The nonautonomous reduction in tissue size upon targeted activation of the PI3K/PTEN and TSC/TOR pathways does not rely on Dp53 activity and apoptosis and is not affected by nutrient restriction.

<p>(A) <i>ci>GFP</i>, <i>ci>Rheb</i>, and <i>ci>Dp110</i> wing imaginal discs labelled with TUNEL to visualize apoptotic cells (in red or white). The <i>ci</i> domain is labelled with GFP (in green), and the boundary between A and P cells is marked by a red line. (B) Histogram plotting the quantification of the absolute number of TUNEL-positive cells in the A (light green bars) and P (grey bars) compartments of the indicated genotypes. Error bars indicate the standard deviation. Number of wing discs analyzed per genotype ≥ 10. ***<i>p</i> < 0.001. (C, D) Cuticle preparations of <i>en>GFP</i>, <i>Rheb</i> (C), and <i>ci>GFP</i>, <i>Dp110</i> (D) adult wings either in a wild-type background, in a heterozygous background for <i>H99</i> (a deficiency that uncovers <i>reaper</i>, <i>hid</i>, and <i>grim</i> proapoptotic genes), or coexpressing a dominant negative form of Dp53 (Dp53^Ct). The blue line marks the boundary between the A and P compartments. The domains of transgene expression are marked with a blue asterisk. (C’, D’, E) Histograms plotting tissue size (C’, D’, E) and cell density values (C’, E) of the transgene-expressing compartment (blue bars) and the adjacent cell populations (white bars) of adult wings with the indicated genotypes normalized as a percent of the control wings. Error bars show the standard deviation. Number of wings analyzed per genotype ≥ 10. ***<i>p</i> < 0.001; **<i>p</i> < 0.01; *<i>p</i> < 0.05. (F) Cuticle preparations of <i>ci>GFP</i> adult wings of well-fed (100 g/L yeast food, top) and starved (20 g/L yeast food, bottom) animals. Note the reduction in wing size caused by starvation. (G, H) Histograms plotting tissue size of the transgene-expressing compartment (blue bars) and the adjacent cell populations (white bars) of adult wings with the indicated genotypes normalized as a percent of the control wings. Error bars show the standard deviation. Number of wings analyzed per genotype ≥ 10. ***<i>p</i> < 0.001; **<i>p</i> < 0.01; *<i>p</i> < 0.05. Quantification was made in well-fed (100 g/L yeast food, left histogram) and starved (20 g/L yeast food, right histogram) animals of identical genotypes.</p

A role of Dally in tissue growth.

<p>(A) Activation of the PI3K/PTEN, TSC/TOR, or Yorkie pathways in a defined cell population (in blue) increases cell and/or tissue size in an autonomous manner and induces a nonautonomous reduction in both cell size and number in neighboring cell populations (in grey). Whereas the autonomous and nonautonomous effects on tissue size are mediated by Dally, the effects on cell size are Dally independent. The nonautonomous reduction in tissue size is a consequence of reduced Dpp signaling, most probably reflecting increased number of Dpp molecules bound to the overgrowing (Dally overexpressing) tissue and a consequent reduction in the number of available Dpp molecules to the neighboring cell population. (B) Within the feeding animal, the PI3K/PTEN and TSC/TOR pathways sense nutrient conditions and modulate both cell and tissue size. Whereas the effects on tissue size are mediated by the glypican Dally, which modulates the spreading of the Dpp morphogen throughout the tissue, the effects on cell size are Dally independent.</p

Dally Proteoglycan Mediates the Autonomous and Nonautonomous Effects on Tissue Growth Caused by Activation of the PI3K and TOR Pathways

<div><p>How cells acquiring mutations in tumor suppressor genes outcompete neighboring wild-type cells is poorly understood. The phosphatidylinositol 3-kinase (PI3K)–phosphatase with tensin homology (PTEN) and tuberous sclerosis complex (TSC)-target of rapamycin (TOR) pathways are frequently activated in human cancer, and this activation is often causative of tumorigenesis. We utilized the Gal4-UAS system in <i>Drosophila</i> imaginal primordia, highly proliferative and growing tissues, to analyze the impact of restricted activation of these pathways on neighboring wild-type cell populations. Activation of these pathways leads to an autonomous induction of tissue overgrowth and to a remarkable nonautonomous reduction in growth and proliferation rates of adjacent cell populations. This nonautonomous response occurs independently of where these pathways are activated, is functional all throughout development, takes place across compartments, and is distinct from cell competition. The observed autonomous and nonautonomous effects on tissue growth rely on the up-regulation of the proteoglycan Dally, a major element involved in modulating the spreading, stability, and activity of the growth promoting Decapentaplegic (Dpp)/transforming growth factor β(TGF-β) signaling molecule. Our findings indicate that a reduction in the amount of available growth factors contributes to the outcompetition of wild-type cells by overgrowing cell populations. During normal development, the PI3K/PTEN and TSC/TOR pathways play a major role in sensing nutrient availability and modulating the final size of any developing organ. We present evidence that Dally also contributes to integrating nutrient sensing and organ scaling, the fitting of pattern to size.</p></div

Dally acts as a bridge between nutrient sensing and wing scaling.

