The nuclear envelope proteome.
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Hepatic nuclear envelope cytochrome P-450 in rats fed 2-acetylaminofluorene. Effect of dietary fats and butylated hydroxytoluene.
Early losses of nuclear envelope (NE) cytochrome P-450 occur during 2-acetylaminofluorene (AAF)-hepatocarcinogenesis. Butylated hydroxytoluene (BHT), which protects against AAF in rats fed high fat diets, preserves NE P-450 suggesting that the latter may be required for AAF detoxification. AAF-hepatocarcinogenesis is enhanced by increasing the amount or degree of unsaturation of dietary fats. The present studies show that in rats fed AAF in high fat diets BHT preservation of NE P-450 is considerably shorter (3 weeks) with polyunsaturated than with saturated fat (9 weeks). In rats fed AAF in a low fat diet, where BHT does not modify the low tumor incidence, there is a bimodal effect on NE P-450. In controls and AAF-fed rats, BHT causes significant induction of NE P-450 at 1-3 weeks. At 9-16 weeks BHT has no effect on controls and only partially preserves NE P-450 in AAF-fed rats. Due to time constraints, livers could not be utilized fresh, but they were kept frozen at -80 degrees C until processed. Thus, specimens from all groups underwent identical treatment for purposes of comparative studies.
2-Acetylaminofluorene↗
Transformation by simian virus 40 induces virus-specific, related antigens in the surface membrane and nuclear envelope.
Nucleus- and mitochondrion-free membranes from hamster lymphocytes transformed by simian virus 40 (SV40), GD248 cells, cause guinea pigs to produce immune sera that reveal the presence in GD248 plasma membranes and mitochondria of two types of glycoprotein that are not detected in membranes of normal lymphocytes [Schmidt-Ullrich, R., Thompson, W. S. & Wallach, D. F. H. (1977) Proc. Natl. Acad. Sci. USA 74, 643-647]. Indirect immune fluorescence of living, SV40-transformed T19 hamster reticulum cells, Balb/c 3T3 mouse fibroblasts, and W18 VA2 human fibroblasts, using the antisera against GD248 membrane, at 4 degrees produced a distinct cell surface fluorescence; however, above 20 degrees , staining at the nuclear perimeter, the SV40 U-antigen reaction, becomes equally prominent. In SV40-transformed cells that had been fixed in cold acetone, as well as in purified GD248 nuclei, thermostable U-antigen staining is dramatic, but there is no reaction for nuclear T-antigen. Rabbit antisera against T19 cells gave immunofluorescence reactions equivalent to those obtained with the antisera against GD248 cells. Normal guinea pig or rabbit sera and cells that had not been transformed by SV40 gave no reaction. Our sera from tumor-bearing hamsters gave only nuclear T-antigen fluorescence. The results indicate the presence of related, SV40-specific antigens in the surface membranes, nuclear envelope, and possibly other intracellular organelles of SV40-transformed cells.
Antigens, Viral↗
The fission yeast transforming acidic coiled coil-related protein Mia1p/Alp7p is required for formation and maintenance of persistent microtubule-organizing centers at the nuclear envelope.
Microtubule-organizing centers (MTOCs) concentrate microtubule nucleation, attachment and bundling factors and thus restrict formation of microtubule arrays in spatial and temporal manner. How MTOCs occur remains an exciting question in cell biology. Here, we show that the transforming acidic coiled coil-related protein Mia1p/Alp7p functions in emergence of large MTOCs in interphase fission yeast cells. We found that Mia1p was a microtubule-binding protein that preferentially localized to the minus ends of microtubules and was associated with the sites of microtubule attachment to the nuclear envelope. Cells lacking Mia1p exhibited less microtubule bundles. Microtubules could be nucleated and bundled but were frequently released from the nucleation sites in mia1delta cells. Mia1p was required for stability of microtubule bundles and persistent use of nucleation sites both in interphase and postanaphase array dynamics. The gamma-tubulin-rich material was not organized in large perinuclear or microtubule-associated structures in mia1delta cells. Interestingly, absence of microtubules in dividing wild-type cells prevented appearance of large gamma-tubulin-rich MTOC structures in daughters. When microtubule polymerization was allowed, MTOCs were efficiently assembled de novo. We propose a model where MTOC emergence is a self-organizing process requiring the continuous association of microtubules with nucleation sites.
Cell Cycle↗
Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins.
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.
Animals↗
Vault mobility depends in part on microtubules and vaults can be recruited to the nuclear envelope.
