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Author(s): Martin Falk [sup.1] , Yana Feodorova [sup.2] [sup.3] , Natalia Naumova [sup.4] [sup.5] , Maxim Imakaev [sup.1] , Bryan R. Lajoie [sup.4] [sup.6] , Heinrich Leonhardt [sup.3] , Boris [...], The nucleus of mammalian cells displays a distinct spatial segregation of active euchromatic and inactive heterochromatic regions of the genome.sup.1,2. In conventional nuclei, microscopy shows that euchromatin is localized in the nuclear interior and heterochromatin at the nuclear periphery.sup.1,2. Genome-wide chromosome conformation capture (Hi-C) analyses show this segregation as a plaid pattern of contact enrichment within euchromatin and heterochromatin compartments.sup.3, and depletion between them. Many mechanisms for the formation of compartments have been proposed, such as attraction of heterochromatin to the nuclear lamina.sup.2,4, preferential attraction of similar chromatin to each other.sup.1,4-12, higher levels of chromatin mobility in active chromatin.sup.13-15 and transcription-related clustering of euchromatin.sup.16,17. However, these hypotheses have remained inconclusive, owing to the difficulty of disentangling intra-chromatin and chromatin-lamina interactions in conventional nuclei.sup.18. The marked reorganization of interphase chromosomes in the inverted nuclei of rods in nocturnal mammals.sup.19,20 provides an opportunity to elucidate the mechanisms that underlie spatial compartmentalization. Here we combine Hi-C analysis of inverted rod nuclei with microscopy and polymer simulations. We find that attractions between heterochromatic regions are crucial for establishing both compartmentalization and the concentric shells of pericentromeric heterochromatin, facultative heterochromatin and euchromatin in the inverted nucleus. When interactions between heterochromatin and the lamina are added, the same model recreates the conventional nuclear organization. In addition, our models allow us to rule out mechanisms of compartmentalization that involve strong euchromatin interactions. Together, our experiments and modelling suggest that attractions between heterochromatic regions are essential for the phase separation of the active and inactive genome in inverted and conventional nuclei, whereas interactions of the chromatin with the lamina are necessary to build the conventional architecture from these segregated phases. Attractions between heterochromatic regions are essential for phase separation of the active and inactive genome in inverted and conventional nuclei, whereas chromatin-lamina interactions are necessary to build the conventional genomic architecture from these segregated phases.

The parameters that control nuclear size and shape are poorly understood. In yeast, unregulated membrane proliferation, caused by deletion of the phospholipid biosynthesis inhibitor SPO7, leads to a single nuclear envelope 'flare' that protrudes into the cytoplasm. This flare is always associated with the asymmetrically localized nucleolus, which suggests that the site of membrane expansion is spatially confined by an unknown mechanism. Here we show that in spo7[DELTA] cells, mutations in vesicle-trafficking genes lead to multiple flares around the entire nucleus. These mutations also alter the distribution of small nucleolar RNA-associated nucleolar proteins independently of their effect on nuclear shape. Both single- and multi-flared nuclei have increased nuclear envelope surface area, yet they maintain the same nuclear/cell volume ratio as wild-type cells. These data suggest that, upon membrane expansion, the spatial confinement of the single nuclear flare is dependent on vesicle trafficking. Moreover, flares may facilitate maintenance of a constant nuclear/cell volume ratio in the face of altered membrane proliferation. doi/ 10.1083/jcb.201006083

Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Set847. Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phospborylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction. hypotbalamo-neurohypophyseal system; central prolactin; hyperprolactinemia; nitric oxide doi: 10.1152/ajpregu.00575.2010

Correct assembly of nuclear pore complexes (NPCs), which directly and indirectly control nuclear environment and architecture, is vital to genomic regulation. We previously found that nucleoporin 153 (Nup153) is required for timely progression through late mitosis. In this study, we report that disruption of Nup153 function by either small interfering RNA--mediated depletion or expression of a dominant-interfering Nup153 fragment results in dramatic mistargeting of the pore basket components Tpr and Nup50 in midbody-stage cells. We find a concomitant appearance of aberrantly localized active Aurora B and an Aurora B--dependent delay in abscission. Depletion of Nup50 is also sufficient to increase the number of midbody-stage cells and, likewise, triggers distinctive mislocalization of Aurora B. Together, our results suggest that defects in nuclear pore assembly, and specifically the basket structure, at this time of the cell cycle activate an Aurora B--mediated abscission checkpoint, thereby ensuring that daughter cells are generated only when fully formed NPCs are present. doi/ 10.1083/jcb.201007124

Many mammalian genes localize near nuclear speckles, nuclear bodies enriched in ribonucleic acid-processing factors. In this paper, we dissect cis-elements required for nuclear speckle association of the heat shock protein 70 (Hsp70) locus. We show that speckle association is a general property of Hsp70 bacterial artificial chromosome transgenes, independent of the chromosome integration site, and can be recapitulated using a 2.8-kilobase HSPA1A gene fragment. Association of Hsp70 transgenes and their transcripts with nuclear speckles is transcription dependent, independent of the transcribed sequence identity, but dependent on the Hsp70 promoter sequence. Transgene speckle association does not correlate with the amount of transcript accumulation, with large transgene arrays driven by different promoters showing no speckle association, but smaller Hsp70 transgene arrays with lower transcript accumulation showing high speckle association. Moreover, despite similar levels of transcript accumulation, Hsp70 transgene speckle association is observed after heat shock but not cadmium treatment. We suggest that certain promoters may direct specific chromatin and/or transcript ribonucleoprotein modifications, leading to nuclear speckle association. doi/ 10.1083/jcb.201004041

