Cytoskeletal Morphology Research Articles

Quantitative kinetics of damage and recovery of cytoskeletal structure by means of image analysis

The cytoskeleton is a network of proteins which structurally and dynamically organise the cytoplasm of living cells. Microtubules are among its constituents. Morphological alterations of microtubules are related to functional impairment. Therefore cytoskeletal morphology is a valuable indicator of cell injury and functionality. This paper focuses on the comparison between normal and altered cytoskeletal microtubules by means of image analysis and classification with the aim of replacing visual assessment. Morphology has been quantified by the extraction of some descriptors yielded by spatial differentiation, fractal analysis and Fourier analysis followed by non-linear filtering. The principal component analysis of these descriptors has led to image recognition and has been applied to hepatocytes and fibroblasts exposed to some xenobiotics. In the case of hepatocytes, images have been ranked according to the severity of cytoskeletal damage, a dose–response relation has been derived from the regression of the first principal component and the percentage of structural recovery after exposure has been estimated.

Structural polymorphism of the cytoskeleton: A model of linker-assisted filament aggregation

The phase behavior of charged rods in the presence of interrod linkers is studied theoretically as a model for the equilibrium behavior underlying the organization of actin filaments by linker proteins in the cytoskeleton. The presence of linkers in the solution modifies the effective interrod interaction and can lead to interfilament attraction. Depending on the composition and physical properties of the system, such as linker-binding energies, filaments will orient either perpendicular or parallel to each other, leading to network-like or bundled structures. We show that such a system can have one of three generic phase diagrams, one dominated by bundles, another by networks, and the third containing both bundle and network-like phases. The first two diagrams can be found over a wide range of interaction energies, whereas the third diagram occurs only for a narrow range. These results provide theoretical understanding of the classification of linker proteins as bundling proteins or crosslinking proteins. In addition, they suggest possible mechanisms by which the cell may control cytoskeletal morphology.

GPR56 is a GPCR that is overexpressed in gliomas and functions in tumor cell adhesion

GPR56 (also known as TM7XN1) is a newly discovered orphan G-protein-coupled receptor (GPCR) of the secretin family that has a role in the development of neural progenitor cells and has been linked to developmental malformations of the human brain. GPR56 diverges from other secretin-like family members in that it has an extremely large N-terminal extracellular region (381 amino acids) and contains a novel feature among this new subclass, consisting of four cysteine residues that define a GPCR proteolytic site (GPS motif) located just before the first transmembrane spanning domain. The rest of the amino-terminal domain contains a large number of possible N- and O-linked glycosylation sites similar to mucin-like proteins. These features suggest a role in cell-cell, or cell-matrix interactions. Here, we demonstrate upregulation of GPR56 in glioblastoma multiforme tumors using functional genomics. Immunohistochemistry studies confirmed the expression of GPR56 protein in a majority of glioblastoma/astrocytoma tumor samples with undetectable levels of expression in normal adult brain tissue. Immunofluorescence analysis of human glioma cells using anti-GPR56 antibodies demonstrate that GPR56 is expressed on the leading edge of membrane filopodia and colocalizes with alpha-actinin. Purified recombinant GPR56 extracellular domain protein inhibits glioma cell adhesion and causes abnormal cytoskeletal morphology and cell rounding. These results indicate that the extracellular domain may compete for unidentified ligand(s), and block the normal function of GPR56 in cell attachment. In reporter assays, overexpression of GPR56 activates the NF-kappaB, PAI-1 and TCF transcriptional response elements. These pathways have been implicated in cytoskeletal signaling, adhesion and tumor biology. The above results indicate that GPR56 serves as an adhesion GPCR and is involved in adhesion signaling.

