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Results for “cytoskeleton”

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At least 19 recordsLinked to original sources

PubMed1989

cAMP-independent and cAMP-dependent protein phosphorylations by isolated goldfish xanthophore cytoskeletons: evidence for the association of cytoskeleton with a carotenoid droplet protein.

Triton-insoluble cytoskeleton of nonpigment cells has bound protein kinase that phosphorylates, with or without added cAMP, tubulins and the intermediate filament proteins p60, p56, p53, and p45a to give multiple charge variants. In the absence of 8-Br-cAMP, Triton-insoluble cytoskeletons from xanthophores also phosphorylate p60, p56, and p45a, but not p53; tubulin phosphorylation may also be reduced. In the presence of 8-Br-cAMP, p53, as well as several other peptides, are phosphorylated. One of these latter peptides was identified as the carotenoid droplet (pigment organelle) protein p57, whose phosphorylation and dephosphorylation precede pigment dispersion and aggregation respectively (Lynch et al.: J. Biol. Chem. 261:4204-4211, 1986). The amount of pp57 produced depends on the state of pigment distribution in the xanthophores used to prepare the cytoskeletons for labeling. With cytoskeletons from xanthophores with aggregated pigment, pp57 is a major labeled phosphoprotein seen in two-dimensional gels. With cytoskeletons prepared from xanthophores with dispersed pigment, the yield of labeled pp57 is greatly reduced (by at least 90%). Together with earlier results, we propose that, in the aggregated state, p57 serves to bind carotenoid droplets to the cytoskeletons, most likely the microtubules. The significance of other cAMP-dependent phosphorylation reactions is unknown but may be related to cAMP-induced cytoskeleton rearrangement in intact xanthophores.

Animals↗

PubMed1992

Detergent-resistant cytoskeleton of the surface-activated platelet differs from the suspension-activated platelet cytoskeleton.

This study contrasts the protein composition of the detergent-resistant cytoskeleton of platelets fully spread on glass with the cytoskeletal composition of resting platelets and platelets aggregated in suspension with thrombin. Complete Triton X-100-insoluble cytoskeletons were isolated from spread, resting, and suspension-activated platelets in the presence of protease inhibitors, solubilized in sodium dodecyl sulfate/EDTA and analyzed on reduced, one-dimensional polyacrylamide gels. The protein composition of the cytoskeletons differed both qualitatively and quantitatively. Most notable were more extensive incorporation of total protein, talin, and vinculin into the cytoskeleton of spread platelets than the cytoskeleton of suspension-activated platelets. Varying the concentration and time of exposure to thrombin during suspension activation did not mimic the cytoskeletal changes of surface activation. Scanning electron microscopy, measurement of lipid phosphorus content, and varying the duration of Triton extraction did not show incomplete solubilization or nonspecific trapping of constituents in the spread platelet cytoskeleton. Proteolysis of talin was minimal in suspension-activated platelets and in platelets spread for 50 minutes. The differences in the detergent-resistant cytoskeletons of surface- and suspension-activated platelets indicate significant divergence in the physiologies of platelet spreading on surfaces and platelet activation in suspension.

Adult↗

PubMed1990

[Cytoskeleton--function and pathology: studies on the pathology of the intermediate filament cytoskeleton of liver cells].

Microfilaments, microtubules and intermediate filaments (IF) are major filamentous components of the cytoskeleton and play a role in the modulation of cell shape, in cellular movements, cellular stability, intracellular organisation as well as cell-to-cell and cell-to-stroma interactions. Particular interest was concentrated in the last few years on IF because of their cell type-specificity and, consequently, their suitability as cell markers in diagnostic pathology. Despite their apparent stability, IF are dynamic structures which may be modified under pathologic conditions. In recent years, pathologic alterations related to the IF cytoskeleton have been described in a diversity of chronic and degenerative disorders, including alcoholic hepatitis and neurologic diseases (e.g. M. Alzheimer, M. Parkinson). Our studies were particularly devoted to the elucidation of the pathogenesis of severe alcoholic liver injury (alcoholic hepatitis), which is associated with inflammation, liver cell degeneration and necrosis and morphologically characterized by the appearance of cytoplasmic hyaline inclusions (i.e., Mallory bodies). In the present review morphologic, immunologic and biochemical studies on nature and pathogenesis of Mallory bodies are summarized. Moreover, similarities between Mallory bodies and other cytoskeleton-related inclusion bodies suggest common routes of pathogenesis. Consequently, studies along these lines may not only lead to the understanding of mechanisms involved in alcoholic injury but may also provide information on general principles of cell damage as well as on regulation and function of the IF cytoskeleton.

