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PubMed1983

Nuclear envelope invaginations in hamster facial motor neurons during development and aging.

Morphometric measurements of nuclear envelope invaginations (NEI) in hamster facial motor neurons were made. These showed that the amount of NEI increased markedly between fetal life and birth to a high level that was maintained during the period of accelerated neuronal somal growth. Subsequently, the amount of NEI decreased to a plateau of low incidence that persisted through maturity and aging. This sequence of NEI formation and subsidence differs from that previously observed in a similar developing and aging series of pyramidal neurons in the same species. This may indicate, as discussed, that the presence of NEI reflects stage-specific functions that may differ depending on neuronal type.

Aging↗
PubMed1997

Calcium-induced restructuring of nuclear envelope and endoplasmic reticulum calcium stores.

The spatial organization of endoplasmic reticulum (ER) and nuclear envelope (NE) calcium stores is important for the regulation of localized calcium signals and sustained calcium gradients. Here, we have used a lumenal GFP fusion protein and shown that, in resting cells, large molecules can rapidly diffuse across the cell within the lumenal storage space defined by the ER and NE membranes. Increases in cytosolic calcium concentration reversibly fragmented ER tubules and prevented lumenal diffusion. However, the integrity of the NE was maintained, and a significant fraction of NE lumenal protein accumulated in an NE-associated vesicle. These dynamic properties of ER-NE calcium stores provide insights into the spatiotemporal control of calcium signaling.

3T3 Cells↗
PubMed2002

Organellar proteomics: the prizes and pitfalls of opening the nuclear envelope.

Proteomic studies have the potential to comprehensively define the composition of organelles but are limited by the organellar cross-contamination that arises during subcellular fractionation. Comparative proteomics of organellar subfractions can mitigate these problems, as demonstrated by a recent study involving the nuclear envelope.

Animals↗
PubMed2000

Nuclear envelope associated protein that binds telomeric DNAs.

Rana temporaria oocytes at the 6th diplotene stage of maturation contain a special structure, the karyosphere capsule, with chromosomes covered and detached from the nuclear envelope (NE), though at the previous stage the telomeres were attached to the membrane, as characteristic of germ cells. The DNA-protein complexes from band shift assays with proteins extracted from oocyte NEs and telomeric DNA fragment (T(2)G(4))(130) were isolated and injected into a guinea pig. In the present paper the only protein of 70 kDa recognized by antibody (AB) in the NE is named the Membrane Telomere Binding Protein (MTBP). Western blots with guinea pig AB and AB against telobox peptide from TRF2 show that protein of 60 kDa (probably TRF1) belongs to the chromatin, but MTBP (TRF2 according to immunoprecipitation) belongs to the NE. In the somatic cell nuclei both proteins are present and recognized by AB against telobox peptide, but AB raised recognize only MTBP/TRF2 due to the epitope different from telobox. Combined in situ hybridization with the vertebrate telomeric DNA sequences (T(2)AG(3))(135) and immunocytochemistry with the MTBP AB showed them to be colocalized within the mouse nucleus. As it was shown by immunofluorescense of NE spread, MTBP is organized in a distinct pattern that looks like a network made of double-dots. Electron microscope immunogold staining with both ABs showed that the protein is localized on the outer surface of the oocyte NE within cup-like structures attached to the membrane. This is the first clear evidence of a protein, which could be responsible for the attachment of telomeres to the nuclear membrane.

Animals↗
PubMed2001

Nuclear envelope and chromatin compositional differences comparing undifferentiated and retinoic acid- and phorbol ester-treated HL-60 cells.

The human leukemic cell line (HL-60) can be induced to differentiate in vitro to granulocytic form with retinoic acid (RA), or to monocytic/macrophage form with phorbol ester (TPA). The granulocytic form acquires nuclear lobulation, nuclear envelope-limited chromatin sheets (ELCS), and cytoskeletal polarization, none of which are acquired following treatment with TPA. Immunoblotting analyses and capillary zone electrophoresis demonstrated that following RA treatment: lamins A/C and B1, and vimentin decreased to negligible amounts; LAP2 beta, lamin B2 and emerin remained essentially unchanged; lamin B receptor (LBR) increased markedly; histone subtypes H1.4 and 1.5 exhibited dephosphorylation. Following TPA treatment: lamins A/C and B1, B2 and vimentin increased in amount; LAP2 beta and emerin remained essentially unchanged; LBR increased markedly; histone subtypes H1.4 and 1.5 exhibited dephosphorylation. Emerin, which was cytoplasmic in undifferentiated or granulocytic cells, localized into the nuclear envelope following TPA. Normal human granulocytes revealed compositional differences compared to granulocytic forms of HL-60, namely increased vimentin and appearance of histone subtype H1.3. A working hypothesis for nuclear lobulation postulates a combination of: increased nuclear envelope deformability due to lamins A/C and B1 deficiency; an increase in nuclear surface area/volume; an increase in chromatin-nuclear envelope interactions.

