Effect of organ site on nuclear matrix protein composition

The nuclear matrix has been linked to several important cellular functions within cells, such as DNA organization and replication, as well as regulation of gene expression. It has been reported that the nuclear matrix protein composition is altered in cells grown on different extracellular matrices in vitro. This study examined the nuclear matrix protein composition of tumors produced by MAT-LyLu (MLL) rat prostate tumor cells implanted at different organ sites within the rat. When high resolution two-dimensional gels were utilized to compare nuclear matrix protein composition to the prostate orthotopic tumor, it was found that there were distinct protein differences depending upon where the tumor grew. In particular, there were 14 proteins found in the lung, six proteins found in intramuscular, 17 proteins is the heart, and five proteins in the tail vein tumor tissue that were not present in the prostate orthotopic tumor tissue. Therefore, this study adds evidence to support that the nuclear matrix composition of a cell is dependent, at least in part, by the extracellular matrix and/or different cellular environments and may have a role in site-specific differences in tumor properties. © 1996 Wiley-Liss, Inc.     

The yeast nuclear pore complex: composition, architecture, and transport mechanism

An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.     

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma cells is differentiation-stage dependent

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma (EC) cells was compared with that of clonal derivatives of P19 EC differentiated in vitro, and with that of P19 EC cells induced to differentiate with retinoic acid (RA). Several major differences in nuclear matrix protein composition were found between the cell lines tested. Some polypeptides were found to occur only in EC cells, whereas others proved to be restricted to one or more of the differentiated derivatives. During RA treatment of EC cells a transient expression of some matrix proteins was observed. Several new proteins appeared, and others disappeared. Our data indicate that the protein composition of the nuclear matrix is a sensitive gauge for the differentiation state of cells.     

Identification of a nuclear protein matrix

The structural framework of the rat liver nucleus has been identified and consists of a nuclear protein matrix. This matrix is 98.4% protein, 0.1% DNA, 1.2% RNA, and 0.5% phospholipid. The nuclear protein matrix is composed primarily of three acidic polypeptide fractions in the molecular weight range of 60–70,000 daltons.     

The insulator binding protein CTCF associates with the nuclear matrix

Nuclear DNA is organized into chromatin loop domains. At the base of these loops, matrix-associated regions (MARs) of the DNA interact with nuclear matrix proteins. MARs act as structural boundaries within chromatin, and MAR binding proteins may recruit multiprotein complexes that remodel chromatin. The potential tumor suppressor protein CTCF binds to vertebrate insulators and is required for insulator activity. We demonstrate that CTCF is associated with the nuclear matrix and can be cross-linked to DNA by cisplatin, an agent that preferentially cross-links nuclear matrix proteins to DNA in situ. These results suggest that CTCF anchors chromatin to the nuclear matrix, suggesting that there is a functional connection between insulators and the nuclear matrix. We also show that the chromatin-modifying enzymes HDAC1 and HDAC2, which are intrinsic nuclear matrix components and thought to function as corepressors of CTCF, are incapable of associating with CTCF. Hence, the insulator activity of CTCF apparently involves an HDAC-independent association with the nuclear matrix. We propose that CTCF may demarcate nuclear matrix-dependent points of transition in chromatin, thereby forming topologically independent chromatin loops that may support gene silencing.     

Protein composition of the chromosomal scaffold and interphase nuclear matrix

Residual protein structures were prepared from isolated chromosomes and interphase nuclei of in vitro cultured bovine liver cells and the protein compositions were analysed. Chromosomes with minimal cytoplasmic contamination were obtained by a simple procedure using a pH 8 isolation medium containing Triton X-100 and polyamines, and residual protein-DNA complexes were prepared by extraction with 2 M NaCl. Residual protein structures were also obtained by digesting isolated chromosomes with staphylococcal nuclease. Protein compositions of both structures as obtained by SDS-polyacrylamide gel electrophoresis were essentially the same. Residual protein structures were prepared from isolated nuclei by the same procedures. The major nuclear matrix proteins, i.e., the lamins A, B, and C, were not found in the chromosomes and chromosome scaffolds. On the other hand, the residual chromosome structures contained two major polypeptides of 37 and 83 kilodalton relative molecular weights that were absent from the nuclear matrix preparations. A few polypeptides with the same or very similar electrophoretic mobilities were found in the residual structures of both the nuclei and the chromosomes.     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Cortisol-induced stimulation of nuclear matrix protein phosphorylation

The rat liver nuclear matrix specifically binds steroids and responds to hormone binding by phosphorylation modification of protein constituents. This process is suggested to be part of nuclear nonhistone protein modification underlying modulation of chromatin structure and selective gene expression.     

Three-dimensional immunogold labeling of nuclear matrix proteins in permeabilized cells.