Inhibitory effects of anticancer peptide from Mercenaria on the BGC-823 cells and several enzymes

An anti-cancer peptide was purified from the Mercenaria (Meretrix meretrix Linnaeus) by the method of chromatography on Sephadex G-25 and FPLC, and its molecular weight was determined to be 3147Da by the way of MALDI-TOF mass spectrum. The effects of this peptide on human gastric gland carcinoma cells (BGC-823) and their cytoskeletal morphology were investigated. The results showed that the peptide could inhibit the proliferation of BGC-823 cells and obviously destroy the skeletal structures of the cells. When the concentration of the peptide reached 4.0μg/ml, the inhibition percentage of the cell growth was about 60%. The effects of this anticancer peptide on the activities of superoxide dismutase (SOD), alkaline phosphatase (ALP) and tyrosinase were studied. The results showed that the peptide activated ALP and SOD, but inhibit the tyrosinase activity. When the concentration of the peptide reached to 0.5μg/ml, the relative activities of SOD, ALP and tyrosinase were determined to be 188.5%, 122.0% and 27.5%, respectively.

Small G-protein Rho is involved in the maintenance of cardiac myocyte morphology

The use of small membrane-permeable sequences or protein transduction domains (PTDs) can facilitate the transport of proteins into many cell types. In preliminary studies with the application of three PTDs (penetratin, modified penetratin, and the HIV TAT transduction domains) to cardiac myocytes, we found that the TAT and penetratin sequences showed high efficiency of uptake and low toxicity. Rho has been previously shown to be an important regulator of cytoskeletal organization and morphology in other non-cardiac cell types. To evaluate a role for Rho in cardiac myocyte morphology, we used the TAT-PTD to deliver a RhoA-specific inhibitor, the C3 exoenzyme, to cultured cardiac myocytes. We showed that this incubation with TAT-C3 abolished the basal levels of RhoA activity, demonstrating the efficacy of this treatment. Incubation with TAT-C3 also altered cardiac myocyte morphology so that TAT-C3-treated cells produced multiple projections from the major cell body. This was accompanied by a statistically significant increase in cell size, albeit to a lesser extent than the changes accompanying exposure to the hypertrophic agent, endothelin-1. Furthermore, the change in size of TAT-C3-treated cells was not accompanied by the induction of atrial natriuretic factor (ANF) expression that accompanies the hypertrophy of cardiac myocytes. These results reveal a role for RhoA in the maintenance of normal myocyte morphology.

The emergence of integrins: a personal and historical perspective.

The emergence of integrins: a personal and historical perspective.

Macrophages from lupus-prone MRL mice are characterized by abnormalities in Rho activity, cytoskeletal organization, and adhesiveness to extracellular matrix proteins.

Macrophages (mphi) from prediseased mice of the major murine models of lupus have an identical defect in cytokine expression that is triggered by serum and/or apoptotic cells. It is striking that cytokine expression in the absence of serum and apoptotic cells is equivalent to that of nonautoimmune mice. Here, we show that mphi from prediseased lupus-prone MRL/MpJ (MRL/+) or MRL/MpJ-Tnfrsf6(lpr) (MRL/lpr) mice also have reversible abnormalities in morphology, cytoskeletal organization, and adhesive properties. In the presence of serum, MRL mphi adhered in increased numbers to a variety of extracellular matrix proteins compared with mphi from two nonautoimmune strains. However, in the absence of serum, adhesion by MRL mphi was similar to that of nonautoimmune mphi. Increased adhesion by MRL mphi was also observed in the presence of apoptotic, but not necrotic, cells. The morphology and actin-staining pattern of adherent MRL mphi were consistent with reduced activity of Rho, a cytoskeletal regulator. Indeed, MRL mphi cultured in the presence of serum had markedly decreased levels of active Rho compared with nonautoimmune mphi. It is remarkable that when cultured in the absence of serum, MRL mphi displayed normal Rho activity and cytoskeletal morphology. Addition of a Rho inhibitor to normal mphi reproduced the morphologic and cytoskeletal abnormalities observed in MRL mphi. Taken together, our findings support the hypothesis that mphi from MRL and other systemic lupus erythematosus-prone mice have an apoptotic, cell-dependent, autoimmune phenotype that affects a broad range of mphi functions, including cytokine gene expression and Rho-dependent cytoskeletal regulation.

