Browsing by Subject "Drosophila melanogaster"
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Item Biochemical Characterizations of Nicastrin, Barium Blockage of Potassium Channels and Calcium Blockage of Cyclic Nucleotide-Gated Channels(2015-08-07) Lam, Yee Ling; Rosenbaum, Daniel M.; Jiang, Youxing; Brautigam, Chad A.; Rizo-Rey, José; Yu, GangThis dissertation is divided into three independent chapters dedicated to research work in biochemical characterizations of nicastrin, barium blockage of potassium channels and calcium blockage of cyclic nucleotide-gated channels. The first chapter describes studies on an intramembrane protease called γ-secretase. This protease complex is known to generate the pathogenic peptide species in Alzheimer's disease, a neurodegenerative disease affecting billions around the globe. In particular, the study focused on understanding one subunit of the protease complex, the substrate receptor nicastrin. While the ultimate goal of crystallizing nicastrin was not achieved, the purification processes have revealed that glycosylation is important for folding or trafficking of the protein and disulfide bonds are crucial to maintain the tertiary structure of nicastrin. The second chapter focuses on a classic blocking phenomenon in potassium channels known as barium blocking. Previous studies on barium blocking have fueled our understanding on the ion conduction pore of potassium channels. A model of blockage has been proposed but has yet to be proven. This study recapitulated properties of barium blocking in the potassium channel NaK2K and provided structural evidence for the model of barium block. The crystal structure of NaK2K in the presence of sodium and barium showed a barium-binding pattern different from that of a structure in the presence of potassium and barium. The difference in binding explains the different blocking behaviors in the presence and absence of low concentrations of external potassium. The third chapter details a study on the weak calcium block of monovalent ion current in Drosophila cyclic nucleotide-gated (CNG) channel. Calcium blockage in the CNG channel arises from preferential but slow conduction of calcium. This blocking phenomenon is observed in all CNG channels, but to various extents. In this study, a threonine immediately outside of the selectivity filter was found to modulate the calcium block of Drosophila CNG channels, likely by causing the channel to adopt a selectivity filter structure different from canonical CNG channels. This threonine is unique to the CNG channels of Drosophila and a few insects, whereas most other CNG channels have a proline at the equivalent position. These findings have allowed us to identify a structure-function relationship for calcium blockage in Drosophila CNG channels.Item Causes of Naive Ethanol Avoidance in Drosophila Melanogaster(2016-06-22) Ritz, Morgan Paige; Rothenfluh, Adrian; Rodan, Aylin; Self, David W.BACKGROUND: Alcohol dependence is a pressing public health concern, yet little is still known about its molecular causes. Although current studies have started to understand human addiction, Drosophila research is used as a tool to carry out more genetic and behavioral approaches that are crucial in learning about the addiction process. OBJECTIVE: The aim of this project was to understand the mechanisms of ethanol avoidance in Drosophila. METHODS: I applied quantitative ingestion assays to determine the amounts of food flies ate, with or without supplemented ethanol. I also used a choice assay, the FRAPPE, to determine whether naive flies exhibited preference for 15% ethanol. To interfere with neuronal function, I used the Gal4/UAS system, which allows for tissue specific manipulation of the activity of both neurons and genes. RESULTS: On average, Drosophila flies ate less sucrose when ethanol was added. One reason for this was that fewer flies initiated feeding. Upon silencing of gustatory neurons that perceive aversive tastes, flies showed less aversion to ethanol-containing food in the choice FRAPPE assay. As I increased the starvation time, almost all flies initiated feeding, but consumption amounts were still lowered when ethanol was supplemented. Additional feeding experiments where flies were only exposed to ethanol odor, but were unable to touch it, suggested that ethanol odor also suppresses food intake. I corroborated this with ethanol vapor exposures of defined intensity and duration: during the first minute of exposure, ethanol vapor stimulated food intake, but beyond that, it caused a suppression. Mutation in the ics gene affected ethanol-induced food suppression, but had no effect on the initial ethanol-induced stimulation of food intake. CONCLUSION: Drosophila flies show multimodal suppression of food intake by ethanol. Both the taste and smell of ethanol can reduce sucrose consumption. Interestingly, ethanol odor initially enhanced, but with continued exposure suppressed food intake. This suppression was abolished in ics mutants. This gene, whose human ortholog is linked to alcohol abuse disorders, is therefore critical for alcohol aversion, explaining how ics mutant flies show high, naive preference for ethanol-containing food.Item Characterization of Ataxin 2-Binding Protein 1 During Female Germ Cell Differentiation in Drosophila Melanogaster(2010-11-02) Yilmaz, Ömür; Buszczak, MichaelIn the Drosophila ovary, the molecules that promote the continued stepwise development of germline stem cells after their exit the niche remain largely unknown. During Drosophila oogenesis, a germline stem cell (GSC) divides asymmetrically to produce a renewing stem cell and a differentiated daughter that will progress through different stages of