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At least 523 records · Page 29Linked to original sources
PubMed1994

Electron microscopic and new microscopic studies of hepatocyte cytoskeleton: physiological and pathological relevance.

The cytoskeleton is involved ina wide variety of cell functions and therefore is implicated in the pathogenesis of various liver diseases. However, the relationship between localization of the cytoskeleton and hepatocyte function has not yet been fully elucidated. In the present study distribution of the cytoskeleton in relation to the physiology and pathology of hepatocytes was studied by electron microscopy and new microscopic techniques, including confocal laser scanning microscopy, time-lapse video microscopy and high sensitivity digital imaging microscopy. The results suggested the usefulness of the new microscopic techniques in the analysis of pathophysiological roles of cytoskeleton in the liver.

Animals↗
PubMed1994

Phosphorylated HSP27 associates with the activation-dependent cytoskeleton in human platelets.

The three prominently phosphorylated 29-kD proteins in thrombin-activated human platelets are forms of the mammalian 27-kD heat-shock protein (HSP27). Though the function of HSP27 is not yet known, its phosphorylation is highly correlated with platelet secretion, and recent evidence in nonhematopoietic cells suggests that HSP27 regulates cortical actin filament assembly. Therefore, the subcellular location and phosphorylation state of HSP27 in resting and thrombin-activated platelets was studied. Platelets were fractionated by established Triton X-100 lysis methods followed by differential centrifugation to obtain the 14,000g fraction (low-speed cytoskeleton), 100,000g fraction (membrane skeleton), and the 100,000g supernatant fraction containing soluble cytosolic proteins. In resting platelets, HSP27 was present principally in the 100,000g supernatant fraction. Platelet activation with thrombin led to translocation of the majority of HSP27 to the low-speed cytoskeleton. This association was reversible by DNase, supporting the idea that HSP27 is a specific component of the actin cytoskeleton. Immunofluorescence studies similarly showed HSP27 is cytoplasmic in resting platelets but colocalizes with actin in fully spread, glass-activated platelets. Immunoprecipitation studies showed a small amount of constitutively phosphorylated HSP27 in resting platelets, but phosphorylation of the majority of HSP27 after thrombin activation. After activation, virtually all phosphorylated HSP27 was found in the low-speed cytoskeletal fraction, and each of the three phosphorylated forms of HSP27 were present by two-dimensional autoradiography. Furthermore, in time-course studies, the phosphorylation of HSP27 occurred just before localization of HSP27 to the low-speed pellet. These results show that, after platelet activation, HSP27 is first phosphorylated and then translocated from the cytoplasm to the assembling cytoskeleton, and suggest that HSP27 phosphorylation may be important to the binding of HSP27 to cytoskeletal components and the cytoskeletal rearrangements characteristic of platelet activation.

Actins↗
PubMed1994

Rapid and transient translocation of PLC-gamma 1 to the cytoskeleton of thrombin-stimulated platelets. Evidence for a role of tyrosine kinases.

Cytoskeleton reorganization has been suggested to play an important role in platelet signal transduction. A number of signalling molecules are found to relocalize to this fraction upon thrombin stimulation. In this paper, we show that PLC-gamma 1, a key enzyme of the inositol lipid metabolism, is also translocated to the platelet cytoskeleton upon thrombin stimulation. Interestingly, its translocation is very rapid and transient, and correlates with the increase in PLC activity previously measured in the cytoskeleton by our group. Using a potent tyrosine kinase inhibitor, tyrphostin AG-213, we show a significant inhibition of the translocation of PLC-gamma 1, indicating an involvement of tyrosine kinases in its relocation. Thus, our results demonstrate for the first time a rapid and transient tyrosine kinase-dependent translocation of PLC-gamma 1 to the cytoskeleton of thrombin-stimulated platelets.

Biological Transport, Active↗
PubMed1994

Modulation of the axonal microtubule cytoskeleton by myelinating Schwann cells.

The Trembler PNS myelin-deficient mutant mouse offers a unique model for the study of axon-glial interactions. Previous work in our laboratory on Trembler mouse sciatic nerve established that myelinating Schwann cells exert a profound effect on the underlying neuronal cytoskeleton. Demyelinated axon segments exhibited decreases in the rate of slow axonal transport, axonal caliber, and neurofilament phosphorylation, as well as increases in neurofilament density. The present study considers effects on the microtubule cytoskeleton. At least two aspects of the microtubule cytoskeleton in Trembler PNS axons were altered by demyelination. First, the stability of the Trembler axonal microtubule cytoskeleton is decreased, as measured by decreased levels of insoluble tubulin (Sahenk and Brady, 1987). Second, the composition and phosphorylation of axonal microtubule-associated proteins, including tau, MAP 1A, and MAP 1B, are changed in Trembler demyelinated nerves. Further, the fraction of axonal tubulin moving at slow component b rates was increased (de Waegh and Brady 1990, 1991). These results provide further evidence that cell-cell interactions between myelinating glia and their underlying axons extend beyond a structural role, actively influencing biochemical and physiological properties of the axon.

