Necdin interacts with the ribonucleoprotein hnRNP U in the nuclear matrix

Necdin is expressed predominantly in terminally differentiated neurons, and its ectopic expression suppresses cell proliferation. We screened a cDNA library from neurally differentiated embryonal carcinoma P19 cells for necdin-binding proteins by the yeast two-hybrid assay. One of the positive clones contained cDNA encoding a carboxyl-terminal portion of heterogeneous nuclear ribonucleoprotein U (hnRNP U), a nuclear matrix-associated protein that interacts with chromosomal DNA. We isolated cDNA encoding full-length mouse hnRNP U to analyze its physical and functional interactions with necdin. The necdin-binding site of hnRNP U was located near a carboxyl-terminal region that mediated the association between hnRNP U and the nuclear matrix. In postmitotic neurons, endogenously expressed necdin and hnRNP U were detected in the nuclear matrix and formed a stable complex. Ectopically expressed necdin was concentrated in the nucleoli, but coexpressed hnRNP U recruited necdin to the nucleoplasmic compartment of the nuclear matrix. Furthermore, under the same conditions necdin and hnRNP U cooperatively suppressed the colony formation of transfected SAOS-2 cells. These results suggest that necdin suppresses cell proliferation through its interaction with hnRNP U in the specific subnuclear structure.     

Macromolecular domains containing nuclear protein p107 and U-snRNP protein p28: Further evidence for an in situ nuclear matrix

Summary Polyclonal antibodies have been produced which react with a nuclear protein having a molecular weight of 107kD and a pl of 8.7–8.8 (designated p107). This protein is shown to be a component of the residual ribonucleoprotein (RNP) network of the nuclear matrix. P107 localized exclusively to the nuclear interior but not within nucleolar or chromatin domains. We have taken advantage of this unique probe to examine whether the RNP network of the isolated nuclear matrix has a physical counterpart in situ. We show that RNA, p107, divalent cations and the 28 kD Sm antigen of U-snRNPs are components of in situ macromolecular assemblies. While the morphology and intranuclear distribution of these assemblies are insensitive to the removal of chromatin, they are markedly altered by degradation of RNA. Digestion in situ of RNA in the presence of EDTA followed by extraction with high ionic strength buffers solubilized the components of these assemblies. Electron microscopic and immunobiochemical data are presented which support the concept that the residual RNP network of the nuclear matrix is an isolate of a pre-existing structure, and that perturbations in this internal network can be created by RNA degradation, depletion of essential metal ions and proteolysis.Abbreviations CRLM polyclonal chicken antibody raised against rat liver nuclear matrix - Sm monoclonal antibody specific for the 28 kd protein antigen of U1, U2, U4, U5 and U6 snRNPs - hnRNP ribonucleoprotein particles containing hnRNA - snRNP ribonucleoprotein particles containing snRNA - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediaminetetraacetic acid - VRC vanadium ribonucleoside complex - BSA bovine serum albumin - DMSO dimethylsulfoxide - HS high salt buffer - LS low salt buffer     

Domains of the human androgen receptor and glucocorticoid receptor involved in binding to the nuclear matrix

Steroid receptors have been reported to bind to the nuclear matrix. The nuclear matrix is operationally defined as the residual nuclear structure that remains after extraction of most of the chromatin and all soluble and loosely bound componnets. To obtain insight in the molecular mechanism of the interaction of steroid receptors with the nuclear matrix, we studied the binding of several deletion mutants of the human androgen receptor (hAR) and the human glucocorticoid receptor (hGR) to the nuclear matrix. Receptor binding was tested for two different nuclear matrix preparations: complete matrices, in which most matrix proteins are retained during the isolation procedure, and depleted matrices, which consist of only a subset of these proteins. The results show that the C-terminal domain of the hAR binds tightly to both depleted and complete matrices. In addition, at least one other domain of the hAR binds to complete matrices but not to depleted matrices. In contrast to the hAR, the hGR binds only to complete matrices. For this interaction both the DNA-binding domain and the C-terminal domain of the hGR are required, whereas the N-terminal domain is not. We conclude that specific protein domains of the hAR and the hGR are involved in binding to the nuclear matrix. In addition, our results indicate that the hAR and the hGR are attached to the nuclear matrix through different molecular interactions.     

