The nuclear lamin protein family in higher vertebrates. Identification of quantitatively minor lamin proteins by monoclonal antibodies

The nuclear lamina, a structure closely apposed to the inner nuclear membrane, is believed to provide a framework important for nuclear envelope integrity and interphase chromatin organization. So far, in mammalian and avian species three major constituents of the lamina, lamins A, B, and C, have been identified. These proteins migrate to characteristic positions on two-dimensional gels, lamin B being more acidic than lamins A and C. Here, we show that the composition of the nuclear lamina in avian and mammalian cells is more complex than previously assumed. When analyzed on two-dimensional gels, the major 66-kDa chicken "lamin B" protein can readily be identified. However, an additional 68-kDa protein migrates to a similarly acidic position. Based on the following evidence, both proteins can be considered as two distinct members of the lamin protein family. First, peptide mapping experiments and immunological criteria demonstrate that these two proteins are not related to each other or to lamin A via postsynthetic modifications or precursor-product relationships. Second, as determined by immunocytochemical techniques, both proteins are located exclusively at the nuclear periphery. Third, both proteins display the biochemical properties characteristic of lamin proteins, i.e. they are resistant to extraction of nuclei with nonionic detergents, nucleases, and high salt. Fourth, both proteins are immunologically related to previously characterized lamin proteins: the major 66-kDa chicken "lamin B" protein shares at least two epitopes with lamin A. However, contrary to what current nomenclature might suggest, this 66-kDa chicken "lamin B" protein is not related to rat liver lamin B, but to a minor component of rat liver pore-complex lamina preparations that had not previously been recognized as a lamin protein. Conversely, the minor 68-kDa component of chicken lamina preparations that had not previously been considered to be a lamin protein is immunologically related to rat liver lamin B. Thus, in addition to demonstrating the existence of quantitatively minor lamin proteins in higher vertebrates, our results caution against assigning structural homologies between lamin proteins from different species on the basis of gel electrophoresis analyses.     

小眼虫的核骨架研究

小眼虫细胞经轻度超声处理、选择性抽提后,利用DGD包埋-去包埋剂电镜技术及Westernblot分析技术对其核骨架、核纤层进行了研究.结果显示;细胞核内存在一个不被DNase所降解和热三氯醋酸所去除的纤维蛋白性网架结构;核的周围有一层明显的核纤层结构;Westernblot分析表明其核纤层有两种阳性蛋白成分,一为相当于高等真核细胞核纤层蛋白laminB的强阳性成分,另一为相当于laminA的弱阳性成分,但无相当于laminC的成分.本文认为小眼虫这种低等的单细胞真核生物已具有了核骨架、核纤层结构,其核纤层的蛋白组成应该代表了核纤层进化历程中早期的一个阶段.       

Transfection of human lamins A and C into mouse embryonal carcinoma cells possessing only lamin B

The peripheral lamina of eukaryotic nuclei is composed of polypeptides called lamins that vary in number from one to four according to organism, cell type, and differentiated state of the cells. Early embryonic cells and stem cells of mammals generally possess only lamin B while lamins A and C appear later during differentiation. To study the role of the late appearance of lamins A and C in the differentiated phenotype, we have performed transfection of cDNAs coding for human lamins A or C into mouse embryonal carcinoma (EC) cell lines F9 and P19 lacking these two lamins. Transient transfections have shown that lamins A or C could be expressed, translocated to the peripheral lamina, and distributed into daughter cell nuclei after mitosis. These results demonstrated that EC cells devoid of lamins A and C nevertheless possessed the appropriate mechanisms for the localization and mitotic redistribution of exogenous lamins A and C.     

Characterization of lamin proteins in BHK cells

Lamins are structural proteins found in rat liver nuclear envelope and are major constituents of the nuclear matrix. 2-D gel electrophoresis indicates that BHK cell nuclear matrix is composed of four major proteins (62 kD, 68 kD, 70 kD and 72 kD). Three of these proteins are very similar to lamins A, B and C of rat liver nuclear envelope according to their molecular mass and isoelectric points. An anti-serum specific to BHK matrix proteins has been raised. On 2-D immunoblot, this serum detects all the 62, 68 and 72 kD polypeptide isovariants but only one of the two isovariants of the 70 kD polypeptide. Rat lamins A, B and C react with the anti-BHK matrix serum. However, when a monoclonal antibody to rat liver lamins A, B and C is used (Burke, B, Tooze, J & Warren, G, EMBO j 2 (1983) 361 [23]), only the 72 kD (lamin A-like) and the 62 kD (lamin C-like) BHK polypeptides are detected. Our results suggest that although a strong similarity exists between BHK and rat lamins, there is no identical cross-reactivity between the two species.     