Animals↗

PubMed1989

A cytoskeleton-dependent pathway for induction of IL-2 production and a cytoskeleton-independent pathway for IL-2-mediated signal transduction.

We have used the synthetic microtubule inhibitor Tubulozole C in order to study the role of the microtubule system in human lymphocyte activation. Microtubule disruption prior to activation with phytohemagglutinin (PHA) resulted in a drastic reduction of IL-2 production. Similarly, using OKT3 or PHA as stimulators, a substantial decrease in proliferation was observed. Although IL-2 receptor analysis performed on the stimulated and antitubular-treated lymphocytes showed a 2-fold decrease in high-affinity and a 100-fold decrease in low-affinity IL-2 receptor expression, a proliferative response to externally added rIL-2 was noticed. This occurred provided the triggering agent was excluded or added in suboptimal concentrations. These results indicate that intact microtubules are necessary for PHA/OKT3-induced proliferation and IL-2 production, but not for IL-2-induced proliferation.

Antibodies, Monoclonal↗

PubMed1989

Interaction between phosphatidylserine and the isolated cytoskeleton of human blood platelets.

Binding experiments were performed to demonstrate a direct interaction between cytoskeletons from human blood platelets and phosphatidylserine. A centrifugation technique using radiolabeled phosphatidylserine-vesicles and Triton X-100 insoluble residues from unstimulated human platelets was used to assess the binding. Interaction between cytoskeleton and phospholipid is demonstrated to be specific for phosphatidylserine. No binding was observed for phosphatidylcholine. The binding of phosphatidylserine was saturable and dependent on the concentration of cytoskeleton used. The interaction between phosphatidylserine and the cytoskeleton appeared to be completely reversible. The existence of a reversible and specific interaction between phosphatidylserine and the cytoskeleton of unstimulated platelets would suggest a role for the cytoskeleton in the maintenance of the asymmetric distribution of this lipid in the plasma membrane. We have previously shown (Comfurius et al. (1985) Biochim. Biophys. Acta 815, 143-148) that in activated platelets a strong correlation exists between degradation of platelet cytoskeletal proteins by the endogenous calcium-dependent proteinase (calpain) and exposure of phosphatidylserine at their outer surface. Nevertheless, hydrolysis of the isolated cytoskeleton by calpain did not result in a change in the parameters of the binding between phosphatidylserine and cytoskeleton. Also, sulfhydryl oxidation of the cytoskeleton by diamide did not affect its binding properties for phosphatidylserine, in spite of the fact that diamide treatment of platelets results in exposure of phosphatidylserine at the outer surface. Exposition of phosphatidylserine upon activation of platelets cannot be directly ascribed to a change in affinity or number of binding sites of the modified cytoskeleton as measured in model systems. However, it cannot be excluded that topological rearrangements of the cytoskeleton as occur within the cell during platelet activation lead to a decreased contact between cytoskeleton and lipid, irrespective of the binding parameters.

Binding Sites↗

PubMed1991

Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton.

Previous studies indicated a correlation between the formation of EDTA-resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA-resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA-resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet-fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

Antibodies, Monoclonal↗

PubMed1988

Aggregation-related association of lipid with the cytoskeleton of rabbit and human platelets prelabeled with [3H]palmitic acid. Similar effects of adenosine diphosphate- and thrombin-induced aggregation.

