Ultrastructural and Molecular Characterization of Platelet-derived growth factor Beta-Positive Leptomeningeal Cells in the Adult Rat Brain.

The leptomeninges, referring to the arachnoid and pia mater and their projections into the perivascular compartments in the central nervous system, actively participate in diverse biological processes including fluid homeostasis, immune cell infiltrations, and neurogenesis, yet their detailed cellular and molecular identities remain elusive. This study aimed to characterize platelet-derived growth factor beta (PDGFR-ß)-expressing cells in the leptomeninges in the adult rat brain using light and electron microscopy. PDGFR-ß+ cells were observed in the inner arachnoid, arachnoid trabeculae, pia mater, and leptomeningeal sheath of the subarachnoid vessels, thereby forming a cellular network throughout the leptomeninges. Leptomeningeal PDGFR-ß+ cells were commonly characterized by large euchromatic nuclei, thin branching processes forming web-like network, and the expression of the intermediate filaments nestin and vimentin. These cells were typical of active fibroblasts with a well-developed rough endoplasmic reticulum and close spatial correlation with collagen fibrils. Leptomeningeal PDGFR-ß+ cells ensheathing the vasculature in the subarachnoid space joined with pial PDGFR-ß+ cells upon entering the cortical parenchyma, yet perivascular PDGFR-ß+ cells in these penetrating vessels underwent abrupt changes in their morphological and molecular characteristics: they became more flattened with loss of immunoreactivity for nestin and vimentin and deficient collagen deposition, which was indicative of inactive fibroblasts termed fibrocytes. In the cortical parenchyma, PDGFR-ß immunoreactivity was almost exclusively localized to larger caliber vessels, and significantly decreased in capillary-like microvessels. Collectively, our data identify PDGFR-ß as a novel cellular marker for leptomeningeal fibroblasts comprising the leptomeninges and perivascular adventitial cells of the subarachnoid and penetrating large-sized cortical vasculatures.

In Vitro Assembly Kinetics of Cytoplasmic Intermediate Filaments: A Correlative Monte Carlo Simulation Study.

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 interact end-to-end with other ULFs to form short filaments, which further anneal with ULFs and with each other to increasingly longer filaments. This assembly concept was derived from time-lapse electron and atomic force microscopy data. We previously have quantitatively verified this concept through the generation of time-dependent filament length-profiles and an analytical model that describes assembly kinetics well for about the first ten minutes. In this time frame, filaments are shorter than one persistence length, i.e. ~1 µm, and thus filaments were treated as stiff rods associating via their ends. However, when filaments grow several µm in length over hours, their flexibility becomes a significant factor for the kinetics of the longitudinal annealing process. Incorporating now additional filament length distributions that we have recorded after extended assembly times by total internal reflection fluorescence microscopy (TIRFM), we developed a Monte Carlo simulation procedure that accurately describes the underlying assembly kinetics for large time scales.

Vimentin filament organization and stress sensing depend on its single cysteine residue and zinc binding.

The vimentin filament network plays a key role in cell architecture and signalling, as well as in epithelial-mesenchymal transition. Vimentin C328 is targeted by various oxidative modifications, but its role in vimentin organization is not known. Here we show that C328 is essential for vimentin network reorganization in response to oxidants and electrophiles, and is required for optimal vimentin performance in network expansion, lysosomal distribution and aggresome formation. C328 may fulfil these roles through interaction with zinc. In vitro, micromolar zinc protects vimentin from iodoacetamide modification and elicits vimentin polymerization into optically detectable structures; in cells, zinc closely associates with vimentin and its depletion causes reversible filament disassembly. Finally, zinc transport-deficient human fibroblasts show increased vimentin solubility and susceptibility to disruption, which are restored by zinc supplementation. These results unveil a critical role of C328 in vimentin organization and open new perspectives for the regulation of intermediate filaments by zinc.

Gelsolin is a potential cellular target for cotinine to regulate the migration and apoptosis of A549 and T24 cancer cells.

