The nuclear matrix from cells of different origin. Evidence for a common set of matrix proteins

We compared the protein composition of the nuclear matrix isolated from several murine embryonal carcinoma cells and mature tissues by two-dimensional gel electrophoresis. Two nuclear matrix fractions were investigated: the "peripheral" nuclear matrix (matrix proteins that remain insoluble after reduction), and the "internal" nuclear matrix (matrix proteins released by reduction). The two subfractions have completely different protein compositions. Although numerous differences in nuclear matrix protein composition among different cell types were observed, a limited set of polypeptides common to all mouse cell types was identified. A majority of these common proteins was also present in cells from other mammalian species (i.e. rat and human). For this set of proteins, we coin the term "minimal matrix." As expected, lamin B, known to be expressed throughout differentiation, is part of the common set of peripheral nuclear matrix proteins. Lamins A and C are not because these proteins were absent from undifferentiated embryonal carcinoma cells. Since these common nuclear matrix proteins occur in all mammalian nuclear matrices analyzed so far, it is likely that they have a basic role in nuclear organization and function.     

Salt dissociation of nuclear particles containing DNA-like RNA. Distribution of phosphorylated and nonphosphorylated species.

Electrophoresis of proteins from nuclear particles containing DNA-like RNA gave a pattern with 45 bands. The possibility that some of these proteins arose by contamination with ribosomes, chromatin, or soluble nuclear proteins was examined and eliminated. The fate of the proteins of the particles was studied after partial dissociation with 0.25 and 0.70 M NaCl. The individual proteins were released progressively and in different quantities. A group of easily released species (75 and 95% removed with 0.25 and 0.70 M NaCl) was demonstrated. This group contained 8 species between 29,000 and 39,000 daltons which represented approximately one-half of the total number of molecules. It is suggested that they are bound to repetitive sequences of the RNA. At least 30 and 60% of the other proteins were released at 0.25 and 0.70 M NaCl, respectively. There were no specific proteins tightly bound to the RNA, unless the nature of the remaining species is different from that of the released ones of the same molecular weight. The phosphorylated proteins were more tightly bound to the RNA than the nonphosphorylated species of similar molecular weight. In several instances, the 32-P radioactivity was associated with quantitatively minor bands of proteins.     

Evidence that the Arabidopsis nuclear gibberellin signalling protein GAI is not destabilised by gibberellin

Plant growth is regulated by bioactive gibberellin (GA), although there is an unexplained diversity in the magnitude of the GA responses exhibited by different plant species. GA acts via a group of orthologous proteins known as the DELLA proteins. The Arabidopsis genome contains genes encoding five different DELLA proteins, the best known of which are GAI and RGA. The DELLA proteins are thought to act as repressors of GA-regulated processes, whilst GA is thought to act as a negative regulator of DELLA protein function. Recent experiments have shown that GA induces rapid disappearance of nuclear RGA, SLR1 and SLN1 (DELLA proteins from rice and barley), suggesting that GA signalling and degradation of DELLA proteins are coupled. However, RGL1, another Arabidopsis DELLA protein, does not disappear from the nucleus in response to GA treatment. Here, we present evidence suggesting that GAI, like RGL1, is stable in response to GA treatment, and show that transgenic Arabidopsis plants containing constructs that enable high-level expression of GAI exhibit a dwarf, GA non-responsive phenotype. Thus, GAI appears to be less affected by GA than RGA, SLR1 or SLN1. We also show that neither of the two putative nuclear localisation signals contained in DELLA proteins are individually necessary for nuclear localisation of GAI. The various DELLA proteins have different properties, and we suggest that this functional diversity may explain, at least in part, why plant species differ widely in their GA response magnitudes.     

