Browsing by Subject "Drosophila Proteins"
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Item Apoptosis Determinants in Drosophila Melanogaster(2007-12-17) Chew, Su Kit; Abrams, John M.Apoptosis is a form of programmed cell death (PCD) that is governed by a core set of genes conserved across diverse metazoan phyla. Cells dying by apoptosis exhibit a characteristic series of morphological and biochemical changes that is also conserved. This form of PCD plays pivotal roles in homeostatic regulation of cell numbers, developmental sculpting of organs, damage and infection responses; conversely, its disregulation has profound implications in diseases such as cancers, immune disorders infertility and dystrophies. Common parallels in the regulation of the core apoptosis machinery have been elucidated in human and experimental model organisms, though many fundamental questions in our understanding of its regulation remain. A conserved node in the apoptosis pathway is the apoptosome, comprising the apical caspase and its adaptor protein. To understand the functions of this node, I generated a null allele of the apical caspase Dronc in the experimental model organism Drosophila melanogaster. Dronc is required for developmentally regulated apoptosis in multiple tissues during embryogenesis and larval development. Failure of apoptosis correlated with tissue hyperplasia. Notably, the removal of Dronc eliminated the cellular apopototic response to stresses in cells. In some of the stress contexts tested, Dronc depletion partially rescued cell viability to the same levels as pan-caspase inhibition by small peptide inhibitors, suggesting that Dronc functions map specifically to caspase activation and apoptosis. These and similar observations in its adaptor protein Dark point to the apoptosome as a key node for apoptosis in Drosophila. From these observations, I sought to use the induced apoptosis cellular response as a means to identify novel components and regulators in the apoptosis pathway. I optimized a cell culture system for high-throughput cell-based screening using RNA interference (RNAi) mediated gene silencing and a synthetic antagonist of inhibitors of apoptosis proteins (IAPs). From a genome-wide Drosophila RNAi library, I identified 42 potential genes required for apoptosis, of which I characterized 13 highly validated targets for their requirements in multiple stress contexts. One of these hits, Tango7, regulates pro-Dronc protein and represents an unprecedented point of apoptosis regulation. Collectively, my studies bolster the model for the crucial requirement of the apoptosome in apoptosis and identify new regulation entry-points into the apoptosis pathway.Item Bcl-2 Function in Drosophila(2007-12-17) Galindo, Kathleen A.; Abrams, John M.Bcl-2 family members are pivotal regulators of programmed cell death (PCD). In mammals, pro-apoptotic Bcl-2 family members initiate early apoptotic signals by causing the release of cytochrome c from the mitochondria, a step necessary for the initiation of the caspase cascade. Worms and flies do not show a requirement for cytochrome c during apoptosis, but both model systems express pro- and anti-apoptotic Bcl-2 family members. Drosophila encodes two Bcl-2 family members, Debcl (pro-apoptotic) and Buffy (anti-apoptotic). To understand the role of Debcl in Drosophila apoptosis, we produced an authentic null allele at the Debcl locus. Although gross development and lifespans were unaffected, we found that debcl was required for pruning cells in the developing central nervous system. debcl genetically interacted with the ced-4/Apaf-1counterpart, dark, but was not required for killing by RPR proteins. Surprisingly, in a model of caspaseindependent cell death, we found that heterologous killing by Murine Bax required debcl to exert its pro-apoptotic activity. DebclKO mutants were also significantly affected for mitochondrial density. Taken together, these findings suggest that evolutionary functions impacting mitochondrial properties represent ancient activities which preceded the evolution of these proteins as central regulators of PCD.Item Biochemical Analysis of the Drosophila RNAi Pathway(2009-01-14) Jiang, Feng; Liu, QinghuaRNA interference is post-transcriptional gene silencing mediated by (21-26 nt) miRNAs and siRNAs. In Drosophila, the RNase III enzymes Dicer-1 and Dicer-2 generate miRNAs and