oogenesis to produce a mature egg. Numerous factors regulate the balance between GSC and a daughter cell, the cystoblast (CB). The Dpp/BMP signaling pathway from the niche silences bam transcription, a key differentiation factor, in the GSC. Bam protein is expressed in the cystoblast and forms a complex with its partner Bgcn, to antagonize the Pumilio-Nanos (Pum-Nos) complex through nos 3’UTR. Repression of Pum-Nos activity by the Bam-Bgcn complex permits CB differentiation. The CB goes through four incomplete divisions to give rise to a 16 cell cyst that buds off from the germarium to form an egg chamber. A number of genes have been found to have roles at 16 cell stage germ cell cysts but the molecular events that govern the intermediate stages (2-, 4- and 8-cell cysts) have remained elusive. In this study, I investigated the function of Drosophila homolog of the human disease gene Ataxin 2-binding protein 1 (A2BP1) during germline cyst differentiation. Through phenotypic analysis I showed that strong A2BP1 mutants display cystic tumors and mitosis to meiosis transition defects while weaker alleles have germline counting defects. Also, by genetic and biochemical analysis, I found that A2BP1 interacts with itself and Bruno, a known translational repressor. In addition, I examined the relationship between A2BP1 protein and other differentiation genes by expression analysis and showed that A2BP1 expression bridges the expression of the early differentiation factor Bam with late markers such as Bruno, Orb and Rbp9. The expression of A2BP1 is lost in bam, snf and mei-P26 mutants but is still present in rbp9 and arrest mutants. These observations might indicate existence of a linear hierarchy between these differentiation genes. In summary, my studies revealed A2BP1 defines a new tier within the genetic hierarchy that promotes the differentiation of single germ cells into mature 16-cell cysts during Drosophila oogenesis.Item Characterization of Drosophila Scap: Analysis of Mutants and Evidence for a Retention Factor(2011-08-10) Özdemir, Cafer; Rawson, Robert B.The SREBP pathway is one of the major regulators of lipid homeostasis and it is highly conserved among metazoans. SREBP is a transcription factor whose precursor is an endoplasmic reticulum (ER) transmembrane protein. In order to be activated it must travel to the Golgi apparatus via interaction with an escort protein, Scap. Scap, in turn can interact with components of the coatamer protein complex II (COPII) when lipid levels fall. In the Golgi, SREBP is cleaved sequentially by two proteases, S1P and S2P. By contrast to mammalian cells, which cannot survive without S2P or Scap, flies lacking Scap or S2P can activate SREBP. These mutants survive owing to non-canonical mechanisms of SREBP activation. Scap has a intrinsic tendency to travel to Golgi. In vertebrates, the ER retention factor, Insig, anchors the Scap:SREBP complex to the ER membrane when de novo lipid synthesis is not required. In Drosophila dSREBP pathway there is no Insig orthologues. However, our data suggest that there should be an analogous component that retains dScap in the ER. In order to discover the putative retention factor and other modifiers of the dSREBP, I set up a high through-put genome-wide screen. Employing luciferase as reporter, knocking-down each gene in genome through RNA interference will reveal the genes that modulate the activity of dSREBP.Item Characterization of Factors Controlling Germline Stem Cell Maintenance in Drosophila Melanogaster(2009-09-04) Park, Joseph Kwang; McKearin, DennisDuring Drosophila oogenesis, a germline stem cell (GSC) divides asymmetrically to produce a renewed stem cell and a differentiated daughter cystoblast (CB) that will progress through the 14 stages of oogenesis to produce a mature egg. A myriad of factors regulate GSC maintenance; extrinsic signals from the somatic niche integrate with intrinsic GSC factors to control CB differentiation pathway. In particular, studies of a key differentiation factor, bam, underscored the important paradigm of maintaining GSC by preventing the initiation of the CB differentiation pathway. The Dpp/BMP signaling pathway from the GSC niche promotes quiescence of bam transcription in the GSC. In the differentiating CB, bam transcription is initiated and Bam protein, together with its protein partner Bgcn, functions to antagonize the translational repression mediated by Pumilio-Nanos (Pum-Nos) complexes. Repression of Pum-Nos activity by Bam-Bgcn complexes permits CB differentiation, presumably through the derepression of CB-promoting mRNAs. In this study, I investigated the transcription- and translation-dependent mechanisms controlling the GSC to CB transition. Through genome-wide expression profiling experiments, I showed that there are only minimal transcriptional differences between the GSC and CB. This supports previous studies that highlight the importance of translational repression in maintaining a GSC state. However, some of the transcriptional differences between the undifferentiated GSC and differentiated germ cells were uncovered by expression profiling experiments of germ cells lacking Stonewall (Stwl), a protein required for GSC maintenance through epigenetic regulation of CB-promoting genes. Data from gene profiling experiments of stwl bam and bam mutant germ cells suggest that a suite of genes is normally repressed by Stwl to maintain a GSC fate. In addition, I examined whether putative "stemness" genes identified from mammalian systems also affected Drosophila GSC fate using a pilot screen in a hypomorphic bam background. Components from the COP9 signalosome complex (CSN) and the SCF E3 ubiquitin ligase complex were identified as dominant