MK2-Dependent p38b Signalling Protects Drosophila Hindgut Enterocytes against JNK-Induced Apoptosis under Chronic Stress

The integrity of the intestinal epithelium is crucial for the barrier function of the gut. Replenishment of the gut epithelium by intestinal stem cells contributes to gut homeostasis, but how the differentiated enterocytes are protected against stressors is less well understood. Here we use the Drosophila larval hindgut as a model system in which damaged enterocytes are not replaced by stem cell descendants. By performing a thorough genetic analysis, we demonstrate that a signalling complex consisting of p38b and MK2 forms a branch of SAPK signalling that is required in the larval hindgut to prevent stress-dependent damage to the enterocytes. Impaired p38b/MK2 signalling leads to apoptosis of the enterocytes and a subsequent loss of hindgut epithelial integrity, as manifested by the deterioration of the overlaying muscle layer. Damaged hindguts show increased JNK activity, and removing upstream activators of JNK suppresses the loss of hindgut homeostasis. Thus, the p38/MK2 complex ensures homeostasis of the hindgut epithelium by counteracting JNK-mediated apoptosis of the enterocytes upon chronic stress

A universal method for automated gene mapping

Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost

The Drosophila SH2B Family Adaptor Lnk Acts in Parallel to Chico in the Insulin Signaling Pathway

Insulin/insulin-like growth factor signaling (IIS) plays a pivotal role in the regulation of growth at the cellular and the organismal level during animal development. Flies with impaired IIS are developmentally delayed and small due to fewer and smaller cells. In the search for new growth-promoting genes, we identified mutations in the gene encoding Lnk, the single fly member of the SH2B family of adaptor molecules. Flies lacking lnk function are viable but severely reduced in size. Furthermore, lnk mutants display phenotypes reminiscent of reduced IIS, such as developmental delay, female sterility, and accumulation of lipids. Genetic epistasis analysis places lnk downstream of the insulin receptor (InR) and upstream of phosphoinositide 3-kinase (PI3K) in the IIS cascade, at the same level as chico (encoding the single fly insulin receptor substrate [IRS] homolog). Both chico and lnk mutant larvae display a similar reduction in IIS activity as judged by the localization of a PIP3 reporter and the phosphorylation of protein kinase B (PKB). Furthermore, chico; lnk double mutants are synthetically lethal, suggesting that Chico and Lnk fulfill independent but partially redundant functions in the activation of PI3K upon InR stimulation

A reverse genetic screen in Drosophila using a deletion-inducing mutagen

We report the use of the cross-linking drug hexamethylphosphoramide (HMPA), which introduces small deletions, as a mutagen suitable for reverse genetics in the model organism Drosophila melanogaster. A compatible mutation-detection method based on resolution of PCR fragment-length polymorphisms on standard DNA sequencers is implemented. As the spectrum of HMPA-induced mutations is similar in a variety of organisms, it should be possible to transfer this mutagenesis and detection procedure to other model systems

Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance

<p>Abstract</p> <p>Background</p> <p>Insulin and insulin-like growth factors (IGFs) signal through a highly conserved pathway and control growth and metabolism in both vertebrates and invertebrates. In mammals, insulin-like growth factor binding proteins (IGFBPs) bind IGFs with high affinity and modulate their mitogenic, anti-apoptotic and metabolic actions, but no functional homologs have been identified in invertebrates so far.</p> <p>Results</p> <p>Here, we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2) binds <it>Drosophila </it>insulin-like peptide 2 (Dilp2) and inhibits growth non-autonomously. Whereas over-expressing <it>Imp-L2 </it>strongly reduces size, loss of <it>Imp-L2 </it>function results in an increased body size. <it>Imp-L2 </it>is both necessary and sufficient to compensate Dilp2-induced hyperinsulinemia <it>in vivo</it>. Under starvation conditions, <it>Imp-L2 </it>is essential for proper dampening of insulin signaling and larval survival.</p> <p>Conclusion</p> <p>Imp-L2, the first functionally characterized insulin-binding protein in invertebrates, serves as a nutritionally controlled suppressor of insulin-mediated growth in <it>Drosophila</it>. Given that Imp-L2 and the human tumor suppressor IGFBP-7 show sequence homology in their carboxy-terminal immunoglobulin-like domains, we suggest that their common precursor was an ancestral insulin-binding protein.</p

