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Triple negative breast cancer (TNBC) has poor prognosis and neither established biomarkers nor therapeutic targets. On the one hand the androgen receptor (AR), a steroid hormone receptor (SR) which is expressed in 10-53% of TNBC and proved to be critical for BC proliferation, has been proposed as a new target in TNBC. On the other hand, we and others have shown that membrane ErbB-2 migrates to the nucleus (nuclear ErbB-2, NErbB-2) where it binds DNA at HER-2 associated sequences (HAS) to regulate BC proliferation and migration. Since we have previously shown a functional interplay between growth factors and SR signaling pathways in BC, we propose the existence of an interaction between AR and ErbB-2 which is involved in NErbB-2+/AR+ BC growth. The experimental model used was the human TNBC cell line MDA-MB-453 which displays high expression levels of AR and NErbB-2. By Western Blot (WB) we found that dihydrotestosterone (DHT) treatment for short times (minutes) did not regulate ErbB-2 phosphorylation status at residues Tyr1221/1222 and 1248 which were constitutively activated. However, DHT led to an increase in ErbB-2 phosphorylation at residue Tyr877 which we have proved to be required for ErbB-2 nuclear migration. The latter effect was blocked by the AR antagonist enzalutamide (enza). Blockage of Src activity with dasatinib inhibited DHT-induced ErbB-2 phosphorylation at Tyr877. By Immunofluorescence and confocal microscopy analyses and subcellular fractionation studies we demonstrated that DHT induced ErbB-2 nuclear migration which was inhibited by enza. By chIP we found that DHT induced ErbB-2 recruitment to a HAS site in ERK5, a gene involved in BC proliferation, and to a HAS site in FKBP5, a classical AR responsive gene. By WB we demonstrated that transfection with an ErbB-2 mutant which is unable to translocate to the nucleus and functions as a dominant negative inhibitor of ErbB-2 nuclear migration (hErbB-2ΔNLS), inhibited FKBP51 up-regulation by DHT. Finally, by microarray and bioinformatics analysis we identified 315 differentially expressed genes (DEGs) in the presence of DHT and NErbB-2 eviction. Enrichment analyses showed that the DEGs belonged to the immune response and interferon pathways. Kaplan-Meier analysis revealed that the expression of 6 genes was significantly associated with overall survival in TNBC patients from the METABRIC cohort: CXCL10, TAP1, STAT1, NMI, HLA-A and NLRC5. Multivariate Cox regression analysis identified the combined expression of the 6 genes as an independent predictor of better clinical outcome in TNBC (HR: 0.56, 95% CI 0.38-0.82, P = 0.003). In conclusion, our findings evidence that DHT-activated AR induces Src-mediated ErbB-2 rapid activation and its migration to the nucleus where it binds to HAS sites in the DNA. Moreover, based on the DEGs of NErbB-2 eviction in presence of DHT we identified a gene signature associated with favorable outcome in TNBC.

The organization of interphase chromosomes in chromosome territories (CTs) was first proposed more than one hundred years ago. The introduction of increasingly sophisticated microscopic and molecular techniques, now provide complementary strategies for studying CTs in greater depth than ever before. Here we provide an overview of these strategies and how they are being used to elucidate CT interactions and the role of these dynamically regulated, nuclear-structure building blocks in directly supporting nuclear function in a physiologically responsive manner.

