A nervous system-specific subnuclear organelle in Caenorhabditis elegans.

We describe here phase-separated subnuclear organelles in the nematode Caenorhabditis elegans, which we term NUN (NUclear Nervous system-specific) bodies. Unlike other previously described subnuclear organelles, NUN bodies are highly cell type specific. In fully mature animals, 4-10 NUN bodies are observed exclusively in the nucleus of neuronal, glial and neuron-like cells, but not in other somatic cell types. Based on co-localization and genetic loss of function studies, NUN bodies are not related to other previously described subnuclear organelles, such as nucleoli, splicing speckles, paraspeckles, Polycomb bodies, promyelocytic leukemia bodies, gems, stress-induced nuclear bodies, or clastosomes. NUN bodies form immediately after cell cycle exit, before other signs of overt neuronal differentiation and are unaffected by the genetic elimination of transcription factors that control many other aspects of neuronal identity. In one unusual neuron class, the canal-associated neurons, NUN bodies remodel during larval development, and this remodeling depends on the Prd-type homeobox gene ceh-10. In conclusion, we have characterized here a novel subnuclear organelle whose cell type specificity poses the intriguing question of what biochemical process in the nucleus makes all nervous system-associated cells different from cells outside the nervous system.

Estimate of the pair correlation functions of nuclei in normal prostate, prostatic intraepithelial neoplasia, and cancer

This study deals with the application of methods of second-order stereology to investigate the spatial distribution of nuclei in normal prostate, prostatic intraepithelial neoplasia and adenocarcinoma. We aimed to identify differences related to the progression of premalignant lesions (PIN) to carcinoma, as well as the spatial changes in relation to tumour grade. Estimation of second-order stereology parameters, such as g(r), (pair correlation function), statistic M, and Clark-Evans aggregation index (CEAI) were employed to investigate the distribution of nuclei. Linear discriminant analysis (LDA) with M and CEAI as model variables was implemented to classify the cancer cases into two groups according to Gleason score. We found that the point processes of the nuclei in prostatic cancer and normal tissues differed by first-order as well as by second-order properties. In the PIN the mean g-values were intermediate between normal and cancer. The LDA indicates that M and CEAI were able to classify into the correct group of Gleason score more than 90% of the cases analysed. Cancer cases showing a higher degree of disorder in the spatial distribution of nuclei were significantly classified into the group of higher Gleason score. The nuclei in both normal and pathological prostate were not Poisson distributed. Additionally, we found that the progression from normal tissue to carcinoma was accompanied by a progressive increase in spatial disorder which is intermediate in pre-malignant lesions (PIN). The parameters employed were able to classify the cancer cases according to the Gleason score

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Postnatal development of Homer1a in the rat hippocampus.

Homer1a (H1a) is an immediate early gene involved in multiple forms of synaptic plasticity. It exhibits a postnatal increase in the rat forebrain (Brakeman et al. (1997) Nature 386:284-288) and reduces the density and size of dendritic spines in hippocampal neurons (Sala et al. (2003) J Neurosci 23:6327-6337). We evaluated hippocampal H1a expression at different postnatal ages (P3, P5, P7, P9, P15, P19, P23, P35, and adult) using Fluorescence In Situ Hybridization (FISH) and qRT-PCR. Maximal electroconvulsive shock (MECS) was used to induce maximal expression relative to home cage (HC) controls. Large scale images and confocal z-stacks from dorsal subiculum (DS), CA1, CA3, and dentate gyrus (DG) were analyzed by both manual and automated methods. In DS, CA1, and CA3 a significant proportion of cells (40%) expressed small but detectable levels of H1a from P3; however, MECS did not up-regulate H1a during the first postnatal week. MECS induced H1a positive cells during the second postnatal week and induction reached adult levels at P9. H1a-Intra Nuclear Foci (INF) size and intensity varied with age, increasing at P19-23 in CA1 and CA3 and from P9 to P23 in DS. In DG, H1a expression exhibited a lamination pattern and an H1a-INF size and intensity gradient across the granule cell layer, consistent with the outside-in maturation of DG granule cells. The developmental progression of H1a corresponds to the synaptic refinement period supporting the conclusion that H1a could play an important role in this process.

