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PubMed1984

The major 67 000 molecular weight protein of the clam oocyte nuclear envelope is lamin-like.

Nuclear envelopes of somatic cells have at least two different major proteins in the 60-70(X 10(3] molecular weight range (lamins A(C) and B) that seem to be involved in chromatin attachment. In contrast, nuclear envelopes from clam germinal vesicles have only a single major protein of the same size class (approximately 67 X 10(3) Mr) and have no chromatin attached to them. The data presented in this report show that this 67 X 10(3) Mr clam protein shares a variety of physical properties with lamins A(C) and B, derived from rat liver nuclei. These properties include similar size, although different isoelectric points; phosphorylated forms; strong tendencies to cross-link by disulphide bonds; presence of carbohydrates, demonstrated by direct incorporation of mannose and labelling with borohydride; and shared epitopes, demonstrated using both monoclonal and polyclonal antibodies. Taken together, these observations identify the clam 67 X 10(3) Mr protein, the major structural protein of a nuclear envelope that lacks attached chromatin, as being lamin-like and demonstrate that it is more closely related to lamin A(C) than to lamin B.

Animals↗
PubMed1992

Regulation of DNA replication by the nuclear envelope.

Recent evidence suggests that the nuclear envelope is directly involved in regulating DNA replication. It does this in at least three ways. First, replication is dependent on assembly of an intact nuclear envelope capable of nuclear transport. Second, the nuclear membrane defines the nucleus as the fundamental unit of replication and determines the timing of initiation. Third, the nuclear membrane is essential for coupling DNA replication to the cell cycle. Thus, regulated DNA replication in eukaryotic cells depends on a structurally intact and functional nuclear envelope.

Animals↗
PubMed1989

Identification of specific polypeptides of the nuclear envelope by iodination of mouse liver nuclei.

A sensitive technique is described for the rapid identification of nuclear-envelope proteins. Mouse liver nuclei (purified on sucrose gradients) were iodinated with Na125I by the immobilized water-insoluble reagent Iodogen. Iodinated nuclei were digested with RNAase A and DNAase I and then salt-extracted to obtain labelled nuclear envelopes. Nuclear envelopes were characterized by morphological and biochemical criteria and by SDS/polyacrylamide-gel electrophoresis. In all, 13 polypeptides of molecular masses 145, 115, 98, 85, 75, 70, 65, 54, 50, 45, 40, 38 and 36 kDa were identified in the labelled nuclear envelopes. The labelled polypeptides were localized to the nuclear envelope by extraction of the envelope with Triton X-100 and different concentrations of salt. Iodination of intact nuclei was shown to be specific for the nuclear envelope by the absence of labelling of histones and cytoplasmic contaminants.

Animals↗
PubMed2006

Nuclear envelope barrier leak induced by dexamethasone.

Nuclear pore complexes (NPCs) are multiprotein channels that span the nuclear envelope. They strongly limit the efficiency of gene transfection by restriction of nuclear delivery of exogenously applied therapeutic macromolecules. NPC dilation could significantly increase this efficiency. Recently, it was shown in oocytes of Xenopus laevis that NPCs dilate from about 82 to 110 nm within min after injection of the glucocorticoid analog dexamethasone (dex). In the present paper we analyzed by means of atomic force microscopy the structural details of NPC dilation and correlated them with functional changes in nuclear envelope permeability. 5-11 min after Dex injection NPC dilation was found at its maximum (approximately 140 nm). In addition, a yet unknown configuration, so-called giant pore, up to 300 nm in diameter, was visualized. Giant pore formation was paralleled by an increase in nuclear envelope permeability tested by electrophysiology and confocal fluorescence microscopy. Even large macromolecules lacking any nuclear localization signal (77 kDa FITC-dextran, molecule diameter up to 36 nm) could gain access to the nucleus. We conclude that dex transiently opens unspecific pathways for large macromolecules. Dex treatment could be potentially useful for improving the efficiency of nuclear gene transfection.

