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PubMed1997

Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis.

The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase alpha, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.

Animals↗
PubMed2000

Monoclonal antibodies to NTF2 inhibit nuclear protein import by preventing nuclear translocation of the GTPase Ran.

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.

Animals↗
PubMed2005

A putative nucleoporin 96 Is required for both basal defense and constitutive resistance responses mediated by suppressor of npr1-1,constitutive 1.

The Arabidopsis thaliana suppressor of npr1-1, constitutive 1 (snc1) mutant contains a gain-of-function mutation in a Toll Interleukin1 receptor-nucleotide binding-Leu-rich repeat-type resistance gene (R-gene), which leads to constitutive activation of disease resistance response against pathogens. In a screen for suppressors of snc1, a recessive mutation, designated mos3 (for modifier of snc1,3), was found to suppress the constitutive pathogenesis-related gene expression and resistance to virulent Pseudomonas syringae maculicola ES4326 and Peronospora parasitica Noco2 in snc1. In addition, mos3 is also compromised in resistance mediated by Resistance to Peronospora parasitica 4 (RPP4), Resistance to Pseudomonas syringae pv maculicola (RPM1), and Resistance to Pseudomonas syringae 4 (RPS4). Single mutant mos3 plants exhibited enhanced disease susceptibility to P. s. pv maculicola ES4326, suggesting that MOS3 is required for basal resistance to pathogens as well. mos3-1 was identified by map-based cloning, and it encodes a protein with high sequence similarity to human nucleoporin 96. Localization of the MOS3-green fluorescent protein fusion to the nuclear envelope further indicates that MOS3 may encode a nucleoporin, suggesting that nuclear and cytoplasmic trafficking plays an important role in both R-gene-mediated and basal disease resistance.

Active Transport, Cell Nucleus↗
PubMed2003

Knockout mice provide novel insights into meiotic chromosome and telomere dynamics.

Meiosis is a succession of two specialized cell divisions that leads to the formation of gametes and thereby compensates for genome doubling at fertilization. During the extended prophase of the first meiotic division chromosomes assemble protein cores (axial elements) that attach their ends to the nuclear envelope. These ends transiently gather at a limited sector of the nuclear periphery (bouquet stage) at a time when meiotic recombination is initiated and when chromosomes initiate stable pairing (synapsis). This review discusses novel insights into the relation between recombinational DNA repair and meiotic telomere dynamics that have arrived from recent studies of transchromosomal mice and knockout mice. Analysis of mice deficient for A-type lamins, histone H2AX, Suv39h HMTases, and the AE protein SYCP3 suggests that entry into prophase I requires heterochromatin integrity and lamin A expression. Initiation of meiotic telomere clustering represents an early recombination-independent event in first meiotic prophase, while exit from the bouquet stage depends on signals that emanate from the progress of recombinational DNA repair as sensed by ATM kinase and relayed through histone H2AX.

Animals↗
PubMed1988

Presence of intranuclear inclusions in the principal cells of the epididymis of the garden dormouse Eliomys quercinus L.

Globular inclusions are present in the nuclei of the columnar epithelial cells lining the ductus epididymidis in the garden dormouse, Eliomys quercinus L. Such inclusions have been reported in the dog where they are very abundant, in the stallion and in man. They have been found to be absent from sheep, cat, rabbit, guinea pig, rat and mouse. We have now found them in the garden dormouse, an ascrotal hibernator, but have been unable to detect them in another hibernator, the thirteen-lined ground squirrel Citellus tridecemlineatus, this time a scrotal mammal. The globular inclusions are first seen in the perinuclear space formed by the two membranes of the nuclear envelope; they are then invested by the inner membrane of the nuclear envelope and transferred into the nucleus. They later disintegrate and the vacuole is finally left with an electron-transparent content. The number of inclusions varies with the hormonal status of the animal: the granules are always present as soon as there is a rise in plasma testosterone. Electron energy-loss spectroscopy in the electron microscope (EELS) shows that inclusions contain C, N, O, Fe, Mg, P and S. This suggests that the intranuclear granules are made of protein cross-linked by disulphur bonds and contain iron, magnesium and phosphorus.

Animals↗
PubMed1976

Some ultrastructural aspects of mitosis and early differentiation in human foetal lung.

In rapidly dividing human foetal lung cells there is evidence of synthesis of extra membrane prior to mitosis. During prophase the nuclear envelope breaks down and is conserved as long double or complex stacks during the following stages of mitosis. It is suggested that the membrane is retained partly for incorporation into the daughter cell nuclear envelopes and partly for the formation of RER. The relevance of the observations to the biochemical data in the literature concerning the formation of intracellular membranes is discussed briefly.

Cell Differentiation↗
PubMed1978

Ultrastructural nuclear changes in human lymphocytes following arabinosylcytosine treatment in vitro.

