RESUMEN

Cell fate determination by lateral inhibition via Notch/Delta signalling has been extensively studied. Most formalised models consider Notch/Delta interactions in fields of cells, with parameters that typically lead to symmetry breaking of signalling states between neighbouring cells, commonly resulting in salt-and-pepper fate patterns. Here, we consider the case of signalling between isolated cell pairs, and find that the bifurcation properties of a standard mathematical model of lateral inhibition can lead to stable symmetric signalling states. We apply this model to the adult intestinal stem cell (ISC) of Drosophila, the fate of which is stochastic but dependent on the Notch/Delta pathway. We observe a correlation between signalling state in cell pairs and their contact area. We interpret this behaviour in terms of the properties of our model in the presence of population variability in contact areas, which affects the effective signalling threshold of individual cells. Our results suggest that the dynamics of Notch/Delta signalling can contribute to explain stochasticity in stem cell fate decisions, and that the standard model for lateral inhibition can account for a wider range of developmental outcomes than previously considered.

Asunto(s)

Comunicación Celular , Linaje de la Célula , Drosophila melanogaster/citología , Animales , Membrana Celular/metabolismo , Sistema Digestivo/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Modelos Biológicos , Receptores Notch/metabolismo , Transducción de Señal

RESUMEN

In epithelial tissues, cells expressing oncogenic Ras (hereafter RasV12 cells) are detected by normal neighbors and as a result are often extruded from the tissue [1-6]. RasV12 cells are eliminated apically, suggesting that extrusion may be a tumor-suppressive process. Extrusion depends on E-cadherin-based cell-cell adhesions and signaling to the actin-myosin cytoskeleton [2, 6]. However, the signals underlying detection of the RasV12 cell and triggering extrusion are poorly understood. Here we identify differential EphA2 signaling as the mechanism by which RasV12 cells are detected in epithelial cell sheets. Cell-cell interactions between normal cells and RasV12 cells trigger ephrin-A-EphA2 signaling, which induces a cell repulsion response in RasV12 cells. Concomitantly, RasV12 cell contractility increases in an EphA2-dependent manner. Together, these responses drive the separation of RasV12 cells from normal cells. In the absence of ephrin-A-EphA2 signals, RasV12 cells integrate with normal cells and adopt a pro-invasive morphology. We also show that Drosophila Eph (DEph) is detected in segregating clones of RasV12 cells and is functionally required to drive segregation of RasV12 cells in vivo, suggesting that our in vitro findings are conserved in evolution. We propose that expression of RasV12 in single or small clusters of cells within a healthy epithelium creates ectopic EphA2 boundaries, which drive the segregation and elimination of the transformed cell from the tissue. Thus, deregulation of Eph/ephrin would allow RasV12 cells to go undetected and expand within an epithelium.

Asunto(s)

Células Epiteliales/fisiología , Receptor EphA2/metabolismo , Proteínas ras/metabolismo , Animales , Comunicación Celular/fisiología , Células Cultivadas , Regulación de la Expresión Génica , Receptor EphA2/genética , Proteínas ras/genética

RESUMEN

We have investigated how cell contractility and adhesion are functionally integrated during epithelial morphogenesis. To this end, we have analysed the role of α-Catenin, a key molecule linking E-Cadherin-based adhesion and the actomyosin cytoskeleton, during Drosophila embryonic dorsal closure, by studying a newly developed allelic series. We find that α-Catenin regulates pulsatile apical contraction in the amnioserosa, the main force-generating tissue driving closure of the embryonic epidermis. α-Catenin controls actomyosin dynamics by stabilising and promoting the formation of actomyosin foci, and also stabilises DE-Cadherin (Drosophila E-Cadherin, also known as Shotgun) at the cell membrane, suggesting that medioapical actomyosin contractility regulates junction stability. Furthermore, we uncover a genetic interaction between α-Catenin and Vinculin, and a tension-dependent recruitment of Vinculin to amniosersoa apical cell membranes, suggesting the existence of a mechano-sensitive module operating in this tissue.

