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Results for “Nuclear envelope”

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At least 19 recordsLinked to original sources
PubMed1977

Ribonucleic acid stimulation of mammalian liver nuclear-envelope nucleoside triphosphatase. A possible enzymic marker for the nuclear envelope.

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.

Adenosine Triphosphatases↗
PubMed1976

An enzymic analysis of a nuclear envelope fraction.

A rat liver nuclear envelope fraction isolated essentially by the technique of Monneron et al. (J. Cell Biol. 55, 104-125 (1972) is characterized by high levels of glucose-6-phosphatase and 5'-nucleotidase. A broadly specific nucleoside triphosphatase activity is present. Cytochromes b5 and P-450 as well as NADPH- and NADH-cytochrome c reductase activities are present but at lower levels than found in microsomes. Cytochrome c oxidase activity is low. RNA polymerase activity is absent from the nuclear envelope fraction. Cytochemistry shows that glucose-6-phosphatase activity is strong and restricted to the nuclear envelope of nuclei. 5'-Nucleotidase shows weak reaction deposit in whole nuclei but in contrast gives clear reaction deposit in isolated nuclear envelopes. Cytochemical reaction deposit due to nucleoside triphosphatase activity is not restricted to the nuclear envelope but is found to a larger extent within the nucleus.

Adenosine Triphosphatases↗
PubMed2026

Small GTPase RAN-driven PNET2 oligomerization and phase separation at the nuclear lamina promote nuclear envelope integrity in plants.

The nuclear envelope is a fundamental organizer of eukaryotic cells, yet how plants regulate its architecture and integrity remains poorly understood. In this study, we identified the plant inner nuclear membrane protein PLANT NUCLEAR ENVELOPE TRANSMEMBRANE 2 (PNET2) as a scaffold that maintains nuclear envelope integrity and genome stability. Loss of PNET2 function compromises nuclear membrane structure and sensitizes cells to DNA damage, whereas overexpression drives aberrant nuclear membrane expansion. Biochemically, PNET2 cooperates with the nuclear lamin protein KAKU4 and CROWDED NUCLEI 1 within the nuclear lamina to promote nuclear membrane remodeling, a process driven by biomolecular condensate formation via their intrinsically disordered regions. We further uncovered a direct interaction between PNET2 and the small GTPase RAN. Structural modeling and biochemical analyses revealed that its active GTP-bound form stimulates PNET2 oligomerization, potentially promoting its phase separation to drive membrane expansion. Genetic analyses showed that PNET2 and RAN function in a shared pathway essential for nuclear membrane integrity. Together, our findings define a regulatory module that orchestrates GTPase signaling to sustain nuclear membrane homeostasis in plants, positioning PNET2 as a nexus linking membrane dynamics, nuclear lamina organization, and genome protection.

PNET2↗
PubMed1979

Localization of oxidized nocotinamide--adenine dinucleotide glycohydrolase in the mouse liver nuclear envelope.

NAD+ glycohydrolase activity located in the nuclear envelope was maximally solubilized by treatment with 0.1--0.2% Triton X-100. The residual activity largely represents the chromatin-associated NAD+ glycohydrolase. Under these conditions the phospholipids were extensively solubilized (over 90%) while leaving the nuclei physically stable, although the nuclear membranes were removed, as shown by electron microscopy. After Triton X-100 treatment, deoxyribonuclease I did not significantly affect the residual NAD+ glycohydrolase activity, although the DNA was completely broken down. This enzyme activity can be released from the nuclear pellet by incubation with phospholipase C. For comparative studies, the glucose 6-phosphatase activity, known to be present in the nuclear envelope, was investigated. Treatment with 0.01% Triton X-100 released 10--20% of the phospholipids, but without solubilizing either glucose 6-phosphatase or NAD+ glycohydrolase. Higher Triton X-100 concentrations (0.1--1.0%) inhibited glucose 6-phosphatase, but not NAD+ glycohydrolase activity. NAD+ glycohydrolase is apparently present in a latent form in the nuclear envelope. Glucose 6-phosphatase, However, shows no such latency.

Animals↗
PubMed1976

Characterization of the NAD+ glycohydrolase associated with the rat liver nuclear envelope.

The localization of NAD+ glycohydrolase [EC 3.2.2.5] (NADase) in purified rat liver nuclei has been examined. Subnuclear fractionation revealed that at least 70% of the NADase in nuclei was associated with the nuclear envelope fraction. The nuclear envelope fraction was practically free of microsomal contamination as judged by electron microscopic morphometry and assays of microsomal marker enzymes. Therefore, NADase was found to be an integral component of the nuclear envelope. The enzymological properties of the nuclear envelope NADase were compared with those of the microsomal enzyme. The nuclear envelope NADase was identical to the microsomal enzyme in its Km for NAD+ (60 muM), pH optimum (pH 6.5), ratio of transglycosidase activity to NADase activity (about 0.5), thermal stability and sensitivity to various inhibitors. Thus, NADase is a common enzymic component of both the nuclear envelope and the endoplasmic reticulum.