<p>(A–D) Cuticle preparations (A, C) and histograms plotting tissue size normalized as a percent of the control wings (B, D) of <i>nub>Rheb</i> (A, B) or <i>nub>Dp110</i> (C, D) adult wings coexpressing either <i>GFP</i> or <i>dally</i>^<i>RNAi</i>. Two independent RNA interference (RNAi) lines were used in B. Error bars show the standard deviation. Number of wings analyzed per genotype ≥ 10. ***<i>p</i> < 0.001. (E) Representative wing discs of the indicated genotypes labeled to visualize <i>dally-lacZ</i> (antibody to β-Gal, green or white) and myrT expression (in red) to mark the transgene-expressing domain. Higher magnification pictures of the squared regions are shown on the right side. Note the reduced expression of <i>dally-lacZ</i> upon depletion of the TSC/TOR or PTEN/PI3K pathways. (F) Representative wing discs of the indicated genotypes and labeled to visualize Ci (red) and DAPI (in blue). Ci labels the anterior compartment. A, anterior compartment; P, posterior compartment. (G) Histogram plotting the P/A size ratio of wing discs of the indicated genotypes. Error bars show the standard deviation. Number of wing discs analyzed per genotype ≥ 10. ***<i>p</i> < 0.001. P/A ratios: hh>2XGFP = 0.55 ± 0.02; hh>GFP, Dp110-RNAi = 0.29 ± 0.06; hh>dally, Dp110-RNAi = 0.42 ± 0.07. (H) Histogram plotting absolute size in a.u. of adult wings of the indicated genotypes. Quantification was made in well-fed (100 g/L yeast food) and starved (20 g/L yeast food) animals. Error bars show the standard deviation. Number of wings analyzed per genotype ≥ 10. ***<i>p</i> < 0.001. ns, not significant.</p

Overexpression of Dally phenocopies the autonomous and nonautonomous effects of targeted activation of the PI3K/PTEN and TSC/TOR pathways.

<p>(A, B) Representative wing discs of the indicated genotypes labeled to visualize <i>dally-lacZ</i> (antibody to β-Gal, green or white) and myrTomato (myrT, in red) expression to mark the transgene-expressing domain. Note stronger expression of <i>dally-lacZ</i> upon activation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways (red brackets). (C, E) Cuticle preparations of male adult wings overexpressing Dally under the control of the <i>ci-gal4</i> (C) or <i>en-gal4</i> (E) drivers. The blue line marks the boundary between the anterior (A) and posterior (P) compartments, and the domains of Dally overexpression are marked with a blue asterisk. (D, F). Histograms plotting tissue size (left) and cell density values (right) of the Dally-expressing domains (blue bars) and the adjacent compartments (white bars) of adult wings overexpressing Dally under the control of the <i>ci-gal4</i> (D) or <i>en-gal4</i> (F) drivers. Values are normalized as a percent of the control (GFP-expressing) wings. Error bars show the standard deviation. Number of wings analyzed per genotype ≥ 10. ***<i>p</i> < 0.001; **<i>p</i> < 0.01. (G, H) Histograms plotting the size of clones (in a.u., G) and the number of cells per clone (H) located in the A or P compartment of <i>ci-gal4</i>, <i>UAS-GFP</i> and <i>ci-gal4</i>, <i>UAS-GFP/UAS-Dally</i> wing discs. Clones were generated at the beginning of the third instar period and quantified 72 h later in late third instar wing discs. Error bars indicate the standard deviation. Number of clones analyzed per genotype ≥ 30. ***<i>p</i> < 0.001. Size of clones (A compartment, a.u.): ci>GFP = 388 ± 141; ci>GFP, Dally = 537 ± 169. Size of clones (P compartment, a.u.): ci>GFP = 348 ± 140; ci>GFP, Dally = 182 ± 89. Number of cells per clone (A compartment): ci>GFP = 20 ± 7; ci>GFP, Dally = 28 ± 12. Number of cells per clone (P compartment): ci>GFP = 23 ± 7; ci>GFP, Dally = 10 ± 4. (I) Wing imaginal discs of <i>ci>GFP</i> and <i>ci>GFP</i>, <i>Dally</i> larvae labelled to visualize pMAD protein (in red or white) and GFP (in green). (J) Average pMAD profiles of wing discs expressing <i>GFP</i> (red line) or <i>GFP</i> and <i>Dally</i> (blue line) under the control of the <i>ci-gal4</i> driver. Profiles were taken along the AP axis and plotted in absolute positions. The standard error to the mean is shown in the corresponding color for each genotype. The AP boundary of both experiments was aligned to allow comparison of the profile in each compartment. Number of wing discs analyzed per genotype ≥ 5. (K) Histogram plotting the total intensity of the pMAD signal in a.u. of the anterior (blue bars) and posterior (white bars) compartments of <i>ci>GFP</i> and <i>ci>Dally</i>, <i>GFP</i> wing discs. Error bars indicate the standard deviation. Number of wing discs analyzed per genotype ≥ 5. ***<i>p</i> < 0.001; **<i>p</i> < 0.01.</p

Targeted activation of the PI3K/PTEN and TSC/TOR pathways induces a nonautonomous reduction in growth rates in adjacent cell populations.