Twitchy, the Drosophila orthologue of the ciliary gating protein FBF1/dyf-19, is required for coordinated locomotion and male fertility

Primary cilia are compartmentalised from the rest of the cell by a ciliary gate comprising transition fibres and a transition zone. The ciliary gate allows the selective import and export of molecules such as transmembrane receptors and transport proteins. These are required for the assembly of the cilium, its function as a sensory and signalling centre and to maintain its distinctive composition. Certain motile cilia can also form within the cytosol as exemplified by human and Drosophila sperm. The role of transition fibre proteins has not been well described in the cytoplasmic cilia. Drosophila have both compartmentalised primary cilia, in sensory neurons, and sperm flagella that form within the cytosol. Here, we describe phenotypes for twitchy the Drosophila orthologue of a transition fibre protein, mammalian FBF1/C. elegans dyf-19. Loss-of-function mutants in twitchy are adult lethal and display a severely uncoordinated phenotype. Twitchy flies are too uncoordinated to mate but RNAi-mediated loss of twitchy specifically within the male germline results in coordinated but infertile adults. Examination of sperm from twitchy RNAi-knockdown flies shows that the flagellar axoneme forms, elongates and is post-translationally modified by polyglycylation but the production of motile sperm is impaired. These results indicate that twitchy is required for the function of both sensory cilia that are compartmentalised from the rest of the cell and sperm flagella that are formed within the cytosol of the cell. Twitchy is therefore likely to function as part of a molecular gate in sensory neurons but may have a distinct function in sperm cells.ISSN:2046-639

Einfluss von Phosphodiesterase 5-Inhibitoren auf die Escherichia coli Hämolysin und Lipopolysaccharid vermittelte Kardiodepression am isolierten Rattenherzen

