Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.     

Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function

The tight association of cytoplasmic intermediate filaments (cIFs) with the nucleus and the isolation of crosslinkage products of vimentin with genomic DNA fragments, including nuclear matrix attachment regions (MARs) from proliferating fibroblasts, point to a participation of cIFs in nuclear activities. To test the possibility that cIFs are complementary nuclear matrix elements, the nuclei of a series of cultured cells were subjected to the Li-diiodosalicylate (LIS) extraction protocol developed for the preparation of nuclear matrices and analyzed by immunofluorescence microscopy and immunoblotting with antibodies directed against lamin B and cIF proteins. When nuclei released from hypotonically swollen L929 suspension cells in the presence of digitonin or Triton X-100 were exposed to such strong shearing forces that a considerable number were totally disrupted, a thin, discontinuous layer of vimentin IFs remained tenaciously adhering to still intact nuclei, in apparent coalignment with the nuclear lamina. Even in broken nuclei, the distribution of vimentin followed that of lamin B in areas where the lamina still appeared intact. The same retention of vimentin together with desmin and glial IFs was observed on the nuclei isolated from differentiating C2C12 myoblast and U333 glioma cells, respectively. Nuclei from epithelial cells shed their residual perinuclear IF layers as coherent cytoskeletal ghosts, except for small fractions of vimentin and cytokeratin IFs, which remained in a dot-to cap-like arrangement on the nuclear surface, in apparent codistribution with lamin B. LIS extraction did not bring about a reduction in the cIF protein contents of such nuclei upon their transformation into nuclear matrices. Moreover, in whole mount preparations of mouse embryo fibroblasts, DNA/chromatin emerging from nuclei during LIS extraction mechanically and chemically cleaned the nuclear surface and perinuclear area from loosely anchored cytoplasmic material with the production of broad, IF-free annular spaces, but left substantial fractions of the vimentin IFs in tight association with the nuclear surface. Accordingly, double-immunogold electron microscopy of fixed and permeabilized fibroblasts disclosed a close neighborhood of vimentin IFs and lamin B, with a minimal distance between the nanogold particles of ca. 30 nm. These data indicate an extremely solid interconnection of cIFs with structural elements of the nuclear matrix, and make them, together with their susceptibility to crosslinkage to MARs and other genomic DNA sequences under native conditions, complementary or even integral constituents of the karyoskeleton.     

Molecular architecture of intermediate filaments

Together with microtubules and actin microfilaments, approximately 11 nm wide intermediate filaments (IFs) constitute the integrated, dynamic filament network present in the cytoplasm of metazoan cells. This network is critically involved in division, motility and other cellular processes. While the structures of microtubules and microfilaments are known in atomic detail, IF architecture is presently much less understood. The elementary 'building block' of IFs is a highly elongated, rod-like dimer based on an alpha-helical coiled-coil structure. Assembly of cytoplasmic IF proteins, such as vimentin, begins with a lateral association of dimers into tetramers and gradually into the so-called unit-length filaments (ULFs). Subsequently ULFs start to anneal longitudinally, ultimately yielding mature IFs after a compaction step. For nuclear lamins, however, assembly starts with a head-to-tail association of dimers. Recently, X-ray crystallographic data were obtained for several fragments of the vimentin dimer. Based on the dimer structure, molecular models of the tetramer and the entire filament are now a possibility.     

Investigation of the morphology of intermediate filaments adsorbed to different solid supports

Morphologically, glutaraldehyde-fixed and -dried intermediate filaments (IFs) appear flexible, and with a 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 of 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 a height of only 3-5 nm, whereas desmin IFs appeared as 8-10 nm height filaments with a helical twist. Neurofilaments were 10-12 nm in height with a pronounced 30-50 nm 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, hydrophilic 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 on this support. These results are straightening the idea that mature IFs are dynamic polymers in vitro and that IFs can be distinguished from each others by their physicochemical properties.     

