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Methionine restriction (MetR) extends lifespan across different species and exerts beneficial effects on metabolic health and inflammatory responses. In contrast, certain cancer cells exhibit methionine auxotrophy that can be exploited for therapeutic treatment, as decreasing dietary methionine selectively suppresses tumor growth. Thus, MetR represents an intervention that can extend lifespan with a complementary effect of delaying tumor growth. Beyond its function in protein synthesis, methionine feeds into complex metabolic pathways including the methionine cycle, the transsulfuration pathway, and polyamine biosynthesis. Manipulation of each of these branches extends lifespan; however, the interplay between MetR and these branches during regulation of lifespan is not well understood. In addition, a potential mechanism linking the activity of methionine metabolism and lifespan is regulation of production of the methyl donor S‐adenosylmethionine, which, after transferring its methyl group, is converted to S‐adenosylhomocysteine. Methylation regulates a wide range of processes, including those thought to be responsible for lifespan extension by MetR. Although the exact mechanisms of lifespan extension by MetR or methionine metabolism reprogramming are unknown, it may act via reducing the rate of translation, modifying gene expression, inducing a hormetic response, modulating autophagy, or inducing mitochondrial function, antioxidant defense, or other metabolic processes. Here, we review the mechanisms of lifespan extension by MetR and different branches of methionine metabolism in different species and the potential for exploiting the regulation of methyltransferases to delay aging. A potential mechanism linking the activity of methionine metabolism and lifespan is regulation of production of the methyl donor S‐adenosylmethionine (SAM), which, after transferring its methyl group, is converted to S‐adenosylhomocysteine (SAH). Methionine metabolism determines the ratio of SAM/SAH metabolites and affects most of the methylation reactions in the cell, which in turn regulate a wide range of processes including ones that were attributed to be responsible for the lifespan extension by MetR.

Despite the deep conservation of the DNA damage response (DDR) pathway, cells in different contexts vary widely in their susceptibility to DNA damage and their propensity to undergo apoptosis as a result of genomic lesions. One of the cell signaling pathways implicated in modulating the DDR is the highly conserved Wnt pathway, which is known to promote resistance to DNA damage caused by ionizing radiation in a variety of human cancers. However, the mechanisms linking Wnt signal transduction to the DDR remain unclear. Here, we use a genetically encoded system in Drosophila to reliably induce consistent levels of DNA damage in vivo, and demonstrate that canonical Wnt signaling in the wing imaginal disc buffers cells against apoptosis in the face of DNA double-strand breaks. We show that Wg, the primary Wnt ligand in Drosophila , activates epidermal growth factor receptor (EGFR) signaling via the ligand-processing protease Rhomboid, which, in turn, modulates the DDR in a Chk2 -, p53 -, and E2F1- dependent manner. These studies provide mechanistic insight into the modulation of the DDR by the Wnt and EGFR pathways in vivo in a highly proliferative tissue. Furthermore, they reveal how the growth and patterning functions of Wnt signaling are coupled with prosurvival, antiapoptotic activities, thereby facilitating developmental robustness in the face of genomic damage.

Stem cells of the Drosophila midgut sense mechanical signals in vivo through the stretch-activated ion channel Piezo, which is expressed on previously unidentified enteroendocrine precursor cells. Stretch-activated ion channel drives cell differentiation The effect of mechanical cues on the behaviour of cells in culture is well documented, but such effects are more difficult to study in vivo . Norbert Perrimon and colleagues find that stem cells of the Drosophila gut sense mechanical signals in vivo through the stretch-activated ion channel Piezo. Piezo is expressed in a subset of enteroendocrine precursor cells. Loss of Piezo reduces the differentiation of the enteroendocrine lineage in adults, while the over expression of this gene in gut stem cells has the reverse effect. Further analysis shows that Piezo activates the calcium signalling pathway in response to mechanical stimuli. Somatic stem cells constantly adjust their self-renewal and lineage commitment by integrating various environmental cues to maintain tissue homeostasis. Although numerous chemical and biological signals have been identified that regulate stem-cell behaviour, whether stem cells can directly sense mechanical signals in vivo remains unclear 1 . Here we show that mechanical stress regulates stem-cell differentiation in the adult Drosophila midgut through the stretch-activated ion channel Piezo. We find that Piezo is specifically expressed in previously unidentified enteroendocrine precursor cells, which have reduced proliferation ability and are destined to become enteroendocrine cells. Loss of Piezo activity reduces the generation of enteroendocrine cells in the adult midgut. In addition, ectopic expression of Piezo in all stem cells triggers both cell proliferation and enteroendocrine cell differentiation. Both the Piezo mutant and overexpression phenotypes can be rescued by manipulation of cytosolic Ca 2+ levels, and increases in cytosolic Ca 2+ resemble the Piezo overexpression phenotype, suggesting that Piezo functions through Ca 2+ signalling. Further studies suggest that Ca 2+ signalling promotes stem-cell proliferation and differentiation through separate pathways. Finally, Piezo is required for both mechanical activation of stem cells in a gut expansion assay and the increase of cytosolic Ca 2+ in response to direct mechanical stimulus in a gut compression assay. Thus, our study demonstrates the existence of a specific group of stem cells in the fly midgut that can directly sense mechanical signals through Piezo.

