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PubMed1997

Does the nuclear envelope contain two types of ligand-gated Ca2+ release channels?

The nuclear envelope is composed of two membranes deliminating a perinuclear space which displays functional properties similar to those of a Ca2+-storing compartment. ATP-driven Ca2+ uptake and InsP3-induced Ca2+ release processes have been described in isolated nuclei. Recently, it was reported that cADP-ribose and InsP3 can trigger a nucleoplasmic Ca2+ increase. It was hypothesized that the inner nuclear membrane possesses Ca2+ channels that are regulated by ryanodine or InsP3. Radio-ligand binding assays and Western blot experiments were performed in order to investigate their presence in sheep cardiac and rat liver nuclear envelopes. Ryanodine receptors (RyR) were not detected in liver nuclear envelopes by either binding assay or Western blot analysis. However, cardiac nuclear envelopes were found to retain a very low level of specific ryanodine binding, which was not detected on immuno-blots obtained with three types of isoform-specific RyR antibodies. In contrast, nuclear InsP3-binding sites were consistently detected in both cardiac and liver nuclear envelopes. Altogether, these results provide evidence for the major contributor InsP3-gated Ca2+ channels in control of Ca2+ release from the perinuclear space in liver and cardiac cells.

Animals↗
PubMed1994

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo.

Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.

Anaphase↗
PubMed1990

Detection of a large cation-selective channel in nuclear envelopes of avian erythrocytes.

To determine whether the nuclear envelope of eukaryotic cells has the capability to regulate ion fluxes, we have used the patch-clamp technique to detect ion channels in this membrane system. Since possible sites for ion channels in the nuclear envelope include not only the nuclear pores, but also both the inner and outer nuclear membranes, we have patched giant liposomes composed of phosphatidylcholine and nuclear envelope fragments isolated from mature avian erythrocytes. A large, cation-selective channel with a maximum conductance of approximately 800 pS in symmetrical 100 mM KCl was detected. This channel is a possible candidate for a nuclear pore.

Animals↗
PubMed1991

A distinct vesicle population targets membranes and pore complexes to the nuclear envelope in Xenopus eggs.

Extracts from Xenopus eggs capable of nuclear envelope assembly in vitro were fractionated by differential and density gradient centrifugation. Nuclear envelope assembly was found to require soluble components in the cytosol and two distinct particulate fractions, which we have called nuclear envelope precursor fractions A and B (NEP-A and NEP-B). Both NEP-A and NEP-B are sensitive to treatments with trypsin, sodium carbonate, and detergents, but can be distinguished from each other by their sensitivities to high salt and N-ethylmaleimide and by their levels of alpha-glucosidase activity. Vesicles in NEP-B bind to chromatin, whereas those in NEP-A do not. NEP-B may therefore be involved in the targeting of membranes to the surface of the chromatin, whereas NEP-A may provide a pool of vesicles that contributes many of the nuclear envelope membranes. NEP-B may also play a role in the assembly of nuclear pore complexes because the density of nuclear pores in the resulting envelope is dependent on the ratio of NEP-B to NEP-A in the reconstituted extract.

Adenosine Triphosphate↗
PubMed1978

Unusual features of the nuclear envelope in human spermatogenic cells.

Different types of human germ cells show unusual features of the nuclear envelope. Spermatogonial nuclei demonstrate two kinds of modifications. The first one is a series of intranuclear flattened cisterns, parallel to each other and to the inner aspect of the nuclear envelope. The second one is a nuclear envelope protrusion into the cytoplasm occupied by a double membrane-limited vesicle. Pores are found on the membrane of the vesicle facing the interior of the nucleus. In spermatocytes the nuclear pores are concentrated over certain areas and completely absent from others. In the regions where they are absent a single cytoplasmic cistern of rough endoplasmic reticulum is closely apposed to the outer membrane of the nuclear envelope. Early modifications of the nuclear surface appear in spermatids before the attachment of the acrosomic vesicle and may indicate an active role of the nuclear envelope in the morphogenesis of the acrosome. In round spermatids nuclear pores are absent from the area which is first related to the Golgi and later covered by the acrosomal cap. Single or multiple layers of cytoplasmic annulate lamellae are closely associated with the nuclear envelope over the pore rich areas. Frequently there are intranuclear accumulations of dense material adjacent to the annulate lamellae-nuclear pore complex. The chromatoid body is usually present on the cytoplasmic side of this complex. In the elongating spermatids most annulate lamellae are free in the cytoplasm, often in relation with Golgi and chromatoid body remnants near the axial filament. Few stacks of annulate lamellae are noted adjacent to the pore rich nuclear regions. It is suggested that the described modifications are related to an active nuclear-cytoplasmic interaction.

