RESUMEN
Agrin, a proteoglycan secreted by motoneurons, is a critical organizer of synaptic differentiation at skeletal neuromuscular junctions. Agrin is widely expressed in the nervous system so other functions seem likely, but none have been demonstrated. To test roles for agrin in interneuronal synapse formation, we studied hippocampi from mutant mice that completely lack the z+ splice form of agrin essential for neuromuscular differentiation and also exhibit severely ( approximately 90%) reduced levels of all agrin isoforms (M. Gautam et al., 1996, Cell 85, 525-535). The brains of neonatal homozygous agrin mutants were often smaller than those of heterozygous and wild-type littermates, but were morphologically and histologically indistinguishable. In particular, antibodies to pre- and postsynaptic components of glutamatergic synapses were similarly coaggregated at synaptic sites in both mutants and controls. Because mutants die at birth due to neuromuscular defects, we cultured neurons to assess later stages of synaptic maturation. In primary cultures, the agrin-deficient neurons formed MAP2-positive dendrites and tau-1-positive axons. Synaptic vesicle proteins, AMPA- and NMDA-type glutamate receptors, GABAA receptors, and the putative synapse-organizing proteins PSD-95, GKAP, and gephyrin formed numerous clusters at synaptic sites. Quantitatively, the number of SV2-labeled contacts per neuron at day 5 and the number of PSD-95 clusters per dendrite length at day 18 in culture showed no significant differences between genotypes. Furthermore, exogenous z+ agrin was unable to induce ectopic accumulation of components of central glutamatergic or GABAergic synapses as it does for neuromuscular cholinergic synapses. These results indicate that the z+ forms of agrin are dispensable for glutamatergic and GABAergic synaptic differentiation in the central nervous system.
Asunto(s)
Agrina/fisiología , Hipocampo/fisiología , Interneuronas/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Agrina/deficiencia , Agrina/genética , Empalme Alternativo , Animales , Diferenciación Celular , Células Cultivadas , Cruzamientos Genéticos , Desarrollo Embrionario y Fetal , Exones , Heterocigoto , Hipocampo/citología , Hipocampo/embriología , Interneuronas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neuronas/citología , Sinapsis/genética , Sinapsis/ultraestructuraRESUMEN
The molecular mechanisms underlying the establishment of a postsynaptic receptor mosaic on CNS neurons are poorly understood. One protein thought to be involved is gephyrin, a peripheral membrane protein that binds to the inhibitory glycine receptor and functions in clustering this receptor at synapses in cultured rat spinal cord neurons. We investigated the possible association of gephyrin with synapses in cultured rat hippocampal neurons, where glutamate and GABA but not glycine are the principal transmitters. Gephyrin immunoreactivity was detected in axons as well as dendrites, changing from a predominantly axonal to a more dendritic distribution with time in culture. Gephyrin staining was not distributed uniformly, but always took the form of clusters. Small clusters of gephyrin (0.2 microns 2), present throughout development, were distributed widely and not restricted to synaptic sites. Larger clusters of gephyrin (0.4-10.0 microns 2, sometimes composed of groups of small clusters), which developed in older cells, were localized to a subset of contacts between axons and dendrites. These large clusters were not present at glutamatergic synapses (marked by immunostaining for GluR1), but were closely associated with GABAergic synapses (marked by immunostaining for GABA and glutamic acid decarboxylase). These results, together with previous findings, suggest that gephyrin may function to anchor GABA and glycine receptors, but not glutamate receptors, at postsynaptic sites on central neurons. They also raise the possibility that gephyrin has additional functions, independent of its role at synapses.
