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PubMed1989

Identification of specific binding proteins for a nuclear location sequence.

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm. Nuclear proteins larger than relative molecular mass 20,000-40,000 are probably actively transported across the envelope through the nuclear pore complex and are directed by specific nuclear location sequences (NLS) in the proteins. NLS mediate the nuclear import of isolated nuclear proteins after microinjection into whole cells and the nuclear accumulation of chimaeric proteins or of non-nuclear proteins conjugated to synthetic peptides. The best-characterized NLS is the simian virus 40 large T-antigen sequence. We have identified two proteins of rat liver by chemical cross-linking that interact with a synthetic peptide containing this sequence: this interaction is specific for a functional NLS, is saturable, and high affinity. The binding proteins are present in a post-mitochondrial supernatant, in nuclei and in a nuclear envelope fraction, which is consistent with a role in the transport of nuclear proteins from the cytoplasm to the nucleus.

Amino Acid Sequence↗
PubMed1982

Comparison of the response of myocardial muscle and capillary endothelial nuclear membranes to the cholesterol probe filipin.

The polyene antibiotic filipin was used as a probe for cholesterol in the nuclear membranes of cardiac muscle and capillary endothelial cells. Samples of perfusion-fixed rabbit and rat myocardium were exposed to filipin in glutaraldehyde fixative, prior to examination by standard freeze-fracture techniques. Quantitative analysis of the responses of the inner and the outer nuclear membranes of the two cell types was carried out by measuring the numerical density of filipin-induced lesions in each. Cardiac muscle cell outer nuclear membranes displayed few or no lesions (less than 0.1 micron-2), and their inner nuclear membranes were completely resistant to the treatment. By contrast, a marked response to filipin was found in the endothelial nuclear envelope, the outer membrane showing an average of 250 lesions micron-2, and the inner membrane 63 lesions micron-2. These results are interpreted as a direct reflection of differences in cholesterol content between the outer and inner nuclear membranes of the two cell types. The significance of the apparent differences in cholesterol content demonstrated between the nuclear envelopes of different cell types is discussed with reference to biochemical data, plasma membrane cholesterol content, nuclear membrane function and the supply of exogenous cholesterol.

Animals↗
PubMed1987

O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins.

Glycoproteins bearing a single N-acetylglucosamine (GlcNAc) residue attached by an O-glycosidic linkage to the polypeptide chain (Holt, G. D., and Hart, G. W. (1986) J. Biol. Chem. 261, 8049-8057) have been found to be enriched in the nuclear and soluble fractions of rat liver. Our goal was to determine the localization and membrane topography of proteins bearing O-linked GlcNAc using galactosyltransferase and wheat germ agglutinin (WGA) as membrane-impermeant probes. Latency of the enzyme mannose-6-phosphatase was used to quantitatively confirm the intactness of the nuclear envelope during incubations with galactosyltransferase or WGA. The O-linked GlcNAc residues of nuclei were fully accessible to modification by galactosyltransferase under conditions where the nuclear envelope mannose-6-phosphatase was 70% latent. Addition of detergent destroyed the permeability barrier but did not increase galactosylation of the O-linked GlcnAc. The major polypeptides bearing O-linked GlcNAc residues on nuclei were peripheral rather than integral membrane proteins with apparent molecular masses ranging from 210 to 54 kDa. The proteins were also detected on sealed nuclei using conjugates of WGA. WGA-rhodamine labeled intact nuclei when examined by immunofluorescence; WGA-peroxidase was used to identify the nuclear glycoproteins after transfer to nitrocellulose. WGA-ferritin selectively labels the cytoplasmic and nucleoplasmic faces of the nuclear pore complex when examined by electron microscopy. Taken together, these data strongly suggest that proteins bearing cytoplasmically oriented O-linked GlcNAc are components of the nuclear pore complex, thereby raising the possibility that cytoplasmic and nucleoplasmic glycoproteins are involved in the assembly or functioning of the nuclear pore.

Acetylglucosamine↗
PubMed1989

Nuclear lamin antigens are developmentally regulated during porcine and bovine embryogenesis.