To investigate the association of lipid with the cytoskeleton of platelets during aggregation, rabbit and human platelets were isolated and labeled with [3H]palmitic acid; lipid extraction showed approximately 80% in phospholipid. Limited aggregation was induced with ADP or thrombin, and the cytoskeleton was isolated after lysis with 1% Triton X-100, 5 mM EGTA. Cytoskeleton from unactivated platelets had approximately 0.03% of the total label in the platelets, but after aggregation with ADP (2 microM) or thrombin (0.1 U/ml) for 20-30 s, 1.5-8% of the label was with the cytoskeleton. Fibrinogen enhanced aggregation and the association of label with the cytoskeleton; incorporation of label increased exponentially as aggregation proceeded, decreased exponentially during deaggregation, and appeared to be related to the number of sites of contact. Inhibitors that increase cyclic AMP inhibited aggregation and cytoskeletal labeling, but aspirin had no effect. Some experiments were done with DNase I and Ca2+ in the Triton X-100 lysis medium to cause actin depolymerization, under conditions in which the Ca2+-dependent protease activity was inhibited. This greatly reduced the association of label with the cytoskeleton at early time points, but when aggregation had proceeded further, a large proportion of the label was not dissociated by this treatment. These findings, electron microscopy, and the enrichment of the cytoskeleton of aggregated platelets with only some of the membrane proteins that were labeled by the 125I-lactoperoxidase method, indicated that with limited aggregation, the 3H-labeled lipid was mainly associated with the cytoskeleton and not with trapped membrane fragments resulting from incomplete lysis. Since the pattern of cytoskeleton labeling ([3H]palmitate) and the selective association of some membrane proteins with the cytoskeleton/lipid complex was the same with ADP and thrombin, the reactions must be dependent on aggregation and not on events associated with the release of granule contents.

Actin Cytoskeleton↗

PubMed1990

Interaction of epidermal growth factor receptors with the cytoskeleton is related to receptor clustering.

Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction, however, is still unknown. Therefore, we have studied the association of mutated EGF receptors with the cytoskeleton. Receptor deletion mutants lacking almost all intracellular amino acid residues displayed no interaction with the cytoskeleton, demonstrating that the cytoplasmic receptor domain is involved in this interaction. Further analysis revealed that receptor-cytoskeleton interaction is independent of receptor kinase activity and the C-terminal 126 amino acid residues, which include the auto-phosphorylation sites. Furthermore, it is shown that the high-affinity receptor subclass is not essential for association of low-affinity receptors to the cytoskeleton. EGF receptor-cytoskeleton interaction was increased, however, by treatment with sphingomyelinase, an enzyme known to induce membrane protein clustering, indicating that EGF receptor clustering may cause the association to the cytoskeleton.

Animals↗

PubMed1991

Stopped-flow measurement of cytoskeletal contraction: Dictyostelium myosin II is specifically required for contraction of amoeba cytoskeletons.

Cytoskeletons provide valuable information on the composition and organization of the cell's contractile machinery, and in many cases these cell models retain the ability to contract. To quantitate contraction rates, we developed a novel stopped-flow assay permitting simultaneous analysis of thousands of Dictyostelium cytoskeletons within milliseconds of mixing with Mg-ATP. Cytoskeletons were placed in one syringe of the stopped flow apparatus and the appropriate buffer was placed in the second syringe. Mixing with Mg-ATP caused an immediate increase in the absorbance at 310 nm. Rapid fixation of the cytoskeletons during the reaction confirmed that this change in absorbance was highly correlated with contraction of the cytoskeletons. This spectroscopic change was used to measure the effects of temperature, pH, ionic strength, and nucleotides on contraction rate. Treatment with high salt and ATP removed most of the myosin, some actin, and small amounts of minor proteins. These extracted cytoskeletons lost the ability to contract, but after the addition of purified Dictyostelium myosin they regained full function. In contrast, rabbit skeletal muscle myosin was unable to restore contractility, even though it bound to the extracted cytoskeletons. Cytoskeletons prepared from a myosin-null mutant did not contract. Upon the addition of purified ameba myosin, however, they became contractile. These results suggest that filamentous Dictyostelium myosin II is essential for contraction, and that the actin cytoskeleton and associated proteins retain their functional organization in the absence of myosin.

