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Results for “Intranuclear Space”

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At least 19 recordsLinked to original sources

PubMed2006

Homeostatic restitution of cell membranes. Nuclear membrane lipid biogenesis and transport of protein from cytosol to intranuclear spaces.

Our studies on homeostatic restitution of cellular and subcellular membranes showed that vesicular intracellular transport is engaged in systematic and coordinated replacement of lipids and proteins in the membranes of the secretory, non-dividing epithelial cells (Slomiany et al., J. Physiol. Pharmacol. 2004; 55: 837-860). In this report, we present evidence on the homeostatic restitution of lipids in the biomembranes that constitute nuclear envelopes. We investigated nuclear membranes lipid synthesis by employing purified intact nuclei (IN), the outer nuclear membrane (ONM), the inner nuclear membrane (INM) and the cell cytosol (CC). In contrast to Endoplasmic Reticulum (ER) which in the presence of CC generates new biomembrane that forms ER vesicles transporting ER products to Golgi, the IN, ONM and INM are not producing transport vesicles. Instead, the newly synthesized lipids remain in the nuclear membranes. The membranes (INM, ONM) of IN incubated with CC become enriched with newly synthesized phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA). The incubation of separated ONM and INM with CC also enriched the membranes with IN specific lipids identified above. Moreover, the incubation of IN or its membranes with CC afforded retention of numerous CC proteins on the nuclear membrane. Here, we concentrated on 30kDa CC protein that displayed affinity to nuclear membrane PIP2. The 30kDa CC protein bound to PIP2 of IN, INM, and ONM. With IN, initially the PIP2-30kDa CC protein complex was detected on ONM, after 30-120 min of incubation, was found on INM and in nuclear contents. At the same time when the 30 kDa protein was released from INM and found in nuclear contents, the PIP2 of INM and ONM became undetectable, while the lipid extract from the membrane displaced from IN contained labeled PI only. Since ONM is an uninterrupted continuum of ER and INM, we speculate that the synthesis of the lipids in the ER, in the region adjacent to nucleus, is defining nuclear outer and inner biomembrane composition, is responsible for transport of the cytosolic protein into the nucleus and, replenishment of ER membrane used for vesicular transport.

Animals↗

PubMed2005

Spectrin repeat proteins in the nucleus.

Spectrin repeat sequences are among the more common repeat elements identified in proteins, typically occurring in large structural proteins. Examples of spectrin repeat-containing proteins include dystrophin, alpha-actinin and spectrin itself--all proteins with well-demonstrated roles of establishing and maintaining cell structure. Over the past decade, it has become clear that, although these proteins display a cytoplasmic and plasma membrane distribution, several are also found both at the nuclear envelope, and within the intranuclear space. In this review, we provide an overview of recent work regarding various spectrin repeat-containing structural proteins in the nucleus. As well, we hypothesize about the regulation of their nuclear localization and possible nuclear functions based on domain architecture, known interacting proteins and evolutionary relationships. Given their large size, and their potential for interacting with multiple proteins and with chromatin, spectrin repeat-containing proteins represent strong candidates for important organizational proteins within the nucleus. Supplementary material for this article can be found on the BioEssays website (http://www.interscience.wiley.com/jpages/0265-9247/suppmat/index.html).

Animals↗

PubMed2005

EAST interacts with Megator and localizes to the putative spindle matrix during mitosis in Drosophila.

We have used immunocytochemistry to demonstrate that the EAST protein in Drosophila, which forms an expandable nuclear endoskeleton at interphase, redistributes during mitosis to colocalize with the spindle matrix proteins, Megator and Skeletor. EAST and Megator also colocalize to the intranuclear space surrounding the chromosomes at interphase. EAST is a novel protein that does not have any previously characterized motifs or functional domains. However, we show by immunoprecipitation experiments that EAST is likely to molecularly interact with Megator which has a large NH2-terminal coiled-coil domain with the capacity for self assembly. On the basis of these findings, we propose that Megator and EAST interact to form a nuclear endoskeleton and as well are important components of the putative spindle matrix complex during mitosis.

Animals↗

PubMed2000

A new look at the nuclear matrix.

