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.     

A constitutive nucleolar protein identified as a member of the nucleoplasmin family.

Using monoclonal antibodies we have localized a polypeptide, appearing on gel electrophoresis with a Mr of approximately 38,000 and a pI of approximately 5.6, to the granular component of the nucleoli of Xenopus laevis oocytes and a broad range of cells from various species. The protein (NO38) also occurs in certain distinct nucleoplasmic particles but is not detected in ribosomes and other cytoplasmic components. During mitosis NO38-containing material dissociates from the nucleolar organizer region and distributes over the chromosomal surfaces and the perichromosomal cytoplasm; in telophase it re-populates the forming nucleoli. With these antibodies we have isolated from a X. laevis ovary lambda gt11 expression library a cDNA clone encoding a polypeptide which, on one- and two-dimensional gel electrophoresis, co-migrates with authentic NO38. The amino acid sequence deduced from this clone defines a polypeptide of 299 amino acids of mol. wt 33,531 which is characterized by the presence of two domains exceptionally rich in aspartic and glutamic acid, one of them flanked by two putative karyophilic signal heptapeptides. Comparison with other protein sequences shows that NO38 is closely related to the histone-binding, karyophilic protein nucleoplasmin: the first 124 amino acids have 58 amino acid positions in common. Protein NO38 also shows striking homologies to the phosphopeptide region of rat nucleolar protein B23 and the carboxyterminal region of human B23. We propose that protein NO38, which forms distinct homo-oligomers of approximately 7S and Mr of approximately 230,000, is a member of a family of karyophilic proteins, the 'nucleoplasmin family'. It is characterized by its specific association with the nucleolus and might be involved in nuclear accumulation, nucleolar storage and pre-rRNA assembly of ribosomal proteins in a manner similar to that discussed for the role of nucleoplasmin in histone storage and chromatin assembly.     

Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation.

DNA (from bacteriophage lambda or Xenopus) is assembled into nucleus-like structures when mixed with an extract from Xenopus eggs. Electron microscopy shows that these in vitro-reconstituted nuclei possess complete double membranes; some, but not all, nuclei have pore complexes. Extracts depleted of their endogenous ATP (by addition of ATPases) cannot assemble nuclear envelopes visible by phase-contrast microscopy. Once synthetic nuclei are assembled, however, they are stable when ATP is subsequently depleted, although their chromatin becomes condensed. About one-fourth of the nuclei assembled in vitro from lambda DNA accumulate nuclear proteins such as nucleoplasmin. ATP depletion blocks nucleoplasmin accumulation both in vitro, in pre-assembled synthetic nuclei, and in vivo, in the nucleus of microinjected oocytes. However, nucleoplasmin previously accumulated by reconstituted nuclei or by the germinal vesicle in microinjected oocytes is retained after ATP depletion.     

Expression and segregation of nucleoplasmin during development in Xenopus

The spatial segregation of informational molecules in the unfertilized egg and embryo has been hypothesized to be a necessary phenomenon for the normal progression of development leading to the determination of cellular phenotypes. This study describes the selection of a monoclonal antibody (Mab: 2G6) that identifies an antigen (Ag: 2G6) which is localized in the germinal vesicle of oocytes and has a discrete pattern of inheritance during embryogenesis. The antigen displayed biochemical and physical characteristics very similar to nucleoplasmin, which is the histone-binding and nucleosome-assembly protein previously described. Immunoblot analysis with purified oocyte nucleoplasmin confirmed this relationship. Indirect immunofluorescence was used to study the temporal expression and spatial distribution of nucleoplasmin. From early cleavage stages through gastrulation, it is preferentially localized in nuclei of blastomeres at the animal pole. By tadpole stages, it was detected only in nuclei of postmitotic cells of the central nervous system and in nuclei of striated muscle. It was not detected in adult tissues. Western blot analysis during embryogenesis revealed at least five immunologically related polypeptides that displayed distinct patterns of expression during development. The different species observed most likely represent different levels of phosphorylation of nucleoplasmin. The more acidic forms, known to be more active in nucleosome assembly, were present during cleavage stages. Analysis of labelled oocyte proteins by two-dimensional immunoblots and autoradiography revealed that synthesis of nucleoplasmin was first detected in stage-2 oocytes, reached 60% maximum levels at stage 3, peaked at stage 4 and was undetectable in stage-6 oocytes. The amount of nucleoplasmin message present does not follow a similar pattern during oogenesis. These results suggest that the message undergoes pronounced changes in translational efficiency during oogenesis. A comparative immunoblot analysis using proteins from a variety of adult tissues revealed that, whereas the polyclonal antisera against amphibian vitellogenic oocyte nucleoplasmin recognized several different, tissue-specific polypeptides, two different monoclonal antibodies (Mab: b7-1D1, Mab: 2G6) failed to recognize any of the adult tissues tested. We conclude that nucleoplasmin is a family of closely related proteins with distinct embryonic and adult members.     

