Distribution of intermediate filaments in amphibian oxyntic cells

Summary Intermediate filaments of toad oxyntic cells were isolated and analysed by SDS-PAGE. The major proteins of the residue were identified as actin and a 51,000 dalton polypeptide. Immunological crossreactivity between toad oxyntic cell intermediate filament components and anti-prekeratin, was shown by double immunodiffusion tests and indirect immunofluorescence. The immunofluorescent decoration of oxyntic cells and the electron microscope images are coincident in locating the intermediate filaments mainly at the cortical and perinuclear basal zones. Furthermore, the cortical zone appears especially rich in prekeratin-like material at its adluminal third. This results in a cup-like structure that encloses the cell portion occupied by the tubulovesicular system, which does not contain intermediate filaments. The translocation of membranes occurring during the secretory cycle of the oxyntic cell, has been attributed to a system of contractile proteins. The disposition of the prekeratin-like material suggests a role for intermediate filaments in the generation of movement, produced by actin and myosin interaction, by providing a fixed plane for the anchoring of actin microfilaments.     

A direct interaction between actin and vimentin filaments mediated by the tail domain of vimentin

The assembly and organization of the three major eukaryotic cytoskeleton proteins, actin, microtubules, and intermediate filaments, are highly interdependent. Through evolution, cells have developed specialized multifunctional proteins that mediate the cross-linking of these cytoskeleton filament networks. Here we test the hypothesis that two of these filamentous proteins, F-actin and vimentin filament, can interact directly, i.e. in the absence of auxiliary proteins. Through quantitative rheological studies, we find that a mixture of vimentin/actin filament network features a significantly higher stiffness than that of networks containing only actin filaments or only vimentin filaments. Maximum inter-filament interaction occurs at a vimentin/actin molar ratio of 3 to 1. Mixed networks of actin and tailless vimentin filaments show low mechanical stiffness and much weaker inter-filament interactions. Together with the fact that cells featuring prominent vimentin and actin networks are much stiffer than their counterparts lacking an organized actin or vimentin network, these results suggest that actin and vimentin filaments can interact directly through the tail domain of vimentin and that these inter-filament interactions may contribute to the overall mechanical integrity of cells and mediate cytoskeletal cross-talk.     

Characterization of intermediate filaments and their structural organization during epithelium formation in pigmented epithelial cells of the retina in vitro

Summary Retinal pigmented epithelial cells of chicken have circumferential microfilament bundles (CMBs) at the zonula adherens region. Isolated CMBs are polygons filled with a meshwork composed primarily of intermediate filaments; they show three major components of 200000, 55000, and 42000 daltons in SDS-gel electrophoresis. Here we have characterized the 55000-dalton protein immunochemically and ultrastructurally. Immunoblotting and immunofluorescence microscopy have shown that the 55000-dalton protein is an intermediate filament protein, vimentin.Vimentin filaments changed their distribution during differentiation of pigmented epithelial cells in culture. The protein in the elongated cells showed a fibroblast-type pattern of intermediate filaments. During epithelium formation, the filaments were uniformly distributed and formed a finer meshwork at the apical level. In pigmented epithelial cells that differentiated and matured in culture, vimentin and actin exhibited their characteristic behavior after treatment with colcemid. In the central to basal region of the cell, intermediate filaments formed thick perinuclear bundles. In the apical region, however, intermediate filaments changed in organization from a nonpolarized meshwork to a polarized bundle-like structure. Simultaneously, new actin bundles were formed, running parallel to the intermediate filaments. This suggests that there is some interaction between microfilaments and intermediate filaments in the apical region of these cells.     

Substrate stiffness regulates solubility of cellular vimentin

The intermediate filament protein vimentin is involved in the regulation of cell behavior, morphology, and mechanical properties. Previous studies using cells cultured on glass or plastic substrates showed that vimentin is largely insoluble. Although substrate stiffness was shown to alter many aspects of cell behavior, changes in vimentin organization were not reported. Our results show for the first time that mesenchymal stem cells (hMSCs), endothelial cells, and fibroblasts cultured on different-stiffness substrates exhibit biphasic changes in vimentin detergent solubility, which increases from nearly 0 to 67% in hMSCs coincident with increases in cell spreading and membrane ruffling. When imaged, the detergent-soluble vimentin appears to consist of small fragments the length of one or several unit-length filaments. Vimentin detergent solubility decreases when these cells are subjected to serum starvation, allowed to form cell–cell contacts, after microtubule disruption, or inhibition of Rac1, Rho-activated kinase, or p21-activated kinase. Inhibiting myosin or actin assembly increases vimentin solubility on rigid substrates. These data suggest that in the mechanical environment in vivo, vimentin is more dynamic than previously reported and its assembly state is sensitive to stimuli that alter cellular tension and morphology.     