The concept of the nuclear matrix, a karyoskeletal structure that serves as a support for the genome and its activities, has stimulated many studies of the association of nuclear components and functions with this structure. However, certain experimental findings are not consistent with the existence of the nuclear matrix in vivo, including our inability to visualise a corresponding structure in intact cells, the demonstrated mobility in vivo of chromatin and messenger ribonucleoprotein particles, which are claimed to be bound to the nuclear matrix, the paradoxical extractability from nuclei in low ionic strength buffers of enzymes that are found in the 2 M NaCl-insoluble matrix, and the extractability, in conditions which reproduce the intranuclear milieu, of regions of DNA (matrix or scaffold attachment regions, MAR/SARs) postulated to be bound to the nuclear matrix in vivo. This review considers the nuclear matrix model in the light of sometimes overlooked evidence that each step in its isolation may cause nuclear components to bind to it by new liaisons that do not exist in vivo. This is illustrated by experiments where nuclear-targeted green fluorescent protein is found in the nuclear matrix, and raises the possibility that MAR/SARs actually bind to DNA-binding proteins or multiprotein complexes, including replicational, transcriptional and processing machinery, and topoisomerases that are incorporated into the nuclear matrix during its preparation. Considering that the nuclear lamina forms a rigid exoskeleton, the necessity for internal skeletal structures is raised; the major roles that macromolecular crowding, phase partitioning, and charge effects are likely to play in organisation of the intranuclear space may provide new models for the compartmentalisation of proteins and functions into different nuclear domains and of chromosomes into territories.

DNA↗

PubMed2003

Cross-linking of cellular proteins by tissue transglutaminase during necrotic cell death: a mechanism for maintaining tissue integrity.

Tissue transglutaminase (tTG) is a Ca(2+)-dependent enzyme which cross-links proteins via epsilon(gamma-glutamyl)lysine bridges. There is increasing evidence that tTG is involved in wound repair and tissue stabilization, as well as in physiological mechanisms leading to cell death. To investigate the role of this enzyme in tissue wounding leading to loss of Ca(2+) homoeostasis, we initially used a model involving electroporation to reproduce cell wounding under controlled conditions. Two cell models were used whereby tTG expression is regulated either by antisense silencing in ECV 304 cells or by using transfected Swiss 3T3 cells in which tTG expression is under the control of the tet regulatory system. Using these cells, loss of Ca(2+) homoeostasis following electroporation led to a tTG-dependent formation of highly cross-linked proteinaceous shells from intracellular proteins. Formation of these structures is dependent on elevated intracellular Ca(2+), but it is independent of intracellular proteases and is near maximal after only 20 min post-wounding. Using labelled primary amines as an indicator of tTG activity within these 'wounded cells', we demonstrate that tTG modifies a wide range of proteins that are present in both the perinuclear and intranuclear spaces. The demonstration of entrapped DNA within these shell structures, which showed limited fragmentation, provides evidence that the high degree of transglutaminase cross-linking results in the prevention of DNA release, which may serve to dampen any subsequent inflammatory response. Comparable observations were shown when monolayers of cells were mechanically wounded by scratching. In this second model of cell wounding, redistribution of tTG activity to the extracellular matrix was also demonstrated, an effect which may serve to stabilize tissues post-trauma, and thus contribute to the maintenance of tissue integrity.

3T3 Cells↗

PubMed2004

Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila.

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.

Animals↗

PubMed2003

An uncertainty principle in chromosome positioning.

Chromosomes are non-randomly positioned in the mammalian interphase nucleus. It is not known how patterns of chromosome positions are established or to what degree spatial arrangements of chromosomes change during the cell cycle, especially during mitosis. Two reports have applied in vivo microscopy to track chromosomes in space and time. The results highlight the inherently imperfect and probabilistic nature of chromosome positioning in the cell nucleus.

Animals↗

PubMedDate not supplied

Interstitial Cajal-like cells (ICLC) in human resting mammary gland stroma. Transmission electron microscope (TEM) identification.

