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PubMed2000

"Atypical p-ANCA" in IBD and hepatobiliary disorders react with a 50-kilodalton nuclear envelope protein of neutrophils and myeloid cell lines.

BACKGROUND & AIMS: Atypical "antineutrophil cytoplasmic antibodies" (ANCA) are present in patients with ulcerative colitis (UC), primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH). Recently, we showed that atypical p-ANCA react with nuclear envelope proteins of neutrophils. Based on this observation, we aimed to characterize the nuclear antigen recognized by atypical p-ANCA. METHODS: We prepared cytoplasmic and nuclear extracts of human neutrophils, human HL-60, and murine 32D myeloid cells. Proteins were resolved by 1- and 2-dimensional gel electrophoresis. Reactive proteins were detected by immunoblotting with sera from 118 individuals (UC, 25; PSC, 28; AIH, 35; disease and normal controls, 30). Atypical p-ANCA (n = 64) were affinity-purified against the reactive protein and investigated for their immunofluorescence pattern using confocal microscopy. RESULTS: Immunoblotting showed reactivity to a myeloid-specific 50-kilodalton nuclear protein with an isoelectric point of pH 6.0 detected in 92% (59 of 64) of the patients with inflammatory bowel or hepatobiliary diseases and atypical p-ANCA. Affinity-purified antibodies against the 50-kilodalton protein gave a nuclear rim-like fluorescence on myeloid cells examined by immunofluorescence microscopy. Affinity-purified antibodies did not recognize antigens in nonmyeloid cells. CONCLUSIONS: Atypical p-ANCA in UC, PSC, or AIH recognize a 50-kilodalton myeloid-specific nuclear envelope protein.

Adolescent↗
PubMed1988

A role for the nuclear envelope in controlling DNA replication within the cell cycle.

In eukaryotes the entire genome is replicated precisely once in each cell cycle. No DNA is re-replicated until passage through mitosis into the next S-phase. We have used a cell-free DNA replication system from Xenopus eggs to determine which mitotic changes permit DNA to re-replicate. The system efficiently replicates sperm chromatin, but no DNA is re-replicated in a single incubation. This letter shows that nuclei replicated in vitro are unable to re-replicate in fresh replication extract until they have passed through mitosis. However, the only mitotic change which is required to permit re-replication is nuclear envelope permeabilization. This suggests a simple model for the control of DNA replication in the cell cycle, whereby an essential replication factor is unable to cross the nuclear envelope but can only gain access to DNA when the nuclear envelope breaks down at mitosis.

Animals↗
PubMed1995

[The roles of two kinds of membrane vesicles in the formation of annulate lamellae and nuclear envelopes in a cell-free system from Xenopus egg extracts].

The eggs of Xenopus laevis were activated to complete their second meiosis and enter interphase by calcium ionophore A 23187. Then they were centrifuged at 10,000 g to get activated Xenopus egg extracts. Chromatin structure could form in such an extracts after exogenous Lambda DNA was added, and nuclear envelopes (NEs) could assemble around the chromatin. Furthermore, annulate lamellae (AL), a NE-like membrane structure, could form in chromatin-free regions simultaneously. After large amounts of observation and analysis at ultrastructural level, we propose that both AL and NEs assemble in the extracts by the fusion of membrane vesicles. There were two kinds of membrane vesicles in Xenopus egg extracts, small rough vesicles with diameters of 200 nm and large smooth vesicles. They had many differences in morphology, size and membrane structure. However, they both participate in the formation of AL and NEs, 200 nm membrane vesicles first fused each other to become parallel double membrane cisternae, nuclear pore complexes assembled in the double membranes in the mean time. The fusion of large smooth vesicles with the double membrane cirternae led to the growth of AL and the maturity of NEs. During the formation of NEs, large smooth vesicles were easily observed to link two neighboring double membrane fragments by fusing with them, so that integrated nuclear envelope could form. 200 nm small rough vesicles could attach to the surface of chromatin to start the assembly of nuclear envelopes, while others could fuse each other at chromatin-free regions and these fusion resulted in the formation of annulate lamellae.

Animals↗
PubMed2002

Ran GTPase cycle and importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos.

