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M Bornens

Publications and source records attributed to M Bornens.

At least 91 records · Page 5Linked to original sources
PubMed1985

The Golgi apparatus remains associated with microtubule organizing centers during myogenesis.

In vitro myogenesis involves a dramatic reorganization of the microtubular network, characterized principally by the relocalization of microtubule nucleating sites at the surface of the nuclei in myotubes, in marked contrast with the classical pericentriolar localization observed in myoblasts (Tassin, A. M., B. Maro, and M. Bornens, 1985, J. Cell Biol., 100:35-46). Since a spatial relationship between the Golgi apparatus and the centrosome is observed in most animal cells, we have decided to follow the fate of the Golgi apparatus during myogenesis by an immunocytochemical approach, using wheat germ agglutinin and an affinity-purified anti-galactosyltransferase. We show that Golgi apparatus in myotubes displays a perinuclear distribution which is strikingly different from the polarized juxtanuclear organization observed in myoblasts. As a result, the Golgi apparatus in myotubes is situated close to the microtubule organizing center (MTOC), the cis-side being situated at a fixed distance from the nuclear envelope, a situation which suggests the existence of a structural association between the Golgi apparatus and the nuclear periphery. This is supported by experiments of microtubule depolymerization by nocodazole, in which a minimal effect was observed on Golgi apparatus localization in myotubes in contrast with the dramatic scattering observed in myoblasts. In both cell types, electron microscopy reveals that microtubule disruption generates individual dictyosomes; this suggests that the connecting structures between dictyosomes are principally affected. This structural dependency of the Golgi apparatus upon microtubules is not apparently accompanied by a reverse dependency of MTOC structure or function upon Golgi apparatus activity. Golgi apparatus modification by monensin, as effective in myotubes as in myoblasts, is without apparent effect on MTOC localization or activity and on microtubule stability. The main result of our study is to show that in a cell type where the MTOC is dissociated from centrioles and where antero-posterior polarity has disappeared, the association between the Golgi apparatus and the MTOC is maintained. The significance of such a tight association is discussed.

Animals↗
PubMed1984

Vimentin filaments and centrosomes. Are they associated?

HeLa cells were examined by immunofluorescence using anti-vimentin and anti-centrosphere anti-bodies, and by transmission electron microscopy (TEM), after vimentin redistribution induced by the action of nocodazole or taxol. A redistribution of vimentin bundles in the centriolar area was observed after nocodazole treatment, although no direct interaction between centrioles and vimentin filaments could be detected. After taxol treatment, the juxtanuclear accumulation of vimentin filaments and the centrioles were rarely observed in the same area. Our results do not support the concept of a direct association between centrioles and vimentin filaments.

Alkaloids↗
PubMed1982

Solubilization of nuclear structures by the polyanion heparin.

Rat liver nuclei were treated with different concentrations of the polyanion heparin, at low ionic strength and in the absence of divalent cations. Heparin at the optimal concentration (Heparin:DNA greater than or equal to 1) completely solubilized the intranuclear structures, allowing the preparation of pure nuclear membranes. The majority of the nuclear proteins, including all of H1 and 70% of the core histone oligomers, were solubilized. By contrast, suboptimal concentrations of heparin (heparin:DNA less than 0.3) resulted in the selective solubilization of non-histone proteins and the partial solubilization of chromatin without dissociation of histones from DNA. Among the RNA- and DNA-associated non-histone proteins revealed by the heparin fractionation procedure, some are noteworthy. A DNA-associated protein of 40,000 daltons was found to be associated with core histone oligomer. Three proteins of Mr = 62,000, 70,000, and 74,000, identical with that of the lamina proteins, were distinguished by their presence in all the fractions obtained, their predominance in the less soluble fractions, and their tight association with DNA and RNA. More than the solubilization of the histones, their extraction seems to be a prerequisite for chromatin solubilization by heparin.

Animals↗
PubMed1982

In vivo and in vitro effects of the mitochondrial uncoupler FCCP on microtubules.

FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone), a potent uncoupler of oxidative phosphorylation, induces the complete disruption of cellular microtubules. A further analysis of this effect on BHK21 cells has shown that a decrease in the number of microtubules can be observed 15 min after adding FCCP and there is complete disruption after 60 min. Regrowth of microtubules was initiated 30 min after removal of FCCP, in marked contrast with the rapid reversion observed when microtubules are disrupted by nocodazole. A similar delay was required for the recovery of mitochondrial function as assessed by rhodamine 123 labelling. The effect of FCCP on microtubules was partially inhibited by preincubation of the cells with NaN3, suggesting that FCCP acts on microtubules through mitochondria. FCCP did not depolymerize microtubules of cells permeabilized with Triton X-100. In vitro polymerisation of microtubule protein was only slightly diminished by concentrations of FCCP which provoke complete disassembly in vivo. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the microtubules polymerized in vitro in the presence of FCCP showed a reduced amount of high mol. wt. proteins, mainly MAP 2, associated with them. In an attempt to reproduce the mitochondrial effects of FCCP in vitro, we checked the effects of alkaline pH and calcium on microtubule protein polymerization in the presence of FCCP. FCCP did not influence the calcium inhibitory effect but did significantly increase the inhibitory effect of alkaline pH. We conclude that FCCP could depolymerise microtubules in vivo through a dual operation: increasing the intracellular pH by the disruption of the mitochondrial H+ gradient and decreasing the stability of microtubules by impairing the binding of microtubule-associated proteins.

