Cell type-specific expression of nuclear lamina proteins during development of Xenopus laevis

The cell type-specific expression of the major nuclear lamina polypeptides ("lamins") during development of Xenopus was studied using two monoclonal antibodies (L(0)46F7: specific for LIII, the single lamin of oocytes; PKB8: specific for LI and LII of some somatic cells). In the oocyte, LIII localizes in the nuclear polymer, but upon nuclear envelope breakdown it is solubilized to a form sedimenting at 9 S. In early embryos, LIII contributes to nuclear lamina formation until its depletion. Correspondingly, LI and LII begin to be expressed at a specific point in embryogenesis and appear to be integrated with LIII into a common lamina structure. Later in development, LIII reappears as a prominent nuclear lamina protein but only in certain cells (neurons, muscle cells, and diplotene oocytes). We conclude that amphibian lamins represent a family of proteins expressed in relation to certain programs of cell differentiation.     

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.     

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.     

A cell free system to study reassembly of the nuclear envelope at the end of mitosis

We described a cell free system involving total homogenates of metaphase CHO cells, which yields telophase-like assembly of nuclear envelopes around mitotic chromosomes. During formation of the nuclear envelope in vitro, the three major lamina polypeptides (lamins A, B, and C) assemble around chromosomes and become dephosphorylated, similar to their behavior in vivo during telophase. Nuclear lamina and envelope assembly apparently do not require free ATP and are strongly inhibited by gamma-S-ATP, supporting the notion that these processes are regulated by protein dephosphorylation. Immunological depletion of disassembled lamins from the initial assembly system results in strong inhibition of subsequent nuclear envelope assembly, directly demonstrating that the lamins are involved in this process.     

A monoclonal antibody against nuclear lamina proteins reveals cell type-specificity in Xenopus laevis

Immunofluorescence microscopy shows that the monoclonal murine antibody PKB8 stains the nuclear lamina of various somatic cells from vertebrates as diverse as mammals, birds and amphibia. It also decorates the nuclear periphery of oocytes from rat and chicken but does not react with spermatocytes, spermatids and spermatozoa. Immunoblotting experiments demonstrate reaction with lamina polypeptides A, B and C of rat, with lamina polypeptide A of chicken, and with lamina polypeptides LI and LII of erythrocytes of the frog, Xenopus laevis. Antibody PKB8 does, however, not bind, on blotted polypeptides and on sections through ovaries, to the pore complex-lamina polypeptide of Mr 68000 present in Xenopus oocytes. These results reveal the existence of a common antigenic determinant in all three lamina polypeptides of mammals, in one lamina polypeptide of chicken and in two amphibian lamina polypeptides. The immunological data also indicate that, in Xenopus laevis, pore complex-lamina polypeptides of somatic cells and oocytes are distinct. The Mr 68000 protein of Xenopus oocytes is also different from polypeptides LI and LII of somatic Xenopus cells by tryptic peptide mapping. The results suggest that nuclear pore complex-lamina polypeptides represent a family of related polypeptides containing regions highly conserved during evolution and that these polypeptides can be differentially expressed in cells of at least one species, Xenopus laevis.     

Immunolocalization of lamins in the thick nuclear lamina of human synovial cells

While in the great majority of cells the nuclear lamina is not resolved as a distinct structure separating the chromatin from the nuclear envelope, a demonstrable nuclear lamina ("fibrous lamina") of 30 to 300 nm thickness, interposed between the inner nuclear membrane and the peripheral chromatin, is characteristic for certain types of cells of vertebrates and invertebrates. We have examined whether the thick (50-70 nm) fibrous lamina of human synovial cells from patients suffering from rheumatoid arthritis indeed contains the lamins found in the indiscernible lamina structures present in most normal cells. We have observed, by electron microscopic immunolocalization, that both the A and the B type lamins occur throughout the entire nuclear lamina of these cells and that this structure is also resistant to treatments with nucleases and high salt buffers. This shows that the thick fibrous lamina only seen in certain vertebrate cells is compositionally related to the "masked" nuclear lamina of most other cells which usually is identified only upon removal of the adjacent nuclear structures.     

Independent expression and assembly properties of heterologous lamins A and C in murine embryonal carcinomas.

The majority of cells derived from adult mammalian tissues contain three major species of nuclear lamin proteins, A, B and C. In contrast, embryonic cells including undifferentiated murine embryonal carcinomas, contain only B-type lamins, A and C appearing only after differentiation. Human lamins A or C have been introduced by transfection into undifferentiated P19 embryonal carcinomas. Twenty-four hours after transfection, both of these proteins were found to independently associate with the nuclear envelope as judged by immunofluorescence microscopy and at the same time were associated with a salt-resistant structure having solubility properties similar to those of the nuclear lamina. Biosynthetic experiments indicated that heterologous lamin A underwent processing to its mature molecular weight, an event which in adult type cells occurs after assembly into the lamina. Observations on mitotic cells demonstrate that either of the two human lamins will, independent of the other, become dispersed throughout the cytoplasm during prophase and subsequently reassemble at the nuclear periphery during telophase. Nuclear lamins A and C are not, however, equivalent in their abilities to incorporate into the nuclear lamina in these cells. Experiments involving cells arrested in S phase using thymidine suggest that lamin C, but not lamin A, requires progression through the cell cycle and probably mitosis for assembly into the nuclear lamina of P19 EC cells.     

