Manabu Tsuda

The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila

Wingless (Wg) is a member of the Wnt family of growth factors, secreted proteins that control proliferation and differentiation during development. Studies in Drosophila have shown that responses to Wg require cell-surface heparan sulphate, a glycosaminoglycan component of proteoglycans,. These findings suggest that a cell-surface proteoglycan is a component of a Wg/Wnt receptor complex. We demonstrate here that the protein encoded by the division abnormally delayed (dally) gene is a cell-surface, heparan-sulphate-modified proteoglycan,. dally partial loss-of-function mutations compromise Wg-directed events, and disruption of dally function with RNA interference produces phenotypes comparable to those found with RNA interference of wg or frizzled (fz)/Dfz2 (ref. 7). Ectopic expression of Dally potentiates Wg signalling without altering levels of Wg and can rescue a wg partial loss-of-function mutant. We also show that dally, a regulator of Decapentaplegic (Dpp) signalling during post-embryonic development, has tissue-specific effects on Wg and Dpp signalling. Dally can therefore differentially influence signalling mediated by two growth factors, and may form a regulatory component of both Wg and Dpp receptor complexes.

The RING-finger scaffold protein Plenty of SH3s targets TAK1 to control immunity signalling in Drosophila.

Imd‐mediated innate immunity is activated in response to infection by Gram‐negative bacteria and leads to the activation of Jun amino‐terminal kinase (JNK) and Relish, a nuclear factor‐κB transcription factor responsible for the expression of antimicrobial peptides. Plenty of SH3s (POSH) has been shown to function as a scaffold protein for JNK activation, leading to apoptosis in mammals. Here, we report that POSH controls Imd‐mediated immunity signalling in Drosophila. In POSH‐deficient flies, JNK activation and Relish induction were delayed and sustained, which indicated that POSH is required for properly timed activation and termination of the cascade. The RING finger of POSH, possessing ubiquitin‐ligase activity, was essential for termination of JNK activation. We show that POSH binds to and degrades TAK1, a crucial activator of both the JNK and the Relish signalling pathways. These results establish a novel role for POSH in the Drosophila immune system.

The calcineurin regulator sra plays an essential role in female meiosis in Drosophila.

Modulatory calcineurin-interacting proteins (MCIPs)--also termed regulators of calcineurin (RCNs), calcipressins, or DSCR1 (Downs syndrome critical region 1)--are highly conserved regulators of calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase . Although overexpression experiments in several organisms have revealed that MCIPs inhibit calcineurin activity , their in vivo functions remain unclear. Here, we show that the Drosophila MCIP sarah (sra) is essential for meiotic progression in oocytes. Eggs from sra null mothers are arrested at anaphase of meiosis I. This phenotype was due to loss of function of sra specifically in the female germline. Sra is physically associated with the catalytic subunit of calcineurin, and its overexpression suppresses the phenotypes caused by constitutively activated calcineurin, such as rough eye or loss of wing veins. Hyperactivation of calcineurin signaling in the germline cells resulted in a meiotic-arrest phenotype, which can also be suppressed by overexpression of Sra. All these results support the hypothesis that Sra regulates female meiosis by controlling calcineurin activity in the germline. To our knowledge, this is the first unambiguous demonstration that the regulation of calcineurin signaling by MCIPs plays a critical role in a defined biological process.

Calcineurin and Its Regulator Sra/DSCR1 Are Essential for Sleep in Drosophila

Sleep is a fundamental biological process for all animals. However, the molecular mechanisms that regulate sleep are still poorly understood. Here we report that sleep-like behavior in Drosophila is severely impaired by mutations in sarah (sra), a member of the Regulator of Calcineurin (RCAN) family of genes. Sleep reduction in sra mutants is highly correlated with decreases in Sra protein levels. Pan-neural expression of sra rescues this behavioral phenotype, indicating that neuronal sra function is required for normal sleep. Since Sra regulates calcineurin (CN), we generated and examined the behavior of knock-out mutants for all Drosophila CN genes: CanA-14F, Pp2B-14D, and CanA1 (catalytic subunits), and CanB and CanB2 (regulatory subunits). While all mutants show at least minor changes in sleep, CanA-14FKO and CanBKO have striking reductions, suggesting that these are the major CN subunits regulating sleep. In addition, neuronal expression of constitutively active forms of CN catalytic subunits also significantly reduces sleep, demonstrating that both increases and decreases in CN activity inhibit sleep. sra sleep defects are suppressed by CN mutations, indicating that sra and CN affect sleep through a common mechanism. Our results demonstrate that CN and its regulation by Sra are required for normal sleep in Drosophila and identify a critical role of Ca2+/calmodulin-dependent signaling in sleep regulation.