Distinct LIM domains of Hic-5/ARA55 are required for nuclear matrix targeting and glucocorticoid receptor binding and coactivation

Hydrogen peroxide-inducible clone-5 (Hic-5), belongs to the group III LIM domain protein family and contains four carboxyl-terminal LIM domains (LIM1-LIM4). In addition to its role in focal adhesion signaling, Hic-5 acts in the nucleus as a coactivator for some steroid hormone receptors such as the glucocorticoid receptor (GR) and androgen receptor (AR). Based upon its effect on AR transactivation, Hic-5 has also been designated as ARA55. Here, we report mapping studies of Hic-5/ARA55 functional domains and establish that LIM3 and LIM4 are necessary for maximal effects on GR transactivation. However, results from yeast two-hybrid assays demonstrated that these two LIM domains together, while necessary, are not sufficient to interact with the tau2 transactivation domain of GR. LIM4 also functions as a nuclear matrix targeting sequence (NMTS) for Hic-5/ARA55, as it is both necessary and sufficient to target a heterologous protein to the nuclear matrix. Thus, as suggested from previous analysis of LIM domain-containing proteins, separate but highly related LIM domains serve distinct functions.     

Dynamic continuity of nuclear and mitotic matrix proteins in the cell cycle

The eukaryotic cell nucleus is a membrane-enclosed compartment containing the genome and associated molecules supported by a highly insoluble filamentous network known as the nucleoskeleton or nuclear matrix. The nuclear matrix is believed to play roles in maintaining nuclear architecture and organizing nuclear metabolism. Recently, advances in microscopic techniques and the availability of new molecular probes have made it possible to localize functional domains within the nuclear matrix and demonstrate dynamic interactions between both soluble and insoluble components involved in the control of multiple nuclear transactions. Like the cytoplasm and its skeleton, the nucleoplasm is highly structured and very crowded with an equally complex skeletal framework. In fact, there is growing evidence that the two skeletal systems are functionally contiguous, providing a dynamic cellular matrix connecting the cell surface with the genome. If we impose cell cycle dynamics upon this skeletal organization, it is obvious that the genome and associated nuclear matrix must undergo a major structural transition during mitosis, being disassembled and/or reorganized in late G2 and reassembled again in daughter nuclei. However, recent evidence from our laboratory and elsewhere suggests that much of the nuclear matrix is used to form the mitotic apparatus (MA). Indeed, both facultative and constitutive matrix-associated proteins such as NuMA, CENP-B, CENP-F, and the retinoblastoma protein (Rb) associate within and around the MA. During mitosis, the nuclear matrix proteins may either become inert “passengers” or assume critical functions in partitioning the genome into newly formed G1 nuclei. Therefore, we support the view that the nuclear matrix exists as a dynamic architectural continuum, embracing the genome and maintaining cellular regulation throughout the cell cycle. © 1996 Wiley-Liss, Inc.     

Necdin is required for terminal differentiation and survival of primary dorsal root ganglion neurons

Necdin is expressed predominantly in postmitotic neurons and serves as a growth suppressor that is functionally similar to the retinoblastoma tumor suppressor protein. Using primary cultures of dorsal root ganglion (DRG) of mouse embryos, we investigated the involvement of necdin in the terminal differentiation of neurons. DRG cells were prepared from mouse embryos at 12.5 days of gestation and cultured in the presence of nerve growth factor (NGF). Immunocytochemistry revealed that necdin accumulated in the nucleus of differentiated neurons that showed neurite extension and expressed the neuronal markers microtubule-associated protein 2 and synaptophysin. Suppression of necdin expression in DRG cultures treated with antisense oligonucleotides led to a marked reduction in the number of terminally differentiated neurons. The antisense oligonucleotide-treated cells did not attempt to reenter the cell cycle, but underwent death with characteristics of apoptosis such as caspase-3 activation, nuclear condensation, and chromosomal DNA fragmentation. Furthermore, a caspase-3 inhibitor rescued antisense oligonucleotide-treated cells from apoptosis and significantly increased the population of terminally differentiated neurons. These results suggest that necdin mediates the terminal differentiation and survival of NGF-dependent DRG neurons and that necdin-deficient nascent neurons are destined to caspase-3-dependent apoptosis.     