Vaults are ribonucleoproteins that may function in intracellular transport processes. We investigated the intracellular distribution and dynamics of vaults in non-small cell lung cancer cells in which vaults are labeled with the green fluorescent protein. Immunofluorescence experiments showed that vaults are dispersed throughout the cytoplasm; a small fraction is found in close proximity to microtubules. Immunoprecipitation experiments corroborated these results showing co-precipitation of MVP and beta-tubulin. Using quantitative fluorescence-recovery after photobleaching (FRAP), we demonstrated that vault mobility over longer distances in part depends on intact microtubules; vaults moving slower when microtubules are depolymerized by nocodazole. Biochemical fractionation indicated a small fraction of MVP associated with the nucleus, however, no GFP-tagged vaults could be observed inside the nucleus. We observed an accumulation of vaults at the nuclear envelope upon treatment of cells with the protein synthesis inhibitor cycloheximide. Analysis of nucleo-cytoplasmic transport using a fluorescent substrate containing a classical NLS and NES expressed in MVP+/+ and MVP-/- mouse embryonic fibroblasts indicated no differences in nuclear import/export kinetics, suggesting no role for vaults in these processes. We hypothesize that a subset of vaults moves directionally via microtubules, possibly towards the nucleus.
Active Transport, Cell Nucleus↗
Presence of neuropeptide Y and the Y1 receptor in the plasma membrane and nuclear envelope of human endocardial endothelial cells: modulation of intracellular calcium.
The aims of the present study were to investigate the presence and distribution of NPY and the Y1 receptor in endocardial endothelial cells (EECs), to verify if EECs can release NPY, and to determine if the effect of NPY on intracellular calcium is mediated via the Y1 receptor. Immunofluorescence, 3-D confocal microscopy and radioimmunoassay techniques were used on 20-week-old human fetal EECs. Our results showed that NPY and the Y1 receptor are present in human EECs (hEECs) and that their distributions are similar, the fluorescence labelling being higher in the nucleus and more particularly at the level of the nuclear envelope when compared with the cytosol. Using radioimmunoassay, we demonstrated that EECs are a source of NPY and can secrete this peptide upon a sustained increase of intracellular calcium ([Ca]i). Using fluo-3 and 3-D confocal microscopy technique, superfusion of hEECs as well as EECs isolated from rat adult hearts with increasing concentrations of NPY induced a dose-dependent, sustained increase in free cytosolic and nuclear Ca2+ levels. This effect of NPY on EEC [Ca]i was completely reversible upon washout of NPY and was partially blocked by BIBP3226, a selective Y1 receptor antagonist. The results suggest that NPY and Y1 receptors are present in the EECs of 20-week-old human fetal heart and they share the same distribution and localization inside the cell. In addition, EECs are able to secrete NPY in response to an increase in [Ca]i, and the Y1 receptor as well as other NPY receptors seem to participate in mediating the effects of NPY on [Ca]i in these cells. Thus, NPY released by EECs may modulate excitation-secretion coupling of these cells.
Aniline Compounds↗
Modifications of the nuclear envelope of BHK cells after infection with herpes simplex virus type 1.
Numerous discrete lesions, which we have termed blebs, appeared in the nucleus of BHK cells 10 to 15 h after infection with herpes simplex virus type 1 (HSV-1). They were formed in the inner portion of the nuclear envelope by the apposition of two thickened lamellae overlying a vacuole. As demonstrated by electron microscopic studies, blebs were regular and associated with the peripheral lamina in the nucleus, averaging 3.5 blebs per micron2. They appeared to be associated with an enrichment of the 155K major capsid protein in the nuclear membrane subfractions as compared with the protein composition of nuclei and plasma membrane fractions. We propose that blebs represent the site of assembly of capsid proteins before DNA insertion and eventual envelopment.
Animals↗
Effect of divalent cations and chelators on metaphase to telophase progression and nuclear envelope formation in Chinese hamster cells.
Chinese hamster DON cells in log phase were treated with Colcemid in the G2 period with or without divalent cation chelating agents. The metaphase cells were isolated and incubated in two ways: 1) without Colcemid but with chelating agents or La3+ and observed for metaphase to telophase progression, and 2) with Colcemid, with or without chelating agents and the rate of micronuclei formation in the absence of anaphase monitored. The effect of the chelating agents on cellular 45Ca2+ during metaphase to telophase progression was also studied. The results indicate that Ca2+ and possibly Mg2+ ions are involved in the regulation of certain segments of mitosis. The reduction of environmental and plasma membrane associated Ca2+ with the chelators and La3+ promoted the metaphase to telophase progression as well as nuclear envelope and micronuclei formation.
Animals↗
Translocation of Bax and Bid to mitochondria, endoplasmic reticulum and nuclear envelope: possible control points in apoptosis.
