Presence of snake-like chromatin in epithelial cells of keratoconjunctivitis sicca followed by a large number of micronuclei

OBJECTIVE: To evaluate the number of micronuclei in snake-like chromatin (SLC) cells in the conjunctival epithelium of keratoconjunctivitis sicca (KCS) patients. To elucidate possible correlations between SLC cell numbers and KCS intensity. STUDY DESIGN: Impression cytology specimens from the bulbar conjunctiva of healthy controls and KCS patients were harvested and divided into 3 groups: group 1, controls; group 2, KCS SLC-negative; and group 3, KCS SLC-positive. The number of micronuclei (MNi) in SLC-negative and SLC-positive epithelial cells of each group was counted. RESULTS: The number of MNi in SLC-negative cells of groups 1 and 2 did not exceed 1 MNi/1,000 cells. A significant increase in the frequency of micronuclei in the upper bulbar conjunctiva was noted in SLC-positive (14.75 +/- 8.09 MNi/1,000 cells) as well as SLC-negative cells (4.0 +/- 3.83 MNi/1,000 cells) of group 3. CONCLUSION: We demonstrate here that the presence of MNi in the conjunctival epithelium of KCS patients could be a characteristic feature accompanying SLC cells. The fact that increased numbers of SLC cells correlates with impaired values in clinical test as well as decreased goblet and epithelial cell densities confirms that the presence of SLC cells correlates with KCS intensity.     

Nuclear envelope proteins and chromatin arrangement: a pathogenic mechanism for laminopathies

The involvement of the nuclear envelope in the modulation of chromatin organization is strongly suggested by the increasing number of human diseases due to mutations of nuclear envelope proteins. A common feature of these diseases, named laminopathies, is the occurrence of major chromatin defects. We previously reported that cells from laminopathic patients show an altered nuclear profile, and loss or detachment of heterochromatin from the nuclear envelope. Recent evidence indicates that processing of the lamin A precursor is altered in laminopathies featuring pre-mature aging and/or lipodystrophy phenotype. In these cases, pre-lamin A is accumulated in the nucleus and heterochromatin is severely disorganized. Here we report evidence indicating that pre-lamin A is mis-localized in the nuclei of Emery-Dreifuss muscular dystrophy fibroblasts, either bearing lamin A/C or emerin mutations. Abnormal pre-lamin A-containing structures are formed following treatment with a farnesyl-transferase inhibitor, a drug that causes accumulation of pre-lamin A. Pre-lamin A-labeled structures co-localize with heterochromatin clumps. These data indicate that in almost all laminopathies the expression of the mutant lamin A precursor disrupts the organization of heterochromatin domains. Our results further show that the absence of emerin expression alters the distribution of pre-lamin A and of heterochromatin areas, suggesting a major involvement of emerin in pre-lamin A-mediated mechanisms of chromatin remodeling.     

Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function

The tight association of cytoplasmic intermediate filaments (cIFs) with the nucleus and the isolation of crosslinkage products of vimentin with genomic DNA fragments, including nuclear matrix attachment regions (MARs) from proliferating fibroblasts, point to a participation of cIFs in nuclear activities. To test the possibility that cIFs are complementary nuclear matrix elements, the nuclei of a series of cultured cells were subjected to the Li-diiodosalicylate (LIS) extraction protocol developed for the preparation of nuclear matrices and analyzed by immunofluorescence microscopy and immunoblotting with antibodies directed against lamin B and cIF proteins. When nuclei released from hypotonically swollen L929 suspension cells in the presence of digitonin or Triton X-100 were exposed to such strong shearing forces that a considerable number were totally disrupted, a thin, discontinuous layer of vimentin IFs remained tenaciously adhering to still intact nuclei, in apparent coalignment with the nuclear lamina. Even in broken nuclei, the distribution of vimentin followed that of lamin B in areas where the lamina still appeared intact. The same retention of vimentin together with desmin and glial IFs was observed on the nuclei isolated from differentiating C2C12 myoblast and U333 glioma cells, respectively. Nuclei from epithelial cells shed their residual perinuclear IF layers as coherent cytoskeletal ghosts, except for small fractions of vimentin and cytokeratin IFs, which remained in a dot-to cap-like arrangement on the nuclear surface, in apparent codistribution with lamin B. LIS extraction did not bring about a reduction in the cIF protein contents of such nuclei upon their transformation into nuclear matrices. Moreover, in whole mount preparations of mouse embryo fibroblasts, DNA/chromatin emerging from nuclei during LIS extraction mechanically and chemically cleaned the nuclear surface and perinuclear area from loosely anchored cytoplasmic material with the production of broad, IF-free annular spaces, but left substantial fractions of the vimentin IFs in tight association with the nuclear surface. Accordingly, double-immunogold electron microscopy of fixed and permeabilized fibroblasts disclosed a close neighborhood of vimentin IFs and lamin B, with a minimal distance between the nanogold particles of ca. 30 nm. These data indicate an extremely solid interconnection of cIFs with structural elements of the nuclear matrix, and make them, together with their susceptibility to crosslinkage to MARs and other genomic DNA sequences under native conditions, complementary or even integral constituents of the karyoskeleton.     

