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PubMed2002

SUMO-1 protease-1 regulates gene transcription through PML.

During a screen to identify c-Jun activators, we isolated a cysteine protease, SuPr-1, that induced c-Jun-dependent transcription independently of c-Jun phosphorylation. SuPr-1 is a member of a new family of proteases that hydrolyze the ubiquitin-like modifier, SUMO-1. SuPr-1 hydrolyzed SUMO-1-modified forms of the promyelocytic leukemia gene product, PML, and altered the subcellular distribution of PML in nuclear PODs (PML oncogenic domains). SuPr-1 also altered the distribution of other nuclear POD-associated proteins, such as CBP and Daxx, that act as transcriptional regulators. SuPr-1 action on transcription was enhanced by PML, and SuPr-1 failed to activate transcription in PML-deficient fibroblasts. Our studies establish an important role for SUMO proteases in transcription.

Amino Acid Sequence↗
PubMed2003

Monochloramine induces reorganization of nuclear speckles and phosphorylation of SRp30 in human colonic epithelial cells: role of protein kinase C.

Intestinal epithelial cells are constantly stimulated by reactive oxidant metabolites (ROMs) in inflamed mucosa. Monochloramine (NH2Cl), a cell-permeant ROM, is particularly relevant to the pathogenesis of inflammation in the gastrointestinal tract. Nuclear speckles, a unique nuclear subcompartment, accumulate a family of proteins, namely, serine- and arginine-rich (SR) proteins. They play important roles in regulation of pre-mRNA splicing. Currently, little is known about the link between inflammatory stimulation and the pre-mRNA splicing process, although gene expression is changed in inflamed tissues. The present study was designed to investigate whether stimulation of human colonic epithelial cells (HT-29 and Caco-2 cell lines) with NH2Cl affects nuclear speckles and their components. By indirect immunofluorescence, nuclear speckles have been shown to undergo rapid aggregation after NH2Cl stimulation. By utilizing Western blotting, SRp30 (a subset of SR proteins) in intestinal epithelial cells was found to be phosphorylated after NH2Cl treatment, whereas other SR proteins were not responsive to NH2Cl stimulation. The cytotoxic effect of NH2Cl was excluded by both negative lactate dehydrogenase assay and propidium iodide staining. Therefore, NH2Cl-induced morphological changes on nuclear speckles and phosphorylated SRp30 do not result from intestinal epithelial injury. Furthermore, the effect of NH2Cl on nuclear speckles and SRp30 was blocked by bisindolylmaleimide I, a selective PKC inhibitor. Together, the available data suggest that stimulation of intestinal epithelial cells with NH2Cl results in a consequent change on pre-mRNA splicing machinery via a distinctive signal pathway involving activation of PKC. This effect may contribute to oxidant-induced pathophysiological changes in the gastrointestinal tract.

Caco-2 Cells↗
PubMed1986

[Interaction of subunits of cAMP-dependent protein kinase with structural elements of the cell nucleus].

Using the method of protein transfer from polyacrylamide gel to nitrocellulose filters with subsequent incubation of filter-adsorbed protein with [32P]DNA, it was found that the catalytic subunit of cAMP-dependent protein kinase from porcine brain is capable of interacting with DNA to form a stable complex. This complex is resistant even to 2 M NaCl. The ability of the catalytic subunit to interact with DNA depends on the degree of enzyme nativity. The regulatory subunit of cAMP-dependent protein kinase does not bind to DNA both in the presence and absence of cAMP. The 125I-labeled regulatory subunit can interact with some chromatin proteins, in particular, with histone H1 and core histones. An essential role in this binding belongs to electrostatic and hydrophobic interactions.

Animals↗
PubMedDate not supplied

The nuclear tubular invaginations are dynamic structures inside the nucleus of HeLa cells.

