Disappearance and reformation of the nuclear lamina structure during specific stages of meiosis in oocytes

The nuclear lamina is a rigid, proteinaceous layer underlying the inner nuclear membrane of eucaryotic cells. It is present in somatic cell nuclei, disappears during mitosis, and is absent from male meiotic cells. We have investigated the disappearance and reformation of the nuclear lamina during meiosis in oocytes, using immunofluorescence and electron microscopy. We find that the status of the nuclear lamina during meiosis of oocytes differs from the reversible depolymerization seen in mitosis in two respects. First, the lamina disappears during meiotic prophase without affecting the structure of the nuclear membranes or the nuclear pores. Second, the proteins of the dissociated lamina are undetectable by immunological methods in pachytene oocytes, whereas they persist in the cytoplasm during mitosis.     

Nuclear mechanotransduction: response of the lamina to extracellular stress with implications in aging

Mechnotransduction, the phenomenon by which cells respond to applied force, is necessary for normal cell processes and is implicated in the pathology of several diseases including atherosclerosis. The exact mechanisms which govern how forces can affect gene expression have not been determined, but putative direct force effects on the genome would require transduction through the nuclear lamina. In this study we show that nuclei in cells exposed to shear stress significantly change shape, upregulate nuclear lamins and move lamins from the nuclear interior to the nuclear periphery. We hypothesize that the augmentation of the nuclear lamina at the nuclear periphery protects the nuclear interior from the force and allows a nuclear adaptation to shear stress. We also investigate the shear stress response of nuclei in cells that have been transfected with lamin A Delta50, which significantly stiffens nuclei. Lamin A Delta50 causes the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS) and models many aspects of normal aging. We find that the presence of lamin A Delta50 in only 30% of cells greatly reduces the response of the nuclear lamina in all cells in the flow field. We suggest that cells expressing lamin A Delta50 lack the ability to adapt to flow and may prevent neighboring cells from adapting as well. These results provide insight into the development of cardiovascular disease both in patients with HGPS and in normal aging.     

A fractionated cell-free system for analysis of prophase nuclear disassembly

We describe a cell-free system in which a postribosomal supernatant (s140) from metaphase Chinese hamster ovary (CHO) cells induces prophase-like changes in isolated CHO cell nuclei, including chromatin condensation, and nuclear envelope and lamina disassembly. These events are strongly promoted by gamma-S-ATP and an ATP-regenerating system, and do not take place with an s140 derived from G2-phase cells. The metaphase cell s140 also induces disassembly of an isolated nuclear lamina fraction that is depleted of membranes, chromatin, and nuclear pore complexes. Disassembly of the isolated lamina is accompanied by phosphorylation of the major lamina proteins (lamins A, B, and C) to levels characteristic of metaphase cells. Kinetic analysis of lamina depolymerization indicates that cooperativity may be involved in this process. The biochemical properties of in vitro lamina disassembly suggest that the activity that depolymerizes the lamina during mitosis is soluble in metaphase cells, and support the notion that this activity is a lamin protein kinase.     

Characterization of the nuclear envelope, pore complexes, and dense lamina of mouse liver nuclei by high resolution scanning electron microscopy

We have used high resolution scanning electron microscopy (SEM) to study the nuclear envelope components of isolated mouse liver nuclei. The surfaces of intact nuclei are covered by closely packed ribosomes which are distinguishable by SEM from nuclear pore complexes. After removal of nuclear membranes with the nonionic detergent Triton X-100, the pore complexes remain attached to an underlying, peripheral nuclear lamina, as described by others. The surface of this dense lamina is composed of particulate granules, 75-150 A in diameter, which are contiguous over the entire periphery. We did not observe the pore-to-pore fibril network suggested by other investigators, but such a structure might be the framework upon which the dense lamina is formed. Morphometric analysis of pores and pore complexes shows their size, structure, and density to be similar to that of other mammalian cells. In addition, several types of pore complex-associated structures, not previously reported by other electron microscope (EM) techniques, are observed by SEM. Our studies suggest that the major role of the dense lamina is associated with the distribution, stability, and perhaps, biogenesis of nuclear pore complexes. Treatment of isolated nuclei with a combination of Triton X-100 and sodium deoxycholate removes membranes, dense lamina, and nuclear pore complexes. The resulting "chromatin nuclei" retain their integrity despite the absence of any limiting peripheral structures.     

Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis

We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis.     

Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution

This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex- lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis.     

Heat-induced morphological and biochemical changes in the nuclear lamina from Ehrlich ascites tumor cells in vivo

Membrane-depleted nuclei from Ehrlich ascites tumor (EAT) cells isolated at low ionic strength in the presence of EDTA exhibit highly decondensed chromatin fibers and a loss of morphologically identifiable nucleoli. Treatment of these nuclei with nucleases and 2 M NaCl followed by low-speed centrifugation permitted the facile isolation of the nuclear lamina layer. Under the same conditions, but after heat-shock treatment of the living cells, the chromatin appears in a more condensed state, the nucleoli are well-defined, and the nuclear lamina layer was destabilized in concert with the appearance of an internal nuclear matrix and nucleolar skeleton. Furthermore, we also found both an increase in the protein mass as well as the appearance of a relatively large number of new proteins in this fraction, which are phosphorylated. The major proteins of the nuclear lamina, the lamins, and the residual vimentin remained insoluble. These heat-shock-induced changes were also accompanied by a dephosphorylation of lamins A and C but not of lamin B.     

Immunological analysis of nuclear lamina proteins

Antibodies have been obtained against specific fractions of the nuclear lamina from chick red blood cells. Immunofluorescent staining of acrylamide gels from nuclear lamina preparations revealed a spectrum of at least 8–10 proteins cross-reacting immunologically with each other. These proteins are not the result of proteolysis in the course of preparation. The antigens are localized in the nuclear periphery and do not extend into the chromatin. Interspecies cross-reactions enabled us to localize the antigens in the envelope of Xenopus oocyte nuclei. In this case any association with the chromatin is unlikely. During mitosis the antigens are released from the nuclear lamina and are recovered from the postmicrosomal supernatant. The molecular weights of the nuclear lamina proteins do not change during mitosis.     

Distribution of nuclear matrix proteins in interphase CHO cells and rearrangements during the cell cycle. An ultrastructural study

The nuclear matrix contains a group of residual non-histone proteins which remain structurally organized after extensive extraction of isolated nuclei with a high salt buffer, nucleases and a non-ionic detergent. Electron microscopic examination shows that the nuclear matrix is composed of a pore-complex lamina, an intranuclear network and residual nucleoli. In CHO cells biochemical analyses performed by one-dimensional SDS-PAGE show three major nuclear matrix polypeptides with molecular weights between 60 and 70 kDa. Polyclonal antibodies produced against these polypeptides were used to determine their nuclear distribution. Using immunoblotting, these proteins were found in whole nuclei, nuclear matrix, and in the intranuclear network but not in the pore-complex lamina. In order to determine the relationship between these structural proteins and the organization of the nucleus, the proteins were localized in situ. Ultrastructural detection was carried out by immunogold staining of thin sections of Lowicryl K4M-embedded cells. In interphase nuclei all condensed chromatin clumps were labelled. The nucleolus and the interchromatin granules were never immunogold-stained. During mitosis, the label was found to be associated with the chromosomes. This study shows that unlike the lamins, these 60-70 kDa nuclear matrix proteins are associated with the condensed chromatin throughout the cell cycle.     

A combined ultrastructural approach to the study of nuclear matrix thermal stabilization.

Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37 degrees C of isolated nuclei. If purified nuclei were heated for 45 min at 37 degrees C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-amine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.     