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.     

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.     

Nuclear lamin antigens are developmentally regulated during porcine and bovine embryogenesis

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

Isolation and characterization of nuclear lamina from Ehrlich ascites tumor cells

We have developed a simple and rapid method for isolation of purified nuclear lamina from Ehrlich ascites tumor cells. The procedure employs chromatin structures prepared from whole cells at low ionic strength and is carried out under conditions that minimize the formation of artifactual protein-DNA complexes. When the isolation is performed in the presence of EDTA, nuclear lamina without distinct pore complexes is obtained. In the absence of EDTA, intact pore complexes and a large amount of vimentin 100 A filaments are seen associated with nuclear lamina. The main nuclear lamina proteins are characterized using gel electrophoresis, immunoblotting, and two-dimensional peptide mapping. An extensive structural homology is found between lamin A and lamin C, whose peptide maps differ by only one major spot, whereas lamin B has apparently unrelated pattern.     

Caspase-3 is required in the apoptotic disintegration of the nuclear matrix

Apoptotic breakdown of cellular structures is largely mediated by caspases. One target of degradation is a proteinaceous framework of the nucleus termed the nuclear matrix. We compared the apoptotic changes of the nuclear matrix in staurosporine-treated caspase-3-deficient MCF-7 cells transfected with intact CASP-3 gene (MCF-7c3) or an empty vector (MCF-7v) as a control. Nuclear Mitotic Apparatus protein (NuMA), lamin A/C and lamin B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. In both cell lines, staurosporine induced rapid cytoplasmic shrinkage and partial chromatin condensation. MCF-7c3 cells formed apoptotic bodies, whereas MCF-7v cells did not. NuMA and lamins were actively cleaved in MCF-7c3 cells following caspase-3 activation, but only minimal or no cleavage was detected in MCF-7v cells. Interestingly, lamin B but not lamin A/C was relocated into cytoplasmic granules in apoptotic MCF-7v cells. Pancaspase inhibitor, z-VAD-fmk, prevented the apoptotic changes, while caspase-3 inhibitor, z-DEVD-fmk, induced lamin B granules in both cell lines. These results show that caspase-3 is involved in the cleavage of NuMA and lamins either directly or by activating other proteases. This may be essential for disintegration of the nuclear structure during apoptosis.     

Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery

Antibodies directed against nuclear envelope lamin proteins have been used in conjunction with three-dimensional light and electron microscope methodologies to determine the spatial organization of lamins in diploid interphase nuclei and to relate this organization to the positions of chromatin in the nuclear periphery. Using Drosophila early embryos, Drosophila Kc cells, and human HeLa cells, it is qualitatively and quantitatively observed that lamins are organized as a highly discontinuous, apparently fibrillar network that leaves large voids in the nuclear periphery containing little or no lamin. Using fluorescence microscopy to compare and quantitate the relationship between chromatin and the lamin network, it is found that although there is a strong tendency for the most peripheral chromatin to be positioned directly underneath a lamin fiber, only a small fraction of the chromatin in the nuclear periphery is sufficiently close to a lamin fiber to possibly be in direct contact.     

On the cell-free association of lamins A and C with metaphase chromosomes

Nuclear envelopes have previously been shown to assemble spontaneously around endogenous chromosomes in cell-free homogenates of mitotic Chinese hamster ovary cells. In order to further analyze the mechanisms underlying nuclear envelope reformation and the functions of the individual nuclear lamin polypeptides, a fractionated cell-free nuclear envelope reassembly system involving purified chromosomes and either a postchromosomal supernatant or a cytosol fraction from mitotic cells has been devised. Results obtained with this fractionated system show that lamins A and C will associate with the surfaces of chromosomes in the absence of lamin B and membranes, this association being inhibitable by ATP-gamma-S. However, in the absence of membranes chromatin decondensation never occurs. Using the reversible swelling of chromosomes in low ionic strength buffers lacking divalent cations as the basis of a simple assay, it is demonstrated that the association of lamins A and C with the surfaces of chromosomes has a pronounced and easily observable effect on chromatin organization.     