In the present work, we have investigated the effect of cotinine, the major metabolite of nicotine on the A549 and T24 cell lines in the context of structural and quantitative changes of F-actin, gelsolin and vimentin. The chosen cell lines constitute the established experimental models for lung and bladder cancers, respectively, in the case of which, smoking cigarettes is one of the key factor increasing their incidence rate significantly. In order to evaluate the impact of cotinine on the viability and proliferation of A549 and T24 cells, the MTT assay was performed. The organization and distribution of F-actin, gelsolin and vimentin were examined using conventional and confocal fluorescence microscopy. The levels of F-actin and gelsolin as well as the percentages of apoptotic and dead cells were assessed using the image-based cytometer. The ultrastructural changes of cotinine-treated A549 and T24 cells were visualized under the transmission electron microscopy. We have shown here that cotinine enhances the survival and proliferation rate of A549 and T24 cells. We have also found that in A549 cells, but not in T24 cell line, cotinine acted stimulating on the vimentin filament network. Furthermore, the increase in the fluorescence intensity of gelsolin upon the addition of cotinine to the T24 cells was found to be correlated with the lack of apoptosis induction as well as the increase of migration potential of these cells. On the other hand, the cotinine-induced decrease in the fluorescence intensity of gelsolin was associated with the increase in the percentages of apoptotic A549 cells and the decreased migratory ability of these cells. Based on the obtained results, we propose that the gelsolin is an important cellular target for cotinine, through which this compound influences on the basic processes involved in neoplastic transformation and metastasis, such as migration and apoptosis.

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.

Stabilization of vimentin coil2 fragment via an engineered disulfide.

Cytoskeletal intermediate filaments (IFs) assemble from the elementary dimers based on a segmented α-helical coiled-coil (CC) structure. Crystallographic studies of IF protein fragments remain the main route to access their atomic structure. To enable crystallization, such fragments must be sufficiently short. As a consequence, they often fail to assemble into the correct CC dimers. In particular, human vimentin fragment D3 corresponding to the first half of coil2 (residues 261-335) stays monomeric in solution. We have induced its dimerization via introducing a disulfide link between two cysteines engineered in the hydrophobic core of the CC close to its N-terminus. The 2.3 Å crystal structure of the D3st (stabilized) fragment reveals a mostly parallel α-helical bundle structure in its N-terminal half which smoothly continues into a left-handed CC towards the C-terminus. This provides a direct evidence for a continuously α-helical structure of the coil2 segment and disproves the previously suggested existence of linker L2 separating it into two left-handed CCs. The general principles of CC dimer stabilization by disulfide introduction are also discussed.

Inroads into the structure and function of intermediate filament networks.

Although intermediate filaments are one of three major cytoskeletal systems of vertebrate cells, they remain the least understood with respect to their structure and function. This is due in part to the fact that they are encoded by a large gene family which is developmentally regulated in a cell and tissue type specific fashion. This article is in honor of Ueli Aebi. It highlights the studies on IF that have been carried out by our laboratory for more than 40 years. Many of our advances in understanding IF are based on conversations with Ueli which have taken place during adventurous and sometimes dangerous hiking and biking trips throughout the world.

Colocalization of serum amyloid a with microtubules in human coronary artery endothelial cells.

Serum amyloid A (SAA) acts as a major acute phase protein and represents a sensitive and accurate marker of inflammation. Besides its hepatic origin, as the main source of serum SAA, this protein is also produced extrahepatically. The mRNA levels of SAA become significantly elevated following proinflammatory stimuli, as well as, are induced through their own positive feedback in human primary coronary artery endothelial cells. However, the intracellular functions of SAA are so far unknown. Colocalization of SAA with cytoskeletal filaments has previously been proposed, so we analyzed the colocalization of SAA with all three cytoskeletal elements: actin filaments, vimentin filaments, and microtubules. Immunofluorescent double-labeling analyses confirmed by PLA method revealed a strict colocalization of SAA with microtubules and a very infrequent attachment to vimentin while the distribution of actin filaments appeared clearly separated from SAA staining. Also, no significant colocalization was found between SAA and endomembranes labeled with the fluorescent lipid stain DiO6. However, SAA appears to be located also unbound in the cytosol, as well as inside the nucleus and within nanotubes extending from the cells or bridging neighboring cells. These different locations of SAA in endothelial cells strongly indicate multiple potential functions of this protein.

Vimentin and laminin are altered on cheek pouch microvessels of streptozotocin-induced diabetic hamsters.

