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At least 649 records · Page 36Linked to original sources
PubMed1990

Transient increase in vimentin in axonal cytoskeletons during differentiation in NB2a/d1 cells.

The localization of vimentin (Vm) within the Triton-insoluble cytoskeleton was characterized during differentiation of mouse NB2a/dl neuroblastoma cells. Vm staining increased within neurites during the first day of differentiation, and then rapidly declined in both perikarya and neurites. By contrast, immunoreactivity against extensively phosphorylated forms of the high molecular weight neurofilament subunit (NF-H) was absent until the third day after differentiation. Immunoblot analyses confirmed that these alterations reflected specific changes in Vm and NF-H steady-state levels. Metabolic labeling demonstrated a decrease in the rate of Vm synthesis by the third day of differentiation. We conclude that changes in incorporation of intermediate filament species into the axonal cytoskeleton reflect distinct stages in neurite outgrowth and maturation; i.e., the Vm filament system may participate in initial stages of neuritogenesis during which outgrowth is most rapid, while NFPs may subsequently function in the establishment of a stabilized axonal cytoskeleton.

Animals↗
PubMed1987

Concanavalin A binding induces association of possible mating-type receptors with the cytoskeleton in Tetrahymena.

The lectin concanavalin A (conA; 25 micrograms/ml) inhibits conjugation in the ciliate Tetrahymena, and binds to receptors localized at the junction between conjugating cells. We report here that succinyl-conA (30 micrograms/ml) has similar activity, but that two other mannosespecific lectins, lentil and pea lectins, have inhibitory activities more than tenfold lower in this system, indicating that factors other than mannose specificity are essential for biological activity. By using fluorescein-isothiocyanate (FITC)-conA, we have found that extraction of cells with the detergent Triton X-100 removes conA receptors from the extraction-resistant cytoskeleton, but that the binding of conA to its receptor before extraction associates the ligand-receptor complex with the cytoskeleton. Under the hypothesis that the conA receptor may be a mating type receptor, we have used this ligand-induced differential cytoskeletal association, in conjunction with electrophoresis and Western blotting, to identify a glycoprotein with an apparent molecular weight (MW) of 23,000 D which may be a mating type receptor. Our data are consistent with a model in which a direct interaction between the conA receptor and the cytoskeleton, rather than receptor cross-linking, is the biologically significant activity of ligand binding.

Animals↗
PubMed1991

Dystrophin constitutes 5% of membrane cytoskeleton in skeletal muscle.

Dystrophin, which is absent in skeletal muscle of Duchenne muscular dystrophy patients, has not been considered to play a major structural role in the cell membrane of skeletal muscle because of its low abundance (approximately 0.002% of total muscle protein). Here, we have determined the relative abundance of dystrophin in a membrane cytoskeleton preparation and found that dystrophin constitutes approximately 5% of the total membrane cytoskeleton fraction of skeletal muscle sarcolemma. In addition, dystrophin can be removed from sarcolemma by alkaline treatment. Thus, our results have demonstrated that dystrophin is a major component of the subsarcolemmal cytoskeleton in skeletal muscle and suggest that dystrophin could play a major structural role in the cell membrane of skeletal muscle.

Animals↗
PubMed1996

Phase specific association of heterotrimeric GTP-binding proteins with the actin-based cytoskeleton during thrombin receptor-mediated platelet activation.

Subcellular distribution of heterotrimeric GTP-binding proteins during thrombin receptor-mediated platelet activation was examined, revealing two phases of translocation to the cytoskeleton. A part of Gi2 alpha and Gs alpha shows first phase translocation to the low-speed pellet (15000 x g pellet) within 1 min after activation, suggesting involvement in platelet shape change or granule secretion. In the second phase, Gi2 alpha, Gs alpha, Gq alpha, and G beta translocate to the low-speed pellet, depending on platelet aggregation. These translocations correlated with the reorganization of the actin-cytoskeleton and were inhibited by cytochalasin D. Reconstitution experiments also revealed that G proteins are associated with the actin-cytoskeleton during platelet activation.

Actins↗
PubMed1982

Actin in Triton-insoluble cytoskeleton of thyroid.