Asunto(s)

Actomiosina/metabolismo , Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Polaridad Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , alfa Catenina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Adhesión Celular , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Uniones Intercelulares/metabolismo , Mutación/genética , Vinculina/metabolismo

RESUMEN

The extreme phenotypic variability of patients with Gaucher disease (GD) is not completely explained by glucocerebrosidase gene mutations. It has been proposed that genetic modifiers might influence GD phenotype. We examined seven polymorphisms of the glucosylceramide synthase gene (UGCG) and their correlation with severity of GD. Five UGCG variants were significantly associated with disease severity, according to the DS3 scoring system: c.-295C>T, c.-232_-241ins10, c.98+50A>G, c.98+68A>T, and c.861A>G. Heterozygous [N370S]+[L444P] patients with c.[-232_-241ins10;98+50G] haplotype had a significantly lower DS3 score in relation to patients carrying only one of these polymorphisms. Electrophoretic mobility shift assay analysis showed an increased nuclear protein binding ability for the G allele at the cDNA position c.98+50, as well as an altered pattern for the c.-232_-241ins10 allele. The promoter activity of the haplotypes decreased significantly with respect to wild type activity in HepG2 and COS-7 cells (-14% and -16% for the c.[-232_-241ins10;98+50A] haplotype, -44% and -25% for c.[-222nonins;98+50G] haplotypes, and -64% and -75% for c.[-232_-241ins10;98+50G] haplotype, respectively). These data indicate that the c.-232_-241ins10 and c.98+50A>G variants are modifying factors of GD severity, which can partly explain the variability in severity of the disease.

Asunto(s)

Enfermedad de Gaucher/genética , Estudios de Asociación Genética , Glucosiltransferasas/genética , Mutación , Adolescente , Adulto , Anciano , Alelos , Animales , Células COS , Niño , Preescolar , Chlorocebus aethiops , Femenino , Enfermedad de Gaucher/diagnóstico , Enfermedad de Gaucher/terapia , Expresión Génica , Genes Reporteros , Genotipo , Células Hep G2 , Humanos , Lactante , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Regiones Promotoras Genéticas , Adulto Joven

RESUMEN

The Drosophila adult posterior midgut has been identified as a powerful system in which to study mechanisms that control intestinal maintenance, in normal conditions as well as during injury or infection. Early work on this system has established a model of tissue turnover based on the asymmetric division of intestinal stem cells. From the quantitative analysis of clonal fate data, we show that tissue turnover involves the neutral competition of symmetrically dividing stem cells. This competition leads to stem-cell loss and replacement, resulting in neutral drift dynamics of the clonal population. As well as providing new insight into the mechanisms regulating tissue self-renewal, these findings establish intriguing parallels with the mammalian system, and confirm Drosophila as a useful model for studying adult intestinal maintenance.

Asunto(s)

División Celular , Drosophila melanogaster/fisiología , Homeostasis/fisiología , Intestinos/fisiología , Células Madre/fisiología , Animales , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Proliferación Celular , Drosophila melanogaster/citología , Femenino , Intestinos/citología , Células Madre/citología

RESUMEN

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 µM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.

Asunto(s)

Fabaceae/enzimología , Regulación Enzimológica de la Expresión Génica , Ligasas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Fabaceae/química , Fabaceae/genética , Regulación de la Expresión Génica de las Plantas , Inmunohistoquímica , Ligasas/química , Ligasas/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Raíces de Plantas/química , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Transporte de Proteínas

