Browsing by Subject "Drosophila"
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Item Code Within Codes: Codon Usage Regulate Protein Expression, Structure, and Function(2018-07-23) Fu, Jingjing; Jiang, Jin; Liu, Yi; Cobb, Melanie H.; Green, Carla B.Most amino acids are encoded by two to six synonymous genetic codons. Synonymous codons are not used with the same frequency in all organisms, and every organism has its own preferred codon usage bias. Codon usage bias has been shown to positively correlate with tRNA abundance, thus optimal codons are thought to be translated more efficiently and accurately. Consistent with this, strong codon usage biases have been shown to be important for the expression of highly expressed genes in different organisms, and codon optimization has been widely used to enhance heterologous protein expression. Therefore, codon usage can be an important determinant in gene expression. In addition, codon usage has been shown to influence translation elongation rate and protein structure by affecting the co-translational folding process in E. coli, fungi, and insects. In addition to its role in regulating protein translation, codon usage also has a major role in determining the level of gene expression through transcriptional and post-transcriptional processes. As such, gene codon usage has been proposed to be a code within the genetic code that can determine both gene expression levels and protein structures and therefore activity. However, the effects of codon usage in multi-tissue organisms, for example, animals and humans, are not clear. In the first part of the thesis, by codon-optimizing open reading frame of Drosophila period gene, I showed that dper codon usage is critical for its circadian clock function. Optimization of dper codon usage resulted in conformational changes of dPER protein, altered dPER phosphorylation profile and stability, and impaired dPER repressor function in the circadian negative feedback loop. In the second part of the thesis, I reported that changing rare codons to common in KRAS increased translation and mRNA levels. Regulation of mRNA levels is a major mechanism affecting KRAS levels, but the effect was not a product of mRNA stability, but instead transcriptional. Moreover, codon usage also had an impact on the structure of KRAS. Thus, the rare codon bias of KRAS effects more aspects of protein production and function than previously appreciated, which has important implications for other rare codon enriched mammalian genes.Item A Developmental Algorithm for Synapse-Specific Wiring of the Drosophila Visual Map(2017-08-11) Agi, Egemen; Terman, Jonathan R.; Hiesinger, Peter Robin; Krämer, Helmut; Huber, Kimberly M.During brain development, genetic information and environmental input drive neural circuit assembly that requires matching of correct pre- and post-synaptic partners. In cases when environmental input has no instructive role in synaptic partner selection, genetic information alone must suffice to specify synapses in neural circuits. However, how a limited amount of genetic information is translated into developmental algorithms for synapse specification is unclear. A major thrust of the field has been the quest to identify guidance cues and molecular matchmaking codes underlying brain wiring. In this work, I present a complementary approach, in which the characterization of the developmental algorithm based on simple rules is the primary focus, and the molecules executing these rules secondary. I propose that simple rules underlying developmental algorithms can be sufficient to establish seemingly complex wiring diagrams without an elaborate matchmaking code between synaptic partners. I used Drosophila visual map, which is a genetically encoded neural circuit, as a model system to test my hypothesis. During visual map formation, around 4800 photoreceptors simultaneously project to their correct target layer 'lamina' in the brain to find their correct synaptic partners. I developed a 2-photon microscopy-based, intravital imaging technique with which I could observe the development of individual photoreceptor growth cones at the spatiotemporal resolution of filopodial dynamics over 24 hours during visual map formation. Based on these imaging data, I spearheaded a group effort to formulate and computationally test simple rules that are sufficient for photoreceptors to sort to their correct partners without a requirement for precise matchmaking codes. A key prediction of the model was that the post-synaptic partners may not act as target cues for the pre-synaptic photoreceptors. In the second part of my thesis, I tested this hypothesis by ablating and blocking membrane dynamics of post-synaptic partners. My findings indicate that indeed post-synaptic partners of photoreceptors do not act as target cues for photoreceptors, but are necessary during a preceding step in the developmental algorithm to ensure correct wiring. In brief, results I presented in this work support the idea that correct synaptic partner selection can be achieved through a developmental algorithm based on simple rules that sorts correct cells together prior to synapse formation.