Morphological and mechanical stability of bladder cancer cells in response to substrate rigidity

BackgroundMorphology of cells can be considered as an interplay between the accessibility of substrate anchoring sites, cytoskeleton properties and cellular deformability. To withstand tension induced by cell's environment, cells tend to spread out and, simultaneously, to remodel actin filament organization.MethodsIn this context, the use of polyacrylamide hydrogel substrates with a surface coated with laminin allows to trace remodeling of actin cytoskeleton during the interaction of cells with laminin-rich basement membrane. Reorganization of actin cortex can be quantified by a surface spreading area and deformability of single cells.ResultsIn our study, we demonstrated that morphological and mechanical alterations of bladder cancer cells in response to altered microenvironment stiffness are of biphasic nature. Threshold-dependent relations are induced by mechanical properties of cell microenvironment. Initially, fast alterations in cellular capability to spread and to deform are followed by slow-rate changes. A switch provided by cellular deformability threshold, in the case of non-malignant cells, triggers the formation of thick actin bundles accompanied by matured focal adhesions. For cancer cells, cell spreading and deformability thresholds switch between slow and fast rate of changes with weak reorganization of actin filaments and focal adhesions formation.ConclusionsThe presence of transition region enables the cells to achieve a morphological and mechanical stability, which together with altered expression of vinculin and integrins, can contribute to invasiveness of bladder cancers.General significanceOur findings show that morphological and mechanical stability is directly related to actin filament organization used by cancer cells to adapt to altered laminin-rich microenvironment.     

Cytoskeleton and vesicle mobility in astrocytes

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.     

Intermediate filaments regulate astrocyte motility.

Intermediate filaments (IFs) compose, together with actin filaments and microtubules, the cytoskeleton and they exhibit a remarkable but still enigmatic cell-type specificity. In a number of cell types, IFs seem to be instrumental in the maintenance of the mechanical integrity of cells and tissues. The function of IFs in astrocytes has so far remained elusive. We have recently reported that glial scar formation following brain or spinal cord injury is impaired in mice deficient in glial fibrillary acidic protein and vimentin. These mice lack IFs in reactive astrocytes that are normally pivotal in the wound repair process. Here we show that reactive astrocytes devoid of IFs exhibit clear morphological changes and profound defects in cell motility thereby revealing a novel function for IFs.     

The nanomechanical properties of rat fibroblasts are modulated by interfering with the vimentin intermediate filament system

The contribution of the intermediate filament (IF) network to the mechanical response of cells has so far received little attention, possibly because the assembly and regulation of IFs are not as well understood as that of the actin cytoskeleton or of microtubules. The mechanical role of IFs has been mostly inferred from measurements performed on individual filaments or gels in vitro. In this study we employ atomic force microscopy (AFM) to examine the contribution of vimentin IFs to the nanomechanical properties of living cells under native conditions. To specifically target and modulate the vimentin network, Rat-2 fibroblasts were transfected with GFP-desmin variants. Cells expressing desmin variants were identified by the fluorescence microscopy extension of the AFM instrument. This allowed us to directly compare the nanomechanical response of transfected and untransfected cells at high spatial resolution by means of AFM. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. Expression of the non-filament forming GFP-DesL345P mutant led to a collapse of the endogenous vimentin network in the perinuclear region that was accompanied by localized stiffening. Correlative confocal microscopy indicates that the expression of desmin variants specifically targets the endogenous vimentin IF network without major rearrangements of other cytoskeletal components. By measuring functional changes caused by IF rearrangements in intact cells, we show that IFs play a crucial role in mechanical behavior not only at large deformations but also in the nanomechanical response of individual cells.     

