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PubMed2002

Electron microscopic observations of karyogamy in the fish egg.

In the initial step of pronuclear association in fertilized fish eggs, the female and male pronuclei (containing large nucleolus-like bodies) were juxtaposed in the center of the blastodisc and formed nucleoplasmic projections along adjacent surfaces. After contact of the pronuclei, small internuclear bridges joining them were formed by fusion at several regions of the nuclear envelope projections. No specific site of fusion or breakdown of nuclear envelopes was recognized in the pronuclei during karyogamy. In the advanced stage, clumps of condensing chromatin appeared in the nucleoplasm of the newly fused pronuclei. The number and diameter of the internuclear bridges increased gradually by progressive fusion in many regions, finally yielding a spherical zygote nucleus. Following complete formation of the zygote nucleus, the pronuclear envelope began to break down concomitantly with shrinkage of the nucleoplasm, which was highly convoluted around the entire nuclear surface. The nucleoplasm containing chromosomes then mingled with the perinuclear cytoplasm.

Animals↗
PubMed1991

Postmitotic reassembly of the cell nucleus in whole cells: an electron-spectroscopic study.

By electron-spectroscopic imaging it is possible to visualize selectively the distribution of phosphorus-rich structures such as nucleosomes, ribosomes or other ribonucleoprotein particles. Using this method we re-examined assembly of the nucleus in telophase of dividing onion root cells and human HeLa cells. Our observations disagree considerably with conclusions drawn from work with cell-free systems. We consistently observed reassembly of nuclear envelope cisternae from vesicles in the cytoplasm without direct contact with chromatin. The preassembled envelope cisternae then enclosed the telophase chromosome mass, contacting the chromatin in some tracts, but also trapping cytoplasmic material such as ribosomes between chromosomes and envelope. Until a late stage in telophase the re-forming nuclear envelope left large gaps between the nuclear and the cytoplasmic compartments. Exclusion of cytoplasmic material from the re-assembling nucleus was facilitated by prenucleolar material, which accumulated in the deep furrows of the chromosomes and interchromosomal spaces. This material expanded considerably while the envelope was still open, in this way displacing cytoplasm non-selectively from the future nucleus. The model we propose for reassembly of the nucleus in whole cells does not postulate contact with and complete enclosure of chromosomes by the re-forming envelope, and suggests a decisive role for expanding prenucleolar material in the process of nucleocytoplasmic compartmentalization.

Cell Nucleolus↗
PubMed1999

Taxol affects nuclear lamina and pore complex organization and inhibits import of karyophilic proteins into the cell nucleus.

Treatment of human carcinoma cells with Taxol induces focal unraveling of the nuclear lamina and extensive clustering or ectopic localization of the nuclear pore complexes. These striking aberrations develop when the cells are transferred to drug-free medium and are allowed to complete mitosis. As could be confirmed by terminal deoxynucleotidyl transferase-mediated nick end labeling assays, 4,6-diamidino-2-phenylindole staining, 5-bromo-2-deoxyuridine incorporation, and examination of the nuclear lamins by Western blotting, the malformation of the nuclear envelope is not a consequence of apoptosis or G1 arrest. In fact, Taxol-treated cells possessing a defective nuclear envelope remain alive and replication-competent for at least 24 h, undergoing programmed death 72 h after removal of the drug. While still in the nonapoptotic state, these cells lose the ability to import karyophilic proteins into the nucleus. Diminished nucleocytoplasmic transport through the nuclear pore complex can be readily demonstrated by in vitro assays involving digitonin-permeabilized cells or in vivo monitoring of nuclear factor-kappaB translocation upon stimulation with tumor necrosis factor-alpha. These observations reveal novel cellular targets of antimicrotubule drugs and may pave the way for improved schemes of anticancer treatment.

Apoptosis↗
PubMed1976

[Electron microscopic study of the nuclear membrane of SPEV cells during mitosis. II. Reconstruction of the nuclear membrane (anaphase and telophase)].

Reformation of the nuclear envelope of the PKLV cells starts in the early anaphase when numerous contacts between membranous elements and chromosomes become visible. The nuclear envelope reforms, first, on the periphery of chromosomes and in chromosome's centromeric regions, and later - in the telomeric regions. The latest reconstruction of nuclear envelope occurs in spaces where microtubules come up close to the "nuclei", The appearance of normal pore complexes in the late telophase is preceded by the appearance, in the early and middle telophase, of pores with unusual structure.

