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Berlin Brandenburg

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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 18 December 2012, Vol.109(51), pp.20895-900
    Description: Cytoplasmic dynein is the major motor protein responsible for microtubule minus-end-directed movements in most eukaryotic cells. It transports a variety of cargoes and has numerous functions during spindle assembly and chromosome segregation. It is a large complex of about 1.4 MDa composed of six different subunits, interacting with a multitude of different partners. Most biochemical studies have been performed either with the native mammalian cytoplasmic dynein complex purified from tissue or, more recently, with recombinant dynein fragments from budding yeast and Dictyostelium. Hardly any information exists about the properties of human dynein. Moreover, experiments with an entire human dynein complex prepared from recombinant subunits with a well-defined composition are lacking. Here, we reconstitute a complete cytoplasmic dynein complex using recombinant human subunits and characterize its biochemical and motile properties. Using analytical gel filtration, sedimentation-velocity ultracentrifugation, and negative-stain electron microscopy, we demonstrate that the smaller subunits of the complex have an important structural function for complex integrity. Fluorescence microscopy experiments reveal that while engaged in collective microtubule transport, the recombinant human cytoplasmic dynein complex is an active, microtubule minus-end-directed motor, as expected. However, in contrast to recombinant dynein of nonmetazoans, individual reconstituted human dynein complexes did not show robust processive motility, suggesting a more intricate mechanism of processivity regulation for the human dynein complex. In the future, the comparison of reconstituted dynein complexes from different species promises to provide molecular insight into the mechanisms regulating the various functions of these large molecular machines.
    Keywords: Cytoplasmic Dyneins -- Chemistry
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    In: PLoS ONE, 2014, Vol.9(4)
    Description: Mechanical and structural properties of K8/K18 and vimentin intermediate filament (IF) networks have been investigated using bulk mechanical rheometry and optical microrheology including diffusing wave spectroscopy and multiple particle tracking. A high elastic modulus G 0 at low protein concentration c , a weak concentration dependency of G 0 ( G 0 ∼ c 0.5±0.1 ) and pronounced strain stiffening are found for these systems even without external crossbridgers. Strong attractive interactions among filaments are required to maintain these characteristic mechanical features, which have also been reported for various other IF networks. Filament assembly, the persistence length of the filaments and the network mesh size remain essentially unaffected when a nonionic surfactant is added, but strain stiffening is completely suppressed, G 0 drops by orders of magnitude and exhibits a scaling G 0 ∼ c 1.9±0.2 in agreement with microrheological measurements and as expected for entangled networks of semi-flexible polymers. Tailless K8Δ/K18ΔT and various other tailless filament networks do not exhibit strain stiffening, but still show high G 0 values. Therefore, two binding sites are proposed to exist in IF networks. A weaker one mediated by hydrophobic amino acid clusters in the central rod prevents stretched filaments between adjacent cross-links from thermal equilibration and thus provides the high G 0 values. Another strong one facilitating strain stiffening is located in the tail domain with its high fraction of hydrophobic amino acid sequences. Strain stiffening is less pronounced for vimentin than for K8/K18 due to electrostatic repulsion forces partly compensating the strong attraction at filament contact points.
    Keywords: Research Article ; Biology And Life Sciences ; Physical Sciences
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: PLoS ONE, 2011, Vol.6(4), p.e19202
    Description: Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several µm long and therefore they exceed the field of view of many electron (EM) or atomic force microscopy (AFM) techniques. Thereby quantitative studies become extremely laborious and time-consuming. To overcome these difficulties, we prepared fluorescently labeled vimentin for visualization by total internal reflection fluorescence microscopy (TIRFM). In order to investigate if the labeling influences the assembly properties of the protein, we first determined the association state of unlabeled vimentin mixed with increasing amounts of labeled vimentin under low ionic conditions by analytical ultracentrifugation. We found that bona fide tetrameric complexes were formed even when half of the vimentin was labeled. Moreover, we demonstrate by quantitative atomic force microscopy and electron microscopy that the morphology and the assembly properties of filaments were not affected when the fraction of labeled vimentin was below 10%. Using fast frame rates we observed the rapid deposition of fluorescently labeled IFs on glass supports by TIRFM in real time. By tracing their contours, we have calculated the persistence length of long immobilized vimentin IFs to 1 µm, a value that is identical to those determined for shorter unlabeled vimentin. These results indicate that the structural properties of the filaments were not affected significantly by the dye. Furthermore, in order to analyze the late elongation phase, we mixed long filaments containing either Alexa 488- or Alexa 647-labeled vimentin. The ‘patchy’ structure of the filaments obtained unambiguously showed the elongation of long IFs through direct end-to-end annealing of individual filaments.