RESUMEN

Globins constitute a superfamily of proteins widespread in all kingdoms of life, where they fulfill multiple functions, such as efficient O(2) transport and modulation of nitric oxide bioactivity. In plants, the most abundant Hbs are the symbiotic leghemoglobins (Lbs) that scavenge O(2) and facilitate its diffusion to the N(2)-fixing bacteroids in nodules. The biosynthesis of Lbs during nodule formation has been studied in detail, whereas little is known about the green derivatives of Lbs generated during nodule senescence. Here we characterize modified forms of Lbs, termed Lba(m), Lbc(m), and Lbd(m), of soybean nodules. These green Lbs have identical globins to the parent red Lbs but their hemes are nitrated. By combining UV-visible, MS, NMR, and resonance Raman spectroscopies with reconstitution experiments of the apoprotein with protoheme or mesoheme, we show that the nitro group is on the 4-vinyl. In vitro nitration of Lba with excess nitrite produced several isomers of nitrated heme, one of which is identical to those found in vivo. The use of antioxidants, metal chelators, and heme ligands reveals that nitration is contingent upon the binding of nitrite to heme Fe, and that the reactive nitrogen species involved derives from nitrous acid and is most probably the nitronium cation. The identification of these green Lbs provides conclusive evidence that highly oxidizing and nitrating species are produced in nodules leading to nitrosative stress. These findings are consistent with a previous report showing that the modified Lbs are more abundant in senescing nodules and have aberrant O(2) binding.

Asunto(s)

Fabaceae/química , Hemo/química , Leghemoglobina/química , Especies de Nitrógeno Reactivo/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Espectrofotometría Ultravioleta

RESUMEN

The activity of Wnt and Notch signalling is central to many cell fate decisions during development and to the maintenance and differentiation of stem cell populations in homeostasis. While classical views refer to these pathways as independent signal transduction devices that co-operate in different systems, recent work has revealed intricate connections between their components. These observations suggest that rather than operating as two separate pathways, elements of Wnt and Notch signalling configure an integrated molecular device whose main function is to regulate transitions between cell states in development and homeostasis. Here, we propose a general framework for the structure and function of the interactions between these signalling systems that is focused on the notion of 'transition states', i.e. intermediates that arise during cell fate decision processes. These intermediates act as checkpoints in cell fate decision processes and are characterised by the mixed molecular identities of the states involved in these processes.

Asunto(s)

Receptores Notch/metabolismo , Células Madre/fisiología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Comunicación Celular , Drosophila/metabolismo , Homeostasis , Receptores Notch/genética , Proteínas Wnt/genética

RESUMEN

• Aluminum (Al) toxicity is a major limiting factor of crop production on acid soils, but the implication of oxidative stress in this process is controversial. A multidisciplinary approach was used here to address this question in the forage legume Lotus corniculatus. • Plants were treated with low Al concentrations in hydroponic culture, and physiological and biochemical parameters, together with semiquantitative metabolic and proteomic profiles, were determined. • The exposure of plants to 10 µM Al inhibited root and leaf growth, but had no effect on the production of reactive oxygen species or lipid peroxides. By contrast, exposure to 20 µM Al elicited the production of superoxide radicals, peroxide and malondialdehyde. In response to Al, there was a progressive replacement of the superoxide dismutase isoforms in the cytosol, a loss of ascorbate and consistent changes in amino acids, sugars and associated enzymes. • We conclude that oxidative stress is not a causative factor of Al toxicity. The increased contents in roots of two powerful Al chelators, malic and 2-isopropylmalic acids, together with the induction of an Al-activated malate transporter gene, strongly suggest that both organic acids are implicated in Al detoxification. The effects of Al on key proteins involved in cytoskeleton dynamics, protein turnover, transport, methylation reactions, redox control and stress responses underscore a metabolic dysfunction, which affects multiple cellular compartments, particularly in plants exposed to 20 µM Al.