<p>(A) Quantification of tissue size (in arbitrary units [a.u.]) of both anterior (A) and posterior (P) domains of wing imaginal discs of the indicated genotypes in three distinct time points during development (in hours [h] AEL). Error bars show the standard deviation. Number of wing discs analyzed per genotype ≥ 15. ***<i>p</i> < 0.001; **<i>p</i> < 0.01. (B) Examples of <i>ci>GFP</i> (upper panel) and <i>ci>GFP</i>, <i>Dp110</i> (lower panel) wing imaginal discs in the developmental time points shown in panel A. The transgene-expressing domain is marked with GFP (in green), and the disc is labelled with DAPI (in blue). The A and P compartments are indicated. (C, D) Histograms plotting the size of clones (in a.u., C) and the number of cells per clone (D) located in the A or P compartment of <i>ci-gal4</i>,<i>UAS-GFP</i> and <i>ci-gal4</i>, <i>UAS-GFP/UAS-Dp110</i> wing discs. Clones were generated at the beginning of the third instar period and quantified 72 h later in late third instar wing discs. Error bars indicate the standard deviation. Number of clones analyzed per genotype ≥ 30. ***<i>p</i> < 0.001. Size of clones (A compartment, a.u.): ci>GFP = 388 ± 141; ci>GFP, Dp110 = 587 ± 249. Size of clones (P compartment, a.u.): ci>GFP = 348 ± 140; ci>GFP, Dp110 = 114 ± 67. Number of cells per clone (A compartment): ci>GFP = 20 ± 7; ci>GFP, Dp110 = 23 ± 8. Number of cells per clone (P compartment): ci>GFP = 23 ± 7; ci>GFP, Dp110 = 9 ± 4. (E) Examples of clones of cells in <i>ci>GFP</i> (left panel) and <i>ci>GFP</i>, <i>Dp110</i> (right panel) wing discs and induced in the developmental time points shown in C and D. The transgene-expressing domain is marked with GFP (in green), and the clones are labeled by the absence of nuclear red fluorescent protein (RFP, red) expression. The A and P compartments are indicated.</p

Structural and functional characterization of Nrf2 degradation by glycogen synthase kinase 3/β-TrCP

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of cellular homeostasis that controls the expression of more than 1% of human genes related to biotransformation reactions, redox homeostasis, energetic metabolism, DNA repair, and proteostasis. Its activity has a tremendous impact on physiology and pathology and therefore it is very tightly regulated, mainly at the level of protein stability. In addition to the very well established regulation by the ubiquitin E3 ligase adapter Keap1, recent advances have identified a novel mechanism based on signaling pathways that regulate glycogen synthase kinse-3 (GSK-3). This kinase phosphorylates specific serine residues in the Neh6 domain of Nrf2 to create a degradation domain that is then recognized by the ubiquitin ligase adapter β-TrCP and tagged for proteasome degradation by a Cullin1/Rbx1 complex. Here we review the mechanistic elements and the signaling pathways that participate in this regulation by GSK-3/β-TrCP. These pathways include those activated by ligands of tyrosine kinase, G protein-coupled, metabotropic, and ionotropic receptors that activate phosphatidyl inositol 3-kinase (PI3K)/ATK and by the canonical WNT signaling pathway, where a fraction of Nrf2 interacts with Axin1/GSK-3. Considering that free Nrf2 protein is localized in the nucleus, we propose a model termed “double flux controller” to explain how Keap1 and β-TrCP coordinate the stability of Nrf2 in several scenarios. The GSK-3/β-TrCP axis provides a novel therapeutic strategy to modulate Nrf2 activity.This study was funded by Grant SAF2013-43271-R of the Spanish Ministry of Economy and Competitiveness (MINECO).Peer reviewe

Additional file 2: Table S2. of Evolution and genome specialization of Brucella suis biovar 2 Iberian lineages

List of genomes used for phylogenetic and comparative genomic analysis.) (DOCX 17 kb

Additional file 7: Figure S2. of Evolution and genome specialization of Brucella suis biovar 2 Iberian lineages

Distribution of SNPs along the genome (SNPs per 0,2 Mb). (PDF 194 kb

Additional file 5: Figure S1. of Evolution and genome specialization of Brucella suis biovar 2 Iberian lineages

Comparative chromosome mapping of 25 Brucella spp. genomes. Genomic alignment of concatenated chromosomes I and II was performed by superstretch approach: DNA seed 10 matches in windows size of 25 bases, minimal stretch length 60 bases, minimal cut-off for stretch identity of 60% in screening windows of 30 bases was used. Each cell in the matrix displays the identity score, with a corresponding color scale. The left-to-right diagonal of the matrix contains those cells representing the comparison of sequences compared to themselves. The value in each cell represent the percentage of repetitive regions for that sequence. The scale goes from black, corresponding with 100% identity, over blue towards white (0% identity). Clustering analysis using UPGMA. All positions containing gaps and missing data were eliminated. (PDF 43 kb