The nuclear lamins, proteins that reside on the inner face of the nuclear envelope, are thought to provide attachment sites for anchoring the chromatin to the nuclear envelope, thus facilitating the overall organization of the nucleus. The composition of the nuclear lamin proteins changes during differentiation and development in a variety of mammalian and nonmammalian tissues. Bovine and porcine oocytes and early embryos were prepared for immunocytochemical detection of nuclear lamins using three different antibodies (recognizing lamin B, lamins A/B/C, or lamins A/C). In both species, germinal vesicle nuclei and early cleavage stage nuclei react positively with the antibodies. However, on nuclei of bovine embryos, the A/C epitope was not detectable at the 16-cell stage, compact morula, spherical blastocyst, or the chorionic cell nuclei of a Day 35 conceptus, but was detectable on both amniotic and embryonic ectodermal cell nuclei of a Day 35 conceptus. All three antibodies reacted with nuclei from two bovine tissue culture cell lines (bovine embryonic cells and Madin-Darby bovine kidney cells) and one porcine kidney cell line. Nuclei in porcine embryos followed a similar pattern, except the loss of the A/C epitope occurred at the 8-cell stage and the epitope was absent from compact morula and spherical blastocyst stage nuclei. All interphase nuclei in both species reacted with both anti-lamin A/B/C and anti-lamin B antibodies, whereas metaphase chromosomes did not react with any of the lamin antibodies tested. The change in recognizing the lamin epitope occurred one cell cycle after the expected transition from maternal control to zygotic control of development. Nuclear transplantation showed that 16-cell stage porcine nuclei, which are lamin A/C negative, acquired the A/C epitope after transfer to an enucleated metaphase II oocyte. These results suggest that the A/C epitope is developmentally regulated.

Animals↗
PubMed1978

Peroxidase activity in monocytes and tissue macrophages of mice.

A description is given of the distribution of peroxidatic (PO) activity in murine monocytes of blood and peritoneal cavity, and in murine macrophages residing in the unstimulated peritoneal cavity as well as in liver, spleen, bone marrow, and small intestine. In the monocytes, PO activity is restricted to some of the cytoplasmic granules; in the tissue (or resident) macrophages present in peritoneal cavity, liver, spleen, and small intestine, the PO activity is located in the nuclear envelope and the rough endoplasmic reticulum. Macrophages in the bone marrow are PO-negative. In the spleen and bone marrow, reticulum cells show PO activity in the nuclear envelope and the RER. Transitional forms between monocytes and tissue macrophages were not observed.

Animals↗
PubMedDate not supplied

Calcium regulation of nuclear pore permeability.

The nuclear envelope is an integral part of the structural framework of the nucleus, and is involved in organizing intranuclear events. It serves as a selective barrier, actively transporting proteins required for normal nuclear function and exporting RNA. The movement of molecules across the nuclear envelope is critical for cellular homeostasis, and it allows cells to respond to external events. The only known pathway for direct communication between the cytoplasm and the nucleoplasm of a cell is through the nuclear pore complex. In the past decade, rapid advances have been made in elucidating the structure and function of the nuclear pore complex. Yet, researchers are just beginning to identify some of the regulatory mechanisms controlling transport through the pore complex. The nucleus is surrounded by a Ca2+ storage compartment, which sequesters and releases Ca2+ in response to intracellular second messengers, Recent evidence suggests that the nuclear Ca2+ store may indirectly regulate passive diffusion through the nuclear pore complex. The evidence for Ca2+ regulation of the nuclear pore complex will be discussed, along with the introduction of the simplest, testable model to describe the observations.

Animals↗
PubMed2000

Effects of altered tonicity by sodium chloride on L-tryptophan binding to hepatic nuclei.