Animals↗
PubMed1994

Cytoskeleton and cell adhesion molecules: critical targets of toxic agents.

Normal development of the nervous system depends upon complex physical interactions between cells and their local environment. These interactions are mediated by several families of cell adhesion molecules (CAMs). Differential expression and function of CAMs are operative in neural tube formation, neuron migration, in post-migratory differentiation, and maintenance of mature neural structure. CAMs also facilitate contact-dependent cell processes, such as formation of cell junctions. Temporal regulation of these molecules during development may provide "windows of vulnerability" to toxicants. In addition to their extracellular binding activities, some CAMs have membrane-spanning domains by which they communicate directly with the cytoskeleton, permitting extracellular signals to be rapidly translated into cell responses via modifications in cytoskeletal organization. These cytologic changes are particularly critical during migration, neurite formation and synaptogenesis. Toxic perturbation of adhesion molecules can have catastrophic effects on morphogenetic processes both directly and via events which depend upon cytoskeletal rearrangement. Toxicants can also act directly upon the cytoskeleton, resulting secondarily in changes of the membrane distribution and function of CAMs. Toxicant-induced changes in CAMs and cytoskeleton may occur contemporaneously. Interference of cell adhesion-cytoskeleton interactions may be a pivotal molecular event dictating developmental consequences of neurotoxicant exposure.

Animals↗
PubMed1993

Hyaluronic acid modulates growth, morphology and cytoskeleton in embryonic chick skin fibroblasts.

The action that hyaluronic acid (HA) exerts on cell proliferation was investigated in embryonic chick skin fibroblasts at different ages (7-14 days) and in different cell-cycle stages evaluated by flow cytometry (cells maintained with and without serum). Proliferation was estimated by 3H-thymidine incorporation and cell counting. The results demonstrated hyaluronic acid inhibits cell multiplication in all different environmental conditions examined. The inhibitory effect of HA is more evident in 14-day than 7-day old fibroblasts. The ability of HA to modulate 3H-thymidine incorporation did not involve a change in the time required for cells entering the S phase of the replicating cycle, but is due to a smaller number of cells entering in this phase. As the relationships between components of the extracellular matrix (ECM) and the cytoskeleton are known, parallel studies were carried out on some cytoskeleton proteins. Furthermore, by modifying the capacity of cells to adhere to the substrate, HA induced alterations in cell shape and in cytoskeleton components involved in these processes. We may hypothesize that HA, binding specific membrane receptors, affects cell adhesion and morphology inducing less receptivity of fibroblasts to mitogenic stimuli by transmembrane interactions with cytoskeleton.

Actinin↗
PubMedDate not supplied

[Cardiomyocyte cytoskeleton in dilated cardiomyopathy and ischemic heart disease].

Summary. Vinculin, the protein of cardiomyocyte cytoskeleton in the myocardium of patients with a dilated cardiomyopathy and ischemic heart disease, was revealed by means of monoclonal antibodies and by a method of indirect immunofluorescence. Cytoskeleton of patients with dilated cardiomyopathy is disorganized, hypertrophic and contains greater amounts of vinculin than cytoskeleton of normal cardiomyocytes. This most likely negatively influences cell contractile capacity. The vinculin content was also higher in cardiomyocyte cytoskeleton of patients with ischemic heart disease but at a less degree than in patients with dilated cardiomyopathy.

Adolescent↗
PubMed1996

Aggregation-dependent, integrin-mediated increases in cytoskeletally associated PtdInsP2 (4,5) levels in human platelets are controlled by translocation of PtdIns 4-P 5-kinase C to the cytoskeleton.