Treatment of bovine thyroid with the non-ionic detergent Triton X-100 extracts most of the cell protein and leaves insoluble residue. This Triton-insoluble cytoskeleton consists of five major polypeptides on sodium dodecyl sulfate polyacrylamide gels. One of these polypeptides is actin. Based on DNase inhibition assay, 30% of the total actin is associated with the cytoskeleton as the filamentous form. Thyroid actin from the cytoskeleton has been solubilized by dialysis against a low ionic strength buffer at pH 8.0 and purified to homogeneity by a polymerizing-depolymerizing cycle. The overall purification was about 144-fold with a yield of 10%. Bovine thyroid actin is very similar to actins from other tissues on the basis of: (1) comigration with rabbit skeletal muscle actin during gel electrophoresis in sodium dodecyl sulfate, (2) its amino acid composition, which includes about 1 mole of 3-methylhistidine per 42,000 g, (3) its ability to bind and inhibit pancreatic deoxyribonuclease I, and (4) its ability to form 7-8 nm microfilaments which is similar to that of skeletal filamentous actin. Thyroid actin contains beta- and gamma-isoactins, with isoelectric points more alkaline than the alpha-actin of rabbit skeletal muscle.

Actins↗
PubMed1986

Involvement of actin filaments in budding of measles virus: studies on cytoskeletons of infected cells.

Cytoskeletons were prepared from measles virus infected HeLa cells to investigate the involvement of cytoskeletal filaments in virus budding at the plasma membrane. The cytoskeletons retained nearly 80% of measles virus hemagglutinin, the major viral polypeptides, including P, NP, and M, and 2 to 12% of the total cell bound infectivity. As demonstrated with platinum- and carbon-shadowed cytoskeletons, all stages of budding, i.e., virus specific strands, stub-like protrusions, and completely rounded virus particles, are associated with actin filaments composing the outer part of the cytoskeletal network. As shown with ultrathin sections of flat embedded extracted cells, actin filaments identified with heavy meromyosin almost exclusively protrude into virus particles with their barbed ends and are in close association with viral nucleocapsids. The data support previous suggestions that actin is involved in virus budding and show that budding itself is possibly the result of a vectorial growth of actin filaments.

Actins↗
PubMed1987

Association of bluetongue virus with the cytoskeleton.

Analysis of the distribution of [35S]methionine-labeled virus proteins following lysis of bluetongue virus (BTV)-infected cells with nonionic detergents showed that a major proportion of the virus-specific proteins was located in the insoluble nuclear-cytoskeletal fraction. Neither the proportion nor the species of virus protein associated with the cytoskeleton was altered following treatment of infected cells with microtubule or microfilament disrupting drugs (colchicine and cytochalasin B, respectively). Electron microscopic examination of BTV-infected cells revealed cytoplasmic virus-specified tubules, viral inclusion bodies (VIB), and progeny virus particles. Whole-mount transmission electron microscopy of nonionic detergent-extracted cells demonstrated the association of VIB, virus particles, and tubules with the cytoskeleton. The identity of virus particles was confirmed with an immunogold labeling technique using a neutralizing monoclonal antibody to BTV protein VP2. Several lines of evidence indicate that virus particles, VIB, and tubules bind to intermediate filaments in BTV-infected cells. These structures remained cytoskeleton associated in infected cells treated with colchicine or cytochalasin B. Linear arrays of filament-associated virus particles were formed around VIB following colchicine-induced juxtanuclear aggregation of intermediate filaments. Virus particles were associated with filaments approximately 10 nm in diameter. Filaments associated with virus particles reacted with an anti-vimentin monoclonal antibody in an immunogold labeling procedure.

Animals↗
PubMed1983

Many cytoskeletal proteins associate with the hela cytoskeleton during translation in vitro.

Observations that cytoskeletal proteins assemble in vivo close to the time and site of synthesis have been confirmed and extended by an in vitro translation system. HeLa cytoskeletons prepared with Triton in a translation-extraction buffer without reticulocyte or wheat germ lysate efficiently incorporate 35S-methionine into polypeptides, and are stable during this translation. Cytoskeletal proteins translated in this way associate with the HeLa cytoskeleton independent of the concentration of soluble proteins. These associations are puromycin-resistant before the proteins are complete; the protein associations made in vitro show only minor differences from those made in vivo. The protein associations are not simply a consequence of protein solubility in the buffers used, as the associations require initiation in vivo. These results indicate that many cytoskeletal proteins associate with the cytoskeleton during translation.

