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PubMed2005

Nuclear envelope remodeling during mouse spermiogenesis: postmeiotic expression and redistribution of germline lamin B3.

Lamins are members of a multigene family of structural nuclear envelope (NE) proteins. Differentiated mammalian somatic cells express lamins A, C, B1, and B2. The composition and organization of the nuclear lamina of mammalian spermatogenic cells differ significantly from that of somatic cells as they express lamin B1 as well as two short germ line-specific isoforms, namely lamins B3 and C2. Here we describe in detail the expression pattern and localization of lamin B3 during mouse spermatogenesis. By combining RT-PCR, immunoblotting, and immunofluorescence microscopy, we show that lamin B3 is selectively expressed during spermiogenesis (i.e., postmeiotic stages of spermatogenesis). In round spermatids, lamin B3 is distributed in the nuclear periphery and, notably, also in the nucleoplasm. In the course of spermiogenesis, lamin B3 becomes redistributed as it concentrates progressively to the posterior pole of spermatid nuclei. Our results show that during mammalian spermiogenesis the nuclear lamina is composed of B-type isoforms only, namely the ubiquitous lamin B1 and the germline-specific lamin B3. Lamin B3 is the first example of a mammalian lamin that is selectively expressed during postmeiotic stages of spermatogenesis.

Age Factors↗
PubMed1980

[Fragmentation of polyploid nuclei in rat trophoblast giant cells. II. Formation of deep folds in the nuclear envelope--the beginning of fragmentation].

Using light and electron microscopy, the nuclear envelope (NE) of the trophoblast giant cells has been examined at the beginning of the spontaneous fragmentation of nuclei on the last days of pregnancy (not long before their degeneration). In the course of the polyploid nucleus division, deep and narrow invaginations appear in the NE, frequently running through the whole nucleus, from one pole towards the other, and separating it into fragments. Short finger-like outgrowings are seen extending from the long invaginations perperdicularly or at a certain angel. Both the nuclear membrances, having numerous nuclear pores, are involved in the formation of these invaginations and shorter extentions. Local enlargements are seen produced in the perinuclear space, mainly in the area of NE invaginations. Narrow folds, separating the giant nucleus into fragments, are filled with the cytoplasm rich in ribosomes. In the nuclear fragments so produced, association between the earlier dispersed chromatin and the NE becomes stronger. Large accumulations of the condensed chromatin are mainly distributed under the NE, only a few minute chromatin blocks being found in the center of the nucleus filled up with diffuse chromatin.

Animals↗
PubMed1985

The isolation of nuclear envelopes. Effects of thiol-group oxidation and of calcium ions.

The effects of (a) oxidative cross-linking of protein thiol groups and (b) the presence or absence of Ca2+ ions on rat liver nuclear-envelope isolation were studied. Two envelope-isolation procedures were compared: a well characterized low-ionic-strength method and a recently developed high-ionic-strength method. The latter method seems preferable to the former in respect of lower intranuclear contamination of the envelopes, suppression of endogenous serine proteinase, and maintenance of high specific activities of envelope-associated enzymes. In both procedures, however, the presence of Ca2+ gave rise to a rapid, apparently irreversible, contamination of the envelopes by intranuclear material. This effect was half-maximal at 20 microM-Ca2+. In addition, the envelopes became contaminated with intranuclear material by a Ca2+-independent mechanism, apparently resulting from N-ethylmaleimide-sensitive intermolecular disulphide-bond formation. This oxidative process seemed to have two major kinetic components (half-life, t1/2, approx. 2 min and 10 min). In view of these findings, it is recommended that (i) for most purposes, nuclear envelopes be isolated by the newly developed high-ionic-strength procedure, (ii) irrespective of the method used, Ca2+-chelators be included in all the buffers, (iii) thiol-group oxidation be prevented or reversed during the procedure.

Animals↗
PubMed2004

Importin alpha associates with membranes and participates in nuclear envelope assembly in vitro.