The Role of Vimentin Intermediate Filaments in Cortical and Cytoplasmic Mechanics

The mechanical properties of a cell determine many aspects of its behavior, and these mechanics are largely determined by the cytoskeleton. Although the contribution of actin filaments and microtubules to the mechanics of cells has been investigated in great detail, relatively little is known about the contribution of the third major cytoskeletal component, intermediate filaments (IFs). To determine the role of vimentin IF (VIF) in modulating intracellular and cortical mechanics, we carried out studies using mouse embryonic fibroblasts (mEFs) derived from wild-type or vimentin^−/− mice. The VIFs contribute little to cortical stiffness but are critical for regulating intracellular mechanics. Active microrheology measurements using optical tweezers in living cells reveal that the presence of VIFs doubles the value of the cytoplasmic shear modulus to ∼10 Pa. The higher levels of cytoplasmic stiffness appear to stabilize organelles in the cell, as measured by tracking endogenous vesicle movement. These studies show that VIFs both increase the mechanical integrity of cells and localize intracellular components.     

Association of mitochondria with intermediate filaments and of polyribosomes with cytoplasmic actin

Summary Anti-mitochondrial autoantibody and fluorescent derivatives of insulin stain phase-dense mitochondria in acetone-fixed monolayers of fibroblasts. Double fluorochrome studies show mitochondria in close topographic association with intermediate filaments. In cells treated with vinblastine or colchicine, mitochondria are relocated in sites closely associated with coils of perinuclear intermediate filaments. In contrast, autoantibody to polyribosomes stains granules aligned in the long axis of well spread embryonic cells, in the direction of actin-containing fibrils, an arrangement that is lost in cells pretreated with the actin filament disrupting drug cytochalasin B. In more mature fibroblasts, antiribosomal antibody reacts with phase-dense rough endoplasmic reticulum and this staining pattern is not affected by cytochalasin B. The observations suggest that mitochondria are associated with intermediate filaments and that free polyribosomes, but not polyribosomes attached to rough endoplasmic reticulum, are associated with cytoplasmic actin.Supported by a grant from the Anti-Cancer Council of Victoria. We thank Mrs. I. Burns for technical assistance and Dr. H.A. Ward and staff for preparation of fluorescent conjugates     

The Association of Tau-Like Proteins with Vimentin Filaments in Cultured Cells

There is increasing evidence that the different polymers that constitute the cytoskeleton are interconnected to form a three-dimensional network. The macromolecular interaction patterns that stabilize this network and its intrinsic dynamics are the basis for numerous cellular processes. Within this context,in vitrostudies have pointed to the existence of specific associations between microtubules, microfilaments, and intermediate filaments. It has also been postulated that microtubule-associated proteins (MAPs) are directly involved in mediating these interactions. The interactions of tau with vimentin filaments, and its relationships with other filaments of the cytoskeletal network, were analyzed in SW-13 adenocarcinoma cells, through an integrated approach that included biochemical and immunological studies. This cell line has the advantage of presenting a wild-type clone (vim+) and a mutant clone (vim−) which is deficient in vimentin expression. We analyzed the cellular roles of tau, focusing on its interactions with vimentin filaments, within the context of its functional aspects in the organization of the cytoskeletal network. Cosedimentation experiments of microtubular protein with vimentin in cell extracts enriched in intermediate filaments, combined with studies on the direct interaction of tau with nitrocellulose-bound vimentin and analysis of tau binding to vimentin immobilized in single-strand DNA affinity columns, indicate that tau interacts with the vimentin network. These studies were confirmed by a quantitative analysis of the immunofluorescence patterns of cytoskeleton-associated tubulin, tau, and vimentin using flow cytometry. In this regard, a decrease in the levels of tau associated to the cytoskeletal network in the vim− cell mutant compared with the wild-type clones was observed. However, immunofluorescence data on SW-13 cells suggest that the absence of a structured network of vimentin in the mutant vim− cells does not affect the cytoplasmic organization formed by microtubules and actin filaments, when compared with the wild-type vim+ cells. These studies suggest that tau associates with vimentin filaments and that these interactions may play a structural role in cells containing these filaments.     

Disruption of the keratin filament network during epithelial cell division

The behaviour of keratin filaments during cell division was examined in a wide range of epithelial lines from several species. Almost half of them show keratin disruption as described previously: by immunofluorescence, filaments are replaced during mitosis by a 'speckled' pattern of discrete cytoplasmic dots. In the electron microscope these ' speckles ' are seen as granules around the cell periphery, just below the actin cortical mesh, with no detectable 10 nm filament structure inside them and no keratin filament bundles in the rest of the cytoplasm. A time course of the filament reorganization was constructed from double immunofluorescence data; filaments are disrupted in prophase, and the filament network is intact again by cytokinesis. The phenomenon is restricted to cells rich in keratin filaments, such as keratinocytes; it is unrelated to the co-existence of vimentin in many of these cells, and vimentin is generally maintained as filaments while the keratin is restructured. Some resistance to the effect may be conferred by an extended cycle time. Filament reorganization takes place within minutes, so that a reversible mechanism seems more likely than one involving de novo protein synthesis, at this metabolically quiet stage of the cell cycle.     