The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution. How the Drosophila lymph gland hemocytes develop and are regulated at a single-cell level is unclear. Here, the authors use single-cell RNA sequencing to show heterogeneity of developing hemocytes in the lymph gland and how they react to wasp infestation, and compare hemocytes from two independent origins.

Genome-wide screens in Drosophila cells have offered numerous insights into gene function, yet a major limitation has been the inability to stably deliver large multiplexed DNA libraries to cultured cells allowing barcoded pooled screens. Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in Drosophila S2R+ cells. Under basal growth conditions, 1235 genes were essential for cell fitness at a false-discovery rate of 5%, representing the highest-resolution fitness gene set yet assembled for Drosophila, including 407 genes which likely duplicated along the vertebrate lineage and whose orthologs were underrepresented in human CRISPR screens. We additionally performed context-specific fitness screens for resistance to or synergy with trametinib, a Ras/ERK/ETS inhibitor, or rapamycin, an mTOR inhibitor, and identified key regulators of each pathway. The results present a novel, scalable, and versatile platform for functional genomic screens in invertebrate cells. Genes are made up of DNA and carry the instructions necessary to build an organism. Humans have over 20,000 genes, while other animals, such as fruit flies, have about 14,000. An ongoing challenge in biology is to identify the role of every gene in the human body. Since most of them are conserved in the fruit fly, this insect is one of the most extensively studied organisms. Scientists often use a technique called CRISPR to edit genes. It enables researchers to modify DNA sequences to selectively alter the purpose of a gene or even turn it off to find out what it does. CRISPR requires a guide molecule (for example, sgRNAs), which leads the system to a particular DNA sequence to start the process. Often, researchers create many sgRNAs and deliver them to a large pool of cells with the help of viruses, so that each cell gets a different sgRNA that mutates a different gene. When the cells are then treated with a specific drug, the composition of the sgRNAs in the pool changes, depending on which genes are needed to withstand the drug, and which genes – when turned-off – create cells that are resistant to the drug. Although thousands of mutant flies have been created to investigate how a deactivated or faulty gene can affect the health and behavior of the fly, we still lack meaningful information on about half of their genes. This is partly because the viruses used to deliver sgRNAs in mammals do not work in fly cells. Here, Viswanatha et al. developed a simple protocol to generate cell pools of CRISPR mutants, which uses a new strategy that uses bacteria to deliver DNA to fly cells. This allowed to identify over 1,000 genes necessary for cells to multiply properly, many of which had not been studied before. The technique was also used in combination with drugs to examine the interactions between genes and drugs – an approach that could be further adapted to examine interactions between genes and nutrients, or between genes. This new approach will open doors to systematically uncover the purpose of every gene in the fly. A better understanding of what genes do could help to identify potential genetic weaknesses in certain types of cancer or other diseases, which may lead to the development of more effective treatments. Moreover, the method is likely to work in other insects, for example, mosquitos, where it may uncover new genes involved in mosquito-borne diseases such as malaria or Zika virus.

Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns. Fruit flies and other animals have complex body plans containing many different types of cells. To make and maintain these body plans, individual genes must be switched on and off at specific times in particular cells to control how the animal grows. Some of these genes may be switched on for long periods of time, while others may be rapidly switched on and off on repeated occasions. Fluorescent reporter proteins have been extensively used to study gene activity in cells. Typically, this involves linking the gene encoding the fluorescent reporter to a gene of interest, so that when the gene is switched on a fluorescent protein will also be produced. The fluorescent protein emits light of a particular color and measuring this light provides a way to monitor a gene’s activity. Unfortunately, fluorescent proteins tend to break down slowly, and the level of fluorescence emitted cannot fluctuate quickly enough to reflect rapid changes in gene behavior. One way to overcome this limitation is to use destabilized fluorescent proteins that degrade more rapidly inside cells. However, current strategies for creating these proteins cause them to emit less light, making fluorescence more difficult to detect. To address this issue, He et al. developed a new green destabilized protein, adding elements that increase production of the protein so a greater amount of light can be emitted. The green destabilized protein was then combined with a red fluorescent reporter that degrades more slowly to develop a new tool called TransTimer. When the gene linked to the reporter switches on, the green destabilized protein turns on before the red reporter turns on. But, as the gene switches off, the destabilized protein will degrade until only the red signal remains. This allows the ratio of green to red color emitted from the TransTimer to indicate the timing of a gene’s activity. Using this tool, He et al. uncovered new details about the patterns of activity of two signals, known as Notch and STAT, that were largely missed by studies using traditional fluorescent reporters. Further experiments demonstrated that TransTimer can be used to carry out large-scale screens in living fruit flies, which have not been possible with more time consuming live-cell imaging techniques. The fluorescent reporter developed by He et al. will be a useful tool to understand when and where genes are switched on during the lives of fruit flies. In the future, TransTimer could be adapted for use in other model animals or plants.

