The Multifunctional Drosophila melanogaster V-ATPase Is Encoded by a Multigene Family

In animals, V-ATPases are believed to play roles in the plasma membrane,as well as endomembrane. To understand these different functions, it isnecessary to adopt a genetic approach in a physiologically tractable modelorganism. For this purpose, Drosophila melanogaster is ideal,because of the powerful genetics associated with the organism and because ofthe unusually informative epithelial phenotype provided by the Malpighiantubule. Recently, the first animal knockouts of a V-ATPase weredescribed in Drosophila. The resulting phenotypes have generalutility for our understanding of V-ATPase function and suggest a screen fornovel subunits and associated proteins. Genome project resources haveaccelerated our knowledge of the V-ATPase gene family size and the newDrosophila genes vhaSFD, vha100-1, vha100-2, vha100-3, vha16-2,vha16-3, vha16-4, vhaPPA1, vhaPPA2, vhaM9.7.1, and vhaM9.7.2are described. The Drosophila V-ATPase model is thus well-suited toboth forward and reverse genetic analysis of this complex multifunctionalenzyme.     

The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila

The V(0) complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V(0) component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.     

Reduced Gut Acidity Induces an Obese-Like Phenotype in Drosophila melanogaster and in Mice

In order to identify genes involved in stress and metabolic regulation, we carried out a Drosophila P-element-mediated mutagenesis screen for starvation resistance. We isolated a mutant, m2, that showed a 23% increase in survival time under starvation conditions. The P-element insertion was mapped to the region upstream of the vha16-1 gene, which encodes the c subunit of the vacuolar-type H⁺-ATPase. We found that vha16-1 is highly expressed in the fly midgut, and that m2 mutant flies are hypomorphic for vha16-1 and also exhibit reduced midgut acidity. This deficit is likely to induce altered metabolism and contribute to accelerated aging, since vha16-1 mutant flies are short-lived and display increases in body weight and lipid accumulation. Similar phenotypes were also induced by pharmacological treatment, through feeding normal flies and mice with a carbonic anhydrase inhibitor (acetazolamide) or proton pump inhibitor (PPI, lansoprazole) to suppress gut acid production. Our study may thus provide a useful model for investigating chronic acid suppression in patients.     

果蝇胰岛素样肽8(Dilp8)的功能及作用机制

果蝇Drosophila作为一种模式生物,具有生长周期短、繁殖能力强和研究成本低等优点。而且果蝇有65%的基因与人类同源,特别是其遗传背景简单的特点,使其在生物生长发育研究、病理机制研究和基因表达调控等研究中发挥重要作用。目前在果蝇中已发现8种胰岛素样肽,即果蝇胰岛素样肽1-8(Drosophila insulin-like peptide 1-8, Dilp1-8),而对于果蝇胰岛素信号通路的研究主要集中在其调控机体生长发育和能量代谢的方面,这些功能主要通过Dilp1-7来发挥作用。对于Dilp8的功能及其发挥作用的分子机制知之甚少。本文总结了自Dilp8被发现以来,人们对于其功能的研究结果。Dilp8主要在幼虫成虫盘和成年雌果蝇的卵巢中表达,其主要作用是调节果蝇的组织生长和发育时间,使果蝇生长为具有相对固定体型和一定对称性的个体。当果蝇幼虫在生长过程中受到损伤时,Dilp8会通过延缓发育时间来缓解异常生长。Dilp8被激活后,在中枢神经系统与其受体富含亮氨酸重复序列的G蛋白偶联受体3(leucine-rich repeat-containing G protein-coupled ...     

Probing Subunit-Subunit Interactions in the Yeast Vacuolar ATPase by Peptide Arrays

Background

Vacuolar (H⁺)-ATPase (V-ATPase; V₁V_o-ATPase) is a large multisubunit enzyme complex found in the endomembrane system of all eukaryotic cells where its proton pumping action serves to acidify subcellular organelles. In the plasma membrane of certain specialized tissues, V-ATPase functions to pump protons from the cytoplasm into the extracellular space. The activity of the V-ATPase is regulated by a reversible dissociation mechanism that involves breaking and re-forming of protein-protein interactions in the V₁-ATPase - V_o-proton channel interface. The mechanism responsible for regulated V-ATPase dissociation is poorly understood, largely due to a lack of detailed knowledge of the molecular interactions that are responsible for the structural and functional link between the soluble ATPase and membrane bound proton channel domains.