Suppressors of bam, Su(bam). Since both the CSN and SCF complexes are involved in protein degradation, the suppression of the bam phenotype suggests that they may be involved in stabilizing Bam function through abrogation of Bam turnover. Indeed, though ubiquitinated Bam isoforms were not identified, the abundance of Bam protein was increased in Su(bam) heterozygous animals. In other studies, I examined the requirement for the microRNA pathway in GSC maintenance through the examination of the double-stranded RNA-binding protein Loquacious (Loqs). Loqs enhances Dicer-1's ability to process pre-miRNA hairpin moieties to mature miRNA duplexes. Loqs is required for viability and germline mosaic analysis of loqs GSCs indicates an intrinsic, cell-autonomous requirement for the miRNA pathway in GSC maintenance. Loqs localizes to putative RNP complexes and a specialized region of the oocyte cytoplasm, termed the pole plasm. These data suggest that Loqs is a component of protein-RNA complexes that may be involved in mRNA translational inhibition. In summary, my studies revealed that GSC maintenance is achieved through the repression of CB-promoting factors; epigenetically through the actions of Stwl and other histone-associated proteins and translationally through the actions of the miRNA pathway via Loqs and Dicer-1. My studies provide insights into the understanding of the CB-promoting factors that are held inactive in the GSC and suggest that other stem cell systems may similarly employ multiple layers of repressive mechanisms to maintain a stem cell state.Item Deconstructing Collective Cell Death in a Genetic Model(2015-07-27) Garcia Hughes, Gianella; Rothenfluh, Adrian; Abrams, John M.; Kraus, W. Lee; Krämer, HelmutElimination of cells and tissues by apoptosis is a highly conserved process. In Drosophila, the entire wing epithelium is completely removed shortly after eclosion. The cells that make up this epithelium are collectively eliminated through a highly synchronized form of apoptotic cell death, involving canonical apoptosome genes. Here I present evidence that transcription of the IAP antagonist, head involution defective (hid), is acutely induced in wing epithelial cells prior to this process. hid mRNAs accumulate to levels that exceed a component of the ribosome and likewise, Hid protein becomes highly abundant in these same cells. hid function is required for collective cell death, since loss of function mutants show persisting wing epithelial cells and, furthermore, silencing of the hormone bursicon in the CNS produced collective cell death defective phenotypes manifested in the wing epithelium. Taken together, these observations suggest that acute induction of Hid primes wing epithelial cells for collective cell death and that Bursicon is a strong candidate to trigger this process, possibly by activating the abundant pool of Hid already present. This model of collective cell death in the wing is predictable, easy to observe, and experimentally tractable. Previous studies have shown that mutants in the canonical apoptotic pathways share a late-onset blemishing and persisting cells phenotypes and here I present data of two other possible cell death gene candidates. First, I show that a compound deletion, 33B(del), which removes vps33B and part of fur1, generates late-onset blemishes and persisting cells in the wing blade. These phenotypes cannot be rescued by a vps33B genomic rescue, and the possibility exists that the phenotypes are due to fur1. Finally, I show that silencing of CTCF, a protein involved in chromosomal looping and 3D genomic organization, also generates late-onset blemishes and persisting cells in the wing blade. These results, together with the binding sites annotation in the Reaper region, suggest that CTCF might be coordinating the expression of the IAP antagonists, hid in particular, by chromosomal looping. Taken together, these studies contribute to the characterization of collective cell death in Drosophila.Item Defining Genes and Circuits Affecting Naïve and Experience-Dependent Alcohol Preference in Drosophila melanogaster(2015-07-22) Ojelade, Shamsideen Adeniyi; Krämer, Helmut; Terman, Jonathan R.; Self, David W.; Rothenfluh, AdrianDespite alcohol being one of the most used and abused drugs in the world, the molecular mechanisms underlying alcohol abuse disorders remain largely unknown. In this dissertation, I utilized the model system Drosophila melanogaster to identify genes and circuits affecting ethanol-induced behaviors. From an unbiased genetic screen, I identified a novel gene that affects ethanol consumption in both flies and humans. Ras suppressor 1 (Rsu1) is required in the adult Drosophila nervous system for normal sensitivity to ethanol-induced sedation, and acts upstream of Rac1 and downstream of integrin to regulate the actin cytoskeleton. In a two bottle choice assay called the capillary feeding (Café) assay, loss of Rsu1 causes immediate heightened alcohol preference compared to wild type's initial naïve aversion. In contrast, flies specifically lacking Rsu1 in the mushroom bodies show normal initial aversion to alcohol, but then fail to acquire ethanol preference like normal flies do. Our data show that not only is Rsu1 required for normal alcohol responses, it suggests that different anatomical brain structures in flies control distinct alcohol behavioral responses. In humans, we find that polymorphisms in RSU1 are associated with brain activation in the ventral striatum during reward anticipation in adolescents and alcohol consumption in both adolescents and adults. Together, these data suggest a conserved role for integrin/Rsu1/Rac1/actin signaling in modulating reward-related phenotypes, including