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PubMed1982

[Relationships among the postnuclear band, chromatin and nuclear envelope of ram spermatids. Concept of a partial model to explain the displacements of the manchette].

After alkaline extraction, the postnuclear band appeared to be composed of two parts, the intranuclear part contiguous to the chromatin and the extranuclear part apposed to the external membrane of the nuclear envelope. This membrane was discontinuous inside the postnuclear band, revealing that the band might be a zone of nuclear envelope remodeling. A model is proposed to explain the pulling of the manchette by intranuclear forces and the formation of the redundant nuclear envelope during spermiogenesis.

Animals↗
PubMed2006

Yeast nuclear envelope subdomains with distinct abilities to resist membrane expansion.

Little is known about what dictates the round shape of the yeast Saccharomyces cerevisiae nucleus. In spo7Delta mutants, the nucleus is misshapen, exhibiting a single protrusion. The Spo7 protein is part of a phosphatase complex that represses phospholipid biosynthesis. Here, we report that the nuclear protrusion of spo7Delta mutants colocalizes with the nucleolus, whereas the nuclear compartment containing the bulk of the DNA is unaffected. Using strains in which the nucleolus is not intimately associated with the nuclear envelope, we show that the single nuclear protrusion of spo7Delta mutants is not a result of nucleolar expansion, but rather a property of the nuclear membrane. We found that in spo7Delta mutants the peripheral endoplasmic reticulum (ER) membrane was also expanded. Because the nuclear membrane and the ER are contiguous, this finding indicates that in spo7Delta mutants all ER membranes, with the exception of the membrane surrounding the bulk of the DNA, undergo expansion. Our results suggest that the nuclear envelope has distinct domains that differ in their ability to resist membrane expansion in response to increased phospholipid biosynthesis. We further propose that in budding yeast there is a mechanism, or structure, that restricts nuclear membrane expansion around the bulk of the DNA.

Cell Nucleolus↗
PubMed2000

Dynamic associations of heterochromatin protein 1 with the nuclear envelope.

To study the dynamics of mammalian HP1 proteins we have microinjected recombinant forms of mHP1alpha, M31 and M32 into the cytoplasm of living cells. As could be expected from previous studies, the three fusion proteins were efficiently transported into the nucleus and targeted specific chromatin areas. However, before incorporation into these areas the exogenous proteins accumulated in a peripheral zone and associated closely with the nuclear envelope. This transient association did not occur when the cells were treated with deacetylase inhibitors, indicating an acetylation-inhibited interaction. In line with these observations, recombinant HP1 proteins exhibited saturable binding to purified nuclear envelopes and stained the nuclei of detergent-permeabilized cells in a rim-like fashion. Competition experiments with various M31 mutants allowed mapping of the nuclear envelope-binding site within an N-terminal region that includes the chromodomain. A His(6)-tagged peptide representing this region inhibited recruitment of LAP2beta and B-type lamins around the surfaces of condensed chromosomes, suggesting involvement of HP1 proteins in nuclear envelope reassembly.

Acetylation↗
PubMed1989

Persistence of major nuclear envelope antigens in an envelope-like structure during mitosis in Drosophila melanogaster embryos.

Using monoclonal antibodies, we followed the fate of three different nuclear envelope proteins during mitosis in Drosophila early embryos by indirect immunofluorescence microscopy. Two of these proteins, lamin and otefin, a newly characterized nuclear envelope polypeptide with an apparent Mr of 53,000, are apparently present in an envelope-like structure that is present throughout mitosis. Immunoelectron microscopy of interphase nuclei indicates that otefin, like lamin, is not a component of nuclear pore complexes. In contrast with lamin and otefin, gp188, a putative pore complex component, was completely redistributed through the surrounding cytoplasm during prophase in comparable early embryo specimens and was present in an envelope only in interphase. Together with previous morphological studies by other workers, these data suggest that the entire mitotic apparatus including condensed chromosomes and spindle is enclosed by an envelope throughout mitosis during early embryogenesis in Drosophila. This 'spindle envelope', as it has been named by others, contains both lamin and otefin but probably not pore complex proteins.

Animals↗
PubMed2006

SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope.