Methodology/Principal Findings

To gain insight into where some of the stator subunits of the V-ATPase associate with each other, we have developed peptide arrays from the primary sequences of V-ATPase subunits. By probing the peptide arrays with individually expressed V-ATPase subunits, we have identified several key interactions involving stator subunits E, G, C, H and the N-terminal domain of the membrane bound a subunit.

Conclusions

The subunit-peptide interactions identified from the peptide arrays complement low resolution structural models of the eukaryotic vacuolar ATPase obtained from transmission electron microscopy. The subunit-subunit interaction data are discussed in context of our current model of reversible enzyme dissociation.     

Na,K-ATPase and V-ATPase in ovarian follicles of Drosophila melanogaster.

Uncovering the cause and meaning of bioelectric phenomena in developing systems requires investigations of the distribution and activity of ion-transport mechanisms. In order to identify and localize ion pumps in ovarian follicles of Drosophila, we used immunofluorescence microscopy, immunoelectron microscopy, subcellular fractionation, immunoblots, and acridine-orange staining. We applied various antibodies directed against the Na,K-pump (Na,K-ATPase) and against vacuolar-type proton pumps (V-ATPase). During all phases of oogenesis, Na,K-ATPase were found in apical and lateral follicle-cell membranes and, during rapid follicle growth (beginning with stage 10), also in nurse-cell membranes and in the oolemma. V-ATPase were detected in various cytoplasmic vesicles and in yolk spheres and, beginning with stage 10, also in apical follicle-cell membranes and in the oolemma. Given these and earlier results, we propose that: 1) V-ATPase coupled to secondary active antiporters represent the ouabain-intensitive potassium pumps described previously; 2) both Na,K-ATPase and V-ATPase are involved in bioelectric phenomena as well as in osmoregulation and follicle growth, especially during stages 10-12; 3) organelle-associated V-ATPase play a role in vesicle acidification and in yolk processing; and 4) the channel-forming protein ductin is a component of both V-ATPase and gap junctions in ovarian follicles of Drosophila.     

The Drosophila melanogaster gene vha14 encoding a 14-kDa F-subunit of the vacuolar ATPase

A Drosophila melanogaster (Dm) cDNA (vha14) encoding the 14-kDa F-subunit of the vacuolar H⁺-ATPase (V-ATPase) has been cloned via homology with the corresponding Manduca sexta (Ms) gene. Its deduced translation product is a 124-amino-acid polypeptide sharing 90% identity with the Ms polypeptide and 50% identity with an analogous polypeptide of Saccharomyces cerevisiae, and a more distant similarity to a subunit of the Na⁺-transporting ATPase of Enterococcus hirae. Homology was also found with expressed sequence tags from man, Arabidopsis thaliana, Caenorhabditis elegans and C. briggsiae, Oryza sativa and Plasmodium falciparum, indicating that the subunit is phylogenetically conserved. The Dm gene (vha14) is present as a single copy at cytological position 52B on the second chromosome, and gives rise to an mRNA species of 0.65 kb. Expression of the latter shows relatively little variation during development, or between adult head, thorax and abdomen, suggesting that the F-subunit is a relatively ubiquitous component of the V-ATPase.     

Enzymatic studies of DNA repair in Drosophila melanogaster

Thus far, our studies in Drosophila have concentrated primarily on the various enzymes involved in the in vitro repair of modified or nonconventional DNA substrates. In some cases, our findings have led us to investigate events that may not have a bearing on DNA repair, but rather may be associated with developmental signals important to the maturation of the organism. As appealing as some of these models seem, however, they must await confirmation through detailed genetic studies before any substantial conclusions can be drawn. This combination of genetic and biochemical knowledge makes Drosophila an exciting organism for an eventual detailed understanding of the developmental expression and cellular location of DNA-repair systems.     