Importin alpha is well known as an adaptor that functions with Importin beta in the nuclear import of proteins containing specific nuclear localization signals (NLSs). We show here that either an excess or a lack of Importin alpha blocks nuclear envelope (NE) assembly in vitro, and our data suggest that soluble Importin alpha functions in NE assembly in conjunction with NLS-containing partner proteins. Surprisingly, a significant proportion of Importin alpha is found to fractionate with Xenopus egg membranes. We demonstrate that membrane association of Importin alpha is regulated by phosphorylation. Using mutant forms of Importin alpha that either do not bind membranes or are not released from them by phosphorylation, we provide evidence that membrane-associated Importin alpha is required for NE formation. Unlike other functions of Importin alpha, this membrane-associated activity does not require interaction with NLS proteins.

Active Transport, Cell Nucleus↗
PubMed1991

Assembly/disassembly of the nuclear envelope membrane: cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro.

Dissociation and association of membranes with chromatin at the beginning and end of mitosis are critical in controlling nuclear dynamics during these stages of the cell cycle. Employing purified membrane and cytosolic fractions from Xenopus eggs, a simple assay was developed for the reversible binding of nuclear membrane vesicles to chromatin. We have shown, using phosphatase and kinase inhibitors, that membrane-chromatin association is regulated by a phosphatase/kinase system. In interphase, the balance in this system favors dephosphorylation, possibly of a membrane receptor, which then mediates chromatin binding. At mitosis the membrane receptor is phosphorylated, causing release of chromatin-bound membrane. Purified MPF kinase does not directly cause membranes to dissociate from chromatin. Rather, binding of membranes to chromatin at mitosis appears to be regulated indirectly by MPF through its action on a phosphatase/kinase system that directly modulates the phosphorylation state of a nuclear membrane component.

Animals↗
PubMed1994

The protein product of the oncogene bcl-2 is a component of the nuclear envelope, the endoplasmic reticulum, and the outer mitochondrial membrane.

The protein product of the oncogene bcl-2 is a potent inhibitor of apoptotic cell death. The Bcl-2 protein has variously been reported to reside in the nuclear envelope and endoplasmic reticulum or exclusively in the inner membrane of mitochondria. We therefore undertook a detailed analysis of the intracellular distribution of Bcl-2 by immunofluorescence, immunogold electron microscopy, and subcellular fractionation in three mouse cell lines expressing a human bcl-2 transgene and measured its importation into isolated mitochondria. By these methods, the protein was localized to the nuclear envelope, the endoplasmic reticulum, and the outer mitochondrial membrane. Any proposal for the mechanism by which Bcl-2 inhibits apoptosis must therefore accommodate the fact that Bcl-2 localizes to cytoplasmic membranes facing the cytosol.

Animals↗
PubMed2006

Spectral shift of fluorescent dye FM4-64 reveals distinct microenvironment of nuclear envelope in living cells.

We report a distinct microenvironment within the nuclear envelope (NE) in living cells revealed by a spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br). The dye readily translocated to the NE at physiological temperature where it exhibited enhanced fluorescence when excited at 620-650 nm in contrast to 480-520 nm excitation in the endocytic pathway and in the endoplasmic reticulum (ER). In vitro data indicated that the dye reveals an enrichment of negatively charged lipids, presumably due to local phospholipid synthesis. Dual-excitation imaging of FM4-64 in relation to lamina-associated polypeptide-1-green fluorescent protein during mitosis suggested that the disassembly of NE preserves microscale lipid complexes in the ER. Convolutions of NE in cancer or primary cells were readily visualized, and killing of tumor cells by T cells was marked by sudden loss of the long-wavelength excited fluorescence in the NE coincident with apoptosis. This report of FM4-64 as the first vital dye sensitive to the NE environment opens new ways for real-time visualization and functional studies of the NE.

Cell Survival↗
PubMed1982

Similarities between rabbit hepatocyte membrane and nuclear envelope as regards the binding of homologous cytophilic IgG.

Nuclei isolated from rabbit liver are able to bind cytophilic monomeric and polymeric homologous IgG while non-cytophilic rabbit IgG are not bound by nuclei even after its aggregation. The present finding suggests that the rabbit hepatocyte nuclear envelope is similar to the liver cell membrane as regards the specific binding of monomeric cytophilic IgG via a selective binding site for the Fc fragment of IgG. Therefore, the Fc receptor is not restricted to the external membrane of the hepatocyte but exists also on the nuclear envelope.