Enhancement of pancreatic islet cell monolayer growth by endothelial cell matrix and insulin

Summary The roles of glucose and insulin in the promotion of DNA synthesis in pancreatic islet cell monolayers were assessed using a variety of in vitro conditions. Several substrates including collagen, poly-l-lysine, Matrigel, and the extracellular matrix produced by cultured bovine endothelial cells (BCEM) were compared for their ability to promote monolayer growth. Islets grown on BCEM in combination with medium RPMI 1640 supplemented with 22.2 mM glucose or 10 μg/ml insulin gave the best results as determined by new DNA synthesis. The new-form monolayers were free of contaminating, fibroblasts. These results suggest that insulin is critical to pancreatic islet growth when the cells are attached to biocompatible matrices.     

Direct protein-protein interaction between PLCgamma1 and the bradykinin B2 receptor--importance of growth conditions

Recently, we have described a novel protein-protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)gamma1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCgamma1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCgamma1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCgamma1. Finally we also identified bradykinin-induced PLCgamma1 recruitment and activation in primary culture renal mesangial cells.     

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.     

Mouse submandibular salivary epithelial cell growth and differentiation in long-term culture: Influence of the extracellular matrix

Summary The adult mouse submandibular salivary gland provides a good model system to study gene regulation during normal and abnormal cell behavior because it synthesizes functionally distinct products ranging from growth factors and digestive enzymes to factors of relevance to homeostatic mechanisms. The present study describes the long-term growth and differentiation of submandibular salivary epithelial cells from adult male mice as a function of the culture substratum. Using a two-step partial dissociation procedure, it was possible to enrich for ductal cells of the granular convoluted tubules, the site of epidermal growth factor synthesis. Long-term cell growth over a period of 2 to 3 mo. with at least 3 serial passages was obtained only within three-dimensional collagen gels. Cells grew as ductal-type structures, many of which generated lumens with time in culture. Electron microscopic analysis in reference to the submandibular gland in vivo revealed enrichment for and maintenance of morphologic features of granular convoluted tubule cells. Reactivity with a keratin-specific monoclonal antibody established the epithelial nature of the cells that grew within collagen. Maintenance of cell differentiation, using immunoreactivity for epidermal growth factor as criterion, was determined by both cytochemical and biochemical approaches and was found to be dependent on the collagen matrix and hormones. Greater than 50% of the cells in primary collagen cultures contained epidermal growth factor only in the presence of testosterone and triiodothyronine. In contrast, cells initially seeded on plastic or cycled to plastic from collagen gels were virtually negative for epidermal growth factor. Biochemical analysis confirmed the presence of a protein with an apparent molecular weight of 6000 which comigrated with purified mouse epidermal growth factor. Epidermal growth factor was also present in detectable levels in Passage 1 cells. This culture system should permit assessment of whether modulation of submandibular gland ductal cell growth can be exerted via a mechanism that in itself includes epidermal growth factor and its receptor and signal transduction pathway. This work was supported by Public Health Service grant DE07766 from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD.     