Nuclear pore complexes (NPCs) control the movement of molecules across the nuclear envelope (NE). We investigated the molecular interactions that exist at the interface between the NPC scaffold and the pore membrane. We show that key players mediating these interactions in mammalian cells are the nucleoporins Nup155 and Nup160. Nup155 depletion massively alters NE structure, causing a dramatic decrease in NPC numbers and the improper targeting of membrane proteins to the inner nuclear membrane. The role of Nup155 in assembly is likely closely linked to events at the membrane as we show that Nup155 interacts with pore membrane proteins Pom121 and NDC1. Furthermore, we demonstrate that the N terminus of Pom121 directly binds the [beta]-propeller regions of Nup155 and Nup160. We propose a model in which the interactions of Pom121 with Nup155 and Nup160 are predicted to assist in the formation of the nuclear pore and the anchoring of the NPC to the pore membrane. doi/ 10.1083/jcb.201007098

Kinesin-1 and dynein are recruited to the nuclear envelope by the Caenorhabditis elegans klarsicht/ ANC-1/Syne homology (KASH) protein UNC-83 to move nuclei. The mechanisms of how these motors are coordinated to mediate nuclear migration are unknown. Time-lapse differential interference contrast and fluorescence imaging of embryonic hypodermal nuclear migration events were used to characterize the kinetics of nuclear migration and determine microtubule dynamics and polarity. Wild-type nuclei display bidirectional movements during migration and are also able to roll past cytoplasmic granules, unc-83, unc-84, and kinesin-1 mutants have severe nuclear migration defects. Without dynein, nuclear migration initiates normally but lacks bidirectional movement and shows defects in nuclear rolling, implicating dynein in resolution of cytoplasmic roadblocks. Microtubules are highly dynamic during nuclear migration. EB1::green fluorescence protein imaging demonstrates that microtubules are polarized in the direction of nuclear migration. This organization of microtubules fits with our model that kinesin-1 moves nuclei forward and dynein functions to move nuclei backward for short stretches to bypass cellular roadblocks. doi/ 10.1083/jcb.201004118

In metazoa, new nuclear pore complexes (NPCs) form at two different cell cycle stages: at the end of mitosis concomitant with the reformation of the nuclear envelope and during interphase. However, the mechanisms of these assembly processes may differ. In this study, we apply high resolution live cell microscopy to analyze the dynamics of single NPCs in living mammalian cells during interphase. We show that nuclear growth and NPC assembly are correlated and occur at a constant rate throughout interphase. By analyzing the kinetics of individual NPC assembly events, we demonstrate that they are initiated by slow accumulation of the membrane nucleoporin Pom121 followed by the more rapid association of the soluble NPC subcomplex Nup107-160. This inverse order of recruitment and the overall much slower kinetics compared with postmitotic NPC assembly support the conclusion that the two processes occur by distinct molecular mechanisms. doi/ 10.1083/jcb.201007076

The mitochondrial genome of basal animals is generally more slowly evolving than that of bilaterians. This difference in rate complicates the study of relationships among members of these lineages and the discovery of cryptic species or the testing of morphological species concepts within them. We explore the properties of mitochondrial and nuclear ribosomal genes in the cnidarian order Actiniaria, using both an ordinal- and familial-scale sample of taxa. Although the markers do not show significant incongruence, they differ in their phylogenetic informativeness and the kinds of relationships they resolve. Among the markers studied here, the fragments of 12S rDNA and 18S rDNA most effectively recover well-supported nodes; those of 16S rDNA and 28S rDNA are less effective. The general patterns we observed are similar to those in other hexacorallians, although Actiniaria alone show saturation of transitions for ordinal-scale analyses. doi: 10.1093/icb/icq081

A cold-sensitive [gamma]-tubulin allele of Aspergillus nidulans, mipAD159, causes defects in mitotic and cell cycle regulation at restrictive temperatures that are apparently independent of microtubule nucleation defects. Time-lapse microscopy of fluorescently tagged mitotic regulatory proteins reveals that cyclin B, cyclin-dependent kinase 1, and the Ancdc14 phosphatase fail to accumulate in a subset of nuclei at restrictive temperatures. These nuclei are permanently removed from the cell cycle, whereas other nuclei, in the same multinucleate cell, cycle normally, accumulating and degrading these proteins. After each mitosis, additional daughter nuclei fail to accumulate these proteins, resulting in an increase in noncycling nuclei over time and consequent inhibition of growth. Extensive analyses reveal that these noncycling nuclei result from a nuclear autonomous, microtubule-independent failure of inactivation of the anaphase-promoting complex/cyclosome. Thus, [gamma]-tubulin functions to regulate this key mitotic and cell cycle regulatory complex. doi/ 10.1083/jcb.201002105

Most viruses move intracellularly to and from their sites of replication using microtubule-based mechanisms. In this study, we show that nucleocapsids of the baculovirus Autographa californica multiple nucleopolyhedrovirus undergo intracellular motility driven by actin polymerization. Motility requires the viral P78/83 capsid protein and the host Arp2/3 complex. Surprisingly, the virus directs two sequential and coordinated phases of actin-based motility. Immediately after cell entry, motility enables exploration of the cytoplasm and collision with the nuclear periphery, speeding nuclear entry and the initiation of viral gene expression. Nuclear entry itself requires transit through nuclear pore complexes. Later, after the onset of early gene expression, motility is required for accumulation of a subpopulation of nucleocapsids in the tips of actin-rich surface spikes. Temporal coordination of actin-based nuclear and surface translocation likely enables rapid transmission to neighboring cells during infection in insects and represents a distinctive evolutionary strategy for overcoming host defenses. doi/ 10.1083/jcb.201001162

All transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). Despite their enormous size, ~60 MD in vertebrates, they are comprised of only ~30 distinct proteins (nucleoporins or Nups), many of which form subcomplexes that act as building blocks for NPC assembly. One of these evolutionarily conserved subcomplexes, the Nup93 complex, is a major structural component linking the NPC to the membranes of the NE. Using in vitro nuclear assembly assays, we show that two components of the Nup93 complex, Nup188 and Nup205, are dispensable for NPC formation. However, nuclei lacking Nup188 increase in size by several fold compared with wild type. We demonstrate that this phenotype is caused by an accelerated translocation of integral membrane proteins through NPCs, suggesting that Nup188 confines the passage of membrane proteins and is thus crucial for the homeostasis of the different nuclear membranes. www.jcb.org/cgi/doi/10.1083/jcb.200912045

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3. www.jcb.org/cgi/doi/10.1083/jcb.201001124

Introduction Nuclear gene expression in eukaryotes is orchestrated by 3 RNA polymerases (RNAPs) that synthesize RNA molecules from a DNA template. RNAP I generates most ribosomal RNAs (rRNA), RNAP II [...], More than 30 years of research on nuclear RNA polymerases (RNAP I, II, and III) has uncovered numerous factors that regulate the activity of these enzymes during the transcription reaction. However, very little is known about the machinery that regulates the fate of RNAPs before or after transcription. In particular, the mechanisms of biogenesis of the 3 nuclear RNAPs, which comprise both common and specific subunits, remains mostly uncharacterized and the proteins involved are yet to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by nuclear RNAP subunits from the soluble fraction of human cell extracts. Validation of the dataset using a machine learning approach trained to minimize the rate of false positives and false negatives yielded a high-confidence dataset and uncovered novel interactors that regulate the RNAP II transcription machinery, including a set of proteins we named the RNAP II-associated proteins (RPAPs). One of the RPAPs, RPAP3, is part of an 11-subunit complex we termed the RPAP3/R2TP/prefoldin-like complex. Here, we review the literature on the subunits of this complex, which points to a role in nuclear RNAP biogenesis. Key words: RNA polymerase, biogenesis, protein affinity purification, RNA polymerase II-associated proteins (RPAPs), transcription. Plus de 30 annees de recherche sur les ARN polymerases nucleaires (ARNP I, II et III) ont revele l'existence de plusieurs facteurs qui regulent l'activite de ces enzymes durant la transcription. Cependant, on sait peu de choses de la machinerie qui regule le sort des ARNP avant ou apres la transcription. En particulier, les mecanismes de biogenese des trois ARNP nucleaires, qui comportent des sous-unites communes et specifiques, demeurent largement peu caracterises et les proteines impliquees restent encore a decouvrir. A l'aide d'un protocole de purification de proteines par affinite couple a la spectroscopie de masse, nous avons recemment identifie un reseau d'interactions a haute densite forme par les sousunite s des ARNP nucleaires a partir de la fraction soluble d'extraits de cellules humaines. La validation de ces donnees par une approche d'apprentissage automatique concu pour minimiser le taux de faux positifs et de faux negatifs a permis de generer un ensemble de donnees a haut degre de confiance et de reveler de nouveaux interacteurs qui regulent la machinerie de transcription par l'ARNP II, notamment un ensemble de proteines associees a l'ARNP appelees RPAP (<< RNAP II-associated proteins >>). Une de ces RPAP, la RPAP3, fait partie d'un complexe de 11 sous-unites appele complexe de type RPAP3-R2TP/prefoldine. Nous faisons ici une revue de la litterature traitant des sous-unites constituant ce complexe, qui s'oriente vers un role dans la biogenese des ARNP nucleaires. Mots-cles: ARN polymerase, biogenese, purification de proteines par affinite, proteines associees a l'ARNP II (RPAP), transcription. [Traduit par la Redaction]

Although the role of liganded nuclear receptors in mediating coactivator/corepressor exchange is well-established, little is known about the potential regulation of chromosomal organization in the 3-dimensional space of the nucleus in achieving integrated transcriptional responses to diverse signaling events. Here, we report that ligand induces rapid interchromosomal interactions among specific subsets of estrogen receptor [alpha]-bound transcription units, with a dramatic reorganization of nuclear territories, which depends on the actions of nuclear actin/myosin-I machinery and dynein light chain 1. The histone lysine demethylase, LSD1, is required for these ligand-induced interactive loci to associate with distinct interchromatin granules, long thought to serve as 'storage' sites for the splicing machinery, some critical transcription elongation factors, and various chromatin remodeling complexes. We demonstrate that this 2-step nuclear rearrangement is essential for achieving enhanced, coordinated transcription of nuclear receptor target genes. enhancement of gene expression | nuclear architecture | chromosomal movement | interchromosomal interactions | SC35 domains

The functions and the importance of the nucleolus present in the germinal vesicle are discussed. The maternal nucleolus is shown to be extremely important for life and early embryonic development of the mammals.