Animals↗
PubMed1998

Papillary thyroid carcinoma oncogene (RET/PTC) alters the nuclear envelope and chromatin structure.

Current evidence suggests the papillary thyroid carcinoma oncogene (RET/PTC) generates papillary thyroid carcinomas in one genetic step. We tested a resulting prediction that RET/PTC expression in thyroid epithelium should be sufficient to cause the changes in nuclear morphology diagnostic of this tumor. Primary cultures of human thyroid epithelial cells were infected with a RET/PTC retroviral construct. Morphological scoring by two independent cytopathologists shows RET/PTC expression by immunohistochemistry to be highly associated (p << 0.0001) with an irregular nuclear contour and a euchromatic appearance compared with non-expressing cells in the same cultures. The altered nuclear morphology is not due to gene transfer or transformation per se as primary thyroid cell cultures infected with a retroviral H-RAS construct differ from RET/PTC-infected cells by showing round nuclear envelopes and coarser chromatin, as determined by the independent scoring of two cytopathologists (p << 0.0001). In addition, RET/ PTC-transfected cells appear to disperse, whereas RAS-transfected cells grow as discrete colonies. The results provide additional support for the hypothesis that RET/PTC is sufficient to cause papillary thyroid carcinomas. A signaling pathway downstream of RET/ PTC leads to restructuring of the nuclear envelope and chromatin, and the signal does not depend entirely, if at all, on a RAS pathway.

3T3 Cells↗
PubMed2002

Three proteins required for early steps in the protein secretory pathway also affect nuclear envelope structure and cell cycle progression in fission yeast.

The Ran GTPase is an essential protein that has multiple functions in eukaryotic cells. Fission yeast cells in which Ran is misregulated arrest after mitosis with condensed, unreplicated chromosomes and abnormal nuclear envelopes. The fission yeast sns mutants arrest with a similar cell cycle block and interact genetically with the Ran system. sns-A10, sns-B2 and sns-B9 have mutations in the fission yeast homologues of S. cerevisiae Sar1p, Sec31p and Sec53p, respectively, which are required for the early steps of the protein secretory pathway. The three sns mutants accumulate a normally secreted protein in the endoplasmic reticulum (ER), have an increased amount of ER membrane, and the ER/nuclear envelope lumen is dilated. Neither a post-ER block in the secretory pathway, nor ER proliferation caused by overexpression of an integral ER membrane protein, results in a cell cycle-specific defect. Therefore, the arrest seen in sns-A10, sns-B2 and sns-B9 is most likely due to nuclear envelope defects that render the cells unable to re-establish the interphase organization of the nucleus after mitosis. As a consequence, these mutants are unable to decondense their chromosomes or to initiate of the next round of DNA replication.

Active Transport, Cell Nucleus↗
PubMed2002

Plant RanGAPs are localized at the nuclear envelope in interphase and associated with microtubules in mitotic cells.

In animals and yeast, the small GTP-binding protein Ran has multiple functions - it is involved in mediating (i) the directional passage of proteins and RNA through the nuclear pores in interphase cells; and (ii) the formation of spindle asters, the polymerization of microtubules, and the re-assembly of the nuclear envelope in mitotic cells. Nucleotide binding of Ran is modulated by a series of accessory proteins. For instance, the hydrolysis of RanGTP requires stimulation by the RanGTPase protein RanGAP. Here we report the complementation of the yeast RanGAP mutant rna1 with Medicago sativa and Arabidopsis thaliana cDNAs encoding RanGAP-like proteins. Confocal laser microscopy of Arabidopsis plants overexpressing chimeric constructs of GFP with AtRanGAP1 and 2 demonstrated that the fusion protein is localized to patchy areas at the nuclear envelope of interphase cells. In contrast, the cellular distribution of RanGAPs in synchronized tobacco cells undergoing mitosis is characteristically different. Double-immunofluorescence shows that RanGAPs are co-localized with spindle microtubules during anaphase, with the microtubular phragmoplast and the surface of the daughter nuclei during telophase. Co-assembly of RanGAPs with tubulin correlates with these in vivo observations. The detected localization pattern is consistent with the postulated function of plant RanGAPs in the regulation of nuclear transport during interphase, and suggests a role for these proteins in the organization of the microtubular mitotic structures.