Animals↗
PubMed1978

Abnormalities of the nuclear envelope in porcine muscle affected with congenital myofibrillar hypoplasia.

The nuclear ultrastructure of muscle fibres in congenital myofibrillar hypoplasia of piglets was studied in order to provide more information on the pathological changes present. In the initial stages of the disease, muscle fibres with a small reduction of myofibrils often had an increased number of nuclear pores. In the more advanced stages of the disease, the following nuclear abnormalities were observed in muscle fibres showing an extensive disintegration of myofibrils: 1) large dilations of the perinuclear cisternae, 2) small nuclear protrusions projecting into these dilations and 3) appositions of stray membranes of the disrupted endoplasmic reticulum to the nuclear envelope. The increase in the number of nuclear pores may be significant in the development of pathological changes in congenital myofibrillar hypoplasia which is thought to be a hereditary disease. The observed abnormalities of the nuclear envelope appear to be characteristic for muscle fibres undergoing degeneration.

Animals↗
PubMed1979

Properties of mammalian nuclear-envelope nucleoside triphosphatase.

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.

Adenine↗
PubMed1979

Dynamics of the nuclear envelope during cell cycle in plants.

Stereology of Allium cepa root meristem cells was done to evaluate changes in the nuclear envelope during cell cycle. A naturally synchronous population was labelled as binucleate by caffeine inhibition of cytokinesis. Growth of the nuclear envelope preferentially occurs from mid G2 to the next mid G1, most probably in relation to the reforming sister nuclei after mitosis. On the other hand, the number of nuclear pores doubles from mid G1 to mid G2, their growth rate being higher in the first half of interphase (from mid G1 to mid S). Hence, the new nuclear envelope probably lacks nuclear pores, which appear later.

Cell Cycle↗
PubMed2025

Unraveling Plant Nuclear Envelope Composition Using Proximity Labeling Proteomics.

The nuclear envelope (NE) defines the eukaryotic cell and functions in a myriad of fundamental cellular processes including but not limited to signal transduction, lipid metabolism, chromatin organization, and nucleocytoplasmic transportation. Although the general structure of the NE is well-conserved across eukaryotic kingdoms, its composition and functions vary substantially between species and remain largely unknown in plants. In this chapter, we describe a proximity-labeling-based proteomic approach to profile novel NE components in the model organism Arabidopsis. This method is generally suitable for the identification of protein components in subcellular compartments or protein complexes that are poorly accessible to traditional mass spectrometry approaches and can be easily applied to other plant species. In addition to giving a step-by-step detailed description of the proximity labeling proteomics procedure in plant samples, we also provide guidelines on the appropriate use of controls and statistical analysis to achieve a highly specific selection of probed candidates.

Proteomics↗
PubMed1977

Nuclear envelope transport capacity and the cell cycle in yeast (Saccharomyces cerevisiae).

Changes in the nuclear envelope transport capacity, as measured by the number of nuclear pore complexes/unit nuclear volume/cell, were followed during the Saccharomyces cerevisiae cell cycle using data obtained by freeze-fracture electron microscopy. Pore number per unit nuclear volume decreased sharply in early G0, remained steady from mid-GO through S to G2, and showed a further slight decrease at M and G1. These periods of decline apparently resulted from nuclear enlargement without sufficient formation of new nuclear pore complexes to maintain the pore number to nuclear volume ratio. However, marked nuclear pore formation did accompany both increases in nuclear volume. The significance of these changes in relation to other events in the cell cycle is discussed. The validity of using nuclear pore number/unit nuclear volume and other pore number data as indices of nuclear envelope transport capacity and cell activity is critically examined.

Biological Transport↗
PubMed1979

The lectin binding sites on the membranes of the nuclear envelope, mitochondria and the cell surface of human lymphoblastoid cells.

The membranes of the cell surface, the endoplasmic reticulum, outer and inner mitochondrial leaflet and nuclear envelope were isolated from three human lymphoblastoid cell lines. Membrane components were separated by dodecyl sulfate polyacrylamide gel electrophoresis and the gels incubated with the radioiodinated lectins from lentil, castor bean, scarlet runner bean, gorse seed and Roman snail. After gel slicing and counting, the molecular weights of the lectin binding sites were determined. About 20 glycoproteins were identified as constituents of the plasma membrane, a similar glycoprotein distribution was observed in the endoplasmic reticulum. The outer mitochondrial membrane contained some impurities from the plasma membrane, the inner mitochondrial membrane lacked specific lectin receptors. Two prominent glycoproteins with molecular weights of 70 000 and 60 000 were identified with the castor bean lectin in the nuclear envelope.