Changes in the transcriptional state of genes have been correlated with their repositioning within the nuclear space. Tethering reporter genes to the nuclear envelope alone can impose repression and recent reports have shown that, after activation, certain genes can also be found closer to the nuclear periphery. The molecular mechanisms underlying these phenomena have remained elusive. Here, with the use of dynamic three-dimensional tracking of a single locus in live yeast (Saccharomyces cerevisiae) cells, we show that the activation of GAL genes (GAL7, GAL10 and GAL1) leads to a confinement in dynamic motility. We demonstrate that the GAL locus is subject to sub-diffusive movement, which after activation can become constrained to a two-dimensional sliding motion along the nuclear envelope. RNA-fluorescence in situ hybridization analysis after activation reveals a higher transcriptional activity for the peripherally constrained GAL genes than for loci remaining intranuclear. This confinement was mediated by Sus1 and Ada2, members of the SAGA histone acetyltransferase complex, and Sac3, a messenger RNA export factor, physically linking the activated GAL genes to the nuclear-pore-complex component Nup1. Deleting ADA2 or NUP1 abrogates perinuclear GAL confinement without affecting GAL1 transcription. Accordingly, transcriptional activation is necessary but not sufficient for the confinement of GAL genes at the nuclear periphery. The observed real-time dynamic mooring of active GAL genes to the inner side of the nuclear pore complex is in accordance with the 'gene gating' hypothesis.

Diffusion↗
PubMed1998

cut11(+): A gene required for cell cycle-dependent spindle pole body anchoring in the nuclear envelope and bipolar spindle formation in Schizosaccharomyces pombe.

The "cut" mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11(+) suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11(+) encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.

Amino Acid Sequence↗
PubMed1987

Expression and nuclear envelope localization of biologically active fusion glycoprotein gB of herpes simplex virus in mammalian cells using cloned DNA.

Herpes simplex virus (HSV) is known to bud from the inner membrane of the nuclear envelope. The structural gene for the glycoprotein gB, which is essential for virus entry and cell fusion induced by HSV type 1, has been cloned in a transient expression vector containing the adenovirus major late promoter, tripartite leader sequence, and VARNA (a special RNA in adenovirus-infected cells) genes. Synthesis and glycosylation of glycoprotein gB was observed in COS-1 cells transfected with the vector containing the gB gene. Removal of a 3' fragment of the cloned gene resulted in the synthesis and secretion of a truncated gB glycoprotein. Immunofluorescence studies revealed that the expressed glycoprotein was localized in the nuclear envelope as well as in the cell surface. The expressed gB-1 glycoprotein was biologically active and induced fusion of cells to produce polykaryons. These data show that HSV glycoprotein gB expressed from cloned gene can be used as a model to study targeting of proteins into the nuclear envelope as well as cell fusion induced by the virus.

Animals↗
PubMed2003

AKAP149 is a novel PP1 specifier required to maintain nuclear envelope integrity in G1 phase.

Reassembly of the nuclear envelope (NE) at the end of mitosis requires targeting of the B-type lamin protein phosphatase, PP1, to the envelope by A-kinase anchoring protein AKAP149. We show here that NE-associated AKAP149 is a novel PP1-specifying subunit involved in maintaining nuclear architecture through G1 phase. PP1 remains associated with NE-bound AKAP149 during G1 but is released from AKAP149 upon S phase entry, as AKAP149 becomes serine-phosphorylated. NE-associated AKAP149 inhibits PP1 activity towards glycogen phosphorylase but enhances PP1 phosphatase activity towards B-type lamins, indicating that AKAP149 is a B-type lamin specifying subunit of PP1. In vivo dissociation of PP1 from NE-bound AKAP149 in G1-phase nuclei triggers phosphorylation and depolymerization of A- and B-type lamins. The lamins solubilize intranuclearly without affecting the inner nuclear membrane or pore complex distribution. This correlates with the induction of a G1 arrest and, ultimately, apoptosis. We propose that AKAP149-regulated PP1 activity at the NE during G1 is required to maintain nuclear integrity and cell survival.

A Kinase Anchor Proteins↗
PubMed1994

Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion.

