RESUMEN
This study extends the observations on the defects in pseudopod formation of ABP-120+ and ABP-120- cells by a detailed morphological and biochemical analysis of the actin based cytoskeleton. Both ABP-120+ and ABP-120- cells polymerize the same amount of F-actin in response to stimulation with cAMP. However, unlike ABP-120+ cells, ABP-120- cells do not incorporate actin into the Triton X-100-insoluble cytoskeleton at 30-50 s, the time when ABP-120 is incorporated into the cytoskeleton and when pseudopods are extended after cAMP stimulation in wild-type cells. By confocal and electron microscopy, pseudopods extended by ABP-120- cells are not as large or thick as those produced by ABP-120+ cells and in the electron microscope, an altered filament network is found in pseudopods of ABP-120- cells when compared to pseudopods of ABP-120+ cells. The actin filaments found in areas of pseudopods in ABP-120+ cells either before or after stimulation were long, straight, and arranged into space filling orthogonal networks. Protrusions of ABP-120- cells are less three-dimensional, denser, and filled with multiple foci of aggregated filaments consistent with collapse of the filament network due to the absence of ABP-120-mediated cross-linking activity. The different organization of actin filaments may account for the diminished size of protrusions observed in living and fixed ABP-120- cells compared to ABP-120+ cells and is consistent with the role of ABP-120 in regulating pseudopod extension through its cross-linking of actin filaments.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Proteínas Portadoras/genética , Dictyostelium/ultraestructura , Proteínas de Microfilamentos/genética , Seudópodos/ultraestructura , Actinas/metabolismo , Amanitinas/metabolismo , Animales , AMP Cíclico/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Dictyostelium/efectos de los fármacos , Dictyostelium/genética , Eliminación de Gen , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , Modelos Biológicos , Fagocitos/ultraestructura , Polietilenglicoles/farmacologíaRESUMEN
Mouse L929 cells were exposed to the nonionic detergent Brij 58. As has been shown in some other cell types, protein leaked from Brij 58 exposed cells only after a lag phase. In the current study we have extended the observations of the kinetics of protein efflux using cultured L cells subjected to treatment with buffers containing Brij 58. The results show that while the cells become permeable essentially at first exposure to the detergent, proteins do not escape immediately. This lag in efflux is at least partly dependent on the concentration of detergent such that a greater lag is seen in cells exposed to the lowest concentrations of Brij. Data are presented that are most readily interpreted as protein leakage having occurred fairly rapidly from individual cells and that show that the time course of protein efflux results, to a large extent, from different sensitivities of individual cells to the detergent. The permeabilized suspension cells consist of only two types, whereas the conversion of cells from one type to the other occurs through the loss of protein to the permeabilization medium. Only two bands are seen in continuous density gradients and there is a conversion of the more dense type to the less dense with longer exposure to detergent. Moreover, the less dense cells contained about half of the protein per cell as the bottom banding cells, and the proteins of the more dense cells appear to be the sum of those released into the permeabilization medium plus those found in the less dense cells.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Cetomacrogol/farmacología , Proteínas/metabolismo , Animales , Tampones (Química) , Línea Celular , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Fluoresceínas/metabolismo , Cinética , Células L , RatonesRESUMEN
We have examined a salt-soluble, transcriptionally competent gene-enriched fraction of chicken erythrocyte chromatin and compared it to bulk chromatin using the unique microanalytical capabilities of Electron Spectroscopic Imaging (ESI). The salt-soluble fraction is enriched 48 fold in beta-globin gene sequences and is also enriched in histones that are post-synthetically modified, including acetylation and ubiquitination. Differences between the two fractions are also apparent in the distribution of the two major forms of nucleoprotein structures, including (1) particles which present a circular profile and possess protein and DNA content nearly identical to that of the canonical nucleosome and account for 89% of particles in the bulk fraction but account for only 66% of the particles in the competent fraction, and (2) u-shaped particles which possess about 20% less protein mass than particles of circular profile and are about 10x more prevalent in the transcriptionally competent fraction than in the bulk. Additionally, elongated particles with protein and DNA content similar to the u-shaped objects are also seen in the competent fraction.