Asunto(s)

Aluminio/toxicidad , Lotus/efectos de los fármacos , Lotus/metabolismo , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/metabolismo , Ácidos Carboxílicos/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas/genética , Lotus/genética , Lotus/crecimiento & desarrollo , Metabolómica , Fenómenos Fisiológicos de la Nutrición/efectos de los fármacos , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Isoformas de Proteínas/metabolismo , Proteoma/metabolismo , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética

RESUMEN

Early in the development of the imaginal wing disc of Drosophila, the LIM-HD gene tailup (islet), together with the HD genes of the iroquois complex, specify the notum territory of the disc. Later, tailup has been shown to act as a prepattern gene that antagonizes formation of sensory bristles on the notum of this fly. It has been proposed that Tailup downregulates the expression of the proneural genes achaete and scute by interfering with factors needed to activate these genes in the dorsocentral and scutellar regions of the disc. By means of a clonal analysis performed with tailup null alleles, here we show that, on the one hand, tailup is necessary to prevent formation of extra macrochaetae on most of the 11 sites where these landmark bristles arise on the fly notum. On the other hand, tailup is required to activate achaete and scute at the dorsocentral region, probably by acting as an hexameric complex with the cofactor Chip and the transcriptional activator Sspd on the dorsocentral enhancer of the achaete-scute complex. In contrast, in the scutellar region Tailup acts downstream of achaete-scute, antagonizing the proneural function of these genes probably in cooperation with Chip. We conclude that tailup acts on bristle development by several, even antagonistic, mechanisms.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Alas de Animales/embriología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Tipificación del Cuerpo/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Elementos de Facilitación Genéticos/genética , Genes de Insecto , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Factores de Transcripción/metabolismo , Alas de Animales/anatomía & histología , Alas de Animales/citología

RESUMEN

The neuron-like UR61 cell is a stable PC12 subline that contains a mouse N-ras oncogene. Dexamethasone (Dex) treatment induces a neuron-like differentiation, which is associated with neuritogenesis and nuclear expression of the glucocorticoid receptor and c-Jun. In differentiated UR61 cells, small ubiquitin-like modifiers 1 (SUMO-1) is concentrated in a new category of SUMO-1 nuclear bodies (SNBs) distinct from promyelocytic leukemia (PML) bodies by their large size and absence of PML protein. SNBs are 1 to 3 mum in diameter and exhibit a fine granular texture by electron microscopy. They are free of splicing factors and transcription foci and show spatial associations with Cajal bodies. In addition to SUMO-1 and the E2-conjugating enzyme Ubc9, which is essential for sumoylation, SNBs concentrate the transcriptional regulators CBP, CREB, and c-Jun. Moreover, transfection experiments demonstrate that SNBs accumulate the active conjugating form of SUMO-1 but not the conjugation defective variant of SUMO-1, supporting that SNBs are sites of sumoylation. Our results suggest that SNBs play a role in the control of the nucleoplasmic concentration of transcription regulators involved in neuroprotection and survival of the UR61 cells.

Asunto(s)

Proteína de Unión a CREB/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Proteína SUMO-1/aislamiento & purificación , Animales , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestructura , Células Cultivadas , Dexametasona , Regulación de la Expresión Génica , Neuronas/química , Células PC12 , Ratas , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo

RESUMEN

This paper studies the molecular organization, neuronal distribution and cellular differentiation dynamics of the giant fibrillar centers (GFCs) of nucleoli in rat sensory ganglia neurons. The GFC appeared as a round nucleolar domain (1-2 microm in diameter) partially surrounded by the dense fibrillar component and accompanied by numerous small FCs. By immunocytochemistry, the GFC concentrated the upstream binding factor, which may serve as a marker of this structure, and also contain RNA polymerase I, DNA topoisomerase I, SUMO-1 and Ubc9. However, they lack ubiquitin-proteasome conjugates and 20S proteasome. Transcription assay with 5'-fluorouridine incorporation revealed the presence of nascent RNA on the dense fibrillar component of the neuronal nucleolus, but not within the low electron-density area of the GFC. The formation of GFCs is neuronal size dependent: they were found in 58%, 30% and 0% of the large, medium and small neurons, respectively. GFCs first appeared during the postnatal period, concomitantly with a stage of neuronal growth, myelination and bioelectrical maturation. GFCs were not observed in segregated nucleoli induced by severe inhibition of RNA synthesis. We suggest that the formation of GFCs is associated with a high rate of ribosome biogenesis of the transcriptionally more active large-size neurons.