Cell Fractionation↗
PubMed1985

Transmembrane modulation of the concanavalin A inhibition of 5'-nucleotidase is not due to a direct association of the enzyme with the cytoskeleton.

The inhibition of the cell surface enzyme 5'-nucleotidase by concanavalin A is being studied as a model for understanding transmembrane modulation of cell surface functions. Nucleotidase of 13762 MAT-C1 ascites rat mammary adenocarcinoma cells is inhibited by concanavalin A in a noncooperative process. When cells are treated with the cytoplasmic effectors cytochalasins, colchicine, energy poisons, calcium plus ionophore or hypotonic buffers, the concanavalin A inhibition of the enzyme becomes cooperative. 5'-Nucleotidase of isolated MAT-C1 microvilli is also inhibited by concanavalin A in a noncooperative process; however, treatment of the microvilli with the same cytoplasmic effectors does not induce cooperativity. Since previous studies in several systems have suggested an association of nucleotidase with actin-containing microfilaments or the cell cytoskeleton, one explanation for the cooperativity changes is that they result from a change in the association of the enzyme with the cytoskeleton. However, Triton X-100 extractability of nucleotidase is the same for MAT-C1 cells exhibiting cooperative or noncooperative concanavalin A inhibition. Moreover, enzyme from cells exhibiting cooperative inhibition can be extracted into the zwitterionic detergent Zwittergent in a cooperative form, while enzyme exhibiting noncooperative behavior can be extracted into Zwittergent in a noncooperative form. Gel filtration and rate-zonal sucrose density gradient centrifugation showed little discernible size or sedimentation difference between enzyme samples exhibiting noncooperative and cooperative inhibition. These results indicate that changes in the cooperativity of the concanavalin A inhibition of nucleotidase are not a result of changes in the association of the enzyme with the cytoskeleton. These studies emphasize the caution which must be exercised in interpreting the effects of cytoskeletal perturbants on cell surface functions.

5'-Nucleotidase↗
PubMed1995

Rapid protein tyrosine phosphorylation in the cytoskeleton of stimulated human platelets.

Upon activation platelets show elevated protein tyrosine phosphorylation, and translocation of the protein tyrosine kinase pp60c-src from the plasma membrane to the cytoskeleton occurs. We therefore investigated whether tyrosine phosphorylation also increases in the cytoskeletal compartment. Here we show that almost identical patterns of phosphotyrosine-containing proteins are detectable in the cytoskeleton after platelet stimulation with compounds that directly (phorbol 12-myristate, 13-acetate) or indirectly (thrombin, vasopressin, collagen, ADP) activate protein kinase C. The apparent molecular masses of the proteins phosphorylated at tyrosine residues are 145, 130, 100, 85, 80, 60, 56, 54 and 38 kDa. Elevation of cyclic AMP by prostaglandin E1 had no effect. Concentrations of thrombin as low as 0.01 units per ml are able to cause tyrosine phosphorylation of multiple proteins. The time course of protein tyrosine phosphorylation for thrombin- and vasopressin-stimulated platelets revealed a rapid increase in the cytoskeleton within 5 to 20 s following activation consistent with a role in early events of platelet function.

Blood Platelets↗
PubMed1994

Neutrophil control of formylmethionyl-leucyl-phenylalanine induced mobilization of secretory vesicles and NADPH-oxidase activation: effect of an association of the ligand-receptor complex to the cytoskeleton.

The stimulus formylmethionyl-leucyl-phenylalanine (FMLP) interacts with neutrophils and generates signal(s) in the cells that induces mobilization of the secretory vesicles as well as activation of the superoxide anion/hydrogen peroxide generating NADPH-oxidase. Binding, at 15 degrees C, of FMLP to its neutrophil surface receptor is followed by an association of the ligand-receptor complex to the cell cytoskeleton, and this association occurs concomitant with a desensitization of the cells with respect to activation of the NADPH-oxidase. Other stimuli can still activate the oxidase (in fact even induce a primed response), indicating that the observed phenomenon is stimulus specific and could not be accounted for by an effect on the oxidase itself, but rather that the association of the ligand-receptor complex to the cytoskeleton eliminates the capacity of the complex to generate the signal(s) that activates the NADPH-oxidase. The cytoskeleton associated ligand-receptor complex generates, however, the signal(s) responsible for mobilization of the secretory vesicles, to the plasma membrane, and this mobilization occurs without any increase in the intracellular concentration of free Ca2+.