siRNAs, respectively. Nascent miRNA and siRNA duplexes are assembled into distinct RNA induced silencing complexes termed miRISC and siRISC, of which AGO1 and AGO2 are the respective catalytic subunits. My dissertation project is focused on identifying new RNAi components and understanding mechanisms of RISC assembly by biochemical reconstitution. Our group previously identified a novel dsRNA-binding protein named R2D2 which functioned in complex with Dicer-2 to process dsRNA into siRNA. Only the Dicer-2/R2D2 complex, but neither Dicer-2 nor R2D2 alone, efficiently interact with duplex siRNA. Furthermore, the tandem dsRNA binding domains of R2D2 are required for siRNA binding. Therefore, although R2D2 is dispensable for siRNA production, it is required for incorporating siRNA onto the siRISC complex. Generation of recombinant AGO2 protein is essential for in vitro reconstitution of the RNAi pathway. We believe that the unique poly glutamine repeat region of fly AGO2 may be problematic for expression. Thus, a series of truncated AGO2 baculoviruses that remove some or all polyQ repeats of AGO2 were generated. Co-expression with AGO1 increases the expression level of AGO2 by at least 10 fold. Affinity purified full length and one truncated form of AGO2 show minimal RISC activity, i.e. could be programmed with single stranded siRNA and perform sequence specific cleavage of mRNA. Most interestingly, adding purified recombinant Dicer-2/R2D2 complex to recombinant Ago2 generated dsRNA and siRNA initiated RISC activity. Catalytic mutant of Ago2 is unable to reconstitute RISC activity with recombinant Dicer-2/R2D2 complex, showing that the RISC activity is specific. Therefore, the three component system, Dicer-2, R2D2, and Ago2, can reconstitute the RNAi pathway of Drosophila. By a bioinformatics approach, a novel protein named Loquacious (Loqs) was identified with considerable sequence homology to R2D2. Loqs and Dicer-1 interact with each other by co-immunoprecipitation in S2 cell extract. Recombinant Loqs could enhance miRNA production by Dicer-1 by increasing its affinity for the pre-miRNA substrate. Furthermore, depleting Loqs or Dicer-1 by dsRNA knockdown resulted in reduction of the miRNA-generating activity and accumulation of pre-miRNA in S2 cells. To study the physiological function of loqs in flies, we obtained a piggyback (PB) fly strain in which the PB transposon was inserted into the first exon and before the translation start site of loqs gene. Pre-miRNAs accumulate in the loqs PB flies, indicating they are defective for miRNA biogenesis. However, while both siRISC and miRISC activities are greatly reduced in dcr-1 null extract, these activities are not affected in loqs null extract, indicating that loqs is not essential for miRISC assembly. To test whether the known components are sufficient to reconstitute the miRNA pathway, recombinant AGO1 protein was expressed using the insect cell expression system. It is generally believed that siRISC slices, whereas miRISC represses translation of cognate mRNA in animals. However, recombinant AGO1 can be programmed by single stranded miRNA into a minimal miRISC and sequence specifically cleaves complementary mRNA in vitro. Furthermore, the catalytic activity of AGO1 is dependent on the consensus catalytic "DDH" motif. My present studies suggest that recombinant Dicer-1, Loqs and AGO1 are not sufficient to reconstitute the miRNA pathway, indicating that there are other unknown components to be discovered.Item Centrosomin Self-Assembly and Centrosomal Protein Recruitment(2005-08-11) Bauer, Ruth Anne; Megraw, TimothyCentrosomes, the major microtubule organizing centers in animal cells, are important for mitotic spindle formation. Normally, each cell has two centrosomes which migrate to opposite sides of the nuclear envelope prior to entry into mitosis. Centrosomin (Cnn) is a major centrosomal protein that is important for nucleation and organization of bipolar spindle microtubules at mitosis. Cnn protein localizes to the pericentriolar matrix and from there other centrosomal proteins 'load' onto the centrosome, including gamma-tubulin. Centrosomes are non-functional without the addition of Cnn since it is responsible for recruitment of other centrosomal proteins. There are two conserved motifs in Cnn protein, currently of unknown function. One of these motifs is most likely responsible for