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PubMed1991

Stimulatory effect of okadaic acid, an inhibitor of protein phosphatases, on nuclear envelope breakdown and protein phosphorylation in mouse oocytes and one-cell embryos.

Treatment of one-cell mouse embryos with okadaic acid (OA), which is an inhibitor of protein phosphatases 1 and 2A, induces a concentration-dependent precocious nuclear envelope breakdown (NEBD) of the pronuclei; at 10 microM okadaic acid, NEBD starts to occur after 1 hr and the embryos become committed to NEBD after about 45 min. Correlated with NEBD is the conversion of a protein of Mr 32,000 (p32) to more highly phosphorylated forms. One-cell embryos cultured continuously in OA-containing medium do not cleave, whereas one-cell embryos incubated for 15-60 min prior to transfer to OA-free medium reveal a time-dependent inhibition in their ability to cleave. OA treatment of oocytes that are arrested from resuming spontaneous maturation by either a phosphodiesterase inhibitor or biologically active phorbol diester results in germinal vesicle breakdown and the maturation-associated changes in the pattern of protein phosphorylation, which include the apparent phosphorylation of p32. Results of these experiments implicate protein phosphatases in the G2 to M transition of the cell cycle in both meiotic and mitotic cells.

Animals↗
PubMed1990

Identification of two HSP70-related Xenopus oocyte proteins that are capable of recycling across the nuclear envelope.

Two 70-kD polypeptides, B3 and B4, are present in equivalent concentrations in the nucleus and cytoplasm of Xenopus oocytes. The objectives of this study were to determine if they (a) are members of the 70-kD family of heat shock proteins, and (b) recycle between the nuclear and cytoplasmic compartments. Evidence based on high-affinity binding to ATP, cross-reactivity of B3/B4-specific antibodies with rat hsc70, and a comparison of cyanogen bromide cleavage peptide maps with hsc70, verified that B3 and B4 are members of the 70-kD family of heat-shock proteins. Nuclear uptake studies were performed by microinjecting 125I-labeled B3/B4, rat hsc70, and BSA into the cytoplasm of oocytes, and examining their subsequent intracellular distributions. By 6 h postinjection, the nuclear concentration of B3/B4 and hsc70 were approximately 24-fold greater than BSA controls. It was also found that B3/B4-coated gold particles as large as 120A in diameter were able to enter the nucleus by passing through the pores. Nuclear efflux was analyzed by microinjecting the iodinated proteins directly into the oocyte nuclei. 2 h after nuclear injection, at least 46% of the B3/B4 and 60% of the hsc70 were found in the cytoplasmic fractions, compared with less than 10% for the BSA controls. Cell fusion experiments, in which labeled, anucleate oocyte vegetal hemispheres were fused, under oil, with nucleate unlabeled animal hemispheres, demonstrated that cytoplasmic B3 and B4 could enter the nucleus after equilibration was reached, arguing against the existence of separate nuclear and cytoplasmic populations. Collectively, these results show that B3, B4, and rat hsc70 are transported across the nuclear envelope and recycle between the nucleus and cytoplasm.

Amino Acid Sequence↗
PubMed1969

Crystalline inclusions in the nuclear envelope and granular endoplasmic reticulum of the fish spinal cord.

Crystalline inclusions were found in the nuclear envelope and granular endoplasmic reticulum of spinal cord oligodendroglia of the common guppy (Poecilia reticulata) and the lungfish (Polypterus enlicheri). A considerably increased incidence of these inclusions was noted in guppies with congenital and hereditary (sex-linked, recessive) lordosis. Identical crystalline inclusions were observed in protoplasmic and fibrous astrocytes, ependymal cells, and capillary endothelial cells of the spinal cord of the lordotic fish. The oligodendroglia in these fish also revealed a prominent alteration of the endoplasmic reticulum and Golgi apparatus, with the accumulation of dense (secretion?) granules and large amounts of electron-opaque material in markedly dilated sacs of the endoplasmic reticulum. The authors postulate that this alteration is caused by a genetic defect in the control mechanism governing the elaboration of this material in the lordotic guppy, with subsequent stasis and crystallization of this material.

Animals↗
PubMed1986

Distribution of microtubules during the breakdown of the nuclear envelope of the Xenopus oocyte: an immunocytochemical study.

