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PubMed2005

Alterations of nuclear envelope and chromatin organization in mandibuloacral dysplasia, a rare form of laminopathy.

Autosomal recessive mandibuloacral dysplasia [mandibuloacral dysplasia type A (MADA); Online Mendelian Inheritance in Man (OMIM) no. 248370] is caused by a mutation in LMNA encoding lamin A/C. Here we show that this mutation causes accumulation of the lamin A precursor protein, a marked alteration of the nuclear architecture and, hence, chromatin disorganization. Heterochromatin domains are altered or completely lost in MADA nuclei, consistent with the finding that heterochromatin-associated protein HP1beta and histone H3 methylated at lysine 9 and their nuclear envelope partner protein lamin B receptor (LBR) are delocalized and solubilized. Both accumulation of lamin A precursor and chromatin defects become more severe in older patients. These results strongly suggest that altered chromatin remodeling is a key event in the cascade of epigenetic events causing MADA and could be related to the premature-aging phenotype.

Adolescent↗
PubMed1994

Association of cytoplasmic dynein with manchette microtubules and spermatid nuclear envelope during spermiogenesis in rats.

During spermiogenesis, the shape of the spermatid nucleus, which is spherical, changes and it becomes the sperm head. A microtubular structure called a manchette is thought to be involved in this morphogenetic process. In this report, we demonstrate the localization of cytoplasmic dynein and manchette development by a double immunofluorescence technique using anti-bovine brain MAP 1C and anti-tubulin. Before step 6 of the Leblond and Clermont staging, the microtubules showed a fine reticular network, and the dynein staining was homogeneous. In step 6, the microtubular network was concentrated around the nucleus. The manchette developed in step 7 spermatids, and was fully formed, with a skirt-like appearance, covering the nuclear surface in step 8. Dynein fluorescence was associated with the microtubular manchette in steps 7-10. During these steps, the nucleus was protruded from the cytoplasm. In steps 11-13, the most active stages in nuclear shaping, the dynein was densely localized at the nuclear surface covered by the manchette. As the nucleus acquired a shape similar to the mature spermatozoon at step 14, the dynein fluorescence was localized only at the concave side of the nuclear caudal edge. The manchette became narrower and elongated. In step 15, the manchette extended into the elongated cytoplasm, diminishing during steps 16-18. The localization of the dynein was limited to the ventral aspect of the caudal head in these steps. There was little dynein fluorescence in mature spermatozoa. Immunoelectron microscopy showed positive reactions in the nuclear envelope and the inner region of the microtubular manchette. These observations suggest that cytoplasmic dynein, possibly bound to the nuclear envelope, and manchette microtubules are involved in the protrusion of the spermatid nucleus from the cytoplasm.

Animals↗
PubMed1999

[Alteration of ryanodine receptors in cardiac sarcoplasmic reticulum and nuclear envelope of rats during sepsis].

To investigate changes of ryanodine receptors in the sarcoplasmic reticulum (SR) and the nuclear envelope (NE) of rat cardiac myocytes during sepsis induced by cecal ligation and puncture (CLP), myocardial SR and NE were fractionated with density gradient centrifugation and the characteristic of ryanodine receptor was assayed with a method of radioreceptor binding assay. The result showed that Bmax of ryanodine receptors in cardiac SR was increased by 23% during early sepsis (9 h after CLP), but decreased by 38% during late sepsis (18 h after CLP). Bmax of ryanodine receptors in cardiac NE, on the other hand, was increased by 100% and 160% during early and late sepsis respectively. Kd of ryanodine binding to SR and NE remained unchanged during sepsis. These results demonstrated up-regulation of ryanodine receptors in SR occurred during early sepsis and down-regulation of these receptors in SR occurred during late sepsis, while up-regulation of ryanodine receptors in NE occurred during both the early and the late sepsis.

Animals↗
PubMed1990

A 41-42 bp tandemly repeated sequence isolated from nuclear envelopes of chicken erythrocytes is located predominantly on microchromosomes.