Animals↗
PubMed2006

The karyopherin Kap95 regulates nuclear pore complex assembly into intact nuclear envelopes in vivo.

Nuclear pore complex (NPC) assembly in interphase cells requires that new NPCs insert into an intact nuclear envelope (NE). Our previous work identified the Ran GTPase as an essential component in this process. We proposed that Ran is required for targeting assembly factors to the cytoplasmic NE face via a novel, vesicular intermediate. Although the molecular target was not identified, Ran is known to function by modulating protein interactions for karyopherin (Kap) beta family members. Here we characterize loss-of-function Saccharomyces cerevisiae mutants in KAP95 with blocks in NPC assembly. Similar to defects in Ran cycle mutants, nuclear pore proteins are no longer localized properly to the NE in kap95 mutants. Also like Ran cycle mutants, the kap95-E126K mutant displayed enhanced lethality with nic96 and nup170 mutants. Thus, Kap95 and Ran are likely functioning at the same stage in assembly. However, although Ran cycle mutants accumulate small cytoplasmic vesicles, cells depleted of Kap95 accumulated long stretches of cytoplasmic membranes and had highly distorted NEs. We conclude that Kap95 serves as a key regulator of NPC assembly into intact NEs. Furthermore, both Kap95 and Ran may provide spatial cues necessary for targeting of vesicular intermediates in de novo NPC assembly.

Alleles↗
PubMed1987

Kinetochores on chromosomes enclosed within the nuclear envelope.

The kinetochore plate which develops after nuclear envelope breakdown in normal cells can be seen to be formed on condensed chromosomes still enclosed in the nuclear envelope in fused multinucleate cells where some nuclei show delayed envelope breakdown caused by nuclear interaction. This suggests that neither nuclear envelope breakdown nor assembly of microtubules is directly related to the formation of the kinetochore plate. Furthermore, it can be clearly observed in these cells that the kinetochores do not have any special association with the nuclear envelope.

Chromosomes, Human↗
PubMed1995

Structural and functional organization of the nuclear envelope.

The double-membrane nuclear envelope is punctuated by pores where the two membranes are joined. These pores are stabilized by the elaborate nuclear pore complex, which is anchored to the inner membrane by the nuclear lamina, as well as to other nuclear and cytoskeletal structures. Recent experiments have identified proteins involved in the stability of this organization as well as in the function of the nuclear pore complex, which we relate here to newer aspects of nuclear envelope structure.

Animals↗
PubMed1989

The CaaX motif of lamin A functions in conjunction with the nuclear localization signal to target assembly to the nuclear envelope.

While the nuclear lamin proteins (A, B, and C) assemble specifically at the surface of the nuclear membrane, their sequences do not reveal stretches of hydrophobic amino acids that might explain their association with the nuclear membranes. However, the A and B lamin proteins possess Ras-like C-terminal CaaX sequence motifs, which in Ras proteins are sites of hydrophobic modifications required for membrane association and function. From the analysis of single and double lamin A mutants affecting the CaaX motif, the nuclear localization signal, and higher-order assembly properties, we propose that the CaaX motif functions as a nonspecific, low affinity membrane probe for proteins ultimately segregated to specific cellular membrane systems. Committed association with specific membranes requires additional interactions with membrane-resident factors.

Amino Acid Sequence↗
PubMed2006

The inner nuclear membrane protein Lem2 is critical for normal nuclear envelope morphology.

The inner nuclear membrane (INM) of eukaryotic cells is characterized by a unique set of transmembrane proteins which interact with chromatin and/or the nuclear lamina. The number of identified INM proteins is steadily increasing, mainly as a result of proteomic and computational approaches. However, despite a link between mutation of several of these proteins and disease, the function of most transmembrane proteins of the INM remains unknown and depletion of many of these proteins from a variety of systems did not produce an obvious phenotype in the affected cells. Here, we report that depletion of the conserved INM protein Lem2 from human cell lines leads to abnormally shaped nuclei and severely reduces cell survival. We suggest that interactions of Lem2 with lamins or chromatin are critical for maintaining the integrity of the nuclear envelope.

