Cytoskeleton disruption in chondrocytes from a rat osteoarthrosic (OA) -induced model: its potential role in OA pathogenesis

Morphological and functional changes of chondrocytes are typical in OA cartilage. In this work, we have described noteworthy changes in intermediate filaments cytoskeleton evidenced by transmission electron microscopy. Alterations in the distribution as well as in the content of vimentin, actin, and tubulin have been described by specific fluorescence labelling of each cytoskeletal component and confocal analysis. Normal vs OA cartilages showed a reduction in the percentage of labelled chondrocytes of 37.1% for vimentin, 4.7% for actin, and 20.1% for tubulin. Statistical analysis of fluorescence intensities (mean % +/- SEM) between normal and OA rat cartilage revealed a highly significant difference in vimentin, a significant difference in tubulin, and a non-significant difference in actin. Moreover, by western blot, altered electrophoretic patterns were observed mainly for vimentin and tubulin in OA cartilage in comparison with normal cartilage. These results allow us to suggest that substantial changes in vimentin and tubulin cytoskeleton of chondrocytes might be involved in OA pathogenesis.     

Distribution of Cytoskeletal Proteins in Neomycin-Induced Protrusions of Human Fibroblasts

The organization of actin, tubulin, and vimentin was studied in protruding lamellae of human fibroblasts induced by the aminoglycoside antibiotic neomycin, an inhibitor of the phosphatidylinositol cycle. Neomycin stimulates the simultaneous protrusion of lamellae in all treated cells, and the lamellae remain extended for about 15–20 min, before gradually withdrawing. The pattern and distribution of actin, tubulin, and vimentin during neomycin stimulation were analyzed by fluorescence and electron microscopy. F-actin in the newly formed lamellae is localized in a marginal band at the leading edge. Tubulin is colocalized with F-actin in the marginal band, but the newly formed lamellae are initially devoid of microtubules. Over a period of 10 to 20 min after the addition of neomycin, microtubules grow into the lamellae from the adjacent cytoplasm, while the intensity of tubulin staining of the marginal band decreases. Distribution of vimentin remains unchanged in neomycin-treated cells and vimentin filaments do not enter the new protrusions. Treatment of cells with colchicine and Taxol do not inhibit neomycin-induced protrusion but protrusions are no longer localized at the ends of cell processes and occur all around the cell periphery. We conclude that actin filaments are the major component of the cytoskeleton involved in generating protrusions. Microtubules and, possibly, intermediate filaments control the pattern of protrusions by their interaction with actin filaments.     

Organization of the cytoskeleton and the focal contacts of bovine aortic endothelial cells cultured on type I and III collagen.

We investigated the organization of the cytoskeleton and the focal contacts of bovine aortic endothelial cells cultured on type I and III collagen. The influence of these collagens on cell morphology and the distribution pattern of actin, vimentin, talin, and vinculin was analyzed by light microscopy, conventional electron microscopy, immunofluorescence, and immunogold labeling after lysis-squirting. Whereas the morphology of the endothelial cells is not markedly influenced, the structure of the cytoskeleton and the focal contacts of the cells are altered by the different collagen types. Stress fibers are more distinct in cells grown on type I collagen; cells on type III collagen show a more diffuse distribution of actin molecules. Intermediate filaments seem not to be affected by the collagens. The areas of focal contacts are larger in cells on type I collagen. Additionally, the labeling pattern of talin and vinculin is denser in focal contacts of cells grown on type I collagen. These results suggest an important role of the type of collagen in mediation of the organization of the microfilament system and the adhesion structures of bovine aortic endothelial cells in culture.     