Human lymphocytes were separated by centrifugation and grown for 48--72 h in TC 199 medium. Synchronous cells, obtained by the addition of colchicine, were treated with arabinosylcytosine (Ara-C), an antimitotic substance which is known to produce nuclear changes in vivo and in vitro. In this case, alterations in the nuclear envelope closely resembling the malignancy associated changes described in human leukaemic cells were observed. The results allow us to suggest that such alterations may be due to a cellular derangement involving the renewal of the nuclear envelope in th G1 phase of the mitotic cycle.

Cell Nucleus↗
PubMed2000

Serine base-exchange in rat liver nuclei.

It has been shown that the incorporation of [(14)C]serine into phosphatidylserine (PS) in isolated rat liver nuclei is intrinsic to this organelle as attested by marker enzyme activity. Serine incorporation into PS was the highest in nuclei depleted of the outer membrane of the nuclear envelope (nucleoplasts) and negligible in the outer membrane. Trypsin treatment of nucleoplasts caused a strong inactivation of PS synthesis and only a moderate one of the NAD pyrophosphorylase activity, the marker enzyme of the inner nuclear membrane. We suggest that the serine base-exchange enzyme is located in the inner membrane of the nuclear envelope and accessible from the periplasmic surface of this membrane.

Animals↗
PubMed2003

The C. elegans hook protein, ZYG-12, mediates the essential attachment between the centrosome and nucleus.

The centrosome and nucleus are intimately associated in most animal cells, yet the significance of this interaction is unknown. Mutations in the zyg-12 gene of Caenorhabditis elegans perturb the attachment of the centrosome to the nucleus, giving rise to aberrant spindles and ultimately, DNA segregation defects and lethality. These phenotypes indicate that the attachment is essential. ZYG-12 is a member of the Hook family of cytoskeletal linker proteins and localizes to both the nuclear envelope (via SUN-1) and centrosomes. ZYG-12 is able to bind the dynein subunit DLI-1 in a two-hybrid assay and is required for dynein localization to the nuclear envelope. Loss of dynein function causes a low percentage of defective centrosome/nuclei interactions in both Drosophila and Caenorhabditis elegans. We propose that dynein and ZYG-12 move the centrosomes toward the nucleus, followed by a ZYG-12/SUN-1-dependent anchorage.

Alternative Splicing↗
PubMed1981

Microtubule-organizing centres and assembly of the double-spiral microtubule pattern in certain heliozoan axonemes.

The double-spiral microtubule pattern is established by a self-linkage procedure when axopodial axonemes reassemble after cold treatment in multinucleate Echinosphaerium nucleofilum and mononucleate Actinophrys sol. Nuclei are related spatially to axoneme morphogenesis in both organisms but in rather different ways. Microtubules grow out in all directions from discrete clumps of dense material situated close to nuclei in E. nucleofilum as axonemal assembly begins. Each clump acts as a microtubule-organizing centre (MTOC) in so far as it is associated spatially with the assembly of microtubules for a single axoneme. The dense material spreads along the sides of a developing axoneme for several micrometers, where it probably promotes further microtubule assembly as the double-spiral pattern is established. Pattern is generated as microtubules that are randomly oriented to begin with become more closely juxtaposed and aligned with each other. There are indications that juxtaposition is brought about by the contractile action of a filamentous meshwork that interconnects the microtubules. Final positioning and alignment appears to be accomplished by a 'zippering' together of adjacent portions of microtubules that proceeds in both directions along the lengths of developing axonemes as self-linkage is effected. Considerable numbers of more or less radially oriented microtubules remain and project from the surface membrane of the single central nucleus during cold treatment of A. sol. Additional tubules assemble and become associated similarly with the nuclear envelope immediately after cold treatment. Initially these microtubules are not arranged in a double-spiral pattern, which is subsequently generated by procedures similar to those outlined above for E. nucleofilum. It is suggested that the surface of the nuclear envelope may act as an MTOC.

Animals↗
PubMed1979

In vitro studies of rickettsia-host cell interactions: ultrastructural changes induced by Rickettsia rickettsii infection of chicken embryo fibroblasts.