Cell Line↗
PubMed2006

Association of PP1 with its regulatory subunit AKAP149 is regulated by serine phosphorylation flanking the RVXF motif of AKAP149.

Reformation of the nuclear envelope at the end of mitosis involves the recruitment of the B-type lamin phosphatase PP1 to nuclear membranes by A-kinase anchoring protein AKAP149. PP1 remains associated to AKAP149 throughout G1 but dissociates from AKAP149 when AKAP149 is phosphorylated at the G1/S transition. We examine here the role of phosphorylation of serines flanking the RVXF PP1-binding motif of AKAP149, on PP1 anchoring. The use of AKAP149 peptides encompassing the RVXF motif and five flanking serines, either wild type (wt) or bearing S-->A or S-->D mutations, specifically shows that phosphorylation of S151 or S159 abolishes PP1 binding to immobilized AKAP149. Peptides with S151 or S159 as the only wt serine residue trigger dissociation of PP1 from immunoprecipitated AKAP149, whereas S151/159D mutants are ineffective. Furthermore, immunoprecipitated AKAP149 from purified G1-phase nuclear envelopes binds PKA and PKC in overlay assays. PKA binding to AKAP149 in vitro is unaffected by the presence of PKC or PP1, and similarly, PKC binding is independent of PKA or PP1. The immunoprecipitated AKAP149 complex contains PKA and PKC activities. Both AKAP149-associated PKA and PKC serine-phosphorylate immunoprecipitated AKAP149 in vitro; however, only PKC-mediated phosphorylation promotes dissociation of PP1 from the AKAP. The results suggest a putative temporally and spatially controlled mechanism promoting release of PP1 from AKAP149. AKAP149 emerges as a scaffolding protein for multiple protein kinases and phosphatases that may be involved in the integration of intracellular signals that converge at the nuclear envelope.

A Kinase Anchor Proteins↗
PubMed1964

ELECTRON MICROSCOPY OF MITOSIS IN A RADIOSENSITIVE GIANT AMOEBA.

Various aspects of the ultrastructure of the dividing nuclei in the large radiosensitive amoeba Pelomyxa illinoisensis are demonstrated. Evidence of nuclear envelope breakdown is presented, and membrane fragments are traced throughout metaphase to envelope reconstruction in anaphase and telophase. Annuli in the nuclear envelope and its fragments are shown throughout mitosis. During metaphase and anaphase some 15 to 20 mitochondria are aligned at each end of the spindle, and are called polar mitochondria. The radioresistant amoebae Pelomyxa carolinensis and Amoeba proteus do not have polar mitochondria, and Pelomyxa illinoisensis is unique in this regard. The shape of the P. illinoisensis interphase nucleoli differs from that in the two radioresistant species, and certain aspects of nucleolar dissolution in the prophase vary. Helical coils in the interphase nucleoplasm are similar to those in the radioresistant amoebae. A "blister" phase in the flatly shaped telophase nuclei of P. illinoisensis is described which is interpreted to be the result of a rapid nuclear expansion leading to the formation of the normal spherical interphase nuclei.

Amoeba↗
PubMed1997

Mediators of nuclear protein import target karyophilic proteins to pore complexes of cytoplasmic annulate lamellae.

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes of Xenopus laevis. In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin beta and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central "transporter" and closely resemble those of "transport intermediates" found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.

Animals↗
PubMed2003

Effects of charged cluster mutations on the function of herpes simplex virus type 1 UL34 protein.