This study was concerned with the effects of NaCl administered in vivo or added in vitro to isolated nuclei on [(3)H]tryptophan binding to rat hepatic nuclei assayed in vitro. Hypertonic (10.7%) NaCl administered in vivo to rats caused at 10 min a marked decrease in in vitro binding (total and specific) of [(3)H]tryptophan to hepatic nuclei. In vitro incubation of isolated hepatic nuclei, but not of isolated nuclear envelopes, with added NaCl (particularly at 0.125 x 10(-4) M and 0.25 x 10(-4) M) revealed significant inhibition of [(3)H]tryptophan binding. However, isolated hepatic nuclear envelopes prepared after in vitro incubation of isolated nuclei with added NaCl did show inhibition of [(3)H]tryptophan binding (total and specific) compared with controls. Other salts (KCl, MgCl(2), NaHCO(3), NaC(2)H(3)O(2), NaF, or Na(2)SO(4)), at similar concentrations to that of NaCl except for MgCl(2), when added to isolated nuclei did not appreciably inhibit nuclear tryptophan binding. Kinetic studies of in vitro nuclear [(3)H]tryptophan binding in the presence of 0.125 x 10(-4) M NaCl revealed that binding decreased at 0.5 h and continued to 2 h compared with nuclear [(3)H]tryptophan binding with controls (without NaCl addition). The results obtained in vivo in rats and those obtained in vitro with isolated hepatic nuclei revealed NaCl-induced inhibitory effects on [(3)H]tryptophan binding to hepatic nuclei. Although the inhibitory effects were similar under the two different experimental conditions, the mechanism for each may be different in that the NaCl concentration in hepatic cells after administration of NaCl in vivo was appreciably higher than the low levels added in vitro to the isolated hepatic nuclei.

Animals↗
PubMed1983

Congenital dyserythropoietic anemia type I: a freeze-fracture and thin section electron microscopic study.

Congenital dyserythropoietic anemia type I was diagnosed in a Japanese patient. Electron microscopic examination of thin sections revealed the features of erythroblasts peculiar to CDA type I; namely "spongy appearance" of nucleus and "internuclear chromatin bridge" etc. However, the widening of nuclear pores, which has been reported as another peculiar feature of CDA type I erythroblasts, were not confirmed. Aberrations of erythroblast nuclear membrane which have not been hitherto noticed were as follows: The nuclear envelopes of erythroblasts frequently lacked heterochromatin application in wide areas where they often showed a marked invagination or evagination. This is associated with the invasion of cytoplasmic organelles into the nuclear territory and occasionally with the extrusion of the nucleolus into the cytoplasm. There were no nuclear pores on the invaginated or the evaginated portions of the nuclear envelope where the perinuclear cistern was compressed to lose the cisternal space, or widened to contain some membrane debris. The findings with thin sectionings were confirmed with freeze-fracturing. Both findings suggest that an aberration may reside in the erythroblast nuclear membrane itself, but not in nuclear pores.

Adult↗
PubMed1998

The nuclear basket of the nuclear pore complex is part of a higher-order filamentous network that is related to chromatin.

We have achieved well-preserved nuclear basket structures in amphibian nuclear envelope spreads dried by the critical point method after tannic acid fixation, and we have compared these images with those obtained from conventional sections of amphibian oocyte nuclei. In cross sections, bundles of filaments from adjacent nuclear pore complexes were interconnected at regular distances, building up a higher-order network. Sometimes these bundles were observed to extend inward to amplified nucleoli located near the nuclear envelope. Furthermore, immunoelectron analysis indicated that DNA and histones were localized at these intranuclear filaments, suggesting a close relationship between chromatin and nuclear pore complexes. A model is proposed by which the intranuclear filaments associated with adjacent nuclear pore complexes create a regular higher-order network, which extends into the nucleus.

Animals↗
PubMed1978

Distribution of nuclear pores and perinuclear dense substances in spermatocytes I of some Oedionychina fleabeetles.

The nuclear envelope of growing postpachytene spermatocyte I differs notably in structure between the fleabeetles Omophoita cyanipennis and Oedionychus bicolor. The former species shows a more conventional structure with an even and probably random distribution of nuclear pores, and a strongly electron-opaque layer of fibrogranular material (FM) separated from the outer nuclear membrane by an intermediate layer of about 40 nm thickness. Peripherally from the FM layer, a continuous corona of granular dense material (GM) is accumulated around the nucleus. In Oedionychus, all nuclear pores are clustered in "nuclear sieves", i.e., shallow, cup-like indentations of the nuclear envelope. The sieves are filled with an electron-opaque substance resembling the FM of the Omophoita spermatocytes. This substance is kept at a distance of about 40 nm from the outer nuclear membrane. GM is produced only at the sives, and is thus discontinuous. The sieves with their contents are called nuclear sieve complexes (NSC).

Animals↗
PubMed2005

Spectrins and the Golgi.