Xenopus oocytes were stained by anti-tubulin and anti-MAP1 antibodies during the first meiotic cell division. In the prophase-blocked oocytes, only few microtubules are present around the upper part of the nuclear envelope. These microtubules are resistant to cold, calcium and antimitotic drug treatments. At this stage, monoclonal anti-MAP1 antibody and polyclonal anti-centrosome antibody reveal punctate staining of the nucleus and nucleoli. During the progesterone-induced maturation, a microtubular network appears at the basal part of the disrupting nucleus. Anti-MAP1 and anti-centrosome antibodies stain a dense layer at the basal part of this microtubular array. Microtubules present in this array are cold, calcium- and antimitotic drug sensitive. Anti-MAP1 and anti-tubulin antibodies stain the whole metaphase II spindle, whereas only the poles of the metaphase II spindle are stained by the anti-centrosome antibody.

Animals↗
PubMedDate not supplied

Nuclear transport kinetics in microarrays of nuclear envelope patches.

Optical Single Transporter Recording (OSTR) is a technique for analyzing membrane transport kinetics at high sensitivity, selectivity, and spatial resolution. Cellular membranes are firmly attached to microarrays of small test compartments (TCs) with diameters between approximately 0.1 and 100 microm and depths between approximately 10 and 100 microm. This permits to generate either "small" membrane patches containing few transporters or "large" patches containing many transporters. Transport of substrates across membrane patches is recorded by confocal microscopy. The present article reviews recent applications of OSTR to the nuclear pore complex (NPC). The results show that the transport functions of the NPC, previously studied almost exclusively in intact and permeabilized cells, are conserved in isolated nuclei and can be fully reconstituted in purified nuclear envelopes by addition of recombinant transport factors. This opens new avenues to the analysis of nuclear transport including the export of nucleic-acid-protein and ribosomal particles.

Active Transport, Cell Nucleus↗
PubMed2001

Role for phosphatidylinositol in nuclear envelope formation.

PtdIns is a minor membrane phospholipid that is important in signal transduction. Recently, derivatives of PtdIns phosphorylated at the 3-position of the inositol ring have been implicated in the regulation of constitutive membrane traffic and in membrane fusion events. Assembly of the nuclear envelope (NE), a crucial step in the progress of mitosis, is also likely to involve membrane fusion reactions. We therefore investigated the role of PtdIns and phosphoinositide 3-kinase (PI-3K) activity in NE formation in vitro. GTP-induced NE formation was blocked by wortmannin and LY294002, two specific inhibitors of PI-3K, suggesting a role for PtdIns phosphorylated at the 3-position. PtdIns-specific phospholipase C mimicked GTP hydrolysis as an inducer of NE formation. This induction was dependent on a membrane vesicle subfraction (MV1) that was highly enriched in PtdIns, as determined by heteronuclear two-dimensional NMR spectroscopy. On the basis of these results, we suggest that the MV1 population serves as a source of membranes rich in PtdIns that might facilitate fusion, possibly through the production of the membrane-destabilizing lipid diacylglycerol.

Androstadienes↗
PubMed1998

Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association.

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101- amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1-SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.

Amino Acid Sequence↗
PubMed2005

Dystonia and the nuclear envelope.

Mutations in torsinA cause dominantly inherited early-onset torsion dystonia in humans. In this issue of Neuron, Goodchild et al. show that torsinA knockout and knockin mice have similar phenotypes, which suggests that the mutant torsinA allele causes disease because it has decreased function. The experiments also highlight the possible role of nuclear envelope dynamics in maintaining normal neuronal function.

Animals↗
PubMed1961

Microtubulation of the inner membrane of the nuclear envelope.

In the course of a light and electron microscopy study of spermatogenesis in the European crayfish, Astacus fluviatilis, spermatocytes of abnormal appearance were observed in two instances in individuals that had passed the mating period. The electron microscope showed that the inner membrane of the nuclear envelope of these cells was erupting into a mass of microtubules, 15 to 18 mmicro in diameter and 0.5 micro or more in length, while the outer membrane transformed into cytoplasmic vesicles. Stages in the formation of these novel processes were followed. The plasma membrane of the affected cells was seen in some cases to erupt into similar although shorter microtubules. It is concluded that the phenomenon is part of a degenerative process in which the spermatocytes are being absorbed by sustentacular cells. It is suggested that the observations provide further evidence for a fundamental functional as well as a morphological similarity between the membranes bounding the nucleus and the plasma membrane.