interaction with gamma-tubulin and other centrosomal proteins which make the centrosome capable for microtubule nucleation. Cnn full-length and half proteins were expressed in E. coli and purified in vitro. The properties of these Cnn proteins show self-assembly and recruitment of centrosomal proteins. These activities of Cnn in vitro are novel and will help further the investigation of Cnn protein function in the context of biological systems. Cnn fusion proteins show characteristics similar to centrosomal 'satellite' or 'flare' particles described in animal cells. It is likely that these satellites communicate with the actin cytoskeleton in syncytial Drosophila embryos.Item Communal Cell Death and P53 Mediated Transcriptional Control in Drosophila Melanogaster(2011-08-10) Link, Nichole Lynn; Abrams, John M.Apoptosis is essential for all metazoan development. The key component that functions in apoptosis, the apoptosome, is a molecular machine that initiates caspase activation and is conserved throughout the animal kingdom. Drosophila strains that are mutated for genes encoding the apoptosome show pronounced defects in programmed cell death (PCD). Using a characteristic phenotype associated with mosaic animals, we conducted a screen in Drosophila to discover new regulators or effectors of the apoptosome. Using this model, we also discovered a unique communal form of cell death where large regions of epithelial cells are eliminated within minutes. We also produced 'saturation tile' arrays by digital optical chemistry for an unbiased sampling of transcriptional activity in the Drosophila genome. We found that the scope of unannotated transcriptional activity is extensive and widespread. A dominant population of noncanonical transcripts was stress-responsive and required p53, a master regulator of conventional stress-responsive target genes in vertebrates and invertebrates. This prompted us to examine stimulus dependent activity surrounding a single p53 enhancer in our tiled region. Through genetic analyses, we showed that this enhancer coordinates stimulus dependent induction of multiple genes spanning over 300kb throughout the Reaper region. Surprisingly, this same enhancer regulated a gene positioned across the centromere at distances over 20Mb and also controlled at least one gene mapping to a different chromosome. Chromosome conformation capture analyses placed this enhancer in close proximity to these distant targets in vivo through specific DNA looping and these interactions were influenced by p53. Therefore, a single p53 enhancer is necessary and sufficient for long range, multigenic regulation in cis and in trans.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 Direct Redox Regulation of F-Actin Assembly and Disassembly by MICAL(2013-04-08) Hung, Ruei-Jiun 1982-; Hiesinger, Peter Robin; Terman, Jonathan R.; Rosen, Michael K.; Yin, Helen L.How guidance cues present outside of cells exert their precise effects on the internal actin cytoskeleton is poorly understood. Such effects are critical for diverse cellular behaviors including polarity, morphology, adhesion, motility, process elongation, navigation, and connectivity. Semaphorins, for example, are one of the largest families of these guidance signals and play critical roles in neurobiology, angiogenesis, immunology, and cancer. One interesting characteristic of the Semaphorins is that they inhibit the movement of cells (and their membranous processes) through their ability to disrupt actin cytoskeletal organization. However, despite considerable progress in the identification of Semaphorin receptors and their signal transduction pathways, the molecules linking them to the precise control of the actin cytoskeleton have remained mysterious. During my graduate studies, I sought to better understand a family of unusual proteins called the MICALs (which includes one Drosophila Mical and three vertebrate MICALs), which associate with the Semaphorin cell-surface receptor Plexin and are important for Semaphorins to exert their effects. Nothing was known, however, regarding the specific role of the MICALs in these Semaphorin-dependent events. Not long after I began my graduate work, my colleagues and I noticed that Mical was necessary for proper actin cytoskeletal organization and sufficient to reorganize the actin cytoskeleton in vivo. Therefore, to better understand the role that Mical