Several isoforms of spectrin membrane skeleton proteins have been localized to the Golgi complex. Golgi-specific membrane skeleton proteins associate with the Golgi as a detergent-resistant cytoskeletal structure that likely undergoes a dynamic assembly process that accommodates Golgi membrane dynamics. This review discusses the potential roles for this molecule in Golgi functions. In particular, it will focus on a recently identified distant cousin to conventional erythroid spectrin variously named Syne-1, Nesprin, myne, Enaptin, MSP-300, and Ank-1. Syne-1 has the novel ability to bind to both the Golgi and the nuclear envelope, a property that raises several intriguing and novel insights into Golgi structure and function. These include (1) the facilitation of interactions between Golgi and transitional ER sites on the nuclear envelope of muscle cells, and (2) an ability to impart localized specificity to the secretory pathway within large multinucleate syncytia such as skeletal muscle fibers.

Animals↗
PubMed2005

Nuclear entry of nonviral vectors.

Nonviral gene delivery is limited to a large extent by multiple extracellular and intracellular barriers. One of the major barriers, especially in nondividing cells, is the nuclear envelope. Once in the cytoplasm, plasmids must make their way into the nucleus in order to be expressed. Numerous studies have demonstrated that transfections work best in dividing populations of cells in which the nuclear envelope disassembles during mitosis, thus largely eliminating the barrier. However, since many of the cells that are targets for gene therapy do not actively undergo cell division during the gene transfer process, the mechanisms of nuclear transport of plasmids in nondividing cells are of critical importance. In this review, we summarize recent studies designed to elucidate the mechanisms of plasmid nuclear import in nondividing cells and discuss approaches to either exploit or circumvent these processes to increase the efficiency of gene transfer and therapy.

Active Transport, Cell Nucleus↗
PubMed1983

Evidence for a unique profile of phosphatidylcholine synthesis in late mitotic cells.

Evidence is presented that the structural rearrangements in late mitosis are accompanied by an alteration in membrane lipid synthesis. This evidence was derived from analyzing phospholipid classes after rapid-labeling, as well as from determining the intracellular site of incorporation of choline by HeLa S3 cells as they progressed from metaphase into early interphase (G1). Compared with postmitotic cell data, the recent mitotic cell data indicate a specific two- to threefold increase in the net synthesis of phosphatidylcholine (PC) species, which appeared to contain the more saturated fatty acids. Since this was observed with glycerol, choline, and orthophosphate labelings, and not with methyl labeling, it appears that the CDP-choline plus diacylglycerol pathway rather than the phosphatidylethanolamine to PC pathway was augmented. Electron microscope autoradiography of anaphase, telophase, and early G1 cells demonstrated that the reformed nuclear envelope was the incorporation site of a significant proportion of the newly synthesized PC. This incorporation occurred by early telophase prior to chromosome decondensation. The potential significance of PC metabolism with regard to membrane rearrangements, such as nuclear envelope reformation, is discussed.

Choline↗
PubMed2002

Spc24 interacts with Mps2 and is required for chromosome segregation, but is not implicated in spindle pole body duplication.

Mps2 (monopolar spindle protein) is a coiled-coil protein found at the spindle pole body (SPB) and at the nuclear envelope that is required for insertion of the SPB into the nuclear envelope. We identified three proteins that interact with Mps2 in a two-hybrid screen: Bbp1, Ynl107w and Spc24. All three proteins contain coiled-coil motifs that appear to be required for their interaction with Mps2. In this work, we verified the Mps2-Spc24 interaction by co-immunoprecipitation in vivo and by the in vitro interaction of recombinant proteins. Previous two-hybrid screens with Spc24 as bait had identified Spc25 and Ndc80 as putative interacting partners, and we verified these interactions in vivo by purification of TAP-tagged derivatives of Spc24 and Ndc80. Finally, we found that spc24 thermosensitive mutants had a chromosome segregation defect, but no apparent defect in SPB duplication. These results are consistent with recently published data showing that Spc24, Spc25 and Ndc80 are peripheral kinetochore com-ponents required for chromosome segregation. The Mps2-Spc24 interaction may contribute to the localization of Spc24 and other kinetochore components to the inner plaque of the SPB.

Chromosome Segregation↗
PubMed1994

Initiation of DNA synthesis by nuclei from scrape-ruptured quiescent mammalian cells in high-speed supernatants of Xenopus egg extracts.

