polylysine (PL)-coated mica were investigated by scanning force microscopy (SFM) in solution. From the polymer statistical analysis of linear DNA, we could distinguish between re-arrangements or trapping of the DNA on the surface. Conditions of re-arrangements to an almost equilibrated state can be achieved at appropriate PL surface concentrations. We could show that the ability of re-arrangements depends on the salt concentration of the adsorption/imaging buffer. Comparing the statistical analysis of the linear DNA with SFM images of scDNA suggested that irregular scDNA conformations are formed under conditions of trapping, whereas plectonemic structures are favoured under conditions of surface re-arrangements. Salt-dependent changes in the scDNA conformation over the range of 10-100 mM NaCl, as characterised by the parameters writhe and the superhelix radius r, are observable only under conditions that enable surface re-arrangements. The measured values of writhe suggest that the scDNA loses approximately one-half of the supercoils during the binding to the surface. At the same time r increases systematically with decreasing writhe, thus the scDNA topology remains determined by the constraints on supercoiling during the binding to PL-coated mica
    Type of Publication: Journal article published
    PubMed ID: 14602930
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  • 2
    Keywords: IN-VITRO ; MICROSCOPY ; VITRO ; PROTEINS ; MICE ; IDENTIFICATION ; LENGTH ; MICA ; intermediate filaments ; INTERMEDIATE-FILAMENTS ; Jun ; vimentin ; LIVING CELLS ; ATOMIC-FORCE MICROSCOPY ; ARCHITECTURE ; electron microscopy ; intermediate filament ; ELECTRON-MICROSCOPY ; DESMIN ; SWITZERLAND ; SOLID SUPPORTS ; VIMENTIN FILAMENTS ; ULF ; MATURE ; AFM ; NEUROFILAMENTS ; adsorption ; glutaraldehyde fixation ; supramolecular architecture ; surface interaction
    Abstract: Morphologically, glutaraldehyde-fixed and -dried intermediate filaments (IFs) appear flexible, and with it width of 8 12 nm when observed by electron microscopy. Sometimes, the filaments are even unraveled on the carbon-coated grid and reveal a protofilamentous architecture. In this study, we have used atomic force microscopy to further investigate the morphology of IFs in a more physiological environment. First, we have imaged hydrated glutaraldehyde-fixed IFs adsorbed to a graphite support. in such conditions, human vimentin and desmin IFs appeared compact with a height or 5 8 nm and revealed either a beading repeat or a helical morphology. Second, we have analyzed the architecture of hydrated vimentin, desmin, and neurofilament IFs adsorbed to mica, graphite, and hydrophilic glass without the presence of fixative. On mica, vimentin IFs had it height of only 3 5 nm, whereas desmin IFs appeared as 8 10nm height filaments with a helical twist. Neurofilaments were 10 -12nm in height with a pronounced 30 50nm beading along their length. On graphite, the different IFs were either not adsorbing properly or their architecture was modified yielding, for example. broad. flattened filaments. Finally, hydrophilie glass Was the surface which seemed to best preserve the architecture of the three IFs, even if, in some cases, unraveled vimentin filaments were observed oil this support, These results are straightening the idea that mature IFs are dynamic polymers in vitro and that IFs can be distinguished front each others by their physicochemical properties. (c) 2005 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15890275
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  • 3
    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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  • 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: IN-VITRO ; Germany ; MICROSCOPY ; DISEASE ; PROTEIN ; PROTEINS ; DYNAMICS ; BIOLOGY ; fibroblasts ; DAMAGE ; LENGTH ; vimentin ; ATOMIC-FORCE MICROSCOPY ; SMOOTH-MUSCLE ; electron microscopy ; molecular biology ; DEPENDENCE ; DESMIN ; desmin and vimentin intermediate filament ; ELASTIC LIGHT-SCATTERING ; F-ACTIN SOLUTIONS ; MICRORHEOLOGY ; persistence length ; rheology ; strain stiffening ; THERMAL FLUCTUATIONS