Senescence-associated alterations of cytoskeleton: extraordinary production of vimentin that anchors cytoplasmic p53 in senescent human fibroblasts

The cytoskeleton of senescent cells was systematically studied using senescent and young fibroblasts. In the cell senescence, skin fibroblasts extraordinarily produced vimentin in contrast to actin and tubulin, which were down-regulated. Among the focal adhesion proteins, paxillin and c-Src decreased also. Senescent cells developed a long and dense vimentin network, long and thin actin fibers, and numerous small focal contact sites, which contrasted with young cells with short and thick actin stress fibers and prominently large focal adhesions. Noticeably, senescent fibroblasts markedly produced p53 molecules and anchored them to vimentin-cytoskeleton in the cytoplasm. The vimentin-anchored p53 was detected with antibody PAb240 that specifically recognizes a conformation variant of p53. A GFP-tagged wild type p53 cDNA was expressed by transfection and shown also to be retained in the cytoplasm in senescent cells, suggesting that p53 is structurally modified to be recognized by PAb240 and anchored to vimentin filaments. We discuss the correlation of the marked alteration of cytoskeleton and senescent cells diminished proliferation and migration, as well as the significance of cytoskeletal anchorage of tumor suppressor p53.     

Antibodies to tubulin and actin bind to the surface of a human monocytic cell line, U937

Intermittent reports of cytoskeleton proteins (actin and tubulin) on the cell surface have appeared over the last 13 years. Whereas most have concentrated on lymphocytes, this study provides evidence for the presence of these proteins on the surface of a human cultured monocyte-like cell line, U937. Both actin and tubulin were detected on the surface of U937 cells by flow cytometry, using an indirect staining procedure based on biotin-streptavidin-phycoerythrin, chosen for greater sensitivity. By use of this procedure, the majority of viable unstimulated U937 cells stained positively for actin and tubulin, although the level of fluorescence intensity was low. With an antibody specific for tyrosine-tubulin, most of the surface tubulin was also found to be tyrosinylated. For vimentin, an intermediate filament protein abundantly present in the cytoplasm of U937 cells, no staining could be detected. Confirmation of the flow cytometry data for surface actin and tubulin on unstimulated U937 cells was achieved by direct vesualization using a confocal laser scanning microscope. When U937 cells were activated with PMA and LPS, a marked reduction in the level of cell surface actin and tubulin occurred. The role of cell surface actin and tubulin on unstimulated U937 cells, in terms of monocyte function, remains to be elucidated.     

Integrating the actin and vimentin cytoskeletons. adhesion-dependent formation of fimbrin-vimentin complexes in macrophages.

Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that fimbrin interacts with a vimentin subunit. A fimbrin-binding site was identified in the NH(2)-terminal domain of vimentin and the vimentin binding site at residues 143-188 in the CH1 domain of fimbrin. Based on these observations, we propose that a fimbrin-vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.     

Endothelin-1 combined with extracellular matrix proteins promotes the adhesion and chemotaxis of amelanotic melanocytes from human hair follicles in vitro

During the repigmentation of vitiliginous skin, amelanotic melanocytes (AMMCs) migrate from the outer root sheath (ORS) of the hair follicles into depigmented skin. It has been shown that endothelin-1 (ET-1) is an important cytokine in the migration of epidermal melanocytes, produced by keratinocytes particularly after irradiation with ultraviolet B (UVB). To further examine the role of ET-1 on the migration of AMMCs, we investigated the effects of ET-1 to the adhesion and chemotaxis of human AMMCs combined with extracellular matrix proteins (ECMP) and observed the effects on the actin and tubulin cytoskeleton of AMMC by ET-1. Human AMMCs were treated with different concentrations of ET-1 (0.1-100 nM) to observe adhesion on culture dishes coated with fibronectin (FN), laminin (LN) and collagen IV (CIV). In addition, chemotaxis on FN, LN and CIV coated micropore filters, with various concentrations of ET-1 as attractants, was investigated using a Boyden chemotaxis chamber. Cellular microfilaments and microtubules were immunostained with Rhodamine labeled actin and FITC labeled beta-tubulin. The effects of ET-1 on cytoskeleton were observed with laser confocal microscopy. The study demonstrated that ET-1 increases human AMMCs adhesion on FN in a dose-dependent manner, but minor increases are found on the coated surface with LN and CIV. ET-1 also induces chemotaxis of AMMCs on CIV, LN and FN in a dose-dependent manner. The greatest effect was seen with CIV. Minor chemotactic effects were observed with non-coated surfaces. A concentration of >or=10nM ET-1 induced an apparent increase in stress fibers underneath the cell membrane, but no effects were found on tubulin. ET-1 has various effects on the adhesion and chemotaxis of AMMCs on various ECMP, which could be partly due to a modulation and reorganization of the actin cytoskeleton.     

Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells: alterations underlying surface bleb formation

Several in vitro and in vivo studies have suggested that surface bleb formation during oxidative cell injury is related to alteration in cytoskeleton organization. Various cell lines different in origin and growth characteristics were exposed to 2-methyl-1,4-naphthoquinone (menadione) which is known to induce bleb formation and cytotoxicity by generating considerable amounts of oxygen-reactive species. Treated cells were analyzed by means of immunocytochemistry and electron microscopy in order to investigate the morphological and molecular features underlying bleb generation. The results obtained indicate that menadione-induced bleb formation is a widely observed phenomenon present mainly in round or mitotic cells. Surface blebs appear free of organelles and contain only few ribosomes and amorphous material. Occasionally, they undergo detachment from the cell surface as large cytoplasmic vesicles. Bleb surfaces with protein clusters as well as bald blisters with an almost exclusive localization of intramembrane particles on their narrow base were detected using freeze-fracture techniques. Immunocytochemical investigations performed on menadione-exposed cells revealed that some surface proteins (collagen IV, sialo-proteins, beta 2 microglobulin and fibronectin) and adhesion molecules (vinculin) underwent changes in their expression over the bleb surface. Moreover, different behavioural characteristics of actin microfilaments, vimentin and keratin intermediate filaments and microtubules was observed. Alpha-actinin, vimentin and microtubular proteins (tubulin, MAPs and tau) were detected within the blebs. On the other hand, actin and keratin filaments appeared to be absent. The results presented here demonstrate that cytoskeletal structures and the microfilament system in particular, represent important targets in menadione-induced morphological changes in cultured cells. These changes appear to lead to the redistribution of several cytoskeletal and membrane proteins as well as dissociation of the cytoskeleton network from its anchoring domains in the plasma membrane thus generating sites of structural weakness where blebs would arise and progressively grow. Experimental evidence supporting a crucial role of thiol oxidation and elevation of cytoplasmic calcium concentration in bleb formation is also provided.     

Effect of extracellular matrix on adhesion, viability, actin cytoskeleton and K+ currents of cells expressing human ether à go-go channels

Ether à go-go (EAG) potassium channels possess oncogenic properties and have gained great interest as research tools for cancer detection and therapy. Besides, EAG electrophysiological properties are regulated through the cell cycle and determined by cytoskeletal interactions. Thus, because of the pivotal role of extracellular matrix (ECM) and cytoskeleton in cancer progression, we studied the effect of ECM components on adhesion, viability, actin organization and EAG currents in wild-type CHO cells (CHO-wt) and cells expressing human EAG channels (CHO-hEAG). At short incubation times, adhesion and viability of CHO-hEAG cells grown on collagen, heparin or poly-lysine were lower than CHO-wt cells, however, only CHO-hEAG sustained growing under total serum starvation. CHO-hEAG cells grown on poly-lysine did not organize their cytoskeleton but when grown on collagen or fibronectin displayed lamellipodia and stress fibers, respectively. Interestingly, EAG expressing cells displayed special actin structures suggesting a dynamic actin cytoskeleton, such structures were not exhibited by wild-type cells. EAG current density was significantly lower in cells grown on collagen at short incubation times. Finally, we studied potential associations between hEAG channels and integrins or actin filaments by confocal microscopy. No association between beta1-integrins and hEAG channels was found, however, a very strong co-localization was observed between hEAG channels and actin filaments, supported by immunoblot experiments in which hEAG channels were found in the insoluble fraction (associated to cytoskeleton). Our results suggest ECM components as potential modulators of oncogenic human-EAG expressing cells and emphasize the relationship between potassium channels, cytoskeleton, ECM and cancer.     

Reduced number and LH content of marginated secretory granules in pituitary gonadotropes of streptozocin-induced diabetic male rats.