US3 of herpes simplex virus type 1 encodes a promiscuous protein kinase that phosphorylates and alters localization of lamin A/C in infected cells

The herpes simplex virus type 1 (HSV-1) US3 gene encodes a serine/threonine kinase that, when inactivated, causes capsids to aggregate aberrantly between the inner and outer nuclear membranes (INM and ONM, respectively) within evaginations/extensions of the perinuclear space. In both Hep2 cells and an engineered cell line derived from Hep2 cells expressing lamin A/C fused to enhanced green fluorescent protein (eGFP-lamin A/C), lamin A/C localized mostly in a reticular pattern with small regions of the INM devoid of eGFP-lamin A/C when they were either mock infected or infected with wild-type HSV-1(F). Cells infected with HSV-1(F) also contained some larger diffuse regions lacking lamin A/C. Proteins UL31 and UL34, markers of potential envelopment sites at the INM and perinuclear virions, localized within the regions devoid of lamin A/C and also in regions containing lamin A/C. Similar to previous observations with Vero cells (S. L. Bjerke and R. J. Roller, Virology 347:261-276, 2006), the proteins UL34 and UL31 localized exclusively in very discrete regions of the nuclear lamina lacking lamin A/C in the absence of US3 kinase activity. To determine how US3 alters lamin A/C distribution, US3 was purified and shown to phosphorylate lamin A/C at multiple sites in vitro, despite the presence of only one putative US3 kinase consensus site in the lamin A/C sequence. US3 kinase activity was also sufficient to invoke partial solubilization of lamin A/C from permeabilized Hep2 cell nuclei in an ATP-dependent manner. Two-dimensional electrophoretic analyses of lamin A/C revealed that lamin A/C is phosphorylated in HSV-infected cells, and the full spectrum of phosphorylation requires US3 kinase activity. These data suggest that US3 kinase activity regulates HSV-1 capsid nuclear egress at least in part by phosphorylation of lamin A/C.     

Lamin A is part of the internal nucleoskeleton of human erythroleukemia cells

Nuclear lamins are the most abundant components of the nuclear lamina, a 10–50-nm-thick fibrous layer underlying the inner nuclear envelope membrane. Nevertheless, a number of recent investigations performed on epithelial and fibroblast cells have suggested that nuclear lamins are also present within the nucleoplasm and could be important constituents of the nucleoskeleton. We have studied the subnuclear distribution of lamins A and B1 in human erythroleukemia cells by using immunoblotting analysis and immunofluorescent staining of fractionated nuclei. In intact cells and isolated nuclei, antibodies to lamins A and B1 mainly stained the nuclear periphery, although some immunoreactivity was detected in the nuclear interior. However, when chromatin was removed by nuclease digestion and extraction with nonionic detergent or 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 or scaffold. Preferential localization of lamin A to the inner part of the nucleus was also demonstrated by the presence of the majority of lamin A in the solubilized inner nuclear network subfraction. In contrast, lamin B1 was mainly recovered in the fraction corresponding to the nuclear periphery. Double labeling experiments showed that lamin A, but not lamin B1, colocalized with coiled and GATA-1 bodies. Thus, our results support the hypothesis that lamin A, but not lamin B1, may be a component of an internal nucleoskeleton in human erythroleukemia cells. J. Cell. Physiol. 178:284–295, 1999. © 1999 Wiley-Liss, Inc.     

The highly conserved nuclear lamin Ig-fold binds to PCNA: its role in DNA replication

This study provides insights into the role of nuclear lamins in DNA replication. Our data demonstrate that the Ig-fold motif located in the lamin C terminus binds directly to proliferating cell nuclear antigen (PCNA), the processivity factor necessary for the chain elongation phase of DNA replication. We find that the introduction of a mutation in the Ig-fold, which alters its structure and causes human muscular dystrophy, inhibits PCNA binding. Studies of nuclear assembly and DNA replication show that lamins, PCNA, and chromatin are closely associated in situ. Exposure of replicating nuclei to an excess of the lamin domain containing the Ig-fold inhibits DNA replication in a concentration-dependent fashion. This inhibitory effect is significantly diminished in nuclei exposed to the same domain bearing the Ig-fold mutation. Using the crystal structures of the lamin Ig-fold and PCNA, molecular docking simulations suggest probable interaction sites. These findings also provide insights into the mechanisms underlying the numerous disease-causing mutations located within the lamin Ig-fold.     