We have previously shown that ionotropic glutamate receptors in the caudal portion of the nucleus tractus solitarii (NTS), especially in the commissural NTS, play a prominent role in the mediation of tracheobronchial cough and that substance P potentiates this reflex. This NTS region could be a site of action of some centrally acting antitussive agents and a component of a drug-sensitive gating mechanism of cough. To address these issues, we investigated changes in baseline respiratory activity and cough responses to tracheobronchial mechanical stimulation following microinjections (30-50 nl) of centrally acting antitussive drugs into the caudal NTS of pentobarbitone-anesthetized, spontaneously breathing rabbits. [D-[Ala.sup.2],N-Me-[Phe.sup.4],[Gly.sup.5]-ol]-en-kephalin (DAMGO) and baclofen decreased baseline respiratory frequency because of increases in the inspiratory time only at the higher concentration employed (5 mM and 1 mM, respectively). DAMGO (0.5 mM) and baclofen (0.1 mM) significantly decreased cough number, peak abdominal activity, peak tracheal pressure, and increased cough-related total cycle duration. At the higher concentrations, these agents suppressed the cough reflex. The effects of these two drugs were counteracted by specific antagonists (10 mM naloxone and 25 mM CGP-35348, respectively). The neurokinin-1 ([NK.sub.1]) receptor antagonist CP-99,994 (10 mM) abolished cough responses, whereas the [NK.sub.2] receptor antagonist MEN 10376 (5 mM) had no effect. The results indicate that the caudal NTS is a site of action of some centrally acting drugs and a likely component of a neural system involved in cough regulation. A crucial role of substance P release in the mediation of reflex cough is also suggested. [micro]-opioid receptors; [GABA.sub.B] receptors; neurokinin receptors; control of breathing; airway defensive reflexes

Redistribution of blood from the viscera to the peripheral vasculature is the major cardiovascular response designed to restore thermoregulatory homeostasis after an elevation in body core temperature. In this study, we investigated the role of the hypothalamic paraventricular nucleus (PVN) in the reflex decrease in renal blood flow that is induced by hyperthermia, as this brain region is known to play a key role in renal function and may contribute to the central pathways underlying thermoregulatory responses. In anesthetized rats, blood pressure, heart rate, renal blood flow, and tail skin temperature were recorded in response to elevating body core temperature. In the control group, saline was microinjected bilaterally into the PVN; in the second group, muscimol (1 nmol in 100 nl per side) was microinjected to inhibit neuronal activity in the PVN; and in a third group, muscimol was microinjected outside the PVN. Compared with control, microinjection of muscimol into the PVN did not significantly affect the blood pressure or heart rate responses. However, the normal reflex reduction in renal blood flow observed in response to hyperthermia in the control group (~70% from a resting level of 11.5 ml/min) was abolished by the microinjection of muscimol into the PVN (maximum reduction of 8% from a resting of 9.1 ml/min). This effect was specific to the PVN since microinjection of muscimol outside the PVN did not prevent the normal renal blood flow response. The data suggest that the PVN plays an essential role in the reflex decrease in renal blood flow elicited by hyperthermia. hyperthermia

The ability to form associations between predictive environmental events and rewarding outcomes is a fundamental aspect of learned behavior. This apparently simple ability likely requires complex neural processing evolved to [...]

Specific proteins in the nucleus act as transcription factors upon activation through binding of small molecules (all-trans-retinoic acid, thyroid hormone, vitamin D, and others). The activated (liganded) receptors bind to specific DNA elements as heterodimers, each in combination with the retinoic acid-X receptor (RXR). 9-Cis-retinoic acid binds to RXR with high affinity and activates it. Though 9-cis-retinoic acid was initially found in animal tissues, in later work 9-cis-retinoic acid could not be detected. A search for a ligand for RXR in tissues showed that unsaturated fatty acids, particularly linoleic, linolenic, and doco-sahexaenoic acids, bound to and activated RXR as specific ligands, although with low affinity. A critical experiment demonstrated that, at least in developing mouse skin, 9-cis-retinoic acid is not the ligand for RXR. Key words: all-trans-refinoic acid, 9-cis-retinoic acid, docosahexaenoic acid, fatty acids, heterodimers, linoleic acid, linolenic acid, oleic acid, retinoic acid-X receptor doi: 10.1301/nr.2006.dec.532-538

Mechanical function of the intervertebral disc is maintained through the interaction between the hydrated nucleus pulposus, the surrounding annulus fibrosus, and the superior and inferior endplates. In disc degeneration the normal transfer of load between disc substructures is compromised. The objective of this study was to explore the mechanical role of the nucleus pulposus in support of axial compressive loads over time. This was achieved by measuring the elastic slow ramp and viscoelastic stress-relaxation mechanical behaviors of cadaveric sheep motion segments before and after partial nucleotomy through the endplate (keeping the annulus fibrosus intact). Mechanics were evaluated at five conditions: Intact, intact after 10,000 cycles of compression, acutely after nucleotomy, following nucleotomy and 10,000 cycles of compression, and following unloaded recovery. Radiographs and magnetic resonance images were obtained to examine structure. Only the short time constant of the stress relaxation was altered due to nucleotomy. In contrast, cyclic loading resulted in significant and large changes to both the stiffness and stress relaxation behaviors. Moreover, the nucleotomy had little to no effect on the disc mechanics after cyclic loading, as there were no significant differences comparing mechanics after cyclic loading with or without the nucleotomy. Following unloaded recovery the mechanical changes that had occurred as a consequence of cyclic loading were restored, leaving only a sustained change in the short time constant due to the trans-endplate nucleotomy. Thus the swelling and redistribution of the remaining nucleus pulposus was not able to fully restore mechanical behaviors. This study reveals insights into the role of the nucleus pulposus in disc function, and provides new information toward the potential role of altered nucleus pulpous function in the degenerative cascade. [DOI: 10.1115/1.2354210] Keywords: intervertebral disc, nucleus pulposus, endplate, stress relaxation, cyclic loading

Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI/SNF family of ATPases/helicases, and it possesses a [C.sub.3]H[C.sub.4] RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein, which, together with Mms2-Ubc13, promotes replication through DNA lesions via an error-free postreplicational repair pathway. Genetic evidence in yeast has indicated a role for Rad5 as a ubiquitin ligase in mediating the Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Here we show that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6-Rad18 and Mms2-Ubc13 complexes, and we show that SHPRH protein is a ubiquitin ligase indispensable for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raises the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans. postreplication repair | translesion synthesis | tumor suppressor

It is important to observe and study cancer cells' cycle progression in order to better understand drug effects on cancer cells. Time-lapse microscopy imaging serves as an important method to measure the cycle progression of individual cells in a large population. Since manual analysis is unreasonably time consuming for the large volumes of time-lapse image data, automated image analysis is proposed. Existing approaches dealing with time-lapse image data are rather limited and often give inaccurate analysis results, especially in segmenting and tracking individual cells in a cell population. In this paper, we present a new approach to segment and track cell nuclei in time-lapse fluorescence image sequence. First, we propose a novel marker-controlled watershed based on mathematical morphology, which can effectively segment clustered cells with less oversegmentation. To further segment undersegmented cells or to merge oversegmented cells, context information among neighboring frames is employed, which is proved to be an effective strategy. Then, we design a tracking method based on modified mean shift algorithm, in which several kernels with adaptive scale, shape, and direction are designed. Finally, we combine mean-shift and Kalman filter to achieve a more robust cell nuclei tracking method than existing ones. Experimental results show that our method can obtain 98.8% segmentation accuracy, 97.4% cell division tracking accuracy, and 97.6% cell tracking accuracy. Index Terms--Cell segmentation, cell tracking, Kalman filter, mean shift, watershed.

Cytosolic accumulation of beta-catenin in response to a Wnt signal is a crucial signaling event, but it is only an intermediate step in the transduction of the signal into the nucleus, to its ultimate destinations, the Wnt target genes. It is observed that many of the central cytosolic Wnt regulators including APC, Axin, Dsh, and the Wnt regulatory protein kinases also localize to the nucleus, where they may, directly or indirectly, interact with beta-catenin and modulate its transcriptional activity.

Stimulation of cardiac mechanoreceptors during volume expansion elicits reflex compensatory changes in sympathetic nerve activity (SNA). The hypothalamic paraventricular nucleus (PVN) and nucleus of the tractus solitarius (NTS) are autonomic regions known to contribute to this reflex. Both of these nuclei project to the rostral ventrolateral medulla (RVLM), critical in the tonic generation of SNA. Recent reports from our laboratory show that these pathways 1) are activated following cardiac mechanoreceptor stimulation, and 2) produce nitric oxide, known to influence SNA. The aims of the present study were to determine whether 1) the activated neurons within the PVN and NTS were nitrergic and 2) these neurons projected to the RVLM. Animals were prepared, under general anesthesia, by microinjection of a retro-gradely transported tracer into the pressor region of the RVLM and the placement of a balloon at the right venoatrial junction. In conscious rats, the balloon was inflated to stimulate the cardiac mechanoreceptors or was left uninflated. Balloon inflation elicited a significant increase in Fos-positive neurons in the parvocellular PVN (sevenfold) and NTS (fivefold). In the PVN, 51% of nitrergic neurons and 61% of RVLM-projecting nitrergic neurons were activated. In the NTS, these proportions were 8 and 18%, respectively. The data suggest that nitrergic neurons within the PVN and, to a lesser extent, in the NTS, some of which project to the RVLM, may contribute to the central pathways influencing SNA elicited by cardiac mechano-receptor stimulation. hypothalamic paraventricular nucleus; nucleus tractus solitarius; cardiac mechanoreceptors; Fos; nitric oxide synthase

The anterior interpositus nucleus (AIN) is the proposed site of memory formation of eyeblink conditioning. A large part of the underlying molecular events, however, remain unknown. To elucidate the molecular mechanisms, we examined transcriptional changes in the AIN of mice trained with delay eyeblink conditioning using microarray, quantitative real-time RT-PCR, and in situ hybridization techniques. Microarray analyses suggested that transcriptionally up-regulated gene sets were largely different between early (3-d training) and late (7-d) stages. Quantitative real-time RT-PCR aided by laser microdissection indicated that the expression of representative EARLY genes (Sgk, IkBa, and Plekhf1) peaked at 1-d training in both the paired and unpaired conditioning groups, and was maintained at a higher level in the paired group than in the unpaired group after 3-d training. In situ hybridization revealed increased expression of these genes in broad cerebellar areas, including the AIN, with no hemispheric preferences. In contrast, the expression of representative LATE genes (Vamp1, Camk2d, and Prkcd) was selectively increased in the AIN of the 7-d paired group, with dominance in the ipsilateral AIN. Increased Vamp1 mRNA expression was restricted to the ipsilateral dorsolateral hump, a subregion of the AIN. These expression patterns of two distinct subsets of genes fit well with the two-stage learning theory, which proposes emotional and motor learning phases, and support the notion that AIN has a crucial role in memory formation of eyeblink conditioning. gene expression | interpositus nucleus

G-protein-coupled receptors (GPCRs) comprise a wide family of monomeric heptahelical glycoproteins that recognize a broad array of extracellular mediators including cationic amines, lipids, peptides, proteins, and sensory agents. Thus far, [...]

Cytosolic [Ca.sup.2+] is a versatile secondary messenger that regulates a wide range of cellular activities. In the past decade, evidence has accumulated that free [Ca.sup.2+] within the nucleus also plays [...]

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 [...]

The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm of interphase eukaryotic cells, and it mediates all trafficking between these 2 cellular compartments. As [...]