The small GTPase Ran has been found to play pivotal roles in several aspects of cell function. We have investigated the role of the Ran GTPase cycle in spindle formation and nuclear envelope assembly in dividing Caenorhabditis elegans embryos in real time. We found that Ran and its cofactors RanBP2, RanGAP, and RCC1 are all essential for reformation of the nuclear envelope after cell division. Reducing the expression of any of these components of the Ran GTPase cycle by RNAi leads to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins. Ran, RanBP2, and RanGAP are also required for building a mitotic spindle, whereas astral microtubules are normal in the absence of these proteins. RCC1(RNAi) embryos have similar abnormalities in the initial phase of spindle formation but eventually recover to form a bipolar spindle. Irregular chromatin structures and chromatin bridges due to spindle failure were frequently observed in embryos where the Ran cycle was perturbed. In addition, connection between the centrosomes and the male pronucleus, and thus centrosome positioning, depends upon the Ran cycle components. Finally, we have demonstrated that both IMA-2 and IMB-1, the homologues of vertebrate importin alpha and beta, are essential for both spindle assembly and nuclear formation in early embryos.

Aneuploidy↗
PubMed1990

Isolation and characterization of the Drosophila nuclear envelope otefin cDNA.

We have recently identified and characterized a 53-kDa inner nuclear membrane-associated protein in Drosophila and termed it otefin. Here we report the isolation and characterization of cDNA and genomic clones of the otefin gene. Based on sequence analysis, we deduced that the primary translation product has a calculated mass of 45 kDa, contains many serine and threonine residues, and is mostly hydrophilic. However, in the carboxyl terminus, there is a hydrophobic region which may serve as a membrane anchoring domain. RNA blot analysis indicated that the otefin gene codes for a single poly(A+) transcript of 1.6 kilobases and that relatively large amounts of this transcript are present during developmental stages in which many nuclear divisions occur. Polyclonal antibodies raised against the cDNA translation product react with a 58-kDa mammalian nuclear envelope protein, demonstrating evolutionary conservation.

Amino Acid Sequence↗
PubMed1992

Chloroquine inhibits the insulin binding and the imprinting of nuclear envelope in Tetrahymena.

Chloroquine possessing lysosomotrop effects inhibits the development of insulin imprinting both on the plasma membrane and the nuclear envelope. Simultaneously chloroquine can inhibit the insulin binding in the nucleus itself but not in the plasma membrane. There is a dose dependent increase of binding of labelled insulin to the nuclear envelope. The control related binding of nuclei pretreated with insulin or chloroquine is similar and have a direct ratio, independently of the applied concentrations.

Animals↗
PubMed2003

Accumulation of beta (m), a structural member of X,K-ATPase beta-subunit family, in nuclear envelopes of perinatal myocytes.

Recently discovered muscle-specific beta(m) protein is structurally closely related to the X,K-ATPase beta-subunits. However, it has a number of unique properties such as predominant localization in intracellular stores and lack of association with known X,K-ATPase alpha-subunits on heterologous coexpression. In this study, the primary structure of mouse beta(m) was determined and developmental regulation of the gene (ATP1B4) was analyzed. The expression is first detected at day 14 of gestation, is sharply increased at day 16, and reaches its maximum at day 18. After birth, the expression quickly decreases and is hardly detectable in adult mice. A more detailed subcellular localization study was undertaken, and its results indicate that beta(m) not only is located in sarcoplasmic reticulum but is concentrated in nuclear envelopes of both prenatal and postnatal skeletal muscles. Immunohistochemical studies show that beta(m) is specific to myocytes and, at the subcellular level, many nuclear envelopes are intensively labeled in both fetal and newborn skeletal muscles. Accordingly, beta(m) is detected by immunoblotting in purified nuclei and nuclear membranes from neonatal skeletal muscles. On transfection of human rhabdomyosarcoma cell line RD, green fluorescent protein-tagged beta(m) resides intracellularly with significant enrichment in nuclear envelopes, whereas beta(m) with transmembrane domain deleted localizes in both cytoplasm and nucleoplasm. Nuclear beta(m) apparently is not in association with Na,K-ATPase because we never detected its alpha-subunit in myonuclear membranes. These results indicate that beta(m) has a specialized function in mammalian perinatal myocytes, different from functions of other X,K-ATPase beta-subunits. The unique temporospatial distribution of beta(m) protein expression suggests its important role in development of growing skeletal muscle.

Adenosine Triphosphatases↗
PubMed2001

Roles of Ran-GTP and Ran-GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system.