In many calcium-imaging studies, the nuclear envelope appears to maintain a gradient of free calcium between the nucleus and cytosol. This issue was examined by loading amphibian sympathetic neurons with the calcium indicator fluo 3 via whole-cell patch clamping. Confocal optical sectioning allowed acquisition of independent calibration curves for the nucleus and cytoplasm. Cells were loaded with free calcium levels ranging from 10 nM to 50 microM, using 10 mM BAPTA to control free calcium. The nuclear fluorescence was usually about 130% brighter than the cytoplasmic fluorescence. Had the increased nuclear fluorescence been due to a calcium gradient, then, as fluo 3 was saturated with calcium in both compartments, the fluorescence gradient should have gradually disappeared. Instead, with free-calcium in the pipette set at 50 microM, about five times the level required to nearly saturate fluo 3, the nuclear/cytoplasmic (N/C) fluorescence ratio was not decreased but instead increased slightly. Perfusion of the patch pipette was used in conjunction with imaging to confirm that cytoplasmic fluo 3 was saturated with calcium. After loading cells with 10 nM free calcium, the patch pipette was perfused with high calcium (10 microM). Again, the N/C fluorescence ratio increased at high calcium. The effectiveness of patch-pipette perfusion in changing cellular free calcium levels was indicated by the degree of fluorescence increase--both nuclear and cytosolic compartments showed a roughly 20-fold increase in fluorescence, that is, most of the dynamic range observed in test droplets. To confirm further that cytoplasmic fluo 3 was saturated, cells were perfused with manganese, which binds with very high affinity to fluo 3. Manganese rapidly entered the cytoplasm and nucleus, causing a large increase in fluorescence, but the N/C fluorescence ratio remained relatively constant. Because free manganese in the pipette was 50,000 times the amount required to saturate fluo 3, the greater nuclear fluorescence probably results from additional fluo 3 in the nucleus rather than from calcium or manganese gradients. To gauge further the permeability of the nuclear envelope, the diffusion of calcium was visualized. Under voltage clamp, calcium channels were opened for periods ranging from 5 to 200 msec. Peak calcium levels were observed within 2 microns of the plasma membrane, and declined as calcium diffused into the cell. The nuclear fluorescence increased more than cytosolic fluorescence, but this apparent "amplification" was eliminated by correcting for autofluorescence. Use of cells cultured on glass coverslips and a high-NA microscope objective allowed a satisfactory correction.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Nuclear envelope removal/maintenance determines the structural and functional remodelling of embryonic red blood cell nuclei in activated mouse oocytes.

Nuclei of embryonic red blood cells (e-RBC) from 12-day mouse fetuses are arrested in G0 phase of the cell cycle and have low transcriptional activity. These nuclei were transferred with help of polyethylene glycol (PEG)-mediated fusion to parthenogenetically activated mouse oocytes and heterokaryons were analysed for nuclear structure and transcriptional activity. If fusion proceeded 25-45 min after oocyte activation, e-RBC nuclei were induced to nuclear envelope breakdown and partial chromatin condensation, followed by formation of nuclei structurally identical with pronuclei. These 'pronuclei', similar to egg (female) pronuclei, remained transcriptionally silent over several hours of in vitro culture. If fusion was performed 1 h or later (up to 7 h) after activation, the nuclear envelope of e-RBC nuclei remained intact and nuclear remodelling was less spectacular (slight chromatin decondensation, formation of nucleolus precursor bodies). These nuclei, however, reinforced polymerase-II-dependent transcription within a few hours of in vitro culture. Our present experiments, together with our previous work, demonstrate that nuclear envelope breakdown/maintenance are critical events for nuclear remodelling in activated mouse oocytes and that somatic dormant nuclei can be stimulated to renew transcription at a time when the female pronucleus remains transcriptionally silent.

Animals↗
PubMed1970

The formation of multivesicular bodies from the nuclear envelope.

Cells of the gas gland of the perch Perca fluviatilis L., stimulated to increased generation of gas by the repeated emptying of the swim-bladder, were examined in the electron microscope. Intense activity of the nuclear envelope was demonstrated. Simple vesicles originating from the external nuclear membrane and the so-called multivesicular bodies derived from the outpocketings of both membranes of the nuclear envelope were observed. The multivesicular bodies were filled with numerous fine vesiculae arising from the active proliferation of their internal membrane. The authors offer two alternative mechanisms of formation of fine vesiculae inside the multivesicular bodies and the mechanism of the tearing away of these bodies from the nuclear envelope.

Air Sacs↗
PubMed2001

BAF is required for emerin assembly into the reforming nuclear envelope.

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envelope assembly in living HeLa cells. During telophase, emerin accumulates briefly at the 'core' region of telophase chromosomes, and later distributes over the entire nuclear rim. Barrier-to-autointegration factor (BAF), a protein that binds nonspecifically to double-stranded DNA in vitro, co-localized with emerin at the 'core' region of chromosomes during telophase. An emerin mutant defective for binding to BAF in vitro failed to localize at the 'core' in vivo, and subsequently failed to localize at the reformed NE. In HeLa cells that expressed BAF mutant G25E, which did not show 'core' localization, the endogenous emerin proteins failed to localize at the 'core' region during telophase, and did not assemble into the NE during the subsequent interphase. BAF mutant G25E also dominantly dislocalized LAP2beta and lamin A from the NE, but had no effect on the localization of lamin B. We conclude that BAF is required for the assembly of emerin and A-type lamins at the reforming NE during telophase, and may mediate their stability in the subsequent interphase.

