24 research outputs found
Vimentin Sâglutathionylation at Cys328 inhibits filament elongation and induces severing of mature filaments in vitro
Vimentin intermediate filaments are a significant component of the
cytoskeleton in cells of mesenchymal origin. In vivo, filaments assemble and
disassemble and thus participate in the dynamic processes of the cell. Posttranslational
modifications (PTMs) such as protein phosphorylation regulate
the multiphasic association of vimentin from soluble complexes to insoluble
filaments and the reverse processes. The thiol side chain of the single vimentin
cysteine at position 328 (Cys328) is a direct target of oxidative modifications
inside cells. Here, we used atomic force microscopy, electron
microscopy and a novel hydrogenâdeuterium exchange mass spectrometry
(HDex-MS) procedure to investigate the structural consequences of S-nitrosylation
and S-glutathionylation of Cys328 for in vitro oligomerisation of
human vimentin. Neither modification affects the lateral association of tetramers
to unit-length filaments (ULF). However, S-glutathionylation of
Cys328 blocks the longitudinal assembly of ULF into extended filaments. Snitrosylation
of Cys328 does not hinder but slows down the elongation.
Likewise, S-glutathionylation of preformed vimentin filaments causes their
extensive fragmentation to smaller oligomeric species. Chemical reduction of
the S-glutathionylated Cys328 thiols induces reassembly of the small fragments
into extended filaments. In conclusion, our in vitro results suggest Sglutathionylation
as a candidate PTM for an efficient molecular switch in
the dynamic rearrangements of vimentin intermediate filaments, observed
in vivo, in response to changes in cellular redox status. Finally, we demonstrate
that HDex-MS is a powerful method for probing the kinetics of
vimentin filament formation and filament disassembly induced by PTMs
Deconstructing the Late Phase of Vimentin Assembly by Total Internal Reflection Fluorescence Microscopy (TIRFM)
Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several Âľm 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 Âľm, 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 âpatchyâ structure of the filaments obtained unambiguously showed the elongation of long IFs through direct end-to-end annealing of individual filaments
Withaferin A Alters Intermediate Filament Organization, Cell Shape and Behavior
Withaferin A (WFA) is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF) into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects
Impact of ion valency on the assembly of vimentin studied by quantitative small angle X-ray scattering
The assembly kinetics of intermediate filament (IF) proteins from tetrameric complexes to single filaments and networks depends on the protein concentration, temperature and the ionic composition of their environment. We systematically investigate how changes in the concentration of monovalent potassium and divalent magnesium ions affect the internal organization of the resulting filaments. Small angle X-ray scattering (SAXS) is very sensitive to changes in the filament cross-section such as diameter or compactness. Our measurements reveal that filaments formed in the presence of magnesium chloride differ distinctly from filaments formed in the presence of potassium chloride. The principle multi-step assembly mechanism from tetramers via unit-length filaments (ULF) to elongated filaments is not changed by the valency of ions. However, the observed differences indicate that the magnesium ions free the head domains of tetramers from unproductive interactions to allow assembly but at the same time mediate strong inter-tetrameric interactions that impede longitudinal annealing of unit-length filaments considerably, thus slowing down filament growth
Dominant cataract formation in association with a vimentin assembly disrupting mutation
Cataracts are characterized by an opacification of the eye lens, often caused by protein misfolding and aggregation. The intermediate filament protein vimentin, which is highly expressed in lens fiber cells and in mesenchymal tissues, is a main structural determinant in these cells forming a membrane-connected cytoskeleton. Additional functions of vimentin remain to be identified. Here, we demonstrate that a mutation in VIM causes a dominant, pulverulent cataract. We sequenced the complete human VIM gene in 90 individuals suffering from congenital cataract and found a G596A change in exon 1 in a single individual, causing the missense mutation E151K in coil 1B of vimentin. The mutant vimentin formed an aberrant vimentin cytoskeleton and increased the proteasome activity in transfected cells. Furthermore, this mutation causes a severe kinetic defect in vimentin assembly both in vitro and in vivo. Hence, in conjunction with available mouse and cell culture models, our results reveal for the first time an important functional role for vimentin in the maintenance of lens integrity. Finally, this invites novel therapy approaches for cataracts.We acknowledge partial support by the Deutsche Forschungsgemeinschaft (grant numbers Ma 1316/7 and He 1853/5-1) and by the Bonner Forum Biomedizin to T.M.M. M.M. is supported by a fellowship from DFG (GRK804
WFA has no effect on the <i>in vitro</i> assembly of human recombinant vimentin.
<p>(A) Recombinant human vimentin (0.2 mg/ml) was assembled for 10 min at 37°C in (i) 50 mM NaCl; (ii) with 0.25% DMSO; (iii) with 50 ΟM WFA; and (iv) for 30 min with 50 ΟM WFA at a protein concentration of 0.5 mg/ml. The filaments were fixed with glutaraldehyde and visualized by negative stain electron microscopy. The arrows in (ii) indicate lateral annealing and apparent fusion of individual filaments. (scale bars, 0.2 Οm). (B) Viscometric analysis of vimentin assembly in the absence (ctrl) and presence of 50 ΟM WFA at 37°C in 50 mM NaCl. (C) Centrifugation assay of vimentin assembled in the absence (c) and presence of WFA (w). VIF were assembled for the indicated times (5 to 15 min) in 160 mM NaCl then centrifuged for 5 min at 10 psi in an Airfuge. Samples were separated by SDS-PAGE and stained with Coomassie. The position of vimentin is indicated (55 kDa).</p
Keratin IF are less sensitive to WFA than VIF.
<p>Human lung cancer cells, A549, were treated for 3 hrs with DMSO [ctrl] (A), 4.0 ÎźM WFA (B), and 6.0 ÎźM WFA (C), followed by staining with vimentin (Aâ˛, Bâ˛, Câ˛) and pan-cytokeratin antibodies (Aâ˛â˛, Bâ˛â˛, Câ˛â˛). Scale bars =10 Îźm.</p
WFA treatment inhibits cell motility.
<p>(A) The average speed of BJ-5ta fibroblasts was calculated before treatment (white bar), during incubation with 2 ÎźM WFA for 4 hrs (black bars) and after the cells were allowed to recover in fresh medium (gray bars). (B and C) Cells were treated with 2 ÎźM WFA for 3 hrs and then placed into fresh medium followed by fixation and processing for immunofluorescence with vimentin antibodies after 6h rs (B) and 9 hrs (C). Scale bars =10 Îźm.</p
WFA treatment induces an increase in the phosphorylation of vimentin serine-38.
<p>BJ-5ta cells were treated for 3 hrs with DMSO (A) or 2 ÎźM (B) WFA, then fixed and double labeled with vimentin (AⲠand Bâ˛) and pSer38 vimentin (Aâ˛â˛ and Bâ˛â˛) antibodies. Scale bars =10 Îźm. Arrow: a region depicted at higher magnification in the inset showing vimentin particles stained with pSer38 vimentin antibody. (C) Whole cell lysates of cells treated with DMSO (ctrl) or 2 ÎźM WFA for 60 min, 120 min, and 180 min, were separated by SDS-PAGE and stained with anti-vimentin and anti-vimentin pSer38 vimentin antibodies.</p