Durch Mikroorganismen, wie Bakterien, Viren oder Pilze kommt es zu Primärinfektionen, in deren Folge sich eine Sepsis entwickeln kann. Die häufigsten Sepsisverursacher sind Bakterien, wobei der Tatsache, dass das Krankheitsbild der Sepsis nicht an die Anwesenheit intakter Bakterien in der Zirkulation gebunden ist sondern die hämatogene Streuung sezernierter Exotoxine und endotoxinhaltiger Zellfragmente zur Induktion einer Sepsis ausreichen, besondere Bedeutung beigemessen werden muss. Die während einer Sepsis auftretenden kardiovaskulären Veränderungen tragen entscheidend zur Induktion des septischen Multiorganversagens sowie des septischen Schocks bei. Es ist dem Herzen im Zuge einer akuten septischen Kardiomyopathie häufig nicht möglich, seine Pumpleistung so zu erhöhen, wie es zur Aufrechterhaltung eines adäquaten Blutdrucks bei stark erniedrigtem systemischem Widerstand nötig wäre. Die septische Kardiomyopathie kann durch die Wirkung kardiodepressiver Zytokine wie TNF-alpha und Il-1beta hervorgerufen werden; daneben sind Mikrozirkula-tionsstörungen innerhalb des Myokards, welche auch ohne globale Minderperfusion zu einer hypoxisch bedingten Pumpinsuffizienz führen können, als mögliche Ursache anzusehen. Zyklische Nukleotide stellen dabei einen wichtigen Bestandteil in der Regulation von koronarer Vasomotorik und kardialer Kontraktilität dar, überdies spielen sie auch eine Rolle in der Pathophysiologie der Sepsis. Die vorliegende Arbeit behandelt die Frage, ob Phosphodiesterase (PDE5) -Inhibitoren den kardiodepressiven Effekt gram-negativer Bakterien respektive deren Endo- und Exotoxinen zumindest teilweise aufheben und so zu einem günstigeren Verlauf der Sepsis beitragen können. Dazu wurden isolierte Rattenherzen mit Escherichia coli Hämolysin (ECH) und Lipopolysaccharid (LPS) perfundiert. Die dadurch generierte Kardiodepression sowie Vasokonstriktion im Koronargefäßsystem sollten mit Hilfe von Sildenafil und Zaprinast inhibiert werden, was partiell gelang. Desweiteren wurde durch die Aufarbeitung von Ventrikelpräparaten eine verminderte Freisetzung kardio-depressiver Mediatoren, insbesondere von TNF-alpha und Cysteinyl-Leukotrienen (Cys-LT), nachgewiesen. Eine Beeinflussung der Genexpression hingegen konnte nicht gezeigt werden. Für die LPS-Versuche stellt sich der protektive Effekt folgendermaßen dar: Der durch die PDE5-Inhibitoren erhöhte cGMP-Spiegel führt zu einer koronaren Vasodilatation, welche Mikrozirkulationsstörungen vorbeugt, was zur Verbesserung der Myokardversorgung und einer verminderten Freisetzung von TNF-alpha führt. Durch diese zeitige Intervention in die teilweise selbstunterhaltenden Mechanismen können der TNF-alpha abhängige kardiodepressive Effekt, aber auch die Aktivierung der cNOS/iNOS, des Sphingomyelinasesignalwegs, der freien Radikalliberation und der Apoptose antagonisiert werden. Im Falle der ECH-Versuche steht ebenfalls die vasodilatative Wirkung der PDE5-Inhibitoren im Koronargefäßsystem im Vordergrund. Diese beruht sowohl auf einer prolongierten cGMP Wirkung als auch auf einer herabgesetzten Cys-LT Liberation. Die verbesserte Perfusion verhindert die sich selbstunterhaltende, ischämisch bedingte Cys-LT Freisetzung und verringert so eine weitere Erhöhung des koronaren Gefäßwiderstandes. Infolge der Senkung des koronaren Gefäßwiderstandes wird die Kardiodepression nahezu vollständig aufgehoben. Die gewonnenen Ergebnisse deuten auf ein protektives Potential der PDE5-Inhibitoren bezüglich der septischen Kardiomyopathie hin.Microorganisms like bacteria, viruses and fungi cause primary infections, which can lead to sepsis. In fact, sepsis is primarily caused by bacteria. Induction of septicemia is not dependent on the presence of intact bacterial organisms in the blood circulation. Hematogenic spreading of secreted bacterial exotoxins as well as cellular fragments containing endotoxins can also lead to sepsis. Cardiovascular alterations due to sepsis crucially contribute to multiple organ dysfunction syndrome and to septic shock. The heart of septic patients is frequently unable to increase cardiac output adequately to compensate the systemic fall in blood pressure. On the one hand septic cardiomyopathy can be induced by cardiodepressive cytokines like TNF-alpha or Il-1beta and on the other hand by microcirculatory dysfunctions within the myocardium, which even without global ischemia may lead to a decreased cardiac output due to hypoxia. Cyclic nucleotides play an important role in coronary vasoconstriction and cardial depression and also in pathophysiology of sepsis. The present study investigates in how far the phosphodiesterase (PDE5) inhibitors Sildenafil and Zaprinast could be able to inhibit the cardiodepressive effect of gram–negative bacteria and their endo- and exotoxins respectively, which then could lead to an improvement of the prognosis. Isolated rat hearts were perfused with Escherichia coli hemolysin (ECH) and lipopolysaccharides (LPS) respectively which lead to cardiodepression and coronary vasoconstriction. After treatment with Sildenafil and Zaprinast respectively this effect could be partially decreased. Analysis of samples gained from ventricular tissue revealed a decreased liberation of cardiodepressive mediators, such as TNF-alpha and cysteinyl-leukotrienes (Cys-LT). Alterations in gene expression could not be shown. The protective effect of the PDE5-inhibitors in the LPS perfused rat hearts can be explained in the following way: PDE5-inhibitor elevate the cGMP levels which lead to coronary vasodilatation inhibiting microcirculatory dysfunction. This improves myocardial supply and decreases TNF-alpha liberation. The early intervention in the partially self engaging mechanisms inhibit the TNF-alpha dependent cardiodepressive effect, activation of the cNOS/iNOS in the sphingomyelinase signaling pathway, free radical liberation and apoptosis. The main effect of PDE5-inhibotor treatment on ECH perfused hearts is a coronary vasodilatation due to a prolonged cGMP impact and a decreased Cys-LT liberation. PDE5-inhibitors decrease self engaging, ischemic induced Cys-LT liberation, which in the end leads to a minimized increase in coronary vascular resistance. Due to the decrease of the coronary resistance cardiodepression can be abolished nearly completely. All results gained in this study recommend a beneficial potential of PDE5-inhibitors in septic cardiomyopathy

Madm (Mlf1 adapter molecule) cooperates with Bunched A to promote growth in Drosophila

