A genetic analysis of nitric oxide-mediated signaling during chronological aging in the yeast

In mammals, NO•, a signaling molecule is implicated in the regulation of vasodilation, neurotransmission and immune response. It is believed that NO• is a signaling molecule also in unicellular organism like yeast and may be involved in the regulation of apoptosis and sporulation. It has been reported that NO• is produced during chronological aging (CA) leading to an increase of the superoxide level, which in turn mediates apoptosis. Since this conclusion was based on indirect measurements of NO• by the Griess reaction, the role of NO• signaling during CA in the yeast remains uncertain. We investigated this issue more precisely using different genetic and biochemical methodologies. We used cells lacking the factors influencing nitrosative stress response like flavohemoglobin metabolizing NO•, S-nitrosoglutathione reductase metabolizing S-nitrosoglutathione and the transcription factor Fzf1p mediating NO• response. We measured the standard parameters describing CA and found an elevation in the superoxide level, percentage of death cells, the level of TUNEL positive cells and a decrease in proliferating potential. These observations showed no significant differences between wild type cells and the disruptants except for a small elevation of the superoxide level in the Δsfa1 mutant. The intracellular NO• level and flavohemoglobin expression decreased rather than increased during CA. Products of general nitrogen metabolism and protein tyrosine nitration were slightly decreased during CA, the magnitude of changes showing no differences between the wild type and the mutant yeast. Altogether, our data indicate that apoptosis during yeast CA is mediated by superoxide signaling rather than NO• signaling

The Drosophila F-box protein Archipelago controls levels of the Trachealess transcription factor in the embryonic tracheal system

AbstractThe archipelago gene (ago) encodes the F-box specificity subunit of an SCF(skp-cullin-f box) ubiquitin ligase that inhibits cell proliferation in Drosophila melanogaster and suppresses tumorigenesis in mammals. ago limits mitotic activity by targeting cell cycle and cell growth proteins for ubiquitin-dependent degradation, but the diverse developmental roles of other F-box proteins suggests that it is likely to have additional protein targets. Here we show that ago is required for the post-mitotic shaping of the Drosophila embryonic tracheal system, and that it acts in this tissue by targeting the Trachealess (Trh) protein, a conserved bHLH-PAS transcription factor. ago restricts Trh levels in vivo and antagonizes transcription of the breathless FGF receptor, a known target of Trh in the tracheal system. At a molecular level, the Ago protein binds Trh and is required for proteasome-dependent elimination of Trh in response to expression of the Dysfusion protein. ago mutations that elevate Trh levels in vivo are defective in binding forms of Trh found in Dysfusion-positive cells. These data identify a novel function for the ago ubiquitin-ligase in tracheal morphogenesis via Trh and its target breathless, and suggest that ago has distinct functions in mitotic and post-mitotic cells that influence its role in development and disease

Immune Cell Production Is Targeted by Parasitoid Wasp Virulence in a Drosophila–Parasitoid Wasp Interaction

The interactions between Drosophila melanogaster and the parasitoid wasps that infect Drosophila species provide an important model for understanding host–parasite relationships. Following parasitoid infection, D. melanogaster larvae mount a response in which immune cells (hemocytes) form a capsule around the wasp egg, which then melanizes, leading to death of the parasitoid. Previous studies have found that host hemocyte load; the number of hemocytes available for the encapsulation response; and the production of lamellocytes, an infection induced hemocyte type, are major determinants of host resistance. Parasitoids have evolved various virulence mechanisms to overcome the immune response of the D. melanogaster host, including both active immune suppression by venom proteins and passive immune evasive mechanisms. We identified a previously undescribed parasitoid species, Asobara sp. AsDen, which utilizes an active virulence mechanism to infect D. melanogaster hosts. Asobara sp. AsDen infection inhibits host hemocyte expression of msn, a member of the JNK signaling pathway, which plays a role in lamellocyte production. Asobara sp. AsDen infection restricts the production of lamellocytes as assayed by hemocyte cell morphology and altered msn expression. Our findings suggest that Asobara sp. AsDen infection alters host signaling to suppress immunity

The <em>Archipelago</em> Ubiquitin Ligase Subunit Acts in Target Tissue to Restrict Tracheal Terminal Cell Branching and Hypoxic-Induced Gene Expression

