The nuclear matrix prepared by amine modification

The nucleus is spatially ordered by attachments to a nonchromatin nuclear structure, the nuclear matrix. The nuclear matrix and chromatin are intimately connected and integrated structures, and so a major technical challenge in nuclear matrix research has been to remove chromatin while retaining a native nuclear matrix. Most methods for removing chromatin require first a nuclease digestion and then a salt extraction to remove cut chromatin. We have hypothesized that cut chromatin is held in place by charge interactions involving nucleosomal amino groups. We have tested this hypothesis by chemically modifying amino groups after nuclease digestion. By using this protocol, chromatin could be effectively removed at physiological ionic strength. We compared the ultrastructure and composition of this nuclear matrix preparation with the traditional high-salt nuclear matrix and with the third nuclear matrix preparation that we have developed from which chromatin is removed after extensive crosslinking. All three matrix preparations reveal internal nuclear matrix structures that are built on a network of branched filaments of about 10 nm diameter. That such different chromatin-removal protocols reveal similar principles of nuclear matrix construction increases our confidence that we are observing important architectural elements of the native structure in the living cell.     

Ultrastructural alterations during ischemia and reperfusion in human hearts during cardiac surgery

Needle biopsies taken from human hearts during cardiac surgery were investigated with the electron microscope. Cardiac tissue obtained before induction of ischemia was compared with tissue removed at the end of the ischemic period during total cardiopulmonary bypass with mild cardiac hypothermia and elective cardiac arrest (cardioplegia) and with tissue obtained after onset of coronary reperfusion. Ischemia led to changes in mitochondrial, nuclear, and vascular structures. The mitochondrial changes consisted of: loss of dense matrix granules, clearing of the matrix, fragmentation of cristae, appearance of amorphous densities, vacuolation and formation of myelin figures. The nuclear changes were: swelling of the nucleus, margination of chromatin, clumping of chromatin and, later, shrinkage of the nucleus. Study of reperfused tissue showed that amorphous densities, vacuolation and formation of myelin figures in mitochondria and nuclear shrinkage are probably irreversible. A good correlation was found between the duration of ischemia and the degree of ultrastructural damage. The degree of damage and the recovery upon reperfusion also correlated well. Full reversibility of ischemic changes was noted up to 40 min of ischemia. Between 40 and 60 min a number of cells appeared irreversibly damaged. The proportion of irreversibly damaged cells increased markedly with ischemia times exceeding 60 min.     

Cytoskeletal involvement in cAMP-induced sensitization of chromatin to nuclease digestion in transformed Chinese hamster ovary K1 cells.

The cAMP-induced reverse transformation of CHO-K1 cells, which restores fibroblastic morphology, normal nuclear structure, specific membrane structures and biochemical activities, and cell growth regulation, also restores the sensitivity of nuclear chromatin digestion by DNase I to that resembling the normal fibroblast. All of these aspects of the reverse-transformation reaction require integrity of the cytoskeleton. The nuclease-sensitivity effect is achieved only when the entire cell rather than the isolated nucleus is incubated with cAMP derivatives, indicating linkage between the cytoskeleton and nuclear components. Evidence is presented to show that the DNA sensitization to digestion involves interaction between DNA and other chromatin components and affects different regions of the genome in specific ways. Normal fibroblasts display greater endogenous nuclease activity than the transformed cell. The data are interpreted in terms of a genetic regulatory system extending from the membrane to the nucleus and utilizing the cAMP-induced cytoskeleton.     