Review: the dynamics of the nuclear lamins during the cell cycle-- relationship between structure and function

The nuclear lamins are members of the intermediate filament (IF) family of proteins. The lamins have an essential role in maintaining nuclear integrity, as do the other IF family members in the cytoplasm. Also like cytoplasmic IFs, the organization of lamins is dynamic. The lamins are found not only at the nuclear periphery but also in the interior of the nucleus, as distinct nucleoplasmic foci and possibly as a network throughout the nucleus. Nuclear processes such as DNA replication may be organized around these structures. In this review, we discuss changes in the structure and organization of the nuclear lamins during the cell cycle and during cell differentiation. These changes are correlated with changes in nuclear structure and function. For example, the interactions of lamins with chromatin and nuclear envelope components occur very early during nuclear assembly following mitosis. During S-phase, the lamins colocalize with markers of DNA replication, and proper lamin organization must be maintained for replication to proceed. When cells differentiate, the expression pattern of lamin isotypes changes. In addition, changes in lamin organization and expression patterns accompany the nuclear alterations observed in transformed cells. These lamin structures may modulate nuclear function in each of these processes.     

Review: The Dynamics of the Nuclear Lamins during the Cell Cycle— Relationship between Structure and Function

The nuclear lamins are members of the intermediate filament (IF) family of proteins. The lamins have an essential role in maintaining nuclear integrity, as do the other IF family members in the cytoplasm. Also like cytoplasmic IFs, the organization of lamins is dynamic. The lamins are found not only at the nuclear periphery but also in the interior of the nucleus, as distinct nucleoplasmic foci and possibly as a network throughout the nucleus. Nuclear processes such as DNA replication may be organized around these structures. In this review, we discuss changes in the structure and organization of the nuclear lamins during the cell cycle and during cell differentiation. These changes are correlated with changes in nuclear structure and function. For example, the interactions of lamins with chromatin and nuclear envelope components occur very early during nuclear assembly following mitosis. During S-phase, the lamins colocalize with markers of DNA replication, and proper lamin organization must be maintained for replication to proceed. When cells differentiate, the expression pattern of lamin isotypes changes. In addition, changes in lamin organization and expression patterns accompany the nuclear alterations observed in transformed cells. These lamin structures may modulate nuclear function in each of these processes.     

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.     

Lysosome-mediated processing of chromatin in senescence

Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C–negative, but strongly γ-H2AX–positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression.     

Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract

The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.     

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.     

Dynamics of lamin A/C in porcine embryos produced by nuclear transfer

This study was conducted to investigate the presence of lamin A/C in porcine nuclear transfer embryos and to determine whether lamin A/C can serve as a potential marker for nuclear reprogramming. First, lamin A/C was studied in oocytes and embryos produced by fertilization or parthenogenetic oocyte activation. We found that lamin A/C was present in the nuclear lamina of oocytes at the germinal vesicle stage while it was absent in mature oocytes. Lamin A/C was detected throughout preimplantation development in both in vivo-derived and parthenogenetic embryos. Incubation of the activated oocytes in the presence of alpha-amanitin (an inhibitor of RNA polymerase II), or cycloheximide (a protein synthesis inhibitor) did not perturb lamin A/C assembly, indicating that the assembly resulted from solubilized lamins dispersed in the cytoplasm. In nuclear transfer embryos, the lamin A/C signal that had previously been identified in fibroblast nuclei disappeared soon after fusion. It became detectable again after the formation of the pronucleus-like structure, and all nuclear transfer embryos displayed lamin A/C staining during early development. Olfactory bulb progenitor cells lacked lamin A/C; however, when such cells were fused with enucleated oocytes, the newly formed nuclear envelopes stained positive for lamin A/C. These findings suggest that recipient oocytes remodel the donor nuclei using type A lamins dispersed in the ooplasm. The results also indicate that lamin A/C is present in the nuclear envelope of pig oocytes and early embryos and unlike in some other species, its presence after nuclear transfer is not an indicator of erroneous reprogramming.