ethanol consumption in flies and humans. Using a modified Café paradigm, we investigated whether dopamine plays a role in both the aversive and experience-dependent properties of alcohol. I show that distinct subsets of DA neurons innervating the Fan-shaped body (FSB) and Mushroom body (MB) mediate naïve alcohol aversion (NAA) and experience-dependent alcohol preference (EDAP) respectively in flies. Furthermore, Rac1-dependent actin alteration in these anatomical structures (FSB and MB) also mirror dopaminergic-induced neuronal activity in these circuits suggesting that dopamine functions upstream of Rac1-signaling to affect alcohol preference in flies. Taken together, my dissertation suggests a conserved role for dopamine and the integrin/Rsu1/Rac1/Cofilin/Actin signaling pathway in modulating drug-induced behavioral plasticity across phyla, and highlights Drosophila as an effective model for integrative translational research.Item Fatty Acid Auxotrophy in Drosophila Larvae Lacking SREBP(2006-08-11) Kunte, Amit Sudhakar; Brown, Michael S.; Goldstein, Joseph L.A rapid increase in size is a major characteristic of larval development in Drosophila melanogaster. Such growth presumably requires the concomitant production of membrane lipids and is also accompanied by a significant accumulation of neutral lipid stores. Growing larvae must accumulate fatty acids to permit the synthesis of these lipids. Interestingly, wild type Drosophila can grow in the complete absence of exogenous fatty acids. This dissertation reports the finding that a lipogenic transcription factor, dSREBP (Drosophila Sterol Regulatory Element Binding Protein), is essential for the maintenance of this prototrophy. Drosophila larvae lacking dSREBP demonstrate a profound growth deficit in the second instar and die before reaching third instar. This is accompanied by transcriptional deficits in fatty acid synthetic genes. The growth deficit and lethality can be reversed by supplementing the culture medium with fatty acids. The most effective fatty acid, oleate, rescues 80 percent of dSREBP mutants to adulthood. Thus, a lack of dSREBP renders larvae auxotrophic for fatty acids. A reporter system demonstrates that dSREBP is active in tissues known to be involved in lipid metabolism- the fat body, oenocytes and anterior midgut. Finally, as expected of an end-product inhibited metabolic pathway, dSREBP activity can be suppressed by dietary supplementation with lipids. Thus, the dSREBP pathway coordinates endogenous synthesis with the dietary provision of exogenous lipids. These results establish Drosophila as a viable model for the genetic study of the SREBP pathway and provide the first evidence that, at an organismal level, the essential role of the pathway is the accumulation of lipids. The auxotrophic mutants and other reagents described here should be useful tools for further study of the SREBP pathway in particular and fatty acid metabolism in general.Item Feedback Regulation of Wnt Signaling by Naked Cuticle (Nkd) During Drosophila Embryogenesis(2008-05-12) Chan, Chih-Chiang; Wharton, Keith A.Wnt/beta -catenin signals are essential for many developmental and physiological processes in animals. Deregulation of the Wnt signaling pathway in mammals can cause diseases such as birth defects, cancer, osteoporosis, and diabetes. In Drosophila, the naked cuticle (nkd) gene antagonizes the Wnt/beta -catenin signaling in every segment of the embryo. Nkd is a modular, evolutionarily conserved protein that uses an EF-hand motif and adjacent sequences to target the cytoplasmic Wnt signal transducer Disheveled (Dsh). The mechanism by which Nkd antagonizes Wnt signaling in Drosophila embryos is not well understood. The abundance and bulk distribution of Dsh is not altered in nkd mutants as compared to wild type embryos, and overexpression of Nkd transgenes in nkd mutants did not alter Dsh distribution or abundance by confocal microscopy. Nkd transgenes lacking Dsh-binding regions were mostly able to rescue nkd mutants, suggesting that the Dsh-binding regions of Nkd contribute little to Nkd activity, at least when the transgenes were overexpressed. In this thesis, I have investigated non-Dsh binding regions that are critical for Nkd function. Our lab's findings indicate that a conserved 30 amino acid motif is essential for Nkd nuclear localization and function. Substitution of the 30aa motif with a heterologous nuclear localization sequence (NLS) rescued some nkd mutants to adulthood. In support of Nkd's role in the nucleus, Nkd binds to Importin-alpha 3, an adaptor for the canonical nuclear import apparatus. I identified that Nkd associates with Importin-alpha 3 via a motif ("D6") that is conserved between D. melanogaster and D. pseudoobscura. NkdΔD6, lacking the Importin-alpha 3-binding motif, was defective in nuclear localization and in rescuing nkd mutants. RNAi knockdown of importin-alpha 3 prevented the nuclear localization of Nkd. The findings that Nkd possesses two NLSs, each of which is required for function, and that Nkd associates with a component of the nuclear import apparatus, suggest that Nkd antagonizes the Wnt/beta -catenin signaling in the nucleus. Furthermore, I also addressed the function of the N-terminus of Drosophila Nkd. Unlike mammalian Nkd homologs that have N-terminal myristoylation consensus sequences responsible for membrane association, the N-terminus of Drosophila Nkd, also conserved in mosquito Nkd, lack such a sequence. Nonetheless, Nkd's N-terminus was required for function and membrane association. Substitution of the N-terminus with heterologous myristoylation sequences did not restore nkd function, indicating that the mechanism by which Drosophila Nkd associates with