plays in actin cytoskeletal rearrangements, I took a biochemical approach, and purified the Mical protein. Utilizing biochemical and imaging approaches with purified proteins, I found that Mical directly binds to actin filaments (F-actin) and is able to induce the rapid disassembly of F-actin. Thus, my results revealed that Mical is a novel F-actin disassembly factor that provides a molecular conduit through which F-actin disassembly can be precisely achieved in response to Semaphorins. So I next wondered how Mical induces F-actin disassembly. Interestingly, the MICALs belong to a class of flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in oxidationreduction (Redox) reactions. Although MICALs have no known substrate/s, my in vivo and in vitro results revealed that Mical employs its Redox region to bind F-actin and disassembles filaments in an NADPH-dependent manner. Moreover, this Mical-treated actin failed to repolymerize even after removal of Mical, indicating that Mical stably modifies actin to alter polymerization. Mass spectrometric analyses revealed that F-actin subunits were directly modified by Mical on their conserved pointed-end that is critical for filament assembly. Specifically, Mical post-translationally oxidized a conserved amino acid (Methionine 44) within a region of actin that is critical for actin-actin contacts, simultaneously severing filaments and decreasing polymerization. Thus, my thesis observations reveal a novel and specific oxidation dependent signaling mechanism that selectively regulates actin dynamics and cellular behaviors.Item Dissection of Mechanisms Regulating the Drosophila Hedgehog Pathway(2012-07-20) Shi, Qing; Jiang, JinHedgehog (Hh) signaling is essential for both embryonic development and adult tissue homeostasis. Malfunction of Hh signaling pathway causes many human disorders including birth defects and cancers. In Drosophila, the G-protein-coupled-receptor-like protein Smoothened (Smo) transduces the Hh signal across the plasma membrane, and an intracellular Hh signaling complex (HSC) containing the kinesin-related protein Costal2 (Cos2), the serine/threonine protein kinase Fused (Fu) and a PEST-domain containing protein suppressor of Fused (Sufu) relays the Hh signal downstream from Smo to the Zn finger transcription factor Cubitus interruptus (Ci). Our previous studies have demonstrated that Hh transduces signal by regulating the subcellular localization and conformational state of Smo, but how Smo relays the signal to cytoplasmic signaling components remains poorly understood. In this study, we show that Hh-induced Smo conformational change promotes the recruitment of Cos2/Fu complex and Fu dimerization. We find that induced dimerization through the Fu kinase domain activates Fu by inducing multi-site phosphorylation of its activation loop (AL), and phospho-mimetic mutations of AL suffice to activate the Hh pathway. Moreover, we find that activated Fu regulates Ci by both promoting its transcriptional activator activity and inhibiting its proteolysis into a repressor form. We provide evidence to suggest that activated Fu exerts the regulation by interfering with the formation of Ci-Sufu and Ci-Cos2-kinase complexes that normally inhibit Ci activity and promote its processing. In the rest part of the study, we further explore additional mechanisms regulating Ci activity. We have identified and characterized three types of functional regulatory elements in Ci, including a transcriptional repression domain in the N-terminal region of Ci, multiple Ser/Thr motifs in the amino-(N-) and carboxy-(C-) terminal regions of Ci serving as HIB/SPOP E3 ligase-specific degrons, and finally a novel PY-NLS around the N-terminal highly conserved domain of Ci.Item Dynamic Scaffolding in a G-Protein Signaling Cascade(2009-06-18) Mishra, Prashant; Ranganathan, RamaThe InaD scaffold organizes a multi-protein complex that is essential for proper visual signaling in Drosophila photoreceptor cells. Here we show that one of the InaD PDZ domains (PDZ5) exists in a redox-dependent equilibrium between two conformations- a reduced form which is similar to the structure of other PDZ domains, and an oxidized form in which the ligand-binding site is distorted through formation of a strong intramolecular disulfide bond. We demonstrate transient light-dependent formation of this disulfide bond in vivo, and find that