RhoA-Binding Kinase α Translocation Is Facilitated by the Collapse of the Vimentin Intermediate Filament Network

The regulation of morphological changes in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). The putative effector of RhoA, RhoA-binding kinase α (ROKα), is a serine/threonine kinase that has been implicated in the reorganization of actin filaments and in myosin contractility. Here, we show that ROKα also directly affects the structural integrity of IFs. Overexpression of active ROKα, like that of RhoA, caused the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kinase-inactive ROKα inhibited the collapse of vimentin IFs induced by RhoA in HeLa cells. In vitro, ROKα bound and phosphorylated vimentin at its head-rod domain, thereby inhibiting the assembly of vimentin. ROKα colocalized predominantly with the filamentous vimentin network, which remained intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also resulted in filamentous vimentin collapse and concomitant ROKα translocation to the cell periphery. ROKα translocation did not occur when the vimentin network remained intact in vinblastine-treated cells at 4°C or in the presence of the dominant-negative RhoAN19 mutant. Transient translocation of ROKα was also observed in cells subjected to heat shock, which caused the disassembly of the vimentin network. Thus, the translocation of ROKα to the cell periphery upon overexpression of RhoAV14 or growth factor treatment is associated with disassembly of vimentin IFs. These results indicate that Rho effectors known to act on microfilaments may be involved in regulating the assembly of IFs. Vimentin when phosphorylated also exhibits reduced affinity for the inactive ROKα. The translocation of ROKα from IFs to the cell periphery upon action by activated RhoA and ROKα suggests that ROKα may initiate its own cascade of activation.     

Crystal structure of the human lamin A coil 2B dimer: implications for the head-to-tail association of nuclear lamins

Nuclear intermediate filaments (IFs) are made from fibrous proteins termed lamins that assemble, in association with several transmembrane proteins of the inner nuclear membrane and an unknown number of chromatin proteins, into a filamentous scaffold called the nuclear lamina. In man, three types of lamins with significant sequence identity, i.e. lamin A/C, lamin B1 and B2, are expressed. The molecular characteristics of the filaments they form and the details of the assembly mechanism are still largely unknown. Here we report the crystal structure of the coiled-coil dimer from the second half of coil 2 from human lamin A at 2.2A resolution. Comparison to the recently solved structure of the homologous segment of human vimentin reveals a similar overall structure but a different distribution of charged residues and a different pattern of intra- and interhelical salt bridges. These features may explain, at least in part, the differences observed between the lamin and vimentin assembly pathways. Employing a modeled lamin A coil 1A dimer, we propose that the head-to-tail association of two lamin dimers involves strong electrostatic attractions of distinct clusters of negative charge located on the opposite ends of the rod domain with arginine clusters in the head domain and the first segment of the tail domain. Moreover, lamin A mutations, including several in coil 2B, have been associated with human laminopathies. Based on our data most of these mutations are unlikely to alter the structure of the dimer but may affect essential molecular interactions occurring in later stages of filament assembly and lamina formation.     

Differential sensitivity of vimentin and nuclear lamins from Ehrlich ascites tumor cells toward Ca2+ -activated neutral thiol proteinase