Interleukin-33 (IL-33) is the most attractive novel cytokine identified as an IL-1family member. IL-33 was first named NF-HEV (nuclear factor from high endothelial venules), as itwas known to interact with nuclear chromatin although its exact intracellular functions are still tobe clarified. IL-33 is now recognized as the specific ligand for the orphan IL-1 receptor familymember ST2 and to be involved in polarization of T cells towards T helper 2-cell phenotype and inactivation of mast cells, basophils, eosinophils and natural killer cells. It is essential for IL-33 to beextracellularly released in order to bind to the ST2 receptor and consequently play a crucial role ininflammatory, infectious and autoimmune diseases. However, like the IL-1 family members, IL-1beta and IL-18, IL-33 mRNA is translated without a signal sequence for secretion. Additionally, IL-33 cannot be released by the processing and secretion mechanism shared by IL-1beta and IL-18as IL-33 is not a substrate of caspase-1 and does not require proteolysis for activation. In contrast,IL-33 can be inactivated by apoptotic caspases. Accordingly, IL-33 is proposed to be released asan alarmin from necrotic cells but deleted during apoptosis. Besides the known autocrine,paracrine mechanisms of cellular interaction with cytokines, release by necrotic cells is anotherpathway for a cytokine to display its function, which we suggest might be called 'necrocrine'.

Insulator bodies are novel nuclear stress foci that can be used as a proxy to monitor the chromatin-bound state of insulator proteins., Chromatin insulators assist in the formation of higher-order chromatin structures by mediating long-range contacts between distant genomic sites. It has been suggested that insulators accomplish this task by forming dense nuclear foci termed insulator bodies that result from the coalescence of multiple protein-bound insulators. However, these structures remain poorly understood, particularly the mechanisms triggering body formation and their role in nuclear function. In this paper, we show that insulator proteins undergo a dramatic and dynamic spatial reorganization into insulator bodies during osmostress and cell death in a high osmolarity glycerol–p38 mitogen-activated protein kinase–independent manner, leading to a large reduction in DNA-bound insulator proteins that rapidly repopulate chromatin as the bodies disassemble upon return to isotonicity. These bodies occupy distinct nuclear territories and contain a defined structural arrangement of insulator proteins. Our findings suggest insulator bodies are novel nuclear stress foci that can be used as a proxy to monitor the chromatin-bound state of insulator proteins and provide new insights into the effects of osmostress on nuclear and genome organization.

Take a look at a textbook illustration of a cell and you will immediately be able to locate the nucleus, which is often drawn as a spherical or ovoid shaped structure. But not all cells have such nuclei. In fact, some disease states are diagnosed by the presence of nuclei that have an abnormal shape or size. What defines nuclear shape and nuclear size, and how does nuclear geometry affect nuclear function? While the answer to the latter question remains largely unknown, significant progress has been made towards understanding the former. In this review, we provide an overview of the factors and forces that affect nuclear shape and size, discuss the relationship between ER structure and nuclear morphology, and speculate on the possible connection between nuclear size and its shape. We also note the many interesting questions that remain to be explored.

We recently reported on a new wrinkle of complexity in how eukaryotic genes are regulated by providing evidence for a hitherto unknown nuclear function of the signaling kinase, Protein Kinase C-theta (PKC-θ). This chromatin-anchored complex positively regulates inducible immune genes and negatively regulates target miRNA genes. These data challenge the traditional view of mammalian signaling kinases and provides new avenues for therapeutic drug design.

Gender differences in cardiovascular mortality are well documented and oestrogen replacement therapy in post-menopausal women is associated with improved outcomes from cardiovascular events. Indeed, oestrogen therapy has been shown to restore endothelial function in post-menopausal women and reduce the development of atherosclerotic lesions. Despite extensive evidence on the beneficial effect of oestrogen, relatively little is known regarding the molecular signalling mechanism(s) by which oestrogen exerts some of its vascular effects. While the nuclear function of the oestrogen receptor is clearly established, previous studies regarding the membrane and cytoplasmic actions of oestrogen remain inconclusive. Cross-coupling of the oestrogen receptor to phosphoinositide 3-kinase signalling suggests a potentially critical non-nuclear action of the oestrogen receptor and considerably broadens our understanding of the cellular effects of oestrogen.