transgenic flies expressing a mutant InaD in which PDZ5 is locked in the reduced state display severe defects in termination of visual responses and visually-mediated reflex behavior. These studies demonstrate a novel conformational switch mechanism for PDZ domain function and suggest that InaD behaves more like a dynamic machine rather than a passive scaffold, regulating signal transduction at the millisecond timescale through cycles of conformational change.Item EGFR and Akt Signaling in Rhabdomyosarcoma Pathogenesis(2018-07-25) Granados, Valerie Ann; Amatruda, James F.; Olson, Eric N.; Lum, Lawrence; Galindo, ReneRhabdomyosarcoma is an aggressive soft-tissue malignancy comprised microscopically of neoplastic skeletal muscle-lineage precursors that fail to exit the cell-cycle and fuse into syncytial muscle - the underlying pathogenetic mechanisms for which remain unclear. We previously identified that misregulated myoblast fusion signaling via the TANC1 adaptor molecule promotes neoplastic transformation in RMS cells. As TANC1 is not presently pharmacologically targetable, here we have turned to our Drosophila RMS-related model to identify myoblast fusion-related elements potentially targetable in RMS. Genetic modifier screening against the fly model revealed that decreased Epidermal Growth Factor Receptor (EGFR) activity, which regulates myoblast fusion programming in flies, suppresses PAX-FOXO1 (PF)-induced lethality. As EGFR is pharmacologically targetable, we demonstrate that EGFR inhibitors antagonize RMS in a ERMS-RD cell line, but that other RMS cell lines are resistant. Further interrogation finds that EGFR inhibitor-sensitive cells exhibit marked down-regulated activation of the Akt intracellular signaling transducer, but not MEK/MAPK or STAT3, suggesting that Akt promotes and/or sustains RMS. We then demonstrate that Akt pharmacologic inhibition antagonizes RMS in vitro and in vivo, including RMS cells resistant to EGFR inhibition. We additionally find that sustained Akt1 activity promotes RMS cell terminal differentiation-arrest. Together, these findings point towards Akt activity as a broad RMS underpinning and therapeutic vulnerability.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 Genetic Analysis of Fic-Mediated BiP AMPylation in Photoreceptors(2019-04-10) Moehlman, Andrew Terry; Smith, Dean P.; Orth, Kim; Cobb, Melanie H.; Thomas, Philip J.; Krämer, HelmutIn response to environmental, developmental, and pathological stressors, cells engage homeostatic pathways to maintain their function. The Unfolded Protein Response protects cells from the accumulation of misfolded proteins in the ER. Depending on ER stress levels, the ER-resident Fic protein catalyzes AMPylation or deAMPylation of BiP, the major ER chaperone. This work elucidates a critical role of the reversible AMPylation of BiP in maintaining the Drosophila visual system in response to constant light-induced stress. In response to extended light exposure, flies mutant for fic display loss of synaptic function and disintegration of rhabdomeres, closely stacked plasma membrane microvilli that house the proteins of the phototransduction cascade. These phenotypes are replicated in BiP mutants lacking the Thr366 AMPylation site. Strikingly, these degeneration-like phenotypes are reversible: photoreceptors in both fic and bip mutants regain their structure and function within 72 hours once returned to a standard light:dark cycle. In fic mutants, these phenotypes are preceded by a dysregulation of the IREI and PERK-mediated ER stress responses detected with specific reporters. These findings indicate that Fic-mediated AMPylation of BiP is required for neurons to adapt to transient stress demands, and that this process may require proper activation of the Unfolded Protein Response. Furthermore, I have helped identify multiple cell membrane transporters involved in synaptic signaling between photoreceptors and lamina neurons. These transporters are involved directly and indirectly in the recycling of the neurotransmitter histamine in the synaptic cleft.Item Genetics of Stress-Induced Responses in Drosophila(2006-08-11) Akdemir, Fatih; Abrams, John M.Apoptosis is a highly conserved process responsible for elimination of cells during normal development and after cellular damage. Apical caspases that initiate caspase cascades are stimulated upon interaction with adaptor