We have previously shown the existence of ICLC in human resting mammary gland stroma by means of methylene blue (vital) staining and c-kit immunopositivity (immunofluorescence and immunohistochemistry). In addition, we reported the phenotype characteristics of these ICLC in vitro (primary cell cultures). Since the identification of ICLC outside the gut requires, at this moment, the obligatory use of TEM, we used this technique and provide unequivocal evidence for the presence of ICLC in the intralobular stroma of human resting mammary gland. According to the 'platinum standard' (10 TEM criteria for the certitude diagnosis of ICLC), we found interstitial cells with the following characteristics: 1. location: among the tubulo-alveolar structures, in the non-epithelial space; 2. caveolae: approximately 2.5% of cell volume; 3. mitochondria: approximately 10% of cell volume; 4. endoplasmic reticulum: either smooth or rough, approximately 2-3% of cell volume; 5. cytoskeleton: intermediate and thin filaments, as well as microtubules are present; 6. myosin thick filaments: undetectable; 7. basal lamina: occasionally found; 8. gap junctions: occasionally found; 9. close contacts with targets: nerve fibers, capillaries, immunoreactive cells by 'stromal synapses'; 10. characteristic cytoplasmic processes: i) number: frequently 2-3; ii) length: several tens of mum; iii) thickness: uneven caliber, 0.1-0.5 microm, with dilations, but very thin from the emerging point; iv) aspect: moniliform, usually with mitochondria located in dilations; v) branching: dichotomous pattern; vi) Ca(2+) release units: are present; vii) network labyrinthic system: overlapping cytoplasmic processes. It remains to be established which of the possible roles that we previously suggested for ICLC (e.g. juxta- and/or paracrine secretion, uncommited progenitor cells, immunological surveillance, intercellular signaling, etc.) are essential for the epithelium/stroma equilibrium in the mammary gland under normal or pathological conditions.

Cells, Cultured↗

PubMed2004

Theoretical evaluation of dielectric absorption of microwave energy at the scale of nucleic acids.

A theoretical model is proposed for the evaluation of dielectric properties of the cell nucleus between 0.3 and 3 GHz, as a function of its nucleic acids (NA) concentration (CNA). It is based on literature data on dielectric properties of DNA solutions and nucleoplasm. In skeletal muscle cells, the specific absorption rate (SAR) ratio between nucleoplasm and cytoplasm is found to be larger than one for CNA above 30 mg/ml. A nearly linear relationship is found between CNA and this nucleocytoplasmic SAR ratio. Considering the nanoscale of the layer of condensed counterions and bound water molecules at the NA-solution interface, the power absorption per unit volume is evaluated at this precise location. It is found to be between one and two orders of magnitude above that in muscle tissue as a whole. Under realistic microwave (MW) exposure conditions, however, these SAR inhomogeneities do not generate any significant thermal gradient at the scale considered here. Nevertheless, the question arises of a possible biological relevance of nonnegligible and preferential heat production at the location of the cell nucleus and of the NA molecules.

Absorption↗

PubMed2005

Comment on "Theoretical evaluation of dielectric absorption of microwave energy at the scale of nucleic acids".

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Absorption↗

PubMed2005

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.

Cell Nucleus↗

PubMed2005

Structural organization and cellular localization of tuftelin-interacting protein 11 (TFIP11).

Tuftelin-interacting protein (TFIP11) was first identified in a yeast two-hybrid screening as a protein interacting with tuftelin. The ubiquitous expression of TFIP11 suggested that it might have other functions in non-dental tissues. TFIP11 contains a G-patch, a protein domain believed to be involved in RNA binding. Using a green fluorescence protein tag, TFIP11 was found to locate in a novel subnuclear structure that we refer to as the TFIP body. An in vivo splicing assay demonstrated that TFIP11 is a novel splicing factor. TFIP11 diffuses from the TFIP body following RNase A treatment, suggesting that the retention of TFIP11 is RNA dependent. RNA polymerase II inhibitor (-amanitin and actinomycin D) treatment causes enlargement in size and decrease in number of TFIP bodies, suggesting that TFIP bodies perform a storage function rather than an active splicing function. The TFIP body may therefore represent a new subnuclear storage compartment for splicing components.

Adenoviridae↗

PubMed2005

Predicting protein subnuclear location with optimized evidence-theoretic K-nearest classifier and pseudo amino acid composition.

The nucleus is the brain of eukaryotic cells that guides the life processes of the cell by issuing key instructions. For in-depth understanding of the biochemical process of the nucleus, the knowledge of localization of nuclear proteins is very important. With the avalanche of protein sequences generated in the post-genomic era, it is highly desired to develop an automated method for fast annotating the subnuclear locations for numerous newly found nuclear protein sequences so as to be able to timely utilize them for basic research and drug discovery. In view of this, a novel approach is developed for predicting the protein subnuclear location. It is featured by introducing a powerful classifier, the optimized evidence-theoretic K-nearest classifier, and using the pseudo amino acid composition [K.C. Chou, PROTEINS: Structure, Function, and Genetics, 43 (2001) 246], which can incorporate a considerable amount of sequence-order effects, to represent protein samples. As a demonstration, identifications were performed for 370 nuclear proteins among the following 9 subnuclear locations: (1) Cajal body, (2) chromatin, (3) heterochromatin, (4) nuclear diffuse, (5) nuclear pore, (6) nuclear speckle, (7) nucleolus, (8) PcG body, and (9) PML body. The overall success rates thus obtained by both the re-substitution test and jackknife cross-validation test are significantly higher than those by existing classifiers on the same working dataset. It is anticipated that the powerful approach may also become a useful high throughput vehicle to bridge the huge gap occurring in the post-genomic era between the number of gene sequences in databases and the number of gene products that have been functionally characterized. The OET-KNN classifier will be available at www.pami.sjtu.edu.cn/people/hbshen.

