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.     

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.     

The MAN antigens are non-lamin constituents of the nuclear lamina in vertebrate cells

The characterization of the human antiserum designated MAN has led to the identification of a subset of non-lamin proteins that are exclusively located at the nuclear periphery in all vertebrate cell types examined, from human to fish. Immunoreactive protein species were whown to comprise three major polypeptides of M _r 78000, 58000 and 40000. These antigens co-partitioned with the nuclear lamina during in situ isolation of nuclear matrices from lamin A/C-positive and-negative mammlian cells. Using double immunofluorescence, the spatial relationship of MAN antigens to type-A and type-B lamins was further examined throughout the cell cycle of lamin A/C-positive mammalian cells. In interphase HeLa and 3t3 cells, MAN antigens colocalized with both types of lamins at the periphery of the nucleus, but were absent from intranuclear foci of lamin B. As HeLa cells proceeded into mitosis, MAN antigens were seen to segregate from lamins A/C and coredistribute with lamin B. Lamins A/C disassembled during late prophase/early prometaphase and reassociated with chromatin in telophase/cytokinesis. In contrast, MAN antigens and lamin B dispersed late during prometaphase and reassembled on chromosomes in anaphase. Altogether, our data suggest that MAN antigens may play key functions in the maintenance of the structural integrity of the nuclear compartment in vertebrate cells.     

Remodelling of the nuclear periphery during muscle cell differentiation in vitro

We have examined the composition and ultrastructure of the nuclear periphery during in vitro myogenesis of the rat myoblast cell line, L6E9. Immunofluorescence labelling and immunoblotting showed that lamins A/C and B were all present in undifferentiated cells, but that they increased significantly before extensive cell fusion had occurred, with lamins A/C increasing proportionately more. Electron microscopic observations were consistent with these results, showing an increase in the prominence of the lamina during differentiation. On the other hand, immunofluorescence labelling suggested that the P1 antigen began to disappear from the nuclear periphery as the cells were fusing, after the increase in lamin quantity, and was no longer detectable in multinucleated cells. Unexpectedly, however, P1 was readily detected in isolated nuclei, whether prepared from myoblast or differentiated cultures, as well as in both myoblast and myotube nuclear matrices. It appears probable, therefore, that the fading of P1 labelling is due to masking of the epitope by a soluble factor recruited to the nuclear periphery as cells differentiate. These data, together with evidence that the genome is substantially rearranged during L6E9 myogenesis [Chaly and Munro, 1996], suggest that L6E9 cells are a useful model system in which to study the interrelationship of nuclear envelope organization, chromatin spatial order, and nuclear function. © 1996 Wiley-Liss, Inc.     

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.     

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.     

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.     

Expression of nuclear lamins during mouse preimplantation development

The expression of nuclear lamins during mouse preimplantation development was studied by immunofluorescence, immunoblotting and immunoprecipitation. Two sera were used, specific either for lamin B or lamins A and C. Both sera gave a positive staining of the nuclear periphery throughout preimplantation development (fertilized eggs to late blastocysts). Immunoblots revealed that the three lamins were present in eggs and blastocysts. However, lamin A from eggs was found to have a higher apparent Mr than lamin A from blastocysts and other mouse cells. Using immunoprecipitation, synthesis of lamin A was detected in eggs while synthesis of lamin B was detected in 8-cell embryos and blastocysts, indicating that at least some of the lamins used during early development do not come from a store in the egg. These results are discussed in relation to the possible role of lamins during cell differentiation.     

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.     

A chromatin binding site in the tail domain of nuclear lamins that interacts with core histones

Interaction of chromatin with the nuclear envelope and lamina is thought to help determine higher order chromosome organization in the interphase nucleus. Previous studies have shown that nuclear lamins bind chromatin directly. Here we have localized a chromatin binding site to the carboxyl-terminal tail domains of both A- and B-type mammalian lamins, and have characterized the biochemical properties of this binding in detail. Recombinant glutathione-S-transferase fusion proteins containing the tail domains of mammalian lamins C, B1, and B2 were analyzed for their ability to associate with rat liver chromatin fragments immobilized on microtiter plate wells. We found that all three lamin tails specifically bind to chromatin with apparent KdS of 120-300 nM. By examining a series of deletion mutants, we have mapped the chromatin binding region of the lamin C tail to amino acids 396- 430, a segment immediately adjacent to the rod domain. Furthermore, by analysis of chromatin subfractions, we found that core histones constitute the principal chromatin binding component for the lamin C tail. Through cooperativity, this lamin-histone interaction could be involved in specifying the high avidity attachment of chromatin to the nuclear envelope in vivo.     

Developmental changes in the organization of the nuclear lamina in mouse liver

We have compared the organization of the nuclear lamina in adult and fetal mouse liver. Western blot analysis of the expression of lamins with specific antibodies indicates that lamin B is expressed throughout liver development, unlike lamins A and C which are absent in fetal liver. Using [125I]lamin in blot binding assays, we have observed that lamin B binds to at least three membrane proteins (96, 54 and 34 kDa) and to lamins A and C in adult nuclear envelopes, but only to the 54 and 34 kDa proteins and lamin B itself in fetal nuclear envelopes, where lamin B appears to be hyperphosphorylated.     

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.     

Structural basis for dimerization of LAP2alpha, a component of the nuclear lamina

Lamina-associated polypeptides (LAPs) are important components of the nuclear lamina, the dense network of filaments that supports the nuclear envelope and also extends into the nucleoplasm. The main protein constituents of the nuclear lamina are the constitutively expressed B-type lamins and the developmentally regulated A- and C-type lamins. LAP2alpha is the only non-membrane-associated member of the LAP family. It preferentially binds lamin A/C, has been implicated in cell-cycle regulation and chromatin organization, and has also been found to be a component of retroviral preintegration complexes. As an approach to understanding the role of LAP2alpha in cellular pathways, we have determined the crystal structure of the C-terminal domain of LAP2alpha, residues 459-693. The C-terminal domain is dimeric and possesses an extensive four-stranded, antiparallel coiled coil. The surface involved in binding lamin A/C is proposed based on results from alanine-scanning mutagenesis and a solid-phase overlay binding assay.     

The nuclear lamina. Both a structural framework and a platform for genome organization

The inner face of the nuclear envelope of metazoan cells is covered by a thin lamina consisting of a one-layered network of intermediate filaments interconnecting with a complex set of transmembrane proteins and chromatin associating factors. The constituent proteins, the lamins, have recently gained tremendous recognition, because mutations in the lamin A gene, LMNA, are the cause of a complex group of at least 10 different diseases in human, including the Hutchinson-Gilford progeria syndrome. The analysis of these disease entities has made it clear that besides cytoskeletal functions, the lamina has an important role in the "behaviour" of the genome and is, probably as a consequence of this function, intimately involved in cell fate decisions. Furthermore, these functions are related to the involvement of lamins in organizing the position and functional state of interphase chromosomes as well as to the occurrence of lamins and lamina-associated proteins within the nucleoplasm. However, the structural features of these lamins and the nature of the factors that assist them in genome organization present an exciting challenge to modern biochemistry and cell biology.