Amino Acid Sequence↗
PubMed1998

Thioacetamide effects on the polypeptidic and nucleoporin pattern and chemical composition of rat liver nuclear envelope subfractions.

The effect of the administration of seven doses of the hepatocarcinogen thioacetamide on the chemical composition of rat liver nuclear envelope subfractions: associated chromatin, nuclear membranes and pore complex-lamina fraction, is analyzed. No alteration in DNA, RNA or phospholipid content is observed after the hepatocarcinogen treatment. Electrophoretic studies of each subfraction from thioacetamide treated rats show differences in the relative proportions of some polypeptides when compared with the controls. Examination of the wheat germ agglutinin binding polypeptides of each subfraction reveals a decrease in the stain of two pore complex-lamina nucleoporins of 85 and 164 kDa and an increase in one of 93 kDa; this observation can be due to changes in the quantity and/or in the agglutinin binding capacity of the nucleoporin as a result of thioacetamide administration. In view of the participation of nucleoporins in the nucleocytoplasmic transport, the changes observed suggest a relationship between changes of some O-linked N-acetyl glucosamine polypeptides components of the nuclear pore complex and the altered transport of some RNA species observed after thioacetamide administration.

Animals↗
PubMed1989

Interaction of a nuclear location signal with isolated nuclear envelopes and identification of signal-binding proteins by photoaffinity labeling.

The nuclear envelope (NE) separates the two major compartments of eukaryotic cells, the nucleus and the cytoplasm. Recent studies suggest that the uptake of nuclear proteins into the nucleus is initiated by binding of nuclear location signals (NLSs) contained within these proteins to receptors in the NE, followed by translocation through the nuclear pore complex. To examine the binding step without interference from intranuclear events, we have used a system consisting of (i) purified rat liver NEs fixed onto glass slides and (ii) the prototype simian virus 40 large T antigen (SV40 T) NLS conjugated to nonnuclear carrier proteins, and we have visualized the receptor-ligand interaction by indirect immunofluorescence. In this system, incubation of isolated NEs with the wild-type SV40 T NLS conjugate with carrier proteins resulted in binding that was signal sequence-dependent, could be competitively blocked with excess conjugated and unconjugated wild-type peptide, did not require ATP, and was not affected by the transport-inhibiting lectin wheat germ agglutinin. In contrast, only minimal binding was observed with a mutant SV40 T NLS conjugate. These results are consistent with those obtained in other, more complex in vitro systems and suggest that binding of the SV40 T NLS is receptor-mediated. Binding is largely abolished by extraction of the NE with the nonionic detergent Triton X-100, suggesting that the receptor is soluble in detergent. We find in the Triton X-100 supernatant four major NLS-binding proteins with apparent molecular masses of 76, 67, 59, and 58 kDa by photoaffinity labeling with a highly specific crosslinker, azido-NLS. The reduced complexity of the system described here should be useful for the functional study of other potential NLSs for the identification and isolation of their binding sites and for the screening of antibodies raised against these binding sites.

Affinity Labels↗
PubMed1996

Specific interactions of chromatin with the nuclear envelope: positional determination within the nucleus in Drosophila melanogaster.

Specific interactions of chromatin with the nuclear envelope (NE) in early embryos of Drosophila melanogaster have been mapped and analyzed. Using fluorescence in situ hybridization, the three-dimensional positions of 42 DNA probes, primarily to chromosome 2L, have been mapped in nuclei of intact Drosophila embryos, revealing five euchromatic and two heterochromatic regions associated with the NE. These results predict that there are approximately 15 NE contacts per chromosome arm, which delimit large chromatin loops of approximately 1-2 Mb. These NE association sites do not strictly correlate with scaffold-attachment regions, heterochromatin, or binding sites of known chromatin proteins. Pairs of neighboring probes surrounding one NE association site were used to delimit the NE association site more precisely, suggesting that peripheral localization of a large stretch of chromatin is likely to result from NE association at a single discrete site. These NE interactions are not established until after telophase, by which time the nuclear envelope has reassembled around the chromosomes, and they are thus unlikely to be involved in binding of NE vesicles to chromosomes following mitosis. Analysis of positions of these probes also reveals that the interphase nucleus is strongly polarized in a Rabl configuration which, together with specific targeting to the NE or to the nuclear interior, results in each locus occupying a highly determined position within the nucleus.