Calcium↗
PubMed1991

PAC 1: an epitope associated with two novel glycoprotein components of isolated postsynaptic densities and a novel cytoskeleton-associated polypeptide.

A monoclonal antibody has been raised which recognizes an epitope, PAC 1 (postsynaptic density and cytoskeleton enriched), which is specifically associated with two novel glycoprotein components of forebrain postsynaptic density preparations and a novel neuronal cytoskeletal-associated polypeptide. The monoclonal antibody has been used to study the cellular and subcellular localization of these molecules and for the partial characterization of all three PAC 1 antigens in the rat. The PAC 1 epitope is present on two concanavalin A binding glycoproteins of apparent molecular weights 130,000 (pgp130) and 117,000 (pgp117). Both species are enriched in preparations of rat forebrain postsynaptic densities and to a lesser extent in synaptic membranes. The epitope is also expressed by a polypeptide of 155,000 mol. wt, cp155. This molecule is highly enriched in cytoskeleton rather than membrane preparations. Enzymic removal of N-linked carbohydrate lowers the molecular weights of the PAC 1 glycoproteins pgp130 and pgp117 by 11,000 and 14,000 respectively, and suggests that cp155 is not glycosylated. Detergent, alkaline and salt extractions of postsynaptic densities and synaptic membranes indicate that pgp130 and pgp117 are integral membrane glycoproteins and are tightly bound components of postsynaptic density preparations. Immunocytochemical studies of adult rat forebrain show prominent staining of pyramidal cell dendrites and perikarya. There is no evidence of glial staining. Electron microscope studies show staining of microtubules together with punctate deposits of plasma membrane-associated reaction product. Several criteria have been used to show that pgp130 and pgp117 do not correspond to other known neuronal glycoproteins of similar molecular weight. We conclude that the PAC 1 epitope is expressed by two novel synaptic glycoproteins which are very probably integral components of the postsynaptic density and by a novel neuronal cytoskeleton-associated protein.

Animals↗
PubMed1979

Nucleus-anchoring cytoskeleton in chicken red blood cells.

Cytoskeleton of chicken erythrocytes was studies studied after extraction of the cells with Triton X-100. In phase contrast microscopy the extracted cells were seen as ghost-like structures with preserved morphology, distinct nucleus and surrounding plasma membrane remnant. In electron microscopy, dense matrix-like nucleus, fibrillar plasma membrane residue and filaments, ca. 10 nm in diameter, traversing the cytoplasmic domain, were seen. Distinct bands of molecular weight 68000, 53000, 32000 and bands of both higher and lower molecular weight were obtained by polyacrylamide gel electrophoresis of the extracted cells. These results indicate that intermediate filaments, forming the nucleus-anchoring cytoskeleton, are present in nucleated chicken erythrocytes as part of cellular cytoskeleton.

Animals↗
PubMed1992

Isolation of cDNA clones encoding proteins of complex structures: analysis of the Trypanosoma brucei cytoskeleton.

We have adapted a group of well-known procedures in order to devise a simple method that allows the isolation of specific cDNAs encoding proteins located in different regions of the Trypanosoma brucei cytoskeleton. cDNA clones were isolated by screening a lambda gt11 expression library with a polyspecific, polyclonal antiserum against a complex immunogen, in this case the complete cytoskeleton. The fusion proteins produced by the clones were then used as an affinity immunoadsorbant to select monospecific polyclonals. The monospecific antisera isolated were used as probes to identify and localize different cytoskeleton proteins by Western blotting and immunofluorescence. This method proved particularly useful for the molecular identification of minor components in a complex structure. It should prove applicable to the molecular analysis of other organelles or protein complexes.

Animals↗
PubMed1985

The effect of preparative protocol on the cytoskeleton ultrastructure observed in extracted whole-mount BHK cells.