A preembedment labeling procedure is described for the three-dimensional (3D) labeling of nuclear matrix proteins in permeabilized cells. The procedure is based on the use of ultra-small (1 nm) gold particles as a marker system. This marker penetrates the nucleus more efficiently than the conventionally used 5-10 nm colloidal gold probes. Dehydration is performed by freeze-substitution to preserve the ultrastructure of the cell as optimally as possible. During freeze-substitution the samples are stained by uranyl ions to stain the cellular material throughout the resin section. The 3D gold-labeled and uranyl-stained specimen is embedded in Epon resin and semi-thin (0.2-0.5 microns) sections are made for stereo electron microscopy. The applicability of this method is illustrated by the localization of nuclear matrix-associated nuclear bodies in permeabilized interphase and mitotic HeLa cells.     

Bone formation: The nuclear matrix reloaded

In this issue of Cell, Grosschedl and colleagues (Dobreva et al., 2006) report that the nuclear matrix protein Satb2 represses Hoxa2 expression and acts with other regulatory proteins to promote osteoblast differentiation. This work suggests a molecular mechanism that enables the integration of patterning and differentiation during bone formation.     

The murine nuclear matrix protein specificcaly binds to centromeric satellite DNA

The nuclear matrix (NM) of mouse contains a protein (miSat BP) that can specifically bind to mouse centromeric minor satellite DNA as shown by gel shift assay. The ion-exchange chromatography on DEAE-Sepharose was used as the first miSat BP purification. MiSat BT was eluted by 0.2 M NaCl. Antibodies against p70, a human NM protein of 70 kDa described earlier as a protein recognizing human alphoid DNA, produce hypershift effect when added to the retardation incubation mix. Immunoblotting of NM and an active NM fraction (0.2 M NaCl) with these antibodies revealed a protein with 70 kDa in both preparations. This antigen retained in NM in situ being associated with residual DNA as shown by indirect immunofluorescent staining. In the untreated interphase nucleus most of miSat BP granules were shown to be colocalized with prekinetochores. We suggest that miSat BP is capable of recognizing the minor satellite DNA due to its structural features, but it does not belong to a group of constitutive centromeric proteins.     

hnRNA and its attachment to a nuclear protein matrix

In this study, DNA-depleted nuclear protein matrices are isolated from HeLa S3 cells. These nuclear matrices consist of peripheral laminae, residual nucleoli, and internal fibrillar structures. High molecular weight, heterogeneous nuclear RNA (hnRNA) is quantitatively associated with these structures and can be released intact only by affecting the integrity of the matrices. It is, therefore, concluded that hnRNA is part of a highly organized nuclear structure. By irradiation of intact cells or isolated nuclear matrices with ultraviolet light, proteins tightly associated with hnRNA can be induced to cross-link with the RNA. Performing the cross-linking in vivo is an extra guarantee that only hnRNA-protein (hnRNP) complexes existing in the intact cell are covalently linked. Such hnRNP complexes were isolated and purified under conditions that completely dissociate nonspecific RNA-protein complexes. By comparison of the hnRNP found in nuclear matrices and the published data on the composition of hnRNP particles, it was found that the so-called hnRNP "packaging" proteins (32,000-38,000 mol wt) were not efficiently cross-linked to hnRNA by UV irradiation. They were, however, present in the matrix preparations, bound to hnRNA, because they were released from nuclear matrices after ribonuclease treatment of these structures. On the other hand, two major hnRNPs (41,500 and 43,000 mol wt) were efficiently cross-linked to hnRNA. These proteins were not released by ribonuclease treatment, which suggests that they are involved in the binding of hnRNA to the nuclear matrix.     

Three-dimensional electron microscopy analysis reveals endopolygeny-like nuclear architecture segregation in Plasmodium oocyst development

The genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the production of haploid parasites in the Plasmodium life cycle: the oocyst stage in mosquitoes and the liver and blood stages in mammalian hosts. The Plasmodium oocyst stage plays an important role in the production of the first generation of haploid parasites. Nuclear division is the most important event that occurs during the proliferation of all eukaryotes. However, obtaining the details of nuclear division at the oocyst stage is challenging owing to difficulties in preparation. In this study, we used focused-ion-beam-milling combined with scanning-electron-microscopy to report the 3D architecture during nuclear segregations in oocyst stage. This advanced technology allowed us to analyse the 3D details of organelle segregation inside the oocyst during sporogony formation. It was revealed that multiple nuclei were involved with several centrosomes in one germ nucleus during sporozoite budding (endopolygeny). Our high-resolution 3D analysis uncovered the endopolygeny-like nuclear architecture of Plasmodium in the definitive host. This nuclear segregation was different from that in the blood stage, and its similarity to other apicomplexan parasite nuclear divisions such as Sarcocystis is discussed.     

Nuclear dreams: The malignant alteration of nuclear architecture

Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets. J. Cell. Biochem. 70:172–180, 1998. © 1998 Wiley-Liss, Inc.     

SnRNP core protein enrichment in the nuclear matrix

The D protein (16 kDa) is part of a protein core, common to U1, U2, U5, U4/U6 small nuclear RNA containing ribonucleoprotein particles. Monoclonal antibodies reactive with the D protein were used in quantitative dot blotting and Western blotting to demonstrate that this protein was a component of salt resistant nuclear structures and was enriched greater than 3 to 5-fold in RNAase-protected nuclear matrix preparations.