Adult↗
PubMed1978

Isolation of nuclear envelopes with polyanions.

Optimal conditions for the isolation of nuclear envelopes by the action of heparin on nuclei are established and a morphological and biochemical study of such isolated envelopes is presented. An almost 100% yield of pure nuclear envelopes can be obtained by a single sedimentation step after incubation of nuclei with heparin for 40 min at 4 degrees C. The nuclear membrane pellet obtained in this way contains whole envelopes with a preserved perinuclear space and with ribosomes present on the outher leaflet. A single band with an apparent buoyant density of 1.18 is obtained by sucrose density gradient analysis. The chemical composition of the pellet is similar to that of the purified membranes and corresponds to 62% proteins, 34% phospholipids, 3% RNA, and 0.5% DNA. The presence of low concentrations of sodium phosphate (2-10 mM) is critical for a complete solubilization of the chromatin. A less rapid and complete solubilization is obtained with the potassium salt. Low concentrations of Mg++ (1-3 mM) counteract chromatin solubilization by heparin mainly at the level of chromatin-nuclear membrane association. The presence of EDTA in the medium leads to isolated nuclear envelopes on which neither ribosomes nor nuclear pores are visible, indicating the pore structure is dependent on the presence of Ca++ or Mg++. A comparison with other polyanions indicates a decisive advantage of heparin. However, pure nuclear envelopes can also be obtained by the action of dextran sulfate (mol wt 500,000) on nuclei incubated for 5 min at 37 degrees C, in the presence of phosphate ions.

Animals↗
PubMed2001

The nuclear envelope in muscular dystrophy and cardiovascular diseases.

Considerable interest has been focused on the nuclear envelope in recent years following the realization that several human diseases are linked to defects in genes encoding nuclear envelope specific proteins, most notably A-type lamins and emerin. These disorders, described as laminopathies or nuclear envelopathies, include both X-linked and autosomal dominant forms of Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction system defects, limb girdle muscular dystrophy 1B with atrioventricular conduction disturbances, and Dunnigan-type familial partial lipodystrophy. Certain of these diseases are associated with nuclear structural abnormalities that can be seen in a variety of cells and tissues. These observations clearly demonstrate that A-type lamins in particular play a central role, not only in the maintenance of nuclear envelope integrity but also in the large-scale organization of nuclear architecture. What is not obvious, however, is why defects in nuclear envelope proteins that are found in most adult cell types should give rise to pathologies associated predominantly with skeletal and cardiac muscle and adipocytes. The recognition of these various disorders now raises the novel possibility that the nuclear envelope may have functions that go beyond housekeeping and which impact upon cell-type specific nuclear processes.

Animals↗
PubMed1995

Analysis of nucleocytoplasmic transport and nuclear envelope structure in yeast disrupted for the gene encoding the nuclear pore protein Nup1p.

Nup1p and Nsp1p are structurally related nuclear pore complex (NPC) proteins which contain many degenerate repeat sequences of the FSFG type in their central domains. To find out whether this similarity also reflects a functional overlap at the NPC, we analyzed delta nup1 cells in comparison to ts nsp1 cells for defects in nucleocytoplasmic transport. When the NUP1 gene was disrupted in two different laboratory yeast strains, haploid delta nup1 progeny was viable, showing that NUP1 is not essential for vegetative cell growth; delta nup1 strains, however, exhibited a strongly reduced growth rate at 37 degrees C as compared to 23 degrees C. When analyzed by thin section electron microscopy, delta nup1 cells show a normal nuclear envelope morphology and the number and appearance of nuclear pore complexes apparently was not altered. Whereas delta nup1 cells grown at 37 degrees C could still accumulate a lacZ reporter protein carrying a nuclear localization sequence (NLS) inside the nucleus, poly(A)+ RNA export was significantly inhibited. Our data suggest that Nup1p which is not physically associated with Nsp1p, is required for efficient nucleocytoplasmic transport reactions.

Biological Transport↗
PubMed1980

The nuclear envelope lamina is reversibly depolymerized during mitosis.