Herpes simplex virus type 1 (HSV-1) is a DNA virus that acquires an envelope by budding into the inner nuclear membrane of an infected cell. Recombinant HSV-1 lacking the U(L)34 gene cannot undergo this event. U(L)34 and U(L)31, another viral protein, colocalize in an infected cell and are necessary and sufficient to target both proteins to the inner nuclear envelope. In order to define and characterize sequences of U(L)34 that are necessary for primary envelopment to occur, a library of 19 U(L)34 charged cluster mutants and a truncation mutant lacking the putative transmembrane domain (DeltaTM) were generated. Mutants in this library were analyzed in a complementation assay for their ability to function in the production of infectious virus. Seven of the mutants failed to complement a U(L)34-null virus. The remainder of the mutants complemented at or near wild-type U(L)34 levels. Failure of a mutant protein to function might be the result of incorrect subcellular localization. To address this possibility, confocal microscopy was used to determine the localization of the U(L)34 protein in charged cluster mutants and DeltaTM. In transfection-infection experiments, all of the functional U(L)34 mutants and four of the six noncomplementing mutants localized to the inner nuclear envelope in a manner indistinguishable from that of wild-type U(L)34. All of the noncomplementing U(L)34 mutants mediated proper localization of U(L)31. Charged clusters critical for U(L)34 function are dispersed throughout the protein sequence and do not correlate well with highly conserved regions of the protein. These data suggest that U(L)34 has at least one function in addition to mediating proper localization of U(L)31 in infected cells and provide further support for the role of U(L)34 in mediating proper localization of U(L)31 in infected cells.

Amino Acid Sequence↗
PubMed2005

High-resolution near-field optical imaging of single nuclear pore complexes under physiological conditions.

Scanning near-field optical microscopy (SNOM) circumvents the diffraction limit of conventional light microscopy and is able to achieve optical resolutions substantially below 100 nm. However, in the field of cell biology SNOM has been rarely applied, probably because previous techniques for sample-distance control are less sensitive in liquid than in air. Recently we developed a distance control based on a tuning fork in tapping mode, which is also well-suited for imaging in solution. Here we show that this approach can be used to visualize single membrane protein complexes kept in physiological media throughout. Nuclear envelopes were isolated from Xenopus laevis oocytes at conditions shown recently to conserve the transport functions of the nuclear pore complex (NPC). Isolated nuclear envelopes were fluorescently labeled by antibodies against specific proteins of the NPC (NUP153 and p62) and imaged at a resolution of approximately 60 nm. The lateral distribution of epitopes within the supramolecular NPC could be inferred from an analysis of the intensity distribution of the fluorescence spots. The different number densities of p62- and NUP153-labeled NPCs are determined and discussed. Thus we show that SNOM opens up new possibilities for directly visualizing the transport of single particles through single NPCs and other transporters.

Animals↗
PubMed1984

Changes in nuclear antigens during reactivation of chick erythrocyte nuclei in heterokaryons.

Reactivation of dormant chick erythrocyte (CE) nuclei was studied by fusing chick red cells with rat myoblasts, HeLa cells, and chick fibroblasts. Heterokaryons representing different stages of nuclear reactivation were fixed and examined for nuclear antigens using polyclonal patient autoantisera reacting with mammalian (human, mouse, and rat) nuclear envelope, nucleoplasm, and chromatin (DNA) antigens. Reactivation of CE nuclei was associated with marked changes in nuclear antigenicity. In rat myoblast and HeLa heterokaryons the CE nuclei acquired mammalian nucleoplasmic and nuclear envelope antigens in the corresponding nuclear subcompartments. Drastic changes in nuclear antigenicity were noted also in heterokaryons stained with DNA antisera. The compact chromatin of nuclei in mature chick erythrocytes showed little binding of DNA antibodies. Isolated nuclei on the other hand gave a strong immunofluorescence. CE nuclei in heterokaryons were strongly positive during the early stages of nuclear reactivation but then exhibited decreased reactivity. An unexpected finding was a marked reduction in the capacity of mammalian nuclei in heterokaryons to bind DNA-antibodies. This observation is discussed in relation to the previous finding that in CE-heterokaryons these nuclei often show reduced transcription and replication. The present results indicate that in heterokaryons both types of nuclei exchange macromolecules of regulatory importance via the common cytoplasm.

Animals↗
PubMed2001

A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim.