The meeting ‘The Dynamic Nucleus: Questions and Implications’ took place at Imperial College, London, UK, between June 27 and 29, 2002. It was sponsored by the MRC Clinical Sciences Centre in London and was organized by Niall Dillon and Ana Pombo. ![][1] Over the course of two and a half days, hypotheses and results about the organization and function of the eukaryotic nucleus were discussed intensely in the congenial atmosphere of this meeting. The topics covered ranged from DNA repair to nuclear actin, from nucleolar assembly to the deposition of histone variants, and from technical aspects of fluorescence signal separation to the analysis of chromatin dynamics in living cells. Many nuclear domains have previously been identified (Fig. 1) and recent advances in live‐cell imaging techniques have opened the door for real‐time analyses of the diverse range of nuclear functions associated with these structures. Nearly every major nuclear process was discussed, yet the most novel ideas dealt with the dynamics of nuclear structures and events. New ways to monitor dynamics, paired with the fact that nuclear organization now attracts experts from a wide range of fields, left participants uniformly excited about the potential offered by the marriage of live fluorescence imaging and molecular genetics. A few highlights of the work presented at the meeting follow. Figure 1. Diagram of a mammalian cell nucleus showing many of the nuclear domains that have been identified so far. Although each of these domains can be easily observed by light or electron microscopy approaches, remarkably, a large number of their protein constitutents are highly dynamic. For a detailed overview of these compartments see Spector (2001). ### Rapid diffusion of nuclear factors A large number of studies that monitor fluorescence recovery kinetics of green fluorescent protein (GFP) fusion proteins after photobleaching (FRAP; see Fig. 2) have taught us that nuclear factors are highly dynamic, … [1]: /embed/graphic-1.gif

Posttranscriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify transcriptome-wide ∼26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in mRNA processing. Upon HuR knockdown, mRNA levels and protein synthesis of thousands of target genes were downregulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knockdown triggered strong and specific upregulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing.

Production of estradiol-17β and cAMP was assayed during maturation of the follicle-enclosed oocytes of Xenopus laevis and Rana temporaria stimulated by pituitary suspension or hCG, correspondingly. In some samples, the nuclear function was suppressed by pretreatment with AD. The level of estradiol-17β in the oocytes of the both species was very low and was not affected by hormonal or AD treatment, while the level of cAMP (follicle + medium) increased. When maturation was stimulated by low concentrations of gonadotropic hormones inducing complete or near 100% maturation, the cAMP level increased 1.4- to 3.9-fold. Both higher concentrations of the gonadotropic hormones and pretreatment of the follicles with AD increased the cAMP level still more. Combination of these treatments led to a greater increase of cAMP and to inhibition of oocyte maturation. We conclude that cAMP, rather than estradiol-17β, is involved in the AD-induced inhibition of amphibian oocyte maturation stimulated by gonadotropins. © 1995 Wiley-Liss, Inc.

The microscope has been indispensable to the last century of chromatin structure research. Microscopy techniques have revealed that the three-dimensional location of chromatin is not random but represents a further manifestation of a highly compartmentalized cell nucleus. Moreover, the structure and location of genetic loci display cell type-specific differences and relate directly to the state of differentiation. Advances to bridge imaging with genetic, molecular and biochemical approaches have greatly enhanced our understanding of the interdependence of chromatin structure and nuclear function in mammalian cells. In this review we discuss the current state of chromatin structure research in relationship to the variety of microscopy techniques that have contributed to this field.

In the past 20 years cell biologists have studied the cell nucleus extensively, aided by advances in cell imaging technology and microscopy. Consequently, the volume of image data of the cell nucleus – and the compartments it contains – is growing rapidly. The spatial organisation of these nuclear compartments is thought to be fundamentally associated with nuclear function. However, the rules that oversee nuclear architecture remain unclear and controversial. As a result, there is an increasing need to replace qualitative visual assessment of microscope images with quantitative and automated methods. Such tools can substantially reduce manual labour and more importantly remove subjective bias. Quantitative methods can also increase the accuracy, sensitivity and reproducibility of data analysis. In this paper, we describe image processing and analysis methodology for the investigation of nuclear architecture, and the application of these methods to quantitatively explore the promyelocytic leukaemia (PML) nuclear bodies (NBs). PML NBs are linked with numerous nuclear functions including transcription and protein degradation. However, we know very little about the three-dimensional (3-D) architecture of PML NBs in relation to each other or within the general volume of the nucleus. Finally, we emphasise methods for the analysis of replicate images (of a given nuclear compartment across different cell nuclei) in order to aggregate information about nuclear architecture.