The transport of secretory granules towards the subplasmalemmal (SPL) region of the luteinizing-hormone (LH) gonadotrope is controlled by the LH-releasing-hormone-dependent pathway. The SPL granules contain the most readily releasable LH. To test the effect of diabetes on both the number and LH content of marginated granules, we studied by indirect immunogold-labelling pituitaries from control and streptozocin (STZ)-treated male rats. On electron micrographs we measured the areas of the gonadotrope nucleus and cytoplasm, counted all secretory granules, and counted and measured secretory granules in selected SPL regions. Furthermore, we counted gold particles (IG) on (a) the secretory granules of the SPL regions, (b) the intergranular SPL cytoplasm and (c) the region outside the cell. Finally, in order to evaluate possible diabetes-related changes of the pituicyte cytoskeleton, we measured by densitometry actin, tubulin, vimentin, and desmin in immunocytochemically stained pituitary sections. In diabetic compared with control cells of the studied pituitary region, we observed: (a) cytoplasmic atrophy; (b) the number of secretory granules per unit area increased in the total cytoplasm, and decreased in the SPL cytoplasm (lowered regulated secretion); (c) decreased IG labelling in the SPL granules (reduced amount of hormone transported by each granule towards the cell membrane); (d) decreased IG labelling in the integranular SPL cytoplasm (reduced constitutive secretion), and (e) strongly increased actin and desmin, yet unchanged tubulin and vimentin immunoreactivity. Our data indicate that both regulated and constitutive secretion are possibly reduced in gonadotropes of diabetic male rats. The cytoskeletal alterations may also contribute to the reduced regulated secretion.     

Regulation of the association of alpha 6 beta 4 with vimentin intermediate filaments in endothelial cells

The adhesion of microvascular endothelial cells to their underlying basement membrane is important for the maintenance of vascular integrity. Most integrins function in endothelial cell adhesion by forming a transmembrane link between their basement membrane ligand and the actin microfilament cytoskeleton. The alpha 6 beta 4 laminin-binding integrin, however, associates with vimentin intermediate filaments (IFs) in microvascular endothelial cells and therefore is likely to uniquely contribute to the barrier function of the endothelium. In this study, we examined the regulation of alpha 6 beta 4-vimentin IF association. We first tested the requirement for alpha 6 beta 4-laminin interactions and actin microfilament assembly. We found that alpha 6 beta 4 associated with vimentin IFs when cells were adherent to either laminin 5 or fibronectin, indicating that this association can occur independent of alpha 6 beta 4-ligand interactions. Additionally, we found that alpha 6 beta 4 was associated with vimentin IFs prior to cell spreading, indicating that changes in the microfilament cytoskeleton associated with changes in cell shape are also not required. Thus, although the association of alpha 6 beta 4 with vimentin IFs may strengthen cell adhesion by providing endothelial cells with an additional transmembrane linkage between the basement membrane and the cytoskeleton, this association is not itself regulated by alpha 6 beta 4-mediated adhesion. Finally, we tested the role of plectin in the association of alpha 6 beta 4 with vimentin IFs. Plectin is known to bind in vitro to both IFs and the beta 4 cytoplasmic domain (beta 4 tail), suggesting that it may be important for this linkage. Therefore, we generated deletion mutants of the beta 4 tail and compared the ability of alpha 6 beta 4 containing these deletions to associate with vimentin IFs. We targeted the two regions of the beta 4 tail known to bind to plectin IN VITRO: the N-terminal and C-terminal plectin binding sites. We found that deletion of the N-terminal binding site inhibited the association of alpha 6 beta 4 with vimentin IFs. Thus, plectin-beta 4 tail interactions may play an important role in connecting alpha 6 beta 4 with vimentin IFs and may prove to be important targets in the regulation of this association in endothelial cells.     

Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.     