Adenosine Triphosphate↗

PubMed1991

Association of various T cell-surface molecules with the cytoskeleton. Effect of cross-linking and activation.

The association of various surface molecules with the cytoskeleton in resting peripheral blood T cells was examined by assaying the capacity of detergent to solubilize them. Cytoskeletal association was assessed by staining T cells with a fluorescein-conjugated mAb, resuspending the cells in buffer with or without the nonionic detergent, NP-40, and determining the capacity of the detergent to remove the mAb from the cell surface by using flow microfluorimetry. MAb to CD3, the TCR, and CD45 were completely removed from the cell surface by detergent. In contrast, 7 to 50% of mAb to CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules were resistant to detergent solubilization, demonstrating that a fraction of these molecules was constitutively associated with the cytoskeleton. The effect of cross-linking these molecules with a mAb and a secondary goat anti-mouse Ig was also examined. Cross-linking CD3 or the TCR induced cytoskeletal association of these molecules. In addition, cross-linking increased the fraction of CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules that was associated with the cytoskeleton. In contrast, cross-linking CD45 did not induce an association with the cytoskeleton. The effect of T cell activation on the cytoskeletal association of these molecules was also examined. Stimulation of T cells with ionomycin and PMA greatly increased the expression of CD2 and CD44 without increasing the number of molecules associated with the cytoskeleton. Stimulation with PMA alone had no effect on the expression of CD2 or CD44, but was found to decrease the percentage of these molecules associated with the cytoskeleton. Stimulation with ionomycin and PMA increased both the expression of class I MHC molecules and the number of molecules associated with the cytoskeleton proportionally. Finally, stimulation with ionomycin and PMA decreased CD3 expression, but increased the number of CD3 molecules associated with the cytoskeleton. The data establish a pattern of cytoskeletal association of T cell-surface molecules that is a characteristic of each individual molecule and can be altered by cross-linking. Moreover, the results indicate that the association of various T cell surface molecules with the cytoskeleton is a dynamic process that varies with the state of activation and or differentiation of the cells.

Adult↗

PubMed1990

Differential association of membrane-bound and non-membrane-bound polysomes with the cytoskeleton.

We report here a differential release of specific mRNAs from the cytoskeleton by cytochalasin D treatment. Non-membrane-bound polysomal mRNAs, such as histone mRNA and c-fos mRNA, are readily released from the cytoskeleton of HeLa cells during cytochalasin D treatment. Over 90% of H3 and H4 histone mRNA is associated with the cytoskeleton in control cells and only 25% in cells treated with cytochalasin D (40 micrograms/ml). In contrast, the membrane-bound polysomal mRNAs for HLA-B7 and chorionic gonadotropin-alpha are inefficiently released from the cytoskeletal framework by cytochalasin D alone; approximately 98% of the HLA-B7 mRNA in control cells is associated with the cytoskeleton, whereas approximately 65% of the HLA-B7 mRNA is retained on the cytoskeleton in cells treated with cytochalasin D (40 micrograms/ml). Disruption of polysome structure with puromycin during cytochalasin D treatment results in the efficient release of HLA-B7 mRNA from the cytoskeleton. Under these conditions, only 25% of the HLA-B7 mRNA remains associated with the cytoskeletal framework. Thus, membrane-bound polysomes appear to be attached to the cytoskeleton through a cytochalasin D-sensitive site as well as through association with the nascent polypeptide and/or ribosome. These results demonstrate a complex association of polysomes with the cytoskeleton and elements of the endoplasmic reticulum.

Actin Cytoskeleton↗

PubMed1989

Ligand-induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells.