Thrombin-stimulated aggregation of human platelets promotes an increase in the phosphatidylinositol 4-phosphate (PtdIns 4-P) 5-kinase (PIPkin) activity in the cytoskeleton. This phenomenon is associated with translocation of PIPkin isoform C to the cytoskeleton and with an increase in the amount of phosphatidylinositol bisphosphate (PtdInsP2) bound to the cytoskeletal pellet. All three of these effects are prevented if the platelets are not stirred or if RGD-containing peptides are present, demonstrating that they require integrin activation. All three are also abolished by pretreatment with okadaic acid, which also prevents the aggregation-dependent translocation of pp60(c-src) to the cytoskeleton. The results point to the existence of a cytoskeletally associated PtdInsP2 pool under the control of integrin-mediated signals that act via PIPkin C and suggest that a common, okadaic acid-sensitive mechanism may underlie the aggregation-dependent translocation of certain signalling molecules to the platelet cytoskeleton.

Blood Platelets↗
PubMed1996

[The reaction of the cytoskeleton and smooth endoplasmic reticulum of Mauthner's neurons in goldfish to partial denervation and prolonged sensory stimulation].

Structure of smooth endoplasmic reticulum and cytoskeleton of goldfish Mauthner neurons were studied together with the behavior of fish after partial deafferentation of one of the dendrites with following long-term natural stimulation of another one and soma. Strong disturbance of cytoskeleton and proliferation of smooth endoplasmic reticulum especially of its transversely localised component accompanies exhaustion of Mauthner neurons caused by stimulation in normal absolute afferentation. On behavioral level it is manifests in significant suppression of fish motor activity. Similar stimulation of partly deafferentated Mauthner neurons turns to be more sparing both for fish behaviour and Mauthner neurons structure. Proliferation of endoplasmic reticulum was not in fact observed and in the cytoskeleton bundles of filaments were detected which implies improvement of its physico-chemical characteristics and increase of resistance to external factors. Probably, this relative resistance of partly deafferentated Mauthner neurons of fish to long-term natural stimulation is conditioned predominantly by postsynaptic mechanisms and is likely linked with reorganization in cytoskeleton, caused by preliminary change of Mauthner neurons innervation.

Animals↗
PubMed1998

Rap1b association with the platelet cytoskeleton occurs in the absence of glycoproteins IIb/IIIa.

Platelet membrane glycoproteins (GP) IIb/IIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann's thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and raplb is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

Blood Platelets↗
PubMed1998

Stress-induced fractal rearrangement of the endothelial cell cytoskeleton causes apoptosis.

BACKGROUND: Apoptosis, a mechanism of cell death prominent in critical illnesses including disseminated inflammation and multiorgan dysfunction syndrome, is characterized by morphologic changes including cell shrinkage, condensation of organelles, blebbing, and chromatin fragmentation. These phenomena suggest substantial changes in cytoskeletal structure. We hypothesized that stress-induced apoptosis in endothelial cells is, in part, a consequence of a critical cytoskeletal rearrangement. METHODS: Porcine aortic endothelial cells in culture, surrogates for the microvasculature in vivo, were exposed sequentially to Escherichia coli endotoxin (25 micrograms/mL; 18 hours) to induce the inflammatory response and then to sodium arsenite (160 mumol/L; 120 minutes) to induce the heat-shock response, a well-characterized model of stress-induced apoptosis. Laser confocal micrographs of fluorescein isothio-cyanate-labeled phalloidin-stained cells were analyzed to calculate the border fractal dimension of the cytoskeleton. Other cells were exposed to cytochalasin D, a fungal metabolite, which interferes with polymerization of actin from its globular to its filamentous form, and similarly were analyzed with respect to fractal dimension, viability (neutral red assay), and manner of death (annexin V fluorescence-activated cell scanning analysis). RESULTS: Induction of the inflammatory or heat-shock responses caused subtle and distinct rearrangement of the actin cytoskeleton. When these stimuli were applied in sequence, a synergistic interaction led to profound cytoskeletal collapse. Reversal of the sequence did not induce the cytoskeletal disruption. Cytochalasin D alone induced a dose-dependent cytoskeletal collapse indistinguishable from that caused by the acute phase-heat shock sequence that caused cell death by apoptosis. The effect of lower doses of Cytochalasin D could be potentiated by subsequent induction of the heat-shock response. CONCLUSIONS: Sequential stresses that mimic pathophysiologic "two-hit" stimuli induce a characteristic fractal rearrangement of the actin cytoskeleton. Because cytochalasin D-induced rearrangement of this cytoskeleton produced apoptosis indistinguishable from the stress-induced apoptosis, we conclude that the cytoskeletal rearrangement is likely a critical event in the pathway to apoptosis. This disruption of intracellular interconnections mirrors endotoxin-induced disruption in signals among organs and supports the mechanistic hypothesis that multiorgan dysfunction syndrome generally reflects disruption of signals and connections at several levels of biologic organization.