The nuclear envelope lamina is a supramolecular protein assembly associated with the nucleoplasmic surface of the inner nuclear membrane, which contains three predominant polypeptide components in mammalian cells (lamins A, B and C). We previously demonstrated by immunofluorescence microscopy that the lamina is reversibly disassembled during cell division, coincident with the disassembly and reconstruction of the mitotic nuclear envelope architecture. In this paper, these immunocytochemical observations are extended with cell fractionation and immunoprecipitation studies performed on synchronized populations of tissue culture cells. With these techniques, we have established that during mitosis, lamina A and C occur in a soluble and nonmembrane-associated state. In contrast, the mitotic lamin B may be associated with membrane fragments derived from the disassembled interphase nuclear envelope. From sedimentation analysis on sucrose gradients, we have determined that all three lamins are monomeric at periods of mitotic lamina disassembly. These results, together with quantitative immunoprecipitation studies, demonstrate that the lamina is reversibly depolymerized during cell division. Attendant with the depolymerized state of the lamina, the mitotic lamins (which are phosphoproteins) have a distinctly more acidic isoelectric point and a substantially higher level of phosphorylation compared to their interphase counterparts. This indicates that reversible enzymatic phosphorylations of the lamins may be involved in modulating the state of polymerization of the lamina and its reversible mitotic disassembly.

Animals↗
PubMed1987

The incorporation of fatty acids into triacylglycerols of isolated neuronal nuclear envelopes: the influence of thiol reducing reagents and chromatin.

Using [3H]arachidonate, ATP, coenzyme A, MgCl2, EGTA and CMP, triacylglycerols were labelled in an isolated neuronal nuclear fraction, N1 (from immature rabbit cerebral cortex). When the radioactive nuclear fraction N1 was subfractionated, 75% of the labelled triacylglycerol product was located in the nuclear envelope fraction, E, indicating that the fatty acid incorporation was taking place at the nuclear membrane. However, when nuclear envelope fraction E was first isolated and then incubated with radioactive fatty acid, a significant incorporation into triacylglycerol was found only when nuclear envelope fractions had been prepared in the presence of dithiothreitol or mercaptoethanol. The use of the thiol compounds during the isolation of nuclear envelope fraction E led to specific incorporation rates (based on phospholipid content) which were at best 45-56% of the corresponding values seen for the parent nuclear N1 fraction. This was seen for nuclear envelope fractions isolated by two different procedures. Specific rates for acyl-CoA synthetase and diacylglycerol generation (by cholinephosphotransferase) were measured in nuclear envelope fractions and found to be similar to specific rates for these enzymes in the nuclear N1 fraction. The deficiency in triacylglycerol labelling in nuclear envelope fractions was likely due to impaired diacylglycerol acyltransferase activity. Higher specific rates of triacylglycerol labelling (82-90% of N1 values) were seen in nuclear envelope fractions assayed very shortly after preparation and in another subfraction of nuclear fraction N1 which contained small amounts of phospholipid and high concentrations of nucleates and protein. These data suggest that triacylglycerol formation may be maintained by the presence of chromatin, while in its absence there is a loss of acylation activity in nuclear envelope fractions.

Animals↗
PubMed2004

Nuclear envelope breakdown requires overcoming the mechanical integrity of the nuclear lamina.

In prophase cells, lamin B1 is the major component of the nuclear lamina, a filamentous network underlying the nucleoplasmic side of the nuclear membrane, whereas lamin A/C is dissociated from the scaffold. In vivo fluorescence microscopy studies have shown that, during the G2/M transition, the first gap in the nuclear envelope (NE) appears before lamin B1 disassembly and is caused by early spindle microtubules impinging on the NE. This result suggests that the mechanical tearing of the NE by microtubules plays a central role to the progression of mitosis. To investigate whether this microtubule-induced NE deformation is sufficient for NE breakdown, we assess the mechanical resilience of a reconstituted lamin B1 network. Quantitative rheological methods demonstrate that human lamin B1 filaments form stiff networks that can resist much greater deformations than those caused by microtubules impinging on the NE. Moreover, lamin B1 networks possess an elastic stiffness, which increases under tension, and an exceptional resilience against shear deformations. These results demonstrate that both mechanical tearing of the lamina and biochemical modification of lamin B1 filaments are required for NE breakdown.

Actins↗
PubMed1982

Action of insulin at the nuclear envelope.