Caspase cleavage of MST1 promotes nuclear translocation and chromatin condensation

MST1, mammalian STE20-like kinase 1, is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. MST1 is capable of inducing apoptotic morphological changes such as chromatin condensation upon overexpression. In this study, we show that MST1 contains two functional nuclear export signals (NESs) in the C-terminal domain, which is released from the N-terminal kinase domain upon caspase-mediated cleavage. Full-length MST1 is excluded from the nucleus and localized to the cytoplasm. However, either truncation of the C-terminal domain, point mutation of the two putative NESs, or treatment with leptomycin B, an inhibitor of the NES receptor, results in nuclear localization of MST1. Staurosporine treatment induces chromatin condensation, MST1 cleavage, and nuclear translocation. Staurosporine-induced chromatin condensation is partially inhibited by expressing a kinase-negative mutant of MST1, suggesting an important role of MST1 in this process. Significantly, MST1 is more efficient at inducing chromatin condensation when it is constitutively localized to the nucleus by mutation of its NESs. Moreover, inhibition of MST1 nuclear translocation by mutation of its cleavage sites reduces its ability to induce chromatin condensation. Taken together, these results suggest that truncation of the C-terminal domain of MST1 by caspases may result in translocation of MST1 into the nucleus, where it promotes chromatin condensation.     

Nuclear matrix proteins in human colon cancer.

The nuclear matrix is the nonchromatin scaffoldingof the nucleus. This structure confers nuclear shape, organizes chromatin, andappears to contain important regulatory proteins. Tissue specific nuclear matrixproteins have been found in the rat, mouse, and human. In this study we comparedhigh-resolution two-dimensional gel electropherograms of nuclear matrix proteinpatterns found in human colon tumors with those from normal colon epithelia.Tumors were obtained from 18 patients undergoing partial colectomy foradenocarcinoma of the colon and compared with tissue from 10 normal colons. Wehave identified at least six proteins which were present in 18 of 18 colontumors and 0 of 10 normal tissues, as well as four proteins present in 0 of 18tumors and in 10 of 10 normal tissues. These data, which corroborate similarfindings of cancer-specific nuclear matrix proteins in prostate and breast,suggest that nuclear matrix proteins may serve as important markers for at leastsome types of cancer.     

Association of glucocorticoid receptors with prostate nuclear sites for androgen receptors and with androgen response elements

Ventral prostate glands of intact normal rats contained low levels (2500 molecules/cell) of high-affinity (dissociation constant (Kd) 0.57 nmol/l) glucocorticoid receptors (GR). Levels of GR increased 2.8-fold 1 day after castration, and 4.3-fold 3 days after castration. Nuclear GR increased from a normal value of 1150 molecules/nucleus to 5200 molecules/nucleus 3 days after castration. The greater increase in intranuclear GR was in that associated with oligomeric chromatin. Although nuclear GR never approached the normal population of nuclear androgen receptors (AR; approximately 16000 molecules/nucleus), the selective rise in chromatin-associated receptors ensured that almost 60% of chromatin sites remained occupied. GR associated with prostate nuclear structures in a similar manner to AR, and exogenous GR bound saturably and with high affinity (Kd 100 pmol/l) to a similar number of sites as did AR. Both steroid receptors apparently competed for the same sites. In DNA-cellulose competition analyses, synthetic oligonucleotides containing glucocorticoid response elements or putative androgen response elements competed similarly against immobilized non-specific DNA for both AR and GR. In view of these data and information from other sources, it is probable that the role of GR in the prostate should be assessed with a view to understanding its action under conditions of androgen deprivation.     

The human brm protein is cleaved during apoptosis: the role of cathepsin G

The human brm (hbrm) protein (homologue of the Drosophila melanogaster brahma and Saccharomyces cervisiae SNF-2 proteins) is part of a polypeptide complex believed to regulate chromatin conformation. We have shown that the hbrm protein is cleaved in NB4 leukemic cells after induction of apoptosis by UV-irradiation, DNA damaging agents, or staurosporine. Because hbrm is found only in the nucleus, we have investigated the nature of the proteases that may regulate the degradation of this protein during apoptosis. In an in vitro assay, the hbrm protein could not be cleaved by caspase-3, -7, or -6, the "effector" caspases generally believed to carry out the cleavage of nuclear protein substrates. In contrast, we find that cathepsin G, a granule enzyme found in NB4 cells, cleaves hbrm in a pattern similar to that observed in vivo during apoptosis. In addition, a peptide inhibitor of cathepsin G blocks hbrm cleavage during apoptosis but does not block activation of caspases or cleavage of the nuclear protein polyADP ribose polymerase (PARP). Although localized in granules and in the Golgi complex in untreated cells, cathepsin G becomes diffusely distributed during apoptosis. Cleavage by cathepsin G removes a 20-kDa fragment containing a bromodomain from the carboxyl terminus of hbrm. This cleavage disrupts the association between hbrm and the nuclear matrix; the 160-kDa hbrm cleavage fragment is less tightly associated with the nuclear matrix than full-length hbrm.     