Secondary chicken embryo fibroblasts infected with the Sheila Smith strain of Rickettsia rickettsii and grown in monolayer culture undergo rapid morphological alterations. Transmission electron microscopic examination of cells at intervals after infection showed several progressive host cell lesions, including widespread dilatation of the rough endoplasmic reticulum and outer nuclear envelope and the accumulation of electron-dense material within the cisternae of intracellular membranes. Dilatation of the rough endoplasmic reticulum is a common, early reversible manifestation of other forms of cell injury. However, the severity of the damage to the host cell resulting from the progressive distention of intracellular membranes and the subsequent formation of small segments of membrane-bound host cytoplasm within the cisternae of these membranes is unknown. Early in the infection cycle, the rickettsiae were found free in the host cell cytoplasm, within invaginations of the nuclear envelope, occasionally free in the space between the outer and inner nuclear membranes, and in the host nucleoplasm, but not within cisternae formed by swollen endoplasmic reticulum. As a consequence of intracisternal swelling and fusion of intracellular membranes later in the infection cycle, the majority of the rickettsiae were found surrounded by host cytoplasm bound by host-derived internal membranes and appeared to persist in this state until cell lysis. The overall cytopathological changes in cells infected with R. richettsii appear dramatic and, from other studies in our laboratory, are significantly different from those observed in cells infected with Rickettsia prowazekii.

Animals↗
PubMed1998

The nucleus of HeLa cell contains tubular structures for Ca2+ signalling.

It has long been assumed that Ca2+ are translocated from the cytosol to the cell nucleus by a long distance to activate transcription machinery buried deep in the nucleoplasm. However, this model has been recently challenged. When HeLa cells were loaded with fluo-3, highly fluorescent spots of approximately 2 microns in diameter were observed in the cell nucleus while the fluo-3 signals were low in their neighbouring nucleoplasm as determined by confocal microscopy. These fluorescent spots were devoid of but usually associated with chromatin on their boundary. When cells were stimulated by ionomycin (1 microM), the fluo-3 fluorescence in these spots increased faster than that in their neighbouring nucleoplasm. In another experiment, optical sections with hot spot(s) were used to construct 3-D images to study the morphology of the hot spots. Views of reconstruction from different angles indicated that the hot spots formed a tubular structure with a connection to the nucleocytoplasmic interface. Moreover, injection of calcium green-dextran (70 kDa), a Ca(2+)-sensitive indicator conjugated with an inert molecule of large molecular size, into the cytosol leads to a formation of signals also in a tubular shape inside the nucleoplasm. This suggests that the 'channels' are real inside the nucleus and they are derived from an invagination of the double-membraned nuclear envelope. Taken together, our results indicate (1) tubular structures are found inside the cell nucleus; (2) they are extended from the cytosol into the nucleus through the invagination of the double membraned nuclear envelope; (3) molecules of molecular size up to 70 kDa could penetrate into these 'tunnels'; (4) Ca2+ can be released or transported into the cell nucleus through these tubular structures after ionomycin stimulation; and (5) the structures are usually associated with chromatin.

Aniline Compounds↗
PubMed1981

Distribution of kinetochore (centromere) antigen in mammalian cell nuclei.

Antigens associated with mammalian centromeres were localized at the high and electron microscopic levels using the peroxidase-labeled antibody method. The antibody used was of a type naturally occurring in the sera of patients with scleroderma. At the light microscopic level, it reacts specifically with the centromere regions of chromosomes in a variety of mammalian species and strains in discrete foci in interphase nuclei. We find that the number of foci approximates the number of chromosomes present in the various cell types. At the ultrastructural level, the antigenic foci are confirmed to lie in the kinetochore regions of each chromosome. In interphase nuclei, the antigenic foci were usually associated either with the inner surfaces of the nuclear envelope or with the nucleoli. These observations indicate that the centromere regions of the chromosomes in interphase are not randomly distributed within the nucleus but are usually fixed either to the inner surface of the nuclear envelope or to nucleoli.

Animals↗
PubMed1978

Association of virus specific replicative ribonucleic acid with nuclear membrane in chick embryo cells infected with japanese encephalitis virus.

Using high resolution electron microscopic autoradiography and velocity sedimentation, RNA synthesis was examined in chick embryo cells infected with Japanese encephalitis virus (JEV). RNA was labelled with 3H-uridine for 5 min or 10 min at 15 h after infection in the presence of actinomycin D and D-glucosamine. Microautoradiography showed significantly numbers of silver grains on the nuclear membranes of 5 min pulse-labelled thin cell sections. The RNA species in membrane fractions obtained from the nucleus and cytoplasm of the infected cells were analysed by sucrose density gradient sedimentation. Radioactive 23S replicative form RNA and 8-12S RNA were obtained from the outer membrane fractions of the nuclear envelope. Labelled 42S virus RNA was obtained from the fractions containing large vesicle membranes and plasma membranes. These results suggest that JEV-RNA synthesis is initiated in the perinuclear region in close association with the outer membranes of the nuclear envelope.

Animals↗
PubMed2003

ANChors away: an actin based mechanism of nuclear positioning.