Actins↗
PubMed1996

Effects of electroporation on the tubulin cytoskeleton and directed migration of corneal fibroblasts cultured within collagen matrices.

Electroporation provides a useful method for loading fibroblasts with fluorescent probes for the cytoskeleton, but the possible deleterious effects of this loading technique on cell motility are unknown. We have used conventional and confocal microscopy of living cells and immunohistochemistry to examine the migration and cytoskeleton of chick embryo corneal fibroblasts electroporated while cultured within collagen gels. Fibroblasts cultured in collagen (1 mg/ml) are successfully electroloaded (0.5-1.0 kVcm-1/960 microF in DMEM/F12/20 mM Hepes, pH 7.2) with dextran (4-150 kDa) and immunoglobulin, but subsequently display uncoordinated pseudopodia and hence are unable to migrate effectively in any one direction. The lack of directed movement is due to depolymerization of microtubules and/or a perinuclear collapse of vimentin filaments, seemingly caused by millimolar levels of Ca2+ ions derived from culture medium following electroporation. Fibroblasts loaded in a buffer which resembles intracellular fluid (< or = 10 microM Ca2+) maintain their cytoskeleton and continue to migrate, when returned to culture medium within 10 min. Using this novel approach, we have loaded fibroblasts migrating through extracellular matrix (ECM) with rhodamine phalloidin and monitored the behavior of the labeled actin cortex by confocal microscopy. During migration phalloidin-actin accumulates near the base of pseudopodia and at the rear of the cell where it is subsequently left behind. We conclude that electroporation is a valuable technique for loading fibroblasts to study migration within ECM, provided that the conditions used support stability of the tubulin cytoskeleton.

Actins↗
PubMed1990

Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos.

Heat shock has a dramatic effect on the organization of the cytoplasm, causing the intermediate filament cytoskeleton to aggregate at the nucleus. This has previously been shown in cultured Drosophila and mammalian cells. In this paper we analyze the heat lability of the intermediate filament cytoskeleton in early Drosophila embryos by indirect immunofluorescence. At all stages of embryogenesis tested, the intermediate filament cytoskeleton, which is maternally provided, is severely disturbed by 30 min heat shock at 37 degrees C. After the nuclei have migrated to the subcortical cytoplasm, it collapses around them. Nuclei in all heat-shocked embryos are considerably enlarged and become displaced. Embryos before cellular blastoderm stage, in which heat shock protein synthesis is not inducible, are irreversibly arrested in development by heat shock. Embryos at or after cellular blastoderm, which do synthesize heat shock proteins in response to stress, are also immediately arrested in development but continue development when returned to 25 degrees C. We discuss the possibility that cytoplasmic events such as the intermediate filament cytoskeleton rearrangement may be involved in heat shock-mediated phenocopy induction.

Animals↗
PubMed2000

Helicobacter pylori vacuolating cytotoxin (VacA) alters cytoskeleton-associated proteins and interferes with re-epithelialization of wounded gastric epithelial monolayers.

In previous studies, we demonstrated that Helicobacter pylori vacuolating cytotoxin (VacA) inhibits gastric epithelial cell proliferation and inhibits epidermal growth factor (EGF)-activated signal transduction. Cell proliferation and migration, both essential for mucosal healing are dependent on the cell cytoskeleton. Other investigators demonstrated that VacA induces vacuolation of eukaryotic cells. Since in some cells, control of actin cytoskeleton involves GTP-binding proteins of Rho family, in this study we examined whether VacA affects wound re-epithelialization, cell cytoskeleton-associated proteins Rho, Rac1 in a gastric epithelial (RGM1) cell monolayer wound model, and whether these changes correlate with vacuolation. VacA treatment significantly inhibited wound re-epithelialization, cell proliferation vs control. VacA-induced cell vacuolation strongly correlated with inhibition of wound re-epithelialization. Furthermore, VacA reduced Rac-1 protein expression and distribution, and C3-mediated ADP-ribosylation of Rho. These findings suggest that VacA may interfere with repair of gastric mucosal injury and ulcer re-epithelialization by altering cytoskeleton-dependent cell functions and signaling.

ADP Ribose Transferases↗
PubMed1983

Phosphorylation controls brush border motility by regulating myosin structure and association with the cytoskeleton.