OBJECTIVE: Normal endothelial cells respond to shear stress by elongating and aligning in the direction of fluid flow. Hyperglycemia impairs this response and contributes to microvascular complications, which result in deleterious effects to the endothelium. This work aimed to evaluate cheek pouch microvessel morphological characteristics, reactivity, permeability, and expression of cytoskeleton and extracellular matrix components in hamsters after the induction of diabetes with streptozotocin. METHODS: Syrian golden hamsters (90-130 g) were injected with streptozotocin (50 mg/kg, i.p.) or vehicle either 6 (the diabetes mellitus 6 group) or 15 (the diabetes mellitus 15 group) days before the experiment. Vascular dimensions and density per area of vessels were determined by morphometric and stereological measurements. Changes in blood flow were measured in response to acetylcholine, and plasma extravasation was measured by the number of leakage sites. Actin, talin, α-smooth muscle actin, vimentin, type IV collagen, and laminin were detected by immunohistochemistry and assessed through a semiquantitative scoring system. RESULTS: There were no major alterations in the lumen, wall diameters, or densities of the examined vessels. Likewise, vascular reactivity and permeability were not altered by diabetes. The arterioles demonstrated increased immunoreactivity to vimentin and laminin in the diabetes mellitus 6 and diabetes mellitus 15 groups. DISCUSSION: Antibodies against laminin and vimentin inhibit branching morphogenesis in vitro. Therefore, laminin and vimentin participating in the structure of the focal adhesion may play a role in angiogenesis. CONCLUSIONS: Our results indicated the existence of changes related to cell-matrix interactions, which may contribute to the pathological remodeling that was already underway one week after induction of experimental diabetes.

Vimentin and laminin are altered on cheek pouch microvessels of streptozotocin-induced diabetic hamsters

OBJECTIVE: Normal endothelial cells respond to shear stress by elongating and aligning in the direction of fluid flow. Hyperglycemia impairs this response and contributes to microvascular complications, which result in deleterious effects to the endothelium. This work aimed to evaluate cheek pouch microvessel morphological characteristics, reactivity, permeability, and expression of cytoskeleton and extracellular matrix components in hamsters after the induction of diabetes with streptozotocin. METHODS: Syrian golden hamsters (90-130 g) were injected with streptozotocin (50 mg/kg, i.p.) or vehicle either 6 (the diabetes mellitus 6 group) or 15 (the diabetes mellitus 15 group) days before the experiment. Vascular dimensions and density per area of vessels were determined by morphometric and stereological measurements. Changes in blood flow were measured in response to acetylcholine, and plasma extravasation was measured by the number of leakage sites. Actin, talin, α-smooth muscle actin, vimentin, type IV collagen, and laminin were detected by immunohistochemistry and assessed through a semiquantitative scoring system. RESULTS: There were no major alterations in the lumen, wall diameters, or densities of the examined vessels. Likewise, vascular reactivity and permeability were not altered by diabetes. The arterioles demonstrated increased immunoreactivity to vimentin and laminin in the diabetes mellitus 6 and diabetes mellitus 15 groups. DISCUSSION: Antibodies against laminin and vimentin inhibit branching morphogenesis in vitro. Therefore, laminin and vimentin participating in the structure of the focal adhesion may play a role in angiogenesis. CONCLUSIONS: Our results indicated the existence of changes related to cell-matrix interactions, which may contribute to the pathological remodeling that was already underway one week after induction of experimental diabetes.

Vimentin intermediate filaments: the central base in sinus endothelial cells of the rat spleen.

The ultrastructural distribution of vimentin intermediate filaments (IFs) and localizations of the related proteins in sinus endothelial cells of the rat spleen was examined by confocal laser scanning and electron microscopy with detergent extraction, myosin-fragment 1 decoration, and immunogold labeling to elucidate their functions in endothelial cells. Vimentin IFs were extremely abundant over stress fibers in the basal part of the endothelial cells. Some of them were intermingled with actin filaments in stress fibers, and were associated with coated vesicles. Plectin was predominantly localized in the layers of vimentin and stress fibers of the endothelial cells, but rarely in the vicinity of adherens junctions in the lateral part and focal adhesions in the basal part of the cells. Neither plakoglobin nor desmoplakin, which is coupled VE-cadherin to vimentin IFs, was detected in sinus endothelial cells. Vinculin was localized in the basal membranes of the endothelial cells. These data suggest that abundant vimentin IFs are associated with stress fibers by plectin in the basal part of the cells and form cytoskeletal cores of sinus endothelial cells only partially supported by the ring-shaped basal lamina to have roles in scaffolding and the mechanical stabilization of the endothelial cells. Furthermore, taken in connection with recently revealed functions of vimentin and plectin, vimentin might play a cytoskeletal core of sinus endothelial cells.