molecules. The Drosophila adaptor protein Dark, a homolog of nematode Ced-4 and mammalian Apaf-1, regulates the apical caspase Dronc, through interactions involving respective caspase recruitment domains (CARD). Dronc is the only caspase in the fly genome with a caspase recruitment domain. Here I pursue functional characterization of dronc and dark animals especially to find out whether they are required for all programmed cell death and whether they have distinct functions. dronc mutants have extensive hyperplasia of hematopoietic tissues and adult structures lacking dronc are disrupted for fine patterning. In diverse models of metabolic injury, dronc- cells are completely insensitive to induction of cell killing. I also show the generation and functional characterization of dark null mutant animals. Using in vivo and ex vivo assays, I demonstrate a global apoptogenic requirement for dark and show that a required focus of dark- organismal lethality maps to the central nervous system. Finally I show functional similarities of dronc and dark null mutants by a diverse set of experiments. Together these findings illustrate broad requirements for Dark and Dronc in adaptive responses during stress-induced apoptosis and in normal cell death. Treatment of cells with DNA-damaging agents upregulates the transcription of many genes, many of which are not functionally characterized. In a series of independent studies I characterize an ionizing radiation (IR)-induced gene, CG17836, designated as xrp1. Xrp1 is robustly responsive to IR, and it is a nuclear protein with DNA-binding activity as inferred from domain structure. I have characterized two different loss-of-function mutants of xrp1. In a loss-of-heterozygosity (LOH) assay xrp1 mutant animals display higher genomic instability than wild types after IR challenge. Even though xrp1 is not required for apoptosis or cell cycle arrest after IR treatment in animals, surprisingly, its overexpression in cell culture prevents cell proliferation. Thus, Xrp1 might maintain genomic stability by modulating cell cycle checkpoints upon IR exposure.Item Hedgehog Signaling Plays a Conserved Role in Inhibiting Fat Formation(2006-08-11) Gao, Xiaohuan; Graff, Jonathan M.The involvement of hedgehog (Hh) signaling in cell determination and differentiation in a wide variety of tissues in both invertebrates and vertebrate has been well established. However, relative little is known about its function in formation of adipose tissues. To address this question, Drosophila and mammalian models were used to analyze its potential role. Components of the Hh pathway were expressed in the Drosophila fat body. Activating Hh signaling specifically in fat body inhibited fly fat formation. Conversely, blocking Hh signaling specifically in fat body stimulated fly fat formation. Analysis in mammalian models suggested the presence of functional Hh signaling in murine developing fat, adult fat and in mammalian adipogenic models. Down-regulation of Hh signaling marked the stage of terminal differentiation. In 3T3-L1 preadipocyte cell line, addition of recombinant murine sonic Hh (Shh) potently inhibited adipogenic differentiation dose-dependently, resulting in decreased intracellular triglyceride accumulation and reduced mRNA levels of established adipogenic genes. Treatment of KAAD-cyclopamine, an antagonist of Hh signaling, promoted adipogenesis. Activating or blocking Hh signaling genetically produced similar effects as pharmacological treatment. Additional study in multipotent cell lines, NIH3T3 and C3H10T1/2, reinforced the inhibitory role of Hh signaling in adipogenesis. However, the inhibition was effective only when Hh signaling was activated during early stage of adipogenesis. Epistasis tests suggested Hh signaling functioned upstream of PPARgamma . Mechanistic studies showed that Hh signaling might act as a molecular switch, likely mediated by anti-adipogenic transcrition factors such as GATA2, to divert preadipocytes as well as multipotent mesenchymal prescursors away from adipogenesis to osteogenesis. My study on the function of Hh signaling in fat formation of both invertebrates and vertebrates suggested that Hh signaling played a conserved role in inhibiting fat formation and highlighted the potential of the Hh pathway as a therapeutic target for osteoporosis, lipodystrophy, diabetes and obesity.Item Histone Demethylase