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 Filopodial Dynamics and Synapse Specification in the Drosophila Visual System(2017-11-27) Ozel, Mehmet Neset; Terman, Jonathan R.; Meeks, Julian P.; Danuser, Gaudenz; Huber, Kimberly M.; Hiesinger, Peter RobinHow is the synaptic specificity achieved in neural circuits comprised of hundreds of different types of neurons? My dissertation aims to advance our knowledge on this overarching question using the complex visual processing circuitry of D. melanogaster. This system not only provides excellent genetic amenability but also a model where almost all connectivity can be built without environmental input, i.e. it is genetically hardwired. Nearly three decades of research has identified a vast array of genes required for various steps of synapse specification. However, it remained unclear how these genes implement the developmental rules that result in the final connectivity and we understand very little of what actually goes wrong between a particular genetic perturbation and the resulting miswired circuit. To that end, I focused on the actual subcellular substrate of connectivity: axonal growth cones. To gain access to the details of their dynamic behavior during development, I developed an imaging technique which allows the monitoring of intact, developing fly brains over long periods in high temporal and spatial resolution. Using live imaging and the axonal terminals of R7 photoreceptor as a model, I performed a detailed analysis of growth cone dynamics during various steps of synaptic specification, in wild-type and perturbed conditions. Interestingly, I found that none of the perturbations that were previously tied to 'layer specific targeting' of R7 axons were actually required for the recognition of or targeting to a specific layer; instead, all displayed a loss of stabilization with various timings of onset. High speed live analysis revealed the stochastic filopodial dynamics of these axons as crucial mediators of this stabilization. First, as the substrate of attachment to the target layer during early development (Chapter 2); second, as the searching agents for postsynaptic partners during synapse formation (Chapter 3). In brief, my research provided a valuable bridge between the genetic factors that instruct the synapse specific wiring of the brain and how they regulate the dynamic properties of axonal growth cones and synaptic terminals in distinct ways to achieve that final outcome.Item In Pursuit of a Molecular Fountain of Youth: The Identification and Characterization of Lifespan Regulators in Drosophila(2012-07-20) Stenesen, Drew Stanness; Graff, Jonathan M.Over the past century, average human lifespan has experienced steady increase despite lack of substantial intervention or understanding of the aging process. In fact, many organisms have the latent potential to live much longer than they normally do. This indicates lifespan determination is an active process subject to regulation. Components of this impending longevity are beginning to unravel through dietary and genetic studies in model systems. To date, several pathways indicate human lifespan extension through direct molecular intervention may be feasible, however, important limitations persist. A common thread among these conserved lifespan regulators is metabolism. Therefore, further insight into lifespan extending mechanisms may lie within tissues governing important metabolic processes. Here we describe a multi-tiered, strategy to identify Drosophila melanogaster mutants with extended lifespan based upon enrichment for insertions in genes that are expressed in metabolic tissues. Our results indicate metabolically relevant tissues are a rich source of genetic longevity regulation. We identified a regulator of G protein signaling (RGS) domain containing sorting nexin, termed snazarus (sorting nexin lazarus, snz). Flies with insertions into the 5' untranslated region of snz live up to twice as long as controls. Transgenic expression of UAS-Snz from the snz Gal4 enhancer trap insertion, active in metabolic tissues, rescued lifespan extension. Notably, old snz mutant flies remain active and fertile indicating that snz mutants have prolonged youthfulness, a goal of aging research. Since mammals have snz-related genes, it is possible that the functions of the snz family may be conserved to humans. Next, we identified the two key adenosine monophosphate (AMP) biosynthetic pathways as regulators of Drosophila longevity. We found that heterozygous mutation of anabolic components of the de novo as well as the salvage AMP biosynthesis pathways extend lifespan. These pathway mutations, and caloric restriction, increased adenosine mono- and diphosphate to adenosine triphosphate (ATP) ratios. Consistent with the altered ratios, lifespan extension was dependent on functional adenosine monophosphate-activated protein kinase (AMPK). Supplementing the diets of adult mutants with adenine restored adenosine nucleotide ratios and rescued lifespan extension. These data establish de novo and salvage AMP biosynthesis as determinants of adult lifespan. The dosage sensitivity and enzymatic nature of de novo and salvage AMP biosynthesis, and the conserved aspects of adenosine nucleotide derivatives and lifespan extension, indicate that these pathways are