Recently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X-100-insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady-state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF-binding sites: a high-affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 x 10(4) sites per cell) and a low-affinity site with a KD of 8.5 nM (1.9 x 10(6) sites per cell). Non-equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast-dissociating site, with a dissociation rate constant (k-1) of 1.1 x 10(-3) s-1 (1.0-1.3 x 10(6) sites per cell) and a slow-dissociating site, with a k-1 of 3.5 x 10(-5) s-1 (0.6-0.7 x 10(6) sites per cell). The cytoskeleton of A431 cells was isolated by Triton X-100 extraction. Scatchard analysis revealed that approximately 5% of the original number of receptors were associated with the cytoskeleton predominantly via high-affinity sites (KD = 1.5 nM). This class of receptors is further characterized by the presence of a fast-dissociating component (k-1 = 2.0 x 10(-3) s-1) and a slow-dissociating component (k-1 = 9.1 x 10(-5) s-1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4 degrees C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton-associated receptors appeared to represent low-affinity binding sites (KD = 7 nM). Dissociation kinetics also revealed an increase of fast-dissociating sites. These results indicate that at 4 degrees C EGF induces the binding of low-affinity, fast-dissociating sites to the cytoskeleton of A431 cells.

Carcinoma, Squamous Cell↗

PubMed1990

Palmitic acid-labeled lipids selectively incorporated into platelet cytoskeleton during aggregation.

Previous experiments showed that during the early stages (20-30 seconds) of aggregation induced by adenosine diphosphate (ADP, 2 microM) or thrombin (0.1 U/mL) of rabbit or human platelets prelabeled with [3H]palmitic acid, labeled lipid became associated with the cytoskeleton isolated after lysis with 1% Triton X-100, 5 mM EGTA [ethylene glycol-bis-(beta-aminoethyl ether)]-N,N,N',N'-tetra-acetic acid. The association appeared to be related to the number of sites of contact and was independent of the release of granule contents. We have now investigated the nature of the labeled lipids by thin-layer and column chromatography and found differences between the distribution of the label in intact platelets (both stimulated and unstimulated) and the isolated cytoskeletons. In both species, and with either ADP or thrombin as aggregating agent, 70-85% of the label in both intact platelets and in the cytoskeletons was in phospholipids. The distribution of label among the phospholipids in the cytoskeletons was similar to that in intact platelets except that the percentage of label in phosphatidylcholine was significantly higher in the cytoskeletons of human platelets than in the intact platelets, and the percentage of label in phosphatidylserine/phosphatidylinositol was significantly lower in the cytoskeletons of rabbit platelets and thrombin-aggregated human platelets than in intact platelets. The cytoskeletons contained a lower percentage of label in triacylglycerol, diacylglycerol, and cholesterol ester than the intact platelets. Contrary to a report in the literature, we found no evidence for the incorporation of diacylglycerol and palmitic acid into the cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Membrane-cytoskeleton associations during myogenesis deviate from traditional definitions.

Plasma membrane-cytoskeleton associations involving four membrane proteins (A5, H58, H36, and I20) were studied in developing L8E63 rat skeletal muscle cells using immunofluorescence microscopy and photometry on the basis of three criteria: Triton-insolubility, colocalization with cytoskeletal components, and sensitivity to cytoskeleton-directed drugs. The results presented demonstrate that there are developmental stage-specific associations between membrane proteins and the cytoskeleton during skeletal myogenesis. Several inconsistencies were found with traditional expectations of membrane-cytoskeleton associations. For example, although A5 is Triton-insoluble and sensitive to cytochalasin, its distribution generally does not correspond with any known cytoskeletal structure. Furthermore, the topography of A5 is dependent on the integrity of the plasma membrane. H36 and I20 are completely soluble in Triton and therefore by accepted definitions would not be expected to be associated with any cytoskeletal component. Yet H36 and actin codisrupt in the presence of cytochalasin, while I20, whose distribution does not correspond with microtubules, is uniquely sensitive to their disruption. These results demonstrate that (i) neither Triton-solubility nor colocalization alone predicts all membrane-cytoskeleton associations; some associations between the membrane and cytoskeleton are unstable in nonionic detergent; (ii) the native distribution of proteins in the membrane may not reflect their cytoskeletal associations; and (iii) the topography of some membrane proteins with no apparent association with the cytoskeleton may be greatly influenced by the cell cytoskeleton.