Amino Acid Sequence↗

PubMed2004

Internal organisation of the nucleus: assembly of compartments by macromolecular crowding and the nuclear matrix model.

Many and possibly all macromolecules in the nucleus are segregated into discrete compartments, but the current model that this is achieved by a fibrillar nuclear matrix which structures the nuclear interior and compartments is not consistent with all experimental observations, as reviewed here. New results are presented which suggest that macromolecular crowding forces play a crucial role in the assembly of at least two compartments, nucleoli and PML bodies, and an in vitro system in which crowding assembles macromolecular complexes into structures which resemble nuclear compartments is described. Crowding forces, which are strong in the nucleus due to the high macromolecule concentration (in the range of 100 mg/ml), vastly increase the association constants of intermolecular interactions and can segregate different macromolecules into discrete phases. The model that they play a role in compartmentalisation of the nucleus is generally consistent with the properties of compartments, including their spherical or quasispherical form and their dynamic and mobile nature.

Animals↗

PubMed2004

The role of intranuclear lipids.

The presence of phospholipids as a component of chromatin is now well documented and many enzymes such as sphingomyelinase, sphingomyelin-synthase, reverse sphingomyelin-synthase and phosphatidylcholine-dependent phospholipase C have been described and characterised. Other lipids were demonstrated inside the nucleus especially plasmalogens and cholesterol. The chromatin phospholipids, comprising 10% of that present in the nucleus, show a different metabolism with respect to those present in either microsomes or in nuclear membranes; they increase also during the DNA duplication as shown during both liver regeneration and cell maturation. They appear localised near newly synthesized RNA in decondensed chromatin. Digestion of chromatin with RNase, but not with DNase, causes a loss of phospholipids. The composition of the chromatin phospholipid fraction shows an enrichment in sphingomyelin and phosphatidylserine. In this review the behaviour of single lipids in relation to cell proliferation, cell differentiation and apoptosis is described. Sphingomyelin, the lipid most represented in chromatin with respect to microsomes and nuclear membranes, is localised near to newly synthesized RNA, its presence appearing to protect RNA from RNase digestion. This effect is reversed by sphingomyelinase which digests sphingomyelin and, as a consequence, RNA may be hydrolysed. The amount of sphingomyelin is restored by sphingomyelin-synthase. Sphingomyelin increases during the differentiation process and apoptosis. An increase of sphingomyelinase with consequent decrease in sphingomyelin is observed at the beginning of S-phase of the cell cycle. A possible role in stabilising the DNA double helix is indicated. Phosphatidylserine behaves similarly during differentiation and appears to stimulate both RNA and DNA polymerases. Phosphatidylcholine is implicated in cell proliferation through the activation of intranuclear phosphatidylcholine-dependent phospholipase C and diacylglycerol production. The increase in diacylglycerol stimulates phosphatidylcholine synthesis through the major pathway from cytidyltriphosphate. An inhibition of phosphatidylcholine synthesis is responsible for the initiation of apoptosis. The presence of reverse sphingomyelin-synthase favours the formation of phosphatidylcholine, the donor of phosphorylcholine, from sphingomyelin. Little information has been reported for phospatidylethanolamine, but phosphtidylinositol appears to influence cell differentiation and proliferation. This last effect is due to the action of two enzymes: PI-PLCss1 having a role in the onset of DNA synthesis and PC-PLCgamma1 acting in G2 transit. Phosphoinositides also may have an important role: in membrane-stripped nuclei isolated from mitogen stimulated cells a decrease in PIP and PIP2 followed by an increase in diacylglycerol and a translocation of protein kinase C inside the nucleus is observed. On the other hand, overexpression of the enzyme inositol polysphosphate-1-phosphatase reduced DNA synthesis by 50%. Nevertheless, an enhanced rate of phosphorylation has been demonstrated in cells induced to differentiate. These molecules probably favour RNA transcription, counteracting the inhibition of H1 on RNA polymerase II. Plasmalogens were demonstrated in the nucleus and their increase favours the increased activity of phosphatidylcholine-dependent phospholipase C when DNA synthesis starts. Moreover, two forms of cholesterol has been described in chromatin: one, a less soluble sphingomyelin-linked form and a free fraction. Cholesterol increases during liver regeneration, first as a linked fraction and then, when DNA synthesis starts, as a free fraction. The changes of these components have been summarised in relation to cell function in order to give an overview of their possible roles in the different phases of cell duplication and their influence on cell differentiation and during apoptosis. Finally, the relevance of these molecules as intranuclear signals is discussed and future directions are indicated in clarifying pathological process such as tumour cell transformation and the possibility in finding new therapeutic tools.