Role of extracellular matrix-cell interaction and epidermal growth factor (EGF) on EGF-receptors and actin cytoskeleton arrangement in infantile pituitary cells

Epidermal growth factor (EGF) induces changes in cell morphology, actin cytoskeleton, and adhesion processes in cultured infantile pituitary cells. The extracellular matrix, through integrin engagement, collaborates with growth factors in cell signaling. We have examined the participation of collagen I/III and collagen plus fibronectin in the EGF response of infantile pituitary cells with respect to their cell morphology and actin cytoskeleton. As a comparison, we have used poly-lysine as a substrate. Infantile cells elicit the EGF response when they are associated with extracellular matrix proteins, but no response can be obtained with poly-lysine as the substrate. Cells acquire a flattened shape and organize their actin filaments and vinculin as in focal adhesions. Because the EGF receptor (EGFR) is linked to the actin cytoskeleton in other cells structuring a microdomain in cell signaling, we have investigated this association and substrate adhesion participation in infantile pituitary cells. The proportion of EGFR associated with the actin cytoskeleton is approximately 31%; no difference has been observed between the substrates used. Cells in suspension show actin-associated EGFR, suggesting an association independent of cell adhesion. However, no colocalization of EGFRs with actin fibers has been observed, suggesting an indirect association. Compared with β₁-integrin, which is linked to actin fibers through structural proteins, EGFR binds more strongly with the actin cytoskeleton. This study thus shows cell adhesion dependence on the EGF effect in the actin cytoskeleton arrangement; this is probably favored by the actin fiber/EGFR association that facilitates the cell signaling pathways for actin cytoskeleton organization in infantile pituitary cells.This work was supported by the National Council of Science and Technology of México (grant 44619, and a fellowship to C.T.).     

Essential role of SRC suppressed C kinase substrates in Schwann cells adhesion, spreading and migration

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, while the responses of Schwann cells during adhesion and migration are unknown, so we examined the expression changes of SSeCKS and F-actin in Schwann cells after exposure to fibronectin. Src (sarcoma) suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after Schwann cells adhesion and that SSeCKS increased during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we showed that Schwann cells in which SSeCKS expression was inhibited reduced cellular adhesion, spreading and promoted cellular migration on fibronectin through reorganization of actin stress fibers and blocking formation of focal adhesions. These results demonstrated SSeCKS modulate Schwann cells adhesion, spreading and migration by reorganization of the actin cytoskeleton.     

Changes in Organization and Axonal Transport of Cytoskeletal Proteins During Regeneration

Changes in solubility and transport rate of cytoskeletal proteins during regeneration were studied in the motor fibers of the rat sciatic nerve. Nerves were injured by freezing at the midthigh level either 1-2 weeks before (experiment I) or 1 week after radioactive labeling of the spinal cord with L-[35S]methionine (experiment II). Labeled proteins in 6-mm consecutive segments of the nerve 2 weeks after labeling were analyzed following fractionation into soluble and insoluble populations with 1% Triton at 4 degrees C. When axonal transport of newly synthesized cytoskeleton was examined in the regenerating nerve in experiment I, a new faster component enriched in soluble tubulin and actin was observed that was not present in the control nerve. The rate of the slower main component containing most of the insoluble tubulin and actin together with neurofilament proteins was not affected. A smaller but significant peak of radioactivity enriched in soluble tubulin and actin was also detected ahead of the main peak when the response of the preexisting cytoskeleton was examined in experiment II. It is thus concluded that during regeneration changes in the organization take place in both the newly synthesized and the preexisting axonal cytoskeleton, resulting in a selective acceleration in rate of transport of soluble tubulin and actin.     

Role of caveolin-1 and cytoskeletal proteins, actin and vimentin, in adipogenesis of bovine intramuscular preadipocyte cells

We investigated the involvement of caveolin-1 and the cytoskeletal proteins, actin and vimentin, in the adipogenesis of bovine intramuscular preadipocyte (BIP) cells. Immunoblot analysis demonstrated that levels of caveolin-1 and actin gradually increased during adipose conversion in BIP cells, whereas a slight decrease was observed for vimentin. We found that part of the vimentin was clearly distributed to caveolin-1-enriched membrane fractions in BIP cells, but actin was not. During adipogenesis of BIP cells, treatment with the tubulin depolymerizer, nocodazole, significantly increased intracellular triglyceride accumulation compared to non-treated cells. Immunocytochemical analysis showed that actin microfilaments were significantly disrupted in nocodazole-treated cells. Also, a decrease in the localization of vimentin in caveolin-1-enriched fractions and a failure of vimentin to co-immunoisolate with caveolin-1 were observed in nocodazole-treated cells. These results suggest that a rearrangement of cytoskeletal proteins has a role in the intracellular accumulation of lipid droplets during adipogenesis of BIP cells.     