Purification of a novel, nucleoplasmin-like protein from somatic nuclei.

We have purified a nucleoplasmin-like protein from the nuclei of somatic Xenopus laevis cells. This protein possesses a number of the distinctive features of nucleoplasmin isolated from oocytes or unfertilized eggs. The protein is recognized by both monoclonal and polyclonal antisera raised against egg nucleoplasmin. The protein has an oligomeric structure, which must be heated in SDS to completely dissociate, is acidic, phosphorylated and efficiently promotes the in vitro formation of chromatin. We have partially characterized this novel protein and because of its resemblance to nucleoplasmin isolated from oocytes or unfertilized eggs we have named this protein nucleoplasmin S.     

A Novel Karyoskeletal Protein: Characterization of Protein NO145, the Major Component of Nucleolar Cortical Skeleton in Xenopus Oocytes

The nucleolus is a ubiquitous, mostly spheroidal nuclear structure of all protein-synthesizing cells, with a well-defined functional compartmentalization. Although a number of nonribosomal proteins involved in ribosome formation have been identified, the elements responsible for the shape and internal architecture of nucleoli are still largely unknown. Here, we report the molecular characterization of a novel protein, NO145, which is a major and specific component of a nucleolar cortical skeleton resistant to high salt buffers. The amino acid sequence of this polypeptide with a SDS-PAGE mobility corresponding to M(r) 145,000 has been deduced from a cDNA clone isolated from a Xenopus laevis ovary expression library and defines a polypeptide of 977 amino acids with a calculated mass of 111 kDa, with partial sequence homology to a synaptonemal complex protein, SCP2. Antibodies specific for this protein have allowed its recognition in immunoblots of karyoskeleton-containing fractions of oocytes from different Xenopus species and have revealed its presence in all stages of oogenesis, followed by a specific and rapid degradation during egg formation. Immunolocalization studies at the light and electron microscopic level have shown that protein NO145 is exclusively located in a cage-like cortical structure around the entire nucleolus, consisting of a meshwork of patches and filaments that dissociates upon reduction of divalent cations. We propose that protein NO145 contributes to the assembly of a karyoskeletal structure specific for the nucleolar cortex of the extrachromosomal nucleoli of Xenopus oocytes, and we discuss the possibility that a similar structure is present in other cells and species.     

The function of the nuclear envelope in nuclear protein accumulation

The mechanism by which proteins accumulate in the cell nucleus is not yet known. Two alternative mechanisms are discussed here: (a) selective unidirectional entry of karyophilic proteins through the nuclear pores, and (b) free diffusion of all proteins through the nuclear pores and specific binding of nuclear proteins to nondiffusible components of the nucleoplasm. We present experiments designed to distinguish between these alternatives. After mechanical injury of the Xenopus oocyte nuclear envelope, nuclear proteins were detected in the cytoplasm by immunohistochemical methods. In a second approach, nuclei from X. borealis oocytes were isolated under oil, the nuclear envelopes were removed, and the pure nucleoplasm was injected into the vegetal pole of X. laevis oocytes. With immunohistochemical methods, it was found that each of five nuclear proteins rapidly diffuses out of the injected nucleoplasm into the surrounding cytoplasm. The subsequent transport and accumulation in the intact host nucleus could be shown for the nuclear protein N1 with the aid of a species-specific mAb that reacts only with X. borealis N1. Purified and iodinated nucleoplasmin was injected into the cytoplasm of Xenopus oocytes and its uptake into the nucleus was studied by biochemical methods.     

Ultrastructure of trophocyte nuclei in polytrophic ovarioles of Chrysopa perla (Neuroptera)]

The light and electron microscope and autoradiographic studies (H3-uridin incorporation) were carried out on the trophocyte nuclei of imago polytrophic ovarioles of Chrysopa perla (Neuroptera), from the trophocyte differentiation up to their degeneration. Like the oocytes, one of the seven nurse cells o every ovariole chamber contains extrachromosomal DNA bodies. This nurse cell is formed during differential mitoses in the germarium as one of two prooocytes. In contrast to extrachromosomal DNA of oocytes the trophocyte DNA bodies are less active structures. Several (2--4) complex nucleoli develop in the trophocytes of Chrysopa in the early stages of oogenesis. They consist of three main components: the chromatin mass, fibrillar bodies and granular strands. Such nucleoli grow, through increasing in number of fibrillar bodies and granular strands. They are most developed by the start of the vitellogenesis. At the middle vitellogenesis the general nucleolar structure modify due to the beginning of trophocyte degeneration. The consecutive stages of nuclear degeneration are described. The trophocyte nucleoli synthesize RNA still in germarium. The most intensive RNA synthesis is observed at the beginning of the vitellogenesis to decrease by the beginning of trophocyte degeneration.     

