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.     

Cell type-specific differences in protein composition of nuclear pore complex-lamina structures in oocytes and erythrocytes of Xenopus laevis

Nuclear envelopes from oocytes of Xenopus laevis are rich in pore complexes and contain a major polypeptide of apparent molecular weight (M_r) 68,000. A rapid extraction procedure using buffer containing 1% ($\text{v}\text{v}$) Triton X-100 and 1.0 m-KCl allows the preparation of insoluble nuclear envelope skeletons showing only residual pore complex structures, with some interconnecting filament material, and one major polypeptide; i.e. that of M_r 68,000. This skeletal protein, which is not found in nuclear contents, reveals, on two-dimensional gel electrophoresis, a series of distinct isoelectric variants focusing in the pH range from 6.4 to 6.6. In living oocytes, this protein is continuously synthesized, as demonstrated by incorporation of labelled amino acids, and phosphorylated, A similar prominent skeletal protein has been found in nuclear envelopes of oocytes of other amphibia; however, slight but significant differences in electrophoretic mobility can be noted between different amphibian species.For comparison, nucleocortical lamina structures containing few pore complexes have been isolated, using similar extraction procedures, from various somatic cells of X. laevis, including erythrocytes. Laminae from these cells contain two major polypeptides, one (L_I) of M_r 72,000 focusing at approximately pH 5.35 and another (L_II) of M_r 69,000 focusing in several variants between pH 6.20 and 6.35. Similarly extracted “pore complex-lamina” fractions from rat liver contain a polypeptide of similar size and electrical charge as protein L_I from Xenopus and, in addition, two other polypeptides (M_r values: 74,000 and 62,000) both focusing between pH 6.6 and 6.9.It is concluded that the pore complex-lamina structure of the oocyte nucleus is assembled by only one major protein of M_r 68,000. The results also show that the protein composition of this insoluble nucleocortical structure can be different in different cells of the same organism. The compositional differences of these nuclear envelope skeletons are discussed in relation to the relative proportions of pore complex and interporous (lamina) material in the nuclear envelopes of the specific cells. It is suggested that the M_r 68,000 protein predominant in oocyte nuclear envelopes contributes, as an architectural component, to the formation of the highly organized nuclear pore complex.     

Immunological localization of a major karyoskeletal protein in nucleoli of oocytes and somatic cells of Xenopus laevis

Oocyte nuclei of Xenopus laevis contain two major karyoskeletal proteins characterized by their resistance to extractions in high salt buffers and the detergent Triton X-100, i.e. a polypeptide of 68,000 mol wt which is located in the core complex-lamina structure and a polypeptide of 145,000 mol wt enriched in nucleolar fractions. Both proteins are also different by tryptic peptide maps and immunological determinants. Mouse antibodies were raised against insoluble karyoskeletal proteins from Xenopus oocytes and analyzed by immunoblotting procedures. Affinity purified antibodies were prepared using antigens bound to nitrocellulose paper. In immunofluorescence microscopy of Xenopus oocytes purified antibodies against the polypeptide of 145,000 mol wt showed strong staining of nucleoli, with higher concentration in the nucleolar cortex, and of smaller nucleoplasmic bodies. In various other cells including hepatocytes, Sertoli cells, spermatogonia, and cultured kidney epithelial cells antibody staining was localized in small subnucleolar granules. The results support the conclusion that this "insoluble" protein is a major nucleus-specific protein which is specifically associated with--and characteristic of--nucleoli and certain nucleolus-related nuclear bodies. It represents the first case of a positive localization of a karyoskeletal protein in the nuclear interior, i.e. away from the pore complex-lamina structure of the nuclear cortex.     

Immunological localization of the major architectural protein associated with the nuclear envelope of the Xenopus laevis oocyte

The nuclear envelope (NE) of Xenopus laevis contains a major architectural protein which is resistant to extraction in high salt buffer and non-ionic detergent and is characterized by a polypeptide molecular weight (MW) of 68000. Two different antisera which showed specific binding of antibodies (IgG) to this polypeptide, as demonstrated by ‘immunoblotting’ techniques, were used for immunolocalization at the electron microscopic level. Whereas antibodies of serum I reacted only with the nuclear lamina structure, antibodies of serum II, which were raised against undenatured karyoskeletal protein from oocytes, showed additional strong reaction in nuclear pore complexes. This first positive localization of a polypeptide in the nuclear pore complex suggests that MW 68000 polypeptide contributes as a major karyoskeletal component to the structure of both the lamina and the pore complex.     