Ran is a small signaling GTPase that is involved in nucleocytoplasmic transport. Two additional functions of animal Ran in the formation of spindle asters and the reassembly of the nuclear envelope in mitotic cells have been recently reported. In contrast to Ras or Rho, Ran is not associated with membranes. Instead, the spatial sequestering of its accessory proteins, the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1, appears to define the local concentration of RanGTP vs. RanGDP involved in signaling. Mammalian RanGAP is bound to the nuclear pore by a mechanism involving the attachment of small ubiquitin-related modifier protein (SUMO) to its C terminus and the subsequent binding of the SUMOylated domain to the nucleoporin Nup358. Here we show that plant RanGAP utilizes a different mechanism for nuclear envelope association, involving a novel targeting domain that appears to be unique to plants. The N-terminal WPP domain is highly conserved among plant RanGAPs and the small, plant-specific nuclear envelope-associated protein MAF1, but not present in yeast or animal RanGAP. Confocal laser scanning microscopy of green fluorescent protein (GFP) fusion proteins showed that it is necessary for RanGAP targeting and sufficient to target the heterologous protein GFP to the plant nuclear rim. The highly conserved tryptophan and proline residues of the WPP motif are necessary for its function. The 110-aa WPP domain is the first nuclear-envelope targeting domain identified in plants. Its fundamental difference to its mammalian counterpart implies that different mechanisms have evolved in plants and animals to anchor RanGAP at the nuclear surface.

Amino Acid Sequence↗
PubMed1984

Permeability properties of mammalian cell nuclei in living cells and in vitro.

The present study evaluates the role of the nuclear envelope in mammalian cells by applying two different approaches using either intact cells of mouse liver or isolated nuclei. In one approach the nuclei were studied with microelectrodes. The transmembrane voltage drop produced by passing current through an impaling microelectrode was measured with a second impaling microelectrode. In the second approach, the permeability of the nuclear envelope was studied by injection of a series of fluorescent probes. Lucifer Yellow CH and a variety of exogenous proteins labelled by conjugation with Lucifer Yellow VS were delivered into either the cytoplasm or the nucleus in situ. The fluorescence of the probe was followed either with a video camera or photographically. The results agree with the idea that the mammalian nuclear envelope is permeable to rather large molecules. Molecules with estimated radii below 2.4 nm seem to exchange rapidly, whereas molecules with estimated radii of 2.8 nm or above are excluded. The low electrical resistance of the envelope yields an estimate of pore radius, in the range of 3.4-6.5 nm.

Animals↗
PubMed2004

Visualization of a highly organized intranuclear network of filaments in living mammalian cells.

For 30 years, the mammalian cell nucleus has been hypothesized to contain a filamentous framework, the nuclear matrix or karyoskeleton, which regulates nuclear structure and function. However, such an organized network of filaments has never been observed in living cells. Here we show that human Cdc14B phosphatase in living cells tightly associates with long filaments that begin at the nucleolar periphery and extend to the nuclear envelope, frequently making close connections with nuclear pore complexes. We demonstrate that Cdc14B contains a bipartite signal that directs it to the intranuclear filaments, and we also detect a small amount of Cdc14B on interphase and mitotic centrosomes. Furthermore, we show that Cdc14B is critical for the maintenance of proper nuclear structure together with polo-like kinase Plk1. This work provides the first direct evidence for the existence of an intranuclear filamentous framework in living mammalian cells and implicates Cdc14B in the control of mammalian nuclear architecture.

Animals↗
PubMed1984

Effects of prolonged antitubulin culture on annulate lamellae in mouse alpha L929 fibroblasts.

The fine structure of alpha L929 fibroblasts cultured in colchicine or vinblastine sulfate for periods as long as 48 hr was compared to control cells not exposed to antitubulins . In response to prolonged antitubulin culture, several changes in cell ultrastructure were noted: Control fibroblasts contain cytoplasmic annulate lamellae (AL), but prolonged exposure to either vinblastine sulfate or colchicine results in enhanced development of AL. Single pore complexes are present in the rough-surfaced endoplasmic reticulum (rER) in both control and antitubulin-treated cells, but stacked porous cytomembranes also occur under both conditions. Polyribosomes often are closely associated or continuous with the pore complexes. Many antitubulin-treated cells become multinucleate. Some nuclei in both control and antitubulin-treated cells contain large and multiple nucleoli. The large and multiple nucleoli are either attached directly to the inner membrane of the nuclear envelope or to infoldings of the nuclear envelope. Antitubulin-treated cells, after 48-hr exposure, appear also to contain enhanced quantities of smooth-surfaced endoplasmic reticulum (sER) and cytoplasmic filaments (and in some cells, lysosomes and rER as well) when compared to untreated cells. In both control and colchicine-treated cells, AL can exhibit continuity with either rER or sER. Further, all three membrane systems may at times be continuous, but the quantity of these membranes appears to be greater in colchicine-treated cells than in control cells. The results are discussed with respect to possible functional significance.