POSH, a scaffold protein for JNK signaling, binds to ALG-2 and ALIX in Drosophila

Plenty of SH3s (POSH) functions as a scaffold protein for the Jun N‐terminal kinase (JNK) signal transduction pathway, which leads to cell death in mammalian cultured cells and Drosophila. Here, we show that POSH forms a complex with Apoptosis‐linked gene‐2 (ALG‐2) and ALG‐2‐interacting protein (ALIX/AIP1) in a calcium‐dependent manner. Overexpression of ALG‐2 or ALIX in developing imaginal eye discs resulted in roughened or melanized eyes, respectively. These phenotypes were enhanced by co‐overexpression of POSH. We found that overexpression of either gene could induce ectopic JNK activation, suggesting that POSH/ALG‐2/ALIX may function together in the regulation of the JNK pathway.

A gain-of-function screen identifies wdb and lkb1 as lifespan-extending genes in Drosophila.

The insulin/insulin-like growth factor (IGF) and the target of rapamycin (TOR) signaling pathways are known to regulate lifespan in diverse organisms. However, only a limited number of genes involved in these pathways have been examined regarding their effects on lifespan. Through a gain-of-function screen in Drosophila, we found that overexpression of the wdb gene encoding a regulatory subunit of PP2A, and overexpression of the lkb1 gene encoding a serine/threonine kinase, reduced organ size and extended lifespan. Overexpression of wdb also reduced the level of phosphorylated AKT, while overexpression of lkb1 increased the level of phosphorylated AMPK and decreased the level of phosphorylated S6K. Taken together, our results suggest that wdb- and lkb1-dependent lifespan extension is mediated by downregulation of S6K, a downstream component of the insulin/IGF and TOR signaling pathways.

Loss of Trx-2 enhances oxidative stress-dependent phenotypes in Drosophila.

Overexpression of thioredoxin (TRX) confers oxidative stress resistance and extends lifespan in mammals and insects. However, less is known about phenotypes associated with loss of TRX. We investigated loss‐of‐function phenotypes of Trx‐2 in Drosophila, and found that the mutant flies are hyper‐susceptible to paraquat, a free radical generator, but not to hydrogen peroxide. They contain a high amount of protein carbonyl, which dramatically increases with age. Trx‐2 mutants express high levels of anti‐oxidant genes, such as superoxide dismutase, catalase, and glutathione synthetase. This is the first demonstration of biochemical and physiological consequences caused by loss of Trx‐2 in Drosophila.

MachiBase: a Drosophila melanogaster 5′-end mRNA transcription database

MachiBase (http://machibase.gi.k.u-tokyo.ac.jp/) provides a comprehensive and freely accessible resource regarding Drosophila melanogaster 5′-end mRNA transcription at different developmental states, supporting studies on the variabilities of promoter transcriptional activities and gene-expression profiles in the fruitfly. The data were generated in conjunction with the recently developed high-throughput genome sequencer Illumina/Solexa using a newly developed 5′-end mRNA collection method.

Insulin-degrading enzyme antagonizes insulin-dependent tissue growth and Aβ-induced neurotoxicity in Drosophila

Insulin‐degrading enzyme (IDE) is implicated in the pathogenesis of type 2 diabetes mellitus (DM2) and Alzheimers disease (AD). Here we provide genetic evidence that Drosophila Ide (dIde) antagonizes the insulin signaling pathway and human Aβ‐induced neurotoxicity in Drosophila. In this study, we also generated a dIde knockout mutant (dIde KO ) by gene targeting, and found that loss of IDE increases the content of the major insect blood sugar, trehalose, thus suggesting a conserved role of IDE in sugar metabolism. Using dIde KO as a model, further investigations into the biological functions of IDE and its role in the pathogenesis of DM2 and AD can be made.

Impaired energy metabolism in a Drosophila model of mitochondrial aconitase deficiency.

Aconitase catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle, and its deficiency in humans is associated with an infantile neurodegenerative disorder affecting mainly the cerebellum and retina. Here we investigated the effect of gene knockout and knockdown of the mitochondrial aconitase Acon in Drosophila. Acon-knockout flies were homozygous lethal, indicating that Acon is essential for viability. RNA interference-generated Acon-knockdown flies exhibited a variety of phenotypes, such as reduced locomotor activity, a shortened lifespan, and increased cell death in the developing brain. Metabolomic analysis revealed that acetyl-CoA, citrate/isocitrate, and cis-aconitate were significantly increased, while most metabolites of glycolysis and the TCA cycle were reduced. Reduced triacylglyceride and increased acetyl-CoA suggested that lipids were used as an energy source because of the impaired glycolysis and TCA cycle. The Acon-knockdown model should facilitate further understanding of the pathophysiology of m-aconitase deficiency in humans.