Chromatin architecture and nuclear RNA.

The maintenance of normal chromatin morphology requires ongoing RNA synthesis. We have examined the role of RNA in chromatin organization, using selective detergent extraction of cells, RNA synthesis inhibitors, and enzymatic digestion of nuclear RNA. Comparison of extracted and unextracted cells showed that the important features of chromatin architecture were largely unchanged by the extraction procedure. Normally, chromatin was distributed in small heterochromatic regions and dispersed euchromatic strands. Ribonucleoprotein granules were dispersed throughout the euchromatic regions. Exposure to actinomycin led to the redistribution of chromatin into large clumps, leaving large empty spaces and a dense clustering of the remaining ribonucleoprotein granules. When the nuclei of extracted cells were digested with RNase A, there was a rearrangement of chromatin similar to but more pronounced than that seen in cells exposed to actinomycin. The inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole also inhibits RNA synthesis but by a different mechanism that leaves no nascent RNA chains. The drug had little effect on chromatin after brief exposure but resembled actinomycin in its effect at longer times. We also examined the structure of the nuclear matrix to which most heteronuclear RNA remains associated. Pretreatment of cells with actinomycin or digestion of the nuclear matrix with RNase A caused the matrix fibers to collapse and aggregate. The experiments show a parallel decay of chromatin and of nuclear matrix organization with the depletion of nuclear RNA and suggest that RNA is a structural component of the nuclear matrix, which in turn may organize the higher order structure of chromatin.     

A possible role of cholesterol-sphingomyelin/phosphatidylcholine in nuclear matrix during rat liver regeneration

BACKGROUND/AIMS: Phospholipids and cholesterol in chromatin have been previously demonstrated. The lipid fraction changes during cell proliferation in relation to activation of enzymes of phospholipid metabolism. The aim of the present work is to clarify if chromatin lipids may derive or not from nuclear matrix and if they have different roles. METHODS: The subnuclear fractions were isolated from rat hepatocyte nuclei and the lipid fraction was extracted and analysed by chromatography in normal and regenerating liver. The phosphatidylcholine-sphingomyelin metabolism enzymes activity was assayed, by using radioactive substrates. RESULTS: In nuclear matrix, cholesterol and sphingomyelin are respectively five and three times higher than those present in chromatin; the amount of phosphatidylcholine, which it is enriched in saturated fatty acids, is lower, thus indicating a less fluid structure. The lower content in phosphatidylcholine may be justified by the phosphatidylcholine-dependent phospholipase C activity, which increases during liver regeneration, reaching a peak at the beginning of S-phase, when also cholesterol and sphingomyelin increase. CONCLUSIONS: The nuclear matrix lipids are independent from chromatin lipids; the ratio cholesterol-sphingomyelin/phosphatidylcholine is higher and, as a consequence, nuclear matrix is less fluid in relation to DNA synthesis, suggesting a specific role of nuclear matrix as a structure involved in DNA duplication.     

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-β and ori-γ) being somewhat preferred. We show here that the chromatin in the ori-β and ori-γ regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G₁/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.     