Cell Compartmentation↗
PubMed2006

Identification of regions of leukotriene C4 synthase which direct the enzyme to its nuclear envelope localization.

Leukotrienes (LTs) are fatty acid derivatives formed by oxygenation of arachidonic acid via the 5-lipoxygenase (5-LO) pathway. Upon activation of inflammatory cells 5-LO is translocated to the nuclear envelope (NE) where it converts arachidonic acid to the unstable epoxide LTA4. LTA4 is further converted to LTC4 by conjugation with glutathione, a reaction catalyzed by the integral membrane protein LTC4 synthase (LTC4S), which is localized on the NE and endoplasmic reticulum (ER). We now report the mapping of regions of LTC4S that are important for its subcellular localization. Multiple constructs encoding fusion proteins of green fluorescent protein (GFP) as the N-terminal part and various truncated variants of human LTC4S as C-terminal part were prepared and transfected into HEK 293/T or COS-7 cells. Constructs encoding hydrophobic region 1 of LTC4S (amino acids 6-27) did not give distinct membrane localized fluorescence. In contrast hydrophobic region 2 (amino acids 60-89) gave a localization pattern similar to that of full length LTC4S. Hydrophobic region 3 (amino acids 114-135) directed GFP to a localization indistinguishable from that of full length LTC4S. A minimal directing sequence, amino acids 117-132, was identified by further truncation. The involvement of the hydrophobic regions in the homo-oligomerization of LTC4S was investigated using bioluminescence resonance energy transfer (BRET) analysis in living cells. BRET data showed that hydrophobic regions 1 and 3 each allowed oligomerization to occur. These regions most likely form transmembrane helices, suggesting that homo-oligomerization of LTC4S is due to helix-helix interactions in the membrane.

Amino Acid Sequence↗
PubMed1989

Disassembly of the nuclear envelope of spisula oocytes in a cell-free system.

Nuclei isolated from oocytes of the surf clam Spisula solidissima are disassembled when exposed to extracts from maturing oocytes. In the course of this process the nuclear lamina undergoes a marked reduction in size and the nuclear membrane appears to be fragmented into vesicles. These events are accompanied by extensive phosphorylation of the oocyte 67-kDa lamin and its solubilization. The changes observed are similar to those which occur in vivo in activated Spisula oocytes. Nuclear envelope breakdown in vitro requires ATP and Mg2+, but not Ca2+. It is not affected by protease inhibitors and is inhibited by alkaline phosphatase.

Adenosine Triphosphate↗
PubMed1990

The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains.

The lamin B receptor is a previously identified integral membrane protein in the nuclear envelope of turkey erythrocytes that associates with the nuclear intermediate filament protein lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). In the present report, we use cell fractionation and antibodies against the lamin B receptor to localize it to an 8-M urea-extracted membrane fraction of chicken liver nuclei, supporting an inner nuclear membrane localization. We deduced the amino acid sequence of the chicken lamin B receptor from overlapping clones obtained by screening cDNA libraries with a probe generated by the polymerase chain reaction with primers based on the partial protein sequence of the isolated protein. The mature lamin B receptor has a calculated molecular mass of 73,375 D and eight segments of hydrophobic amino acids that could function as transmembrane domains as determined by hydropathy analysis. Preceding the first putative transmembrane segment is a highly charged 204-residue-long amino terminal region that contains two consensus sites for phosphorylation by protein kinase A. Since the lamin B receptor has been shown to be phosphorylated by protein kinase A in vitro and in vivo and this phosphorylation affects lamin B binding (Applebaum, J., G. Blobel, and S. D. Georgatos. 1990. J. Biol. Chem. 265:4181-4185), it is likely that this amino terminal region faces the nucleoplasm. The amino terminal region also contains three DNA-binding motifs that are found in gene regulatory proteins and histones, suggesting that the lamin B receptor may additionally play a role in gene regulation and/or chromatin organization.