A comparative study of the susceptibility of vimentin and nuclear lamins from cultured Ehrlich ascites tumor (EAT) cells to degradation by Ca2+ -activated neutral thiol proteinase (calpain) has been undertaken. While pure vimentin was degraded very quickly at physiological ionic strength by purified calpain, isolated lamin B was digested comparatively slowly and purified lamins A/C were fairly resistant to proteolytic degradation. Similar digestion patterns were obtained from vimentin and lamin B with intermediary breakdown products close in size to the corresponding alpha-helical rod domains. To exclude the possibility that the low susceptibility of isolated lamins to Ca2+-dependent proteolytic degradation was due to irreversible denaturation during their isolation and purification, Triton cytoskeletons were prepared and their nuclear lamina as well as vimentin filaments were exposed to relatively large quantities of purified calpain. Under these conditions, not only vimentin filaments but also lamins A and B were digested while lamin C remained intact to a high degree. The major breakdown products of vimentin and lamins were identified as polypeptides which were 35 to 45 amino acids longer than the corresponding alpha-helical rod domains. Most of the vimentin-derived material and all high molecular weight polypeptides originating from lamins remained associated with the Triton cytoskeletons as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with immunoblotting. Indirect immunofluorescence and electron microscope analysis of the calpain-digested Triton cytoskeletons revealed that they still contained a laminalike structure around the nuclear chromatin and numerous structurally altered intermediate filaments in the cytoplasmic remnant, although all vimentin had been degraded with the formation of 40/41 kDa polypeptides as major digestion products. In untreated Triton cytoskeletons, the vimentin filaments seemed to be in direct physical contact with the nuclear lamina, whereas in digested Triton cytoskeletons there was a distinct gap between structurally altered filaments and the nuclear surface. This shows that vimentin filaments and the nuclear lamina are differentially susceptible to degradation by calpain under certain ionic conditions and suggests that both filamentous structures are intimately associated with each other.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphological analysis of glutaraldehyde-fixed vimentin intermediate filaments and assembly-intermediates by atomic force microscopy

Atomic force microscopy (AFM) was used to study the morphology of vimentin intermediate filaments (IFs) and their assembly intermediates. At each time after initiation of IF assembly in vitro of recombinant mouse vimentin, the sample was fixed with 0.1% glutaraldehyde and then applied to AFM analysis. When mature vimentin IFs were imaged in air on mica, they appeared to have a width of approximately 28 nm, a height of approximately 4 nm and a length of several micrometers. Taking into account the probe tip's distortion effect, the exact width was evaluated to be approximately 25 nm, suggesting that the filaments flatten on the substrate rather than be cylindrical with a diameter of approximately 10 nm. Vimentin IFs in air clearly demonstrated approximately 21-nm repeating patterns along the filament axis. The three-dimensional profiles of vimentin IFs indicated that the characteristic patterns were presented by repeating segments with a convex surface. The repeating patterns close to 21 nm were also observed by AFM analysis in a physiological solution condition, suggesting that the segments along the filaments are an intrinsic substructure of vimentin IFs. In the course of IF assembly, assembly intermediates were analyzed in air. Many short filaments with a full-width and an apparent length of approximately 78 nm (evaluated length approximately 69 nm) were observed immediately after initiation of the assembly reaction. Interestingly, the short full-width filaments appeared to be composed of the four segments. Further incubation enabled the short full-width filaments to anneal longitudinally into longer filaments with a distinct elongation step of approximately 40 nm, which corresponds to the length of the two segments. To explain these observations, we propose a vimentin IF formation model in which vimentin dimers are supercoiling around the filament axis.     

衣藻(Chlamydomonas sp)中间纤维及核纤层存在的证实(简报)

衣藻(Chlamydomonas sp)是属于绿藻门的最低等单细胞植物,为典型的真核生物。迄今以衣藻为材料所作的有关细胞骨架方面的研究多集中在微管蛋白(tubulin)。C.J.Miller等曾以衣藻(Chlamydomonas reinhardtii)全蛋白与几种中间纤维抗体进行免疫印迹实验有阳性反应,但是衣藻中是否存在中间纤维与核纤层是不清楚的问题。衣藻中间纤维与核纤层的形态研究更未见报道。目前认为中间纤维-核纤     

Cytoskeletal proteins connecting intermediate filaments to cytoplasmic and nuclear periphery