Morphological alterations in blood vessel endothelia of rat brain after administration of CCNU (lomustine)

Studies were performed on adult Wistar strain rats which were given four one-week spaced, intragastric doses of 2.5 mg or (the last dose) 5.0 mg CCNU. The animals were sacrificed one week after the last dose of the cytostatic drug. Cell nuclei of blood vessel endothelia in the parietal cerebral cortex and in the frontal-lateral part of the thalamus were examined. Karyo- and cytophotometric measurements were performed on sections subjected to Feulgen's reaction, using an automatic microscope image analyzer Morphoquant (Carl Zeiss, Jena). In CCNU-administered rats, blood vessel endothelia in the brain exhibited morphological alterations in the form of edema, increased DNA content in cell nuclei, modified distribution and density of chromatin lumps (nuclear chromatin lumps shifted towards the center of cell nucleus in the parietal cortex and in the thalamus, increased density of nuclear chromatin in the thalamus). Morphological alterations, changes in DNA content and in the nuclear chromatin status were of a similar type in blood vessel endothelia in the two examined structures but were somewhat more intense in endothelia of the cortex than in thalamus endothelia.     

Thyroid hormones induce sumoylation of the cold shock domain-containing protein PIPPin in developing rat brain and in cultured neurons

We previously identified a cold shock domain (CSD)-containing protein (PIPPin), expressed at high level in brain cells. PIPPin has the potential to undergo different posttranslational modifications and might be a good candidate to regulate the synthesis of specific proteins in response to extracellular stimuli. Here we report the effects of T(3) on PIPPin expression in developing rat brain. We found that a significant difference among euthyroid and hypothyroid newborn rats concerns sumoylation of nuclear PIPPin, which is abolished by hypothyroidism. Moreover, T(3) dependence of PIPPin sumoylation has been confirmed in cortical neurons purified from brain cortices and cultured in a chemically defined medium (Maat medium), with or without T(3). We also report that about one half of unmodified as well as all the sumoylated form of PIPPin could be extracted from nuclei with HCl, together with histones. Moreover, this HCl-soluble fraction remains in the nucleus even after treatment with 0.6 M KCl, thus suggesting strong interaction of PIPPin with nuclear structures and perhaps chromatin.     

Localization of the heme-binding protein in the cytoplasm and of a heme-binding protein-like immunoreactive protein in the nucleus of rat liver parenchymal cells: immunocytochemical evidence of the subcellular distribution corroborated by radioimmunoassay and immunoblotting

The abundant heme-binding protein of the liver, probably identical with Z-protein or liver fatty acid-binding protein, has an apparent molecular weight of 14,000 Da and is presumably involved in the intracellular transport of a variety of compounds. The cellular and subcellular distribution of HBP in the liver was studied in adult male and female rats by postembedding immunocytochemistry using the protein A-gold technique. By light microscopic examination heme-binding protein is present exclusively in parenchymal cells and not found in the sinusoidal lining cells or other cells in portal tracts. Immunoreactivity for heme-binding protein is uniformly strong throughout the liver lobule in female rats but is markedly reduced in the pericentral region in male animals. By immunoelectron microscopy heme-binding protein immunoreactivity is localized in cytoplasm and nuclear matrix. The mitochondria and peroxisomes and the secretory apparatus are free of the label. In nuclei, gold labeling is confined to the interchromatin region (euchromatin) and nucleoli; condensed chromatin (heterochromatin) and nucleolus-associated chromatin are negative. The subcellular localization was substantiated by radioimmunoassay and immunoblotting of nuclear and cytosolic fractions. Immunoblotting shows that the heme-binding protein-like immunoreactive protein in the nucleus has a slightly larger molecular weight than that in the cytoplasm.     