Actins↗

PubMed1992

Shortening of the calycal process actin cytoskeleton is correlated with myoid elongation in teleost rods.

Light activates the elongation of rods within teleost retinas. Rod cell elongation is mediated by actin-dependent length changes of the myoid portion of the inner segment. The actin cytoskeleton of the inner segment consists of filament bundles, which run parallel to the long axis of the rod, from the calycal processes, through the ellipsoid and into the myoid. In isolated rod inner/outer segments (RIS-ROS), myoid elongation was found to occur in the absence of net polymerization of actin into filaments. Outgrowth of actin filaments within the myoid was counterbalanced by a shortening of actin filaments within the calycal processes. In this study, we have further examined light-activated modifications of the rod cytoskeleton using rhodamine-phalloidin to stain actin filaments within retinal cryosections as well as in isolated RIS-ROS. In RIS-ROS isolated from dark-adapted green sunfish, the phalloidin-stained calycal processes appeared as long, brush-like structures, averaging 4.2 microns in length. In light-cultured RIS-ROS populations, the calycal process actin cytoskeleton shortened from 4.2 microns to 1.7 microns. In control, dark-cultured populations, RIS-ROS that did not elongate maintained long calycal process actin cytoskeletons. However, in cases where dark-cultured RIS-ROS did elongate, despite the absence of a light stimulus, myoid elongation was accompanied by a shortening of the calycal process actin cytoskeleton, suggesting that the two events are correlated with one another. In light-adapted green sunfish and in light-cultured retinas from green sunfish and the Midas cichlid, the calycal process cytoskeleton of intact rods shortened by 40-60%. Within the two-tiered retina of green sunfish, shortening of the calycal process cytoskeleton, from 5.1 microns to 2.1-3.1 microns, was only evident in the shorter, inner tier of rods. The calycal process actin cytoskeleton did not appear to shorten within the longer, outer tier of rods; here, stained processes were short, averaging 2.3 microns in length, within dark-adapted retinas. Using scanning and transmission electron microscopy, we present evidence to suggest that the plasmalemmal surface of the calycal processes shortens along with the cytoskeletal actin core. We conclude that calycal processes of teleost rods are dynamic structures which shorten during light-activated myoid elongation.

Animals↗

PubMed1992

Association of a receptor and G-protein-regulated phospholipase C with the cytoskeleton.

Approximately 98% of turkey erythrocyte phospholipase C (PLC) is cytosolic and is released by hypotonic lysis of the cells and extensive washing of the resultant erythrocyte ghosts. Well washed turkey erythrocyte ghosts retain a fraction of tightly associated PLC, which is activated by the P2y-purinergic receptor and G-protein present in ghost membranes. The particulate PLC is sufficient to couple to all the available purinergic receptor-regulated G-protein. In contrast to ghosts, turkey erythrocyte plasma membrane preparations contain no detectable PLC. To investigate the subcellular location of the ghost-associated PLC, cytoskeletons were prepared by Triton X-100 extraction of turkey erythrocyte ghosts. The ghost-associated PLC was quantitatively recovered in cytoskeleton preparations. Cytoskeleton-associated PLC was solubilized by sodium cholate extraction, partially purified, and shown to reconstitute with PLC-free plasma membrane preparations in an agonist and guanine nucleotide-dependent fashion, indicating that the cytoskeleton-associated PLC is G-protein-regulated. Dissociation of erythrocyte ghost cytoskeletons with the actin-binding protein DNase 1 resulted in a dose-dependent inhibition of agonist and guanine nucleotide-stimulated PLC responses in ghosts and caused release of PLC from ghost or cytoskeleton preparations. These data demonstrate the specific association of a receptor and G-protein-regulated PLC with a component of the detergent-insoluble cytoskeleton and indicate that the integrity of the actin cytoskeleton is important for localization and effective coupling of PLC to the relevant G-protein.