Effect of cyclic axial stretch of rat arteries on endothelial cytoskeletal morphology and vascular reactivity

Pulsatile fluid shear stress and circumferential stretch are responsible for the axial alignment of vascular endothelial cells and their actin stress fibers in vivo. We studied the effect of cyclic alterations in axial stretch independent of flow on endothelial cytoskeletal organization in intact arteries and determined if functional alterations accompanied morphologic alterations. Rat renal arteries were axially stretched (20%, 0.5 Hz) around their in vivo lengths, for up to 4 h. Actin stress fibers were examined by immunofluorescent staining. We found that cyclic axial stretching of intact vessels under normal transmural pressure in the absence of shear stress induces within a few hours realignment of endothelial actin stress fibers toward the circumferential direction. Concomitant with this morphologic alteration, the sensitivity (log(EC 50)) to the endothelium-dependent vasodilator (acetylcholine) was significantly decreased in the stretched vessels (after stretching –5.15±0.79 and before stretching –6.71±0.78, resp.), while there was no difference in sodium nitroprusside (SNP) sensitivity. There was no difference in sensitivity to both acetylcholine and SNP in time control vessels. Similar to cultured cells, endothelial cells in intact vessels subjected to cyclic stretching reorganize their actin filaments almost perpendicular to the stretching direction. Accompanying this morphological alteration is a loss of endothelium-dependent vasodilation but not of smooth muscle responsiveness.

Pineal peptide with an inhibiting effect on growth of HeLa S3 tumor cells

The aim of the present study was to assess whether a peptide fraction isolated from calf pineal glands has an effect on proliferation and morphology of HeLa S3 tumor cells. Under the experimental conditions adopted, the results showed that the peptide has a marked inhibitory effect on proliferation of HeLa S3 cells and that permeabilization with calcium phosphate of the plasmatic membrane increases this effect. Moreover, the pineal peptide affects the cytoskeletal morphology of HeLa cells by modifying the distribution of actin. The peptide is probably internalized by the cells and irreversibly modifies the cytoskeletal morphology with consequent inhibition of cellular proliferation.

A functional genomic analysis of cell morphology using RNA interference

BackgroundThe diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies.ResultsWe adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA.ConclusionsUsing RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.

The Cytoskeleton, apoptosis, and gene expression in T lymphocytes and other mammalian cells exposed to altered gravity

Lymphocytes exposed to spaceflight or altered gravity exhibit significant growth attenuation and changes in metabolism, cytoskeletal structure, and gene expression. A significant body of evidence is accumulating to implicate the cytoskeleton in the atypical response of lymphocytes in microgravity. This chapter reviews the effects of spaceflight and altered gravity on cytoskeletal morphology and expression of cytoskeletal genes in T lymphocytes and established T-cell lines. Other mammalian cell types are included for comparison. The effects of simulated shuttle launch vibration are discussed based on research showing that vibration during launch may disrupt the cytoskeleton and alter gene expression. Ground-based vibration studies provide baseline information for distinguishing effects of vibration from effects of the microgravity environment per se. As cytoskeletal disruption is one of several factors known to exacerbate apoptosis, and because of the apparent increase in programmed cell death in space-flown cells, apoptosis is also discussed in the chapter.

Thrombin-mediated permeability of human microvascular pulmonary endothelial cells is calcium dependent.