The molecular mechanism of nuclear envelope (NE) assembly is poorly understood, but in a cell-free system made from Xenopus eggs NE assembly is controlled by the small GTPase Ran [1,2]. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin [1]. Both generation of Ran-GTP by the guanine nucleotide exchange factor RCC1 and GTP hydrolysis by Ran are required for NE assembly, although the roles of the GDP- and GTP-bound forms of Ran in the recruitment of precursor vesicles and their fusion have been unclear. We now show that beads coated with either Ran-GDP or Ran-GTP assemble functional nuclear envelopes in a cell-free system derived from mitotic human cells, forming pseudo-nuclei that actively transport proteins across the NE. Both RCC1 and the GTPase-activating protein RanGAP1 are recruited to the beads, allowing interconversion between Ran-GDP and Ran-GTP. However, addition of antibodies to RCC1 and RanGAP1 shows that Ran-GDP must be converted to Ran-GTP by RCC1 before precursor vesicles are recruited, whereas GTP hydrolysis by Ran stimulated by RanGAP1 promotes vesicle recruitment and is necessary for vesicle fusion to form an intact envelope. Thus, the GTP-GDP cycle of Ran controls both the recruitment of vesicles and their fusion to form NEs.

Animals↗
PubMed1999

Ca(2+)-dependent association of S100A6 (Calcyclin) with the plasma membrane and the nuclear envelope.

Expression of S100A6 (Calcyclin), a member of the S100 family and of Zn(2+)-binding proteins is elevated in a number of malignant tumors. In vitro the protein associates with several actin-binding proteins and annexins in a Ca(2+)-dependent manner. We have now studied the subcellular localization of S100A6 using a new, specific monoclonal antibody. Immunofluorescence microscopy of unfixed, ultrathin, frozen sections demonstrated a dual localization of S100A6 at the nuclear envelope and the plasma membrane of porcine smooth muscle only in the presence of Ca(2+). The same localization was found by immunofluorescence and immunogold electron microscopy as well as by confocal laser scanning microscopy with cultured, fixed, human CaKi-2 and porcine ST interphase cells. Upon cell division, however, S100A6 was found exclusively in the cytoplasm. Cell fractionation studies showed that S100A6 was present in the microsomal fraction in the presence of Ca(2+) and was released from this fraction by the addition of EGTA/EDTA but not by Triton X-100. The data demonstrate that S100A6 is localized both at the plasma membrane and the nuclear envelope in vivo and suggest a Ca(2+)-dependent interaction with annexins or other components of the nuclear envelope.

Animals↗
PubMed1997

Cross-reactivity of anti-nDNA antibodies with nuclear envelope proteins. Isolation of a cDNA encoding the 70 kDa annular protein recognized by autoantibodies from patients with systemic lupus erythematosus.

OBJECTIVES: To determine whether annular rDNA is complexed with the nuclear envelope proteins. METHODS: From a batch of lupus sera with anti-nDNA, we selected a lupus serum containing annular anti-nDNA autoantibodies resistant to DNase digestion and used it to isolate several cDNA clones from a lambda gt11 HeLa cell library. RESULTS: The cloned fusion protein immunoadsorbed the annular anti-nDNA autoantibodies, and the immunoaffinity autoantibodies eluted from the recombinant filters produced an annular pattern around the nucleus in fluorescent assays on HEp-2 cells; by Western blot, they also recognized a 70 kDa protein from HEp-2 cell extracts. Annular-lambda gt11 lysogens generated in E. coli Y1089 produced a fusion protein that recognized annular anti-nDNA autoantibody-containing lupus sera by Western blot. The recombinant filters and annular fusion protein were also recognized by a prototype anti-lamin serum. To determine whether the annular recombinant protein bound DNA, an interaction assay was performed in vitro using DNA minicircles and DNA from HEp-2 cells; this assay resulted in a slowing of the electrophoretic mobility of the DNA. CONCLUSIONS: 1) The annular DNA in eukaryotic cells is complexed with nuclear envelope proteins. 2) Annular anti-nDNA autoantibodies from lupus patients cross-react with perinuclear proteins. 3) Perinuclear proteins recognized by anti-nDNA are lamins. 4) An interaction between DNA and the 70 kDa protein is inducible in vitro. Whether this interaction affects cell function is still unknown.

Antibodies, Antinuclear↗
PubMed1998

Demonstration of equilibrative nucleoside transporters (hENT1 and hENT2) in nuclear envelopes of cultured human choriocarcinoma (BeWo) cells by functional reconstitution in proteoliposomes.

