Selective elimination of acentric double minutes from cancer cells through the extrusion of micronuclei

Several lines of evidences from us or other authors had shown that tumor cells revert their phenotypes and differentiate by the elimination of oncogenes amplified on the acentric double minutes (DMs). The selective incorporation of DMs into the cytoplasmic micronuclei was thought to be involved in this elimination, however, the mechanism by which the content of micronuclei was eliminated from the cells remains to be discovered. In this report, we show the finding and the characterization of the extruded micronuclei in the culture fluid of human COLO 320DM tumor line, and suggest that the extrusion of micronuclei mediates the selective elimination of DMs. The extracellular micronuclei enriched with DMs had an apparently normal cytoplasmic membrane, decondensed chromatin and nuclear lamin protein, and their DNA did not suffer any extensive degradation. These characteristics were closely related to their cytoplasmic counterpart and clearly differentiated from the apoptotic bodies. We also developed a method for purifying the extracellular micronuclei. In this paper, the implications of the micronuclear extrusion are discussed.     

Remodeling the sperm nucleus into a male pronucleus at fertilization

After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male pronucleus. We have investigated the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin-binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed.     

From the nucleus to the plasma membrane: translocation of the nuclear proteins histone H3 and lamin B1 in apoptotic microglia

Nuclear autoantibodies have been found in patients with autoimmune diseases. One possible source for nuclear antigens are apoptotic cells. However, the mechanism of how apoptotic cells make nuclear factors accessible to the immune system is still elusive. In the present study, we investigated the redistribution of nuclear components after UV irradiation in the microglial cell line BV-2 and in primary mouse microglia at the ultrastructural level. We used transmission electron microscopy-coupled electron energy loss spectroscopy (EELS) to measure phosphorus as an indicator for nucleic acids and immunogold labeling to detect histone H3 and lamin B1 in apoptotic cells. EELS revealed elevated concentrations of phosphorus in nuclear and cytoplasmic condensed chromatin compared to the remaining cytoplasm. Furthermore, immunolabeling of lamin B1 and histone H3 was detected in apoptotic microglia not only in the nucleus, but also in the cytoplasm, and even at the plasma membrane. Confocal images of apoptotic microglia, which were not previously permeabilized, showed patches of histone H3 and lamin B1 labeling at the cell surface. The pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) prevented the occurrence of cytoplasmic condensed chromatin in apoptotic microglia. Our findings indicate that nuclear components leak from the nucleus into the cytoplasm in apoptotic microglia. At least histone H3 and lamin B1 reach the cell surface, this may promote autoreactive processes.     

Involvement of nuclear lamins in postmitotic reorganization of chromatin as demonstrated by microinjection of lamin antibodies

The nuclear lamins are major components of a proteinaceous polymer that is located at the interface of the nuclear membrane and chromatin; these lamins are solubilized and dispersed throughout the cytoplasm during mitosis. It has been postulated that these proteins, assembled into the lamina, provide an architectural framework for the organization of the cell nucleus. To test this hypothesis we microinjected lamin antibodies into cultured PtK2 cells during mitosis, thereby decreasing the soluble pool of lamins. The antibody injected was identified, together with the lamins, in cytoplasmic aggregates by immunoelectron microscopy. We show that microinjected cells are not able to form normal daughter nuclei, in contrast to cells injected with other immunoglobulins. Although cells injected with lamin antibodies are able to complete cytokinesis, the chromatin of their daughter nuclei remains arrested in a telophase-like configuration, and the telophase-like chromatin remains inactive as judged from its condensed state and by the absence of nucleoli. These results indicate that lamins and the nuclear lamina structure are involved in the functional organization of the interphase chromatin.     

Phosphorylation on protein kinase C sites inhibits nuclear import of lamin B2

The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.     