In response to inflammation, endothelial cytoskeleton rearrangement, cell contraction, and intercellular gap formation contribute to a loss of capillary barrier integrity and resultant interstitial edema formation. The intracellular signals controlling these events are thought to be dependent on intracellular calcium concentration ([Ca2+]i). We hypothesized that, in human pulmonary microvascular endothelial cells, a thrombin-induced increase in permeability to albumin would be dependent on Ca2+i and subsequent actin cytoskeleton rearrangements. Human lung microvascular endothelial cells, grown on 0.4 micromol/L pore membranes, were activated with 10 nmol/L human thrombin in Hank's balanced salt solution/0.5% fetal bovine serum. Select cultures were pretreated (45 minutes) with 4 micromol Fura-2/AM to chelate Ca2+i. Permeability was assessed as diffusion of bovine serum albumin/biotin across the monolayer. Similarly treated cells were stained with rhodamine-phalloidin to demonstrate actin cytoskeletal morphology. Separately, cells loaded 2 micromol Fura-2/AM were assessed at OD340/380nm after thrombin exposure to detect free Ca2+i. Intracellular Ca2+ levels increased 15-fold (2 seconds) and fell to baseline (10 minutes) after thrombin. Permeability increased 10-fold (30 minutes), and a shift from cortical to actin stress fiber morphology was observed. Chelation of Ca2+i diminished permeability to baseline and reduced the percentage of cells exhibiting stress fiber formation. Thrombin stimulates pulmonary capillary leak by affecting the barrier function of activated pulmonary endothelial cells. These data demonstrate a thrombin-stimulated increase in monolayer permeability, and cytoskeletal F-actin stress fibers were, in part, regulated by endothelial Ca2+i. This early, transient rise in Ca2+i likely activates downstream pathways that more directly affect the intracellular endothelial structural changes that control vascular integrity.

Anodic oxidation and hydrothermal treatment of titanium results in a surface that causes increased attachment and altered cytoskeletal morphology of rat bone marrow stromal cells in vitro.

Previous studies have suggested the usefulness of a new coating method-namely, the forming of a thin hydroxyapatite (HA) layer on commercially pure titanium (cpTi) by anodization and hydrothermal treatment-for use as a dental root implant material. In vivo and in vitro studies confirmed that an HA layer on cpTi (HA/cpTi) implants showed good compatibility with bone tissue, rat bone marrow stromal (RBM) cells, and immune cells. The aim of the present investigation was to further characterize the in vitro early cellular behavior of RBM cells on HA/cpTi implants. Therefore, in this study we performed surface analysis, analysis of cell initial attachment, and analysis of cell morphology and the cytoskeleton. Drops of distilled water or cell culture medium showed smaller contact angles with HA/cpTi than with cpTi. RBM cells were cultured for 30, 60, and 120 min on HA/cpTi and cpTi, and the level of cell adhesion was shown to increase with time on both substrates. However, cell adhesion on HA/cpTi was significantly higher than on cpTi at 60 and 120 min. Especially at 120 min, when compared with cpTi, the cell morphology on the surface of HA/cpTi not only adopted a flattened and spreading form, but also extended filopodium-like processes with irregular edges that were intimately adapted to the surface of the HA microcrystals. The cytoskeleton on HA/cpTi showed well-formed actin filaments that were parallel to each other and the long axis of RBM cells. The actin filaments of RBM cells on the HA/cpTi surface were localized to the periphery (corresponding to the edge of the filopodium-like processes) well after 120 min. This suggests that actin filaments of RBM cells need to be anchored at the HA/cpTi surface and the numerous HA microcrystals precipitated on the HA/cpTi surface. These findings were similar to the scanning electron microscopic morphology. The peripheral anchorage provide sufficient strength of attachment to allow recognization of actin filaments upon HA/cpTi. The surface of HA/cpTi was more hydrophilic and exhibited markedly improved wettability compared to untreated cpTi, and higher levels of early cell attachment were observed on surfaces after anodization and hydrothermal treatment than on surfaces with untreated cpTi. The results of in vitro experiments suggest that this new method for forming a thin HA layer on the surface of cpTi could be useful to ensure excellent cellular behavior on implant surfaces. The characterization of cell morphology on the thin HA layer formed by anodization and hydrothermal treatment on cpTi implant material suggests that physicochemical or biological conditioning of the implant surface involves implant surface topography.