the membrane is different than mammalian Nkds. Therefore, Nkd appears to function in the membrane, in the cytoplasm to target Dsh, and in the nucleus to antagonize Wg signaling.Item Generation of a Novel D. melanogaster Platform to Elucidate Oncogenic Activity of Common Human p53 Missense Mutants(2014-02-04) Jakubowski, Brandon; D'Brot, Alejandro; Abrams, JohnThe tumor suppressor p53 prevents uncontrolled cell growth by three separate mechanisms: inducing apoptosis, initiating cell-cycle arrest, and activating DNA repair mechanisms in response to cell damage. Due to its central role in tumor eradication, it is unsurprising that p53 mutations are found in over half of human cancers. Unlike all other tumor suppressors however, 75% of these are missense mutations, with just six of them accounting for a third of all mutations found in the DNA binding domain of p53. Recent findings indicate that mutations in these "hotspot" locations may encode gain of function oncogenic activity to p53. Given their high prevalence, these mutations suggest a previously underappreciated selective advantage. We sought to decode this novel oncogenic activity of human p53 mutations by exploiting the Drosophila model system. This organism shares a similar p53 regulatory network with humans, as well as many of the same DNA repair and pro-apoptotic target genes. We recently showed that human p53, despite millions of years of evolutionary distance, complements loss of function mutations in the native fly p53 gene. We used six humanized p53 Drosophila strains previously generated in the lab; these contain a human p53 gene insertion, each with one of the six most commonly found missense mutations in patients. To study these mutations, we first profiled the expression patterns of wild type and mutant hp53 in the fly and their ability to rescue dp53 function. Expression levels of p53 were determined by immunofluorescence, while biological function was determined by the use of a GFP biosensor that specifically reports dp53 activity and acridine orange staining to identify dying cells in irradiated embryos. Expression studies demonstrate that the reporter is activated within stem cells in region 1 of the germarium, while its activation was absent in p53 null mutants. This phenotype was recovered with a dp53 insertion rescue. Additionally, two separately generated hp53+ strains show unusually elevated levels of expression compared to the wild type strains, whereas all mutant strains show diminished reporter activation in the region 1 stem cells. Functional studies in the embryo and the wing disc demonstrate that both wild type flies and the dp53 rescue promote cell death after irradiation, while the p53 null mutant does not. The two hp53+ strains rescued the wild type phenotype in the embryo; however, one of the hp53+ strains, named B2, was unable to induce cell death in the wing disc. The missense mutant strains do not exhibit IR-induced apoptosis in the embryo, but preliminary imaging shows they may be able to in the wing disc. We also discovered that, unlike the six hotspot mutants, wild type human p53 localizes to unidentified subnuclear compartments. Importantly, this may allow us to stratify and characterize p53 mutations according to functional differences.Item Genetic Dissection of Heart Development in the Fruit Fly Drosophila Melanogaster(2007-12-18) Yi, Peng; Olson, Eric N.The early morphogenetic mechanisms involved in heart formation are evolutionarily conserved. The Drosophila heart, known as the dorsal vessel, functions as a pulsatile tube-like organ containing an inner layer of contractile cardial cells that adhere tightly to an adjacent layer of pericardial cells. A genetic screen for genes that control Drosophila heart development revealed a cardiac defect in which pericardial and cardial cells dissociate causing loss of cardiac function and embryonic lethality. This phenotype resulted from mutations in the genes encoding HMG-CoA Reductase, downstream enzymes in the mevalonate pathway, and G-protein Ggamma 1, which is geranylgeranylated, thus representing an endpoint of isoprenoid biosynthesis. These findings reveal a cardial cell-autonomous requirement of Ggamma 1 geranylgeranylation for heart formation and suggest the involvement of the mevalonate pathway in congenital heart disease. In addition, we found that the heterotrimeric G proteins Gbeta 13F and G-oalpha 47A together with the RGS (regulator of G protein signaling) protein Loco function in the same pathway as Ggamma 1 to regulate septate junction formation in cardial cells of the Drosophila heart. We also present evidence that the septate junction protein Sinuous interacts with Pericardin, a matrix protein secreted by pericardial cells, providing the molecular basis for cardial-pericardial cell adhesion and serving as a mediator of the actions of the mevalonate pathway and heterotrimeric G protein signaling in Drosophila heart development.Item Illuminating the P53 Regulatory Network in Genetic Models(2011-02-01) Lu, Wan-Jin; Abrams, John M.The tumor suppressor gene p53 is mutated in more than 50% of human cancers, and functions as a central component of stress response machinery that mediates a wide variety of downstream responses. Interestingly, the evolutionary appearance of p53 preceded its role in tumor suppression, suggesting that there may be unappreciated functions for this protein. In order to examine physiologic functions of p53 in vivo, a green fluorescent protein (GFP) reporter was designed to follow the activation of this regulatory network in a genetic model, Drosophila melanogaster. By following the reporter during Drosophila development, physiological activation of the p53 regulatory network in