Protein biomarkers for response to XPO1 inhibition in haematologic malignancies

XPO1 (Exportin-1) is the nuclear export protein responsible for the normal shuttling of several proteins and RNA species between the nucleocytoplasmic compartment of eukaryotic cells. XPO1 recognizes the nuclear export signal (NES) of its cargo proteins to facilitate its export. Alterations of nuclear export have been shown to play a role in oncogenesis in several types of solid tumour and haematologic cancers. Over more than a decade, there has been substantial progress in targeting nuclear export in cancer using selective XPO1 inhibitors. This has resulted in recent approval for the first-in-class drug selinexor for use in relapsed, refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL). Despite these successes, not all patients respond effectively to XPO1 inhibition and there has been lack of biomarkers for response to XPO1 inhibitors in the clinic. Using haematologic malignancy cell lines and samples from patients with myelodysplastic neoplasms treated with selinexor, we have identified XPO1, NF-κB(p65), MCL-1 and p53 protein levels as protein markers of response to XPO1 inhibitor therapy. These markers could lead to the identification of response upon XPO1 inhibition for more accurate decision-making in the personalized treatment of cancer patients undergoing treatment with selinexor.     

Synthesis of nuclear acidic proteins in density-inhibited fibroblasts stimulated to proliferate

Previous work from this laboratory (Rovera and Baserga, 1971) has shown that, when density-inhibited WI-38 human diploid fibroblasts are stimulated to proliferate by a change of medium, the synthesis of nuclear acidic proteins increases within 30 minutes after stimulation; several hours before DNA synthesis begins to increase. Similar results have now been obtained with density-inhibited 3T6 mouse fibroblasts, also stimulated by a change of medium. Gel electrophoretic analysis of nuclear acidic proteins in both WI-38 human diploid fibroblasts and 3T6 mouse fibroblasts stimulated to proliferate indicates that the increased synthesis of nuclear acidic proteins is limited to certain classes of proteins while other classes are totally unaffected. The increase in nuclear acidic proteins synthesis is inhibited when WI-38 cells or 3T6 cells are stimulated in the presence of 5-azacytidine (10 μg/ml), a treatment which also inhibits the subsequent stimulation of DNA synthesis. These results, confirming and extending similar findings previously reported in other models of stimulated DNA synthesis, lend further support to the hypothesis that nuclear acidic proteins may play a critical role in the control of DNA synthesis and cell division in mammalian cells.     

Proteolysis of nuclear proteins by mu-calpain and m-calpain.

Purified calpains are capable of proteolyzing several high Mr nuclear proteins and solubilizing a histone H1 kinase activity from rat liver nuclei upon exposure to 10(-6) - 10(-5) M Ca2+. Major nuclear substrates displayed apparent molecular masses of 200, 130, 120, and 60 kDa on Coomassie Blue-stained SDS-PAGE gels. The nuclear proteins and the H1 kinase were released from Triton-treated nuclei following incubation with buffer containing 0.5 M NaCl. They therefore appeared to be internal nuclear matrix proteins. The nuclear H1 kinase activity solubilized by incubation with m-calpain was eluted in the void volume of a Bio-Gel A-1.5m column, indicating an apparent mass greater than 1,500 kDa. Treatment of the calpain-solubilized kinase with 0.5 M NaCl dissociated it to a form having an apparent mass of 300 kDa (Stokes radius = 5.6 nm), suggesting that the 300-kDa (Stokes radius = 5.6 nm), nuclei by calpain treatment as a large complex containing other internal matrix proteins. Purified human erythrocyte mu-calpain was capable of proteolyzing the nuclear matrix proteins at 10(-6) M Ca2+. In contrast, human erythrocyte multicatalytic protease complex produced little cleavage of the nuclear proteins. Proteolysis of nuclear proteins by either mu-calpain or m-calpain was inhibited by calpastatin. These experiments suggest a physiologic role for the calpains in the turnover of nuclear proteins.     

Crosslinking of nuclear proteins to DNA by cis-diamminedichloroplatinum in intact cells. Involvement of nuclear matrix proteins.

In order to detect the nuclear matrix proteins involved in DNA binding, avoiding possible artifacts derived from the disruption of nuclei, proteins were crosslinked to DNA by the action of cis-diamminedichloroplatinum on intact chicken liver cells and analyzed by two-dimensional gel electrophoresis. At least eleven species of crosslinked proteins were found to derive from the nuclear matrix prepared from the same cell type, and five of these were found also among the proteins crosslinked to DNA in intact liver cells from ox and pig. This subset of common proteins, conserved in different animal species, is likely to have a fundamental role for the anchorage of DNA to the nuclear matrix.     