Mammalian autophagy degrades nuclear constituents in response to tumorigenic stress

During autophagy, double-membrane autophagosomes are observed in the cytoplasm. Thus, extensive studies have focused on autophagic turnover of cytoplasmic material. Whether autophagy has a role in degrading nuclear constituents is poorly understood. We reveal that the autophagy protein LC3/Atg8 directly interacts with the nuclear lamina protein LMNB1 (lamin B1), and binds to LMN/lamin-associated chromatin domains (LADs). Through these interactions, autophagy specifically mediates destruction of nuclear lamina during tumorigenic stress, such as by activated oncogenes and DNA damage. This nuclear lamina degradation upon aberrant cellular stress impairs cell proliferation by inducing cellular senescence, a stable form of cell-cycle arrest and a tumor-suppressive mechanism. Our findings thus suggest that, in response to cancer-promoting stress, autophagy degrades nuclear material to drive cellular senescence, as a means to restrain tumorigenesis. Our work provokes a new direction in studying the role of autophagy in the nucleus and in tumor suppression.     

Paternal cyclophosphamide exposure induces the formation of functional micronuclei during the first zygotic division

Paternal exposures to cancer chemotherapeutics or environmental chemicals may have adverse effects on progeny outcome that are manifested in the preimplantation embryo. The objectives of this study were to determine the impact of paternal exposure to cyclophosphamide, an anticancer alkylating agent, on the formation, chromatin origin and function of micronuclei in cleavage stage rat embryos. Male Sprague-Dawley rats were gavaged with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females to collect pronuclear zygotes and 2 to 8 cell embryos. Micronuclear chromatin structure was characterized using confocal microscopy to detect immunoreactivities for H3K9me3, a marker for maternal chromatin, and lamin B, a nuclear membrane marker. DNA synthesis was monitored using EdU (5-ethynyl-2'-deoxyuridine) incorporation. Fertilization by cyclophosphamide-exposed spermatozoa led to a dramatic elevation in micronuclei in cleavage stage embryos (control embryos: 1% to 5%; embryos sired by treated males: 70%). The formation of micronuclei occurred during the first zygotic division and was associated with a subsequent developmental delay. The absence of H3K9me3 indicated that these micronuclei were of paternal origin. The micronuclei had incomplete peri-nuclear and peri-nucleolar lamin B1 membrane formation but incorporated EdU into DNA to the same extent as the main nucleus. The formation of micronuclei in response to the presence of a damaged paternal genome may play a role in increasing the rate of embryo loss that is associated with the use of assisted reproductive technologies, parenthood among cancer survivors, and paternal aging.     

Spatial distribution of lamin A and B1 in the K562 cell nuclear matrix stabilized with metal ions.

When the nucleus is stripped of most DNA, RNA, and soluble proteins, a structure remains that has been referred to as the nuclear matrix, which acts as a framework to determine the higher order of chromatin organization. However, there is always uncertainty as to whether or not the nuclear matrix, isolated in vitro, could really represent a skeleton of the nucleus in vivo. In fact, the only nuclear framework of which the existence is universally accepted is the nuclear lamina, a continuous thin layer that underlies the inner nuclear membrane and is mainly composed of three related proteins: lamins A, B, and C. Nevertheless, a number of recent investigations performed on different cell types have suggested that nuclear lamins are also present within the nucleoplasm and could be important constituents of the nuclear matrix. In most cell types investigated, the nuclear matrix does not spontaneously resist the extraction steps, but must rather be stabilized before the application of extracting agents. In this investigation, by immunochemical and morphological analysis, we studied the effect of stabilization with different divalent cations (Zn(2+), Cu(2+), Cd(2+)) on the distribution of lamin A and B1 in the nuclear matrix obtained from K562 human erythroleukemia cells. In intact cells, antibodies to both lamin A and B1 mainly stained the nuclear periphery, although some immunoreactivity was detected in the nuclear interior. The fluorescent lamin A pattern detected in Cu(2+)- and Cd(2+)-stabilized nuclei was markedly modified, whereas Zn(2+)-incubated nuclei showed an unaltered pattern of lamin A distribution. By contrast, the distribution of lamin B1 in isolated nuclei was not modified by the stabilizing cations. When chromatin was removed by nuclease digestion and extraction with solutions of high ionic strength, a previously masked immunoreactivity for lamin A, but not for lamin B1, became evident in the internal part of the residual structures representing the nuclear matrix. Our results indicate that when metal ions are used as stabilizing agents for the recovery of the nuclear matrix, the distribution of both lamin A and lamin B1 in the final structures, corresponds to the pattern we have very recently reported using different extraction procedures. This observation strengthen the concept that intranuclear lamins may act as structural components of the nuclear matrix.     