Arrhythmia caused by a Drosophila tropomyosin mutation is revealed using a novel optical coherence tomography instrument

Background

Dilated cardiomyopathy (DCM) is a severe cardiac condition that causes high mortality. Many genes have been confirmed to be involved in this disease. An ideal system with which to uncover disease mechanisms would be one that can measure the changes in a wide range of cardiac activities associated with mutations in specific, diversely functional cardiac genes. Such a system needs a genetically manipulable model organism that allows in vivo measurement of cardiac phenotypes and a detecting instrument capable of recording multiple phenotype parameters.

Methodology and Principal Findings

With a simple heart, a transparent body surface at larval stages and available genetic tools we chose Drosophila melanogaster as our model organism and developed for it a dual en-face/Doppler optical coherence tomography (OCT) instrument capable of recording multiple aspects of heart activity, including heart contraction cycle dynamics, ostia dynamics, heartbeat rate and rhythm, speed of heart wall movement and light reflectivity of cardiomyocytes in situ. We applied this OCT instrument to a model of Tropomyosin-associated DCM established in adult Drosophila. We show that DCM pre-exists in the larval stage and is accompanied by an arrhythmia previously unidentified in this model. We also detect reduced mobility and light reflectivity of cardiomyocytes in mutants.

Conclusion

These results demonstrate the capability of our OCT instrument to characterize in detail cardiac activity in genetic models for heart disease in Drosophila.     

Recent and recurrent selective sweeps of the antiviral RNAi gene Argonaute-2 in three species of Drosophila

Antagonistic host-parasite interactions can drive rapid adaptive evolution in genes of the immune system, and such arms races may be an important force shaping polymorphism in the genome. The RNA interference pathway gene Argonaute-2 (AGO2) is a key component of antiviral defense in Drosophila, and we have previously shown that genes in this pathway experience unusually high rates of adaptive substitution. Here we study patterns of genetic variation in a 100-kbp region around AGO2 in three different species of Drosophila. Our data suggest that recent independent selective sweeps in AGO2 have reduced genetic variation across a region of more than 50 kbp in Drosophila melanogaster, D. simulans, and D. yakuba, and we estimate that selection has fixed adaptive substitutions in this gene every 30-100 thousand years. The strongest signal of recent selection is evident in D. simulans, where we estimate that the most recent selective sweep involved an allele with a selective advantage of the order of 0.5-1% and occurred roughly 13-60 Kya. To evaluate the potential consequences of the recent substitutions on the structure and function of AGO2, we used fold-recognition and homology-based modeling to derive a structural model for the Drosophila protein, and this suggests that recent substitutions in D. simulans are overrepresented at the protein surface. In summary, our results show that selection by parasites can consistently target the same genes in multiple species, resulting in areas of the genome that have markedly reduced genetic diversity.     

Organization of the Rosy Locus in DROSOPHILA MELANOGASTER: Further Evidence in Support of a CIS-Acting Control Element Adjacent to the Xanthine Dehydrogenase Structural Element

The present report summarizes our recent progress in the genetic dissection of an elementary genetic unit in a higher organism, the rosy locus (ry:3--52.0) in Drosophila melanogaster. Pursuing the hypothesis that the rosy locus includes a noncoding control region, as well as a structural element coding for the xanthine dehydrogenase (XDH) peptide, experiments are described that characterize and map a rosy locus variant associated with much lower than normal levels of XDH activity. Experiments are described that fail to relate this phenotype to alteration in the structure of the XDH peptide, but clearly associate this character with variation in number of molecules of XDH per fly. Large-scale fine-structure recombination experiments locate the genetic basis for this variation in the number of molecules of XDH per fly to a site immediately to the left of the XDH structural element within a region previously designated as the XDH control element. Moreover, experiments clearly separate this "underproducer" variant site from a previously described "overproducer" site within the control region. Examination of enzyme activity in electrophoretic gels of appropriate heterozygous genotypes demonstrates the cis-acting nature of this variation in the number of molecules of XDH. A revision of the map of the rosy locus, structural and control elements is presented in the light of the additional mapping data now available.     

CRISPR/Cas9 and Genome Editing in Drosophila

Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.     