Background Mapping of orthologous genes among species serves an important role in functional genomics by allowing researchers to develop hypotheses about gene function in one species based on what is known about the functions of orthologs in other species. Several tools for predicting orthologous gene relationships are available. However, these tools can give different results and identification of predicted orthologs is not always straightforward. Results We report a simple but effective tool, the D rosophila RNAi Screening Center I ntegrative O rtholog P rediction T ool (DIOPT; http://www.flyrnai.org/diopt ), for rapid identification of orthologs. DIOPT integrates existing approaches, facilitating rapid identification of orthologs among human, mouse, zebrafish, C. elegans, Drosophila , and S. cerevisiae . As compared to individual tools, DIOPT shows increased sensitivity with only a modest decrease in specificity. Moreover, the flexibility built into the DIOPT graphical user interface allows researchers with different goals to appropriately 'cast a wide net' or limit results to highest confidence predictions. DIOPT also displays protein and domain alignments, including percent amino acid identity, for predicted ortholog pairs. This helps users identify the most appropriate matches among multiple possible orthologs. To facilitate using model organisms for functional analysis of human disease-associated genes, we used DIOPT to predict high-confidence orthologs of disease genes in Online Mendelian Inheritance in Man (OMIM) and genes in genome-wide association study (GWAS) data sets. The results are accessible through the DIOPT diseases and traits query tool (DIOPT-DIST; http://www.flyrnai.org/diopt-dist ). Conclusions DIOPT and DIOPT-DIST are useful resources for researchers working with model organisms, especially those who are interested in exploiting model organisms such as Drosophila to study the functions of human disease genes.

Over the past decade, numerous reports have underscored the similarities between the metabolism of Drosophila and vertebrates, with the identification of evolutionarily conserved enzymes and analogous organs that regulate carbohydrate and lipid metabolism. It is now well established that the major metabolic, energy-sensing and endocrine signaling networks of vertebrate systems are also conserved in flies. Accordingly, studies in Drosophila are beginning to unravel how perturbed energy balance impinges on lifespan and on the ensuing diseases when energy homeostasis goes awry. Here, we highlight several emerging concepts that are at the nexus between obesity, nutrient sensing, metabolic homeostasis and aging. Specifically, we summarize the endocrine mechanisms that regulate carbohydrate and lipid metabolism, and provide an overview of the neuropeptides that regulate feeding behavior. We further describe the various efforts at modeling the effects of high-fat or -sugar diets in Drosophila and the signaling mechanisms involved in integrating organ function. Finally, we draw attention to some of the cardinal discoveries made with these disease models and how these could spur new research questions in vertebrate systems.

Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. Here, we report that ENDOG released from mitochondria promotes autophagy during starvation, which we find to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans . Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. Our results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response. The role of Endonuclease G in autophagy remains unclear. Here the authors report that ENDOG is released from mitochondria during starvation and promotes autophagy by suppressing mTOR signaling and activating DNA damage response.

Drosophila blood cells, called hemocytes, are classified into plasmatocytes, crystal cells, and lamellocytes based on the expression of a few marker genes and cell morphologies, which are inadequate to classify the complete hemocyte repertoire. Here, we used single-cell RNA sequencing (scRNA-seq) to map hemocytes across different inflammatory conditions in larvae. We resolved plasmatocytes into different states based on the expression of genes involved in cell cycle, antimicrobial response, and metabolism together with the identification of intermediate states. Further, we discovered rare subsets within crystal cells and lamellocytes that express fibroblast growth factor (FGF) ligand branchless and receptor breathless, respectively. We demonstrate that these FGF components are required for mediating effective immune responses against parasitoid wasp eggs, highlighting a novel role for FGF signaling in inter-hemocyte crosstalk. Our scRNA-seq analysis reveals the diversity of hemocytes and provides a rich resource of gene expression profiles for a systems-level understanding of their functions.