Defects in nuclear and cytoskeletal morphology and mitochondrial localization in spermatozoa of mice lacking nectin-2, a component of cell-cell adherens junctions.

Nectin-2 is a cell adhesion molecule encoded by a member of the poliovirus receptor gene family. This family consists of human, monkey, rat, and murine genes that are members of the immunoglobulin gene superfamily. Nectin-2 is a component of cell-cell adherens junctions and interacts with l-afadin, an F-actin-binding protein. Disruption of both alleles of the murine nectin-2 gene resulted in morphologically aberrant spermatozoa with defects in nuclear and cytoskeletal morphology and mitochondrial localization. Homozygous null males are sterile, while homozygous null females, as well as heterozygous males and females, are fertile. The production by nectin-2(-/-) mice of normal numbers of spermatozoa containing wild-type levels of DNA suggests that Nectin-2 functions at a late stage of germ cell development. Consistent with such a role, Nectin-2 is expressed in the testes only during the later stages of spermatogenesis. The structural defects observed in spermatozoa of nectin-2(-/-) mice suggest a role for this protein in organization and reorganization of the cytoskeleton during spermiogenesis.

Tributyltin induces cytoskeletal alterations in the colonial ascidian Botryllus schlosseri phagocytes via interaction with calmodulin

In the colonial ascidian Botryllus schlosseri, tributyltin (TBT), a powerful antifouling biocide, acts as immunotoxic xenobiotic since, at a sublethal concentration (10 μM), it causes an irreversible and significant decrease in in vitro yeast phagocytosis, associated with considerable changes in the shape of phagocytes, which withdraw their pseudopodia and become spherical, due to structural damage of cytoskeletal components. The addition of TBT to the culture medium causes a significant decrease in the amoebocytic index, i.e. the percentage of amoeboid-shaped haemocytes, and prolonged washing in sea water never succeeds in restoring amoeboid shape. In these cytoskeletal alterations, F-actin undergoes extensive depolymerisation, resulting in the absence of FITC-phalloidin fluorescence. Microtubules are not recognisable as single filaments with anti-α-tubulin immunofluorescence, although the centrosome is not affected. The addition of increasing exogenous calmodulin (CaM) concentrations (from 20 to 120 μM) after incubation in TBT determines a significant increase in the amoebocytic index, although it is not able to bring it to that of controls, suggesting that CaM in the medium in any case externally exerts an influence on haemocytes pretreated with TBT. The copresence of TBT and exogenous CaM at concentrations higher than 80 μg/ml restores the amoebocytic index and cytoskeletal morphology. The latter appears complete for microtubules and partial for microfilaments. Experiments with isodynamic mixtures of TBT and specific CaM inhibitors, i.e. chlorpromazine (CPZ) and N-(6-aminohexyl)-5-chloronaphtalene-1-sulfonamide (W-7), reveal the synergistic effect of antagonism, indicating competition for the same site – a Ca 2+-CaM hydrophobic region – by both interacting substances and, therefore, the formation of a TBT-CaM complex. Instead, isodynamic mixtures with thapsigargin, an inhibitor of Ca 2+-ATPase of the endoplasmic reticulum, have an effect of potentiation, suggesting that TBT indirectly interacts with this Ca 2+-ATPase activity. We hypothesise that the main mechanism of action of TBT in B. schlosseri phagocytes is alteration of Ca 2+ homeostasis by means of direct interaction with endogenous CaM, which induces a conformational change preventing the regulative activity of CaM on Ca 2+-ATPase. Consequently, an excess of cytosolic Ca 2+ accumulates which, together with the inhibition of CaM-dependent kinases and Ca 2+-regulated proteins, produces extensive cytoskeletal disorganisation.