Vacuole dynamics in the salivary glands of Drosophila melanogaster during prepupal development

A central function of the Drosophila salivary glands (SGs), historically known for their polytene chromosomes, is to produce and then release during pupariation the secretory glue used to affix a newly formed puparium to a substrate. This essential event in the life history of Drosophila is regulated by the steroid hormone ecdysone in the late‐larval period. Ecdysone triggers a cascade of sequential gene activation that leads to glue secretion and initiates the developmentally‐regulated programmed cell death (PCD) of the larval salivary glands, which culminates 16 h after puparium formation (APF). We demonstrate here that, even after the larval salivary glands have completed what is perceived to be one of their major biological functions – glue secretion during pupariation – they remain dynamic and physiologically active up until the execution phase of PCD. We have used specific metabolic inhibitors and genetic tools, including mutations or transgenes for shi, Rab5, Rab11, vha55, vha68‐2, vha36‐1, syx1A, syx4, and Vps35 to characterize the dramatic series of cellular changes occurring in the SG cells between pupariation and 7–8 h APF. Early in the prepupal period, they are remarkably active in endocytosis, forming acidic vacuoles. Midway through the prepupal period, there is abundant late endosomal trafficking and vacuole growth, which is followed later by vacuole neutralization and disappearance via membrane consolidation. This work provides new insights into the function of Drosophila SGs during the early‐ to mid‐prepupal period.     

Aging Is Not Associated with Proteasome Impairment in UPS Reporter Mice

Background

Covalent linkage of ubiquitin regulates the function and, ultimately, the degradation of many proteins by the ubiquitin-proteasome system (UPS). Given its essential role in protein regulation, even slight perturbations in UPS activity can substantially impair cellular function.

Methodology/Principal Findings

We have generated and characterized a novel transgenic mouse model which expresses a previously described reporter for UPS function. This UPS reporter contains a degron sequence attached to the C-terminus of green fluorescent protein, and is predominantly expressed in neurons throughout the brain of our transgenic model. We then demonstrated that this reporter system is sensitive to UPS inhibition in vivo.

Conclusions/Significance

Given the obstacles associated with evaluating proteasomal function in the brain, our mouse model uniquely provides the capability to monitor UPS function in real time in individual neurons of a complex organism. Our novel mouse model now provides a useful resource with which to evaluate the impact of aging, as well as various genetic and/or pharmacological modifiers of neurodegenerative disease(s).     

Stem cell regulation by JAK/STAT signaling in Drosophila

Stem cells have become one of the "buzz" topics in the last decade or so. One of the best systems to study adult stem cells in vivo is in the model organism, Drosophila melanogaster. One hundred years of genetic analysis, a sequenced and highly annotated genome and genomics makes this a difficult organism to avoid. The JAK/STAT pathway has been shown to regulate stem cells during haematopoiesis and gametogenesis in Drosophila. In this review we cover the current literature and contrast each group of stem cells with respect to JAK/STAT signaling.     

Analysis of SEC9 Suppression Reveals a Relationship of SNARE Function to Cell Physiology

Background

Growth and division of Saccharomyces cerevisiae is dependent on the action of SNARE proteins that are required for membrane fusion. SNAREs are regulated, through a poorly understood mechanism, to ensure membrane fusion at the correct time and place within a cell. Although fusion of secretory vesicles with the plasma membrane is important for yeast cell growth, the relationship between exocytic SNAREs and cell physiology has not been established.

Methodology/Principal Findings

Using genetic analysis, we identified several influences on the function of exocytic SNAREs. Genetic disruption of the V-ATPase, but not vacuolar proteolysis, can suppress two different temperature-sensitive mutations in SEC9. Suppression is unlikely due to increased SNARE complex formation because increasing SNARE complex formation, through overexpression of SRO7, does not result in suppression. We also observed suppression of sec9 mutations by growth on alkaline media or on a non-fermentable carbon source, conditions associated with a reduced growth rate of wild-type cells and decreased SNARE complex formation.

Conclusions/Significance

Three main conclusions arise from our results. First, there is a genetic interaction between SEC9 and the V-ATPase, although it is unlikely that this interaction has functional significance with respect to membrane fusion or SNAREs. Second, Sro7p acts to promote SNARE complex formation. Finally, Sec9p function and SNARE complex formation are tightly coupled to the physiological state of the cell.     