Immunological identification of the karyophilic, histone-binding proteins N1 and N2 in somatic cells and oocytes of diverse amphibia

A polypeptide pair designated N1/N2 (Mr 100 000 and 110 000) is an exceptionally acidic and abundant nuclear protein of oocytes of the toad, Xenopus laevis, and is characterized by a pronounced karyophilia. These proteins have been shown to form specific complexes with free, i.e., non-chromatin-bound histones H3 and H4 (Kleinschmidt & Franke, Cell 29 (1982) 799) [3]. In order to study these proteins and their possible counterparts in other species, antibodies were produced in guinea pigs against proteins N1/N2 purified from Xenopus oocyte nuclei. Using gel electrophoresis, peptide map analysis, immunoblotting techniques and immuno fluorescence microscopy the existence of polypeptides identical in Mr value and charge to polypeptide N1 of oocytes was demonstrated in cultured somatic cells of Xenopus laevis, where it was also highly enriched in cell nuclei, although the cellular concentration was much lower than in oocytes. A similar, if not identical protein, was recognized in nuclei of diverse other cell types including hepatocytes, enterocytes, ovarian follicle cells, and Sertoli cells of testis, of Xenopus, Rana temporaria, R. esculenta, Pleurodeles waltlii but not in erythrocytes and later stages of spermiogenesis. When nuclear proteins from oocytes of different amphibian species were examined with these antibodies it was found that the Mr values of N1/N2 proteins were considerably different in different species, ranging from Mr 110 000 to 190 000. Immunoprecipitation and gel electrophoretic analysis under non-denaturing conditions showed that a significant proportion of these proteins was contained in complexes with histones H3 and H4. The results demonstrate that proteins N1/N2 are not special proteins of oocytes of Xenopus laevis but occur in various other cells of diverse amphibian species. The widespread occurrence of these karyophilic proteins indicates that at least one function of these proteins, i.e., selective binding of the arginine-rich histones H3 and H4, is not exclusive to oocytes but may also contribute to the regulation of histone pools and chromatin formation in other cell types.     

Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations

Nuclear lamin isoforms of vertebrates can be divided into two major classes. The B-type lamins are membrane associated throughout the cell cycle, whereas A-type lamins are recovered from mitotic cell homogenates in membrane-free fractions. A feature of oogenesis in birds and mammals is the nearly exclusive presence of B-type lamins in oocyte nuclear envelopes. In contrast, oocytes and early cleavage embryos of the amphibian Xenopus laevis are believed to contain a single lamin isoform, lamin LIII, which after nuclear envelope breakdown during meiotic maturation is reported to be completely soluble. Consequently, we have reexamined the lamin complement of Xenopus oocyte nuclear envelopes, egg extracts, and early embryos. An mAb (X223) specific for the homologous B-type lamins B2 of mouse and LII of Xenopus somatic cells (Hoger, T., K. Zatloukal, I. Waizenegger, and G. Krohne. 1990. Chromosoma. 99:379-390) recognized a Xenopus oocyte nuclear envelope protein biochemically distinct from lamin LIII and very similar or identical to somatic cell lamin LII. Oocyte lamin LII was detectable in nuclear envelopes of early cleavage embryos. Immunoblotting of fractionated egg extracts revealed that approximately 20-23% of lamin LII and 5-7% of lamin LIII were membrane associated. EM immunolocalization demonstrated that membrane-bound lamins LII and LIII are associated with separate vesicle populations. These findings are relevant to the interpretation of nuclear reconstitution experiments using Xenopus egg extracts.     

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.     

Observation of nuclei reassembled from demembranated Xenopus sperm nuclei and analysis of their lamina components

A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei.Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear memblane,nuclear pore complexes,and decondensed chromatin etc.Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei.Western-blotting results showed that lamin LII was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only egg derived lamin LIII.     

Electron microscopic immunolocalization of a karyoskeletal protein of molecular weight 145 000 in nucleoli and perinucleolar bodies of Xenopus laevis

Amplified nucleoli of Xenopus laevis oocytes contain a major karyoskeletal protein of Mr 145 000 insoluble in low- and high-salt buffer as well as in non-denaturing detergents. Electron microscopic localization on native and high-salt extracted nucleoli using specific murine antibodies against this polypeptide and gold-coupled antibodies for visualization reveals that the Mr 145 000 protein is located in coils of filaments of ca 4 nm diameter. In addition, this protein occurs in the medusoid filament bodies (MFBs) present in the nucleolar cortex and free in the nucleoplasm. In somatic cells of tissues and in A6 kidney epithelial cells grown in vitro the Mr 145 000 polypeptide or an immunologically related protein is also organized in coiled aggregates of filaments 4-12 nm in diameter present both in the periphery of nucleoli and free in the nucleoplasm. We discuss a possible role of this protein as a karyoskeletal support involved in the storage and transport of preribosomal particles.     