Extracted BHK cells (baby hamster kidney) were prepared for electron microscopy by air-drying (with Freon 113), critical-point-drying, and freeze-drying. Variations in the drying procedures had a marked effect on the resultant cytoskeleton ultrastructure. Air drying had to be done in a Freon-saturated atmosphere, residual water had to be removed from the dehydrating solutions and carbon dioxide for critical-point-dried specimens, and freeze-drying had to be done at temperatures lower than -90 degrees C. Failure to exercise these precautions resulted in a cytoskeleton ultrastructure artifact, possibly caused by fusion of the cytoskeleton filaments.

Animals↗
PubMed1996

The compartmentalization of protein synthesis: importance of cytoskeleton and role in mRNA targeting.

Following the synthesis of mRNA molecules in eukaryotic cells, the transcripts are processed in the nucleus and subsequently transported through the nuclear membrane into the cytoplasm before being sequestered into polysomes where the information contained in the RNA molecule is translated into an amino acid sequence. Recent evidence suggests that an association of mRNAs with the cytoskeleton might be important in targeting mechanisms and, furthermore, in the transport of mRNA from the nucleus to its correct location in the cytoplasm. Until recently, polysomes have been considered to exist in two classes, namely free or membrane-bound. There is now compelling evidence, however, that ribosomes, in addition to being associated with endoplasmic reticulum membranes, also are associated with components of the cytoskeleton. Thus, a large number of morphological and biochemical studies have shown that mRNA, polysomes and translational factors are associated with cytoskeletal structures. Although the actual nature and significance of the interaction between components of the translational apparatus and the cytoskeleton is not yet understood in detail, it would seem evident that such interactions are important in both the spatial organization and control of protein synthesis. Recent work has shown that a subcellular fraction, enriched in cytoskeletal components, contains polysomes and these (cytoskeletal-bound) polysomes have been shown to contain specific mRNA species. Thus, a population of cytoskeletal-bound polysomes may provide a specialized mechanism for the sorting, targeting and topographical segregation of mRNAs. In this review, current knowledge of the subcellular compartmentalization of mRNAs is discussed.

Animals↗
PubMed2004

Presence of cytoskeleton proteins in parotid glands and their roles during secretion.

Amylase secretion is induced by the accumulation of cAMP in response to beta-adrenergic stimulation and by the augmentation of intracellular Ca2+ in response to muscarinic-cholinergic stimulation in rat parotid glands. The roles of cytoskeleton and motor proteins in the secretory process are not yet known. We examined the effects of cytoskeleton-modulating reagents on the amylase release induced by isoproterenol (IPR) and carbamylcholine (Cch) in rat parotid acinar cells. The amylase release induced by Cch was decreased by the microtubule-disrupting reagent colchicine (Colch) and the myosin ATPase inhibitor 2,3-butanediene monoxime (BDM), but the release induced by IPR was not. The actin filament-stabilizing reagent jasplakinolide (Jasp) and actin filament-disrupting reagent cytochalasin D (CytoD) decreased the amylase release induced by both the beta-adrenergic and the muscarinic-cholinergic stimulants. Pretreatment with CytoD affected the shape of the acinar cells, which showed an intermediate state between the fusion of the secretory granules with the apical membrane and the retrieval of the membranes only after stimulation with IPR. Myosin and Dynein/dynactin complex were detected in the secretory granule membrane fraction. We concluded from this study that the cytoskeleton played different roles in the beta-adrenergic and the muscarinic-cholinergic secretory processes.

Actins↗
PubMed2006

The cytoskeleton of Entamoeba histolytica: structure, function, and regulation by signaling pathways.

Pathogenesis in the parasite Entamoeba histolytica has been related to motility of the trophozoites. Motility is an important feature in amebas as they perform multiple motile functions during invasion of host tissues. As motility depends on the organization and regulation of the cytoskeleton elements, in particular of the actin cytoskeleton, the study of the molecular components of the machinery responsible for movement has been a key aspect to study in this parasite. Although many of the components have high homology in amino acid sequence and function to those characterized in higher eukaryotic cells, there are important differences to suggest that parasitic organisms may have developed adaptative differences that could be useful as targets to stop invasion. The purpose of this review is to evaluate current knowledge about the cytoskeleton of E. histolytica and the ways in which the parasite controls motility.

Animals↗