To study whether specific DNA sequences are associated with nuclear membranes, residual DNA was extracted from DNase-treated nuclear envelopes prepared from erythrocytes of adult chickens (Gallus domesticus). This DNA was then blunt-end ligated into a bacterial plasmid vector. DNA blot analysis and nucleotide sequence determination revealed that approximately 30% of the cloned fragments consisted of different multiples of a 41-42 bp tandemly repeated, partially symmetrical sequence. In situ hybridization to chicken chromosomes demonstrated that the sequence was located primarily on microchromosomes, although some hybridization was also observed to macrochromosomes 7 and 8. Digestion of chicken DNA with any of a number of restriction enzymes did not completely reduce the intensity of a high molecular weight band to which the repeated sequence hybridized. These results, along with those obtained from in situ hybridization, suggested that many copies of this sequence are organized into large tandem arrays, and are not dispersed in many shorter repetitive blocks throughout the chicken genome. Although the repetitive sequence constituted approximately 10% of the chicken genome, it did not hybridize to quail or turkey DNA.

Animals↗
PubMed2005

Nuclear envelope assembly is promoted by phosphoinositide-specific phospholipase C with selective recruitment of phosphatidylinositol-enriched membranes.

Nuclear envelope (NE) formation in a cell-free egg extract proceeds by precursor membrane vesicle binding to chromatin in an ATP-dependent manner, followed by a GTP-induced NE assembly step. The requirement for GTP in the latter step of this process can be mimicked by addition of bacterial PI-PLC [phosphoinositide (PtdIns)-specific phospholipase C]. The NE assembly process is here dissected in relation to the requirement for endogenous phosphoinositide metabolism, employing recombinant eukaryotic PI-PLC, inhibitors and direct phospholipid analysis using ESI-MS (electrospray ionization mass spectrometry). PtdIns (phosphatidylinositol) species analysis by ESI-MS indicates that the chromatin-bound NE precursor vesicles are enriched for specific PtdIns species. Moreover, during GTP-induced precursor vesicle fusion, the membrane vesicles become partially depleted of the PtdIns 18:0/20:4 species. These data indicate that eukaryotic PI-PLC can support NE formation, and the sensitivity to exogenous recombinant PtdIns-5-phosphatases shows that the endogenous PLC hydrolyses a 5-phosphorylated species. It is shown further that the downstream target of this DAG (diacylglycerol) pathway does not involve PKC (protein kinase C) catalytic function, but is mimicked by phorbol esters, indicating a possible engagement of one of the non-PKC phorbol ester receptors. The results show that ESI-MS can be used as a sensitive means to measure the lipid composition of biological membranes and their changes during, for example, membrane fusogenic events. We have exploited this and the intervention studies to illustrate a pivotal role for PI-PLC and its product DAG in the formation of NEs.

Animals↗
PubMed1995

Formation of the sea urchin male pronucleus in vitro: membrane-independent chromatin decondensation and nuclear envelope-dependent nuclear swelling.

We demonstrate that complete sea urchin male pronuclear development in vitro is a two-step process involving membrane-independent chromatin decondensation and nuclear envelope-dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of approximately 4 microns in diameter. Pronuclear swelling to approximately 7 microns requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca2+ and cytosolic factors, some of which are sensitive to heat and to the sulfhydryl alkylating agent, N-ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations.

Adenosine Triphosphate↗
PubMed2000

Meiotic lamin C2: the unique amino-terminal hexapeptide GNAEGR is essential for nuclear envelope association.

Meiotic lamin C2 is the only A-type lamin expressed during mammalian spermatogenesis. Typical for this short lamin is the unique hexapeptide GNAEGR, which substitutes the nonhelical amino terminus and part of the alpha-helical rod domain present in somatic lamins. Meiotic lamin C2 also lacks a carboxyl-terminal CaaX box, which is modified by isoprenylation and involved in nuclear envelope (NE) association of somatic isoforms. The mechanism by which lamin C2 becomes localized in the NE is totally unknown. Here we demonstrate that the hexapeptide GNAEGR is essential for this process: (i) Its deletion resulted in a diffuse distribution of lamin C2 within nuclei of transfected COS-7 cells; (ii) Mutated somatic lamin C, containing the sequence GNAEGR at its amino terminus, was located at the NE. The mass spectrometric analysis of the amino terminus of lamin C2 revealed that it is modified by myristoylation. Correspondingly, the substitution of the first glycine residue abolishes the NE association of lamin C2. We conclude that NE association of lamin C2 is achieved by a mechanism different from that of somatic lamins.

Amino Acid Sequence↗
PubMed2005

Nesprin-2 is a multi-isomeric protein that binds lamin and emerin at the nuclear envelope and forms a subcellular network in skeletal muscle.