A new lamin in Xenopus somatic tissues displays strong homology to human lamin A

The nuclear lamina of vertebrates is composed of several major polypeptides that range in mol. wt from 60 to 80 kd. In mammals, the three major lamin proteins are designated A, B and C. Two major lamins have been described in Xenopus somatic tissues; two other lamins are expressed primarily in germ cells. We have analysed a cDNA clone encoding a Xenopus lamin that is highly homologous to human lamins A and C. The predicted protein has the carboxy-terminal domain characteristic of human lamin A and is thus a lamin A homologue. Surprisingly, the lamin encoded by the cDNA clone is not one of the known Xenopus lamins. The encoded protein is distinct in size from the oocyte lamin LIII and the two somatic lamins LI and LII. Monoclonal antibodies specific for LII, LIII and LIV (the lamin of male germ cells) do not recognize the protein encoded by the cDNA clone; conversely, a polyclonal antibody against the encoded protein does not recognize any of the known Xenopus lamins. This lamin is expressed late in embryonic development, and is present in all adult somatic cells examined, except erythrocytes. Thus frogs and mammals are similar in having three major somatic lamins that fall into distinct structural classes.     

A human T lymphoblastic cell line lacks lamins A and C

Lamins A, B and C, the three major proteins of nuclear envelope, constitute a class of intermediate filament polypeptides. We have compared the amount of these polypeptides in two human cell lines, epithelial HeLa cells and T lymphoblasts KE 37. It was found that the three lamins were present in roughly equimolar stoichiometry in HeLa cells, while lamin B was the unique lamin component in T lymphoblasts. Moreover, 3-kb mRNA of lamin A and 2.1-kb mRNA of lamin C were detected with a human cDNA probe in HeLa cells but not in T lymphoblasts. These results suggest that (i) lamin B can build up the lamina structure in actively dividing somatic cells by itself, and (ii) lamin expression in lymphoid cells may be subject to important quantitative variations. Comparison of the lamin composition of human cloned T lymphocytes and Epstein-Barr virus-transformed human B lymphocytes confirmed this statement. The lamin B level was nearly equivalent in both cells but the content of lamins A and C varied to a large extent, being low in T cells and high in B cells.     

The stability of the nuclear lamina polymer changes with the composition of lamin subtypes according to their individual binding strengths

The nuclear lamina, which provides a structural scaffolding for the nuclear envelope, consists largely of a polymer of the intermediate filament lamin proteins. Although different cell types contain distinctive relative amounts of the major lamin subtypes (A, C, B1, and B2), the functions of this variation are not understood. We have investigated the possibility that subtype variation affects lamina stability. We find that homotypic and heterotypic binding interactions of lamin B2 are substantially less resistant to chemical dissociation in vitro than those between the other lamin subtypes, whereas lamin A interactions are the most stable. Surprisingly, removal of the central four-fifths of the rod domain did not substantially weaken the interactions of lamins A and B2, suggesting that other regions also strongly contribute to their binding interactions. In contrast, this rod deletion strongly destabilizes the binding interactions of lamins B1 and C. Consistent with the binding studies, lamins are more readily solubilized by chemical extraction from cells enriched for lamin B2 than from cells enriched for lamin A. This suggests that the distinctive ensemble of heterotypic lamin interactions in a particular cell type affects the stability of the lamin polymer, and, correspondingly, could be relevant to tissue-specific properties of the lamina including its involvement in disease.     

Differential effect of pH on solubilization of nuclear lamins A/C and lamin B

Extraction of isolated rat liver nuclear envelopes with 4M urea at various pH values led to differential solubilization of lamin polypeptides. All three lamins A, B and C were solubilized to 70-80% when extraction was performed at pH 8 while only 50-60% of lamins A and C were solubilized at pH 6, leaving lamin B completely insoluble. These results indicated that the interaction of lamin B with the nuclear envelope was strongly dependent upon the ionization state of lamin B and/or of its putative attachment site.     

Nuclear envelope proteins: Identification of lamin B subtypes

The lamins are a group of nuclear envelope proteins thought to form a structural layer at the nuclear periphery. Lamins A, B and C occur in many cell types although lamin C, a subtype of lamin A, is relatively decreased in chicken cells. A subtype of lamin B has been found in chicken cells. This subtype, called lamin B1, is slightly larger and more acidic than the quantitatively major subtype now called lamin B2. The lamin B subtypes have very similar primary sequences and share a distinctive topology. Two lamin B subtypes have not been observed in mammalian tissues but have been found in three avian tissues, erythrocytes of mature chickens and liver and erythrocytes of 11- to 13-day-old embryos. As these tissues differ widely in metabolic activity, both subtypes appear to be constitutive nuclear proteins.     