LSD1 Restricts the Size of the Germline Stem Cell Niche in Drosophila Ovaries(2013-01-16) Eliazer, Susan; Buszczak, MichaelSpecialized microenvironments called niches keep stem cells in an undifferentiated and self-renewing state by producing a variety of factors. The size and signaling output of niches must be finely tuned to ensure proper tissue homeostasis. I use the Drosophila female germline as an excellent model system to study niche development and function. Five to seven somatic cap cells form the ovarian stem cell niche and produce dpp, a BMP homolog necessary for the maintenance of germline stem cells (GSCs). Mutations in Lsd1, a histone demethylase exhibit GSC-like tumor formation. Clonal analysis, cell-type specific knock down and rescue experiments demonstrate that Lsd1 functions within the escort cells that reside immediately adjacent to cap cells (niche). Loss of Lsd1 causes the escort cells to adopt an intermediate fate expressing both escort cell and cap cell markers and enables them to function as ectopic niches for the expanded stem cell population. Temporally restricted gene knock-down experiments suggest that Lsd1 functions both during development, to specify EC fate, and in adulthood, to prevent ECs from forming ectopic niches independent of changes in cell fate. Lsd1 specifically functions to repress dpp, the niche signal in the adult germaria. I have identified engrailed as a direct target of Lsd1 by performing Chromatin Immunoprecipitation (ChIP-seq) analysis in the escort cells of the Drosophila ovary. Engrailed is expressed in the cap cells of wild type germaria and in Lsd1 mutants engrailed transcripts are misexpressed in the escort cells. Knocking down engrailed expression in the escort cells suppresses the Lsd1 mutant phenotype. Moreover, ectopic expression of engrailed in the escort cells displays a GSC-tumor phenotype. Furthermore, I have shown that Engrailed functions upstream of dpp, and activates its expression in the cap cell niche.Item In Vivo Genome-Wide Analyses of the Drosophila p53 Transcriptional Network(2017-07-27) Kurtz, Paula S.; Fontoura, Beatriz; Abrams, John M.; Kraus, W. Lee; Krämer, Helmutp53 is the most commonly mutated gene in human cancers. Despite decades of p53 studies we do not fully understand how p53 suppresses tumors. Similar to human p53, the Drosophila counterpart is a transcription factor that can respond to genotoxic stress and promote adaptive responses at the cellular level. Our lab has leveraged the powerful genetics of Drosophila to study p53 functions in vivo. In the context of the developing fly, p53 robustly activates important apoptotic genes in response to DNA damage to promote cell death. In the embryo model, we discovered an important p53 enhancer that forms chromatin contacts through long genomic distances and enables p53 to activate various genes. How p53 programs are adapted in different cellular contexts is poorly understood. In my dissertation work I examined two layers of p53 regulation, long-range enhancer looping and p53 DNA occupancy. To further examine enhancer looping, I exploited the established embryo model and the well characterized p53 reaper enhancer. At the single cell resolution, I demonstrated that the p53 enhancer can contact multiple targets simultaneously; however these multigenic complexes appear in low frequency. I also have preliminary genome-wide data suggesting in embryos this p53 enhancer contacts additional p53 targets. In addition, through genome-scale analyses I dissected novel p53 programs in a postmitotic model (the Drosophila head). Interestingly, postmitotic p53 programs are distinct from networks described in developing cells. I found that the canonical p53 apoptotic program is unresponsive in Drosophila heads, establishing this system as an ideal in vivo model to study alternate functions of p53. To determine how p53 differential programs are specified, I tested two distinct mechanisms for tissue specific target activation, p53 enhancer looping and DNA binding. Interestingly, I observed no change in enhancer looping to cell death targets in heads. However, I did detect loss of p53 enhancer binding. Lastly, I integrated genome-wide analyses of p53 DNA occupancy and transcriptional control in embryos and heads. Interestingly, I found that at the genome-scale p53 binding landscapes poorly correlate with nearby transcriptional