Active Transport, Cell Nucleus↗
PubMed1985

Involvement of Golgi apparatus and a restructured nuclear envelope during biogenesis and transport of herpes simplex virus glycoproteins.

Following infection of BHK-21 cells with Herpes simplex virus type 1 (HSV-1), progeny nucleocapsids in the nucleus acquire a glycoprotein-rich envelope by budding through host-cell nuclear membranes. To investigate the nature of the glycoprotein products assembled in the virion at the nuclear envelope, infected cells were pulse-labeled with [3H]-mannose, an oligosaccharidal core sugar, or [3H]-fucose, a terminal sugar. After various chase periods, the incorporation of these sugars was monitored by electron microscope radioautography. The results show that HSV glycoproteins accumulate very rapidly in nuclear membranes, where they exist only as core-glycosylated precursors, i.e., containing [3H]-mannose but not [3H]-fucose. [3H]-fucose grains are seen mainly over Golgi membranes and over virions located in the Golgi and in other cytoplasmic vesicular structures. Our data support a model where addition of terminal sugars (e.g., fucose) to HSV-1 glycoprotein precursors can occur at the surface of newly enveloped viral particles as the virions themselves egress from the cell via the Golgi apparatus.

Animals↗
PubMed1982

Origin and maturation of centrioles in association with the nuclear envelope in hypertonic-stressed sea urchin eggs.

Unfertilized sea urchin eggs were parthenogenetically activated via a prolonged hypertonic treatment. The continuous subjection to the osmotic stress turned on the cell cycle and brought about the very slow development of mature centrioles. Within 1 h of the exposure, large osmiophilic aggregates were detected at the nuclear surface and were interpreted as early centriolar precursor forms. Ultrastructural examination of eggs during the 8 h of treatment revealed that the precursor forms systematically converted into mature centrioles which then produced daughter centrioles. All centriolar precursor bodies were associated with microtubules; cartwheel structures were the earliest morphological feature of centrioles detected within the osmiophilic bodies. Nascent centrioles appeared at the nuclear surface and, during their development into centrioles, continually maintained intimate associations with the nuclear envelope. We suggest that the resultant modification of the intracellular environment via the hypertonic stress promotes the activation of centriolar 'information-bearing-residues' or 'seeds' located at the nuclear envelope. The oocyte centrioles, before disappearing from the maturing egg, may have produced these 'seeds' and left them at the nuclear surface.

Animals↗
PubMed1990

The permeability of the nuclear envelope in dividing and nondividing cell cultures.

The objective of this study was to determine whether the permeability characteristics of the nuclear envelope vary during different phases of cellular activity. Both passive diffusion and signal-mediated transport across the envelope were analyzed during the HeLa cell cycle, and also in dividing, confluent (growth-arrested), and differentiated 3T3-L1 cultures. Colloidal gold stabilized with BSA was used to study diffusion, whereas transport was investigated using gold particles coated with nucleoplasmin, a karyophilic Xenopus oocyte protein. The gold tracers were microinjected into the cytoplasm, and subsequently localized within the cells by electron microscopy. The rates of diffusion in HeLa cells were greatest during the first and fifth hours after the onset of anaphase. These results correlate directly with the known rates of pore formation, suggesting that pores are more permeable during or just after reformation. Signal-mediated transport in HeLa cells occurs through channels that are located within the pore complexes and have functional diameters up to 230-250 A. Unlike diffusion, no significant differences in transport were observed during different phases of the cell cycle. A comparison of dividing and confluent 3T3-L1 cultures revealed highly significant differences in the transport of nucleoplasmin-gold across the envelope. The nuclei of dividing cells not only incorporated larger particles (230 A versus 190 A in diameter, including the protein coat), but the relative uptake of the tracer was about seven times greater than that in growth-arrested cells. Differentiation of confluent cells to adipocytes was accompanied by an increase in the maximum diameter of the transport channel to approximately 230 A.

Animals↗
PubMed1992

Distribution of a nuclear envelope antigen during the syncytial mitoses of the early Drosophila embryo as revealed by laser scanning confocal microscopy.