A conserved phosphoprotein that specifically binds nuclear localization sequences is involved in nuclear import [published erratum appears in J Cell Biol 1992 Jul;118(1):215]

We have purified proteins of 70 kD from Drosophila, HeLa cells, and Z. mays that specifically bind nuclear localization sequences (NLSs). These proteins are recognized by antibodies raised against a previously identified NLS-binding protein (NBP) from the yeast S. cerevisiae. All NBPs are associated with nuclei and also present in the cytosol. NBPs are phosphorylated and phosphatase treatment abolished NLS binding. The requirement for NBPs in nuclear protein uptake is demonstrated in semipermeabilized Drosophila melanogaster tissue culture cells. Proper import of a fluorescent protein containing the large T antigen NLS requires cytosol and ATP. In the absence of cytosol and/or ATP, NLS-containing proteins are bound to cytosolic structures and the nuclear envelope. Addition of cytosol and ATP results in movement of this bound intermediate into the nucleus. Anti-NBP antibodies specifically inhibited the binding part of this import reaction. These results indicate that a phosphoprotein common to several eukaryotes acts as a receptor that recognizes NLSs before their uptake into the nucleus.     

Drosophila Melanogaster, Drosophila Simulans: so Similar yet so Different

During the last two decades, the two cosmopolitan species Drosophila melanogaster and Drosophila simulans have been compared with regard to numerous characteristics, ranging from their geographic distribution and ecology to their DNA polymorphism. Various traits have been compared, including morphology, physiology, sexual behavior, allozymes and other proteins, chromosomal inversions, mitochondrial and nuclear DNA, transposable elements, wolbachia etc. Such comparisons reveal similarities and differences between the two species, depending on the trait considered. In most cases, the between-population variability of D. simulans is lower than that of D. melanogaster, but the two species exhibit similar levels of within-population variability. One of the main exceptions is the nucleotide polymorphism of several nuclear regions. Although several hypotheses have been proposed to explain these observations, the evolutionary dynamics of these two species are far from being understood. How have two species sharing a common ancestor in the recent past accumulated so many differences? A brief history of comparisons of the two species, from the first in 1919 by A.H. Sturtevant, and a summary of the hypotheses proposed to explain the similarities and the differences between these species are presented and discussed.     

Mitochondrial ribosomal proteins in Xenopus laevis/X. mulleri interspecific hybrids.

The large subunits of mitochondrial ribosomes were isolated from two related frog species, Xenopus laevis and X. mulleri, and their proteins were compared by two-dimensional polyacrylamide gel electrophoresis. Three of the proteins observed in X. laevis are absent from X. mulleri, and four of the proteins observed in X. mulleri are absent from X. laevis. More than these seven such species-specific proteins may occur.Reciprocal crosses between frogs of the two species gave two groups of F1 hybrids. Nuclear genes in these hybrids derive equally from both species, while mitochondrial DNA (and therefore mitochondrial rRNA) derived exclusively from the maternal species. Electrophoretic analyses of the large subunit proteins of these F1 animals revealed that four of the species-specific proteins are present only when their corresponding species was the mother. While this result is consistent with the coding of these four proteins by mitochondrial DNA, it does not provide evidence against nuclear coding of these proteins. A fifth protein is absent from both F1 hybrids. A sixth is present in both F1 hybrids, and a seventh is present only when its corresponding species was the father. We conclude that at least these latter two mitochondrial ribosomal proteins are encoded by nuclear genes.     

Characterization of the prosome from Drosophila and its similarity to the cytoplasmic structures formed by the low molecular weight heat-shock proteins.

We have identified and characterized a ribonucleoprotein structure from the cytoplasm of Drosophila melanogaster tissue culture cells which is equivalent to the prosome, a recently described ribonucleoprotein particle of duck and mouse cells. During the recovery period following heat shock, the low mol. wt. heat-shock proteins form cytoplasmic ribonucleoprotein particles which co-purify with the Drosophila prosome. Both ribonucleoprotein particles share several structural properties but their protein constituents differ in their metabolism and cellular localization during the heat treatment. We also report the partial nucleotide sequences of several small RNA species associated with the Drosophila prosome. One of them has a strong sequence homology with the U6 mammalian small nuclear RNA.     

Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins

We have used a monoclonal antibody raised against rat liver nuclear proteins to study two cross-reactive proteins in the yeast nucleus. In rat liver, this monoclonal antibody, mAb 414, binds to nuclear pore complex proteins, including one of molecular weight 62,000 (Davis, L. I., and G. Blobel. 1987. Proc. Natl. Acad. Sci. USA. 84:7552-7556). In yeast, mAb 414 cross reacts by immunoblotting with two proteins that have apparent molecular weights of 110,000 and 95,000, and are termed p110 and p95, respectively. Examination of subcellular fractions by immunoblotting shows that both p110 and p95 are located exclusively in the nuclear fraction. The mAb 414 immunoprecipitates several proteins from a crude yeast cell extract, including p110, p95, and a approximately 55-kD protein. Immunoprecipitation from subcellular fractions yields only p110 and p95 from purified nuclei, whereas the approximately 55-kD protein is immunoprecipitated from the soluble fraction. Digestion of purified nuclei with DNase to produce nuclear envelopes releases some of p110, but the majority of p110 is solubilized only after treatment of envelopes with 1 M NaCl. Immunofluorescence localization using yeast cells and isolated nuclei shows a punctate and patchy staining pattern of the nucleus. Confocal laser scanning immunofluorescence microscopy resolves the punctate and patchy staining pattern better and shows regions of fluorescence at the nuclear envelope. Postembedding immunogold electron microscopy using purified nuclei and mAb 414 shows colloidal gold decoration of the yeast nuclear envelope, but resolves pore complexes too poorly to achieve further ultrastructural localization. Immunogold labeling of nuclei followed by embedding suggests decoration of pore complexes. Thus, p110 and/or p95 are localized to the nuclear envelope in yeast, and may be components of the nuclear pore complex.     

Determination of newly synthesized and phosphorylated nuclear proteins in mass-isolated germinal vesicle of Spisula solidissima

The highly specialized nucleus of the oocyte, the germinal vesicle (GV), has been difficult to investigate biochemically because of the small quantities obtainable. A mass isolation procedure was therefore developed using oocytes from Spisula solidissima, which depends on gentle cell lysis and the sedimentation of the large GV by low g-force. The procedure is gentle and fast and can yield several ml of packed nuclei from a single clam. It should facilitate the investigation of the fate of the GV major proteins and RNA species during development. Nuclear proteins, synthesized in the cytoplasm, were shown to segregate immediately into the nuclear compartment. Four high molecular weight proteins were found to be strongly phosphorylated in the GV in addition to the dominant nuclear protein (approx. 47 000 D). The in vitro phosphorylation of these proteins was strongly inhibited by Ca²⁺.     

The binding of [3H]chlorambucil to nuclear proteins of the Yoshida ascites sarcoma.

The binding of [3H]chlorambucil to nuclear proteins, extracted from Yoshida ascites sarcoma cells at 6 h and 24 h after administration of 3H-labelled drug to tumour-bearing animals, has been examined. Both covalent and non-covalent binding was detected. Considerably more drug was found associated with the proteins isolated from the tumour sensitive to the effects of the drug compared with similar proteins isolated from the tumour with an acquired resistance to the effects of alkylating agents. The two-fold difference in binding to total cell protein is attributed to a higher intranuclear protein binding in sensitive cells. In particular the soluble nuclear sap fraction from sensitive cells bound at least five times as much drug as the corresponding fraction from resistant cells. Low levels of binding to histones were demonstrated compared with that to the non-histone chromatin proteins. Binding to the nuclear sap and non-histone chromatin proteins was principally to high molecular weight protein species; these did not appear to represent aggregation products as scans of stained polyacrylamide gels of the extracted protein fractions were unaltered by the treatment of tumour-bearing animals with chlorambucil. Binding to the nuclear proteins from sensitive cells tended to persist over a 24-h period, whereas it was considerably reduced in resistant cells.