Asunto(s)

Nucléolo Celular/ultraestructura , Ganglios Sensoriales/crecimiento & desarrollo , Ganglios Sensoriales/ultraestructura , Neuronas Aferentes/ultraestructura , Proteínas del Complejo de Iniciación de Transcripción Pol1/análisis , Animales , Diferenciación Celular , Nucléolo Celular/química , Ganglios Sensoriales/metabolismo , Masculino , Microscopía Inmunoelectrónica , Neuronas Aferentes/metabolismo , Complejo de la Endopetidasa Proteasomal/análisis , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Ribosómico/análisis , ARN Ribosómico/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína SUMO-1/análisis , Proteína SUMO-1/metabolismo , Transcripción Genética , Ubiquitina/análisis , Ubiquitina/metabolismo

RESUMEN

The nucleolus is a distinct subnuclear compartment that was first observed more than 200 years ago. Nucleoli assemble around the tandemly repeated ribosomal DNA gene clusters and 28S, 18S and 5.8S ribosomal RNAs (rRNAs) are transcribed as a single precursor, which is processed and assembled with the 5S rRNA into ribosome subunits. Although the nucleolus is primarily associated with ribosome biogenesis, several lines of evidence now show that it has additional functions. Some of these functions, such as regulation of mitosis, cell-cycle progression and proliferation, many forms of stress response and biogenesis of multiple ribonucleoprotein particles, will be discussed, as will the relation of the nucleolus to human diseases.

Asunto(s)

Nucléolo Celular/metabolismo , Animales , Nucléolo Celular/química , Nucléolo Celular/genética , Nucléolo Celular/fisiología , Nucléolo Celular/ultraestructura , ADN Ribosómico/análisis , ADN Ribosómico/biosíntesis , Colorantes Fluorescentes , Humanos , Indoles , Microscopía Fluorescente , Mitosis , Modelos Biológicos , Región Organizadora del Nucléolo/fisiología , Región Organizadora del Nucléolo/ultraestructura , Precursores del ARN/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Ribosómico 28S/genética , ARN Ribosómico 28S/metabolismo , ARN Ribosómico 5.8S/genética , ARN Ribosómico 5.8S/metabolismo , ARN Ribosómico 5S/biosíntesis , ARN Ribosómico 5S/genética , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo

RESUMEN

The LIM-HD gene tailup (tup; also known as islet) has been categorised as a prepattern gene that antagonises the formation of sensory bristles on the notum of Drosophila by downregulating the expression of the proneural achaete-scute genes. Here we show that tup has an earlier function in the development of the imaginal wing disc; namely, the specification of the notum territory. Absence of tup function causes cells of this anlage to upregulate different wing-hinge genes and to lose expression of some notum genes. Consistently, these cells differentiate hinge structures or modified notum cuticle. The LIM-HD co-factors Chip and Ssdp are also necessary for notum specification. This suggests that Tup acts in this process in a complex with Chip and Ssdp. Overexpression of tup, together with araucan, a 'pronotum' gene of the iroquois complex (Iro-C), synergistically reinforces the weak capacity of either gene, when overexpressed singly, to induce ectopic notum-like development. Whereas the Iro-C genes are activated in the notum anlage by EGFR signalling, tup is positively regulated by Dpp signalling. Our data support a model in which the EGFR and Dpp signalling pathways, with their respective downstream Iro-C and tup genes, converge and cooperate to commit cells to the notum developmental fate.