The cross-talk between endoplasmic reticulum (ER) and mitochondria was investigated during apoptosis in a breast cancer cell line (MCF-7) in culture. The effect of camptothecin, an inducer of apoptosis and a specific inhibitor of topoisomerase I, was investigated by morphological, immunocytochemical and histochemical techniques for electron microscopy. Our ultrastructural morphological data demonstrate alterations in ER configuration and communication with neighbouring mitochondria early after stimulation by camptothecin. Immunoelectron studies have demonstrated that Bax and Bid translocate from cytoplasm to mitochondria where they initiate mitochondrial dysfunction and cytochrome c release. Bax and Bid were also localized in ER and nuclear envelope. Since ER and mitochondria function as intracellular Ca2+ storage, we hypothesize that Bax and Bid are involved in the emptying of ER Ca2+ pool, triggers secondary changes in mitochondrial Ca2+ levels that contribute to cytochrome c release and cell death.
Apoptosis↗
The importin-beta P446L dominant-negative mutant protein loses RanGTP binding ability and blocks the formation of intact nuclear envelope.
Three of the four independently induced Ketel(D) dominantnegative female sterile mutations that identify the Drosophila importin-beta gene, originated from a C4114--> T transition and the concurrent replacement of Pro446 by Leu (P446L). CD spectroscopy of representative peptides with Pro or Leu in the crucial position revealed that upon the Pro-->Leu exchange the P446L mutant protein loses flexibility and attains most likely an open conformation. The P446L mutation abolishes RanGTP binding of the P446L mutant form of importin-beta protein and results in increased RanGDP binding ability. Notably, the P446L mutant importin-beta does not exert its dominant-negative effect on nuclear protein import and has no effect on mitotic spindle-related functions and chromosome segregation. However, it interferes with nuclear envelope formation during mitosis-to-interphase transition, revealing a novel function of importin-beta.
Active Transport, Cell Nucleus↗
The BiP protein and the endoplasmic reticulum of Schizosaccharomyces pombe: fate of the nuclear envelope during cell division.
A polyclonal antibody was raised to the C-terminal region of fission yeast BiP. The use of this antibody for immunoprecipitation, western blotting and immunofluorescence has confirmed and extended the observations made previously with an epitope-tagged BiP molecule. A fraction of BiP protein is glycosylated in Schizosaccharomyces pombe cells. Pulse-chase experiments showed that this modification occurs rapidly upon synthesis and that the extent of glycosylation does not then change with time. BiP protein is induced by elevated temperatures and by treatment with tunicamycin. The antibody cross-reacts with proteins of similar molecular weight in the yeasts Kluyveromyces lactis and Schizosaccharomyces japonicus. Immunofluorescence of BiP has been used to follow the behaviour of the ER and in particular the nuclear envelope through the cell cycle.
Amino Acid Sequence↗
The polo-like kinase PLK-1 is required for nuclear envelope breakdown and the completion of meiosis in Caenorhabditis elegans.
The Polo-like kinases are key regulatory molecules required during the cell cycle for the successful completion of mitosis. We have cloned a C. elegans homolog of the Drosophila melanogaster polo gene (designated plk-1 for C. elegans polo-like kinase-1) and present the subcellular localization of the PLK-1 protein during the meiotic and mitotic cell cycles in C. elegans oocytes and embryos, respectively. Disruption of PLK-1 expression by RNA-mediated interference (RNAi) disrupts normal oocyte and embryonic development. Inspection of oocytes revealed a defect in nuclear envelope breakdown (NEBD) before ovulation. This defect in NEBD was also observed in oocytes that were depleted of the cyclin-dependent kinase NCC-1 (C. elegans homolog of Cdc2). The plk-1 RNAi oocytes were fertilized; however the resulting embryos were unable to separate their meiotic chromosomes or form and extrude polar bodies. These defects led to embryonic arrest as single cells. genesis 26:26-41, 2000. Published 2000 Wiley-Liss, Inc.
Amino Acid Sequence↗
[Karyosphere and nuclear envelope ultrastructure of oocytes of the common frog Rana temporaria after nuclease treatment and extraction of chromatin and alkali-soluble proteins].
The late oocyte karyosphere of Rana temporaria consists mainly of fibrillar nucleoli and micronucleoli, Fibrillar material containing the pore complexes (pseudomembranes) and modified synaptonemal complexes. Nuclease treatment of the karyosphere reveals regularly repeating bands in the nuclei and micronuclei, the fibrillar material associated with the nuclei being attenuated. After a subsequent treatment with DNase-1, RNase A and 1.5 M Nacl and karyosphere is disintegrated to involve remains of extracted nucleoli and fibrillar material containing pore complexes. 0.025 N NaOH extracted the bulk of the karyosphere and left only the islets if fibrillar material. The nuclear envelope proved to be far more stable to the above treatments. It retained its structure and was loosened partly only after 1.5 M NaCl or 0.025 N NaOH extraction, the pore complexes disappearing only after alkaline extraction. It is assumed that the karyosphere represents a skeletal structure (matrix) of the oocyte nucleus.