Early apoptotic reorganization of spliceosomal proteins involves caspases, CAD and rearrangement of NuMA.

The reorganization of nuclear structures is an important early feature of apoptosis and involves the activity of specific proteases and nucleases. Well-known is the condensation and fragmentation of chromatin; however, much less is understood about the mechanisms involved in the reorganization of structures from the interchromatin space, such as interchromatin granule clusters (IGCs). In this study, we show that the initial enlargement and rounding-up of IGCs correlate with a decrease in mRNA transcription and are caspase-independent, but involve protein phosphatases PP1/PP2A. Subsequently, multiple enlarged IGCs dissociate from chromatin and fuse into a single structure. The dissociation requires caspase activity and involves caspase-activated DNase (CAD). Apoptotic IMR-5 cells, lacking a proper processing of CAD, show multiple enlarged IGCs that remain linked with chromatin. Overexpression of CAD in IMR-5 cells results in the dissociation of IGCs from chromatin, but the fusion into a single structure remains disturbed. Nuclear matrix protein NuMA is reorganized in a caspase-dependent way around fused IGCs. In conclusion, we show here that the apoptotic rearrangement of IGCs, the nuclear matrix and chromatin are closely associated, occur in defined stages and depend on the activity of protein phosphatases, caspases and CAD.

On emerging nuclear order.

Although the nonrandom nature of interphase chromosome arrangement is widely accepted, how nuclear organization relates to genomic function remains unclear. Nuclear subcompartments may play a role by offering rich microenvironments that regulate chromatin state and ensure optimal transcriptional efficiency. Technological advances now provide genome-wide and four-dimensional analyses, permitting global characterizations of nuclear order. These approaches will help uncover how seemingly separate nuclear processes may be coupled and aid in the effort to understand the role of nuclear organization in development and disease.

A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase.

Centrosomes are closely associated with the nuclear envelope (NE) throughout the cell cycle and this association is maintained in prophase when they separate to establish the future mitotic spindle. At this stage, the kinetochore constituents CENP-F, NudE, NudEL, dynein, and dynactin accumulate at the NE. We demonstrate here that the N-terminal domain of the nuclear pore complex (NPC) protein Nup133, although largely dispensable for NPC assembly, is required for efficient anchoring of the dynein/dynactin complex to the NE in prophase. Nup133 exerts this function through an interaction network via CENP-F and NudE/EL. We show that this molecular chain is critical for maintaining centrosome association with the NE at mitotic entry and contributes to this process without interfering with the previously described RanBP2-BICD2-dependent pathway of centrosome anchoring. Finally, our study reveals that tethering of centrosomes to the nuclear surface at the G2/M transition contributes, along with other cellular mechanisms, to early stages of bipolar spindle assembly.

ATP-dependent and independent functions of Rad54 in genome maintenance.

Rad54, a member of the SWI/SNF protein family of DNA-dependent ATPases, repairs DNA double-strand breaks (DSBs) through homologous recombination. Here we demonstrate that Rad54 is required for the timely accumulation of the homologous recombination proteins Rad51 and Brca2 at DSBs. Because replication protein A and Nbs1 accumulation is not affected by Rad54 depletion, Rad54 is downstream of DSB resection. Rad54-mediated Rad51 accumulation does not require Rad54's ATPase activity. Thus, our experiments demonstrate that SWI/SNF proteins may have functions independent of their ATPase activity. However, quantitative real-time analysis of Rad54 focus formation indicates that Rad54's ATPase activity is required for the disassociation of Rad54 from DNA and Rad54 turnover at DSBs. Although the non-DNA-bound fraction of Rad54 reversibly interacts with a focus, independent of its ATPase status, the DNA-bound fraction is immobilized in the absence of ATP hydrolysis by Rad54. Finally, we show that ATP hydrolysis by Rad54 is required for the redistribution of DSB repair sites within the nucleus.

The nucleus introduced.

