Browsing by Subject "Drosophila Proteins"
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Item Apoptosis Determinants in Drosophila melanogaster(2007-12-17) Chew, Su Kit; Abrams, JohnApoptosis 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, JohnBcl-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 ?H?otif. 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(2011-08-26T17:33:49Z) Link, Nichole Lynn; Abrams, JohnApoptosis 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 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. [Keywords: hedgehog, Drosophilia, Fu, Cos2, Ci, Sufu, Smo, HIB, PY-NLS]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 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 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, JonathanThe 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, Micahel Ph.D.Specialized 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 Regulatory Mechanism of the RNAi Pathway(2011-12-14) Liu, Ying; Liu, QinghuaRNA interference (RNAi) is post-transcriptional gene silencing initiated by Dicer, a RNase III that processes double-stranded RNA (dsRNA) precursors into small interfering RNA (siRNA). In Drosophila, Dicer2 and R2D2 coordinately recruit duplex siRNA to the effector RNA-induced silencing complex (RISC), wherein single-stranded siRNA guides the endoribonuclease Argonaute (Ago) to catalyze sequence-specific cleavage of complementary mRNA. It remains unclear as to what constitutes holo-RISC, how is RISC assembled and how is RISC regulated. Here we took a candidate approach to reconstitute for the first time the long double-stranded RNA- and duplex siRNA-initiated RISC activities with the use of recombinant Drosophila Dicer-2, R2D2, and Ago2 proteins. We further employed this core reconstitution system to purify a RNAi regulator that we named C3PO (component 3 promoter of RISC), a complex of Translin and Trax. C3PO is a novel Mg2+ -dependent endoribonuclease that promotes RISC activation by removing the siRNA passenger strand cleavage products. Similar as Drosophila C3PO, human C3PO also degrades passenger strand fragments and facilitates RISC activation. RISC is a multiple-turnover enzyme, wherein single-stranded (ss)-siRNA guides Ago2 to catalyze sequence-specific cleavage of the target mRNA at the effector step. We employed human minimal RISC reconstitution system to purify antoantigen La as a novel activator of the RISC effector step. Biochemical studies indicated that La promotes the multiple-turnover of RISC catalysis by facilitating the release of RISC cleaved products. Moreover, we demonstrated that La is required for efficient RNAi, antiviral defense, and transposon silencing in mammalian and Drosophila cells. Taken together, our findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC. The robust reconstitution system establishes a powerful platform for in-depth studies of the assembly, function, and regulation of RISC. Similar to the discovery of C3PO and La, it can be used to identify novel regulators and study post-translational regulations of RNAi, therefore, connecting RNAi to other cellular signaling pathways. As such, these biomedical studies could have a major and lasting impact on the biological understanding and therapeutic application of RNAi.Item The Role of Double-Stranded RNA Binding Protein R2D2 and Lump in Drosophila Gametogenesis(2010-05-14) Sanders, Charcacia Tiana; Smith, DeanIn 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 The Role of the Scaffolding Protein INAD in Localization of Signaling Complexes to te Rhabdomeres of Drosophil Photoreceptors(2004-12-15) Hahn, Adrienne R.; Ranganathan, RamaOrganization of proteins into macromolecular complexes is one way cells maximize the speed, specificity, and efficiency of signal transduction. In fruit fly photoreceptors InaD, a scaffolding protein containing 5 PDZ domains, organizes proteins involved in the visual signaling pathway into complexes within a microvillar stack of membranes known as the rhabdomere. Light activation of rhodopsin activates a signaling cascade via a Gq-coupled reaction that quickly opens Ca++-selective TRP channels. Subsequent Ca++ influx activates eye protein kinase C (ePKC), and calmodulin, which in turn modulate the activity of other visual proteins. Mutants in which the InaD / TRP association has been disrupted (inaD215) phenocopy the delayed inactivation of mutants lacking ePKC, suggesting that one of the functions of InaD includes localizing ePKC to its downstream targets such as the TRP channel. There are currently two different models for scaffolding proteins: the "beads on a string" or "tethering" model where the order of the binding domains and their respective binding partners is unimportant, and the "specific quaternary structure" model where the specific stereochemical orientation of the domains is vital for proper signaling. The latter model also allows for allosteric regulation of binding. We assess the "beads on a string" vs. the "specific quaternary structure" model for InaD, a scaffolding protein found in the photoreceptor cells of fruit flies, by analyzing the characteristics of the light response in flies expressing two InaD constructs where the order of the PDZ domains has been shuffled. Based on biochemical and electrophysiological data on these mutants, we conclude that the "specific quaternary structure" model applies best to InaD. In addition, we investigate whether the inaD215 phenotype is due to displacement of ePKC from microdomains of calcium initiated by TRP channels, by calcium imaging of photoreceptors expressing visual proteins tagged with CaMgaroo, a Ca++-sensitive derivative of yellow fluorescent protein. After conducting these experiments, we conclude that this is not the case. Rather, the inaD215 phenotype is most likely due to the inability of ePKC to phosphorylate TRP and and attenuate its activity. These results also support the "specific quaternary structure" model for InaD.