    Abstract: We have investigated the viscoelastic properties of the cytoplasmic intermediate filament (IF) proteins desmin and vimentin. Mechanical measurements were supported by time-dependent electron microscopy studies of the assembly process under similar conditions. Network formation starts within 2 min, but it takes more than 30 min until equilibrium mechanical network strength is reached. Filament bundling is more pronounced for desmin than for vimentin. Desmin filaments (persistence length l(p) approximate to 900 nm) are stiffer than vimentin filaments (l(p) approximate to 400 nm), but both IFs are much more flexible than microfilaments. e concentration dependence of the plateau modulus G(0) similar to c(alpha) is much weaker than predicted theoretically for networks of semiflexible filaments. This is more pronounced for vimentin (alpha = 0.47) than for desmin (alpha = 0.70). Both networks exhibit strain stiffening at large shear deformations. At the transition from linear to nonlinear viscoelastic response, only desmin shows characteristics of nonaffine network deformation. Strain stiffening and the maximum modulus occur at strain amplitudes about an order of magnitude larger than those for microfilaments. This is probably attributable to axial slippage within the tetramer building blocks of the IFs. Network deformation beyond a critical strain gamma(max) results in irreversible damage. Strain stiffening sets in at lower concentrations, is more pronounced, and is less sensitive to ionic strength for desmin than for vimentin. Hence, desmin exhibits strain stiffening even at low-salt concentrations, which is not observed for vimentin, and we conclude that the strength of electrostatic repulsion compared to the strength of attractive interactions forming the network junctions is significantly weaker for desmin than for vimentin filaments. These findings indicate that both IFs exhibit distinct mechanical properties that are adapted to their respective cellular surroundings [i.e., myocytes (desmin) and fibroblasts (vimentin)]. (C) 2009 Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 19281820
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  • 6
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    Abstract: Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-alpha-helical carboxy-terminal domain ("tail"), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin "tail" mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length l(p). Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the "tail" mutants (l(p)=1.0-2.0 microm) and the wild-type desmin (l(p)=1.1+/-0.5 microm). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered. In order to explore the importance of the "tail" domain as such for filament network properties, we employed a "tail"-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the "tail" domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle.
    Type of Publication: Journal article published
    PubMed ID: 20171226
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  • 9
    Keywords: ACTIVATION ; RESPONSES ; INFECTION ; RECOGNITION ; DOUBLE-STRANDED-RNA ; BEHAVIOR ; HEPATITIS-C VIRUS ; ADAPTER PROTEIN ; 5'-TRIPHOSPHATE RNA ; INTERFERON INDUCTION ; MDA5
    Abstract: RIG-I is a major innate immune sensor for viral infection, triggering an interferon (IFN)-mediated antiviral response upon cytosolic detection of viral RNA. Double-strandedness and 5'-terminal triphosphates were identified as motifs required to elicit optimal immunological signaling. However, very little is known about the response dynamics of the RIG-I pathway, which is crucial for the ability of the cell to react to diverse classes of viral RNA while maintaining self-tolerance. In the present study, we addressed the molecular mechanism of RIG-I signal detection and its translation into pathway activation. By employing highly quantitative methods, we could establish the length of the double-stranded RNA(dsRNA) to be the most critical determinant of response strength. Size exclusion chromatography and direct visualization in scanning force microscopy suggested that this was due to cooperative oligomerization of RIG-I along dsRNA. The initiation efficiency of this oligomerization process critically depended on the presence of high affinity motifs, like a 5'-triphosphate. It is noteworthy that for dsRNA longer than 200 bp, internal initiation could effectively compensate for a lack of terminal triphosphates. In summary, our data demonstrate a very flexible response behavior of the RIG-I pathway, in which sensing and integration of at least two distinct signals, initiation efficiency and double strand length, allow the host cell to mount an antiviral response that is tightly adjusted to the type of the detected signal, such as viral genomes, replication intermediates, or small by-products
    Type of Publication: Journal article published
    PubMed ID: 21659521
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  • 10
    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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