A 174-kilodalton ATPase/dATPase polypeptide and a glycoprotein of apparently identical molecular weight are common but distinct components of higher eukaryotic nuclear structural protein subfractions

A 174-kilodalton polypeptide of the Drosophila nuclear matrix-pore complex-lamina fraction has been identified as an ATPase/dATPase on the basis of direct UV photoaffinity labeling studies; a polypeptide with similar properties has been found in nuclear envelope fractions prepared from several vertebrate sources (Berrios, M., Blobel, G., and Fisher, P. A. (1983) J. Biol. Chem. 258, 4548-4555). This ATPase/dATPase polypeptide co-migrates on sodium dodecyl sulfate (SDS)-polyacrylamide gels with a glycoprotein also found in all of these fractions. In rat liver, this glycoprotein has been localized to the nuclear pore complex by means of immunoelectron microscopy (Gerace, L., Ottaviano, Y., and Kondor-Koch, C. (1982) J. Cell Biol. 95, 826-837). Following SDS denaturation and reduction/alkylation, chromatography of the Drosophila nuclear matrix-pore complex-lamina fraction on hydroxylapatite columns run in the presence of SDS results in the separation of two quantitatively major 174-kilodalton polypeptides. The peak of glycoprotein elution from the SDS-hydroxylapatite column correlates exactly with that of the early eluting 174-kilodalton species while the photolabeled ATPase/dATPase polypeptide co-chromatographs with the late eluting one. Identical results have been obtained with the rat liver nuclear envelope fraction. The chromatographically separated 174-kilodalton species from both organisms have been further distinguished through the use of polypeptide-specific antisera; finally, the glycoprotein purified from Drosophila embryos is fully sensitive to limited degradation by endoglycosidase H whereas the ATPase/dATPase polypeptide is completely resistant. We have thus established, using material obtained from two widely divergent higher eukaryotes, that the 174-kilodalton ATPase/dATPase is a quantitatively major nuclear matrix-pore complex-lamina component distinct from the nuclear pore complex glycoprotein of apparently identical molecular weight.     

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)     

A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei

A modified procedure for the isolation of a nuclear pore complex-lamina fraction from rat liver nuclei is described. Evidence is provided that the isolated lamina, a 150-A thick, proteinaceous structure, apposes the inner nuclear envelope membrane, connecting nuclear pore complexes and surrounding the entire nucleus.     

Nature of albumin exported from the frog oocyte following microinjection of mouse liver mRNA

Microinjections of mouse liver mRNA into Xenopus laevis oocytes induced efficient export of a polypeptide with an apparent Mr or 68,000 which was immunoprecipitable with anti-mouse albumin antibody. Analysis of the anti-albumin precipitate of the exported protein by two-dimensional gel electrophoresis showed that the electrophoretic behavior precisely coincided with that of authentic mouse serum albumin. This result indicates that the Xenopus oocyte may perform secretory processes similar or identical to those occurring in liver cells with respect to the processing of albumin.     

Vaults. II. Ribonucleoprotein structures are highly conserved among higher and lower eukaryotes.

Vaults are cytoplasmic ribonucleoprotein structures that display a complex morphology reminiscent of the multiple arches which form cathedral vaults, hence their name. Previous studies on rat liver vaults (Kedersha, N. L., and L. H. Rome. 1986. J. Cell Biol. 103:699-709) have established that their composition is unlike that of any known class of RNA-containing particles in that they contain multiple copies of a unique small RNA and more than 50 copies of a single polypeptide of 104,000 Mr. We now report on the isolation of vaults from numerous species and show that vaults appear to be ubiquitous among eukaryotes, including mammals, amphibians (Rana catesbeiana and Xenopus laevis), avians (Gallus Gallus), and the lower eukaryote Dictyostelium discoideum. Electron microscopy reveals that vaults purified from these diverse species are similar both in their dimensions and morphology. The vaults from these various species are also similar in their polypeptide composition; each being composed of a major polypeptide with an approximate mass of 100 kD and several minor polypeptides with molecular masses similar to those seen in the rat. Antibodies raised against rat vaults recognize the major vault protein of all species including Dictyostelium. Vaults therefore appear to be strongly conserved and broadly distributed, suggesting that their function is essential to eukaryotic cells.     

Isolation of mRNA from membrane-bound polysomes synthesizing Sepia officinalis haemocyanin in Xenopus laevis oocytes

Isolation and translation in Xenopus laevis oocytes of the mRNAs from the nuclear pellet and the cytoplasmic supernatant of the branchial glands from Sepia officinalis, homogenized in the presence of vanadyl-ribonucleoside complexes, revealed that the membrane-bound polysomes for haemocyanin sedimented together with the nuclei. Centrifugation on a 15-50% sucrose gradient of these polysomes, released by treatment with Triton X-100, yielded a major peak of heavy ones. The messenger fraction, isolated from this heavy fraction, directed the synthesis of the large 390 000 Mr haemocyanin polypeptide chain in oocytes. A large mRNA on heavy membrane-bound polysomes thus synthesizes the whole haemocyanin chain of S. officinalis.