Age-dependent usage of double-strand-break repair pathways

A DNA double-strand break (DSB) can be repaired by any of several alternative and competing mechanisms. The repaired sequences often differ from the original depending on which mechanism was used so that the cell's "choice" of repair mechanism can have profound genetic consequences. DSBs can accumulate with age , and human diseases that mimic some of the effects of aging, such as increased susceptibility to cancer, are associated with certain defects in DSB repair . The premeiotic germ cells of Drosophila provide a useful model for exploration of the connection between aging and DNA repair because these cells are subject to mortality and other age-related changes , and their DNA repair process is easily quantified. We used Rr3, a repair reporter system in Drosophila, to show that the relative usage of DSB repair mechanisms can change substantially as an organism ages. Homologous repair increased linearly in the male germline from 14% in young individuals to more than 60% in old ones, whereas two other pathways showed a corresponding decrease. Furthermore, the proportion of longer conversion tracts (>156 bp) also increased nearly 2-fold as the flies aged. These findings are relevant to the more general question of how DNA damage and repair are related to aging.     

Tyrosine phosphoproteomics and identification of substrates of protein tyrosine phosphatase dPTP61F in Drosophila S2 cells by mass spectrometry-based substrate trapping strategy

Recent biochemical and genetic approaches have clearly defined the functional role of critical components in tyrosine phosphorylation-dependent signal transduction. These signaling modulators often exhibit evolutionarily conserved functions across various species. It has been proposed that if protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs), and thousands of their substrates could be identified and characterized, it would significantly advance our understanding of the underlying mechanisms that control animal development and physiological homeostasis. The fruit fly Drosophila melanogester has been used extensively as a model organism for investigating the developmental processes, but the state of its tyrosine phosphorylation is poorly characterized. In the current study, we used advanced mass spectrometry (MS)-based shotgun analyses to profile the tyrosine phosphoproteome of Drosophila S2 cells. Using immunoaffinity isolation of the phosphotyrosine (pTyr) subproteome from cells treated with pervanadate followed by enrichment of phosphopeptides, we identified 562 nonredundant pTyr sites in 245 proteins. Both this predefined pTyr proteome subset and the total cell lysates were then used as sample sources to identify potential substrates of dPTP61F, the smallest member in terms of amino acid number and molecular weight in the Drosophila PTP family and the ortholog of human PTP1B and T Cell-PTP, by substrate trapping. In total, 20 unique proteins were found to be specifically associated with the trapping mutant form of dPTP61F, eluted by vanadate (VO4(3-)), and identified by MS analyses. Among them, 16 potential substrates were confirmed as tyrosine phosphorylated proteins, including a receptor PTK PDGF/VEGF receptor, a cytosolic PTK Abl, and several components of SCAR/WAVE complex, which may work in coordination to control actin dynamics. Thus, our data suggest that dPTP61F plays a central role in counteracting PTK-mediated signaling pathways as well as in regulating actin reorganization and remodeling through tyrosine dephosphorylation of critical substrates in Drosophila cells.     

Requirement of V-ATPase for ovulation and embryogenesis in Caenorhabditis elegans

Immunofluorescence analysis indicated that VHA-11, the C subunit of Caenorhabditis elegans V-ATPase, was localized in dot-like structures around the nuclei of early embryonic cells and was also detected in embryonic intestinal cells after comma stage. Vital staining with acridine orange showed that the intestinal cells had acidic compartments generated by V-ATPase, consistent with the intracellular localization of VHA-11. RNA interference could efficiently silence vha-11 gene expression: introduction of vha-11 double strand RNA led to embryonic lethality. Worms injected with the vha-11 double strand RNA produced embryos that became lethal. The development of embryos was arrested at various stages. However, their numbers gradually decreased, and the worms eventually became sterile due to the failure of ovulation. Similar results were obtained for RNA interference of the V-ATPase proteolipid genes. These results suggest that V-ATPases, and thus inside-acidic organelles, are required for ovulation and embryogenesis.