Asunto(s)
Proteínas Portadoras/química , Hipocampo/metabolismo , Proteínas de la Membrana/química , Terminales Presinápticos/metabolismo , Receptores de GABA/metabolismo , Receptores de Glutamato/metabolismo , Animales , Células Cultivadas/química , Histocitoquímica , RatasRESUMEN
We used the technique of scrape loading to introduce phalloidin into mouse embryo fibroblasts in mass culture. Phalloidin almost completely destroyed actin microfilament bundles, but the amount of polymerized cytoskeleton-associated actin was increased approximately two-fold and the amount of monomeric (Triton X-100 extractable) actin was significantly reduced. The major result of the present study is that the rate of actin synthesis in the phalloidin-treated cells was 2-3 times higher than in the control cells. Northern blot and translation in a cell-free system from rabbit reticulocytes showed that the actin mRNA level significantly increased as a result of phalloidin treatment.
Asunto(s)
Actinas/biosíntesis , Faloidina/farmacología , Citoesqueleto de Actina/ultraestructura , Actinas/genética , Animales , Northern Blotting , Células Cultivadas , Citoesqueleto/metabolismo , Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Ratones , ARN Mensajero/genética , Tubulina (Proteína)/metabolismoRESUMEN
Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed.
Asunto(s)
Resistencia a Medicamentos , Linfocitos T/citología , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Northern Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Clonales , ADN/genética , Humanos , Células Asesinas Naturales/citología , Ganglios Linfáticos/inmunología , Linfoma , Ratones , Fenotipo , ARN/genética , Reserpina/farmacología , Linfocitos T/efectos de los fármacos , Vincristina/farmacologíaRESUMEN
Cultured hamster fibroblasts of the DM-15 cell line stained by rhodamine 123 gradually release the dye when placed in dye-free medium. Here we demonstrate that reserpine, verapamil, and trifluoperazine are capable of blocking this release. We also show that reserpine can inhibit the efflux of another dye, phosphine 3R, from DM-15 cells and the release of rhodamine 123 from mouse embryo fibroblasts, four mouse cell lines, and MDCK cells. The three substances that block the release of the dyes are potent inhibitors of the membrane transport system implicated in the phenomenon of multidrug resistance (MDR). By using this system MDR cells can pump many structurally unrelated drugs and dyes, including rhodamine 123 and phosphine 3R, from the cytoplasm to the outer medium. It appears from our results that the membrane transport system responsible for MDR operates slowly in nonresistant cells and can play a role in normal cell physiology.
Asunto(s)
Membrana Celular/metabolismo , Reserpina/farmacología , Trifluoperazina/farmacología , Verapamilo/farmacología , Animales , Transporte Biológico Activo/efectos de los fármacos , Línea Celular Transformada , Membrana Celular/efectos de los fármacos , Cricetinae , Resistencia a Medicamentos , Fibroblastos , Colorantes Fluorescentes , Microscopía Fluorescente , Mitocondrias/metabolismo , Rodamina 123 , RodaminasRESUMEN
Multinucleation of various cultured cells was produced by polyethylene glycol-induced fusion or by cytochalasin-induced block of mitosis. It was found that multinucleation induced by both methods considerably improved deficient spreading of all the tested transformed fibroblastic lines; average substratum area occupied by one cell and divided per number of nuclei was 2.0-2.5 times larger for multinucleated cells than for mononucleated ones. Improved spreading was accompanied by increased area of lamellar cytoplasm, increased number of focal contacts, and, in certain lines, by the appearance of actin bundles; numerous microtubules and intermediate filaments radiated from perinuclear zones into the lamellas of multinucleated cells. The number of cell-associated fibronectin fibrils was not increased by multinucleation. Cycloheximide did not prevent the improvement of spreading, suggesting that this effect was not due to any alterations of protein synthesis. Colcemid considerably decreased the effect of multinucleation but did not abolish it completely. It is suggested that increase of spreading is due to multinucleation-associated alterations of quantitative interrelationships between various cell components. One of these alterations is probably increased density of microtubules per unit length of outer cell edge.