Nesprin-2 is a multi-isomeric, modular protein composed of variable numbers of spectrin-repeats linked to a C-terminal transmembrane domain and/or to N-terminal paired calponin homology (CH) domains. The smaller isoforms of nesprin-2 co-localize with and bind lamin A and emerin at the inner nuclear envelope (NE). In SW-13 cells, which lack lamin A/C, nesprin-2 epitopes and emerin were both mislocalized and formed aggregates in the endoplasmic reticulum (ER). The larger isoforms and other CH-domain-containing isoforms co-localize with heterochromatin within the nucleus and are also present at the outer NE and in multiple cytoplasmic compartments. Nesprin-2 isoforms relocalize during in vitro muscle differentiation of C2C12 myoblasts to the sarcomere of myotubes. Immunogold electron microscopy using antibodies specific for three different epitopes detected nesprin-2 isoforms at multiple locations including intranuclear foci, both membranes of the NE, mitochondria, sarcomeric structures and plasma membrane foci. In adult skeletal muscle, confocal immunolocalization studies demonstrated that nesprin-2 epitopes were present at the Z-line and were also associated with the sarcoplasmic reticulum (SR) in close apposition to SERCA2. These data suggest that nesprin-2 isoforms form a linking network between organelles and the actin cytoskeleton and thus may be important for maintaining sub-cellular spatial organisation. Moreover, its association at the NE with lamin and emerin, the genes mutated in Emery-Dreifuss muscular dystrophy, suggests a mechanism to explain how disruption of the NE leads to muscle dysfunction.

Actin Cytoskeleton↗
PubMed1980

Chromatin-nuclear envelope complex from rat liver: isolation and purification.

We describe a method for the isolation of a fraction of nuclear envelope (NE) from rat liver. The method includes mild treatment of pure nuclei with either endonuclease of DNase I under low ionic strength conditions in the presence of magnesium, which allows the nucleomeric organization of the chromatin (Ch) to be preserved. The NEs were purified by centrifugation in sucrose gradients followed by floatation in sucrose. No more than 3% of the Ch present in the purified Ch-NE complexes was due to the non specific adsorption of Ch to the NE. The main components of the complex (Ch and NE) retained their in situ ultrastructure. The complex consisted of 9--10% DNA, 3--4% RNA, about 63% protein and about 24% phospholipids.

Animals↗
PubMed2001

Both emerin and lamin C depend on lamin A for localization at the nuclear envelope.

Physical interactions between lamins and emerin were investigated by co-immunoprecipitation of in vitro translated proteins. Emerin interacted with in vitro translated lamins A, B1 and C in co-immunprecipitation reactions. Competition reactions revealed a clear preference for interactions between emerin and lamin C. Structural associations between lamins and emerin were investigated in four human cell lines displaying abnormal expression and/or localisation of lamins A and C. In each cell line absence of lamins A and C from the nuclear envelope (NE) was correlated with mis-localisation of endogenous and exogenous emerin to the ER. In two cell lines that did not express lamin A but did express lamin C, lamin C as well as emerin was mis-localised. When GFP-lamin A was expressed in SW13 cells (which normally express only very low levels of endogenous lamin A and mis-localise endogenous emerin and lamin C), all three proteins became associated with the NE. When GFP-lamin C was expressed in SW13 cells neither the endogenous nor the exogenous lamin C was localised to the NE and emerin remained in the ER. Finally, lamins A and C were selectively eliminated from the NE of HeLa cells using a dominant negative mutant of lamin B1. Elimination of these lamins from the lamina led to the accumulation of emerin as aggregates within the ER. Our data suggest that lamin A is essential for anchorage of emerin to the inner nuclear membrane and of lamin C to the lamina.

Adrenal Cortex Neoplasms↗
PubMed1996

Genetic variability of four natural isolates of the Stilpnotia salicis multiple-enveloped nuclear polyhedrosis virus.

Viral genome DNA from four different multiple-enveloped nuclear polyhedrosis virus isolates, obtained from naturally infected larvae of satin moth (Stilpnotia salicis), a pest of poplar tree (Populus) was analysed. Larvae were collected over a period of 11 years, from 1978 to 1989. The genomic DNA restriction patterns pointed to heterogeneity of these wild-type viruses. The differences observed in isolates of several years revealed limited restriction fragment length polymorphism and showed that these viruses contained distinct, but closely related genotypes. The genome size of SsMNPV was established as 128-134 kb, based on HindIII and SacI restriction analysis.