Phosphorylation of the nuclear lamins during interphase and mitosis

The nuclear lamina is a polymeric protein assembly that is proposed to function as an architectural framework for the nuclear envelope. Previous work suggested that phosphorylation of the major polypeptides of the lamina (the "lamins") may induce disassembly of this structure during mitosis. To further investigate the possible involvement of phosphorylation in regulation of lamina structure, we characterized lamin phosphorylation occurring in mammalian tissue culture cells during interphase and mitosis. Phosphorylation occurs continuously throughout all interphase periods (coordinately with nuclear envelope growth), and takes place mainly on the assembled lamina. When the lamina is disassembled during cell division, the lamins are modified with approximately 1-2 molecules of associated phosphate. This level of mitotic phosphorylation is 4-7-fold higher than the average interphase level. Lamin phosphate occurs predominantly as phosphoserine, and is distributed over numerous tryptic peptides, many of which are modified during both interphase and mitotic periods. Significantly, phosphorylation is the only detectable charge-altering postsynthetic modification of the lamins that occurs specifically during mitosis. The results of this study support the notion that phosphorylation is important for regulation of interphase and mitotic lamina structure.     

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)     

Minor lamin polypeptides from rat liver nuclei can be cross-linked into heteropolymers by disulfide bridges

The peripheral lamina of rat liver nuclei is characterized by the presence of three major polypeptides called lamins A, B, and C. Recent studies have identified in rat liver lamina two quantitatively minor polypeptides that have some of the biochemical and immunological properties of the lamins and were tentatively called minor lamin species. We have further characterized these minor lamin polypeptides. Both minor lamin species copurified quantitatively with the major lamins in dissociation-reassociation experiments and shared epitopes with all three major lamins as well as with intermediate filament proteins, including an epitope involved in coiled-coil interactions in lamina and filaments. Minor lamins generated partial peptide maps very similar to each other but completely different from those of lamins A, B, and C. The two minor lamin species could be cross-linked into heteropolymers containing a constant ratio of both polypeptides by exposure to O-phenanthroline - cupric ion complexes, although they did not appear to be cross-linked by disulfide bonds in the native envelope. Preliminary results suggest that the cross-linked minor lamins could be preferentially associated with lamin B. It therefore appears that in addition to the network of lamins A, B, and C, the peripheral lamina is characterized by the presence of two closely juxtaposed minor lamin polypeptides. The molecular interactions between these various polypeptides and their respective roles remain to be identified.     

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.     

Nuclear lamins A and B1: different pathways of assembly during nuclear envelope formation in living cells

At the end of mitosis, the nuclear lamins assemble to form the nuclear lamina during nuclear envelope formation in daughter cells. We have fused A- and B-type nuclear lamins to the green fluorescent protein to study this process in living cells. The results reveal that the A- and B-type lamins exhibit different pathways of assembly. In the early stages of mitosis, both lamins are distributed throughout the cytoplasm in a diffusible (nonpolymerized) state, as demonstrated by fluorescence recovery after photobleaching (FRAP). During the anaphase-telophase transition, lamin B1 begins to become concentrated at the surface of the chromosomes. As the chromosomes reach the spindle poles, virtually all of the detectable lamin B1 has accumulated at their surfaces. Subsequently, this lamin rapidly encloses the entire perimeter of the region containing decondensing chromosomes in each daughter cell. By this time, lamin B1 has assembled into a relatively stable polymer, as indicated by FRAP analyses and insolubility in detergent/high ionic strength solutions. In contrast, the association of lamin A with the nucleus begins only after the major components of the nuclear envelope including pore complexes are assembled in daughter cells. Initially, lamin A is found in an unpolymerized state throughout the nucleoplasm of daughter cell nuclei in early G1 and only gradually becomes incorporated into the peripheral lamina during the first few hours of this stage of the cell cycle. In later stages of G1, FRAP analyses suggest that both green fluorescent protein lamins A and B1 form higher order polymers throughout interphase nuclei.     

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.     

Lamins A and C appear during retinoic acid-induced differentiation of mouse embryonal carcinoma cells

The lamin complement of nuclear matrix isolated from F9 embryonal carcinoma cells was studied during retinoic acid-induced differentiation in culture. Differentiation of the original cells into parietal endoderm-like cells was accompanied by the gradual appearance of lamins A and C while lamin B was present throughout all stages. Lamins were identified by their molecular masses, isoelectric points, recognition by a monoclonal antibody and a polyclonal antiserum, and by peptide mapping. The increase in the amounts of lamins A and C found in the matrix was due to de novo synthesis as no extranuclear pools of these lamins were detected in the undifferentiated cells. These results provide biochemical evidence that, as in amphibian embryogenesis, there are variations in nuclear lamina composition during mammalian development.     

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.     

Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy

The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.     

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.