<div><p>The <em>Drosophila melanogaster</em> gene <em>archipelago</em> (<em>ago</em>) encodes the F-box/WD-repeat protein substrate specificity factor for an SCF (Skp/Cullin/F-box)-type polyubiquitin ligase that inhibits tumor-like growth by targeting proteins for degradation by the proteasome. The Ago protein is expressed widely in the fly embryo and larva and promotes degradation of pro-proliferative proteins in mitotically active cells. However the requirement for Ago in post-mitotic developmental processes remains largely unexplored. Here we show that Ago is an antagonist of the physiologic response to low oxygen (hypoxia). Reducing Ago activity in larval muscle cells elicits enhanced branching of nearby tracheal terminal cells in normoxia. This tracheogenic phenotype shows a genetic dependence on <em>sima</em>, which encodes the HIF-1α subunit of the hypoxia-inducible transcription factor dHIF and its target the FGF ligand <em>branchless (bnl)</em>, and is enhanced by depletion of the <em>Drosophila</em> Von Hippel Lindau (<em>dVHL</em>) factor, which is a subunit of an oxygen-dependent ubiquitin ligase that degrades Sima/HIF-1α protein in metazoan cells. Genetic reduction of <em>ago</em> results in constitutive expression of some hypoxia-inducible genes in normoxia, increases the sensitivity of others to mild hypoxic stimulus, and enhances the ability of adult flies to recover from hypoxic stupor. As a molecular correlate to these genetic data, we find that Ago physically associates with Sima and restricts Sima levels <em>in vivo</em>. Collectively, these findings identify Ago as a required element of a circuit that suppresses the tracheogenic activity of larval muscle cells by antagonizing the Sima-mediated transcriptional response to hypoxia.</p> </div

<i>ago</i>^{Δ<i>3–7/1</i>} larvae display a wide range of tracheal terminal branch phenotypes.

<p>(A,B) Schematic (left) and representative photomicrograph showing branching of LH lateral terminal cells in control (A) and <i>ago</i>^{Δ<i>3–7/1</i>} (B) larvae. Branch termini are indicated with asterisks. (C) Quantification of branch number per LH (left) and LG (right) terminal cell in the indicated genotypes (*p<0.001 relative to control). (D) <i>ago</i>^{Δ<i>3–7/1</i>} larva displaying terminal branch tangling. (E,F) Ganglionic tracheal branches in control (E) and <i>ago</i>^{Δ<i>3–7/1</i>} (F) larvae. Ringlet-shaped branches and tangles occur in approximately 25% of <i>ago</i>^{Δ<i>3–7/1</i>} larvae.</p

<i>ago</i> and <i>dVHL</i> co-regulate terminal branching.

<p>(A,B) VLM12-specific expression of <i>dVHL</i> dsRNA (A) or <i>sima</i> (B) leads to an increase in terminal branch number (black arrows). Double-headed arrow indicates dimension of VLM12. (C,D) Terminal branch phenotypes of <i>agoΔF</i> and <i>dVHL</i> RNAi double-mutant larvae. (C) Schematic (left) and representative photomicrograph showing LF and LH cell terminal branches (black arrows) on VLM12. Double-headed arrow indicates VLM12. (D) Schematic (left) and representative photomicrograph showing ectopic recruitment of LG cell terminal branches to VLM12 (black arrows). Black arrowhead, ectopic LG branch recruited to VLM12; open arrowhead, LG branch following its typical course; double-headed arrow indicates VLM12.</p

Loss of <i>ago</i> deregulates Sima activity and increases Bnl protein levels.

<p>(A,B) Representative images of LG lateral terminal cell branching in <i>ago^Δ3–7/1</i> (A) and <i>ago^Δ3–7/1</i>, <i>sima⁰⁷⁶⁰⁷/+</i> (B) larvae. The main LG branches are indicated with black arrows. (C, left panel) qRT-PCR quantification of <i>dLDH</i> (left) and <i>sima</i> (right) transcription in the body wall muscles of larvae of the indicated genotypes (*p<0.001 relative to control). (C, right panel) qRT-PCR quantification of <i>bnl</i> transcription in the body wall muscles of larvae of the indicated genotypes (*p<3.2×10⁻² relative to control).</p

Ago associates with Sima and limits its abundance in peripheral tissues.

<p>Confocal images of GFP and anti-Sima fluorescence in (A) <i>5053A>sima,nlsGFP</i> and (B) <i>5053A>agoΔF,nlsGFP</i> larval muscles. Nuclear-enriched GFP marks the VLM12 muscle (bracketed by white bars) in the field of surrounding body wall muscle segments. Arrows in (B) indicate Sima-positive nuclei. (C) Anti-Sima immunoblot of wt and <i>ago^Δ3–7</i> 3^rd instar filleted pelts. Molecular weight (MW) markers are indicated. Asterisks denote non-specific bands to indicate loading. (D) Immunoblot of Sima in control (<i>wt</i>) and <i>sima⁰⁷⁶⁰⁷</i> (<i>−/−</i>) whole larval lysates (lanes 1–2), and in normal goat serum (NGS) and anti-Ago immunoprecipitates from normoxic (21% O₂) or hypoxic (5% O₂) larvae.</p