Identification of a short nuclear lamin protein selectively expressed during meiotic stages of rat spermatogenesis

The nuclear lamina is a karyoskeletal structure located at the nuclear periphery and intimately associated with the inner nuclear membrane. It is composed of a multigene family of proteins, the lamins, which show a conspicuous cell type-specific expression pattern. The functional role of lamins has not been definitively established but available information indicates that they are involved in the organization of nuclear envelope and interphase chromatin. Spermatogenesis is characterized, among other features, by stage-specific changes in chromatin organization and function. These changes are accompanied by modifications in the organization and composition of the nuclear lamina. In previous experiments we have determined that rat spermatogenic cells express a lamin closely related, if not identical, to lamin B1 of somatic cells; whereas rat somatic lamins A, C, D and E were not detected. Considering that chromatin reorganizations during spermatogenesis may be directly or indirectly related to changes of the nuclear lamina we have decided to further investigate lamin expression during this process. Here we report on the identification of a 52 kDa protein of the rat which, according to immunocytochemical and biochemical data, appears to be a novel nuclear lamin. Using meiotic stage-specific markers, we have also demonstrated that this short lamin is selectively expressed during meiotic stages of spermatogenesis.     

Selective Entrapment of Extrachromosomally Amplified DNA by Nuclear Budding and Micronucleation during S Phase

Acentric, autonomously replicating extrachromosomal structures called double-minute chromosomes (DMs) frequently mediate oncogene amplification in human tumors. We show that DMs can be removed from the nucleus by a novel micronucleation mechanism that is initiated by budding of the nuclear membrane during S phase. DMs containing c-myc oncogenes in a colon cancer cell line localized to and replicated at the nuclear periphery. Replication inhibitors increased micronucleation; cell synchronization and bromodeoxyuridine–pulse labeling demonstrated de novo formation of buds and micronuclei during S phase. The frequencies of S-phase nuclear budding and micronucleation were increased dramatically in normal human cells by inactivating p53, suggesting that an S-phase function of p53 minimizes the probability of producing the broken chromosome fragments that induce budding and micronucleation. These data have implications for understanding the behavior of acentric DNA in interphase nuclei and for developing chemotherapeutic strategies based on this new mechanism for DM elimination.     

Nuclear envelope dynamics during male pronuclear development

Upon fertilization, the sperm nucleus undergoes reactivation. The poreless sperm nuclear envelope is replaced by a functional male pronuclear envelope and the highly compact male chromatin decondenses. Here some recent evidence is examined: that disassembly of the sperm lamina is required for chromatin decondensation, that remnant portions of the sperm nuclear envelope target the binding of egg membrane vesicles that form the male pronuclear envelope, that functional male pronuclear envelopes containing lamin B receptor assemble prior to lamin import and lamina formation, and that lamina assembly drives male pronuclear swelling. Several unresolved issues are discussed.     

Transmission electron microscope studies of the nuclear envelope in Caenorhabditis elegans embryos

Nuclear membranes and nuclear pore complexes (NPCs) are conserved in both animals and plants. However, the lamina composition and the dimensions of NPCs vary between plants, yeast, and vertebrates. In this study, we established a protocol that preserves the structure of Caenorhabditis elegans embryonic cells for high-resolution studies with thin-section transmission electron microscopy (TEM). We show that the NPCs are bigger in C. elegans embryos than in yeast, with dimensions similar to those in higher eukaryotes. We also localized the C. elegans nuclear envelope proteins Ce-lamin and Ce-emerin by pre-embedding gold labeling immunoelectron microscopy. Both proteins are present at or near the inner nuclear membrane. A fraction of Ce-lamin, but not Ce-emerin, is present in the nuclear interior. Removing the nuclear membranes leaves both Ce-lamin and Ce-emerin associated with the chromatin. Eliminating the single lamin protein caused cell death as visualized by characteristic changes in nuclear architecture including condensation of chromatin, clustering of NPCs, membrane blebbing, and the presence of vesicles inside the nucleus. Taken together, these results show evolutionarily conserved protein localization, interactions, and functions of the C. elegans nuclear envelope.     