The influence of volume exclusion by chromatin on the time required to find specific DNA binding sites by diffusion

Within the nuclei of eukaryotic cells, the density of chromatin is nonuniform. We study the influence of this nonuniform density, which we derive from microscopic images [Schermelleh L, et al. (2008) Science 320:1332-1336], on the diffusion of proteins within the nucleus, under the hypothesis that chromatin density is proportional to an effective potential that tends to exclude the diffusing protein from regions of high chromatin density. The constant of proportionality, which we call the volume exclusivity of chromatin, is a model parameter that we can tune to study the influence of such volume exclusivity on the random time required for a diffusing particle to find its target. We consider randomly chosen binding sites located in regions of low (20th-30th percentile) chromatin density, and we compute the median time to find such a binding site by a protein that enters the nucleus at a randomly chosen nuclear pore. As the volume exclusivity of chromatin increases from zero, we find that the median time needed to reach the target binding site at first decreases to a minimum, and then increases again as the volume exclusivity of chromatin increases further. Random permutation of the voxel values of chromatin density abolishes the minimum, thus demonstrating that the speedup seen with increasing volume exclusivity at low to moderate volume exclusivity is dependent upon the spatial structure of chromatin within the nucleus.     

Remodeling nuclear architecture allows efficient transport of herpesvirus capsids by diffusion

The nuclear chromatin structure confines the movement of large macromolecular complexes to interchromatin corrals. Herpesvirus capsids of approximately 125 nm assemble in the nucleoplasm and must reach the nuclear membranes for egress. Previous studies concluded that nuclear herpesvirus capsid motility is active, directed, and based on nuclear filamentous actin, suggesting that large nuclear complexes need metabolic energy to escape nuclear entrapment. However, this hypothesis has recently been challenged. Commonly used microscopy techniques do not allow the imaging of rapid nuclear particle motility with sufficient spatiotemporal resolution. Here, we use a rotating, oblique light sheet, which we dubbed a ring-sheet, to image and track viral capsids with high temporal and spatial resolution. We do not find any evidence for directed transport. Instead, infection with different herpesviruses induced an enlargement of interchromatin domains and allowed particles to diffuse unrestricted over longer distances, thereby facilitating nuclear egress for a larger fraction of capsids.The nucleus is structured into chromatin and interchromatin compartments, giving it a “sponge-like” appearance, with the chromatin representing the actual sponge material and the interchromatin regions representing the enclosed pores, tubes, tunnels, and cisterna. This conceptual model has important implications for how differently sized molecules could move inside the nucleus. Although small molecules like GFP, streptavidin, fluorescent dextrans, or mRNA can mostly freely diffuse throughout the nucleus (1–3), large macromolecular assemblies, such as 100-nm fluorescent beads and promyelocytic leukemia (PML) or Cajal bodies, are trapped in interchromatin spaces called corrals. These corrals slowly move as a result of chromatin dynamics, and particles can also escape over long time scales (4, 5). Herpesvirus capsids of 125-nm diameter are assembled in the nucleus and must move to the nuclear periphery to exit the nucleus by budding through the nuclear membranes (6). Similarly, large host cargo like ribonucleoprotein (RNP) particles may need to move through the nucleus before they exit the nucleus in the same way (7). Earlier, we and others provided data that suggested that herpesvirus capsids use an active, directed mechanism based on F-actin to transport through the nucleoplasm (8, 9) and/or that nuclear F-actin is involved in capsid assembly. However, we recently showed that herpesviruses do not induce nuclear F-actin in most cells, and, more importantly, that nuclear capsid motility is not dependent on nuclear F-actin (10). This led us to reinvestigate nuclear capsid motility by single particle tracking to address whether molecular motors power it.During herpesvirus infection, the interchromatin domains enlarge and the nuclear volume increases as much as twofold (11). At the same time, viral replication compartments expand, move, and coalesce, but do not mix (12–16). By using a light-sheet modality we dubbed a ring-sheet, we were able to trace intranuclear herpes virus capsids with high spatial and temporal precision and low cytotoxicity. Our data show that nuclear herpesvirus capsids do not use a directed transport mechanism. Instead, we find by single particle tracking and analysis of capsid motility that capsids diffuse in enlarged nuclear spaces. We conclude that the previously described size increase of the interchromatin domain increases the likelihood of capsids to reach the nuclear envelope by diffusion.