Insulin binding sites are present on purified nuclear envelopes from liver and other tissues, and EM autoradiographs and other types of studies indicate that insulin can enter intact target cells and interact with several types of intracellular membranes, including the nuclear envelope. More recent studies indicate that insulin has direct effects on both mRNA efflux from isolated nuclei and nuclear envelope NTPase, the enzyme that regulates mRNA efflux. These studies raise the possibility, therefore, that insulin regulates mRNA levels in target cells by directly influencing nuclear membrane functions as NTPase. Since insulin does not dramatically elevate mRNA levels for all proteins, the question arises as to how insulin selectively increases mRNA for specific mRNAs. One possibility is that there is targeting of specific mRNA molecules for specific pore complexes and that insulin may only influence a certain fraction of the nuclear pores. Thus, continued investigation is needed concerning the role of polypeptide hormones such as insulin in nucleocytoplasmic exchange.

Adipose Tissue↗
PubMed2001

Nuclear envelope organization in papillary thyroid carcinoma.

Papillary thyroid carcinomas (PTCs) have characteristic nuclear shape changes compared to follicular-type thyroid epithelium. We tested the hypothesis that the altered nuclear shape results from altered distribution or expression of the major structural proteins of the nuclear envelope. Lamin A, lamin B1, lamin C, lamin B receptor (LBR), lamina-associated polypeptide 2 (LAP2), emerin, and nuclear pores were examined. PTC's with typical nuclear features by H&E were compared to non-neoplastic thyroid and follicular neoplasms using confocal microscopy, and semi-quantitative immunoblotting. Lamin A/C, lamin B1, LAP2, emerin, and nuclear pores all extend throughout the grooves and intranuclear inclusions of PTC. Their distribution and fluorescent intensity is not predictably altered relative to nuclear envelope irregularities. By immunoblotting, the abundance (per cell) and electrophoretic mobilities of lamin A, lamin B1, lamin C, emerin, and LAP2 proteins do not distinguish PTC, normal thyroid, or follicular neoplasms. These results do not support previously published predictions that lamin A/C expression is related to a loss of proliferative activity. At least three LAP2 isoforms are identified in normal and neoplastic thyroid. LBR is sparse or undetectable in all the thyroid samples. The results suggest that the irregular nuclear shape of PTC is not determined by these nuclear envelope structural proteins per se. We review the structure of the nuclear envelope, the major factors that determine nuclear shape, and the possible functional consequences of its alteration in PTC.

Blotting, Western↗
PubMed1994

Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization.

We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum.

Animals↗
PubMed1986

Interaction of steroids with the nuclear envelope.

Three approaches have been taken to determine the molecular mechanism by which steroid hormones traverse the nuclear envelope on their way to the genome. The first approach involved characterization of steroid binding to nuclear envelope preparations. We have characterized androgen binding to nuclear envelopes isolated from the rat ventral prostate, the rat liver, and androgen-responsive and androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma and glucocorticoid binding to rat liver. Relatively high affinity binding sites for steroids have been identified on nuclear envelopes. Importantly, the number and specificity of the sites correlates with the responsiveness of the tissue to the steroid. In the second approach, we have undertaken to identify the steroid binding site directly. As the characteristics of the rat ventral prostate site resembled those of the nuclear androgen receptor, we have begun purifying that receptor and have found fast protein liquid chromatography to be very effective. By affinity labelling studies, the dexamethasone binding site on the rat liver nuclear envelope has been identified as a peptide of molecular weight of approximately 90,000. The third approach we have used is to identify androgen-dependent peptides in nuclear envelope preparations. In both the rat ventral prostate and an androgen-responsive cell line of the Shionogi mouse mammary carcinoma, we have identified abundant androgen-dependent peptides. The relationship of these peptides to the binding sites identified by the first two approaches and their role in steroid transport is being investigated.

Animals↗
PubMed1983

Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.