Nuclear lamin proteins and the structure of the nuclear envelope: Where is the function?

The nuclear envelope has recently become the object of intense scrutiny because it is the site of nuclear transport and is possibly involved in the organization of the interphase genome, thereby affecting gene expression. The major structural support for the nuclear envelope is the nuclear lamina, composed of the nuclear lamin proteins. They lie on the surface of the inner nuclear membrane and are in direct contact with the chromatin at the edge of the nucleus. The structure of the nuclear lamin proteins has recently been deduced from their cDNAs and shown to have remarkable homologies to the family of cytoplasmic intermediate filaments. However, the lamin proteins have been found to depolymerize in response to metaphase-specific phosphorylation events, and reassemble around daughter chromosomes at the completion of cell division. Little is known of the mechanisms of these dynamics, nor of other post-translational modifications evident in these proteins. In addition, we have as yet no concrete idea of the function of these highly conserved proteins in the cell. This review will summarize our present knowledge of nuclear lamin structure and the new experimental approaches designed to elucidate their function.     

Observation of nuclei reassembled from demembranated Xenopus sperm nuclei and analysis of their lamina components

A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei.Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear memblane,nuclear pore complexes,and decondensed chromatin etc.Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei.Western-blotting results showed that lamin LII was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only egg derived lamin LIII.     

The nuclear matrix of Euglena gracilis (Euglenophyta): A stage of nuclear matrix evolution?

Euglena gracilis cell was extracted sequentially with CSK-Triton buffer, RSB-Magik solution and DNase-As solution. DGD embedment-free electron microscopy showed that in the extracted nucleus there was a residual non-chromatin fibrous network. That it could not be removed by hot trichloroacetic acid further supported the idea that it was a non-histone, non-chromatin fibrous protein network, and should be the internal network of the nuclear matrix. After the sequential extraction, the nuclear membrane was removed, leaving behind a layer of lamina; the chromatin was digested and eluted from the dense chromosomes and residual chromosomal structures that should be chromosomal scaffold were revealed. Western blot analysis with antiserum against rat lamins showed that nuclear lamina of the cell possessed two positive polypeptides, a major one and a minor one, which had molecular masses similar to lamin B and lamin A, respectively. Comparing these data with those of the most primitive eukaryote Archezoa and of higher eukaryotes, it was suggested that the lower unicellular eukaryote E. gracilis already had the nuclear matrix structure, and its nuclear matrix (especially the lamina) might represent a stage of evolutionary history of the nuclear matrix.     

Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein

We have identified an integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD that cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) which contributes the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. We suggest that p56 is a sea urchin LBR homologue that targets membranes to chromatin and later anchors the membrane to the lamina.     

Nuclear lamina at the crossroads of the cytoplasm and nucleus

The nuclear lamina is a protein meshwork that lines the nuclear envelope in metazoan cells. It is composed largely of a polymeric assembly of lamins, which comprise a distinct sequence homology class of the intermediate filament protein family. On the basis of its structural properties, the lamina originally was proposed to provide scaffolding for the nuclear envelope and to promote anchoring of chromatin and nuclear pore complexes at the nuclear surface. This viewpoint has expanded greatly during the past 25 years, with a host of surprising new insights on lamina structure, molecular composition and functional attributes. It has been established that the self-assembly properties of lamins are very similar to those of cytoplasmic intermediate filament proteins, and that the lamin polymer is physically associated with components of the cytoplasmic cytoskeleton and with a multitude of chromatin and inner nuclear membrane proteins. Cumulative evidence points to an important role for the lamina in regulating signaling and gene activity, and in mechanically coupling the cytoplasmic cytoskeleton to the nucleus. The significance of the lamina has been vaulted to the forefront by the discovery that mutations in lamins and lamina-associated polypeptides lead to an array of human diseases. A key future challenge is to understand how the lamina integrates pathways for mechanics and signaling at the molecular level. Understanding the structure of the lamina from the atomic to supramolecular levels will be essential for achieving this goal.