Cell-matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan

Normal human skin fibroblasts were grown in a three-dimensional collagen gel or in monolayer in the presence or absence of high molecular weight hyaluronan (HA) to assess the influence of extracellular HA on cell-matrix interactions. HA incorporated into the collagen gel or added to the culture medium did not modify lattice retraction with time. The effect was independent from HA molecular weight (from 7.5 x 10(5) to 2.7 x 10(6) Da) and concentration (from 0.1 up to 1 mg/ml). HA did not affect shape and distribution of fibroblasts within the gel, whereas it induced the actin filaments to organise into thicker cables running underneath the plasma membrane. The same phenomenon was observed in fibroblasts grown in monolayer. By contrast, vimentin cytoskeleton and cell-substrate focal adhesions were not modified by exogenous HA. The number of fibroblasts attached to HA-coated dishes was always significantly lower compared to plastic and to collagen type I-coated plates. By contrast, adhesion was not affected by soluble HA added to the medium nor by anti-CD44 and anti-RHAMM-IHABP polyclonals. After 24-h seeding on collagen type I or on plastic, cells were large and spread. Conversely, cells adherent to HA-coated surfaces were long, thin and aligned into rows; alcian blue showed that cells were attached to the plastic in between HA bundles. Therefore, normal human skin fibroblasts exhibit very scarce, if any, adhesion to matrix HA, either soluble or immobilised. Moreover, even at high concentration, HA molecules do not exert any visco-mechanical effect on lattice retraction and do not interfere with fibroblast-collagen interactions nor with focal adhesion contacts of fibroblasts with the substrate. This is probably relevant in organogenesis and wound repair. By contrast, HA greatly modifies the organisation of the actin cytoskeleton, suggesting that CD44-mediated signal transduction by HA may affect cell locomotion and orientation, as indicated by the fusiform shape of fibroblasts grown in the presence of immobilised HA. A role of HA in cell orientation could be relevant for the deposition of collagen fibrils in regeneration and tissue remodelling.     

Cholesterol oxides mediated changes in cytoskeletal organisation involves Rho GTPases small star, filled

The small GTPases Rho, Rac, and Cdc42 regulate the actin cytoskeleton in all eukaryotic cells. In this study we have evaluated the effect of cholesterol oxides (7-ketocholesterol and 25-hydroxycholesterol) on cell migration, cell adhesion, and cytoskeletal organisation of lens epithelial cells (LEC). Effects of cholesterol oxides on cytoskeleton were evaluated by immunofluorescence confocal microscopy. The 7-ketocholesterol induced cell arborisation, with bundling of vimentin and tubulin in the cell processes and formation of filopodia and stress fibres. Cells treated with 25-hydroxycholesterol showed a collapse of vimentin filaments towards the nucleus and formation of lamellipodia. In addition, cells treated with 7-ketocholesterol or 25-hydroxycholesterol showed decreased migration. The effects of cholesterol oxides on cytoskeletal proteins involve the activation of the small GTPases Rho, Rac, and Cdc42. Indeed, formation of both filopodia and stress fibres induced by 7-ketocholesterol is inhibited by overexpressing dominant negatives forms of Cdc42 and RhoA, respectively. Similarly, the collapse of vimentin intermediate filament network and the formation of lamellipodia, induced by 25-hydroxycholesterol, is inhibited by overexpressing dominant negatives forms of Rac1. The effects of cholesterol oxides described in this study for LEC are also observed for at least two other cell lines (H36CE and U373), suggesting that this may represent a general mechanism whereby cholesterol oxides induces cytoskeletal disorganisation.     

Mapping the dynamics of shear stress-induced structural changes in endothelial cells

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.