In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins

An in vitro system was developed that provides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold). Selective accumulation of fluorescent nucleoplasmin is observed microscopically within 30 min with rat liver nuclei, Xenopus embryonic nuclei, regrown Xenopus sperm nuclei, or nuclei reconstituted in vitro from bacteriophage lambda DNA. This transport requires the signal domain of nucleoplasmin. Furthermore, the ability of nuclei to accumulate nucleoplasmin directly correlates with their ability to exclude the fluorescent non-nuclear proteins, FITC-immunoglobulin and phycoerythrin. An active transport model would predict that nuclear transport be temperature- and energy- dependent and that inhibition of transport by either low temperature or energy depletion would be reversible. Both predictions were confirmed in our system. Nucleoplasmin accumulation increases with temperature, while the protein is completely excluded at 0 degrees C. The effects of low temperature are reversible. As found for 125I-labeled nucleoplasmin (Newmeyer, D. D., J. M. Lucocq, T. R. Burglin, and E. M. De Robertis, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:501-510), transport of fluorescent nucleoplasmin is inhibited by ATP depletion. This effect is reversed by later ATP addition. Under ATP-depleted conditions non- nuclear proteins continue to be excluded. These results argue for a direct role of ATP in transport rather than for a simple role in preserving envelope integrity. In a first step towards defining the minimum requirements for a transport medium, egg extracts were depleted of membrane vesicles. Membrane-depleted extracts neither support transport nor maintain the integrity of the nuclear envelope.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

Changes in nuclear localization of An3, a RNA helicase,during oogenesis and embryogenesis in Xenopus laevis

The immunolocalization of An3 protein, an ATP-dependent RNA helicase and a member of the DEAD box family, was compared with the localization of fibrillarin, a protein essential for rRNA processing, and snRNPs, which are involved in mRNA splicing reactions, during oogenesis and embryogenesis in Xenopus laevis. Although An3 protein was detected in the cytoplasm of all stages of oocytes, in most stages An3 protein was also present in the nucleus. Prior to stage I An3 protein was uniformly dispersed throughout the entire germinal vesicle; from stages I to V it was in nucleoli. By stage VI nucleolar labeling with anti An3 disappeared and the protein was no longer present within nuclei. An3 reactivity was also present throughout the nuclei of follicle cells surrounding prestage I to stage VI oocytes. Both cytoplasmic and nuclear An3 staining were present in cells of stages 8 to 35 embryos; however, nuclear staining was punctate and uniformly distributed throughout the nucleoplasm. Fibrillarin was diffusely distributed throughout the entire germinal vesicle prior to stage I, localized exclusively to nucleoli of oocytes between stages I and VI and in nucleoli of stages 12 and 35 embryonic cells. Reactivity for snRNPs (anti-Sm) in germinal vesicles of prestage I oocytes was diffuse, and similar to the distribution of An3 and fibrillarin; in later stage oocytes anti-Sm staining was restricted to a population of granules, much fewer in number and more heterogeneous in size than nucleoli. Anti-Sm activity was apparent in nuclei of embryonic cells of stages 8 to 35 embryos. Although colocalization of the Sm epitope and An3 was not observed in developing oocytes and in embryonic cells, Sm reactive material was frequently found in close association with An3-positive nucleoli (oocytes) and nuclear deposits (embryonic cells). In stage IV and V oocytes treated with actinomycin D (4 μg/ml) to inhibit rRNA synthesis, nucleoli, which continued to possess fibrillarin, lacked An3; staining of follicle cell nuclei for An3 was unchanged. Treatment with 200 μg/ml actinomycin D to block mRNA synthesis, inhibited An3 but not fibrillarin staining in nuclei of prestage I oocytes and follicle cells. The changing patterns of An3 reactivity and the differential effects of actinomycin D on such localizations observed here are consistent with a role for An3 in the processing/production of RNA. © 1996 Wiley-Liss, Inc.     