Cell Survival↗
PubMed1988

A histone 1-like antigen is a component of the nuclear envelope.

Rabbit antibodies have been raised against rat liver nuclear envelopes. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titer antiserum specific for the nuclear envelope preparation. Immunocytochemical studies showed that the antiserum stained the nuclear envelopes, but not intra-nuclear components of HEp-2 (human malignant epithelial) cells. When electrophoretically separated peptides were tested by immunoblotting techniques, the rabbit antiserum specifically stained proteins with molecular masses of 26 and 28 kD. These peptides had similar mobilities to purified histone 1 (H1). Indeed purified calf thymus H1 recognized the antiserum. The antigens are not loosely bound to the nuclear envelope, as they could not be extracted with low salt. Therefore, we have established that the 26 and 28 kD nuclear envelope peptides are not contaminants of the nuclear envelope preparation and that they express determinants that are immunologically cross-reactive with purified H1, but not with intra-nuclear H1.

Animals↗
PubMed2005

The nuclear-envelope protein and transcriptional repressor LAP2beta interacts with HDAC3 at the nuclear periphery, and induces histone H4 deacetylation.

Nuclear-envelope proteins have been implicated in diverse and fundamental cell functions, among them transcriptional regulation. Gene expression at the territory of the nuclear periphery is known to be repressed by epigenetic modifications such as histone deacetylation and methylation. However, the mechanism by which nuclear-envelope proteins are involved in such modifications is still obscure. We have previously shown that LAP2beta, an integral nuclear-envelope protein that contains the chromatin-binding LEM domain, was able to repress the transcriptional activity of the E2F5-DP3 heterodimer. Here, we show that LAP2beta's repressive activity is more general, encompassing various E2F members as well as other transcription factors such as p53 and NF-kappaB. We further show that LAP2beta interacts at the nuclear envelope with HDAC3, a class-I histone deacetylase, and that TSA (an HDAC inhibitor) abrogates LAP2beta's repressive activity. Finally, we show that LAP2beta is capable of inducing histone-H4 deacetylation. Our data provide evidence for the existence of a previously unknown repressive complex, composed of an integral nuclear membrane protein and a histone modifier, at the nuclear periphery.

Cell Line, Tumor↗
PubMed1989

The conserved carboxy-terminal cysteine of nuclear lamins is essential for lamin association with the nuclear envelope.

We have analyzed the interaction of soluble nuclear lamins with the nuclear envelope by microinjection of normal and mutated lamins into the cytoplasm of Xenopus laevis oocytes. Our results demonstrate that the conserved cysteine of the carboxy-terminal tetrapeptide Cys Ala/Ser Ile Met of lamins is essential for their association with the nuclear envelope. Removal of this sequence or replacement of the cysteine by serine resulted in Xenopus lamin L1 remaining in a soluble, non-envelope-associated state within the nucleus. Similar mutations of Xenopus lamin A resulted in only partial reduction of nuclear envelope association, indicating that lamin A contains additional signals that can partially compensate for the lack of the cysteine. Mammalian lamin C lacks this tetrapeptide and is not associated with the nuclear envelope in our experimental system. Cloning of the tetrapeptide Cys Ala Ile Met to the carboxy terminus of human lamin C resulted in lamin being found in a nuclear envelope-associated form in oocytes. Mutations at the amino terminus and in the alpha-helical region of lamin L1 revealed that the carboxy terminus mediates the association of lamins with the nuclear envelope; however, this alone is insufficient for maintenance of a stable association with the nuclear envelope.

Amino Acid Sequence↗
PubMed1993

Influence of recipient oocyte cell cycle stage on DNA synthesis, nuclear envelope breakdown, chromosome constitution, and development in nuclear transplant bovine embryos.