Differential transport and integration into the nuclear lamina for lamins A, B, and C

Lamins A, B, and C are the major proteins of the mammalian nuclear lamina and have been well studied in BHK-21 cells. Using in vivo labelling, cell fractionation, and immunoprecipitation, we have found that lamins have different patterns of nuclear transport and solubility. Newly synthesized lamin A is translocated to the nucleus faster than lamin C or B. It is the most tightly bound lamin and cannot be extracted from the lamina by nonionic detergent or high-salt buffers. Lamins B and C migrate more slowly to the nucleus. Partitioning between cytoskeleton and detergent-soluble fractions shows that integration of lamins B and C is not completed before a 1-h chase. For lamin C this process is dependent upon protein synthesis and can be inhibited with cycloheximide. Even though lamins A and C are almost identical, lamin C is never firmly bound to the lamina and can be partially solubilized upon high-salt treatment.     

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.     

Characterization of a second highly conserved B-type lamin present in cells previously thought to contain only a single B-type lamin

Previous analyses of the nuclear lamina of mammalian cells have revealed three major protein components (lamins A, B and C) that have been identified by protein sequence homology as members of the intermediate filament (IF) protein family. It has been claimed that mammalian cells contain either all three lamins or lamin B alone. Using monoclonal antibodies specific for B-type lamins and cDNA cloning we identified a second major mammalian B-type lamin (murine lamin B₂), thus showing that lamin composition in mammals is more complex than previously thought. Lamin B₂ is coexpressed with lamin B₁ (formerly termed lamin B) in all somatic cells and mammalian species that we analysed, including a variety of cells currently believed to contain only a single lamin. This suggests that two B-type lamins are necessary to form a functional lamina in mammalian somatic cells. By cDNA cloning we found thatXenopus laevis lamin L_II is the amphibian homolog of mammalian lamin B₂. Lamin expression during embryogenesis of amphibians and mammals shows striking similarities. The first lamins expressed in the early embryo are the two B-type lamins, while A-type lamins are only detected much later in development. These findings indicate that the genomic differentiation into two B-type lamins occurred early in vertebrate evolution and has been maintained in both their primary structure and pattern of expression.     

Modulators of the eukaryotic heat shock response

The nuclear lamina consists of a proteinaceous layer or meshwork situated subjacent to the inner nuclear membrane. It is a karyoskeletal structure formed by a polymer containing one to three major polypeptides collectively termed the lamins. In all cells examined of vertebrates and invertebrates, the lamins exhibit very similar Mr ranging from 60 000 to 80 000. In vertebrates, two groups of lamins can be distinguished by their isoelectric value, one being near-neutral and the other acidic (isoelectric pH values of 5.6 and lower). The lamins represent a family of polypeptides with regions highly conserved during evolution. In certain species, e.g., the amphibian, Xenopus laevis, they exhibit cell type-specific expression during embryonic development, terminal differentiation of certain somatic cells, and gametogenesis. The nuclear lamina of diverse cell types can be composed of one, two or three different lamin polypeptides, without obvious differences in its morphology.     

Presence of a novel nuclear lamina protein in Raji cells.

In mammalian tissues, the nuclear lamina is composed of the major lamins A, B, and C, and minor lamins D/E. Although lamin B is present in all cell types, lamins A and C are absent from embryonic cells and most undifferentiated cells from hematopoietic lineage. We have investigated the nuclear lamina protein composition of the Raji cell line, lymphoblast-like cells established from a Burkitt lymphoma patient. Lamins A and C were confirmed absent by immunodetection and Northern blot analysis. Besides lamins B and D/E, a protein migrating around 71 kilodaltons was recognized by a serum directed against the nuclear lamina of BHK-21 fibroblasts. Cellular localization by sequential extraction established this 71-kilodalton protein as an exclusive component of the nuclear lamina fraction. These results indicate that the nuclear lamina has a more complex composition than previously thought to be the case for cells devoid of lamins A and C.     