potentially amendable drug targets worth continued exploration.Item Investigations into the Role of Paf1 Complex Proteins in Drosophila Ovaries(2014-10-23) Chaturvedi, Dhananjay; Li, Bing; Olson, Eric N.; Jiang, Jin; Buszczak, MichaelOver the past decade considerable interest has grown in epigenetics and chromatin modifications. The ability of two cells with identical genomes to have entirely different transcriptomes and therefore cellular behaviors has piqued the curiosity of many researchers creating the whole field of Chromatin Biology. The difference in behavior of otherwise identical cells comes from an array of covalent modifications to protein spools wrapped by DNA in each cell. The complex of DNA and proteins is referred to as chromatin. Chromatin modifications influence the expression of regulatory proteins that control effectors of cellular physiology and metabolism. The outcome of protein function within a cell decides its fate from size, shape, function, the ability to divide or the lack thereof. The behavior of cells that can divide to give rise to themselves or progeny with a distinct specified function in an organism is of interest the biomedical community at large. These cells, called stem cells, hold promise in regenerative medicine to treat dystrophic diseases and injury. Work in cell culture systems shows that proteins modifying stem cell chromatin control their fate to a large extent. In this thesis I present to you, my efforts at understanding the role of a chromatin modifying complex: the Paf1 complex in the maintenance and differentiation of in vivo stem cell populations that control the maintenance of Drosophila ovaries.Item The Molecular Basis of Tissue Elasticity and Force Balance During Drosophila Gastrulation(December 2021) Goldner, Amanda Nicole; Collins, James J.; Doubrovinski, Konstantin; Chen, Elizabeth; Douglas, PeterThe mechanics of folding any material rely on two things: the physical forces forming the fold and the material properties of the substance being folded. When working in biological tissue such as an early Drosophila embryo, there is no existing way to directly measure morphogenetic forces, and the relative contributions of forces in various cellular domains remain unknown. To begin, I studied gastrulation in a genetic background where basal membranes never form and cells remain open to the yolk sack throughout the course of VF formation. Strikingly, the VF is still capable of forming in this background. I extensively characterize this phenotype by a combination of electron microscopy and immunofluorescence. My observations rule out a class of popular models of VF formation that would generically predict no folding in the absence of basal membranes. To address this discrepancy, we propose that viscous shear forces play a major role in allowing the furrow to form. We have developed a new computational model that takes cytoplasmic viscous shear into account. In accordance with our observations, our model predicts that basal membranes are dispensable for VF formation. Tissue material properties such as elasticity are also key to fold shape. In vivo tissue deformation experiments show that embryonic tissue is elastic in the stages leading up to gastrulation. Inhibiting F-actin polymerization severely decreases elasticity. I propose that different characteristics of F-actin networks - e.g. branching, remodeling, and crosslinking - are variably responsible for conferring elasticity. It is unclear whether the presence of active forces along actin filaments contributes to tissue elasticity. To this end, I engineered the auxin-inducible degron system to degrade the foremost source of active forces in F-actin networks: myosin II. My design allows us to specifically degrade Drosophila myosin II in under 1hr in vivo. This will allow us to precisely quantify the contribution of myosin II to not only tissue elasticity, but any other feature or developmental process of interest.Item Neuronal Maintenance via a Neuron-Specific Degradation Pathway(2015-01-26) Schmidt, Taylor; Jin, Eugene Jennifer; Ozel, Mehmet Neset; Epstein, Daniel; Marchant, Corey; Hiesinger, RobinBACKGROUND: Neurons can survive for decades via cell maintenance and protein degradation. This process includes the general protein endolysosomal degradation pathway, an integral part of which is the Rab GTPase proteins. Recently, components of a neuron-specific protein degradation pathway were discovered, which include the neuronal vesicle ATPase component V100 and the synaptic vesicle protein neuronal Synaptobrevin (n-Syb). While this neuron-specific degradation pathway has been shown as necessary for neuronal maintenance in adult Drosophila melanogaster fruit flies, it is not known what this neuron-specific degradation pathway does, nor how it interacts with the general protein degradation pathway. Our research aimed to fill this gap in knowledge. Such research may be salient because the misregulation of protein degradation in neurons leads to neurodegenerative diseases like dementia. OBJECTIVE: We hypothesized that neurons either have an increased or a specialized need for protein degradation in comparison to other cells. METHODS: 1. The lab chose a myristoylated protein (myr) to represent general proteins found in every cell, and Synaptotagmin1 (Syt1) to represent neuron-specific proteins. The acidification-sensitive tag mCherry-pHluorin, which changes color with a decrease in pH, was placed on Syt1 and myr to visualize acidification and degradation of the two proteins. 