Asunto(s)

Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Animales , Tipificación del Cuerpo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Alas de Animales/embriología

RESUMEN

Multiple functions have been reported for the transcription factor and candidate tumour suppressor, CTCF. Among others, they include regulation of cell growth, differentiation and apoptosis, enhancer-blocking activity and control of imprinted genes. CTCF is usually localized in the nucleus and its subcellular distribution during the cell cycle is dynamic; CTCF was found associated with mitotic chromosomes and the midbody, suggesting different roles for CTCF at different stages of the cell cycle. Here we report the nucleolar localization of CTCF in several experimental model systems. Translocation of CTCF from nucleoplasm to the nucleolus was observed after differentiation of K562 myeloid cells and induction of apoptosis in MCF7 breast cancer cells. CTCF was also found in the nucleoli in terminally differentiated rat trigeminal ganglion neurons. Thus our data show that nucleolar localization of CTCF is associated with growth arrest. Interestingly, the 180 kDa poly(ADP-ribosyl)ated isoform of CTCF was predominantly found in the nucleoli fractions. By transfecting different CTCF deletion constructs into cell lines of different origin we demonstrate that the central zinc-finger domain of CTCF is the region responsible for nucleolar targeting. Analysis of subnucleolar localization of CTCF revealed that it is distributed homogeneously in both dense fibrillar and granular components of the nucleolus, but is not associated with fibrillar centres. RNA polymerase I transcription and protein synthesis were required to sustain nucleolar localization of CTCF. Notably, the labelling of active transcription sites by in situ run-on assays demonstrated that CTCF inhibits nucleolar transcription through a poly(ADP-ribosyl)ation-dependent mechanism.

Asunto(s)

Nucléolo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Animales , Factor de Unión a CCCTC , Diferenciación Celular/fisiología , Línea Celular Tumoral , Nucléolo Celular/ultraestructura , Células Cultivadas , Proteínas de Unión al ADN/genética , Humanos , Masculino , Neuronas Aferentes/citología , Neuronas Aferentes/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Señales de Clasificación de Proteína , Transporte de Proteínas/fisiología , ARN Polimerasa I/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Ganglio del Trigémino/citología , Dedos de Zinc

RESUMEN

It is well known that the cell nucleus is organized in structural and functional compartments involved in transcription, RNA processing and protein modifications such as conjugation with SUMO-1 and proteolysis. Promyelocytic leukaemia (PML) bodies are dynamic nuclear structures that concentrate PML protein, SUMO-1 and several sumoylated and non-sumoylated protein regulators of nuclear functions. PML bodies and their associated CBP has been involved in neuronal survival. By light and electron microscopy immunocytochemistry and in situ hybridization we reported the presence, in non-pathological conditions, of a large PML-nuclear inclusion (PML-NI) in human supraoptic neurons. This inclusion appears as a single nuclear structure composed of a capsule enriched in PML, SUMO-1 and CBP proteins and a central lattice of filaments immunoreactive for class III beta-tubulin, ubiquitinated proteins and proteasomes. Furthermore, the PML-NI concentrates the SUMO-conjugating enzyme E2 (UBC9). The PML-NI may be considered a nuclear factory involved in sumoylation and proteolysis via ubiquitin-proteasome system, two nuclear pathways engaged in the control of the nucleoplasmic concentration of active transcriptional regulators. Interestingly, the structural and molecular organization of the PML-NI is related to the Marinesco bodies, age-associated ubiquitinated intranuclear inclusions, and to the intranuclear rodlets enriched in class III beta-tubulin, which are nuclear structures markedly decreased in Alzheimer's disease.