A combined ultrastructural approach to the study of nuclear matrix thermal stabilization

Summary Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37° C of isolated nuclei. If purified nuclei were heated for 45 min at 37° C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-ammine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.     

New patterns of nuclear lamina induced by cell fusion

During the eukaryote cell cycle the nuclear envelope displays a series of major morphogenetic changes, the most significant of which include its breakdown and reconstitution as cells move up to, pass through and emerge from division. The three polypeptides, lamins A, B and C, are major components of the nuclear pore complex-lamina fraction of the nuclear envelope and their association with the nuclear membrane or their dispersal in the cytoplasm reflects the existing balance between polymerization and depolymerization in the envelope. We have perturbed the lamina polymerization cycle by means of cell fusion between mitotic and interphase cells, following the redistribution of nuclear lamina protein by means of immunofluorescence techniques. In these heterophasic heterokaryons changes in the distribution of lamina occur as a function of (1) the time elapsed after fusion; (2) the ratio of mitotic to interphase elements in the cell, and (3) the stage in the cell cycle occupied by the interphase partner at the time of fusion. Depolymerization of nuclear lamina occurs most rapidly in cells with high ratios of mitotic to interphase elements, and especially in G1 rather than S-phase nuclei. While lamina depolymerization predominates early after fusion, at later times lamina is deposited around both the original metaphase and interphase nuclear masses and this is associated with the resumption of interphase activity in the form of limited DNA synthesis. These observations lead us to conclude that lamina depolymerization is under positive control mediated by diffusible factors in the cytoplasm of the metaphase partner. Repolymerization is likely to be associated with the inactivation of these factors as the heterokaryons age and, as a result, pass into an interphase-like state.     

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.     

Nuclear mechanics in differentiation and development

The nucleus is by far one of the stiffest organelles within cells of higher eukaryotes. Its mechanical properties are determined by contributions from the nuclear lamina and chromatin. Together they allow a viscoelastic response of the nucleus to applied stresses, where the lamina is thought to behave as an elastic shell, while the nucleoplasm contributes as a largely viscous material. Nuclear mechanics changes during differentiation and development. Altered nuclear mechanics reflects but might also influence global re-arrangements in chromatin architecture, which take place when cells commit themselves into distinct lineages. Thus it is likely that the mechanical characteristics of nuclei significantly contribute to proper differentiation.     

Nuclear entry of the Drosophila melanogaster nuclear lamina protein YA correlates with developmentally regulated changes in its phosphorylation state.

The Drosophila melanogaster YA protein is a maternally provided nuclear lamina component that is essential during the transition from meiosis to mitosis at the beginning of embryogenesis. Localization of YA to the nuclear envelope is required for its function; this localization is cell cycle-dependent during embryogenesis. Here we show that the ability of YA to enter nuclei is modulated during development. In developing egg chambers, YA protein is made but excluded from nuclei of nurse cells and oocytes; upon egg activation, YA acquires the ability to enter nuclei and becomes incorporated into the nuclear lamina in unfertilized eggs and embryos. This localization switch correlates with changes in the phosphorylation state of YA. YA in ovaries is hyperphosphorylated relative to YA in unfertilized eggs and embryos. Through site-directed mutagenesis, we identified 443T, a potential phosphorylation site for both cyclin-dependent protein kinase and mitogen-activated-protein kinase, as one of the sites likely involved in this developmental control. Our results suggest that phosphorylation plays a role in modulating the localization of YA during development. A model for developmental regulation of the nuclear entry of YA is proposed and implications for understanding Drosophila egg activation are discussed.     