Mechanisms for nuclear migration and nuclear anchorage function together to control nuclear positioning. Both tubulin and actin networks play important roles in nuclear positioning. The actin cytoskeleton has been shown to position nuclei in a variety of systems from yeast to plants and animals. It can either act as a stable skeleton to anchor nuclei or supply the active force to move nuclei. Two C. elegans genes and their homologues play important roles in these processes. Syne/ANC-1 anchors nuclei by directly tethering the nuclear envelope to the actin cytoskeleton, and UNC-84/SUN functions at the nuclear envelope to recruit Syne/ANC-1.

Actin Cytoskeleton↗
PubMed1997

Using atomic force microscopy to investigate patch-clamped nuclear membrane.

Nuclear patch clamp is an emerging research field that aims to disclose the electrical phenomena underlying macromolecular transport across the nuclear envelope (NE), its properties as an ion barrier and its function as an intracellular calcium store. The authors combined the patch clamp technique with atomic force microscopy (AFM) to investigate the structure-function relationship of NE. In principle, patch clamp currents, recorded from the NE can indicate the activity of the nuclear pore complexes (NPCs) and/or of ion channels in the two biomembranes that compose the NE. However, the role of the NPCs is still nuclear because the observed NE current in patch clamp experiments is lower than expected from the known density of the NPCs. Therefore, AFM was applied to link patch clamp currents to structure. The membrane patch was excised from the nuclear envelope and, after electrical evaluation, transferred from the patch pipette to a substrate. We could identify the native nuclear membrane patches with AFM at a lateral and a vertical resolution of 3 nm and 0.1 nm, respectively. It was shown that complete NE together with NPCs can be excised from the nucleus after their functional identification in patch clamp experiments. However, we also show that membranes of the endoplasmic reticulum can contaminate the tip of the patch pipette during nuclear patch clamp experiments. This possibility must be considered carefully in nuclear patch clamp experiments.

Animals↗
PubMed1996

Autoantibodies against nucleoporin p62 constitute a novel marker of primary biliary cirrhosis.

BACKGROUND & AIMS: Patients with primary biliary cirrhosis (PBC) frequently produce autoantibodies against constituents of the nuclear envelope. We have recently observed a reactivity of PBC sera with a nuclear envelope antigen at approximately 60 kilodaltons. The aim of this study was to characterize the reactive antigen. METHODS: Sera from 103 patients with liver disease were tested by immunoblotting after the separation of proteins by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) using proteins of whole nuclei and nuclear pore complexes (NPCs). RESULTS: A nuclear immunofluorescence staining with peripheral accentuation was observed in 25 of 43 sera from patients with PBC. In immunoblotting, the sera displaying a peripheral pattern reacted preferentially with two proteins. Twelve sera showed specificity to an antigen of 210 kilodaltons corresponding to gp210, and 14 sera recognized a protein band at 60 kilodaltons. The intensity of the reactive band at 60 kilodaltons was clearly stronger on blots with immobilized proteins of the NPC than on blots with nuclear proteins, thereby indicating that the sera targeted an antigen as being a component of the NPC. Immunoblotting performed after protein separation by two-dimensional PAGE showed that the sera are directed against nucleoporin p62, a functional protein of the NPC. CONCLUSIONS: Anti-p62 antibodies appear to be a novel marker that is specific for PBC.

Autoantibodies↗
PubMed2004

Failure to assemble the peri-nuclear structures in GOPC deficient spermatids as found in round-headed spermatozoa.

Deletion of the GOPC gene encoding mouse GOPC (Golgi-associated PDZ- and coiled-coil motif-containing protein) causes infertile round-headed spermatozoa, which have acrosome-less round heads and deformed tails (Yao et al, 2002). This study investigated how GOPC deficient spermatids fail to assemble the peri-nuclear structures in round-headed spermatozoa during spermiogenesis in GOPC knockout mouse testes. In step 1-8 spermatids, Golgi-derived proacrosomal vesicles that are transported to the perinuclear region formed acrosome-like vesicles of various sizes, called pseudoacrosomes. The marginal ring of the acroplaxome, which is generally formed between the descending edge of a developing acrosome and nuclear envelope in a wild spermatid, was poorly formed between the pseudoacrosome and nuclear envelope. In step 9-11 elongating spermatids, a majority of pseudoacrosomes were detached from the nucleus and disappeared from the perinuclear region by spermiation. Concomitantly, several failures occurred on the nucleus, manchette, postacrosomal sheath (perinuclear theca), and posterior ring. Ectoplasmic specializations were poorly formed, and did not always associate with developing spermatids. Consequently, spermatid nuclear elongation to form round-headed spermatozoa developed was impaired. In addition to these sequential failures, the posterior ring deficiency was attributed to the tail deformation destined to occur during epididymal maturation as reported in an accompanying paper (Suzuki-Toyota et al, 2004 in this issue), its eventual phenotype being reminiscent of the round-headed spermatozoa of human infertile globozoospermia.

Adaptor Proteins, Signal Transducing↗