Nuclear transplantations into metaphase II (MII) and S phase oocyte cytoplasm were performed to investigate the influence of recipient cell cycle stage on nuclear function and development of bovine nuclear transplant (NT) embryos. Rate of inactivation of histone H1 kinase and duration of DNA synthesis in activated oocytes were determined. The proportion of S phase blastomeres in in vivo produced day 5.5 bovine embryos was measured. DNA synthesis was also assessed in NT embryos after transfer into MII and S phase cytoplasm. MII NT embryos were produced by fusing a blastomere into a MII oocyte; the fusion pulse served to activate the oocyte. S NT embryos were produced by fusing a blastomere into an early S phase oocyte electrically activated 4 h prior to fusion. Nuclear envelope structure, chromosome constitution, and extent of development were examined in MII and S NT embryos. Histone H1 kinase activity dropped to baseline within 2 h of electrical activation. A second electrical pulse did not alter H1 kinase activity when delivered 4 h after the first pulse. The frequency of S phase blastomeres in day 5.5 bovine embryos ranged from 79% to 100%, depending on the duration of culture in 3H-thymidine. Nuclear transplantation into MII cytoplasm resulted in a transient drop in DNA synthesis over 3.5 h. DNA synthesis resumed at 4.5 h post activation (hpa), concomittantly with initiation of DNA replication in activated oocytes. In contrast, DNA synthesis was not interrupted after transfer into S phase cytoplasm. DNA synthesis persisted until 13.5 hpa, as in activated oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Sequential PKC- and Cdc2-mediated phosphorylation events elicit zebrafish nuclear envelope disassembly.

Molecular markers of the zebrafish inner nuclear membrane (NEP55) and nuclear lamina (L68) were identified, partially characterized and used to demonstrate that disassembly of the zebrafish nuclear envelope requires sequential phosphorylation events by first PKC, then Cdc2 kinase. NEP55 and L68 are immunologically and functionally related to human LAP2beta and lamin B, respectively. Exposure of zebrafish nuclei to meiotic cytosol elicits rapid phosphorylation of NEP55 and L68, and disassembly of both proteins. L68 phosphorylation is completely inhibited by simultaneous inhibition of Cdc2 and PKC and only partially blocked by inhibition of either kinase. NEP55 phosphorylation is completely prevented by inhibition or immunodepletion of cytosolic Cdc2. Inhibition of cAMP-dependent kinase, MEK or CaM kinase II does not affect NEP55 or L68 phosphorylation. In vitro, nuclear envelope disassembly requires phosphorylation of NEP55 and L68 by both mammalian PKC and Cdc2. Inhibition of either kinase is sufficient to abolish NE disassembly. Furthermore, novel two-step phosphorylation assays in cytosol and in vitro indicate that PKC-mediated phosphorylation of L68 prior to Cdc2-mediated phosphorylation of L68 and NEP55 is essential to elicit nuclear envelope breakdown. Phosphorylation elicited by Cdc2 prior to PKC prevents nuclear envelope disassembly even though NEP55 is phosphorylated. The results indicate that sequential phosphorylation events elicited by PKC, followed by Cdc2, are required for zebrafish nuclear disassembly. They also argue that phosphorylation of inner nuclear membrane integral proteins is not sufficient to promote nuclear envelope breakdown, and suggest a multiple-level regulation of disassembly of nuclear envelope components during meiosis and at mitosis.

Animals↗
PubMedDate not supplied

Transmission electron microscope studies of the nuclear envelope in Caenorhabditis elegans embryos.