Background The TSC-22 domain family (TSC22DF) consists of putative transcription factors harboring a DNA-binding TSC-box and an adjacent leucine zipper at their carboxyl termini. Both short and long TSC22DF isoforms are conserved from flies to humans. Whereas the short isoforms include the tumor suppressor TSC-22 (Transforming growth factor-β1 stimulated clone-22), the long isoforms are largely uncharacterized. In Drosophila, the long isoform Bunched A (BunA) acts as a growth promoter, but how BunA controls growth has remained obscure. Results In order to test for functional conservation among TSC22DF members, we expressed the human TSC22DF proteins in the fly and found that all long isoforms can replace BunA function. Furthermore, we combined a proteomics-based approach with a genetic screen to identify proteins that interact with BunA. Madm (Mlf1 adapter molecule) physically associates with BunA via a conserved motif that is only contained in long TSC22DF proteins. Moreover, Drosophila Madm acts as a growth-promoting gene that displays growth phenotypes strikingly similar to bunA phenotypes. When overexpressed, Madm and BunA synergize to increase organ growth. Conclusions The growth-promoting potential of long TSC22DF proteins is evolutionarily conserved. Furthermore, we provide biochemical and genetic evidence for a growth-regulating complex involving the long TSC22DF protein BunA and the adapter molecule Madm. See minireview at http://jbiol.com/content/9/1/8.ISSN:1478-5854ISSN:1475-492

The Drosophila Forkhead Transcription Factor FOXO Mediates the Reduction in Cell Number Associated with Reduced Insulin Signaling

Background: Forkhead transcription factors belonging to the FOXO subfamily are negatively regulated by protein kinase B (PKB) in response to signaling by insulin and insulin-like growth factor in Caenorhabditis elegans and mammals. In Drosophila, the insulin-signaling pathway regulates the size of cells, organs, and the entire body in response to nutrient availability, by controlling both cell size and cell number. In this study, we present a genetic characterization of dFOXO, the only Drosophila FOXO ortholog. Results: Ectopic expression of dFOXO and human FOXO3a induced organ-size reduction and cell death in a manner dependent on phosphoinositide (PI) 3-kinase and nutrient levels. Surprisingly, flies homozygous for dFOXO null alleles are viable and of normal size. They are, however, more sensitive to oxidative stress. Furthermore, dFOXO function is required for growth inhibition associated with reduced insulin signaling. Loss of dFOXO suppresses the reduction in cell number but not the cell-size reduction elicited by mutations in the insulin-signaling pathway. By microarray analysis and subsequent genetic validation, we have identified d4E-BP, which encodes a translation inhibitor, as a relevant dFOXO target gene. Conclusion: Our results show that dFOXO is a crucial mediator of insulin signaling in Drosophila, mediating the reduction in cell number in insulin-signaling mutants. We propose that in response to cellular stresses, such as nutrient deprivation or increased levels of reactive oxygen species, dFOXO is activated and inhibits growth through the action of target genes such as d4E-BP

Bunched, the Drosophila homolog of the mammalian tumor suppressor TSC-22, promotes cellular growth

<p>Abstract</p> <p>Background</p> <p>Transforming Growth Factor-β1 stimulated clone-22 (TSC-22) is assumed to act as a negative growth regulator and tumor suppressor. TSC-22 belongs to a family of putative transcription factors encoded by four distinct loci in mammals. Possible redundancy among the members of the TSC-22/Dip/Bun protein family complicates a genetic analysis. In <it>Drosophila</it>, all proteins homologous to the TSC-22/Dip/Bun family members are derived from a single locus called <it>bunched </it>(<it>bun</it>).</p> <p>Results</p> <p>We have identified <it>bun </it>in an unbiased genetic screen for growth regulators in <it>Drosophila</it>. Rather unexpectedly, <it>bun </it>mutations result in a growth deficit. Under standard conditions, only the long protein isoform BunA – but not the short isoforms BunB and BunC – is essential and affects growth. Whereas reducing <it>bunA </it>function diminishes cell number and cell size, overexpression of the short isoforms BunB and BunC antagonizes <it>bunA </it>function.</p> <p>Conclusion</p> <p>Our findings establish a growth-promoting function of <it>Drosophila </it>BunA. Since the published studies on mammalian systems have largely neglected the long TSC-22 protein version, we hypothesize that the long TSC-22 protein is a functional homolog of BunA in growth regulation, and that it is antagonized by the short TSC-22 protein.</p