Over 4 decades ago, microelectrode studies of in situ nuclei showed that, under certain conditions, the nuclear envelope (NE) behaves as a barrier opposing the nucleocytoplasmic flow of physiological ions. [...]

Exercise training (ExTr) has been associated with alterations in neural control of the circulation, including effects on arterial baroreflex function. The nucleus tractus solitarius (NTS) is the primary termination site of cardiovascular afferents and critical in the regulation of baroreflex-mediated changes in heart rate (HR) and sympathetic nervous system outflow. The purpose of the present study was to determine whether ExTr is associated with alterations in neurotransmitter regulation of neurons involved in control of cardiovascular function at the level of the NTS. We hypothesized that ExTr would increase glutamatergic and reduce GABAergic transmission in the NTS and that, collectively, these changes would result in a greater overall sympathoinhibitory drive from the NTS in ExTr animals. To test these hypotheses, male Sprague-Dawley rats were treadmill trained or maintained under sedentary conditions for 8-10 wk. NTS microinjections were performed in Inactin-anesthetized animals instrumented to record mean arterial pressure (MAP), HR, and lumbar sympathetic nerve activity (LSNA). Generalized activation of the NTS with unilateral microinjections of glutamate (1-10 mM, 30 nl) produced dose-dependent decreases in MAP, HR, and LSNA that were unaffected by ExTr. Bilateral inhibition of NTS with the GAB[A.sub.A] agonist muscimol (1 mM, 90 nl) produced increases in MAP and LSNA that were blunted by ExTr. In contrast, pressor and sympathoexcitatory responses to bilateral microinjections of the ionotropic glutamate receptor antagonist, kynurenate (40 mM, 90 nl), were similar between groups. Bradycardic responses to bilateral microinjections of the GAB[A.sub.A] antagonist bicuculline (0.1 mM, 90 nl) were attenuated by ExTr. These data indicate that alterations in neurotransmission at the level of the NTS contribute importantly to regulation of HR and LSNA in ExTr animals. In addition to alterations at NTS, these experiments suggest indirectly that changes in other cardiovascular nuclei contribute to the observed alterations in neural control of the circulation following ExTr. microinjection; sympathetic nerve activity; nucleus tractus solitarius; treadmill

The organization of genes within the nucleus may influence transcription. We have analyzed the nuclear positioning of the coordinately regulated [alpha]- and [beta]-globin genes and show that the gene-dense chromatin surrounding the human [alpha]-globin genes is frequently decondensed, independent of transcription. Against this background, we show the frequent juxtaposition of active [alpha]- and [beta]-globin genes and of homologous [alpha]-globin loci that occurs at nuclear speckles and correlates with transcription. However, we did not see increased colocalization of signals, which would be expected with direct physical interaction. The same degree of proximity does not occur between human [beta]-globin genes or between murine globin genes, which are more constrained to their chromosome territories. Our findings suggest that the distribution of globin genes within erythroblast nuclei is the result of a self-organizing process, involving transcriptional status, diffusional ability of chromatin, and physical interactions with nuclear proteins, rather than a directed form of higher-order control.

Nuclear pore complexes (NPCs) govern macromolecular transport between the nucleus and cytoplasm and serve as key positional markers within the nucleus. Several protein components of yeast NPCs have been implicated in the epigenetic control of gene expression. Among these, Nup2p is unique as it transiently associates with NPCs and, when artificially tethered to DNA, can prevent the spread of transcriptional activation or repression between flanking genes, a function termed boundary activity. To understand this function of Nup2p, we investigated the interactions of Nup2p with other proteins and with DNA using immuno-purifications coupled with mass spectrometry and micraarray analyses. These data combined with functional assays of boundary activity and epigenetic variegation suggest that Nup2p and the Ran guanylyl-nucleotide exchange factor, Prp20p, interact at specific chromatin regions and enable the NPC to play an active role in chromatin organization by facilitating the transition of chromatin between activity states.

[beta]-Catenin is the nuclear effector of the Wnt signaling cascade. The mechanism by which nuclear activity of [beta]-catenin is regulated is not well defined Therefore, we used the nuclear marker RanGTP to screen for novel nuclear [beta]-catenin binding proteins. We identified a cofactor of chromosome region maintenance 1 (CRM1)-mediated nuclear export, Ran binding protein 3 (RanBP3), as a novel [beta]-catenin-interacting protein that binds directly to [beta]-catenin in a RanGTP-stimulated manner. RanBP3 inhibits [beta]-catenin-mediated transcriptional activation in both Wnt1- and [beta]-catenin-stimulated human cells. In Xenopus laevis embryos, RanBP3 interferes with [beta]-catenin-induced dorsoventral axis formation. Furthermore, RanBP3 depletion stimulates the Wnt pathway in both human cells and Drosophila melanogaster embryos. In human cells, this is accompanied by an increase of dephosphorylated [beta]-catenin in the nucleus. Conversely, overexpression of RanBP3 leads to a shift of active [beta]-catenin toward the cytoplasm. Modulation of [beta]-catenin activity and localization by RanBP3 is independent of adenomatous polyposis coli protein and CRM1. We conclude that RanBP3 is a direct export enhancer for [beta]-catenin, independent of its role as a CRM1-associated nuclear export cofactor.