Animals↗
PubMed1992

Phospholipids in the nucleus--metabolism and possible functions.

Most of the phospholipids in the nuclear envelope are contained in the double nuclear membrane, and this has an active lipid metabolism consistent with its origins as a component of the endoplasmic reticular system. However, even after removal of the nuclear membrane with detergents, some phospholipids, mostly of unknown location and function, remain. Amongst these are all of the components of what appears to be a nuclear polyphosphoinositide signalling system, distinct from the well-established inositide pathway found in the plasma membrane. The consequences for nuclear function of the activation of these two inositide pathways are discussed, with a detailed consideration of proposed intranuclear functions for protein kinase C, and the maintenance of nuclear Ca2+ homoeostasis.

Animals↗
PubMed1998

Cse1p is required for export of Srp1p/importin-alpha from the nucleus in Saccharomyces cerevisiae.

In metazoan cells, the CAS protein has been shown to function as a recycling factor for the importin-alpha subunit of the classical nuclear localization signal receptor, exporting importin-alpha from the nucleus to allow its participation in multiple rounds of nuclear import. CAS is a member of a family of proteins that bear homology to the larger subunit of the nuclear localization signal receptor, importin-beta, and that are found in all eukaryotes from yeast to humans. Sequence similarity identifies the product of the Saccharomyces cerevisiae CSE1 gene as a potential CAS homologue. Here we present evidence that Cse1p is the functional homologue of CAS: Cse1p is required to prevent accumulation of Srp1p/importin-alpha in the nucleus, it localizes to the nuclear envelope in a pattern typical of nuclear transport receptors, and it associates in vivo with Srp1p in a nucleotide-specific manner. We show further that mutations in CSE1 and SRP1 have specific effects on their association and on the intracellular localization of Cse1p.

Biological Transport↗
PubMed1970

Nuclear membranes from mammalian liver. II. Lipid composition.

The qualitative and quantitative lipid composition of nuclei and nuclear membranes from pig and rat liver were determined. These determinations were compared with the corresponding data obtained for microsomes from the same material after similar treatments. The results indicate that, at least, by far the major part of the nuclear lipids is located in the membranes of the nuclear envelope. The phospholipid pattern of the nuclear membranes and the endoplasmic reticulum (ER) membranes in general is widely identical in both species. As a striking difference in the lipid composition, however, a fourfold increase of esterified cholesterol in the nuclear membranes was found. In a quantitative approach the ratio of total surface area of the nuclear lipids to the total surface area of the nuclear envelope membranes was calculated as being 3.6, a value which fairly approximates the requirements of a bimolecular lipid leaflet model.

Animals↗
PubMed2002

Lamin-dependent localization of UNC-84, a protein required for nuclear migration in Caenorhabditis elegans.

Mutations in the Caenorhabditis elegans unc-84 gene cause defects in nuclear migration and anchoring. We show that endogenous UNC-84 protein colocalizes with Ce-lamin at the nuclear envelope and that the envelope localization of UNC-84 requires Ce-lamin. We also show that during mitosis, UNC-84 remains at the nuclear periphery until late anaphase, similar to known inner nuclear membrane proteins. UNC-84 protein is first detected at the 26-cell stage and thereafter is present in most cells during development and in adults. UNC-84 is properly expressed in unc-83 and anc-1 lines, which have phenotypes similar to unc-84, suggesting that neither the expression nor nuclear envelope localization of UNC-84 depends on UNC-83 or ANC-1 proteins. The envelope localization of Ce-lamin, Ce-emerin, Ce-MAN1, and nucleoporins are unaffected by the loss of UNC-84. UNC-84 is not required for centrosome attachment to the nucleus because centrosomes are localized normally in unc-84 hyp7 cells despite a nuclear migration defect. Models for UNC-84 localization are discussed.

Animals↗