2. The lab generated Drosophila lines to compare acidification and degradation of Syt1 and myr in wild-type versus the following three mutants: rab7 mutants to disrupt general protein degradation, v100 to disrupt the neuron-specific protein degradation, and synaptobrevin also to disrupt neuron-specific degradation. 3. We performed live imaging to visualize acidification and protein degradation at synaptic terminals. Brains of Drosophila pupae from each cross were dissected, mounted onto Petri dishes, and surrounded with a culture medium to be kept alive. A resonant confocal microscope was used to observe the brain's lamina, a layer of neurons between the eye and the brain. At the lamina, we recorded 30-minute videos showing changes in fluorescence representing protein degradation. RESULTS AND CONCLUSION: Preliminary data show that nsyb and v100 mutations may cause defects in the degradation of neuron-specific cargo. Such evidence suggests that the neuron-specific endolysosomal degradation pathway specifically degrades the synaptic vesicle protein Synaptotagmin1. Also, the experiments indicate that disruption of either the neuron-specific or the general endolysosomal degradation pathway has no effect on the acidification of the myristoylated protein. Such evidence implies that the general pathway of protein degradation occurs at synapses, but has no specificity for protein cargo. A greater sample size is needed for future experiments, as well as quantitative analysis.Item Odorant Responses and Courtship Behaviors Influenced by at4 Neurons in Drosophila(2017-02-13) Pitts, Svetlana Vladimirovna; Krämer, Helmut; Smith, Dean P.; Takahashi, Joseph; Meeks, Julian P.In insects, pheromones function as triggers to elicit complex behavior programs, such as courtship and mating. In most species, the neurons tuned to pheromones are localized in a specific subset of olfactory sensilla located on the antenna called trichoid sensilla. In Drosophila there are two classes of trichoid sensilla, at1 sensilla that contain the dendrites of a single neuron that is specifically tuned to the male-specific pheromone 11-cis vaccenyl acetate (cVA), and at4 sensilla that contain three neurons with relatively poorly defined chemical specificity and function. Using a combination of odorant receptor mutant analysis, single sensillum electrophysiology and optogenetics, I have examined the chemical tuning and behavioral consequences of the three at4 olfactory neuron classes. My results indicate that one class, Or65abc neurons, previously reported to be cVA sensors, are unresponsive to cVA pheromone. Or65abc neurons inhibit courtship behaviors. The other two neuron classes, Or88a and Or47b neurons, are sensitive to a diverse array of fly and non-fly odors. Activation of these neurons has little direct impact on courtship behaviors.Item [Southwestern News](1996-06-27) Lyons, MorganItem Transcriptional Regulation of Intestinal Stem Cell Lineage in Drosophila(2017-04-17) Lan, Qing; Jiang, Jin; Kraus, W. Lee; Sadek, Hesham A.; Jiang, HuaqiThe question of how somatic stem cells respond to tissue needs is always intriguing, since aberrant somatic stem cell behaviors may lead to adult tissue degeneration or tumorigenesis. Here, this thesis focuses on the transcriptional regulation of a somatic stem cell lineage: the intestinal stem cell in Drosophila adult gut. The Drosophila adult gut is a dynamic organ. It is maintained by hundreds of somatic gut stem cell evenly distributed throughout the gut epithelium. These multi-potent somatic stem cells undergo self-renewal and differentiation to replenish two mature gut cell types: the absorptive enterocytes and secretory entero-endocrine cells. Through an RNAi screen targeting transcription factors required for stem cell-mediated acute gut regeneration, two novel transcription factors, the FoxA family Fork head (Fkh) and SoxE family sox100b (dSox9), were uncovered and functionally characterized in this thesis. During gut regeneration, transcription factor Fkh and dSox9 are required for stem cell proliferation. During gut homeostasis, Fkh maintains stemness and prevents progenitor from precocious differentiation; dSox9 controls lineage differentiation through Jak-Stat pathway. To further probe mechanisms underlying gut stem cell physiology, ChIP-Seq technique was applied to map chromatin binding sites of gut stem cell regulators (HA tagged) in stem/progenitor cells of dissected fly guts, including transcription factors (FoxA family/Fkh, SoxE family/dSox9, bHLH family/Da), niche pathway downstream factors (Jak-Stat pathway/Stat92E, BMP pathway/Mad, Notch pathway/Su(H), JNK pathway/Kay), and transcriptional regulators (Mediator/Med20, p300/Nej). A set of shared ChIP-Seq peak regions likely functions as enhancers to drive gene expression in gut stem/progenitor cells. This thesis leads to the speculation of a transcriptional network that maintains gut stem/progenitor cell normal physiology in adult Drosophila.