Animals↗

PubMed1987

Role of cell cytoskeleton in Mo-MuLV env transport and processing: implications in ts1 neuropathology.

Treatment of Mo-MuLV-infected cells with cytochalasin B (CB), a microfilament disrupting drug, caused a reduction in virus yield as judged by infectivity assay and reverse transcriptase activity. Pulse-chase experiments with [3H]leucine showed that the env precursor, gPr80env, was inefficiently processed in cells treated with CB. In the presence of monensin, an inhibitor of glycoprotein transport, gPr80env accumulated intracellularly and no gp70 was observed on the cell surface, indicating a complete block in the processing of gPr80env. Pulse-chase studies also showed that gPr80env was not processed in the presence of monensin. SDS-PAGE analysis of TX-100-extracted cell cytoskeletons (TX-insoluble fraction) iodinated and immunoprecipitated with goat anti-gp70 antiserum showed that CB or monensin treatment caused a marked increase of gPr80env in the cytoskeleton-rich fraction. However, the amount of gPr80env associated with the TX-soluble fraction in both CB or monensin-treated and untreated cells labeled with [3H]leucine was about the same. The gPr80env in the TX-100-soluble fraction of the cell was the endoglycosidase H (Endo-H) sensitive mannose-rich form, whereas the cytoskeleton-associated gPr80env was the partially Endo-H-resistant complex carbohydrate form. In the presence of CB or monensin, the complex carbohydrate form of gPr80env accumulated in the cytoskeleton-rich cell fraction. Examination of Mo-MuLV ts1 mutant, which is defective in the processing of env precursor polyprotein, also revealed an accumulation of the complex carbohydrate form of gPr80env in the cytoskeleton-rich fraction and an absence of gp70 on the surface of the cell at the restrictive temperature (39 degrees C). These studies suggest that the cytoskeleton plays a role in the transport and processing of MuLV gPr80env and that oligosaccharide conversion is an important factor in this process. Further, the accumulation of gPr80env on the cytoskeleton of ts1 infected cells at restrictive temperature may play a role in the neurological disorder caused by Mo-MuLV ts1 mutant.

Animals↗

PubMed1992

Cell surface beta-1,4-galactosyltransferase is associated with the detergent-insoluble cytoskeleton on migrating mesenchymal cells.

Cell surface beta-1,4-galactosyltransferase (GalTase) partially mediates a variety of cell interactions with laminin-containing matrices, including mesenchymal cell spreading and migration and neurite initiation, by binding to N-linked oligosaccharides within the E8 domain of laminin. Previous studies using indirect immunofluorescence have suggested that some surface GalTase colocalizes with actin-containing microfilaments in migrating cells. In this study, we present more direct biochemical evidence showing that surface GalTase is associated with the detergent-insoluble cytoskeleton and that this association is dependent upon the integrity of the cytoskeleton, valency of the anti-GalTase antibody, and migratory status of the cell. Two-thirds of the surface GalTase was associated with the detergent-insoluble cytoskeleton when assayed either by monovalent anti-GalTase Fab fragments or by extracting any detergent-soluble GalTase prior to labeling with intact anti-GalTase IgG. However, 80-100% of the surface GalTase could be induced to associate with the cytoskeleton when cross-linked with anti-GalTase IgG prior to detergent extraction. Destabilizing cytoskeleton-protein interactions with high levels of KCl, elevated pH, or cytochalasin B reduced the amount of surface GalTase retained in the detergent-insoluble cytoskeleton fraction. Finally, we have shown previously that laminin induces the expression of GalTase onto lamellipodia of migrating cells, and in this study, we show that the laminin-induced increase in surface GalTase is cytoskeletally associated. Collectively, these data suggest that cell surface GalTase participates in cell spreading and migration on laminin by virtue of its association with the cytoskeleton.

3T3 Cells↗