the female germ line was discovered. It is provoked by the first enzymatic step for meiotic recombination and conserved in both flies and mice. The functional relevance of the p53 activities in the germ line was shown by the meiotic recombination frequency and genetic interactions with a meiotic effector gene, Rad54. Additionally, genotoxic stress selectively activates p53 in germ line stem cells and promotes regeneration of fertility after IR. Activation of p53 was also found in uncontrolled growth of germ cells by blocked differentiation, and surprisingly by overexpression of oncogenic protein in the germ line. Together, my thesis work indicate that the need for controlling growth by the p53 regulatory network is an evolutionary conserved feature, which may serve as a selective pressure to preserve this network. Future studies on the mechanisms of p53 actvities during meiosis and in response to oncogene activation could provide novel insights on its cancer-related functions.Item In Vivo Studies of Ribosomal RNA Transcriptional Regulation within a Drosophila Stem Cell Lineage(2013-09-06) Zhang, Qiao; Chen, Zhijian J.; Cobb, Melanie H.; Jiang, Jin; Buszczak, MichaelAs the first step of ribosome biogenesis, RNA polymerase I-directed ribosomal RNA gene (rRNA) transcription is critical for cell growth, proliferation and cell survival. Upregulated rRNA levels have been observed in many types of cancers. However, the extent to which rRNA transcription is differentially regulated in cells within the same lineage during differentiation in vivo and how changes in rRNA levels affect cell fate determination remains unclear. Here I present the discovery and characterization of a novel Drosophila RNA polymerase I transcriptional regulator Under-developed (Udd). The initial udd1 mutation was discovered in a sterility screen, and was further revealed to disrupt the expression of a gene CG18316. The udd null phenotype was recessive embryonic lethal, and both udd mutant phenotypes were rescued by a transgene carrying the CG18316 ORF. As a nucleolar protein, Udd colocalized with nascent rRNAs and was enriched in the rRNA gene promoter. Disruption of Udd decreased pre-rRNA levels. Moreover, Udd interacted with another two nucleolar proteins which are potential homologs of mammalian rRNA transcription initiation factors, the knockdown of which affected nucleolar expression of Udd and exhibited similar phenotypes to udd mutants. I further observed that the level of rRNA transcription was correlated with the differentiation state of germ cells in Drosophila ovaries. The pre-rRNA level was high in germline stem cells (GSCs), then decreased in early differentiating cysts, and again increased in the later differentiated germ cells. This difference was also demonstrated in undifferentiated ovaries (bam mutants) before and after introduced differentiation. More intriguingly, increasing rRNA synthesis by Tif-IA overexpression led to a mild expansion of GSCs, while downregulation of Pol I transcription in the undifferentiated ovaries filled with GSC-like cells resulted in multicellular cyst formation. Additionally, I observed another cell fate change, an eye-to-antennal transformation, when rRNA transcription was reduced in the undifferentiated eye primordia. These results suggest that rRNA transcription is closely related to differentiation and development, and the modulation of rRNA synthesis could be a part of the differentiation process.Item Mechanisms of Volatile Odorant Detection and Corresponding Behavioral Effects in Drosophila(2011-08-10) Ronderos, David Samuel; Smith, Dean P.One of the central problems in neuroscience concerns the molecular basis of behavior. Animals generate appropriate behavioral responses to environmental stimuli, which are mediated by primary sensory neurons of different modalities including sight, hearing, touch, taste, and smell. Olfaction serves a major role in conveying important information concerning a variety of behavioral determinants such as food sources, danger, and cues from other members of the same species. Drosophila melanogaster provides a tractable model in which to study both the mechanisms of olfaction and subsequent behavioral outputs based on olfactory input. Additionally, basic knowledge pertaining to insect olfaction provides valuable insights into potential targets that may be used to influence insect vectors of diseases (e.g., mosquitoes and malaria or dengue fever), or to improve agriculture by affecting pollination by foraging insects (e.g., honeybees). My research has focused on the molecular mechanisms involved in detection of pheromones, naturally-occurring insect repellents, and food source-derived odorants by primary sensory neurons in Drosophila and the corresponding behaviors generated by these stimuli. To date, the only known volatile pheromone in Drosophila is the male-specific hydrocarbon 11-cis-vaccenyl acetate (cVA). I took a genetic approach to study the role of cVA-induced neuronal activation, and found that activation of the at1 neuronal circuit involved in cVA detection, using a dominant-active form of the odorant binding protein LUSH, is both necessary and sufficient to induce sexually dimorphic mating behavior in Drosophila. In a separate study, I identified novel olfactory sensory neurons that detect the naturally-occurring insect repellent citronellal via a mechanism involving the ion channel TRPA1. Finally, I characterized the first odorant receptor shown to be expressed in neurons of the intermediate class of olfactory sensilla, Or83c, and found that it was highly specific for a sesquiterpene found in citrus fruit peel called farnesol (FOH). Furthermore, FOH detection by Or83c instructs oviposition