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      Deconstructing the Late Phase of Vimentin Assembly by Total Internal Reflection Fluorescence Microscopy (TIRFM)

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          Abstract

          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.

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          Most cited references30

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          Intermediate filaments: from cell architecture to nanomechanics.

          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.
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            Scanning force microscopy of DNA deposited onto mica: equilibration versus kinetic trapping studied by statistical polymer chain analysis.

            This paper reports a study of the deposition process of DNA molecules onto a mica surface for imaging under the scanning force microscope (SFM). Kinetic experiments indicate that the transport of DNA molecules from the solution drop onto the surface is governed solely by diffusion, and that the molecules are irreversibly adsorbed onto the substrate. A statistical polymer chain analysis has been applied to DNA molecules to determine the deposition conditions that lead to equilibrium and those that result in trapped configurations. Using the appropriate conditions, DNA molecules deposited onto freshly cleaved mica, are able to equilibrate on the surface as in an ideal two-dimensional solution. A persistence length of 53 nm was determined from those molecules. DNA fragments that were labeled on both ends with a horseradish peroxidase streptavidin fusion protein were still able to equilibrate on the surface, despite the additional protein-surface interaction. In contrast, DNA molecules deposited onto glow-discharged mica or H+-exchanged mica do not equilibrate on the surface. These molecules adopt conformations similar to those expected for a simple projection onto the surface plane, suggesting a process of kinetic trapping. These results validate recent SFM application to quantitatively analyze the conformation of complex macromolecular assemblies deposited on mica. Under equilibration conditions, the present study indicates that the SFM can be used to determine the persistence length of DNA molecules to a high degree of precision.
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              Viscoelastic properties of vimentin compared with other filamentous biopolymer networks

              The cytoplasm of vertebrate cells contains three distinct filamentous biopolymers, the microtubules, microfilaments, and intermediate filaments. The basic structural elements of these three filaments are linear polymers of the proteins tubulin, actin, and vimentin or another related intermediate filament protein, respectively. The viscoelastic properties of cytoplasmic filaments are likely to be relevant to their biologic function, because their extreme length and rodlike structure dominate the rheologic behavior of cytoplasm, and changes in their structure may cause gel-sol transitions observed when cells are activated or begin to move. This paper describes parallel measurements of the viscoelasticity of tubulin, actin, and vimentin polymers. The rheologic differences among the three types of cytoplasmic polymers suggest possible specialized roles for the different classes of filaments in vivo. Actin forms networks of highest rigidity that fluidize at high strains, consistent with a role in cell motility in which stable protrusions can deform rapidly in response to controlled filament rupture. Vimentin networks, which have not previously been studied by rheologic methods, exhibit some unusual viscoelastic properties not shared by actin or tubulin. They are less rigid (have lower shear moduli) at low strain but harden at high strains and resist breakage, suggesting they maintain cell integrity. The differences between F-actin and vimentin are optimal for the formation of a composite material with a range of properties that cannot be achieved by either polymer alone. Microtubules are unlikely to contribute significantly to interphase cell rheology alone, but may help stabilize the other networks.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2011
                22 April 2011
                : 6
                : 4
                : e19202
                Affiliations
                [1 ]Division Biophysics of Macromolecules, German Cancer Research Center, Heidelberg, Germany
                [2 ]Functional Architecture of the Cell, German Cancer Research Center, Heidelberg, Germany
                Dalhousie University, Canada
                Author notes

                Conceived and designed the experiments: SW HH NM. Performed the experiments: SW ARH MS ES TW. Analyzed the data: SW NM. Contributed reagents/materials/analysis tools: SW TW HH JL NM. Wrote the paper: SW ARH HH JL NM.

                [¤]

                Current address: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States of America

                Article
                PONE-D-10-06632
                10.1371/journal.pone.0019202
                3081349
                21544245
                d585bc76-a03c-4c4e-98d3-6c7fc3499fe2
                Winheim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 9 December 2010
                : 23 March 2011
                Page count
                Pages: 7
                Categories
                Research Article
                Biology
                Biochemistry
                Biophysics
                Molecular Cell Biology
                Chemistry
                Chemical Physics
                Physical Chemistry
                Physics
                Biophysics
                Chemical Physics

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                Uncategorized

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