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.     

上皮细胞分裂过程中中等纤维的变化

应用制备的血清抗体,采用免疫细胞化学方法观察了两株培养上皮细胞的分裂过程中IF的动态变化过程。实验结果显示,在上皮细胞分裂过程中,IF形态结构及空间分布发生了显著变化,不同细胞之间存在差异,分裂的Vero细胞中角蛋白纤维和波形纤维都维持纤维形态,围绕分裂器形成纤维网罩或纤维束环,随着细胞分裂的进行,IF网的空间组织结构和外观发生动态变化;分裂的HeLa细胞中,角蛋白纤维和波形纤维广泛重组形成颗粒状胞质小体,分裂结束后重建IF网。实验结果表明,IF变化具有细胞周期依赖性和一定的细胞特异性。本文对IF在细胞分裂过程中的功能意义作了讨论。     

The Role of Vimentin Intermediate Filaments in Cortical and Cytoplasmic Mechanics

The mechanical properties of a cell determine many aspects of its behavior, and these mechanics are largely determined by the cytoskeleton. Although the contribution of actin filaments and microtubules to the mechanics of cells has been investigated in great detail, relatively little is known about the contribution of the third major cytoskeletal component, intermediate filaments (IFs). To determine the role of vimentin IF (VIF) in modulating intracellular and cortical mechanics, we carried out studies using mouse embryonic fibroblasts (mEFs) derived from wild-type or vimentin^−/− mice. The VIFs contribute little to cortical stiffness but are critical for regulating intracellular mechanics. Active microrheology measurements using optical tweezers in living cells reveal that the presence of VIFs doubles the value of the cytoplasmic shear modulus to ∼10 Pa. The higher levels of cytoplasmic stiffness appear to stabilize organelles in the cell, as measured by tracking endogenous vesicle movement. These studies show that VIFs both increase the mechanical integrity of cells and localize intracellular components.     

Irregular patterns of actin and myosin filaments in human skeletal muscle cells

Summary The ultrastructural study of cross sections of normal skeletal muscle cells showed the existence of irregular patterns of actin filaments in connection with the hexagonal pattern of the myosin filaments. The actin filaments surrounding each myosin filament vary in number from 6 to 11. The most frequent relationship is 9 to 1, followed by 10 to 1 and 8 to 1. The hexagonal pattern of actin filaments was observed only in the 6 to 1 arrays; as the actin filaments increase in number, they tend to form different polygons or circles around the myosin filaments. All described patterns may occur in each sarcomere. The actin to myosin filament ratio varies from 3 to 4 within each individual myofibril. The described variability of the actin filaments arrays leads to several difficulties in an explanation of the mechanism of muscular contraction.Director, Chief of Section, Histology. Profesor Agregado de Embriología e HistologíaProfesor Adjunto de Embriología e HistologíaResidente de Anatomía Patol'ogica de la Ciudad Sanitaria La Paz     

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.     

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.     

A Prestressed Cable Network Model of the Adherent Cell Cytoskeleton

A prestressed cable network is used to model the deformability of the adherent cell actin cytoskeleton. The overall and microstructural model geometries and cable mechanical properties were assigned values based on observations from living cells and mechanical measurements on isolated actin filaments, respectively. The models were deformed to mimic cell poking (CP), magnetic twisting cytometry (MTC) and magnetic bead microrheometry (MBM) measurements on living adherent cells. The models qualitatively and quantitatively captured the fibroblast cell response to the deformation imposed by CP while exhibiting only some qualitative features of the cell response to MTC and MBM. The model for CP revealed that the tensed peripheral actin filaments provide the key resistance to indentation. The actin filament tension that provides mechanical integrity to the network was estimated at ~158 pN, and the nonlinear mechanical response during CP originates from filament kinematics. The MTC and MBM simulations revealed that the model is incomplete, however, these simulations show cable tension as a key determinant of the model response.