Intermediate filaments (IFs), together with microtubules and microfilaments build up the cytoskeleton of most eukaryotic cells. Cytoplasmic IFs form a dense filament network radiating from the nucleus and extending to the plasma membrane. The association between the cytoplasmic and nuclear surfaces appears to provide a continuous link important for the organisation of the cytoplasm, for cellular communication, and possibly for the transport into and out of the nucleus. Cytoplasmic IFs approach the nuclear surface, thin fibrils seem to connect the IFs with the nuclear pore complexes and a direct interaction of cytoplasmic IFs with the nuclear lamin B has been observed by in vitro binding studies. However, none of the components that cross-link IFs to the nucleus has been unambiguously identified. Furthermore, if a direct interaction between cytoplasmic IFs and the nuclear lamin B occurs in vivo, the question of how cytoplasmic IFs get access to the nuclear interior remains to be resolved. The association of IFs with the plasma membranes involves different components, some of which are cell type specific. Two specialised complexes in epithelial cells: the desmosome and the hemidesmosome, serve as attachment sites for keratin filaments. Desmoplakin is considered as the cross-linking component of IFs to the desmosomal plaque, whereas BPAG1 (bullous pemphigoid antigen) would cross-link IFs at the hemidesmosomal plaque. In other cell types the modality of how IFs are anchored to the plasma membrane is less well understood. It involves different components such as the spectrin based membrane skeleton, ankyrin, myosin, plectin and certainly many other still unravelled partners. Association between the IFs and cellular membranes plays an important role in determining cell shape and tissue integrity. Thus, the identification and characterisation of the components involved in these interactions will be crucial for understanding the function of intermediate filaments.     

Regulated docking of nuclear membrane vesicles to vimentin filaments during mitosis

During mitosis, several types of intermediate-sized filaments (IFs) undergo an extensive remodelling in response to phosphorylation by cdc 2 and other protein kinases. However, unlike the nuclear lamins, the cytoplasmic IFs do not seem to follow a fixed disassembly stereotype and often retain their physical continuity without depolymerizing into soluble subunits. To investigate potential interactions between mitotically modified IFs and other cellular structures, we have examined prometaphase-arrested cells expressing the IF protein vimentin. We demonstrate here that vimentin filaments associate in situ and co-fractionate with a distinct population of mitotic vesicles. These vesicles carry on their surfaces nuclear lamin B, the inner nuclear membrane protein p58, and wheat germ agglutinin (WGA)-binding proteins. Consistent with a tight interaction between the IFs and the mitotic membranes, vimentin, nuclear lamin B, and a 180-kD WGA-binding protein are co-isolated when whole mitotic homogenates are incubated with anti-vimentin or anti-lamin B antibodies immobilized on magnetic beads. The vimentin-associated vesicles are essentially depleted of ER, Golgi and endosomal membrane proteins. The interaction of vimentin with lamin B-carrying membranes depends on phosphorylation and is weakened by dephosphorylation during nuclear reassembly in vitro. These observations reveal a novel interaction between IFs and cellular membranes and further suggest that the vimentin filaments may serve as a transient docking site for inner nuclear membrane vesicles during mitosis.     

Keeping the Vimentin Network under Control: Cell–Matrix Adhesion–associated Plectin 1f Affects Cell Shape and Polarity of Fibroblasts

Focal adhesions (FAs) located at the ends of actin/myosin-containing contractile stress fibers form tight connections between fibroblasts and their underlying extracellular matrix. We show here that mature FAs and their derivative fibronectin fibril-aligned fibrillar adhesions (FbAs) serve as docking sites for vimentin intermediate filaments (IFs) in a plectin isoform 1f (P1f)-dependent manner. Time-lapse video microscopy revealed that FA-associated P1f captures mobile vimentin filament precursors, which then serve as seeds for de novo IF network formation via end-to-end fusion with other mobile precursors. As a consequence of IF association, the turnover of FAs is reduced. P1f-mediated IF network formation at FbAs creates a resilient cage-like core structure that encases and positions the nucleus while being stably connected to the exterior of the cell. We show that the formation of this structure affects cell shape with consequences for cell polarization.     

Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers

The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.     

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.     

BHK_(21)细胞的中间纤维-lamina-核骨架体系

以系列选择性抽提技术与显示细胞骨架的整装电镜技术为基础,应用免疫胶体金标记与蛋白质成份的双向电泳分析技术,研究了BHK_(21)细胞的中间纤维-lamina与核骨架(核基质)结构体系及其主要的蛋白成份。BHK_(21)细胞的中间纤维-lamina与核骨架是在结构上相互联系,贯穿于核与质的网络体系。中间纤维单丝直径为10nm,能很好地被抗波形蛋白抗体-金颗粒所标记,生化分析同样说明BHK_(21)细胞中间纤维的主要成份是波形蛋白(vimentin),其分子量为55KD,等电点为5.6。中间纤维网在胞质内呈极性分布,与lamina密切联结。BHK_(21)细胞的lamina能被抗lamin A与C的单克隆抗体-金颗粒标记。双向电泳分析证明,lamina含有三种蛋白成份,即lamin A,B,C,其分子最分别为68KD,70KD与62KD,lamin A,C等电点均为6.9—7.2,而lamin B偏酸,其等电点为5.8。BHK_(21)细胞核骨架纤维网也可以被清晰的显示,其蛋白成份较为复杂,在双向电泳谱上经常出现多个清晰的斑点,很可能含有肌动蛋白(actin)。298KD核基质蛋白的单克隆抗体-金颗粒能准确的标记核骨架纤维。     

The intermediate filament-lamina-nuclear matrix system of BHK-21 cells

We have employed collodial gold immuno-labelling in whole-mount cell and 2-D gel electrophoresis to demonstrate the intermediate filament (IF)-lamina-nuclear matrix (NM) system in BHK-21 (Baby Hamster Kidney) cells. Grown on grids, cells were gently extracted with salt solutions as previously described by S. Penman to preserve intact IF-lamina-NM systems. The extracted samples were fixed, postfixed, dehydrated and dried through the CO2 critical point, then examined under high voltage electron microscope (HVEM). The results revealed that the IF-lamina-NM system is a interconnecting network throughout the cell from cytoplasma to nuclear. The IF unit is 10 nm in diameter. IFs radiate away from the nuclear region into the spreading cytoplasm and the polarity of their distributing is obvious. The IF system closely connected to lamina. Immuno-gold labelling and 2-D gel proved that vimentin, a 55 KD protein (pI 5,6), is the major component of IFs in BHK-21 cells. Lamina can be precisely and specifically labelled with anti-lamin A, C proteins and as well as 2-D gel electrophoresis indicated that there are lamin A, B, C proteins in BHK-21 cells, whose molecular weights are 68 KD, 70 KD, 62 KD respectively. Its components are more complicated, but a few dots of NM proteins can be clearly distinguished in 2-D gel map, in which actin, a 45 KD protein (pI 4.5), might be involved. The nuclear matrix network was also clearly presented under HVEM. Its filaments can be labelled with anti-NM 298 KD protein precisely.     

北京鸭B淋巴细胞的中间纤维—核纤层—核内骨架体系

以北京鸭腔上囊为实验材料,应用细胞分级抽提方法与非树脂包埋－去包埋剂的电镜制样技术相结合,显示出Ｂ细胞中相互连结的中间纤维－核纤层－核内骨架体系的超结构及其分布,中间纤维交织成网络状,纤维直径在９－１１ｎｍ,其成份是分子量为６７ｋＤ,等电点约为６．２的波形蛋白,核纤民支呈片层状结构环绕在核区周围,其主要成份是分子量为６７ｋＤ,等电点偏酸性的Ｌａｍｉｎ Ｂ。核内骨架由粗细不一的纤维形成网络结构,其上     

Lamin B constitutes an intermediate filament attachment site at the nuclear envelope