Differential effects of matrix and growth factors on endothelial and fibroblast motility: application of a modified cell migration assay

Cell migration is crucial in virtually every biological process and strongly depends on the nature of the surrounding matrix. An assay that enables real-time studies on the effects of defined matrix components and growth factors on cell migration is not available. We have set up a novel, quantitative migration assay, which enables unharmed cells to migrate along a defined matrix. Here, we used this so-called barrier-assay to define the contribution of fibronectin (FN) and Collagen-I (Col-I) to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and lysophosphatidic acid (LPA)-induced cell migration of endothelial cells (EC) and fibroblasts. In EC, both FN and Col-I stimulated migration, but FN-induced motility was random, while net movement was inhibited. Addition of bFGF and VEGF overcame the effect of FN, with VEGF causing directional movement. In contrast, in 3T3 fibroblasts, FN stimulated motility and this effect was enhanced by bFGF. This motility was more efficient and morphologically completely different compared to LPA stimulation. Strikingly, directional migration of EC was not paralleled by higher amounts of stable microtubules (MT) or an increased reorientation of the microtubule-organizing centre (MTOC). For EC, the FN effect appeared concentration dependent; high FN was able to induce migration, while for fibroblasts both low and high concentrations of FN induced motility. Besides showing distinct responses of the different cells to the same factors, these results address contradictive reports on FN and show that the interplay between matrix components and growth factors determines both pattern and regulation of cell migration. J. Cell. Biochem. 99: 1536-1552, 2006. (c) 2006 Wiley-Liss, Inc.     

A combinatorial approach for directing the amount of fibronectin fibrils assembled by cells that uses surfaces derivatized with mixtures of fibronectin and cell binding domains

Fibrillar fibronectin (FN) has the crucial role of attracting and attaching cells as well as molecules that mediate tissue repair during wound healing. A previous study demonstrated higher extracellular staining of FN fibrils in cells cultured on surfaces tethered with an equimolar mixture of a FN binding domain and FN's cell binding domain, III1-2 and III9-10 respectively, than on surfaces with III9-10 alone. The effect of varying surface amounts of III1-2 and III9-10 on the quantity of FN fibrils formed by NIH-3T3 fibroblasts was examined. GST tagged III1-2 and III9-10 were conjugated to polyurethane surfaces and ELISAs were used to identify the experimental design space or the range of concentrations of GST-III1-2 and GST-III9-10 that demarcated the limits of protein loading on the surface. When GST-III1-2 was fixed and GST-III9-10 varied within the design space, the amount of FN fibrils measured by immunoblotting detergent insoluble cell lysates was dependent on the ratio of III9-10 to III1-2. When the total protein concentration was fixed and the mixture composition of GST-III1-2 and GST-III9-10 varied such that it optimally covered the design space, a parabolic relationship between FN fibril amount and the ratio of III9-10 to III1-2 was obtained. This relationship had a maximum value when the surface was bonded to equal amounts of III1-2 and III9-10 (P<0.05). Thus the ratio of III9-10 to III1-2 can be utilized to direct the quantity of FN fibrils formed on surfaces.     

Homeotic protein binding sites, origins of replication, and nuclear matrix anchorage sites share the ATTA and ATTTA motifs.

Nuclear matrix organizes the mammalian chromatin into loops. This is achieved by binding of nuclear matrix proteins to characteristic DNA landmarks in introns as well as proximal and distal sites flanking the 5' and 3' ends of genes. Matrix anchorage sites (MARs), origins of replication (ORIs), and homeotic protein binding sites share common DNA sequence motifs. In particular, the ATTA and ATTTA motifs, which constitute the core elements recognized by the homeobox domain from species as divergent as flies and humans, are frequently occurring in the matrix attachment sites of several genes. The human apolipoprotein B 3' MAR and a stretch of the Chinese hamster DHFR gene intron and human HPRT gene intron shown to anchor these genes to the nuclear matrix are mosaics of ATTA and ATTTA motifs. Several origins of replication also share these elements. This observation suggests that homeotic proteins which control the expression level of many genes and pattern formation during development are components of the nuclear matrix. Thus, the nuclear matrix, known as the site of DNA replication, might sculpture the crossroads of the differential activation of origins during development and S-phase and the control of gene expression and pattern formation in embryogenesis.     