Amino Acid Sequence↗
PubMed2003

The actin-based motor myosin Va is a component of the acroplaxome, an acrosome-nuclear envelope junctional plate, and of manchette-associated vesicles.

Protein and vesicle cargos can be mobilized during spermiogenesis by intramanchette transport utilizing microtubule-based protein motors (kinesins and dyneins). However, actin-based unconventional myosin motors may also play a significant role in targeting vesicle cargos to subcellular compartments during sperm development. Here we report that myosin Va, an actin-based motor protein, is a component of the acroplaxome of rodent spermatids. The acroplaxome is an F-actin/keratin-containing scaffold plate with a marginal ring fastening the caudal recess of the developing acrosome to the nuclear envelope during spermatid nuclear shaping. In contrast to the acroplaxome, fluorescently labeled phalloidin does not produce an obvious F-actin signal in the manchette. However, immunogold electron microscopy detects moderate but specific beta-actin immunoreactivity along interconnected tube-like bundles of manchette microtubules. We also show that the membrane of vesicles co-fractionated with intact manchettes by sucrose gradient ultracentrifugation display immunogold-labeled myosin Va. Myosin Va vesicle localization is known to correlate with Rab proteins, monomeric GTPases of the Ras superfamily which recruit myosin Va/VIIa motor proteins through intermediate proteins. RT-PCR analysis demonstrates that transcripts for Rab27a and Rab27b and Slac2-c (a protein that links Rab27a/b to myosin Va/VIIa) are expressed in testis. These results indicate that two independent cytoskeletal tracks, F-actin in the acroplaxome and presumably in the manchette, and manchette microtubules, may facilitate short-range (from the Golgi to the acrosome) and long-range (from the manchette to the centrosome and axoneme) mobilization of appropriate cargos during spermiogenesis.

Acrosome↗
PubMed1985

A monoclonal antibody identifies a 215 000-dalton nuclear envelope protein restricted to certain cell types.

The monoclonal antibody, AGF2.3, was isolated from mice immunised with the human promyeloid cell line HL60. By immunofluorescence and immunoelectron microscopy the antibody was shown to bind to the nuclear envelope in uninduced HL60 cells. Immunofluorescent staining was reduced to very low levels in HL60 cells induced to mature to monocytes or neutrophils by addition of 12-0-tetradecanoylphorbol-13-acetate or dimethyl sulfoxide respectively. Blood neutrophils did not express the antigen. Weak immunofluorescent staining of cell nuclei was observed in peripheral blood lymphocytes and in sections of normal human kidney, tonsil and skin epithelium. The AGF2.3 antigen was strongly expressed on the nuclei of 21/21 haemopoietic cell lines and 21/25 permanent non-haemopoietic cell lines representing various cell types. In contrast, the antigen was not expressed by any of six primary (untransformed) cell cultures. These included fibroblasts, endothelial cells and keratinocytes. The antigen was expressed in the Q10 SV-40 transformed cell line derived from a non-expressing primary fibroblast culture. AGF2.3 antibody precipitated a protein with an apparent subunit molecular weight of approximately 215 kDa from Triton X-100 extracts of HL60 and HeLa cells labelled with 35S-methionine. This protein was not detectable in extracts of primary skin fibroblasts prepared in parallel. We conclude that AGF2.3 antibody recognises a previously undescribed protein associated with the nuclear envelope which is expressed at high levels in most transformed cell lines but which is weakly expressed or absent in normal tissues and primary cell cultures.

Animals↗
PubMed1996

Involvement of mitogen-activated protein kinase and translocation of cytosolic phospholipase A2 to the nuclear envelope in acetylcholine-induced prostacyclin synthesis in rabbit coronary endothelial cells.