We found that urea extraction of turkey erythrocyte nuclear envelopes abolished their ability to bind exogenous 125I-vimentin, while, at the same time, it removed the nuclear lamins from the membranes. After purification of the lamins from such urea extracts, a specific binding between isolated vimentin and lamin B, or a lamin A + B hetero- oligomer, was detected by affinity chromatography. Similar analysis revealed that the 6.6-kD vimentin tail piece was involved in this interaction. By other approaches (quantitative immunoprecipitation, rate zonal sedimentation, turbidometric assays) a substoichiometric lamin B-vimentin binding was determined under in vitro conditions. It was also observed that anti-lamin B antibodies but not other sera (anti- lamin A, anti-ankyrin, preimmune) were able to block 70% of the binding of 125I-vimentin to native, vimentin-depleted, nuclear envelopes. These data, which were confirmed by using rat liver nuclear lamins, indicate that intermediate filaments may be anchored directly to the nuclear lamina, providing a continuous network connecting the plasma membrane skeleton with the karyoskeleton of eukaryotic cells.     

On the relation between distinct components of the cytoskeleton: an epitope shared by intermediate filaments, microfilaments and cytoplasmic foci.

Monoclonal antibodies were generated against detergent-insoluble cytoskeletal proteins isolated from low-density membrane fractions of rat liver. By immunofluorescence, one of the antibodies stains three distinct structures in cultured rat fibroblast and hepatocyte lines as well as the PtK2 rat-kangaroo kidney epithelial line. These structures are: i) many tangled filaments similar to intermediate filaments (IFs), ii) fewer and variable numbers of straight filaments, and iii) punctate cytoplasmic foci, often most intense around the nucleus. All three of these structures are resistant to extraction by non-ionic detergent. Close examination reveals that the tangled and straight filaments are not stained uniformly, but as a series of bright patches. In cells treated with nocodazole, the antibody reacts strongly with a perinuclear filamentous cage. Very few tangled filaments are detected in these cells, however, the straight filaments and punctate cytoplasmic staining are resistant to nocodazole treatment. Double-label immunofluorescence shows that, even though tangled filament distribution and nocodazole sensitivity are similar to the behavior of vimentin IFs, there is only partial coincidence of staining with either vimentin or cytokeratin IFs. The straight filaments coincide with some actin stress fibers, but the punctate cytoplasmic staining is not related to IFs, actin, or tubulin. Thus, this monoclonal antibody stains a novel group of three seemingly unrelated cytoskeletal structures, including a previously undescribed insoluble nonfilamentous pool. Taken as a whole, two hypotheses are consistent with these data. i) The antigen recognized may be a protein which has a large insoluble cytoplasmic pool and binds both IFs and actin, but only binds to a subset of each class of filaments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Engagement of vimentin intermediate filaments in hypotonic stress

Intermediate filaments (IFs) play a key role in the control of cell structure and morphology, cell mechano-responses, migration, proliferation, and apoptosis. However, the mechanisms regulating IFs organization in motile adhesive cells under certain physical/pathological conditions remain to be fully understood. In this study, we found hypo-osmotic–induced stress results in a dramatic but reversible rearrangement of the IF network. Vimentin and nestin IFs are partially depolymerized as they are redistributed throughout the cell cytoplasm after hypo-osmotic shock. This spreading of the IFs requires an intact microtubule network and the motor protein associated transportation. Both nocodazole treatment and depletion of kinesin-1 (KIF5B) block the hypo-osmotic shock–induced rearrangement of IFs showing that the dynamic behavior of IFs largely depends on microtubules and kinesin-dependent transport. Moreover, we show that cell survival rates are dramatically decreased in response to hypo-osmotic shock, which was more severe by vimentin IFs depletion, indicating its contribution to osmotic endurance. Collectively, these results reveal a critical role of vimentin IFs under hypotonic stress and provide evidence that IFs are important for the defense mechanisms during the osmotic challenge.