Differential sensitivity of vimentin and nuclear lamins from Ehrlich ascites tumor cells toward Ca2+ -activated neutral thiol proteinase

A comparative study of the susceptibility of vimentin and nuclear lamins from cultured Ehrlich ascites tumor (EAT) cells to degradation by Ca2+ -activated neutral thiol proteinase (calpain) has been undertaken. While pure vimentin was degraded very quickly at physiological ionic strength by purified calpain, isolated lamin B was digested comparatively slowly and purified lamins A/C were fairly resistant to proteolytic degradation. Similar digestion patterns were obtained from vimentin and lamin B with intermediary breakdown products close in size to the corresponding alpha-helical rod domains. To exclude the possibility that the low susceptibility of isolated lamins to Ca2+-dependent proteolytic degradation was due to irreversible denaturation during their isolation and purification, Triton cytoskeletons were prepared and their nuclear lamina as well as vimentin filaments were exposed to relatively large quantities of purified calpain. Under these conditions, not only vimentin filaments but also lamins A and B were digested while lamin C remained intact to a high degree. The major breakdown products of vimentin and lamins were identified as polypeptides which were 35 to 45 amino acids longer than the corresponding alpha-helical rod domains. Most of the vimentin-derived material and all high molecular weight polypeptides originating from lamins remained associated with the Triton cytoskeletons as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with immunoblotting. Indirect immunofluorescence and electron microscope analysis of the calpain-digested Triton cytoskeletons revealed that they still contained a laminalike structure around the nuclear chromatin and numerous structurally altered intermediate filaments in the cytoplasmic remnant, although all vimentin had been degraded with the formation of 40/41 kDa polypeptides as major digestion products. In untreated Triton cytoskeletons, the vimentin filaments seemed to be in direct physical contact with the nuclear lamina, whereas in digested Triton cytoskeletons there was a distinct gap between structurally altered filaments and the nuclear surface. This shows that vimentin filaments and the nuclear lamina are differentially susceptible to degradation by calpain under certain ionic conditions and suggests that both filamentous structures are intimately associated with each other.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of nuclear lamins A/C in macrophages in in vitro cultures of rat bone marrow precursor cells and human blood monocytes, and in macrophages elicited in vivo by thioglycollate stimulation

Hemopoietic cells from blood and bone marrow of mammals usually do not express lamins A/C but only lamin B, and this feature distinguishes these cells from the vast majority of somatic cells of the adult animal, which reveal lamins A/C as well as lamin B. Here we have cultivated rat bone marrow precursor cells and human monocytes isolated from peripheral blood in tissue culture supplemented with certain growth factors. These conditions allow bone marrow precursor cells and monocytes to differentiate almost quantitatively into accessory cells and/or mature macrophages. The different cell types in the cultures can be identified both morphologically and by other assays. Antibodies specific for mouse A/C lamins, human A/C lamins, or B lamins have been used to define the lamin complement as a function of time in culture and of cell type. A dramatic increase in lamin A/C-positive cells was observed in the first 3 days of culture with both accessory cells and macrophages expressing lamins A/C as soon as such cell types could be identified. Parallel in vivo experiments showed that treatment with thioglycollate caused the percentage of lamin A/C-positive peritoneal macrophages to increase from 5 to 80% between Days 0 and 6.     

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.     

Amino acid sequence and molecular characterization of murine lamin B as deduced from cDNA clones

The nuclear lamina is the karyoskeletal structure, intimately associated with the nuclear envelope, that is widespread among the diverse types of eukaryotic cells. A family of proteins, termed lamins, has been shown to be a prominent component of this lamina, and various members of this family are differentially expressed in different cell types. In mammals, three major lamins (A, B, C) have been identified, and in all cells so far examined lamin B is constitutively expressed while lamins A and C are not, suggesting that lamin B is sufficient to form a functional lamina. Because of this key importance of lamin B, cDNA clones encoding mammalian lamin B were isolated by screening murine cDNA libraries, representing F9 teratocarcinoma cells and fetal liver, with the corresponding cDNA probe of lamin LI of Xenopus laevis. The nucleotide sequence of the murine lamin B mRNA (approximately 2.9 kb) was determined. The deduced amino acid sequence of the encoded polypeptide (587 amino acids; mol. wt. 66760) is highly homologous to X. laevis lamin LI (72.9% identical residues) but displays lower similarity to A-type lamins (53.8% identical amino acid residues with human lamin A). Lamin B also conforms to the general molecular organization principle of the members of the intermediate filament (IF) protein family, i.e., an extended alpha-helical rod domain that is interrupted by two non alpha-helical linkers and flanked by non-alpha-helical head (amino-terminal) and tail (carboxy-terminal) domains. The tail domain, which does not reveal a hydrophobic region of considerable length, contains a typical karyophilic signal sequence and an uninterrupted stretch of eight negatively charged amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)