Cell Membrane↗
PubMed1988

Intracellular free calcium rise triggers nuclear envelope breakdown in the sea urchin embryo.

Cytosolic free calcium has recently been implicated in the regulation of mitosis in plant and animal cells. We have previously found correlations between increases in the levels of intracellular free calcium [Ca2+]i and visible transitions of structure at nuclear envelope breakdown (NEBD) and the onset of anaphase during mitosis in sea urchin embryos and tissue culture cells. To go beyond correlations it is necessary to manipulate [Ca2+]i, and in sea urchin embryos this requires the injection of calcium-chelator buffer solutions as the changes in free calcium in the cell cycle are dependent on intracellular stores. We report here that blocking the increase in [Ca2+]i which just precedes NEBD prevents this from taking place and halts mitosis. Subsequent injections which momentarily increase [Ca2+]i, or a natural recovery of the higher calcium levels, result in NEBD and the successful continuation of mitosis. Similarly, artificially increasing calcium by early injections results in early NEBD. We conclude that the increase in [Ca2+]i preceding NEBD is an essential regulatory step required for entry into mitosis.

Ammonia↗
PubMed1980

Effects of thioacetamide treatment on nuclear envelope nucleoside triphosphatase activity and transport of RNA from rat liver nuclei.

An in vitro system was used for investigating transport of rapidly labeled RNA from liver nuclei isolated from control and thioacetamide-treated rats. An enhanced transport was observed in preparations from thioacetamide-treated animals. Analysis of transport at various temperatures indicated that it proceeded as an energy-requiring process, even in the absence of an exogenously added energy source, and was related to hydrolysis of high-energy nucleoside triphosphate esters. In control preparations, a nucleoside triphosphatase (NTPase) is present in nuclear envelopes, and this TNPase activity appears to be intimately related to in vitro RNA transport. Nuclear membranes were isolated from purified nuclear preparations taken from control and thioacetamide-treated rats and were examined for NPTase activity. The NTPase activity was significantly greater in the preparations from thioacetamide-treated animals. These observations indicate that an early change associated with carcinogen exposure is an enhanced NTPase activity. This may underlie the enhanced RNA transport observed in vitro.

Acetamides↗
PubMed1992

Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte.

During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.

Animals↗
PubMed2005

Interaction between the movement protein of barley yellow dwarf virus and the cell nuclear envelope: role of a putative amphiphilic alpha-helix at the N-terminus of the movement protein.

The open reading frame 4 (ORF 4) gene product of barley yellow dwarf virus (BYDV) may act as a movement protein (MP) by assisting the transport of viral genomic RNA across the nuclear envelope (NE) of host plant cells. To investigate interactions between BYDV MP and the NE, wild-type and mutant open reading frame (ORF 4)-green fluorescent protein (GFP) fusion cistrons were expressed in insect cells. A fusion protein expressed by the wild-type ORF 4-GFP cistron associated with the NE and caused protrusions from its surface. The fusion protein expressed by the mutant ORF 4-GFP cistron lacked a putative amphiphilic alpha-helix at its N-terminus and although associating with the NE, showed decreased levels of protrusions. A peptide homologue of this putative alpha-helix induced an increase of 7 degrees C in the phase transition temperature of dimyrystoyl phosphatidylserine (DMPS) membranes, accompanied by a decrease in membrane fluidity, but exhibited no significant interaction with either dimyristoyl phosphatidylcholine (DMPC) or dimyristoyl phosphatidylethanolamine (DMPE) membranes. These results strongly support the view that BYDV MP may interact with the NE to help transport viral genomic RNA into the nuclear compartment. This function of BYDV MP appears to involve protrusions on the surface of the NE and may require the presence of an N-terminal amphiphilic alpha-helix, which is speculated to destabilize membranes, thereby assisting the entry of BYDV-GAV into the nuclear compartment.

Amino Acid Sequence↗
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↗
PubMed1991

Influence of genotype on diet-induced changes in membrane phosphatidylcholine and phosphatidylethanolamine composition of splenocytes, liver nuclear envelope and liver mitochondria.