Animals↗

PubMed2005

Disruption of microtubules leads to glucocorticoid receptor degradation in HeLa cell line.

The role of microtubules (MTs) in steroid hormone-dependent human glucocorticoid receptor (hGR) activation/translocation is controversial. It was demonstrated recently that colchicine (COL) down-regulates hGR-driven genes in primary human hepatocytes by a mechanism involving inhibition of hGR translocation to the nucleus. To investigate whether inhibition of hGR translocation is the sole reason for its inactivation, we used human cervical carcinoma cells (HeLa) as a model. Herein we present evidence that perturbation of microtubules in HeLa cells leads to rapid time- and dose-dependent degradation of hGR protein. Degradation is proteasome mediated as revealed by its reversibility by proteasome inhibitor MG132. Moreover, degradation was observed for neither wt-hGR nor hGR mutants S226A and K419A in transiently transfected COS-1 cells. On the other hand, c-jun N-terminal kinase (JNK) seems not to be involved in the process because JNK inhibitor 1,9-Pyrazoloanthrone (SP600125) does not reverse hGR degradation. Similarly, another hGR functional antagonist, nuclear factor kappa beta (NFkappaB), did not play any role in the degradation process.

Animals↗

PubMed2004

A role for macromolecular crowding effects in the assembly and function of compartments in the nucleus.

The mechanisms which cause macromolecules to form discrete compartments within the nucleus are not understood. Here, two ubiquitous compartments, nucleoli, and PML bodies, are shown to disassemble when K562 cell nuclei expand in medium of low monovalent cation concentration; their major proteins dispersed as seen by immunofluorescence and immunoelectron microscopy, and nucleolar transcript elongation fell by approximately 85%. These compartments reassembled and nucleolar transcription recovered in the same medium after adding inert, penetrating macromolecules (8 kDa polyethylene glycol (PEG), or 10.5 kDa dextran) to 12% w/v, showing that disassembly was not caused by the low cation concentration. These responses satisfy the criteria for crowding or volume exclusion effects which occur in concentrated mixtures of macromolecules; upon expansion the macromolecular concentration within the nucleus falls, and can be restored by PEG or dextran. These observations, together with evidence of a high concentration of macromolecules in the nucleus (in the range of 100mg/ml) which must cause strong crowding forces, suggest strongly that these forces play an essential role in driving the formation, and maintaining the function of nuclear compartments. This view is consistent with their dynamic and mobile nature and can provide interpretations of several unexplained observations in nuclear biology.

Buffers↗

PubMed2005

Corepressor subnuclear organization is regulated by estrogen receptor via a mechanism that requires the DNA-binding domain.

The restriction of transcription factors to certain domains within the cell nucleus must serve an important regulatory function. The silencing mediator of retinoic acid and thyroid hormone (SMRT) and other members of the corepressor complex are enriched in spherical intranuclear foci, and repress estrogen receptor alpha (ERalpha)-dependent transcriptional activity. When fluorescent protein (FP)-labeled SMRT and ERalpha were co-expressed, the proteins co-localized. The subnuclear organization and positioning of the complexes, however, depended on the ligand state of the receptor. Automated image analysis was used to quantify the ERalpha-dependent change in SMRT organization in randomly selected living cell populations. The results demonstrate that the subnuclear positioning of SMRT is influenced by the ligand-bound ERalpha, and this activity is dependent on the ratio of the co-expressed ERalpha and SMRT. A deletion mutant of ERalpha showed that the receptor DNA-binding domain was necessary for the ligand-dependent positioning of SMRT. These results define important organizational mechanisms that underlie nuclear receptor regulation of gene expression.

Animals↗