Now is an opportune moment to address the confluence of cell biological form and function that is the nucleus. Its arrival is especially timely because the recognition that the nucleus is extremely dynamic has now been solidly established as a paradigm shift over the past two decades, and also because we now see on the horizon numerous ways in which organization itself, including gene location and possibly self-organizing bodies, underlies nuclear functions.

Chromatin movement visualized with photoactivable GFP-labeled histone H4.

The cell nucleus is highly organized with chromosomes occupying discrete, partially overlapping territories, and proteins that localize to specific nuclear compartments. This spatial organization of the nucleus is considered to be dynamic in response to environmental and cellular conditions to support changes in transcriptional programs. Chromatin, however, is relatively immobile when analyzed in living cells and shows a constrained Brownian type of movement. A possible explanation for this relative immobility is that chromatin interacts with a nuclear matrix structure and/or with nuclear compartments. Here, we explore the use of photoactivatable GFP fused to histone H4 as a potential tool to analyze the mobility of chromatin at various nuclear compartments. Selective photoactivation of photoactivatable-GFP at defined nuclear regions was achieved by two-photon excitation with 820 nm light. Nuclear speckles, which are considered storage sites of splicing factors, were visualized by coexpression of a fluorescent protein fused to splicing factor SF2/ASF. The results reveal a constrained chromatin motion, which is not affected by transcriptional inhibition, and suggests an intimate interaction of chromatin with speckles.

Tentative mapping of transcription-induced interchromosomal interaction using chimeric EST and mRNA data.

Recent studies on chromosome conformation show that chromosomes colocalize in the nucleus, bringing together active genes in transcription factories. This spatial proximity of actively transcribing genes could provide a means for RNA interaction at the transcript level. We have screened public databases for chimeric EST and mRNA sequences with the intent of mapping transcription-induced interchromosomal interactions. We suggest that chimeric transcripts may be the result of close encounters of active genes, either as functional products or "noise" in the transcription process, and that they could be used as probes for chromosome interactions. We have found a total of 5,614 chimeric ESTs and 587 chimeric mRNAs that meet our selection criteria. Due to their higher quality, the mRNA findings are of particular interest and we hope that they may serve as food for thought for specialists in diverse areas of molecular biology.

Artificially introduced aneuploid chromosomes assume a conserved position in colon cancer cells.

BACKGROUND: Chromosomal aneuploidy is a defining feature of carcinomas. For instance, in colon cancer, an additional copy of Chromosome 7 is not only observed in early pre-malignant polyps, but is faithfully maintained throughout progression to metastasis. These copy number changes show a positive correlation with average transcript levels of resident genes. An independent line of research has also established that specific chromosomes occupy a well conserved 3D position within the interphase nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated whether cancer-specific aneuploid chromosomes assume a 3D-position similar to that of its endogenous homologues, which would suggest a possible correlation with transcriptional activity. Using 3D-FISH and confocal laser scanning microscopy, we show that Chromosomes 7, 18, or 19 introduced via microcell-mediated chromosome transfer into the parental diploid colon cancer cell line DLD-1 maintain their conserved position in the interphase nucleus. CONCLUSIONS: Our data is therefore consistent with the model that each chromosome has an associated zip code (possibly gene density) that determines its nuclear localization. Whether the nuclear localization determines or is determined by the transcriptional activity of resident genes has yet to be ascertained.

Role of mitogen-activated protein kinase kinase in Porphyromonas gingivalis-induced myocardial cell hypertrophy and apoptosis.

Secreted factors present in the medium following growth of the periodontal pathogen Porphyromonas gingivalis cause increased cardiomyocyte hypertrophy and apoptosis, whereas secreted factors from Actinobacillus actinomycetemcomitans and Prevotella intermedia have no such effects. The purpose of this study was to clarify the role of mitogen-activated protein kinase (MAPK)/extracellular-regulated protein kinase (ERK) pathways in P. gingivalis medium-induced H9c2 myocardial cell hypertrophy and apoptosis. Cellular morphology, DNA fragmentation, nuclear condensation, total mitogen-activated protein kinase/extracellular-regulated protein kinase-1 (ERK-1), total ERK-1 protein, and phosphorylated ERK-1 protein products in cultured H9c2 myocardial cells were measured by actin immunofluorescence, agarose gel electrophoresis, nuclear condensation, and western blotting following stimulation with P. gingivalis spent growth medium or pre-administration of U0126, a potent MEK-1/2 inhibitor. Components of P. gingivalis spent culture medium not only resulted in increased total MEK-1 and ERK-1 protein products, but also caused increased cellular size, DNA fragmentation, and nuclear condensation in H9c2 cells. These three parameters, and the phosphorylated ERK-1 protein products of H9c2 cells treated with P. gingivalis medium, were all significantly reduced after pre-administration of U0126. The results suggest that P. gingivalis-secreted factors may initiate MEK/ERK signal pathways and lead to myocardial cell hypertrophy and apoptosis.