    Keywords: Research Article ; Biology ; Chemistry ; Physics ; Chemistry ; Biophysics ; Physics ; Biochemistry
    E-ISSN: 1932-6203
    Source: PLoS
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  • 4
    Language: English
    In: The Journal of biological chemistry, 25 November 2016, Vol.291(48), pp.24931-24950
    Description: Intermediate filaments (IF) are major constituents of the cytoskeleton of metazoan cells. They are not only responsible for the mechanical properties but also for various physiological activities in different cells and tissues. The building blocks of IFs are extended coiled-coil-forming proteins exhibiting a characteristic central α-helical domain ("rod"). The fundamental principles of the filament assembly mechanism and the network formation have been widely elucidated for the cytoplasmic IF protein vimentin. Also, a comprehensive structural model for the tetrameric complex of vimentin has been obtained by X-ray crystallography in combination with various biochemical and biophysical techniques. To extend these static data and to investigate the dynamic properties of the full-length proteins in solution during the various assembly steps, we analyzed the patterns of hydrogen-deuterium exchange in vimentin and in four variants carrying point mutations in the IF consensus motifs present at either end of the α-helical rod that cause an assembly arrest at the unit-length filament (ULF) stage. The results yielded unique insights into the structural properties of subdomains within the full-length vimentin, in particular in regions of contact in α-helical and linker segments that stabilize different oligomeric forms such as tetramers, ULFs, and mature filaments. Moreover, hydrogen-deuterium exchange analysis of the point-mutated variants directly demonstrated the active role of the IF consensus motifs in the oligomerization mechanism of tetramers during ULF formation. Ultimately, using molecular dynamics simulation procedures, we provide a structural model for the subdomain-mediated tetramer/tetramer interaction via "cross-coiling" as the first step of the assembly process.
    Keywords: Hydrogen-Deuterium Exchange ; Intermediate Filament ; Mass Spectrometry (MS) ; Protein Assembly ; Vimentin ; Molecular Dynamics Simulation ; Protein Multimerization ; Vimentin -- Chemistry
    E-ISSN: 1083-351X
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  • 5
    Language: English
    In: PLoS ONE, 01 January 2016, Vol.11(6), p.e0157451
    Description: Intermediate filament (IF) elongation proceeds via full-width "mini-filaments", referred to as "unit-length" filaments (ULFs), which instantaneously form by lateral association of extended coiled-coil complexes after assembly is initiated. In a comparatively much slower process, ULFs longitudinally...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 6
    Language: English
    In: Biophysical Journal, 22 May 2018, Vol.114(10), pp.2408-2418
    Description: Intermediate filaments (IFs) are principal components of the cytoskeleton, a dynamic integrated system of structural proteins that provides the functional architecture of metazoan cells. They are major contributors to the elasticity of cells and tissues due to their high mechanical stability and intrinsic flexibility. The basic building block for the assembly of IFs is a rod-like, 60-nm-long tetrameric complex made from two antiparallel, half-staggered coiled coils. In low ionic strength, tetramers form stable complexes that rapidly assemble into filaments upon raising the ionic strength. The first assembly products, “frozen” by instantaneous chemical fixation and viewed by electron microscopy, are 60-nm-long “unit-length” filaments (ULFs) that apparently form by lateral in-register association of tetramers. ULFs are the active elements of IF growth, undergoing longitudinal end-to-end annealing with one another and with growing filaments. Originally, we have employed quantitative time-lapse atomic force and electron microscopy to analyze the kinetics of vimentin-filament assembly starting from a few seconds to several hours. To obtain detailed quantitative insight into the productive reactions that drive ULF formation, we now introduce a “stopped-flow” approach in combination with static light-scattering measurements. Thereby, we determine the basic rate constants for lateral assembly of tetramers to ULFs. Processing of the recorded data by a global fitting procedure enables us to describe the hierarchical steps of IF formation. Specifically, we propose that tetramers are consumed within milliseconds to yield octamers that are obligatory intermediates toward ULF formation. Although the interaction of tetramers is diffusion controlled, it is strongly driven by their geometry to mediate effective subunit targeting. Importantly, our model conclusively reflects the previously described occurrence of polymorphic ULF and mature filaments in terms of their number of tetramers per cross section.