Three-dimensional cryo-electron microscopy on intermediate filaments.

Together with microtubules and actin filaments (F-actin), intermediate filaments (IFs) form the cytoskeleton of metazoan cells. However, unlike the other two entities that are extremely conserved, IFs are much more diverse and are grouped into five different families. In contrast to microtubules and F-actin, IFs do not exhibit a polarity, which may be the reason that no molecular motors travel along them. The molecular structure of IFs is less well resolved than that of the other cytoskeletal systems. This is partially due to their functional variability, tissue-specific expression, and their intrinsic structural properties. IFs are composed mostly of relatively smooth protofibrils formed by antiparallel arranged α-helical coiled-coil bundles flanked by small globular domains at either end. These features make them difficult to study by various electron microscopy methods or atomic force microscopy (AFM). Furthermore, the elongated shape of monomeric or dimeric IF units interferes with the formation of highly ordered three-dimensional (3-D) crystals suitable for atomic resolution crystallography. So far, most of the data we currently have on IF macromolecular structures come from electron microscopy of negatively stained samples, and fragmented α-helical coiled-coil units solved by X-ray diffraction. In addition, AFM allows the observation of the dynamic states of IFs in solution and delivers a new view into the assembly properties of IFs. Here, we discuss the applicability of cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) for the field. Both methods are strongly related and have only recently been applied to IFs. However, cryo-EM revealed distinct new features within IFs that have not been seen before, and cryo-ET adds a 3-D view of IFs revealing the path and number of protofilaments within the various IF assemblies.

Divalent cations crosslink vimentin intermediate filament tail domains to regulate network mechanics.

Intermediate filament networks in the cytoplasm and nucleus are critical for the mechanical integrity of metazoan cells. However, the mechanism of crosslinking in these networks and the origins of their mechanical properties are not understood. Here, we study the elastic behavior of in vitro networks of the intermediate filament protein vimentin. Rheological experiments reveal that vimentin networks stiffen with increasing concentrations of Ca(2+) and Mg(2+), showing that divalent cations act as crosslinkers. We quantitatively describe the elastic response of vimentin networks over five decades of applied stress using a theory that treats the divalent cations as crosslinkers: at low stress, the behavior is entropic in origin, and increasing stress pulls out thermal fluctuations from single filaments, giving rise to a nonlinear response; at high stress, enthalpic stretching of individual filaments significantly modifies the nonlinearity. We investigate the elastic properties of networks formed by a series of protein variants with stepwise tail truncations and find that the last 11 amino acids of the C-terminal tail domain mediate crosslinking by divalent ions. We determined the single-filament persistence length, l(P) approximately 0.5 mum, and Young's modulus, Y approximately 9 MPa; both are consistent with literature values. Our results provide insight into a crosslinking mechanism for vimentin networks and suggest that divalent ions may help regulate the cytoskeletal structure and mechanical properties of cells.

Di(n-butyl) phthalate induces vimentin filaments disruption in rat sertoli cells: a possible relation with spermatogenic cell apoptosis.

Phthalate esters have been extensively used as a plasticizer of synthetic polymers. Previous studies have revealed that some phthalate esters including di(n-butyl) phthalate (DBP) induce spermatogenic cell apoptosis, although its mechanism is not yet clear. The present study describes that disruption of Sertoli cell vimentin filaments by DBP administration may relate to spermatogenic cell apoptosis. The present histopathological study revealed that a single oral administration of 500 mg/kg DBP caused progressive detachment and displacement of spermatogenic cells away from the seminiferous epithelium and sloughing of them into the lumen. Degenerative spermatogenic cells characterized by chromatin condensation were frequently observed in DBP-treated rats. Ultrastructurally, the degenerative spermatogenic cells were separated from their neighbours, and a collapse of Sertoli cell vimentin filaments was recognized in DBP-treated rats. Sertoli cell cultures showed the increased number and size of vacuoles in their cytoplasm. In agreement with the in vivo experiment, vimentin filaments clearly showed a gradual collapse in DBP-exposed Sertoli cells in vitro. These in vivo and in vitro experiments indicate that DBP-induced collapse of Sertoli cell vimentin filaments may lead to detachment of spermatogenic cells, and then detached cells may undergo apoptosis because of loss of the support and nurture provided by Sertoli cells.

Atomic structure of vimentin coil 2.