Cell Line↗
PubMed1979

Comparison of the major polypeptides of the erythrocyte nuclear envelope.

The three most abundant nonhistone polypeptides (molecular weights 75,000, 71,000 and 61,000) of the avian erythrocyte nucleus have previously been isolated in the nuclear envelope fraction. They have been separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and peptide-mapped after limited enzymatic digestion. Three enzymes -- chymotrypsin, papain and Staphylococcus aureus protease -- were used. Results obtained with each enzyme indicate strong similarities between the three nuclear envelope polypeptides. The amino acid compositions of the two most abundant polypeptides (P75 and P71) have been determined and found to be similar. Further, they readily yield large fragments upon brief alkaline hydrolysis. For both P75, and P71 the degree and the pattern of alkaline fragmentation are almost identical. A 61,000-dalton polypeptide which appears to be P61 is obtained from P75 and P71 by mild acid hydrolysis. These results establish the close chemical similarity of these predominant polypeptides in the erythrocyte nucleus and suggest that they serve related functions.

Amino Acids↗
PubMed1979

[Action of x-ray irradiation on the nuclear envelope of rat liver cells].

X-irradiation of isolated nuclear envelopes (NE) has revealed their high radiosensitivity, while irradiation of isolated intact nuclei in vitro, in the doses up to 5000 r 18--20 hours after partial hepatectomy, produced no morphological changes in NE. The damaging effect of irradiation on both nuclei and mitochondria (Mt) was revealed only with a decrease in cytochrome-c-oxidase (CO) activity in parallel with an increase in the radiation dose. One hour after the whole body irradiation of rats in the beginning of S-period, the damaging effect was recorded in both NE and Mt at the doses of 50 and 150 t, and was enhanced with the increase of irradiation dose. Morphological changes were observed mostly in the outer nuclear membrane, which lost its distinct outline and disappeared from some nuclear regions. Lethal radiation doses produced a decrease in the number of pore complexes (PC) with their evident segregation from the membranes. After irradiation in a dose of 1200 r, only the residue or "ghosts" of the PCs remained. After irradiation in doses up to 400 r, the CO-activity recovered during the first hour in Mt and during first two hours in the nuclei.

Animals↗
PubMed1979

Cycle specific association of nascent chromatin with nuclear envelope components in Physarum polycephalum.

The action of heparin on isolated nuclei derived from different phases of the mitotic cycle in plasmodia of Physarum polycephalum was studied. Heparin addition at two-fold excess over DNA concentration to nuclei in Mg-free low ionic strength buffer (10 mM TRIS-HC1, 10 mM Na2 HPO4, pH = 8) releases 60-80% of chromatin from S, G2, and mitotic phase nuclei. The RNA/protein ratio of herparin-solubilized cromatin is constant through S and G2 phases, but rises about two-fold at early prophase coincident with nucleolar breakdown. Purified nuclear envelopes were obtained from heparin-treated nuclei by sedimentation according to Bornens procedures (Nature 244, 28, 1973), and examined by transmission electron microscopy. Residual chromatin is seen at all stages with fine network of DNA fibrils in contact with the envelop. Regardless of time in S, 80% of 3H-labeled DNA was released into soluble chromatin with identical 3H/14C ratios. The residual chromatin in nuclear envelopes exhibited a preferential association of early S-DNA in nuclei engaged in early S replication, and late S preferential association in nuclei engaged in late S replication.

Cell Nucleus↗
PubMed1976

Nuclear envelope inclusions demonstrated by freeze-fracture.

Inclusions in the perinuclear space of the nuclear envelope of human diploid (MRC-5) fibroblasts, limpet (Patella vulgata) haemocytes, and yeast (Saccharomyces cerevisiae) as observed with the freeze-fracture technique are described. The significance of these inclusions is discussed and it is tentatively concluded that they represent vesicles engaged in transporting macromolecules between nucleus and cytoplasm. Although the inclusions were infrequently observed, their demonstration in mammalian, invertebrate and lower eukaryotic cell types raises the possibility that this form of nucleocytoplasmic exchange may potentially be adopted under appropriate circumstances by the eukaryotic cell in general.

Animals↗
PubMed1979

Transfer of mouse nuclear envelope specific proteins to nuclei of chick erythrocytes during reactivation in heterokaryons with mouse A9 cells.

