Variability of mammalian liver nuclear-envelope preparations.
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ABSTRACT:
SUBMITTER: Agutter PS
PROVIDER: S-EPMC1162310 | biostudies-other | 1980 Oct
REPOSITORIES: biostudies-other
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Project description:1. The specific activity of rat and pig liver nuclear-envelope nucleoside triphosphatase (EC 3.6.1.3) decreases when the system is depleted of RNA. The activity can be restored by adding high concentrations of yeast RNA to the assay medium. 2. Exogenous RNA also increases the activity of the enzyme in control envelopes (not RNA-depleted). The effect appears to be largely specific for poly(A) and poly(G); it is not stimulated by rRNA or tRNA preparations, ribonuclease-hydrolysed RNA, AMP, or double- or single-stranded DNA. 3. Inhibitors of the enzyme, in concentrations at which half-maximal inhibition of the enzyme is achieved, do not affect the percentage stimulation of the enzyme by yeast RNA. 4. The simulation is abolished by the inclusion of 150 mM-KCl or -NaCl in the assay medium, but not by increasing the assay pH to 8.5. 5. The results are discussed in the light of the possible role of the nucleoside triphosphatase in vivo in nucleo-cytoplasmic ribonucleoprotein translocation. 6. It is proposed that poly(G)-stimulated Mg2+-activated adenosine triphosphatase activity should be adopted as an enzymic marker for the nuclear envelope.
| S-EPMC1164652 | biostudies-other
Project description:The nucleoside triphosphatase activities of the nuclear envelopes from rat liver, pig liver and simian-virus-40-transformed mouse-embryo 3T3 cells were shown to exhibit similar parperties. All three preparations hydrolyse ATP, 2'-dATP, 3'-dATP, GTP, CTP and UTP in the presence of Mg2+, Ca2+, Mn2+ and Co2+ with a pH optimum of 8.0, are sensitive to inhibition by mercurials, arsenicals, quercetin, proflavin and adenosine 5'-[gamma-thio]triphosphate and are partially inactivated by exposure to high ionic strength. The kinetic behaviour is similar for all substrates irrespective of the source of material. The typical Eadie-Hofstee plot, which is concave upwards at pH 8.0 when the ionic strength is 20mM, becomes linear when the pH is increased to 8.5 or the ionic strength to 160mM. The overall evidence, particularly the labelling of only one polypeptide by [gamma-32P]ATP, suggests that under the conditions of preparation and assay used only one class of nucleoside triphosphatase active sites is detectable in nuclear envelopes. The importance of these results for an understanding of the role of the enzyme in vivo is discussed.
| S-EPMC1161204 | biostudies-other
Project description:Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the l I: nker of N: ucleoskeleton and C: ytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153.
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Project description:Analysis of large-scale interphase genome positioning with reference to a nuclear landmark has recently been studied using sequencing-based single cell approaches. However, these approaches are dependent upon technically challenging, time consuming and costly high throughput sequencing technologies, requiring specialized bioinformatics tools and expertise. Here, we propose a novel, affordable and robust microscopy-based single cell approach, termed Topokaryotyping, to analyze and reconstruct the interphase positioning of genomic loci relative to a given nuclear landmark, detectable as banding pattern on mitotic chromosomes. This is accomplished by proximity-dependent histone labeling, where biotin ligase BirA fused to nuclear envelope marker Emerin was coexpressed together with Biotin Acceptor Peptide (BAP)-histone fusion followed by (i) biotin labeling, (ii) generation of mitotic spreads, (iii) detection of the biotin label on mitotic chromosomes and (iv) their identification by karyotyping. Using Topokaryotyping, we identified both cooperativity and stochasticity in the positioning of emerin-associated chromatin domains in individual cells. Furthermore, the chromosome-banding pattern showed dynamic changes in emerin-associated domains upon physical and radiological stress. In summary, Topokaryotyping is a sensitive and reliable technique to quantitatively analyze spatial positioning of genomic regions interacting with a given nuclear landmark at the single cell level in various experimental conditions.