    Keywords: Biology
    ISSN: 0006-3495
    E-ISSN: 1542-0086
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  • 7
    Language: English
    In: PLoS ONE, 01 January 2011, Vol.6(4), p.e19202
    Description: Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several µm long and therefore they exceed the field of view of many electron (EM) or atomic...
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 8
    Language: English
    In: Journal of Molecular Biology, 16 April 2010, Vol.397(5), pp.1188-1198
    Description: 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-α-helical carboxy-terminal domain (“tail”), have been demonstrated to form apparently normal filaments both 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 . Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant R454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the “tail” mutants ( = 1.0–2.0 μm) and the wild-type desmin ( = 1.1 ± 0.5 μm). 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.
    Keywords: Modifications in Desmin Filament and Network Mechanics Due to Mutations ; Electron Microscopy ; Rheology ; Persistence Length ; Strain Stiffening ; Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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  • 9
    Language: English
    In: The Journal of cell biology, 02 February 2015, Vol.208(3), pp.283-97
    Description: Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82-Nup159-Nsp1-Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery.
    Keywords: Nuclear Pore -- Ultrastructure ; Nuclear Pore Complex Proteins -- Ultrastructure ; Saccharomyces Cerevisiae Proteins -- Ultrastructure
    ISSN: 00219525
    E-ISSN: 1540-8140
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  • 10
    Language: English
    In: Journal of Molecular Biology, 26 February 2010, Vol.396(3), pp.719-731
    Description: Half-minilamins, representing amino- and carboxy-terminal fragments of human lamins A, B1 and B2 with a truncated central rod domain, were investigated for their ability to form distinct head-to-tail-type dimer complexes. This mode of interaction represents an essential step in the longitudinal assembly reaction exhibited by full-length lamin dimers. As determined by analytical ultracentrifugation, the amino-terminal fragments were soluble under low ionic strength conditions sedimenting with distinct profiles and -values (1.6–1.8 S) indicating the formation of coiled-coil dimers. The smaller carboxy-terminal fragments were, except for lamin B2, largely insoluble under these conditions. However, after equimolar amounts of homotypic amino- and carboxy-terminal lamin fragments had been mixed in 4 M urea, upon subsequent renaturation the carboxy-terminal fragments were completely rescued from precipitation and distinct soluble complexes with higher -values (2.3–2.7 S) were obtained. From this behavior, we conclude that the amino- and carboxy-terminal coiled-coil dimers interact to form distinct oligomers (i.e. tetramers). Furthermore, a corresponding interaction occurred also between heterotypic pairs of A- and B-type lamin fragments. Hence, A-type lamin dimers may interact with B-type lamin dimers head-to-tail to yield linear polymers. These findings indicate that a lamin dimer principally has the freedom for a “combinatorial” head-to-tail association with all types of lamins, a property that might be of significant importance for the assembly of the nuclear lamina. Furthermore, we suggest that the head-to-tail interaction of the rod end domains represents a principal step in the assembly of cytoplasmic intermediate filament proteins too.
    Keywords: Analytical Ultracentrifugation ; Assembly ; Coiled Coil ; Glycerol Spraying/Rotary Metal Shadowing Electron Microscopy ; Intermediate Filaments ; Biology ; Chemistry
    ISSN: 0022-2836
    E-ISSN: 1089-8638
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