Asunto(s)
Cromatina/ultraestructura , Transcripción Genética , Acetilación , Animales , Pollos , Cromatina/química , Cromatina/metabolismo , ADN/análisis , Microanálisis por Sonda Electrónica , Eritrocitos , Genes , Globinas/genética , Histonas/genética , Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas/análisis , Sales (Química) , SolubilidadRESUMEN
The relationship between histone acetylation and the capacity of H1 histones to cause the 0.15 M NaCl-induced aggregation/precipitation of transcriptionally active/competent gene chromatin fragments was investigated. Previous studies have shown that transcriptionally active/competent, but not repressed, gene chromatin polynucleosomes, which were isolated from chicken erythrocytes, remained soluble in 0.15 M NaCl after being reconstituted with H1 histones. This result suggested that some component of the active/competent gene nucleosome altered the capacity of the H1 histones to condense the chromatin fiber. Recently, Hebbes et al. (Hebbes, T.R., Thorne, A.W., and Crane-Robinson, C. (1988) EMBO J. 7, 1395-1402) demonstrated directly that active, but not repressed, gene chromatin of chicken erythroid cells contain high levels of acetylated histones. Here, we show that the solubility of active/competent gene chromatin fragments in 0.15 M NaCl is dependent on the level of acetylated histone species, with induction of hyperacetylation increasing the solubility of this gene chromatin. Also, we show that lowering the levels of the acetylated histone forms reduces the ability of the active/competent gene chromatin fragments to resist exogenously added H1-histone-induced 0.15 M NaCl aggregation/precipitation. These results suggest that histone acetylation alters the capacity of the H1 histones to form compact higher order chromatin structures such that active/competent gene chromatin is maintained in a less folded state than the bulk of chromatin.
Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , Transcripción Genética , Acetilación , Animales , Southern Blotting , Pollos , ADN/sangre , ADN/genética , ADN/aislamiento & purificación , Eritrocitos/metabolismo , Femenino , Histonas/sangre , Histonas/aislamiento & purificación , Nucleasa Microcócica , Peso Molecular , Hibridación de Ácido NucleicoRESUMEN
The chromatin of several genes was assayed for sensitivity to DNAase I and for solubility as polynucleosomes in 0.15 M NaCl. The degree of solubility of chromatin fragments as polynucleosomes in 0.15 M NaCl correlates well with the sensitivity to DNAase I for several genes. Chromatin of repressed, housekeeping and erythroid-specific genes can be distinguished as distinct groups by the degree to which they display these properties. NaCl precipitation of chromatin fragments stripped and then reconstituted with varying quantities of H1 and H5 (linker) histones indicate that the polynucleosomes of erythroid-specific genes have altered interaction with these histones. Linker histones interacted with bulk chromatin and in the chromatin of the repressed ovalbumin and vitellogenin genes to form salt precipitable structures. Chromatin of erythroid-specific genes (histone H5 and beta-globin) as well as that of the histone H2A.F gene was resistant to linker histone induced precipitation.
Asunto(s)
Cromatina/efectos de los fármacos , Histonas/metabolismo , Animales , Pollos , Desoxirribonucleasa I/metabolismo , Etidio/farmacología , Microscopía Electrónica , Nucleosomas/análisis , Concentración Osmolar , SolubilidadRESUMEN
Mature chicken erythrocyte polynucleosomes which are soluble at physiological ionic strength are enriched in beta-globin DNA sequences. Vitellogenin chromatin, which is not expressed in this tissue, is found in aggregation prone, salt insoluble chromatin. There is a direct correlation between the size of soluble fragments and the degree of globin gene enrichment, with the largest fragments being most highly enriched. The highly globin enriched (about 50 fold) polynucleosomes contain significantly elevated levels of acetylated histones H4, H2A.Z, and H2B, and ubiquitinated (prefix "u") histones H2A and H2B (with a significant relative increase of uH2B over uH2A). These polynucleosomes were complexed with histones H1 and H5 but at a lower level than that found in unfractionated chromatin.
Asunto(s)
Eritrocitos/metabolismo , Genes , Globinas/genética , Histonas/sangre , Nucleosomas/metabolismo , Animales , Fraccionamiento Celular , Pollos , Cromatina/ultraestructura , Nucleoproteínas/sangre , Nucleoproteínas/aislamiento & purificación , Nucleosomas/ultraestructura , Concentración Osmolar , SolubilidadRESUMEN
We [Rocha, E., Davie, J.R., van Holde, K.E., & Weintraub, H. (1984) J. Biol. Chem. 259, 8558-8563] have previously reported that the transcriptionally competent beta-globin gene domain is selectively enriched in chromatin fractions eluted with solutions of approximately physiological ionic strength from micrococcal nuclease digested mature chicken erythrocyte nuclei. In this report, we demonstrate that beta-globin chromatin is eluted as oligonucleosomes while vitellogenin, a transcriptionally inactive gene, is eluted as mononucleosomes as is the bulk of sequences found in this fraction. Following removal of the salt, the eluted chromatin was made 100 mM KCl and separated into aggregation-prone and aggregation-resistant fractions. Globin sequences were present in both fractions and had the greatest enrichment in the aggregation-prone fraction which contained H1 and H5, H1 being more abundant. A procedure is presented in which H1 is selectively removed from the erythrocyte nuclei. Following the selective removal of H1 and subsequent fractionation, globin but not vitellogenin oligonucleosomes were present in the aggregation-resistant chromatin fraction. The results indicate the beta-globin domain is a mosaic of aggregation-resistant and aggregation-prone regions with the latter being associated with H1 and H5. Vitellogenin sequences were associated principally with aggregation-prone regions complexed with H5.