Inbred congenic mice of strains MRL/Mp-lpr/lpr (lpr/lpr) and MRL/Mp(-)+/+ (+/+) were fed nutritionally adequate semipurified diets containing 20% (w/w) fat and differing in linoleic acid content. Levels of linoleic acid (18:2n-6) and arachidonic acid (20:4n-6) in phospholipids of splenocytes, liver mitochondria and liver nuclear envelopes were determined. Membranes of lpr/lpr mice exhibited significantly lower levels of 18:2n-6 and 20:4n-6 in phospholipids compared with the +/+ strain. The high linoleic acid diet increased incorporation of 18:2n-6 and 20:4n-6 in most phospholipid fractions of these membranes. These observations indicate that genotype as well as dietary 18:2n-6 content significantly influenced incorporation of 18:2n-6 and 20:4n-6 into membrane phospholipids. The results also suggest that membrane compositional abnormalities found in the lpr/lpr mice, which develop lymphoma and age faster than +/+ mice, are not restricted to the immune system but also extend to other organs. Differences observed in phospholipid fatty acid composition in splenocytes and liver subcellular membranes for mice fed diets differing in linoleic acid content suggest that the early expression of the lpr gene resulting in progression of autoimmunity may be delayed through dietary manipulation.

Animals↗
PubMed1989

Nuclear envelope breakdown and mitosis in sand dollar embryos is inhibited by microinjection of calcium buffers in a calcium-reversible fashion, and by antagonists of intracellular Ca2+ channels.

Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Matefin/SUN-1 is a nuclear envelope receptor for CED-4 during Caenorhabditis elegans apoptosis.

In Caenorhabditis elegans, the antiapoptotic protein CED-9 is localized at the mitochondria, where it binds the proapoptotic protein CED-4. Induction of apoptosis begins when the proapoptotic protein EGL-1 is expressed and binds CED-9. The binding of EGL-1 to CED-9 releases CED-4 from CED-9 and causes the activation of the caspase CED-3. Upon its release from CED-9, CED-4 rapidly translocates to the nuclear envelope (NE) in a CED-3-independent manner. However, the identity of the NE receptor for CED-4 and its possible role in the execution of apoptosis has remained unknown. Here, we show that the inner nuclear membrane SUN-domain protein matefin/SUN-1 is the NE receptor for CED-4. Our data demonstrate that matefin/SUN-1 binds CED-4 and is specifically required for CED-4 translocation and maintenance at the NE. The role of matefin/SUN-1 in the execution of apoptosis is further suggested by the significant reduction in the number of apoptotic cells in the organism after matefin/SUN-1 down-regulation by RNAi. The finding that matefin/SUN-1 is required for the execution of apoptosis adds an important link between cytoplasmic and nuclear apoptotic events.

Animals↗
PubMed1978

The influence of Triton X-100 on the nuclear envelope of the isolated liver cell nuclei.

In order to obtain more precise information on an eventual presence of extra-membranous lipids in the interior of the nucleus, the effects of Triton X-100 on the lipid content and ultrastructure of isolated rat liver nuclei was investigated. Enzyme markers (a.o. glucose-6-phosphatase) were used to control impurities of the nuclear fractions biochemically along with transmission electron microscopy and qualitative and quantitative light microscopy to check the condition of the nuclei obtained. Treatment of the nuclear fraction with increasing concentrations of Triton X-100 resulted in a decrease of the phospholipid content down to 25% at a Triton X-100/protein ratio of 0.4. A further decrease to 8% was measured at a ratio of 1.5. Electron microscopy of nuclei of the latter group showed nuclei containing outer membrane fragments in 2.5% of their surfaces. The composition of lipids extracted from a nuclear fraction appeared to be markedly changed after treatment with Triton X-100 with an increase of the percentage of neutral lipids and the phospholipids diphosphatidyl-glycerol and spingomyelin. From the chemical and morphological data obtained, the conclusion was drawn that a substantial part of the lipids remaining in the isolated nuclei after treatment with Triton X-100 is localized in both membranes of the nuclear envelope. It cannot however, be excluded that a small portion would be present in the interior of the nuclei.

Animals↗