Animals↗
PubMed1986

A myosin heavy-chain-like polypeptide is associated with the nuclear envelope in higher eukaryotic cells.

A high molecular weight polypeptide, identified as an ATPase subunit by direct ultraviolet photoaffinity labeling, has been shown to be a component of nuclear envelope-enriched fractions prepared from a variety of higher eukaryotes (Berrios, M., G. Blobel, and P. A. Fisher, 1983, J. Biol. Chem., 258:4548-4555). In rat liver as well as Drosophila melanogaster embryos, this polypeptide appears to be a form of myosin heavy chain. This conclusion is based on both immunochemical and immunocytochemical data, as well as on the results of CNBr and chymotryptic peptide map analyses. In Drosophila, the identification of this myosin heavy chain-like polypeptide as a nuclear envelope component has been corroborated in situ by indirect immunofluorescence analyses using permeabilized whole cells, mechanically extruded nuclei, and cryosections obtained from a number of larval tissues. Localization appears to be restricted to the nuclear periphery in a manner similar to that observed for the nuclear lamins and the pore complex glycoprotein. Antibodies directed against the Drosophila nuclear envelope ATPase have also been shown to decorate mammalian and higher plant cell nuclei in situ. Implications for intracellular nuclear mobility and for nucleocytoplasmic exchange of macromolecules in vivo are discussed.

Adenosine Triphosphatases↗
PubMed1999

[Comparative analysis of nucleotide sequences of chromosomal DNA isolated from nuclear envelopes and cores of rosette-like structures of murine interphase chromosomes].

Six DNA fragments of interphase chromosomes isolated from nuclear envelopes of murine hepatocytes were cloned and sequenced. Analysis of their structural-functional organization suggests that these fragments are highly specified protein-nonencoding fractions of a eukaryotic genome. In the evolutionary process, they appear already in archaebacteria and may be "ancestral" for DNA sequences involved in structuring chromosomal domains (rosette-like structures) of tissue-specific genes. In their composition, these fragments have nucleotide sequences homologous to the repeats of the SINE and LINE families and to the satellite DNA of murine centromeres.

Animals↗
PubMed1979

Luteinizing hormone-accelerated redistribution of lysosome-like organelles preceding dissolution of the nuclear envelope in rat oocytes maturing in vitro.

Maturation of the mammalian oocyte is characterized in part by dissolution of the nuclear envelope, or germinal vesicle breakdown (GVB). By fluorescence microscopy after vital uptake of acridine orange (AO), redistribution and perinuclear accumulation of organelles corresponding to lysosomes occur before GVB in rat oocytes undergoing meiotic maturation in vitro. In follicle-enclosed oocytes explanted during the preovulatory gonadotropin surge (GS) and individually cultured as such in chemically defined medium at approximately 22 degrees C, lysosomes aggregated into disperse clusters after 30 min; by 60 min, perinuclear concentration of lysosomes and their essential disappearance from the cortical ooplasm were observed. GVB occurred within 120 min. In contrast, follicle-enclosed oocytes explanted before the GS displayed a generally homogeneous distribution of lysosomes and intact GV for up to 5 h in culture. In oocytes aspirated from follicles before the GS, partially denuded of granulosa cells, and cultivated without added hormone, most lysosomes concentrated around the GV within 60 min, with GVB occurring generally by 120 min. Luteinizing hormone (LH) added in vitro to the isolated preparation at 3 or 30 x 10(-8) M sharply accelerated these events. The effects of LH, not seen with 1.5 x 10(-8) M hormone, were blocked by anti-LH IgG. Up to 60 x 10(-8) M follicle-stimulating hormone or 80 x 10(-8) M prolactin were ineffective in accelerating lysosome redistribution or GVB. After GVB, lysosomes became once again uniformly dispersed and unresponsive, even to 60 x 10(-8) M added LH, a finding consistent with tachyphylaxis of target cells by independent criteria. The present data, all statistically significant at P less than 0.05, demonstrate that mobilization of lysosomes before GVB is a specific response to factors that promote resumption of meiotic maturation of rat oocytes.

Animals↗
PubMed1996

Nuclear envelope acts as a calcium barrier in C6 glioma cells.