Mechanics in human fibroblasts and progeria: Lamin A mutation E145K results in stiffening of nuclei

The lamina is a filamentous meshwork beneath the inner nuclear membrane that confers mechanical stability to nuclei. The E145K mutation in lamin A causes Hutchinson‐Gilford progeria syndrome (HGPS). It affects lamin filament assembly and induces profound changes in the nuclear architecture. Expression of wild‐type and E145K lamin A in Xenopus oocytes followed by atomic force microscopy (AFM) probing of isolated oocyte nuclei has shown significant changes in the mechanical properties of the lamina. Nuclei of oocytes expressing E145K lamin A are stiffer than those expressing wild‐type lamin A. Here we present mechanical measurements by AFM on dermal fibroblasts obtained from a 4‐year‐old progeria patient bearing the E145K lamin A mutation and compared it to fibroblasts obtained from 2 healthy donors of 10 and 61 years of age, respectively. The abnormal shape of nuclei expressing E145K lamin A was analyzed by fluorescence microscopy. Lamina thickness was measured using electron micrographs. Fluorescence microscopy showed alterations in the actin network of progeria cells. AFM probing of whole dermal fibroblasts did not demonstrate significant differences in the elastic moduli of nuclear and cytoplasmic cell regions. In contrast, AFM measurements of isolated nuclei showed that nuclei of progeria and old person's cells are significantly stiffer than those of the young person, indicating that the process of aging, be it natural or abnormal, increases nuclear stiffness. Our results corroborate AFM data obtained using Xenopus oocyte nuclei and prove that the presence of E145K lamin A abnormally increases nuclear stiffness.     

四膜虫细胞的核骨架及类中间纤维

采用非树脂包埋去包埋剂超薄切片结合选择性生抽提方法显示,原生动物四膜虫细胞大核具有发达的核骨架纤维网络,核周是一层完整的核纤层结构,在四膜虫细胞小核中,亦存在核骨架和核纤层。四膜虫细胞皮层中存在水下溶性纤维网架,其中含有类中间纤维蛋白组分,４９ＫＤ蛋白。     

Involvement of the lamin rod domain in heterotypic lamin interactions important for nuclear organization

The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. The lamina is proposed to be an important determinant of nuclear structure, but there has been little direct testing of this idea. To investigate lamina functions, we have characterized a novel lamin B1 mutant lacking the middle approximately 4/5 of its alpha-helical rod domain. Though retaining only 10 heptads of the rod, this mutant assembles into intermediate filament-like structures in vitro. When expressed in cultured cells, it concentrates in patches at the nuclear envelope. Concurrently, endogenous lamins shift from a uniform to a patchy distribution and lose their complete colocalization, and nuclei become highly lobulated. In vitro binding studies suggest that the internal rod region is important for heterotypic associations of lamin B1, which in turn are required for proper organization of the lamina. Accompanying the changes in lamina structure induced by expression of the mutant, nuclear pore complexes and integral membrane proteins of the inner membrane cluster, principally at the patches of endogenous lamins. Considered together, these data indicate that lamins play a major role in organizing other proteins in the nuclear envelope and in determining nuclear shape.     

Nuclear envelope proteins from Spisula solidissima germinal vesicles

Biochemical characterization of the nuclear pore complex requires quantities of highly enriched nuclear pore complex material which could not be obtained with available procedures. We have developed a technique for the mass isolation of nuclear envelopes from germinal vesicles of Spisula solidissima oocytes. The nuclear pore complex is intact after isolation as judged ultrastructurally. The nuclear envelope and the pore complex fibrous lamina fraction are highly purified with respect to nuclear and cytoplasmic protein contaminants. The fibrous lamina pore complex (FLPC) as presently isolated consists of about eight major proteins, three of which are phosphorylated. Comparison of the FLPC of clams with that of rat reveals three proteins of similar molecular weights, which may be pore complex-specific proteins. The clam nuclear envelope has only one protein (67000) in the molecular weight range which is comparable to the three lamina of rat nuclei. The solubility, intermolecular cross-linking and in vitro phosphorylation of this protein resemble that of one of the lamina of rat nuclei. The other lamina of the rat nuclear envelope are not essential proteins of the pore complex because they are not present in the clam FLPC preparation. They also seem non-essential for the maintenance of the fibrous lamina.     

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 α, 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.