Subnuclear organelles: new insights into form and function.

The cell nucleus is a complex and highly dynamic environment with many functionally specialized regions of substructure that form and maintain themselves in the absence of membranes. Relatively little is known about the basic physical properties of the nuclear interior or how domains within the nucleus are structurally and functionally organized and interrelated. Here, we summarize recent data that shed light on the structural and functional properties of three prominent subnuclear organelles--nucleoli, Cajal bodies (CBs) and speckles. We discuss how these findings impact our understanding of the guiding principles of nuclear organization and various types of human disease.

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.

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.

snRNPs are present in the karyosome capsule in the weevil germinal vesicle.

Within the oocyte nucleus of the apple blossom weevil, Anthonomus pomorum (Insecta, Coleoptera) highly condensed and transcriptionaly inactive chromosomes form the karyosome. During its formation, within the nucleoplasm numerous, variably sized spherical inclusions termed nuclear bodies occur. As oogenesis progresses, the karyosome is gradually surrounded by a prominent sheath, the karyosome capsule. The function and molecular composition of both the nuclear bodies and the karyosome capsule are largely unknown. Using cytochemical methods we demonstrate that DNA is confined to the karyosome and there is no extrachromosomal DNA accumulations within the nucleoplasm. In addition, none of the oocyte nucleus subdomains contain argyrophilic proteins. Our immunoEM study revealed that in contrast to similar structures in germinal vesicles in other insect species, the nuclear bodies of A. pomorum do not cross-react with antibodies recognising small nuclear ribonucleoproteins, coilin or the splicing factor SC-35. Unexpectedly, we found that as the karyosome capsule develops, mature small nuclear RNAs and proteins containing the Sm epitope associate with the capsule material. We suggest that the karyosome capsule is a storage site for small nuclear ribonucleoprotein particles, which may be used during early embryonic development.

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.

Identification and characterization of two novel proteins affecting fission yeast gamma-tubulin complex function.

The gamma-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe. To better understand its roles, we have purified the S. pombe gamma-tubulin complex. Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to gamma-tubulin-associated proteins from other organisms. We show that both Mbo1p and Gfh1p localize to microtubule organizing centers. Although cells deleted for either mbo1(+) or gfh1(+) are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification. In addition, mbo1Delta and gfh1Delta cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function. This study expands the known roles of gamma-tubulin complex components in organizing different types of microtubule structures in S. pombe.

Human topoisomerase IIalpha: targeting to subchromosomal sites of activity during interphase and mitosis.

Mammalian topoisomerase IIalpha (topo IIalpha) plays a vital role in the removal of topological complexities left on DNA during S phase. Here, we developed a new assay to selectively identify sites of catalytic activity of topo IIalpha with subcellular resolution. We show that topo IIalpha activity concentrates at replicating heterochromatin in late S in a replication-dependent manner and at centric heterochromatin during G2 and M phases. Inhibitor studies indicate that this cell cycle-dependent concentration over heterochromatin is sensitive to chromatin structure. We further show that catalytically active topo IIalpha concentrates along the longitudinal axis of mitotic chromosomes. Finally, we found that catalytically inert forms of the enzyme localize predominantly to splicing speckles in a dynamic manner and that this pool is differentially sensitive to changes in the activities of topo IIalpha itself and RNA polymerase II. Together, our data implicate several previously unsuspected activities in the partitioning of the enzyme between sites of activity and putative depots.