Specific radial positions of centromeres of human chromosomes X, 1, and 19 remain unchanged in chromatin-depleted nuclei of primary human fibroblasts: evidence for the organizing role of the nuclear matrix

Radial positions of centromeres of human chromosomes X, 1, and 19 were determined in the nuclei of primary fibroblasts before and after removal of 60%-80% of chromatin. It has been demonstrated that the specific radial positions of these centromeres (more central for the chromosome 19 centromere and more peripheral for the centromeres of chromosomes 1 and X) remain unchanged in chromatin-depleted nuclei. Additional digestion of nuclear RNA did not influence this specific distribution. These results strongly suggest that the characteristic organization of interphase chromosomes is supported by the proteinous nuclear matrix and is not maintained by simple repulsing of negatively charged chromosomes.     

A unified matrix hypothesis of DNA-directed morphogenesis,protodynamism and growth control

A theoretical concept is proposed, in order to explain some enigmatic aspects of cellular and molecular biology of eukaryotic organisms. Among these are the C-value paradox of DNA redundancy, the correlation of DNA content and cell size, the disruption of genes at DNA level, the Chromosome field data of Lima de Faria (Hereditas 931, 1980), the quantal mitosis proposition of Holtzeret al. (Curr. Top. Dev. Biol. 7229 1972), the inheritance of morphological patterns, the relations of DNA and chromosome organisation to cellular structure and function, the molecular basis of speciation, etc. The basic proposition of the Unified Matrix Hypothesis is that the nuclear DNA has a direct morphogenic function, in addition to its coding function in protein synthesis. This additional genetic information is thought to be largely contained in the non-protein coding transcribed DNA, and in the untranscribed part of the genome.In this world, seeds of different kinds, sown at the proper time in the land, even in one field, come forth (each) according to its kind.In the biological sense, the term Matrix is used here to signify the integrity of the cell's fibrous networks in nucleus and cytoplasm, during interphase and metaphase. In the philosophical sense, Matrix Hypothesis integrates also the etymological meaning of the term, which stems from mater (i.e. origin), and means also a lattice within a frame of coordinates, or else: Something (as a surrounding or pervading substance or element) within which something else originates or takes form or develops (cf. Webster's Intern. Dict.).—The term Protodynamism was defined earlier (Scherrer, 1966) as meaning the integrity of theorganised movements of the cellular components, excluding mere diffusion.A preliminary version of this assay was published previously (cf. Scherrer, 1985).     

Structural characterization of the N-terminal mineral modification domains from the molluscan crystal-modulating biomineralization proteins, AP7 and AP24

The AP7 and AP24 proteins represent a class of mineral-interaction polypeptides that are found in the aragonite-containing nacre layer of mollusk shell (H. rufescens). These proteins have been shown to preferentially interfere with calcium carbonate mineral growth in vitro. It is believed that both proteins play an important role in aragonite polymorph selection in the mollusk shell. Previously, we demonstrated the 1-30 amino acid (AA) N-terminal sequences of AP7 and AP24 represent mineral interaction/modification domains in both proteins, as evidenced by their ability to frustrate calcium carbonate crystal growth at step edge regions. In this present report, using free N-terminal, C(alpha)-amide "capped" synthetic polypeptides representing the 1-30 AA regions of AP7 (AP7-1 polypeptide) and AP24 (AP24-1 polypeptide) and NMR spectroscopy, we confirm that both N-terminal sequences possess putative Ca (II) interaction polyanionic sequence regions (2 x -DD- in AP7-1, -DDDED- in AP24-1) that are random coil-like in structure. However, with regard to the remaining sequences regions, each polypeptide features unique structural differences. AP7-1 possesses an extended beta-strand or polyproline type II-like structure within the A11-M10, S12-V13, and S28-I27 sequence regions, with the remaining sequence regions adopting a random-coil-like structure, a trait common to other polyelectrolyte mineral-associated polypeptide sequences. Conversely, AP24-1 possesses random coil-like structure within A1-S9 and Q14-N16 sequence regions, and evidence for turn-like, bend, or loop conformation within the G10-N13, Q17-N24, and M29-F30 sequence regions, similar to the structures identified within the putative elastomeric proteins Lustrin A and sea urchin spicule matrix proteins. The similarities and differences in AP7 and AP24 N-terminal domain structure are discussed with regard to joint AP7-AP24 protein modification of calcium carbonate growth.