Animals↗
PubMed1990

Antinuclear antibodies directed to a 200-kilodalton polypeptide of the nuclear envelope in primary biliary cirrhosis. A clinical and immunological study of a series of 150 patients with primary biliary cirrhosis.

Antinuclear antibodies giving a perinuclear fluorescence and directed to a 200-kilodalton polypeptide of the nuclear envelope have been described in primary biliary cirrhosis. The purpose of this study, based on a series of 150 patients with primary biliary cirrhosis, was to ascertain the prevalence of these antibodies and to compare patients with and without these antibodies. Antinuclear antibodies giving a perinuclear fluorescence were demonstrated in 43 of the 150 patients (29%); antibodies directed to the 200-kilodalton polypeptide of the nuclear envelope were found in 40 of these 43 patients. Asthenia, arthralgia, associated extrahepatic diseases, Raynaud's phenomenon, and other antinuclear specificities were significantly less common, and titers of antimitochondrial antibodies were significantly lower in patients with antibodies directed to the 200-kilodalton polypeptide of the nuclear envelope than in patients without these antibodies. Clinical outcome, liver tests, and histological lesions did not significantly differ in patients with and without these antibodies.

Antibodies, Antinuclear↗
PubMed1982

Oligomeric structure of the major nuclear envelope protein lamin B.

Lamins A and B, the two most abundant proteins in the nuclear envelope fraction, can each be converted into disulfide cross-linked homotypic oligomers by oxidation of intrinsic disulfide bonds. The cross-linked polymers of lamin A include dimers, trimers, tetramers, and larger polymers. Those that enter acrylamide gels migrate as expected for their molecular weights. Lamin B has yielded predominantly two oligomers and the number of monomers in these oligomers has been controversial. In the present study, it is demonstrated that migration of the lamin B oligomers is sensitive to the electrophoretic conditions; thus, the oligomers have appeared to be two forms of dimer or two forms of trimer, depending upon conditions. In the present study, a third, smaller oligomer of lamin B has been detected. Because there are three oligomers of lamin B, it is possible that these are a dimer, a trimer, and a tetramer. Support for this model has been obtained by analysis of cross-linked fragments from a mild trypsin digestion of cross-linked lamina proteins. This procedure yields 62,000-, 46,000-, and 30,000-dalton fragments of lamin B. Each fragment also occurs as three homotypic oligomers. The largest oligomer, which appears to be a tetramer, is obtained in high yield under brief, mild cross-linking conditions in several cell types. These results suggest that lamin B occurs as a tetramer in the nuclear envelope.

Animals↗
PubMed2001

Sequential degradation of proteins from the nuclear envelope during apoptosis.

We have produced new antibodies specific for the integral pore membrane protein POM121. Using these antibodies we show that during apoptosis POM121 becomes proteolytically degraded in a caspase-dependent manner. The POM121 antibodies and antibodies specific for other proteins of the nuclear envelope were used in a comparative study of nuclear apoptosis in staurosporine-treated buffalo rat liver cells. Nuclei from these cells were classified in three different stages of apoptotic progression: stage I, moderately condensed chromatin surrounded by a smooth nuclear periphery; stage II, compact patches of condensed chromatin collapsing against a smooth nuclear periphery; stage III, round compact chromatin bodies surrounded by grape-shaped nuclear periphery. We have performed double labeling immunofluorescence microscopy of individual apoptotic cells and quantitative immunoblotting analysis of total proteins from apoptotic cell cultures. The results showed that degradation of nuclear envelope marker proteins occurred in a specific order. POM121 degradation occurred surprisingly early and was initiated before nucleosomal DNA degradation could be detected using TUNEL assay and completed before clustering of the nuclear pores. POM121 was eliminated significantly more rapid compared with NUP153 (a peripheral protein located in the nucleoplasmic basket of the nuclear pore complex) and lamin B (a component of the nuclear lamina). Disappearance of NUP153 and lamin B was coincident with onset of DNA fragmentation and clustering of nuclear pores. By contrast, the peripheral NPC protein p62 was degraded much later. The results suggest that degradation of POM121 may be an important early step in propagation of nuclear apoptosis.

Animals↗