Asunto(s)
Núcleo Celular/fisiología , Transformación Celular Neoplásica , Animales , Virus del Sarcoma Aviar/genética , Línea Celular , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Medios de Cultivo , Cicloheximida/farmacología , Demecolcina/farmacología , Células L/fisiología , Ratones , CodornizRESUMEN
Mitostatic action, cellular uptake and the binding of colchicine and colcemid to tubulin were compared. It was shown that mitostatic action of low doses of colchicine developed only after 24 h incubation of the drug with mouse L fibroblasts, while the colcemid-induced block of mitosis was evident after 2 h incubation. The initial rate of uptake was about 10 times greater for colcemid than for colchicine. Cellular uptake of the drugs reached an equilibrium after 2 and 15-18 h incubation for colcemid and colchicine, respectively, and the plateau values were identical. The kinetics of colchicine and colcemid binding to bovine brain tubulin was studied by the DEAE-filter binding assay. Colcemid binds to tubulin much faster than does colchicine. The rate of colcemid efflux from L cells is much higher than that of colchicine. According to the efflux data, colcemid dissociates readily from a complex with tubulin (t1/2 = 10 min), while the colchicine-tubulin complex is stable for at least 1 h. These results are consistent with previously published data (Frankel, F.R. (1976) Proc. Natl. Acad. Sci. U.S.A. 72, 2798-2802), which showed that colcemid action on cells is more reversible than that of colchicine. We suggest that differences between colchicine and colcemid in the rate of mitostatic action and its reversibility are determined by the differences in parameters of tubulin binding.
Asunto(s)
Colchicina/metabolismo , Demecolcina/metabolismo , Células L/efectos de los fármacos , Mitosis/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Animales , Bovinos , Células L/metabolismo , RatonesRESUMEN
The Colcemid-resistant L--53 cell strain was examined for cross-resistance to metaphase inhibotors (Vincristine, Vinblastine, estradiol-17beta), an antitumor antibiotic (Rubomycin C) and an alkylating agent (Lycurim), compared with the Colcemid-sensitive L cells. The L-53 cells proved to be resistant besides colchicine to Vincristine, Vinblastine and estradiol-17beta concerning their antimitotic effect. The comparison of the viability of L and L-53 cells in the presence of Rubomycin C and Lycurim showed a resistance of the L-53 cells to Rubomycin C, while the effect of Lycurim was the same on both cell lines. The chromosome-mutagenic action of Lycurim was also equal on both cell lines.
Asunto(s)
Antineoplásicos/farmacología , Daunorrubicina/farmacología , Eritritol/análogos & derivados , Células L/efectos de los fármacos , Mesilatos/farmacología , Alquilantes/farmacología , Animales , Línea Celular , Técnicas de Cultivo , Resistencia a Medicamentos , Eritritol/farmacología , Estradiol/farmacología , Ratones , Mitosis/efectos de los fármacos , Mutágenos , Vinblastina/farmacología , Vincristina/farmacologíaRESUMEN
The effect of the non-ionic detergent tween 80 on one colcemid-resistant and one sensitive subline of mouse L cells has been studied. The colcemid-resistant subline was also resistant to colchicine and Vinca alkaloids. Tween 80 at concentrations of 0.01% (v/v) or higher increased the sensitivity of drug-resistant cells to the antimitotic effect of colcemid, colchicine and vinblastine. Tween 80 also potentiated the initial rate and the maximal level of the (3-H)-cholchicine uptake by both sensitive and resistant cells. However, the detergent at concentrations of up to 1% had no effect on (3-H)-colchicine binding by cell homogenates. Thus it appears that the effects of tween 80 were due to an increase in cell membrane permeability to the drugs. The effect was completely reversible. The cells did not become adapted to the sensitizing action of tween 80 even after prolonged incubation in medium containing the detergent. A considerable increase in the permeability to the drug was obtained with doses of tween 80 which were non-toxic and which had no effect on cell proliferation, morphology and locomotion. The increase in membrane permeability caused by tween 80 at these concentrations was selective, the membrane permeability to ions and to (3-H)-2-deowy-D-glucose being, in fact, unchanged.