Nod is a chromokinesin-like protein that plays a critical role in segregating achiasmate chromosomes during female meiosis. The C-terminal half of the Nod protein contains two putative DNA-binding domains. The first of these domains, known as the HMGN domain, consists of three tandemly repeated high-mobility group N motifs. This domain was previously shown to be both necessary and sufficient for binding of the C-terminal half of Nod to mitotic chromosomes in embryos. The second putative DNA-binding domain, denoted HhH(2)/NDD, is a helix-hairpin-helix(2)/Nod-like DNA-binding domain. Although the HhH(2)/NDD domain is not required or sufficient for chromosome binding in embryos, several well-characterized nod mutations have been mapped in this domain. To characterize the role of the HhH(2)/NDD domain in mediating Nod function, we created a series of UAS-driven transgene constructs capable of expressing either a Mid-type Nod-GFP fusion protein or proteins in which the HhH(2)/NDD domain had been altered by site-directed mutagenesis. Although wild-type Nod-GFP localizes to the oocyte chromosomes and rescues the segregation defect in nod mutant oocytes, two of three proteins carrying mutants in the HhH(2)/NDD domain fail to either rescue the nod mutant phenotype or bind to oocyte chromosomes. However, these mutant proteins do bind to the polytene chromosomes in nurse-cell nuclei and enter the oocyte nucleus. Thus, even though the HhH(2)/NDD domain is not essential for chromosome binding in other cell types, it is required for chromosome binding in the oocyte. These HhH(2)/NDD mutants also block the localization of Nod to the posterior pole of stage 9-10A oocytes, a process that is thought to facilitate the interaction of Nod with the plus ends of microtubules (Cgi et al. 2005). This observation suggests that the Nod HhH2/NDD domain may play other roles in addition to binding Nod to meiotic chromosomes.

The mechanism of transport of mRNA-protein (mRNP) complexes from transcription sites to nuclear pores has been the subject of many studies. Using molecular beacons to track single mRNA molecules in living cells, we have characterized the diffusion of mRNP complexes in the nucleus. The mRNP complexes move freely by Brownian diffusion at a rate that assures their dispersion throughout the nucleus before they exit into the cytoplasm, even when the transcription site is located near the nuclear periphery. The diffusion of mRNP complexes is restricted to the extranucleolar, interchromatin spaces. When mRNP complexes wander into dense chromatin, they tend to become stalled. Although the movement of mRNP complexes occurs without the expenditure of metabolic energy, ATP is required for the complexes to resume their motion after they become stalled. This finding provides an explanation for a number of observations in which mRNA transport appeared to be an enzymatically facilitated process. gene expression | live cell imaging | mRNA export | nuclear viscosity

We find that the cell response spectra of lateral geniculate nucleus cells, as well as the reflectance spectra of Munsell color chips, may be modeled by using the cone sensitivity functions of the long and medium cones. We propose a simple model for how the neural signals from the photoreceptors might be combined in the retina to closely approximate the reflectance spectra of Munsell color chips without input from the short cone. vision | color perception

Among replication mutations that destabilize CAG repeat tracts, mutations of RAD27, encoding the flap endonuclease, and CDC9, encoding DNA ligase I, increase the incidence of repeat tract expansions to the greatest extent. Both enzymes bind to proliferating cell nuclear antigen (PCNA). To understand whether weakening their interactions leads to CAG repeat tract expansions, we have employed alleles named rad27-p and cdc9-p that have orthologous alterations in their respective PCNA interaction peptide (PIP) box. Also, we employed the PCNA allele po130-90, which has changes within its hydrophobic pocket that interact with the PIP box. All three alleles destabilize a long CAG repeat tract and yield more tract contractions than expansions. Combining rad27-p with cdc9-p increases the expansion frequency above the sum of the numbers recorded in the individual mutants. A similar additive increase in tract expansions occurs in the rad27-p pol30-90 double mutant but not in the cdc9-p pol30-90 double mutant. The frequency of contractions rises in all three double mutants to nearly the same extent. These results suggest that PCNA mediates the entry of the flap endonuclease and DNA ligase I into the process of Okazaki fragment joining, and this ordered entry is necessary to prevent CAG repeat tract expansions.

In previous work, we showed that telomeres of normal cells are organized within the 3D space of the interphase nucleus in a nonoverlapping and cell cycle-dependent manner. This order is distorted in tumor cell nuclei where telomeres are found in close association forming aggregates of various numbers and sizes. Here we show that c-Myc overexpression induces telomeric aggregations in the interphase nucleus. Directly proportional to the duration of c-Myc deregulation, we observe three or five cycles of telomeric aggregate formation in interphase nuclei. These cycles reflect the onset and propagation of breakage-bridge-fusion cycles that are initiated by end-to-end telomeric fusions of chromosomes. Subsequent to initial chromosomal breakages, new fusions follow and the breakage-bridge-fusion cycles continue. During this time, nonreciprocal translocations are generated, c-Myc-dependent remodeling of the organization of telomeres thus precedes the onset of genomic instability and subsequently leads to chromosomal rearrangements. Our findings reveal that c-Myc possesses the ability to structurally modify chromosomes through telomeric fusions, thereby reorganizing the genetic information. genomic instability | 3D nucleus | breakage-bridge-fusion

An overview of some of the emerging concepts in the study of in vivo genome organization and function is described. The complete clarification of the contribution that nuclear architecture makes to gene expression will be needed to understand physiological processes such as differentiation, development and disease at cellular level.