Animals↗
Aberrant cellular behavior of mutant torsinA implicates nuclear envelope dysfunction in DYT1 dystonia.
Torsion dystonia-1 (DYT1) dystonia, the most common inherited form of dystonia, is caused by a three base pair deletion that eliminates a single amino acid from the disease protein, torsinA. TorsinA is an "AAA" protein thought to reside in the endoplasmic reticulum (ER), yet both its cellular function and the basis for neuronal dysfunction in DYT1 remain unknown. A clue to disease pathogenesis is the fact that mutant, but not wild-type, torsinA forms membranous inclusions in cell culture. To explore the pathobiology of DYT1 dystonia, we generated PC12 neural cell lines that inducibly express wild-type or mutant torsinA. Although in this model torsinA displays some properties consistent with ER localization, mutant torsinA also accumulates in the nuclear envelope (NE), a structure contiguous with cytoplasmic ER. Consistent with this, membranous inclusions formed by mutant torsinA are shown to derive not from the ER, as thought previously, but from the NE. We demonstrate further that torsinA forms different disulfide-linked complexes that may be linked functionally to subcellular localization in the NE versus cytoplasmic ER. Despite mutant TA accumulation in NE structures, nucleocytoplasmic transport of a reporter protein was unaffected. These findings, together with parallel studies failing to demonstrate perturbation of ER function, implicate the NE as a primary site of dysfunction in DYT1. DYT1 dystonia can be added to the growing list of inherited neurological disorders involving the NE.
Active Transport, Cell Nucleus↗
Regulation of cytosol-nucleus pH gradients by K+/H+ exchange mechanism in the nuclear envelope of neonatal rat astrocytes.
In order to study the subcellular heterogeneity of intracellular H+ concentration in reactive astrocytes, the pH in the nucleus and cytosol of cultured astrocytes was measured using a confocal laser scanning microscope (CLSM) and pH indicator dye, 5'(and 6')-carboxyseminaphthofluorescein (carboxy SNAFL-1). The change in intracellular pH was indexed by the fluorescence ratio (F535/F610) at an excitation wavelength of 514.5 nm. The in vitro fluorescence ratio increased as pH decreased. This ratio in the nucleus was significantly lower than that in the cytosol of astrocytes when perfused by HEPES-buffered Hanks' balanced salt solution (HHBSS) at pH 7.4. Acid stimulations of cells (pH 5.0) raised the fluorescence ratio in both nucleus and cytosol. However, the increase in the fluorescence ratio of the nucleus was less than that of cytosol. Treatment with a K+/H+ ionophore, nigericin (20 microM), reversibly nullified this cytosol-nucleus pH gradient. These findings suggest that a buffering mechanism(s) for maintaining of intracellular pH exists between the nucleus and cytosol, and a K+/H+ exchanger may act on the nuclear envelope to eventuate intranuclear pH maintenance in the living cells.
Animals↗
Does a threshold for the effect of dietary omega-3 fatty acids on the fatty acid composition of nuclear envelope phospholipids exist?
Existence of a dietary maximal level or threshold for incorporation of omega 3 fatty acids into membrane phospholipids is of interest as it may further define understanding of the dietary requirement for omega 3 fatty acids. To test whether feeding increasing levels of dietary omega 3 fatty acids continues to increase membrane omega 3 fatty acid content, weanling rats were fed a nutritionally adequate semipurified diet which provided increasing amounts of C20 and C22 omega 3 fatty acids, such as 20:5 omega 3 and 22:6 omega 3. Dietary 20:5 omega 3 and 22:6 omega 3 were provided by substituting a purified shark oil concentrate of high 22:6 omega 3 content for safflower oil high in 18:2 omega 6. After four weeks of feeding, nuclear envelopes from four animals in each diet group were prepared, lipid was extracted and phospholipids separated. Arachidonic acid content in membrane phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine was significantly reduced by feeding increased dietary levels of omega 3 fatty acids. Decline of 20:4 omega 6 level in phospholipid tended to stabilize when the dietary content of total omega 3 fatty acids reached 4-5% of total fatty acids. Above this level, dietary omega 3 fatty acids did not result in a further decrease in membrane content of 20:4 omega 6. Increase in membrane phospholipid content of 20:5 omega 3 occurred as the dietary intake of omega 3 fatty acids increased from 1.1% to 5% of total fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The role of microsomes and nuclear envelope in the metabolic activation of benzo(a)pyrene leading to binding with nuclear macromolecules.
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Animals↗