The equilibrative nucleoside transporters (ENTs) are a newly recognized family of membrane proteins of which hENT1 is the nitrobenzylmercaptopurine ribonucleoside (NBMPR)-sensitive (es) and hENT2 the NBMPR-insensitive (ei) transporter of human cells. BeWo cells exhibit large numbers (>10(7)/cell) of NBMPR-binding sites and high es and ei nucleoside transport activities relative to other cell types. In this work, we have demonstrated that proliferating BeWo cells possess (i) mRNA encoding hENT1 and hENT2 and (ii) hENT1-specific immunoepitopes. We examined NBMPR binding and its inhibition of uridine transport in various BeWo membrane fractions and proteoliposomes derived therefrom to determine if NBMPR binding to intracellular membranes represented interaction with functional es transporters. Unfractionated membranes and fractions enriched 5-fold in plasma membranes relative to postnuclear supernatants exhibited high NBMPR binding activity. Intact nuclei and nuclear envelopes also exhibited abundant quantities of NBMPR-binding sites with affinities similar to those of enriched plasma membranes (Kd = 0.4-0.9 nM). When proteoliposomes were made from octyl glucoside-solubilized membranes, high affinity NBMPR-binding sites were not only observed in crude membrane preparations and plasma membrane-enriched fractions but also in nuclear envelope fractions. Proteoliposomes prepared from either unfractionated membranes or nuclear envelopes exhibited both hENT1-mediated (82-85%) and hENT2-mediated (15-18%) transport of [3H]uridine. These results provided evidence for the presence of functional es and ei transporters in nuclear membranes and endoplasmic reticulum, suggesting that hENT1 and hENT2 may function in the translocation of nucleosides between the cytosol and the luminal compartments of one or both of these membrane types.

Affinity Labels↗
PubMed1992

Different concentrations of thyroid peroxidase and thyroglobulin in the nuclear envelope and the endoplasmic reticulum throughout the cytoplasm.

Thyroid peroxidase (TPO) and thyroglobulin (TG) represent two major glycoproteins of thyroid follicular cells performing biological functions such as iodination, transcytosis of thyroglobulin, and formation of thyroid hormones. They are involved in thyroid autoimmunity and thyroid inborn metabolic disorders. Studying these processes at a molecular level includes the determination of their precise intracellular distribution. An evaluation of the relative concentrations of TG and TPO in different subcellular compartments was carried out in stimulated human follicular cells using thin-frozen sections and the immunogold technique. It is documented that TG is transported from the endoplasmic reticulum and the Golgi apparatus to the follicular lumen by transport vesicles; most of it being present in the expanded endoplasmic reticulum throughout the cytoplasm. On the other hand, gold particles indicating TPO are adjacent to the membranes of the exocytotic pathway. They do not label the basolateral membrane but show the strongest density in the nuclear envelope and the apical membrane. The labeling density of TPO is about four times higher in the nuclear envelope than in the endoplasmic reticulum throughout the cytoplasm. In contrast, TG is concentrated three times higher in the rough endoplasmic reticulum throughout the cytoplasm than in the nuclear cisternae. Our results give the first quantitative evidence that TPO and TG are concentrated in different subcompartments of the endoplasmic reticulum. Because previous studies demonstrated the nuclear envelope as the site where the synthesis of endogenous peroxidase (Brökelmann, J., D. W. Fawcett, Biol. Reprod. 1, 59-71 (1969)) begins, we suggest that synthesis of these functionally related proteins happens in specialized parts of the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗
PubMed2002

Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina.

The mechanism of nuclear envelope breakdown (NEBD) was investigated in live cells. Early spindle microtubules caused folds and invaginations in the NE up to one hour prior to NEBD, creating mechanical tension in the nuclear lamina. The first gap in the NE appeared before lamin B depolymerization, at the site of maximal tension, by a tearing mechanism. Gap formation relaxed this tension and dramatically accelerated the rate of chromosome condensation. The hole produced in the NE then rapidly expanded over the nuclear surface. NE fragments remaining on chromosomes were removed toward the centrosomes in a microtubule-dependent manner, suggesting a mechanism mediated by a minus-end-directed motor.

Animals↗
PubMed1995

Ca(2+)-activated K(+)-channels in the nuclear envelope isolated from single pancreatic acinar cells.

The patch-clamp techniques are applied to the outer membrane of the nuclear envelope isolated from rat pancreatic acinar cells. The nucleus identified under an inverted microscope was removed by cell surgery from enzymatically dispersed single cells. All the patch-clamp techniques, in situ, excised, and whole-material recordings were applied to the envelope. We have found voltage- and Ca(2+)-activated K(+)-channels with an unitary conductance of 200 pS in the outer membrane. The channels are activated by lumen positive potentials and by an increase in luminal Ca2+ concentration. They may play a role for controlling Ca(2+)-release from the lumen of the nuclear envelope (endoplasmic reticulum) to the nucleoplasm and the perinuclear cytoplasm.