The direct addition of insulin to highly purified nuclear envelopes prepared from the livers of diabetic rats resulted in a decrease in the incorporation of 32P into trichloroacetic acid-precipitable proteins. Autoradiography of 32P-labeled envelopes, solubilized in sodium dodecyl sulfate and subjected to electrophoresis, revealed that insulin decreased the phosphorylation of all major protein bands. Insulin produced detectable effects at concentrations between 0.1 and 1 pM, maximal effects at 10 pM, and progressively diminished effects at higher concentrations. Two insulin analogs, desdipeptide proinsulin and desoctapeptide insulin, had approximately 10% and 1%, respectively, the activity of native insulin. When nuclear envelopes were first phosphorylated with [gamma-32P]ATP and insulin was then added with an excess of unlabeled ATP, dephosphorylation was enhanced, suggesting that insulin was regulating nuclear envelope phosphatase activity. The direct addition of insulin to isolated rat liver nuclei in the presence of ATP stimulated the release of previously 14C-labeled trichloroacetic acid-precipitable mRNA-like material, and the direct addition of insulin to nuclear envelopes stimulated the activity of nucleoside triphosphatase, the enzyme that participates in mRNA nucleocytoplasmic transport. Moreover, the dose-response curves for these functions mirrored insulin's inhibition of nuclear envelope phosphorylation. These data suggest, therefore, a mechanism whereby insulin directly inhibits the phosphorylation of the nuclear envelope, leading in turn to the regulation of mRNA metabolism.

Animals↗
PubMed2006

Nuclear envelope transmembrane proteins (NETs) that are up-regulated during myogenesis.

BACKGROUND: The nuclear lamina is a protein meshwork lining the inner nuclear membrane, which contains a polymer of nuclear lamins associated with transmembrane proteins of the inner nuclear membrane. The lamina is involved in nuclear structure, gene expression, and association of the cytoplasmic cytoskeleton with the nucleus. We previously identified a group of 67 novel putative nuclear envelope transmembrane proteins (NETs) in a large-scale proteomics analysis. Because mutations in lamina proteins have been linked to several human diseases affecting skeletal muscle, we examined NET expression during differentiation of C2C12 myoblasts. Our goal was to identify new nuclear envelope and lamina components whose expression is coordinated with muscle differentiation. RESULTS: Using transcriptional microarray analysis, we found that expression of 6 of the NETs significantly increases during myoblast differentiation. We confirmed these results using quantitative RT-PCR, and furthermore, found that all 6 NETs are expressed at high levels in adult mouse skeletal muscle relative to 9 other tissues examined. Using epitope-tagged cDNAs, we determined that the 5 NETs we could analyze (NETs 9, 25, 32, 37 and 39) all target to the nuclear envelope in C2C12 cells. Furthermore, the 3 NETs that we could analyze by immunoblotting were highly enriched in nuclear envelopes relative to microsomal membranes purified from mouse liver. Database searches showed that 4 of the 6 up-regulated NETs contain regions of homology to proteins previously linked to signaling. CONCLUSION: This work identified 6 NETs that are predicted to have important functions in muscle development and/or maintenance from their expression patterns during myoblast differentiation and in mouse tissues. We confirmed that 5 of these NETs are authentic nuclear envelope proteins. Four members of this group have potential signaling functions at the NE, based on their sequence homologies.

Animals↗
PubMed2000

Recruitment of protein phosphatase 1 to the nuclear envelope by A-kinase anchoring protein AKAP149 is a prerequisite for nuclear lamina assembly.

Subcellular targeting of cAMP-dependent protein kinase (protein kinase A [PKA]) and of type 1 protein phosphatase (PP1) is believed to enhance the specificity of these enzymes. We report that in addition to anchoring PKA, A-kinase anchoring protein AKAP149 recruits PP1 at the nuclear envelope (NE) upon somatic nuclear reformation in vitro, and that PP1 targeting to the NE is a prerequisite for assembly of B-type lamins. AKAP149 is an integral membrane protein of the endoplasmic reticulum/NE network. The PP1-binding domain of AKAP149 was identified as K(153)GVLF(157). PP1 binds immobilized AKAP149 in vitro and coprecipitates with AKAP149 from purified NE extracts. Affinity isolation of PP1 from solubilized NEs copurifies AKAP149. Upon reassembly of somatic nuclei in interphase extract, PP1 is targeted to the NE. Targeting is inhibited by a peptide containing the PP1-binding domain of AKAP149, abolished in nuclei assembled with membranes immunodepleted of AKAP149, and restored after reincorporation of AKAP149 into nuclear membranes. B-type lamins do not assemble into a lamina when NE targeting of PP1 is abolished, and is rescued upon recruitment of PP1 to the NE. We propose that kinase and phosphatase anchoring at the NE by AKAP149 plays in a role in modulating nuclear reassembly at the end of mitosis.

A Kinase Anchor Proteins↗