Anodic oxidation and hydrothermal treatment of titanium results in a surface that causes increased attachment and altered cytoskeletal morphology of rat bone marrow stromal cellsin vitro

Previous studies have suggested the usefulness of a new coating method-namely, the forming of a thin hydroxyapatite (HA) layer on commercially pure titanium (cpTi) by anodization and hydrothermal treatment-for use as a dental root implant material. In vivo and in vitro studies confirmed that an HA layer on cpTi (HA/cpTi) implants showed good compatibility with bone tissue, rat bone marrow stromal (RBM) cells, and immune cells. The aim of the present investigation was to further characterize the in vitro early cellular behavior of RBM cells on HA/cpTi implants. Therefore, in this study we performed surface analysis, analysis of cell initial attachment, and analysis of cell morphology and the cytoskeleton. Drops of distilled water or cell culture medium showed smaller contact angles with HA/cpTi than with cpTi. RBM cells were cultured for 30, 60, and 120 min on HA/cpTi and cpTi, and the level of cell adhesion was shown to increase with time on both substrates. However, cell adhesion on HA/cpTi was significantly higher than on cpTi at 60 and 120 min. Especially at 120 min, when compared with cpTi, the cell morphology on the surface of HA/cpTi not only adopted a flattened and spreading form, but also extended filopodium-like processes with irregular edges that were intimately adapted to the surface of the HA microcrystals. The cytoskeleton on HA/cpTi showed well-formed actin filaments that were parallel to each other and the long axis of RBM cells. The actin filaments of RBM cells on the HA/cpTi surface were localized to the periphery (corresponding to the edge of the filopodium-like processes) well after 120 min. This suggests that actin filaments of RBM cells need to be anchored at the HA/cpTi surface and the numerous HA microcrystals precipitated on the HA/cpTi surface. These findings were similar to the scanning electron microscopic morphology. The peripheral anchorage provide sufficient strength of attachment to allow recognization of actin filaments upon HA/cpTi. The surface of HA/cpTi was more hydrophilic and exhibited markedly improved wettability compared to untreated cpTi, and higher levels of early cell attachment were observed on surfaces after anodization and hydrothermal treatment than on surfaces with untreated cpTi. The results of in vitro experiments suggest that this new method for forming a thin HA layer on the surface of cpTi could be useful to ensure excellent cellular behavior on implant surfaces. The characterization of cell morphology on the thin HA layer formed by anodization and hydrothermal treatment on cpTi implant material suggests that physicochemical or biological conditioning of the implant surface involves implant surface topography.

Inhibition of Endothelial Cell Migration, Intercellular Communication, and Vascular Tube Formation by Thromboxane A2

The eicosanoid thromboxane A(2) (TXA(2)) is released by activated platelets, monocytes, and the vessel wall and interacts with high affinity receptors expressed in several tissues including endothelium. Whether TXA(2) might alter endothelial migration and tube formation, two determinants of angiogenesis, is unknown. Thus, we investigated the effect of the TXA(2) mimetic [1S-(1alpha, 2beta(5Z),3alpha(1E,3R), 4alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-o xab icyclo- [2.2.1]heptan-2-yl]-5'-heptenoic acid (IBOP) on human endothelial cell (HEC) migration and angiogenesis in vitro. IBOP stimulation inhibited HEC migration by 50% and in vitro capillary formation by 75%. These effects of IBOP were time- and concentration-dependent with an IC(50) of 25 nM. IBOP did not affect integrin expression or cytoskeletal morphology of HEC. Since gap junction-mediated intercellular communication increases in migrating HEC, we determined whether IBOP might inhibit coupling or connexin expression in HEC. IBOP reduced the passage of microinjected dyes between HEC by 50%, and the effects of IBOP on migration and tube formation were mimicked by the gap junction inhibitor 18beta-glycyrrhetinic acid (1 microM) with a similar time course and efficacy. IBOP (24 h) did not affect the expression or phosphorylation of connexin 43 in whole HEC lysates. Immunohistologic examination of HEC suggested that IBOP may impair functional coupling by altering the cellular distribution of gap junctions, leading to increased connexin 43 internalization. Thus, this finding that TXA(2) mimetics can prevent HEC migration and tube formation, possibly by impairing intercellular communication, suggests that antagonizing TXA(2) signaling might enhance vascularization of ischemic tissue.