Nuclear tubules (NTs) were found in the nucleus of HeLa cells. Although no function has been ascribed to these structures, our previous data has shown that they are the sites of Ca(2+) release with mitochondria shuttled around. In the present study, we further characterized these NTs through different fluorescent dye-labeling and red fluorescent protein transfection experiments. We found that doxorubicin (Dox) is a good indicator to demonstrate the NTs since Dox is fluorescent and DNA is able to quench its fluorescence. By using confocal and electron microscopy, we show that the number and nature of the NTs in HeLa vary from cell to cell, ranging from tubular to intricately branched structures. Additionally, these NTs are double-membrane invaginations of the nuclear envelope and usually lie close to nucleolus. At rest, NTs appeared to be stable and their mouths are always closed. Upon Ca(2+) ionomycin stimulation, various forms of dynamism, including membrane protrusion to the nucleus, enlargement and shrinkage of the NTs, and distortion of the nuclear envelope and NTs were observed over a time scale of minutes. These observations suggest that the NT represents a specialized and dynamic compartment inside the nucleus under the control of Ca(2+).

Acridine Orange↗
PubMed2003

Immunomorphological localization of Vasa protein and pre-mRNA splicing factors in Panorpa communis trophocytes and oocytes.

The distribution of Vasa protein and splicing factors of pre-mRNA has been studied in oogenesis of Panorpa communis. This distribution was analyzed relative to three types of perinuclear bodies (PBs) in trophocyte cytoplasm, PBs and polar granules (PGs) in oocyte. Immunoelectron labeling using antibody against Vasa protein revealed PBs of the 2nd type of P. communis trophocytes as well as oocyte PBs and PGs to contain Vasa protein. From this evidence emerged proposal that PBs of the 2nd type are homologues to the "nuage" of Drosophila, a marker of germ line cells. Besides, we suggest that in P. communis, both trophocytes and oocytes take part in formation of PGs. Using immunoelectron microscopy, we also show small nuclear RNPs both in trophocyte PBs of the 2nd type and in oocyte PBs. The functional significance of coupling in the same structure of Vasa protein and snRNPs is discussed.

Animals↗
PubMed2003

Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum.

Calcium is a second messenger in virtually all cells and tissues. Calcium signals in the nucleus have effects on gene transcription and cell growth that are distinct from those of cytosolic calcium signals; however, it is unknown how nuclear calcium signals are regulated. Here we identify a reticular network of nuclear calcium stores that is continuous with the endoplasmic reticulum and the nuclear envelope. This network expresses inositol 1,4,5-trisphosphate (InsP3) receptors, and the nuclear component of InsP3-mediated calcium signals begins in its locality. Stimulation of these receptors with a little InsP3 results in small calcium signals that are initiated in this region of the nucleus. Localized release of calcium in the nucleus causes nuclear protein kinase C (PKC) to translocate to the region of the nuclear envelope, whereas release of calcium in the cytosol induces translocation of cytosolic PKC to the plasma membrane. Our findings show that the nucleus contains a nucleoplasmic reticulum with the capacity to regulate calcium signals in localized subnuclear regions. The presence of such machinery provides a potential mechanism by which calcium can simultaneously regulate many independent processes in the nucleus.

Active Transport, Cell Nucleus↗
PubMed2002

Nuclear DNA helicase II is recruited to IFN-alpha-activated transcription sites at PML nuclear bodies.

It is known that nuclear DNA helicase II (NDH II) links CREB-binding protein directly to RNA polymerase II holoenzyme, and that this interaction is essential for gene activation by CREB. Here, we report for the first time that some NDH II/RNA helicase A is a component of promyelocytic leukemia nuclear bodies (PML NBs). An autoimmune serum specific for PML NBs was identified and used in immunoprecipitation experiments. NDH II was present in the immunoprecipitates as shown by mass spectrometry and by immunoblotting. Immunofluorescence and ultrastructural studies showed that NDH II colocalizes with a small subset of PML NBs in control cells, however, colocalizes with practically all bodies in interferon-alpha-stimulated cells. After interferon stimulation, more PML NBs were found to contain newly synthesized RNA, as indicated by bromouridine incorporation. PML NBs also contain RNA polymerase II. The association of NDH II with PML NBs was transcriptionally dependent, and NDH II was present in all bodies with nascent RNA. Blocking of mRNA synthesis caused NDH II relocalization from nucleoplasm to nucleoli. Based on the data, we suggest that NDH II recruitment to PML NBs is connected with transcriptional regulation of interferon-alpha-inducible genes attached to PML NBs.