The intestinal epithelial cell brush border (BB) is a useful model for nonmuscle cell motility. We studied regulation of BB motility by analyzing myosin phosphorylation and its association with the cytoskeleton. Our results demonstrate that myosin associates with the cytoskeleton only when it is dephosphorylated. Myosin light chain kinase substrates release myosin, phosphorylated and in the form of filaments, from the cytoskeleton. Although ITP and GTP serve as myosin ATPase substrates, they do not cause BB contraction, myosin release, or phosphorylation. Brush border contraction occurs with ATP or with a mixture of ITP and ATP gamma S. Therefore, phosphorylation regulates myosin association with the cytoskeleton, myosin is not bound at the actin-myosin binding site, and when phosphorylated, myosin forms filaments for movement.

Animals↗
PubMed1997

Effects of the entomopathogenic fungus Metarhizium anisopliae and its secondary metabolites on morphology and cytoskeleton of plasmatocytes isolated from the greater wax moth, Galleria mellonella.

The effects of Metarhizium anisopliae infection and three different secondary metabolites released by the fungus, destruxin A and E and cytochalasin D, on the morphology and cytoskeleton of plasmatocytes of the greater wax moth Galleria mellonella were studied. Plasmatocytes isolated from M. anisopliae infected larvae exhibited impairment of attachment, spreading and cytoskeleton formation accompanied with the occurrence of blebbing and pycnotic nuclei. Plasmatocytes treated with destruxin in vitro exhibited similar morphological and cytoskeleton alterations. The corresponding effects were characterized by inhibition of attachment, spreading and filopodia formation as well as by impaired formation of actin filaments and microtubules. Cytochalasin was shown to affect plasmatocytes in vitro in a different manner than destruxin A and E. The results of our comparative study strongly suggested that the morphology and cytoskeleton alterations of plasmatocytes observed in M. anisopliae infected larvae were predominantly caused by destruxins released by the fungus during mycosis. Its mode of action is discussed with regard to present knowledge about its effects on target cells.

Journal Article↗
PubMed1998

Changes in actin cytoskeleton during volume regulation in C6 glial cells.

Changes in actin cytoskeleton in the C6 rat glial cell line were studied during decrease or increase (abrupt or gradual) of extracellular osmolality. Actin cytoskeleton was visualized by confocal microscopy after FITC-phalloidin labeling. G-actin, Triton-soluble F-actin and Triton-insoluble F-actin subfractions were determined by gel electrophoresis and scanning, and by DNase I inhibition assays. In control conditions C6 glial cells exhibited well-defined stress fibers and a relatively smooth cortical network. Extracellular anisosmotic changes induced a rapid actin cytoskeletal reorganization, which further progressed and was not reversed upon cell volume recovery. Hypotonic shock caused membrane ruffling and a shift towards polymerized actin, whereas hypertonicity (abrupt or gradual) led to a distinct morphological appearance of abundant short actin microfilaments with, however, no detectable alteration in actin subfractions. When anisosmotic cell volume regulation was prevented, cytoskeleton reorganization depended on the osmotic change and the experimental protocol, but was not related to the absence of volume readjustment. Therefore, although involvement of cytoskeletal alterations in transduction of volume regulatory responses cannot be excluded, it is likely that the observed changes in actin cytoskeleton in C6 glial cells are linked with, but do not initiate, cell volume regulatory processes.

Actins↗
PubMed2000

Ankycorbin: a novel actin cytoskeleton-associated protein.

BACKGROUND: Actin cytoskeleton structures are essential for a wide variety of cell functions, including cell shape change, cell motility, cell adhesion, cell polarity and cytokinesis. Many actin filament (F-actin)-binding proteins have been isolated and implicated in the maintenance and reorganization of actin cytoskeleton structures. RESULTS: We purified here a novel protein with a molecular mass of about 125 kDa (p125) from rat liver. We cloned its cDNA from a mouse kidney cDNA library and determined its nucleotide and deduced amino acid sequences. p125 was a protein of 979 amino acids with a calculated Mr of 108 847. p125 contained six ankyrin repeats in the N-terminal region and a domain predicted to form a coiled-coil structure in the C-terminal region. We named p125 ankycorbin (ankyrin repeat- and coiled-coil structure-containing protein). Northern blot analysis indicated that ankycorbin was ubiquitously expressed in all the tissues examined. Immunofluorescence and immunoelectron microscope analyses revealed that ankycorbin was associated with the cortical actin cytoskeleton structures in terminal web and cell-cell adhesion sites and stress fibres. However, ankycorbin did not directly bind to F-actin as estimated by the F-actin co-sedimentation assay. CONCLUSIONS: These results indicate that ankycorbin is indirectly associated with the actin cytoskeleton structures, presumably through an unidentified factor and suggest that it is involved in their maintenance and/or reorganization.

3T3 Cells↗