Animals↗
PubMed1993

Restricted ion flow at the nuclear envelope of cardiac myocytes.

Flow of small ions across the nuclear envelope (NE) is thought to occur without restriction through large diameter nuclear pore complexes (NPCs). However, investigations with electron and fluorescence microscopy, and with patch-clamp and microelectrode electrophysiology, suggest that in many animal and plant cell types small ions move through a barrier having the signature of large conductance nuclear ion channels (NICs). As nucleocytoplasmic transport and gene activity are regulated by cytoplasmic signals and as it has recently been shown by this investigator that cardiac NICs are sensitive to cAMP-dependent processes (1), it was considered relevant to further investigate the effects of various cytosolic signals on NIC activity. Ion species substitution demonstrated that K+ is the major species responsible for NIC currents. The Na-channel blocker tetrodotoxin (TTX, 100 microM) and the Ca-channel blocker diltiazem (100 microM) had no effect, indicating no relation of NICs to Na- or Ca-channels in transit to the cell surface membrane. Zn2+ (100 microM) blocked NIC activity, suggesting a dual role in nucleocytoplasmic transport and gene function. GTP did not produce measurable effect. However, its nonhydrolyzable analogue GTP-gamma-S (10 microM) suppressed NIC activity, suggesting a role for GTP hydrolysis in NIC function. Deoxynucleotides (dNTPs, 200 microM) produced a transient increase in NIC activity, pointing to a modulation of NIC function by nucleic acid substrates. These results indicate a role for NICs in mediating: (a) control of gene activity by transduction and other cytosolic signals, and (b) nuclear demands and response to such signals.

Animals↗
PubMed1996

Interaction between an integral protein of the nuclear envelope inner membrane and human chromodomain proteins homologous to Drosophila HP1.

At the nuclear envelope in higher eukaryotic cells, the nuclear lamina and the heterochromatin are adjacent to the inner nuclear membrane, and their attachment is presumably mediated by integral membrane proteins. In a yeast two-hybrid screen, the nucleoplasmic domain of lamin B receptor (LBR), an integral protein of the inner nuclear membrane, associated with two human polypeptides homologous to Drosophila HP1, a heterochromatin protein involved in position-effect variegation. LBR fusion proteins bound to HP1 proteins synthesized by in vitro translation and present in cell lysates. Antibodies against LBR also co-immunoprecipitated HP1 proteins from cell extracts. LBR can interact with chromodomain proteins that are highly conserved in eukaryotic species and may function in the attachment of heterochromatin to the inner nuclear membrane in cells.

Amino Acid Sequence↗
PubMed2003

Nuclear envelope dynamics in oocytes: from germinal vesicle breakdown to mitosis.

We have recently gained new insight into the mechanisms involved in nuclear envelope breakdown, the irreversible step that commits a cell to the M phase. Results from mammalian cell and starfish oocyte studies suggest that mechanical forces of the cytoskeleton, as well as biochemical disassembly of nuclear envelope protein complexes, play important roles in this process.

Animals↗
PubMed1995

Lipophilic organizing structures of sperm nuclei target membrane vesicle binding and are incorporated into the nuclear envelope.

We report the existence of lipophilic structures (LS's) associated with demembranated sperm nuclei which together act as a pronuclear envelope organizing center. These structures can be visualized as objects which stain with lipophilic dyes but are resistant to solubilization in 0.1% of the non-ionic detergent Triton X (TX)-100. The structures, located at the acrosomal and centriolar poles of the sea urchin sperm nucleus, initiate ATP-dependent membrane vesicle binding in a fertilized egg extract. The lipophilic material in LS's is incorporated into the nuclear envelope during GTP-induced membrane fusion. Removal of the LS's from sperm nuclei with 1% TX-100 abolishes membrane vesicle binding to the nuclei. LS's recovered from supernatants of extracted nuclei can be reconstituted to their original locations on the stripped nuclei. Rebinding of isolated LS's occurs preferentially at the acrosomal pole. Such reconstituted nuclei direct membrane vesicle binding only to the acrosomal pole region and result in incomplete nuclear envelope assembly following membrane fusion. Binding of LS's and subsequently membrane vesicles to both nuclear poles allows complete nuclear envelope formation.

Acrosome↗