Adenosine Triphosphatases↗
PubMed2002

An ATP-dependent step is required for the translocation of microinjected precursor mRNA into nuclear speckles.

Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. In a previous study (Melcák et al., 2001), it has been shown that the pre-spliceosomal assembly on microinjected splicing-competent precursor mRNA takes place in the speckles, and it has been suggested that the targeting of RNA into speckes consists of two interdependent steps, namely the diffusion process, followed by the energy-dependent translocation of RNA into the speckles. In the present study, we confirm the existence of these two steps and show that this latter translocation is ATP dependent.

Adenosine Triphosphate↗
PubMed2026

Nuclear speckles: a fundamental layer of gene regulation.

Within the cell nucleus, non-DNA structures called nuclear bodies interact with chromatin to regulate gene expression and organize our genetic material. Among nuclear bodies, nuclear speckles are prominent. They interact with broad genomic regions, serve as major gene-activating structures, and are implicated in viral infection, cancer, neurodegeneration, stress response, and development. Advances that integrate genomics with imaging are leading to a deeper understanding of how nuclear speckles fit into our current knowledge of chromatin biology, genome organization, and the central dogma of biology. Collectively, these recent studies emphasize nuclear speckles as key gene regulatory structures within the cell nucleus, offering new perspectives on how gene expression dysregulation is linked to disease.

Humans↗
PubMed2006

PV-1 labels trans-cellular openings in mouse endothelial cells and is negatively regulated by VEGF.

The PV-1 protein is endogenously expressed from a single mRNA in the mouse pancreatic MS-1 endothelial cell line as a 60-kDa N-glycosylated and 50-kDa non-glycosylated protein that form DTT sensitive oligomers. In the absence of cell permeabilization, PV-1 antibodies label transcellular openings of variable size, many that penetrate through the cytosol with circular openings on the free and attached surface of the plasma membrane. Intracellular PV-1 is localized in perinuclear aggregates that can extend as a fibrous network through the cytosol and often surround the nuclear compartment. In some cells, PV-1 is organized as a large unipolar spindle-like structure that is often associated with severe deformation of the nucleus. The VEGF-R2 inhibitor SU5614 increased the PV-1 protein levels in a dose-dependent manner and inhibited MS-1 cell growth, without inducing apoptosis. This report provides compelling evidence for a functional role of PV-1 in the formation of large transendothelial channels and modulation of nuclear shape. Moreover, these data suggest the PV-1 protein is negatively regulated by VEGF.

Animals↗
PubMed2003

The DNA topoisomerase I binding protein topors as a novel cellular target for SUMO-1 modification: characterization of domains necessary for subcellular localization and sumolation.

Over the past years, modification by covalent attachment of SUMO (small ubiquitin-like modifier) has been demonstrated for of a number of cellular and viral proteins. While increasing evidence suggests a role for SUMO modification in the regulation of protein-protein interactions and/or subcellular localization, most SUMO targets are still at large. In this report we show that Topors, a Topoisomerase I and p53 interacting protein of hitherto unknown function, presents a novel cellular target for SUMO-1 modification. In a yeast two-hybrid system, Topors interacted with both SUMO-1 and the SUMO-1 conjugating enzyme UBC9. Multiple SUMO-1 modified forms of Topors could be detected after cotransfection of exogenous SUMO-1 and Topors induced the colocalization of a YFP tagged SUMO-1 protein in a speckled pattern in the nucleus. A subset of these Topors' nuclear speckles were closely associated with the PML nuclear bodies (POD, ND10). A central domain comprising Topors residues 437 to 574 was sufficient for both sumolation and localization to nuclear speckles. One SUMO-1 acceptor site at lysine residue 560 could be identified within this region. However, sumolation-deficient Topors mutants showed that sumolation obviously is not required for localization to nuclear speckles.