Nuclear membranes and nuclear pore complexes (NPCs) are conserved in both animals and plants. However, the lamina composition and the dimensions of NPCs vary between plants, yeast, and vertebrates. In this study, we established a protocol that preserves the structure of Caenorhabditis elegans embryonic cells for high-resolution studies with thin-section transmission electron microscopy (TEM). We show that the NPCs are bigger in C. elegans embryos than in yeast, with dimensions similar to those in higher eukaryotes. We also localized the C. elegans nuclear envelope proteins Ce-lamin and Ce-emerin by pre-embedding gold labeling immunoelectron microscopy. Both proteins are present at or near the inner nuclear membrane. A fraction of Ce-lamin, but not Ce-emerin, is present in the nuclear interior. Removing the nuclear membranes leaves both Ce-lamin and Ce-emerin associated with the chromatin. Eliminating the single lamin protein caused cell death as visualized by characteristic changes in nuclear architecture including condensation of chromatin, clustering of NPCs, membrane blebbing, and the presence of vesicles inside the nucleus. Taken together, these results show evolutionarily conserved protein localization, interactions, and functions of the C. elegans nuclear envelope.

Animals↗
PubMed1999

A visual screen of a GFP-fusion library identifies a new type of nuclear envelope membrane protein.

The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism.

Amino Acid Sequence↗
PubMed2000

Yeast nucleoporins involved in passive nuclear envelope permeability.

The vertebrate nuclear pore complex (NPC) harbors an approximately 10-nm diameter diffusion channel that is large enough to admit 50-kD polypeptides. We have analyzed the permeability properties of the Saccharomyces cerevisiae nuclear envelope (NE) using import (NLS) and export (NES) signal-containing green fluorescent protein (GFP) reporters. Compared with wild-type, passive export rates of a classical karyopherin/importin (Kap) Kap60p/Kap95p-targeted NLS-GFP reporter (cNLS-GFP) were significantly faster in nup188-Delta and nup170-Delta cells. Similar results were obtained using two other NLS-GFP reporters, containing either the Kap104p-targeted Nab2p NLS (rgNLS) or the Kap121p-targeted Pho4p NLS (pNLS). Elevated levels of Hsp70 stimulated cNLS-GFP import, but had no effect on the import of rgNLS-GFP. Thus, the role of Hsp70 in NLS-directed import may be NLS- or targeting pathway-specific. Equilibrium sieving limits for the diffusion channel were assessed in vivo using NES-GFP reporters of 36-126 kD and were found to be greater than wild-type in nup188-Delta and nup170-Delta cells. We propose that Nup170p and Nup188p are involved in establishing the functional resting diameter of the NPC's central transport channel.

Biological Transport↗
PubMed1997

Nuclear envelope breakdown in mammalian cells involves stepwise lamina disassembly and microtubule-drive deformation of the nuclear membrane.

We have studied nuclear envelope disassembly in mammalian cells by morphological methods. The first signs of nuclear lamina depolymerization become evident in early prophase as A-type lamins start dissociating from the nuclear lamina and diffuse into the nucleoplasm. While B-type lamins are still associated with the inner nuclear membrane, two symmetrical indentations develop on antidiametric sites of the nuclear envelope. These indentations accommodate the sister centrosomes and associated astral microtubules. At mid- to late prophase, elongating microtubules apparently push on the nuclear surface and eventually penetrate the nucleus. At this point the nuclear envelope becomes freely permeable to large ligands, as indicated by experiments with digitonin-treated cells and by the massive release of solubilized A-type lamins into the cytoplasm. At the prophase/prometaphase transition, the B-type lamina is fragmented, but 'islands' of lamin B polymer can still be discerned on the tips of congressing chromosomes. Finally, at metaphase, the lamin B polymer breaks down into small pieces, which tend to concentrate in the area of the mitotic spindle. Nuclear envelope breakdown is not prevented when the microtubules are depolymerized by nocodazole; however, the mode of nuclear lamina fragmentation in the absence of microtubules is markedly different from the normal one and involves multiple raffles and gaps, which develop rapidly along the entire surface of the nuclear envelope. These data suggest that nuclear envelope disassembly is a stepwise process in which the microtubules play an important part.

Adenocarcinoma↗