The changing distribution of a nuclear envelope antigen recognized by a monoclonal antibody raised against human fibroblast vimentin during the syncytial mitoses of the Drosophila embryo has been studied with a confocal laser scanning microscope. The antigen appears very early as irregular aggregates in the peripheral cytoplasm of the preblastoderm embryo. As the first nuclei reach the periplasm the antigen is localized on the nuclear envelope and the cytoplasmic staining decreases. In addition to the perinuclear labeling we observed intense midzone and polar staining during the mitotic cycle. A possible relationship between polar localization of the antigen and centrosome position is discussed.

Animals↗
PubMed2005

Glucocorticoids remodel nuclear envelope structure and permeability.

The present study describes glucocorticoid induced remodelling of nuclear envelope (NE) structure and permeability. A glucocorticoid analogue, triamcinolone acetonide (TA), is injected into Xenopus laevis oocytes that express an exogeneous glucocorticoid receptor (GR). Electrical, fluorescence and nano-imaging techniques are applied to study the permeability and the structure of the NE at 5 and 60 minutes after injection of TA. A remarkable dilation of nuclear pore complexes (NPCs), a rearrangement of NPC distribution and a significant increase of NE permeability for ions and fluorescent 20 kDa dextran are observed within 5 minutes of TA exposure. At regular distances on local NE patches, NPCs seem to adjoin forming clusters each consisting of several hundred NPCs. Interestingly, at the same time of exposure, hydrophobicity of NPC central channels and NPC-free NE surface increases. The changes in permeability and structure are transient as the NE permeability returns to its initial state within 60 minutes. In conclusion, the NE is a barrier of high plasticity sensitive to hydrophobic molecules. Remodelling of NE structure and permeability is a prerequisite for mediating physiological actions of glucocorticoids.

Animals↗
PubMed1975

Nuclear envelope permeability.

The permeability of the amphibian oocyte nuclear envelope in situ has been determined for three tritiated dextrans. The envelope is a sieve, restricting molecular movement between the cytoplasm and nucleus. The patent radius of its pores is about 45å.

Amphibians↗
PubMed1985

Monoclonal antibodies prepared against the major Drosophila nuclear Matrix-pore complex-lamina glycoprotein bind specifically to the nuclear envelope in situ.

A high molecular weight glycoprotein found associated with a nuclear matrix-pore complex-lamina (NMPCL) preparation obtained from Drosophila melanogaster embryos has been shown by in vitro analyses to be largely confined to this subcellular fraction. In contrast with several of the NMPCL proteins, this glycoprotein remains completely insoluble after treatment with 5 M urea. It has, therefore, been possible to separate the glycoprotein from other NMPCL components by differential urea extraction. The glycoprotein in the 5 M urea-extracted pellet has been solubilized by boiling in sodium dodecyl sulfate and purified to near-homogeneity by sequential steps of chromatography on hydroxylapatite and Sephacryl S-300 (both run in the presence of 0.1% sodium dodecyl sulfate), followed by affinity chromatography on lentil lectin-Sepharose. Over 30 hybridoma cell lines producing antibodies against this glycoprotein have been obtained. Monoclonality has been established for two of these lines (designated AGP-26 and AGP-78), and the antibodies they secrete have been further characterized. Western blot analysis has shown both antibodies to be monospecific (with respect to other Drosophila embryo polypeptides) for the major NMPCL glycoprotein; in addition, antibody AGP-78 has been shown to be weakly cross-reactive with glycoproteins of similar or identical molecular weight found associated with isolated nuclear fractions obtained from Xenopus oocytes, as well as chicken, opossum, and rat livers. Finally, both antibodies AGP-26 and AGP-78 react exclusively with the Drosophila nuclear periphery (nuclear envelope) in situ as demonstrated by indirect immunofluorescence analysis of larval cryosections. Based on these results as well as upon those of biochemical studies reported previously (Berrios, M., Filson, A. J., Blobel, G, and Fisher, P. A. (1983) J. Biol. Chem. 258, 13384-13390), we conclude that the major Drosophila NMPCL glycoprotein is the specific homolog of the high molecular weight glycoprotein recently shown using immunoelectron microscopy to be a distinct component of the rat liver nuclear pore complex (Gerace, L., Ottaviano, Y., and Kondor-Koch, C. (1982) J. Cell Biol. 95, 826-837).

Animals↗