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Project description:The nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process.
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Project description:In order to investigate the impact of using in vitro techniques to generate single cell suspensions of Mycobacterium tuberculosis (Mtb) on macrophage gene expression, we compared uninfected bone marrow derived macrophages to macrophages infected with Mtb that was prepared using gentle sonication followed by low-speed centrifugation (so/sp) or passage through a 5 µm syringe filter (5µmF).
2022-06-23 | GSE206485 | GEO
Project description:We describe a procedure for the preparation of sealed nuclear-envelope vesicles from rat liver nuclei. These vesicles are strikingly similar in their polypeptide composition when compared with those of nuclear envelopes prepared conventionally using deoxyribonuclease I. Subfractionation analysis by means of extraction with high salt and urea show that the components of the nuclear envelope, e.g. the pore-complex/lamina fraction, are present. The residual DNA content is only 1.5%, and typical preparations consist of about 80% vesicles, with the vesicular character of these envelopes shown by microscopic and biochemical studies. The vesicles can be obtained in high yield, are tight and stable for at least two days and are enriched in a nucleoside triphosphatase thought to be involved in nucleocytoplasmic transport processes. Because the vesicles are largely free of components of the nuclear interior, but retain properties of intact nuclei, we believe that they are a valuable model system to study nucleocytoplasmic transport. Although in transport studies with isolated nuclei interference from intranuclear events has to be considered, the nuclear-envelope vesicles provide the possibility of studying translocation alone. Furthermore, the less complex nature of these vesicles compared with whole nuclei should facilitate investigation of the components involved in the regulation of nuclear transport processes.
| S-EPMC1147544 | biostudies-other
Project description:Numerous studies in the past years provided definite evidence that the nuclear envelope is much more than just a simple barrier. It rather constitutes a multifunctional platform combining structural and dynamic features to fulfill many fundamental functions such as chromatin organization, regulation of transcription, signaling, but also structural duties like maintaining general nuclear architecture and shape. One additional and, without doubt, highly impressive aspect is the recently identified key function of selected nuclear envelope components in driving meiotic chromosome dynamics, which in turn is essential for accurate recombination and segregation of the homologous chromosomes. Here, we summarize the recent work identifying new key players in meiotic telomere attachment and movement and discuss the latest advances in our understanding of the actual function of the meiotic nuclear envelope.
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Project description:Hauling and anchoring the nucleus within immobile or motile cells, tissues, and/or syncytia represents a major challenge. In the past 15 years, Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) have emerged as evolutionary-conserved molecular devices that span the nuclear envelope and provide interacting interfaces for cytoskeletal networks and molecular motors to the nuclear envelope. Here, we review the molecular composition of LINC complexes and focus on how their genetic alteration in vivo has provided a wealth of information related to the relevance of nuclear positioning during tissue development and homeostasis with a special emphasis on the central nervous system. As it may be relevant for metastasis in a range of cancers, the involvement of LINC complexes in migration of nonneuronal cells via its interaction with the perinuclear actin cap will also be developed.
| S-EPMC4310828 | biostudies-literature
Project description:Intact nuclear 'ghosts' containing small amounts of DNA were obtained from rat liver. Incubation of radiolabelled dihydrotestosterone with isolated nuclear-envelope fraction from male rat liver resulted in specific binding of the dihydrotestosterone to the membranes. Optimal binding occurred at 20 degrees C after 20h incubation. Storage for 2 weeks at -80 degrees C resulted in little loss of specific binding. Scatchard analysis revealed a class of binding sites with a KD of 23.2 nM. Pronase and heat treatment destroyed the binding site. Androgens and glucocorticoids competed for labelled dihydrotestosterone binding to the ghosts, whereas oestrogens did not compete. Castration 24h before preparation of ghosts did not alter the binding site, and a similar class of binding sites was identified on female rat liver nuclear envelopes.
| S-EPMC1158095 | biostudies-other