Demembranated sperm heads, detergent-isolated somatic nuclei and even naked DNA are efficiently replicated in cytoplasmic extracts of activated amphibian eggs, but only after nuclear assembly and the formation of an intact nuclear envelope. DNA synthesis has not previously been shown to be initiated in high-speed (200,000 g) supernatants of egg cytoplasm because they are depleted of the vesicular material required to support nuclear envelope formation. Here we show that mammalian nuclei prepared by scrape-rupture are able to initiate DNA replication in such high-speed supernatants. These nuclei begin DNA synthesis asynchronously. This asynchrony cannot be attributed to differences in the time taken for nuclear assembly. Instead, we suggest that the asynchrony reflects intrinsic differences between nuclei and that these differences are a major cause of cell cycle variability. Our demonstration of initiation in high-speed supernatants now enables the initiation of eukaryotic DNA synthesis to be studied independently of nuclear assembly.

Animals↗
PubMed2005

Localization of the microtubule end binding protein EB1 reveals alternative pathways of spindle development in Arabidopsis suspension cells.

In a previous study on Arabidopsis thaliana suspension cells transiently infected with the microtubule end binding protein AtEB1a-green fluorescent protein (GFP), we reported that interphase microtubules grow from multiple sites dispersed over the cortex, with plus ends forming the characteristic comet-like pattern. In this study, AtEB1a-GFP was used to study the transitions of microtubule arrays throughout the division cycle of cells lacking a defined centrosome. During division, the dispersed origin of microtubules was replaced by a more focused pattern with the plus end comets growing away from sites associated with the nuclear periphery. The mitotic spindle then evolved in two quite distinct ways depending on the presence or absence of the preprophase band (PPB): the cells displaying outside-in as well as inside-out mitotic pathways. In those cells possessing a PPB, the fusion protein labeled material at the nuclear periphery that segregated into two polar caps, perpendicular to the PPB, before nuclear envelope breakdown (NEBD). These polar caps then marked the spindle poles upon NEBD. However, in the population of cells without PPBs, there was no prepolarization of material at the nuclear envelope before NEBD, and the bipolar spindle only emerged clearly after NEBD. Such cells had variable spindle orientations and enhanced phragmoplast mobility, suggesting that the PPB is involved in a polarization event that promotes early spindle pole morphogenesis and subsequent positional stability during division. Astral-like microtubules are not usually prominent in plant cells, but they are clearly seen in these Arabidopsis cells, and we hypothesize that they may be involved in orienting the division plane, particularly where the plane is not determined before division.

Arabidopsis↗
PubMed1999

Nucleolar segregation during apoptosis of haemopoietic stem cell line FDCP-Mix.

The programmed elimination of cells during apoptosis is distinct from necrosis both morphologically and biochemically. Currently, the morphological description of apoptosis discriminates between the segregation of the nucleolus and the so called 'chromatin condensation'. The latter originates from observations of electron dense material adjacent to the nuclear envelope of apoptotic nuclei. Although there is ample evidence for an involvement of DNA in electron dense marginations, their true nature is still unknown. By studying apoptosis in FDCP-Mix, a pluripotent murine haemopoietic stem cell line, we found morphological and histochemical evidence that electron dense material at the nuclear envelope (NE) has emerged as a result of the segregation of nucleoli in association with the nuclear membrane. The remaining electron dense and homogenous bulk of the nucleolus labels for RNAse-gold, but even more intensely for DNAse-gold, and therefore could possibly be mistaken as 'condensed chromatin' in the light microscope. The labelling of the electron dense material for DNase-gold in FDCP-Mix could be explained by a migration of DNA into the bulk of the nucleoli at an early stage of cell death.

Animals↗
PubMed1985

Analysis of inactive X chromosome structure by in situ nick translation.

Nick translation assays of fixed interphase female fibroblasts with tritiated nucleotides demonstrated a characteristic absence of label over sex chromatin. The chromatin bodies were nearly always peripheral in location and a ribbon of nick translatable DNA was detected between the sex chromatin and the nuclear envelope. High voltage electron microscopy indicated the possibility of a special nuclear envelope attachment region. The apparent resistance of sex chromatin to nick translation did not appear to be due to resistance to DNase I attack.

Cells, Cultured↗