Asunto(s)

Núcleo Celular/metabolismo , Cuerpos de Inclusión Intranucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína SUMO-1/metabolismo , Núcleo Supraóptico/citología , Ubiquitina/metabolismo , Adolescente , Anciano , Proteína de Unión a CREB/metabolismo , Compartimento Celular/fisiología , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestructura , Núcleo Celular/ultraestructura , Cromatina/metabolismo , Femenino , Humanos , Cuerpos de Inclusión Intranucleares/ultraestructura , Masculino , Microscopía Inmunoelectrónica , Persona de Mediana Edad , Neuronas/metabolismo , Neuronas/ultraestructura , ARN Mensajero/análisis

RESUMEN

Even though RAS usually acts as a dominant transforming oncogene, in primary fibroblasts and some established cell lines Ras inhibits proliferation. This can explain the virtual absence of RAS mutations in some types of tumors, such as chronic myeloid leukemia (CML). We report that in the CML cell line K562 Ras induces p21Cip1 expression through the Raf-MEK-ERK pathway. Because K562 cells are deficient for p15INK4b, p16INK4a, p14ARF, and p53, this would be the main mechanism whereby Ras up-regulates p21 expression in these cells. Accordingly, we also found that Ras suppresses K562 growth by signaling through the Raf-ERK pathway. Because c-Myc and Ras cooperate in cell transformation and c-Myc is up-regulated in CML, we investigated the effect of c-Myc on Ras activity in K562 cells. c-Myc antagonized the induction of p21Cip1 mediated by oncogenic H-, K-, and N-Ras and by constitutively activated Raf and ERK2. Activation of the p21Cip1 promoter by Ras was dependent on Sp1/3 binding sites in K562. However, mutational analysis of the p21 promoter and the use of a Gal4-Sp1 chimeric protein strongly suggest that c-Myc affects Sp1 transcriptional activity but not the binding of Sp1 to the p21 promoter. c-Myc-mediated impairment of Ras activity on p21 expression required a transactivation domain, a DNA binding region, and a Max binding region. Moreover, the effect was independent of Miz1 binding to c-Myc. Consistent with its effect on p21Cip1 expression, c-Myc rescued cell growth inhibition induced by Ras. The data suggest that in particular tumor types, such as those associated with CML, c-Myc contributes to tumorigenesis by inhibiting Ras antiproliferative activity.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Leucemia/metabolismo , Leucemia/patología , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , División Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN/metabolismo , Secuencias Hélice-Asa-Hélice , Humanos , Células K562 , Factores de Transcripción de Tipo Kruppel , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas A-raf/metabolismo , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

Acute inflammatory demyelinating polyneuropathy (AIDP) is a type of Guillain-Barré syndrome (GBS) characterized by primary nerve demyelination sometimes with secondary axonal degeneration. Studies on the fine structure of dorsal root ganglia in AIDP are lacking. Our aim was to investigate the cytology and nuclear organization of primary sensory neurons in AIDP with axonal injury using ultrastructural and immunohistochemical analysis. The light cytology of the L5 dorsal ganglion showed the characteristic findings of neuronal axonal reaction. The organization of chromatin, nucleolus, Cajal bodies, and nuclear pores corresponded to transcriptionally active neurons. However, the hallmark of the nuclear response to axonal injury was the formation of numerous nuclear bodies (NBs; 6.37 +/- 0.6, in the AIDP, vs. 2.53 +/- 0.2, in the control, mean +/- SDM), identified as promyelocytic leukemia (PML) bodies by the presence of the protein PML. In addition to PML protein, nuclear bodies contained SUMO-1 and the transcriptional regulators CREB-binding protein (CBP) and glucocorticoid receptor (GR). The presence of proteasome 19S was also detected in some nuclear bodies. We suggest that neuronal PML bodies could regulate the nuclear concentration of active proteins, a process mediated by protein interactions with PML and SUMO-1 proteins. In the AIDP case, the proliferation of PML bodies may result from the overexpression of some nuclear proteins due to changes in gene expression associated with axonal injury.