The interphase nucleus is an active organelle involved in processing genetic information. In higher order eukaryotes, information control is compartmentalized - for example at the scale of inter-chromosome territories and nuclear bodies. Regulatory proteins, nuclear bodies and chromatin assembly are found to be highly dynamic within the nucleus of primary cells and through cellular differentiation programs. In this chapter we describe live-cell fluorescence based techniques and single particle tracking analysis, to probe the spatio-temporal dimension in nuclear function.

Our view of the structure and function of the interphase nucleus has changed drastically in recent years. It is now widely accepted that the nucleus is a well organized and highly compartmentalized organelle and that this organization is intimately related to nuclear function. In this context, chromatin-initially considered a randomly entangled polymer-has also been shown to be structurally organized in interphase and its organization was found to be very important to gene regulation. Relevant and not completely answered questions are how chromatin organization is achieved and what mechanisms are responsible for changes in the positions of chromatin loci in the nucleus. A significant advance in the field resulted from tagging chromosome sites with bacterial operator sequences, and visualizing these tags using green fluorescent protein fused with the appropriate repressor protein. Simultaneously, fluorescence imaging techniques evolved significantly during recent years, allowing observation of the time evolution of processes in living specimens. In this context, the motion of the tagged locus was observed and analyzed to extract quantitative information regarding its dynamics. This review focuses on recent advances in our understanding of chromatin dynamics in interphase with the emphasis placed on the information obtained from single-particle tracking (SPT) experiments. We introduce the basis of SPT methods and trajectory analysis, and summarize what has been learnt by using this new technology in the context of chromatin dynamics. Finally, we briefly describe a method of SPT in a two-photon excitation microscope that has several advantages over methods based on conventional microscopy and review the information obtained using this novel approach to study chromatin dynamics. © 2008 Springer. Fil: Levi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física. Laboratorio de Electrónica Cuántica; Argentina Fil: Gratton, Enrico. University of California at Irvine; Estados Unidos

Mutations in the C. elegans maternal-effect gene clk-1 are highly pleiotropic, affecting the duration of diverse developmental and behavioral processes. They result in an average slowing of embryonic and post-embryonic development, adult rhythmic behaviors, reproduction, and aging.(1) CLK-1 is a highly conserved mitochondrial protein,(2,3) but even severe clk-1 mutations affect mitochondrial respiration only slightly.(3) Here, we review the evidence supporting the regulatory role of clk-1 in physiological timing. We also discuss possible models for the action of CLK-1, in particular, one proposing that CLK-1 is involved in the coordination of mitochondrial and nuclear function. BioEssays 22:48-56, 2000.

A perusal of experimental work on the neurobiology of brain aging shows that several investigations have been carried out on the theoretical bias that the aging process is quantitative in its nature, thus consisting in the progressive generalized weakening of biochemical (metabolic and specific) activities in the neuron, and leading to the impoverishment of brain functions which are typical of the species. In view of the perennity of the neuron, the factors underlying the aging process have been singled out in the deterioration of the nuclear function, especially with regard to protein synthesis, in reduced resistance to and increased formation of toxic wastes, mostly oxygen radicals, and consequent irreversible damage to enzymes, plasma membrane components, etc. Environmental impact on aging is believed to be exerted through these factors. The homogeneous character of these changes has recently been questioned, as in contrast to the ascertained prevailing selectivity of the changes for brain regions and cell types.1 Furthermore, the above interpretative frame of the nature and causation of the brain aging process disagrees with several experimental findings, notably the following.