Animals↗
PubMed1978

Isolation of nuclear envelopes with polyanions.

Optimal conditions for the isolation of nuclear envelopes by the action of heparin on nuclei are established and a morphological and biochemical study of such isolated envelopes is presented. An almost 100% yield of pure nuclear envelopes can be obtained by a single sedimentation step after incubation of nuclei with heparin for 40 min at 4 degrees C. The nuclear membrane pellet obtained in this way contains whole envelopes with a preserved perinuclear space and with ribosomes present on the outher leaflet. A single band with an apparent buoyant density of 1.18 is obtained by sucrose density gradient analysis. The chemical composition of the pellet is similar to that of the purified membranes and corresponds to 62% proteins, 34% phospholipids, 3% RNA, and 0.5% DNA. The presence of low concentrations of sodium phosphate (2-10 mM) is critical for a complete solubilization of the chromatin. A less rapid and complete solubilization is obtained with the potassium salt. Low concentrations of Mg++ (1-3 mM) counteract chromatin solubilization by heparin mainly at the level of chromatin-nuclear membrane association. The presence of EDTA in the medium leads to isolated nuclear envelopes on which neither ribosomes nor nuclear pores are visible, indicating the pore structure is dependent on the presence of Ca++ or Mg++. A comparison with other polyanions indicates a decisive advantage of heparin. However, pure nuclear envelopes can also be obtained by the action of dextran sulfate (mol wt 500,000) on nuclei incubated for 5 min at 37 degrees C, in the presence of phosphate ions.

Animals↗
PubMed1978

Detection of tubulin and actin in various cell lines by an immunoperoxidase technique.

This paper reports on the preparation of immunsera against tubulin and actin, and the purification of anti-tubulin and anti-actin antibodies on immunoadsorbent columns. These purified antibodies were used in an indirect immunoperoxidase assay to visualize microtubules and microfilaments in various cell lines. The specificity of antibodies and the methods of cell fixation required are discussed, as well as some aspects of microtubule and microfilament organization, as visualized by this technique.

Actins↗
PubMed1978

[Role and origin of the centriole].

The centriole could constitute a system of inertia in the cell, providing the system of references controlling the movement of the cell. It would ensure equally the coherence of cell metabolism and its stability. The origin and functioning of the centriole would be linked to a singel physical phenomenon.

Animals↗
PubMed1978

Visualization of tubulin in lymphocytes. I. Comparison of normal and chronic lymphocytic leukemia (CLL) lymphocytes.

Cell structures containing tubulin were studied in peripheral blood lymphocytes from 8 normal donors and 11 patients with CLL using specific antitubulin antibodies revealed by immunoperoxidase assay. The centriole and microtubules were clearly visible in both groups. A "nucleus-associated tubulin-containing structure" was revealed by antitubulin antibodies and was found in virtually all lymphocytes of normal subjects but in a considerably lower number of CLL lymphocytes. The nature of this structure and its relationship to other cell structures are discussed.

Adult↗
PubMed1977

Control of density and microredistribution of concanavalin-A receptors in rat thymocytes at 4 degrees C.

In a previous paper, we reported that at 4 degrees C, a cooperative binding of concanavalin A to rat thymocytes is observed which corresponds to a modification of the membrane leading to the recruitment of receptors and their immobilization. In this paper, we report that both phenomena are modulated at 4 degrees C by drugs such as colchicine and cytochalasin B; cooperative binding of concanavalin A, which reflects receptor recruitment is only slightly modified by each drug alone. when the two drugs are used simultaneously, the binding of concanavalin A to rat thymocytes at low concentrations of the lectin is decreased, while at high concentrations it remains unchanged. The binding of succinyl-concanavalin A to drug-treated cells is lowered at all concentrations of lectin. Also, we have studied the effects of colchicine and cytochalacine B on the binding of horseradish peroxidase to cell-bound concanavalin A, or succinyl-concanavalin A. We have found a decreased amount of horseradish peroxidase binding to concanavalin A bound to cells treated with colchicine or cytochalasine B. In the presence of the two drugs the decrease of peroxidase binding suggested a synergistic action of colchicine and cytochalasin B.

Animals↗
PubMed1977

Antibodies to tubulin in normal nonimmunized animals.

Sera of normal nonimmunized rabbits, pigs, calves, and humans contain tubulin-reactive antibodies. Usually, low amounts of antibodies against tubulin of the IgG class (2.5-4 mg/100 ml of serum from nonimmunized animals) were isolated. Anti-tubulin antibodies were also produced by injecting pig tubulin in complete Freund's adjuvant into rabbits. Slightly higher amounts of anti-tubulin antibody were isolated from sera of immunized rabbits (7 mg/100 ml of serum). The cytoplasmic network of microtubules of Tcc 36 mouse cells in culture was not clearly stained by natural anti-tubulin antibodies, but dense staining of the centrosphere was observed. In contrast, induced anti-tubulin antibodies densely stained cytoplasmic microtubular networks. Vinblastine-induced tubulin paracrystals were equally stained by natural and induced anti-tubulin antibodies.

Animals↗
PubMed1976

Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes.

A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. 97 +/- 2% of the total membrane phosphorus is accounted for as phospholipid phosphorus. Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been indentified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present in the 49 000-60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences. Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight. Membrane phosphorus was distributed between two chromatographic fractions: one containing membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.

Amino Acids↗
PubMed1976

Cooperative binding of concanavalin A to thymocytes at 4 degrees C and micro-redistribution of concanavalin A receptors.

The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.

Animals↗