A balance of signaling by Rho family small GTPases RhoA, Rac1 and Cdc42 coordinates cytoskeletal morphology but not cell survival.

Rho family GTPases are known to be involved in cytoskeletal reorganization. We examined the possibility that these functions may be dictated by a balance of Rho family GTPase signaling. Using transient viral expression of RhoA, Rac1, Cdc42 and their mutants, as well as C3 exoenzyme, we altered cytoskeletal organization under normal growth conditions. Overexpression of wild-type or constitutively active forms of the Rho family GTPases led to their respective activation phenotypes. Overexpression of dominant negative forms of given Rho family GTPases led to a phenotype consistent with activation of the other Rho family GTPase. Treatment with C. difficile toxin A, that inactivates all Rho family GTPases, led to the transient appearance of a variety of activation phenotypes. Previously, we reported that inactivation of Rho led to induction of apoptosis, implying that Rho may play an important role in cell survival signaling. This signaling, however, is not affected by expression of any forms of Rac1 or Cdc42, and only inactivation of Rho led to induction of apoptosis. Rho family GTPases appear to coordinate cytoskeletal organization by a balance of signaling, while cell survival is regulated by a distinct Rho-mediated signaling pathway.

Confocal laser scanning microscopy of chondrocytes in vitro: cytoskeletal changes after quinolone treatment.

The use of quinolone antibiotics would be significant for chronically diseased children (e.g., cystic fibrosis) as a prophylactic long-term treatment. However, quinolones were shown to cause cartilage damage in experimental animals when administered during certain developmental stages. In the present study, the effect of quinolones on chondrocytes was studied in a cell culture model in order to avoid animal experiments, to investigate the influence of single factors, and to open up the possibility to test human tissue. Chondrocytes were obtained from hip joint cartilage of 3 to 4-weeks-old rats and cultured in control medium or quinolone-supplemented medium. It was shown that quinolones heavily disturbed adhesion of chondrocytes to the culture dish, accompanied by changes in cell shape and cytoskeletal morphology. Reduction of filamentous actin (stress fibers) and disintegration of vimentin fibers was demonstrated by immunofluorescence and evaluated by confocal laser scanning microscopy. In contrast, distribution and amount of the adhesion molecule integrin alpha 1 did not change. Results of the present study indicate that quinolones disturb the adherence mechanism of chondrocytes and lead to cytoskeleton changes.

The effects of copper on actin and fibronectin Organization in Mytilus galloprovincialls haemocytes

The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes were studied. The Cu 2+ exposure of mussels caused severe perturbations in haemocyte actin and fibronectin organization with respect to non-exposed organisms. Cytoskeletal actin was analysed by indirect immunofluorescence, using an antitotal actin monoclonal antibody, and by rhodamine-conjugated phalloidin. The majority of haemocytes from Cu 2+-exposed mussels displayed a round morphology, with short and blunt filopodia; they lacked the polarized phenotype which was typical in control samples. The cytoskeleton alteration, more evident after phalloidin staining, resulted in the disappearance of filamentous actin. The actin cortical meshwork also appeared disorganized. The cytoskeletal morphology studied by transmission electron microscopy after negative staining of Triton X-100-treated haemocytes confirmed these observations. The structural organization of actin when analysed by Western blotting showed a larger number of Triton-soluble actin pools in treated mussel haemocytes. Fibronectin was studied by indirect immunofluorescence using a polyclonal antiserum directed against mussel fibronectin. In treated mussels, fibronectin appeared to be strongly disorganized and its levels decreased in both haemocytes and haemolymph. The mechanism(s) of the copper-induced alterations on actin and fibronectin organization in mussel immunocytes is discussed. Copyright