Within a nucleus the eukaryotic genome is probably arranged in a complex but highly-ordered fashion. How this organization relates to nuclear function is, at present, a topic of interest.

This discussion of the biochemical lesions produced in animal cells by ionizing radiation will to all intents and purposes be restricted to a consideration of the immediate response to doses of 1000 rads or less in two rat tissues only, namely the thymus gland and the regenerating liver. The experiments we have carried out with these two tissues have led us to the view that in these organs the ultimate damage to the cell stems from an initial upset of the nuclear thiol-disulphide equilibrium.

The frequency of nuclear pores/μ 2 in isolated nuclei from ischemic rat kidneys decreased by half after 20 min of occlusion of the blood supply to the kidney. The decrease in pore number remained constant through 120 min ischemia. Particles in the concave fracture face of the outer nuclear membrane changed from a random distribution in control nuclei to a reticular pattern after 20 min ischemia, becoming more sparse and clustered through 120 min ischemia. Deterioration of the pore structure also was noted after 120 min ischemia. It is suggested that these changes are related to loss of nuclear function.

In eukaryotic cells, chromatin function varies temporally and spatially with cell cycle and differentiation during development. One way a cell can control its chromatin function is thought to be by changing the configuration of chromatin. Post-translational modifications of chromosomal proteins have been proposed to be important in determining such conformational changes in chromatin structure. Since a minor but significant fraction of chromosomal proteins are reversibly poly(ADP-ribosyl)ated (1, 2) and the modification causes structural changes in chromatin (3, 4), it is assumed that the turnover of poly(ADP-ribose) on proteins has some biological role which is essential to expression of a nuclear function. Although a number of studies have shown that poly(ADP-ribosyl)ation may be involved in DNA repair and replication, gene expression, cell cycling, and differentiation, the physiological function of this modification has not yet been established (5).

Acanthamoeba sp. were cut into nucleated and enucleated portions and exposed to either 14C uracil, 14C orotic acid, 14C adenine or a mixture of all three under aseptic conditions in a complete nutrient medium. Following 3 days of exposure to the radioactive medium, the nucleates and enucleates were autoradiographed. Nucleated cells grow, divide, and incorporate large amounts of all three precursors into RNA. Enucleated cells decrease slightly in size over the 3-day period and fail completely to incorporate radioactivity into RNA. Enucleated cells survive in the nutrient medium for at least several weeks. The data are taken as evidence that RNA synthesis in Acanthamoeba is an exclusively nuclear function.

In the 2-week-old hamster, intranuclear whorls were observed in cells located among the spermatids. The whorls, measuring up to 2.5 μ in diameter, were composed of sheets of microtubules. The distance between the individual sheets was 150 A, the tubular lumen was 80 A, and the tubular wall measured 120 A. It is assumed that the presence of intranuclear whorls indicates changes in nuclear function.

This year's volume of theAnnual Review of Physiologycontains some chapters which continue subjects from last year's volume, namely reproduction; kidneys, water, and electrolytes; respiration; heart; peripheral circulation; alimentary tract; nerve excitation and synaptic transmission; visceral, somatic, and higher functions of the nervous system; and the adenohypophysis and adrenal cortex. Topics not represented in last year's volume are nuclear function and nuclear-cytoplasmic interactions, physiological aspects of genetics, developmental physiology, fuel of muscle metabolism, blood pigments, blood volume regulation, and physiology of vision. Last year's chapter on the pancreas, insulin, and glucagon is followed this year by a chapter on the thyroid. The amount of research work reviewed in these annual volumes is staggering, and the authors of the reviews deserve recognition for undertaking the labor of organizing the mass of new information and making it accessible to their colleagues. Fortunately the modern machinery for handling bibliographic data has facilitated