effects, indicating that p53 enhancers could be generally acting through long distances.Item A Novel Role for Odorant Binding Proteins in Deactivation of Drosophila Olfactory Neurons(2019-07-29) Scheuermann, Elizabeth Anne; Meeks, Julian P.; Smith, Dean P.; Krämer, Helmut; Terman, Jonathan R.In insects, odorant binding proteins are a large and diverse group of low molecular weight proteins secreted into the fluid bathing olfactory and gustatory dendrites. The best-characterized OBP, known as LUSH, is required in Drosophila melanogaster for the detection of physiological levels of the male-specific pheromone cVA. While LUSH acts as a sensitizing factor for pheromone detection, the role of other OBPs encoded in the Drosophila genome is largely unknown. In an effort to characterize members of this family, I used CRISPR-Cas9 to generate and characterize a deletion of two genes encoding the homologous OBPs OS-E and OS-F. These OBPs are nearly 70% identical and their expression is restricted to a small set of antennal chemosensory sensilla. Electrophysiological analysis of the olfactory neurons within these sensilla revealed no major difference in odorant sensitivity or specificity in the mutants but did reveal a striking deactivation defect to a subset of odorants. Surprisingly, other odorants detected by the same receptor are differentially affected by the absence of OS-E and OS-F, revealing an odorant-specific role for these OBPs in deactivation kinetics. Activation kinetics remain normal for the affected odorants in mutants. Genomic rescue experiments revealed that OS-E and OS-F are also functionally redundant, as either OBP is sufficient to revert the mutant phenotype. My findings reveal a new role for OBPs in deactivation of olfactory neurons and expand our understanding of the range of OBP functions.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 Rbfox1 Regulates mRNA Translation to Promote Germ Cell Differentiation(2016-07-12) Carreira-Rosario, Arnaldo; Zinn, Andrew R.; Buszczak, Michael; Conrad, Nicholas; Olson, Eric N.; Rothenfluh, AdrianGerm cells are the only cells that can give rise to an embryo. During differentiation, female germ cells that will give rise to oocytes form a syncytium called a germline cyst. The mechanisms that regulate germline cyst development remain poorly understood. In Drosophila, germline stem cells (GSCs) undergo an asymmetric division, giving rise to a stem cell and a cystoblast that then divides four times to produce a 16-cell germline cyst. This 16-cell cyst will then continue differentiation until it forms a mature oocyte. Drosophila RNA-binding Fox-1 (Rbfox1), also known as Ataxin-2 Binding Protein 1 (A2BP1), mutant females exhibit a germ cell differentiation defect that results in germline cystic tumors. The Rbfox genes encode several isoforms, many of which contain a highly conserved RNA recognition motif (RRM). Disruption of human RBFOX homologs have been linked with a number of different neurological disorders and cancers. Some of these isoforms localize to the nucleus while others localize to the cytoplasm. Nuclear forms have well-established roles in regulating alternative splicing. However the function of Rbfox in the cytoplasm remains unclear. Here, we demonstrate that cytoplasmic Drosophila Rbfox1 regulates germline cyst development. We further show that Rbfox1 represses the translation of mRNAs that contain (U)GCAUG elements within their 3’ UTRs. We have identified pumilio (pum) as a critical Rbfox1 target gene. Pum is an RNA-binding protein essential for germline maintenance across species. During germline cyst differentiation, Rbfox1 silences pum mRNA translation thereby promoting germ cell development. Mis-expression of pum results in the formation of germline cystic tumors that resemble Rbfox1 mutant phenotype. In addition, these cysts breakdown and dedifferentiate back to single, mitotically active cells. Together these results reveal that cytoplasmic Rbfox family members regulate the translation of specific target mRNAs to promote differentiation. In the Drosophila ovary, this activity provides a genetic barrier that prevents germ cells from reverting back to an earlier developmental state. These findings have thus advanced our understanding of germline development and the molecular function of Rbfox proteins, with implications in cellular differentiation and Rbfox-related disorders.