The temporal and spatial changes of intracellular free calcium ([Ca2+]i) within the cytosol and nucleis of C6 glioma cells have been investigated with laser confocal scanning microscopy to evaluate the current view that Ca2+ ions pass freely through nuclear pores by diffusion. Our results indicate that localized cytosolic Ca2+ release, which appeared as puffs, spread with an apparent diffusion rate of 0.35 +/- 0.07 microns/sec (n = 44). This release was followed by an immediate Ca2+ uptake at the resting stage. Following the treatment with thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, release of nuclear Ca2+ from certain nuclear hot zones and nuclear envelope was obtained. Most of the nuclear Ca2+ released were confined to the nuclear boundary, but a slow migration of Ca2+ towards the cytosol was observed. The apparent diffusion rate of this Ca2+ release is 0.015 microns/sec. By contrast, the inward spread into nucleus occurred with a diffusion rate of 0.04 microns/sec. From these diffusion rates and other experimental evidence, we conclude that the movement of Ca2+ at the nucleocytosolic interface is more than a simple diffusion process and the interface is a barrier to Ca2+ movement.

Animals↗
PubMed1999

Spatial localization of chromosome-nuclear envelope interaction sites.

In the interphase nucleus chromosomes are tightly associated with the nuclear envelope (NE) through special granular chromatin particles termed anchorosomes. It remains unclear whether anchorosomes represent constant nuclear structures, persisting throughout the cell cycle, or they appear only in the interphase during the formation of contacts between the chromosomes and NE. In other words, whether specific NE interaction sites do exist in chromosomes or any region can form anchorosome. In this work, we used micrononucleated PK cells, in which almost every micronucleus (MN) is formed by a single chromosome. The spatial distribution and quantitative characteristics of the anchorosomal layer in MN was studied using stereological analysis and three-dimensional computer reconstruction. It was shown that in cells with about 30 MN, the total surface area of NE reaches about 355 microm2, whereas in normal mononuclear cells it is 110 microm2. Hence, the NE surface increases 3-fold during MN formation. In contrast to normal cells, only 80% of the NE surface in MN is covered with anchorosomes, i.e., the total surface area of the anchorosomal layer increases by a factor of 2.5. The 3D reconstruction has demonstrated highly random distribution of anchorosome-free zones, the distribution patterns varying in individual MN. These findings are thought to be evidence for the existence of a limited number of specific chromosomal sites potentially capable of forming contacts with NE.

Animals↗
PubMed1999

Dynamics and mobility of nuclear envelope proteins in interphase and mitotic cells revealed by green fluorescent protein chimeras.

Understanding how membrane proteins are targeted to and retained within the nuclear envelope (NE) and the fate of these proteins during NE disassembly/reassembly in mitosis is central for insight into the function of the NE in nuclear organization and dynamics. To address these issues we have attached green fluorescent protein (GFP) to a well-characterized protein of the inner nuclear membrane, lamin B receptor, believed to be one of the major chromatin docking protein in the NE. We have used this construct in a variety of applications, including dual-color GFP time-lapse imaging, to investigate the mechanisms underlying protein targeting to the NE and NE breakdown and reassembly during mitosis. In this review, we present a summary of the results from such studies and discuss the photobleaching and imaging methodology on which they were derived.

Animals↗
PubMed1975

Isolation of a nuclear ribonucleoprotein network that contains heterogeneous RNA and is bound to the nuclear envelope.

Rapidly labeled polydispersed nuclear RNA is part of a ribonucleoprotein (RNP) network which in turn is tightly bound to the nuclear membrane. The membranous attachment, therefore, established a connection between chromatin and cytoplasm. The ultrastructure of the RNP network comprises fibrils and granules similar to those observed in intact nuclei. When bound to the nuclear membrane it has the composition of 63% protein, 14% RNA, 0.4% DNA, and 22.6% lipids. The proportion of lipids diminishes to 2.2% when nuclear membrane is not present. Chromatin, nucleoli, and ribosomes are minor contaminants since histones and ribosomal proteins are not detectable in polyacrylamide gel electrophoresis. Nuclear disruption at high pressure in a French pressure cell causes fragmentation of the RNP network into a series of polydispersed RNP particles. Fragmentation can be prevented by using mild pressure, or by disrupting nuclei with high salt buffer and digesting the dispersed chromatin with deoxyribonuclease. A RNP network, almost free of membrane, is also obtained if the nucleus is deprived of its envelope by treatment with Triton X-100. Since no polydispersed RNP particles are found following dissolution of the nuclear membrane, it is assumed that the particles are components of the RNP network whose fragmentation occurs as a consequence of two processes: (a) activation of nuclear nucleases and (b) shearing forces.

Animals↗