The nucleolus dynamically alters its shape through the assembly and disassembly of a variety of nucleolar components in proliferating cells. While the nucleolus is known to function in vital cellular events, little is known about how its components are correctly assembled. Through the analysis of a Drosophila mutant that exhibits a reduced number of mushroom body (MB) neurons in the brain, we reveal that the slender lobes (sle) gene encodes a novel nuclear protein that affects nucleolar organization during development. In sle mutant neuroblasts, the nucleolus was packed more tightly, forming a dense sphere, and the nucleolar proteins fibrillarin and Nop60B were abnormally distributed in the interphase nucleolus. Moreover, another nucleolar marker, Aj1 antigen, was localized to the center of the nucleolus in a manner complementary to the Nop60B distribution, and also formed a large aggregate in the cytoplasm. While developmental defects were limited to a few tissues in sle mutants, including MBs and nurse cells, the altered organization of the nucleolar components were evident in most developing tissues. Therefore, we conclude that Sle is a general factor of nuclear architecture in Drosophila that is required for the correct organization of the nucleolus during development. Keywords: Nucleolus; Nucleus; Mushroom body; Cell proliferation; Neuroblast; Drosophila

Spectrin repeat sequences are among the more common repeat elements identified in proteins, typically occurring in large structural proteins. An overview of work regarding various spectrin repeat-containing structural proteins in the nucleus is presented.

The synthesis of two new taxoid derivatives containing the baccatin [14,1-d] dehydrofuran-2-one and -furan-2-one nucleus respectively is described. Esterification of 13-enol derivative and 13-hydroxy compound with N, O-protected norstatine followed by deprotection gave new promising anticancer taxanes.

Byline: E. A. Avetisyan (1), F. A. Adamyan (1), A. A. Petrosyan (1) Keywords: vagus nerve; solitary tract nucleus; vagosensitive neurons; septal nuclei Abstract: Studies on cats anesthetized with a mixture of Nembutal and chloralose were performed to study the descending influences of single, paired, and frequent stimulation of the lateral septal nucleus (LSN) and bed nucleus of the stria terminalis (BNST) on the activity of viscerosensory neurons in the solitary tract nucleus, identified by stimulation of the cervical part of the vagus nerve. Of 70 units recorded in the solitary tract nucleus, 50 were identified as primary and secondary input vagus neurons. Single stimuli applied to the septal nuclei induced initial excitation in 30% (15 units) of vagus neurons. The latent period of these responses was 5--25 msec. Presentation of paired stimuli showed that loss of the ability to respond to the second stimulus occurred at interstimulus intervals of 10--200 and sometimes 300 msec. A total of 34% (17 units) of solitary tract nucleus neurons showed tonic changes in spontaneous activity in response to rhythmic stimulation. Increasing the stimulation frequency to 10--20 Hz led to very different changes in the spontaneous rhythm, i.e., wave-like changes (decreases and increases in frequency, secondary suppression) or complete inhibition, sometimes lasting up to 10 sec after stimulation ended. A small number of units (five) showed a blocking effect of septal discharges on the visceral afferent input in conditions of paired stimulation. These results lead to the conclusion that the LSN and BNST are involved in modulating the activity of bulbar viscerosensory neurons, though their influences are mediated mainly via oligo- and polysynaptic pathways via other limbic structures (hypothalamus, amygdala). Author Affiliation: (1) Laboratory for Autonomic Nervous System Physiology, L. A. Orbeli Institute of Physiology, National Academy of Sciences, 22 Orbeli Street, 375028, Erevan, Armenia Article History: Registration Date: 18/10/2004

The attempts to expand the genetic code that progressed to nucleus containing cells are discussed. The result showed that the transformed yeast cells make an experimental elevation of code complexity possible in mammals.

In stimulated cells, the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) concentrates in the nucleus. Evidence exists for CRM1-dependent, mitogen-activated protein kinase kinase-mediated nuclear export of ERK2, but its mechanism of nuclear entry is not understood. To determine requirements for nuclear transport, we tagged ERK2 with green fluorescent protein (GFP) and examined its nuclear uptake by using an in vitro import assay. GFP-ERK2 entered the nucleus in a saturable, time- and temperature-dependent manner. Entry of GFP-ERK2, like that of ERK2, required neither energy nor transport factors and was visible within minutes. The nuclear uptake of GFP-ERK2 was inhibited by wheat germ agglutinin, which blocks nuclear entry by binding to carbohydrate moieties on nuclear pore complex proteins. The nuclear uptake of GFP-ERK2 also was reduced by excess amounts of recombinant transport factors. These findings suggest that ERK2 competes with transport factors for binding to nucleoporins, which mediate the entry and exit of transport factors. In support of this hypothesis, we showed that ERK2 binds directly to a purified nucleoporin. Our data suggest that GFP-ERK2 enters the nucleus by a saturable, facilitated mechanism, distinct from a carrier- and energy-dependent import mechanism and involves a direct interaction with nuclear pore complex proteins. mitogen-activated protein kinase | import | FXFG motif | nucleoporins

The organization of transcriptionally active ribosomal genes in animal cell nucleoli is investigated in this study in order to address the long-standing controversy with regard to the intranucleolar localization of these genes. Detailed analyses of HeLa cell nucleoli include direct localization of ribosomal genes by in situ hybridization and their indirect localization via nascent ribosomal transcript mappings. On the light microscopy (LM) level, ribosomal genes map in 10-40 fluorescence foci per nucleus, and transcription activity is associated with most foci. We demonstrate that each nucleolar focus observed by LM corresponds, on the EM level, to an individual fibrillar center (FC) and surrounding dense fibrillar components (DFCs). The EM data identify the DFC as the nucleolar subcompartment in which rRNA synthesis takes place, consistent with detection of rDNA within the DFC. The highly sensitive method for mapping nascent transcripts in permeabilized cells on ultrastructural level provides intense and unambiguous clustered immunogold signal over the DFC, whereas very little to no label is detected over the FC. This signal is strongly indicative of nascent 'Christmas trees' of rRNA associated with individual rDNA genes, sampled on the surface of thin sections. Stereological analysis of the clustered transcription signal further suggests that these Christmas trees may be contorted in space and exhibit a DNA compaction ratio on the order of 4-5.5.