We previously showed that acetylcholine (ACh) stimulates production of prostacyclin, measured as immunoreactive 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), in coronary endothelial cells (CEC) of rabbit heart by increasing influx of extracellular Ca2+ through a receptor-operated Ca2+ channel and by activating a pertussis toxin-insensitive G protein. The purposes of this study were to elucidate the type of phospholipase A2 (PLA2) involved in 6-keto-PGF1 alpha production and the mechanism(s) by which ACh activates PLA2 in cultured CEC. In CEC transiently transfected with cytosolic PLA2 but not secretory PLA2 antisense oligonucleotide, ACh failed to increase 6-keto-PGF1 alpha; this was prevented by cotransfection with cPLA2 sense oligonucleotide. ACh increased production of prostacyclin and increased protein kinase C (PKC) activity. The PKC inhibitor calphostin C attenuated the ACh-induced increase in PKC activity but not 6-keto-PGF1 alpha production. Phorbol-12-myristate-13-acetate and phorbol-12, 13-dibutyrate increased PKC activity but failed to alter 6-keto-PGF1 alpha production. ACh enhanced the activity of cPLA2 and p42 mitogen-activated protein kinase (MAPK) in cell lysate prepared from CEC. ACh also caused phosphorylation of p42 MAPK and cPLA2, which was inhibited by AG126 ([alpha-cyano-(3-hydroxy-4-nitro)cinnamonitrile]), a tyrosine kinase inhibitor known to decrease MAPK activity. In addition, ACh stimulated translocation of cPLA2 from cytosol to nuclear envelope; the translocation of cPLA2 was prevented by removal of extracellular calcium but not by AG126 treatment. Okadaic acid, a protein phosphatase inhibitor, increased cPLA2 activity in cell lysate prepared from CEC but did not alter basal 6-keto-PGF1 alpha production in intact CEC; however, ACh-induced 6-keto-PGF1 alpha was enhanced by okadaic acid. These data suggest that ACh stimulates prostacyclin synthesis by activation of cPLA2 in a PKC-independent mechanism and that both cPLA2 translocation to nuclear envelope and phosphorylation by MAPK are required for ACh-induced 6-keto-PGF1 alpha synthesis in CEC.

6-Ketoprostaglandin F1 alpha↗
PubMed2001

Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr.

Human immunodeficiency virus-1 (HIV-1) Vpr expression halts the proliferation of human cells at or near the G2 cell-cycle checkpoint. The transition from G2 to mitosis is normally controlled by changes in the state of phosphorylation and subcellular compartmentalization of key cell-cycle regulatory proteins. In studies of the intracellular trafficking of these regulators, we unexpectedly found that wild-type Vpr, but not Vpr mutants impaired for G2 arrest, induced transient, localized herniations in the nuclear envelope (NE). These herniations were associated with defects in the nuclear lamina. Intermittently, these herniations ruptured, resulting in the mixing of nuclear and cytoplasmic components. These Vpr-induced NE changes probably contribute to the observed cell-cycle arrest.

Active Transport, Cell Nucleus↗
PubMed2006

The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope.

The spindle pole body (SPB) is the sole site of microtubule nucleation in Saccharomyces cerevisiae; yet, details of its assembly are poorly understood. Integral membrane proteins including Mps2 anchor the soluble core SPB in the nuclear envelope. Adjacent to the core SPB is a membrane-associated SPB substructure known as the half-bridge, where SPB duplication and microtubule nucleation during G1 occurs. We found that the half-bridge component Mps3 is the budding yeast member of the SUN protein family (Sad1-UNC-84 homology) and provide evidence that it interacts with the Mps2 C terminus to tether the half-bridge to the core SPB. Mutants in the Mps3 SUN domain or Mps2 C terminus have SPB duplication and karyogamy defects that are consistent with the aberrant half-bridge structures we observe cytologically. The interaction between the Mps3 SUN domain and Mps2 C terminus is the first biochemical link known to connect the half-bridge with the core SPB. Association with Mps3 also defines a novel function for Mps2 during SPB duplication.

Amino Acid Sequence↗
PubMed2006

Emery-Dreifuss muscular dystrophy at the nuclear envelope: 10 years on.

Emery-Dreifuss muscular dystrophy (EDMD) is a neuromuscular degenerative condition with an associated dilated cardiomyopathy and cardiac conduction defect. It can be inherited in either an X-linked or autosomal manner by mutations in the nuclear proteins emerin and lamin A/C, respectively. Traditionally muscular dystrophies were associated with defects in sarcolemma-associated proteins and, therefore, a nuclear connection suggested the existence of novel signalling pathways associated with this group of diseases. Subsequently, other mutations in the lamin A/C gene were attributed to a range of tissue-specific degenerative conditions, collectively known as the 'laminopathies'. Therefore, any proposed hypothesis underlying the molecular mechanism of EDMD needs to include this anomaly. As we celebrate the 10th anniversary of the identification of emerin as a component of the nuclear envelope, I discuss here the available evidence that currently implicates EDMD as arising from perturbations in myogenic regulatory pathways, causing temporal delays in both cell cycle progression and muscle regeneration.