preference in Drosophila via ai2a sensory neurons. These studies advance general knowledge of the architecture and complexity of the Drosophila olfactory system and provide insights into the molecular basis of behaviors in insects.Item Of Apoptosomes and Oncogenes: Repurposing a Death Machine & Deconstructing the Action of P53 Mutations(2014-04-14) D'Brot, Alejandro; Scaglioni, Pier Paolo; Shay, Jerry W.; Yu, HongtaoIt is now well appreciated that the apoptosome, which governs caspase-dependent cell death, also drives nonapoptotic caspase activation to remodel cells. However, determinants that specify whether the apoptosome acts to kill or remodel have yet to be identified. I show here that Tango7 genetically interacts with the apoptotic machinery but instead of regulating cell death, collaborates with the apoptosome to drive caspase-dependent cellular remodeling. Specifically, Tango7 is required for non-apoptotic caspase activity during spermatid remodeling and localizes to the active apoptosome compartment in these cells via its C terminus. Furthermore, Tango7 directly stimulates activity of reconstituted apoptosomes in vitro. These and other data presented here suggest that Tango7 physically recruits the apoptosome to specify this complex for nonapoptotic cellular remodeling. In vivo genetic model systems are powerful tools to deconstruct activity of genes driving human disease. The tumor suppressor p53 is mutated more than any other gene in human cancer, but unlike other tumor suppressors, it acquires missense mutations which encode oncogenic variants. These gain-of-function mutants promote more aggressive and metastatic cancers in vivo but their oncogenic activity is not well understood. To address this problem, I have exploited Drosophila as a platform to study and stratify human p53 (hp53) mutants. I replaced fly p53 with either wild-type hp53 or 5 of the most prevalent hp53 mutations in cancer. In this system, hp53 is under control of endogenous Dp53 regulatory elements and can regulate in vivo transcriptional activation and apoptosis like its fly counterpart. Furthermore, wild-type hp53 forms foci in the germline that localize to the same subnuclear compartment as Dp53 foci. This property is compromised in all of the gain-of-function mutants and can thus be used to distinguish oncogenic variants from wild-type hp53. Future studies aim at finding whether this and other properties shared among the 5 mutants can help stratify oncogenic p53 mutations found in human cancer.Item The Regulation and Function of Drosophila Acinus(2017-12-01) Tyra, Lauren Katherine; Johnson, Jane E.; Smith, Dean P.; Pan, Duojia; Krämer, HelmutAutophagy and growth control are two processes critical to organisms that mutually antagonize and regulate on each other. Several well known connections between these processes have been described, but here I describe a new link. Using Drosophila melanogaster as a model system, my thesis research has identified Acinus and Atg1, already known for their functions in autophagy, as growth suppressors. Further, my data show that this suppression is, at least in part, mediated by Atg1 phosphorylating and thereby inhibiting the pro-growth transcriptional co-activator Yorkie. Genetic gain- and loss-of function experiments indicate that this Atg1 function depends on Acinus. This work provides a new role for Atg1 in inhibition of growth and thereby adding a new regulatory pressure on Yorkie. Furthermore, my data indicate that Acinus’ function in promoting basal autophagy is based on its starvation-independent activation of the Atg1 kinase.Item The Role of a Neuron-Specific V-ATPase in Synapse Specification, Function, and Maintenance(2012-08-13) Williamson, Wallace Ryan; Hiesinger, Peter RobinThe Vacuolar-type (V-) adenosine triphosphatase (ATPase) is a proton-pumping nanomachine consisting of two multi-subunit, reversibly associating protein sectors, the cytosolic V1 sector and the membrane-bound Vo sector. The V1/Vo holoenzyme hydrolyses ATP to translocate protons across biological membranes thereby modulating lumenal and extracellular pH. Additionally, accumulating evidence suggests that the Vo sector has a role in membrane fusion when dissociated from the V1 sector, its proton-pumping partner. Published evidence for this includes a null allele for the neuron-specific a subunit in the Drosophila Vo sector, v100, which leads to defects in synaptic function that are unrelated to pH regulation. My project emerged from the need to explain why v100 has two additional phenotypes that are absent in other synaptic function mutants: functional and structural degeneration in photoreceptor cells and patterning defects in the visual system neuropil. I proposed that v100 has a previously undocumented role on a neuron-specific endo/lysosomal pathway in addition to its documented role in neurotransmitter secretion. To test my hypothesis in the context of only one of the two purported v100 functions, I generated transgenic animals with v100 mutations designed to specifically disrupt either acidification or membrane fusion. Using these genetic tools, I discovered that v100 has an essential role in sorting cargo into an endo-lysosomal pathway that concomitantly requires v100 for the acidification-dependent maturation of degradation-competent organelles. This 'sort-and-degrade' mechanism for v100 defines a neuron-specific degradation pathway that is required for synaptic specification, function, and maintenance. In developmental stages, v100 is required to 'sort-and-degrade' guidance receptors as part of the synapse specification program. In the adult, the 'sort-and-degrade' mechanism provides additional degradative capacity to neurons, a cell type that must