Intermediate filaments (IFs) are essential cytoskeletal components in metazoan cells. They assemble from elementary dimers that are built around the central alpha-helical coiled-coil rod domain representing the IF 'signature'. The rod consists of two similarly-sized parts, coil 1 and coil 2, connected by a non-alpha-helical linker L12. Coil 2 is absolutely conserved in length across all IF types and was initially predicted to consist of a short coiled-coil segment 2A based on a heptad pattern of hydrophobic residues, another linker L2 and a coiled-coil segment 2B. Here we present the crystal structure of human vimentin fragment including residues 261-335 i.e. approximately the first half of coil 2. The N-terminal part of this fragment reveals a parallel alpha-helical bundle characterized by 3.5 consecutive hendecad repeats. It is immediately followed by a regular left-handed coiled coil. The distinct non-helical linker L2 is therefore not observed. Together with the previously determined crystal structure of the major part of segment 2B (Strelkov et al., 2002), we can now build a complete atomic model of the 21nm long vimentin coil 2 dimer being a relatively rigid rod.

Interference of amino-terminal desmin fragments with desmin filament formation.

Short polypeptides from intermediate filament (IF) proteins containing one of the two IF-consensus motifs interfere severely with filament assembly in vitro. We now have systematically investigated a series of larger fragments of the muscle-specific IF protein desmin representing entire functional domains such as coil1 or coil 2. "Half molecules" comprising the amino-terminal portion of desmin, such as DesDeltaC240 and the "tagged" derivative Des(ESA)DeltaC244, assembled into large, roundish aggregates already at low ionic strength, DesDeltaC250 formed extended, relatively uniform filaments, whereas DesDeltaC265 and DesDeltaC300 were soluble under these conditions. Surprisingly, all mutant desmin fragments assembled very rapidly into long thick filaments or spacious aggregates when the ionic strength was raised to standard assembly conditions. In contrast, when these desmin mutants were assembled in the presence of wild-type (WT) desmin, their assembly properties were completely changed: The elongation of the two shorter desmin fragments was completely inhibited by WT desmin, whereas DesDeltaC250, DesDeltaC265 and DesDeltaC300 coassembled with desmin into filaments, but these mixed filaments were distinctly disturbed and exhibited a very different phenotype for each mutant. After transfection into fibroblasts and cardiomyocytes, the truncated mutant Des (ESA)DeltaC244 localized largely to the cytoplasm, as revealed by a tag-specific monoclonal antibody, and also partially colocalized there with the collapsed endogenous vimentin and desmin systems indicating its interference with IF-organizing processes. In contrast, in cells without an authentic cytoplasmic IF system such as line SW13, Des(ESA)DeltaC242 entered the nucleus and was deposited in small dot-like structures in chromatin-free spaces without any noticeable effect on nuclear morphology.

Desmin and vimentin intermediate filament networks: their viscoelastic properties investigated by mechanical rheometry.

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) approximately 900 nm) are stiffer than vimentin filaments (l(p) approximately 400 nm), but both IFs are much more flexible than microfilaments. The concentration dependence of the plateau modulus G(0) approximately 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)].

Towards a molecular description of intermediate filament structure and assembly.

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.

Structural analysis of vimentin and keratin intermediate filaments by cryo-electron tomography.

Intermediate filaments are a large and structurally diverse group of cellular filaments that are classified into five different groups. They are referred to as intermediate filaments (IFs) because they are intermediate in diameter between the two other cytoskeletal filament systems that is filamentous actin and microtubules. The basic building block of IFs is a predominantly alpha-helical rod with variable length globular N- and C-terminal domains. On the ultra-structural level there are two major differences between IFs and microtubules or actin filaments: IFs are non-polar, and they do not exhibit large globular domains. IF molecules associate via a coiled-coil interaction into dimers and higher oligomers. Structural investigations into the molecular building plan of IFs have been performed with a variety of biophysical and imaging methods such as negative staining and metal-shadowing electron microscopy (EM), mass determination by scanning transmission EM, X-ray crystallography on fragments of the IF stalk and low-angle X-ray scattering. The actual packing of IF dimers into a long filament varies between the different families. Typically the dimers form so called protofibrils that further assemble into a filament. Here we introduce new cryo-imaging methods for structural investigations of IFs in vitro and in vivo, i.e., cryo-electron microscopy and cryo-electron tomography, as well as associated techniques such as the preparation and handling of vitrified sections of cellular specimens.

Dissecting the 3-D structure of vimentin intermediate filaments by cryo-electron tomography.

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.