When chick erythrocyte nuclei are introduced into the cytoplasm of mouse A9 cells by cell fusion, proteins present in a fraction of the mouse nuclear envelope begin to appear in the envelope of the chick erythrocyte. The protein uptake was examined using antisera raised in chickens against the 3 major polypeptides of the nuclear pore complex-fibrous lamina fraction from rat liver nuclei. In indirect immunofluorescence studies these antisera give a strong envelope-specific staining with various mammalian but not chicken cells. Eighteen hours after cell fusion the first murine antigens can be observed in the erythrocyte nucleus. Two days after cell fusion the vast majority of the erythrocyte nuclei in cell hybrids contain some antigen and by 3 days the fluorescence of the reactivated erythrocyte nuclei reaches a level comparable to that of the mouse A9 nuclei. The rate of appearance of fluorescence in the chick nuclei depends upon the ratio of A9 cytoplasm to chick nuclei. Antigen uptake by the erythrocyte envelope is inhibited when protein synthesis is blocked suggesting that synthesis of mouse antigen, rather than a redistribution, determines the velocity or erythrocyte envelope reactivation. The early uptake of nucleospecific protein into the reactivating chick erythrocyte may not require any alteration in the nuclear envelope.

Animals↗
PubMed1977

Characterization of the nuclear envelope, pore complexes, and dense lamina of mouse liver nuclei by high resolution scanning electron microscopy.

We have used high resolution scanning electron microscopy (SEM) to study the nuclear envelope components of isolated mouse liver nuclei. The surfaces of intact nuclei are covered by closely packed ribosomes which are distinguishable by SEM from nuclear pore complexes. After removal of nuclear membranes with the nonionic detergent Triton X-100, the pore complexes remain attached to an underlying, peripheral nuclear lamina, as described by others. The surface of this dense lamina is composed of particulate granules, 75-150 A in diameter, which are contiguous over the entire periphery. We did not observe the pore-to-pore fibril network suggested by other investigators, but such a structure might be the framework upon which the dense lamina is formed. Morphometric analysis of pores and pore complexes shows their size, structure, and density to be similar to that of other mammalian cells. In addition, several types of pore complex-associated structures, not previously reported by other electron microscope (EM) techniques, are observed by SEM. Our studies suggest that the major role of the dense lamina is associated with the distribution, stability, and perhaps, biogenesis of nuclear pore complexes. Treatment of isolated nuclei with a combination of Triton X-100 and sodium deoxycholate removes membranes, dense lamina, and nuclear pore complexes. The resulting "chromatin nuclei" retain their integrity despite the absence of any limiting peripheral structures.

Animals↗
PubMed1978

Effects of divalent-cation chelators and chloramphenicol on the spatial relationship of the nuclear envelope to chromatin in micronuclei of Chinese hamster cells.

In the presence of the spindle poison Colcemid in the culture medium to prevent anaphase, approximately 20% of Chinese hamster metaphase cell were converted to micronucleated cells during 7 h. In the micronuclei the chromosome had become enclosed by a nuclear envelope (NE). In the light-microscope the micronuclei were of two kinds: with either visible chromatids or with decondensed chromosomes. In the electron microscope (EM) the spatial relationship of the NE to the chromatin was of two kinds only in the presence of Colcemid. In about 90% of the micronucleated cells the spatial relationship was normal, ie, the NE was immediately adjacent to the chromatin. In the remaining cells, the NE was distended so that the outer NE was separated from the inner one. In the presence of the divalent cation chelator, (ethylenedinitrilo) tetraacetic acid (EDTA) or the Ca2+-chelator [ethylenebis (oxyethylenenitrilo)] tetraacetic acid (EGTA) in addition to Colcemid, the amount of cells with micronuclei increased to 40%. The light-microscope appearance was the same as that found in the absence of the chelating agents. However, after Colcemid plus EGTA, EM revealed that only about 50% of the micronucleated cells had NE that was immediately adjacent to the chromatin and about 10% of them had distended outer NE. In the remaining 40% a third kind of spatial relationship was seen: the NE was intact but most of it was not adjacent to the chromatin. Furthermore, this type of micronucleus often contained mitochondria within the confines of NE. Thus, Ca2+ and possibly Mg2+ may regulate the rate of formation of the NE and also its ultrastructural relation to the chromatin. Mitochondrial function also appears to be involved in this relationship. In the presence of chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, in addition to Colcemid, only about 50% of the micronucleated cells exhibited the normal relationship. The outer NE was separated from the inner NE in about 46% of the micronucleated cells and the third kind of NE-chromatin relationship was observed only in 2%. In the case of the third kind of relationship produced by CAP, inclusion of mitochondria within the micronuclei was not observed, in contrast to the finding with EGTA.

Animals↗