Asunto(s)

Ganglios Sensoriales/metabolismo , Síndrome de Guillain-Barré/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas Nucleares/biosíntesis , Factores de Transcripción/biosíntesis , Anciano , Ganglios Sensoriales/química , Ganglios Sensoriales/ultraestructura , Síndrome de Guillain-Barré/patología , Humanos , Cuerpos de Inclusión Intranucleares/química , Cuerpos de Inclusión Intranucleares/metabolismo , Cuerpos de Inclusión Intranucleares/ultraestructura , Masculino , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/ultraestructura , Proteínas Nucleares/análisis , Proteínas Nucleares/ultraestructura , Proteína de la Leucemia Promielocítica , Factores de Transcripción/análisis , Factores de Transcripción/ultraestructura , Proteínas Supresoras de Tumor

RESUMEN

Neurite outgrowth is a central feature of neuronal differentiation. PC12 cells are a good model system for studying the peripheral nervous system and the outgrowth of neurites. In addition to the dramatic changes observed in the cytoplasm, neuronal differentiation is also accompanied by striking changes in nuclear morphology. The large and sustained increase in nuclear transcription during neuronal differentiation requires synthesis of a large number of factors involved in pre-mRNA processing. We show that the number and composition of the nuclear subdomains called Cajal bodies and gems changes during the course of N-ras-induced neuritogenesis in the PC12-derived cell line UR61. The Cajal bodies found in undifferentiated cells are largely devoid of the survival of motor neurons (SMN) protein product. As cells shift to a differentiated state, SMN is not only globally upregulated, but is progressively recruited to Cajal bodies. Additional SMN foci (also known as Gemini bodies, gems) can also be detected. Using dual-immunogold labeling electron microscopy and mouse embryonic fibroblasts lacking the coilin protein, we show that gems clearly represent a distinct category of nuclear body.

Asunto(s)

Núcleo Celular/química , Cuerpos Enrollados/química , Proteínas del Tejido Nervioso/genética , Neuritas/metabolismo , Proteínas Nucleares/fisiología , Animales , Antiinflamatorios/farmacología , Diferenciación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Citoplasma , Dexametasona/farmacología , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Humanos , Metiltransferasas/antagonistas & inhibidores , Ratones , Microscopía Electrónica , Neuronas Motoras , Atrofia Muscular Espinal , Proteínas Nucleares/genética , Proteínas de Unión al ARN , Proteínas del Complejo SMN

RESUMEN

Intranuclear inclusions composed of tubular filaments constitute a pathological hallmark of oculopharyngeal muscular dystrophy (OPMD). Autosomal dominant OPMD is caused by (GCG) repeat expansions in the gene that encodes for poly(A) binding protein nuclear 1 (PABPN1). The mutation results in the expansion of a polyalanine stretch in the N-terminus of the protein. It has been proposed that mutated PABPN1 induces protein aggregation, which in turn causes the formation of the filamentous nuclear inclusions. Here we report the presence of intranuclear inclusions composed of tubular filaments in oxytocin-producing neurons from normal rat hypothalamus. Like OPMD inclusions, the filamentous structures in neurosecretory neurons accumulate PABPN1, poly(A) RNA, ubiquitin and proteasomes. These inclusions do not contain members of Hsp40 and HDJ-2/DNAJ families of chaperones. The proportion of oxytocin-producing neurons that contain inclusions decreases during parturition and lactation (when synthesis and release of oxytocin is maximal) and increases at 1 day post-weaning (when occurs a drastic reduction in the production of the hormone). Thus, PABPN1 filaments in normal neurons are dynamic structures, the appearance of which correlate with changes in cellular activity. These data provide the first physiological evidence that polyalanine expansions are not essential to induce polymerization of PABPN1 into filamentous nuclear inclusions.

Asunto(s)

Núcleo Celular/metabolismo , Hipotálamo/metabolismo , Neuronas/metabolismo , Sistemas Neurosecretores/metabolismo , Animales , Cuerpos de Inclusión/metabolismo , Distrofia Muscular Oculofaríngea/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero/metabolismo , Ratas , Ubiquitina/metabolismo