often maintain homeostasis for unusually long periods of time. Finally, I provide evidence that the role for V100 in membrane fusion requires a direct, physical interaction with Syntaxin-1, an interaction that can be specifically disrupted in vitro and in vivo. In brief, my results provide mechanistic insight into the acidification-independent role of v100 and reveal the existence of a neuron-specific endo-lysosomal pathway on which v100 functions to 'sort-and-degrade' cargo in order to meet the special needs of a neuron in development, function, and maintenance.Item The Role of Double-Stranded RNA Binding Protein R2D2 and Lump in Drosophila Gametogenesis(2010-05-14) Sanders, Charcacia Tiana; Smith, Dean P.In Drosophila melanogaster, fertility requires the successful development of germline cells into mature gametes. This process is dependent on multiple factors that coordinate migration, proliferation, and differentiation of germline stem cells. Previous studies have shown dsRNA binding proteins have an important role in the induction and maintenance of germ cells. In this study, I investigated the requirement of dsRNA binding proteins, R2D2 and LUMP, in Drosophila melanogaster gametogenesis. I show R2D2 functions in the ovary, specifically in the somatic tissues giving rise to the stalk and other follicle cells critical for establishing the cellular architecture of the oocyte. Most interestingly, the female fertility defects in r2d21 are dramatically enhanced when one copy of the dcr-1 gene is missing. This indicates dicer-1 and r2d21 operate in the same fertility pathway. Furthermore, Dicer-1 protein coimmunoprecipitates with R2D2 antisera, indicating these proteins function in the same protein complex. Thus, r2d21 mutants have reduced viability and defective female fertility that stems from abnormal follicle cell function and Dicer-1 impacts this process. This is the first indication R2D2 functions beyond its known role in RNA interference to include ovarian development in Drosophila. I also studied a second putative double-stranded RNA binding protein (dsRBP). I found that lump is required for male fertility and there is an absence of motile sperm in lump mutant testes squash preparations and the seminal vesicles. The early stages of spermatogenesis, including mitosis, meiosis, and cytokinesis steps are unaffected in lump mutants. This indicated lump is likely required late in sperm development. The spermatid individualization complex is disrupted consistent with an individualization-deficient phenotype. A wildtype genomic rescuing transgene was able to rescue fertility and individualization. However, a lump transgene carrying a point mutation in the first dsRNA binding domain did not rescue fertility or individualization suggesting this domain is essential for lump function. Thus, it is likely that these two dsRNA binding proteins regulate gene expression in the ovary and testes and are essential for normal fertility.Item [Southwestern News](2000-03-31) Stieglitz, HeatherItem Studies of the Hippo Signaling Pathway(2012-08-13) Yue, Tao; Jiang, JinHow multicellular organisms control their growth to reach proper organ size during development is a fascinating question. Recent studies, initially from Drosophila, have identified the Hpo tumor suppressor pathway as a crucial mechanism that controls tissue growth by inhibiting cell growth, proliferation and survival. Deregulation of the Hpo pathway has been implicated in various human cancers. Central to the Hpo pathway is a kinase cassette consisting of four tumor suppressor proteins, the Ste20-like kinase Hpo, the WW domain-containing protein Salvador (Sav), the NDR family kinase Warts (Wts) and the Mob family protein Mats. The kinase activities of Hpo and Wts are facilitated by their regulatory proteins Sav and Mats, respectively. Activated Hpo/Sav complex phosphorylates and activates the Wts/Mats complex, which in turn phosphorylates and inactivates the transcriptional coactivator Yorkie (Yki). Phosphorylation of Yki restricts its nuclear localization through recruiting 14-3-3. When the activity of the Hpo/Wts kinase cassette is compromised, Yki forms complexes with transcription factors including Scalloped (Sd) and translocates to the nucleus to activate Hpo pathway target genes, including cyclin E, diap1, and the microRNA bantam that regulate cell growth, proliferation and survival. To identify novel components of the Hpo signaling pathway, I carried out a genetic modifier screen in which flies carrying GMR-Gal4 and UAS-Yki were crossed to a collection of transgenic RNAi lines from Vienna Drosophila RNAi center (VDRC) and Bloomington stock center, and looked for enhancers or suppressors of the overgrown eye phenotype caused by Yki overexpression. Through this screen, I have found that Echinoid (Ed), an immunoglobulin domain-containing cell adhesion molecule, acts as an upstream regulator of the Hpo pathway. Loss of Ed compromises Yki phosphorylation, resulting in elevated Yki activity that drives Hpo target gene expression and tissue overgrowth. Ed physically interacts with and stabilizes the Hpo-binding partner Sav at adherens junctions. Ed/Sav interaction is promoted by cell-cell contact and requires dimerization of Ed cytoplasmic domain. Overexpression of Sav or dimerized Ed cytoplasmic domain suppressed loss-of-Ed phenotypes. I propose that Ed may link cell-cell contact to Hpo signaling through binding and stabilizing Sav, thus modulating the Hpo kinase activity. Furthermore, the Cul4/WDR40A complex has also been identified as a genetic modifier for the Hippo signaling pathway. However, the exact mechanism by which this complex regulates the Hippo signaling pathway need to be further addressed.