Animals↗
PubMed1995

Isolation and characterization of nuclear envelopes from the yeast Saccharomyces.

We have developed a large scale enrichment procedure to prepare yeast nuclear envelopes (NEs). These NEs can be stripped of peripheral proteins to produce a heparin-extracted NE (H-NE) fraction highly enriched in integral membrane proteins. Extraction of H-NEs with detergents revealed previously uncharacterized ring structures associated with the NE that apparently stabilize the grommets of the nuclear pore complexes (NPCs). The high yields obtained throughout the fractionation procedure allowed balance-sheet tabulation of the subcellular distribution of various NE and non-NE proteins. Thus we found that 20% of endoplasmic reticulum (ER) marker proteins are localized at the NE. Using a novel monospecific mAb made against proteins in the H-NE fraction and found to be directed against the pore membrane protein POM152, we showed that while the majority of POM152 is localized in the NE at the NPC, a proportion of this protein is also present in the ER. This ER pool of POM152 is likely to be involved in the duplication of nuclear pores and NPCs during S-phase. Both the NEs and H-NEs were found to be competent for the in vitro posttranslational translocation of prepro-alpha-factor. They may also be suitable to investigate other ER- and NE-associated functions in cell-free systems.

Antibodies, Monoclonal↗
PubMed1984

Age-dependent changes of nuclear envelope protein phosphokinase and protein phosphatase activities. Significance for altered nucleo-cytoplasmic mRNA translocation during development.

Nuclear envelopes are associated with a protein phosphokinase and a phosphoprotein phosphatase, whose activities are modulated by poly(A) in an opposite manner. The activities of these enzymes were determined in nuclear ghosts from liver and oviduct of quails of different age and of different hormone status. Under optimal conditions, kinase activity was found to increase in immature animals 8-fold in response to diethylstilbestrol; co-administration of progesterone had no marked effect on enzyme activity. After the initial burst, the activity of the enzyme increased only slightly during ageing. Two proteins present in nuclear ghosts of Mr 64 000 and of Mr 106 000 are phosphorylated during the kinase reaction; both the relative and the absolute extents of phosphate incorporation into these proteins alter drastically during ageing or hormone treatment of immature animals. Like the kinase activity, the activity of protein phosphatase increased in immature animals markedly in response to hormone treatment. Thereafter the activity remained constant in liver while in oviducts the phosphatase activity dropped to 30% in both mature and old animals.

Aging↗
PubMed1972

Studies of spermatogenesis in the Hepaticae. 3. Continuity between plasma membrane and nuclear envelope in androgonial cells of Blasia.

An ultrastructural study of late-stage androgonial cells of Blasia pusilla, a thallose liverwort, showed the nearly spherical nuclei often lying close or appressed to the cell walls. In some cells the two membranes comprising the nuclear envelope separated, the inner membrane continuing intact as a limiting boundary of the nucleus and the membrane on the outer, cytoplasmic side recurving away from the nucleus to continue without evident interruption around the periphery of the cell as the plasma membrane. It is believed that Blasia offers the first completely convincing demonstration of the heretofore problematic continuity of cytoplasmic membranes. A possible sequence of events leading to this unusual relationship between nucleus and cytoplasm is suggested. The sequence includes blebbing of the outer membrane of the nuclear envelope and subsequent membrane proliferation, apparent isolation of cytoplasmic ground substance, fusion of internal membrane with the ectoplast, and migration that finally brings the nucleus into flat contact with the wall. While this manifestation of membrane continuity may be anomalous, it is not presently considered the result of cell injury.

Cell Membrane↗
PubMed2006

Use of Xenopus laevis oocyte nuclei and nuclear envelopes in nucleocytoplasmic transport studies.

In this chapter, two techniques for the analysis of transport through the nuclear pore complex are described. In the first technique, nuclei isolated manually from Xenopus laevis oocytes are used to measure the import kinetics of fluorescent substrates by confocal fluorescence microscopy. In the second technique, referred to as optical single transporter recording (OSTR), isolated Xenopus oocyte nuclei, perforated nuclei, or isolated nuclear envelopes are tightly bound to planar transparent substrates containing arrays of nanoscopic-to-microscopic cavities. Transport through membrane patches spanning these cavities is recorded by confocal microscopy. By these means, the transport through single nuclear pore complexes or populations of pore complexes can be quantitatively measured.

Active Transport, Cell Nucleus↗