Active Transport, Cell Nucleus↗
PubMed2003

Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins.

BACKGROUND: Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97) have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. RESULTS: Using confocal microscopy and western analysis techniques, we determined 1) the immunolabeled structures are truly within the nucleus; 2) the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs) and it can be identified on other intermediate filament proteins (IFs) in other cell types; and 3) there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. CONCLUSION: We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

Animals↗
PubMed2005

Rapid, diffusional shuttling of poly(A) RNA between nuclear speckles and the nucleoplasm.

Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22 degrees C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy.

Animals↗
PubMed2005

A global but stable change in HeLa cell morphology induces reorganization of DNA structural loop domains within the cell nucleus.

DNA of higher eukaryotes is organized in supercoiled loops anchored to a nuclear matrix (NM). The DNA loops are attached to the NM by means of non-coding sequences known as matrix attachment regions (MARs). Attachments to the NM can be subdivided in transient and permanent, the second type is considered to represent the attachments that subdivide the genome into structural domains. As yet very little is known about the factors involved in modulating the MAR-NM interactions. It has been suggested that the cell is a vector field in which the linked cytoskeleton-nucleoskeleton may act as transducers of mechanical information. We have induced a stable change in the typical morphology of cultured HeLa cells, by chronic exposure of the cells to the polar compound dimethylsulfoxide (DMSO). Using a PCR-based method for mapping the position of any DNA sequence relative to the NM, we have monitored the position relative to the NM of sequences corresponding to four independent genetic loci located in separate chromosomes representing different territories within the cell nucleus. Here, we show that stable modification of the NM morphology correlates with the redefinition of DNA loop structural domains as evidenced by the shift of position relative to the NM of the c-myc locus and the multigene locus PRM1 --> PRM2 --> TNP2, suggesting that both cell and nuclear shape may act as cues in the choice of the potential MARs that should be attached to the NM.

Cell Nucleus↗
PubMed2000

Induction of DNA double-strand breaks in mammalian cells and yeast.

Induction of DNA double-strand breaks (dsb) and their distribution are dependent on the energy deposition pattern within the cell nucleus (physical structure) and the ultrastructure of the chromosomes and its variation by the cell cycle and gene activities (biological structure). For electron radiation very similar RBE-values are observed for mammalian and yeast cells (AlK, 1.5 keV, 15 keV/micrometer: 2.6 in mammalian cells and 2.2 in yeast; CK 0.278 keV, 23 keV/micrometer: approx. 2.5 in mammalian cells and 3.8 in yeast). In contrast, the RBE-values for the induction of dsb of 4He2+ and light ions in the LET range from about 100 keV/micrometer up to 1000 keV/micrometer are significantly higher for yeast cells compared to mammalian cells. For example, the RBE-value of alpha-particles (120 keV/micrometer) is about 1.2 for mammalian cells whereas for yeast the RBE-value is about 2.5. The yeast chromatin has less condensed fibres compared with mammalian cells. Since a single CK photoelectron can induce only one dsb, the different condensation of the mammalian and yeast chromatin has no influence. However, particles may induce more than one dsb when traversing a chromatin fibre. The probability for the induction of closely neighboured dsb is higher the more condensed the chromatin fibres are. Since small DNA fragments (50 bp up to several kbp) are lost by standard methods of lysis, the underestimation of dsb yields increases with fibre condensation, which is in accordance with the observes dsb yields in mammalian cells and yeast. In order to obtain relevant yields of dsb (and corresponding RBE-values) the measurement of all DNA fragments down to about 50 bp are needed.

DNA Damage↗