The effect of isoproterenol and hydroxyurea on the presence of ubiquitin and protein A24 in the rat salivary gland

The in vivo administration of hydroxyurea for 12 h counteracts DNA synthesis and cell cycling stimulated by 72 h of isoproterenol treatment in rat salivary gland, as determined by fluorescence-activated flow cytometry. Hydroxyurea has little effect on [³H]leucine incorporation (protein synthesis) of the nuclear proteins soluble in 0.35 M NaCl, when examined by polyacrylamide gel chromatography and autoradiography from electro-statically sorted nuclei of (G₀+G₁) and (G₂+M) phases of the in vivo cell cycle. Differential incorporation of [³H]leucine into nuclear proteins was observed during various phases of the cell cycle. Proteins ‘X’ and ‘Z’, observed in stained gel chromatographs of the 0.35 M NaCl-soluble nuclear proteins, were identified by biochemical analyses as ubiquitin and protein A₂₄, respectively. Ubiquitin appeared transiently while A₂₄ increased in gel chromatograms concomitant with progressive quiescence of the salivary gland induced by hydroxyurea.     

Nucleoplasmin: the archetypal molecular chaperone

Nucleoplasmin was the first protein to be described as a molecular chaperone. Studies of nucleoplasmin have resulted in advances in two areas of cell biology. Firstly, the pathway of nucleosome assembly in Xenopus oocytes and eggs has been elucidated and is the only assembly pathway known in detail. Nucleosome assembly represents the major chaperoning function of nucleoplasmin. Secondly, nucleoplasmin has been used to elucidate the transport of proteins into the nucleus, revealing a selective entry mechanism for nuclear proteins, passage through the nuclear pore complex, and a two-step mechanism of transport. The properties and functions of nucleoplasmin are reviewed, together with other proteins which are related either structurally or functionally to nucleoplasmin.     

Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L.

Nuclei were isolated from somatic embryos of carrot (Daucus carota L.) using a buffer system containing non-ionic detergent. To prepare nuclear matrices, the purified membrane-depleted nuclei were digested with DNase I in combination with RNase A, followed by extraction with 1 M NaCl. The DNA residue in the final insoluble fraction was less than 4% of that in isolated nuclei, and most of the residual nuclei retained their sphericity. Electron microscopy revealed that the nuclear matrix was composed of a distinct peripheral layer, an internal matrix structure and some fibrils; residual nucleoli were observed when exogeneous RNase was not incorporated. The proteins extracted from the nuclei and nuclear subfractions were compared by gel electrophoresis, which showed that the residual fraction contained many minor proteins. To identify proteins showing specific localization at the nuclear periphery, we prepared monoclonal antibodies (MAbs) against an ion-exchange chromatography fraction extracted from carrot nuclear matrices. Immunofluorescence microscopy with one of the MAbs, CML-1, showed exclusive staining of the nuclear periphery. The MAb recognized several spots showing microheterogeneity, with a narrow range of pI and molecular mass upon immunoblotting. A complete set of these spots was shown to be conserved in nuclear matrices. On the other hand, MAb CML-13 appeared to react with the nuclear interior as well as the periphery, recognizing a 96-kDa polypeptide of the nuclear matrix. These proteins were thus demonstrated to lie at the nuclear periphery, and to constitute the nuclear matrices in carrot. The 96-kDa polypeptide is suggested to be similar to the 92-kDa nuclear protein reported by Beven et al. in carrot (Beven et al., 1991, J. Cell Sci. 98, 293–302).Abbreviations DEAE diethylaminoethyl - MAb monoclonal antibody - NEPHGE nonequilibrium pH gradient electrophoresis We wish to thank Ms. Akiko Itoh for excellent technical assistance. This work was supported by a Grant-in-Aid (05640738) from the Ministry of Education of Japan.     

Electrostatic interactions at the C-terminal domain of nucleoplasmin modulate its chromatin decondensation activity

The chromatin decondensation activity, thermal stability, and secondary structure of recombinant nucleoplasmin, of two deletion mutants, and of the protein isolated from Xenopus oocytes have been characterized. As previously reported, the chromatin decondensation activity of recombinant, unphosphorylated nucleoplasmin is almost negligible. Our data show that deletion of 50 residues at the C-terminal domain of the protein, containing the positively charged nuclear localization sequence, activates its chromatin decondensation ability and decreases its stability. Interestingly, both the decondensation activity and thermal stability of this deletion mutant resemble those of the phosphorylated protein isolated from Xenopus oocytes. Deletion of 80 residues at the C-terminal domain, containing the above-mentioned positively charged region and a poly(Glu) tract, inactivates the protein and increases its thermal stability. These findings, along with the effect of salt on the thermal stability of these proteins, suggest that electrostatic interactions between the positive nuclear localization sequence and the poly(Glu) tract, at the C-terminal domain, modulate protein activity and stability.     

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.