Browsing by Author "Stern, Michael"
Now showing 1 - 20 of 24
Results Per Page
Sort Options
Item A genetic study of inebriated, a Drosophila gene that physical dual roles in the control of neuronal excitability and the osmotic stress response(2002) Huang, Yanmei; Stern, MichaelThe Drosophila inebriated (ine) gene encodes a putative transporter (Ine) that shares high homology to members of the Na+/Cl- dependent neurotransmitter transporter family. Mutations in the ine gene were found to cause increased neuronal excitability. Research documented in this thesis demonstrated that ine also confers defective osmotic stress response, confirming the dual roles played by certain members of this family in regulating both neuronal excitability and osmotic stress response. In addition, from further investigation of the neuronal phenotypes of ine mutants it was discovered that Ine might act in short-term to affect neuronal excitability, that the transporter can exert its function from either neurons or glia, and that the two isoforms of the transporter, Ine-P1 and Ine-P2, which are identical in major portion of their sequence but differ in their N termini, are both capable of their function in the absence of the other, although the former functions more efficiently. Furthermore, ine overexpression causes phenotypes that closely resemble those of mutants with defective sodium channels. These phenotypes include delayed onset of long-term facilitation, suppression of the leg-shaking phenotypes of Shaker , temperature sensitive paralysis, enhancement of the paralytic (para) mutation, increased failure rate of transmitter release at the larval neuromuscular junction, reduced amplitude of larval nerve compound action potential and failure of compound action potential at restrictive temperature. Taken together, these observations raise the possibility that ine might be involved in a signaling pathway that regulates neuronal sodium channels.Item A PI3-kinase mediated negative feedback regulates neuronal excitability at the Drosophila neuromuscular junction(2009) Howlett, Eric Lane; Stern, MichaelUse-dependent downregulation of neuronal activity (negative feedback) can act as a homeostatic mechanism to maintain neuronal activity at a particular specified value. Disruption of this negative feedback might lead to neurological pathologies such as epilepsy, but the precise mechanisms by which this feedback can occur remain incompletely understood. At one glutamatergic synapse, the Drosophila neuromuscular junction, a mutation in the group II metabotropic glutamate receptor gene ( DmGluRA ) increased motor neuron excitability by disrupting an autocrine, glutamate-mediated negative feedback. I show that DmGluRA mutations increase neuronal excitability by preventing PI3 kinase (PI3K) activation and consequently hyperactivating the transcription factor Foxo. Furthermore, glutamate application increases levels of phospho-Akt, a product of PI3K signaling, within motor nerve terminals in a DmGluRA -dependent manner. Finally, I show that PI3K increases both axon diameter and synapse number via the Tor/S6 kinase pathway, but not Foxo. In humans, PI3K and group II mGluRs are implicated in epilepsy, neurofibromatosis, autism, schizophrenia and other neurological disorders; however, neither the link between group II mGluRs and PI3K, nor the role of PI3K-dependent regulation of Foxo in the control of neuronal excitability, had been previously reported. My work suggests that some of the deficits in these neurological disorders might result from disruption of glutamate-mediated homeostasis of neuronal excitability.Item A two-pronged approach towards the development of novel therapeutics for advanced endometrial cancer(2015-11-24) Engel, Brian Joseph; Carson, Daniel D; Jacot, Jeffrey G; McCrea, Pierre D; Stern, Michael; Wagner, Daniel SEndometrial cancer is the fourth most common cancer among women. The standard of care involves hysterectomy with adjuvant radiation and chemotherapy for advanced disease. Despite these efforts, treatment of advanced and metastatic disease is not very effective. This body of work describes a two-pronged approach to address lack of treatments for advanced endometrial cancer. The first approach was an in-depth study of the mechanism and physiological effect of mucin 1 (MUC1)-driven epidermal growth factor receptor (EGFR) expression and signaling. MUC1 is a large, heavily glycosylated transmembrane protein that functions to lubricate surfaces, provides protection from external insult and plays an important role in embryo implantation. EGFR is a receptor tyrosine kinase that influences cellular proliferation, migration and apoptosis. MUC1 increases EGFR gene expression, mRNA levels, protein levels and signaling in endometrial cancer cell lines. Consequently, MUC1 expression is associated with increased EGF-dependent cellular proliferation, survival and resistance to EGFR inhibitors. In addition, MUC1 and EGFR co-expression is associated with increased cellular proliferation in endometrial tumors. The second approach involved the development and characterization of an advanced three dimensional (3D) hyaluronic acid (HA)-based culture model that is compatible with existing high throughput drug screening methodologies. This system incorporates three layers: an acellular cushion layer; an encapsulated cancer cell layer for growth in 3D; and a collagen-containing layer that supports the growth of stromal cells on top of the hydrogel (2.5D). The robustness of this system was evaluated by incorporating endometrial or prostate cancer cells with associated stromal cells. Both culture systems provided high cancer and stromal cell viability and facilitated paracrine interactions. The response to cytotoxic drugs from cells cultured in 3D HA better matched clinical data than cells grown in 2D and 3D-alginate. These studies provide mechanistic evidence for regulation that occurs in advanced endometrial cancer, as well as an improved platform to screen for effective therapeutics. The 3D culture system could be leveraged to evaluate novel therapeutics for the treatment of advanced endometrial cancer which may include MUC1 and EGF-directed therapies.Item An Investigation of the Biophysical and Biochemical Properties of Perlecan/HSPG2: Implications for Bone Mechanotransduction and Endochondral Ossification(2016-04-26) Martinez, Jerahme R; Stern, Michael; Farach-Carson, Mary CindyPerlecan, also known as heparan sulfate proteoglycan 2 (HSPG2), is a very large secreted proteoglycan ubiquitously expressed in all basement membranes and in the territorial matrix of skeletal tissues. Perlecan is particularly important for the formation and stabilization of tissue layers and its absence is catastrophic for bone and cartilage development. In mineralized bone, perlecan is a key component of pericellular matrix (PCM) surrounding osteocytic processes that preserve fluid flow throughout bone tissue. In addition, recent findings have coupled perlecan expression, along the osteocyte cell surface, to bone mechano-adaptive response. Perlecan’s mechanical properties, important for maintaining the osteocyte cell-bone matrix interface, were studied for the first time. This work demonstrated perlecan functions as a semi-flexible tether that is capable of withstanding physiological loads imposed on cortical bone.The second part of this study examined perlecan in the context of early precartilage condensation. This study demonstrated a novel sub-domain of perlecan, PLN IV-3, located in its fourth domain modulates cell-matrix interactions that are involved in the chondrogenic process. When presented as part of the substratum, PLN IV-3 suppressed focal adhesion kinase (FAK) phosphorylation and signaling through the mitogen-activated protein kinase (MAPK) cascade. This led to reduced cell migration, increased cell-cell adhesion, and decreased cell proliferation, all of which are hallmarks for mesenchyme condensation and subsequent precartilage formation. I hypothesize that this is a possible mechanism by which perlecan functions to coordinate the migration of mesodermal cells and drive chondrocyte differentiation as seen during development. Together, these studies suggest perlecan possesses multifunctional properties that affect bone health throughout various stages of life.Item Characterization of Structure and Function Relationship between Domains of the ER Membrane Protein Atlastin(2014-02-06) Desai, Tanvi; McNew, James A.; Huang, Huey W.; Braam, Janet; Stern, Michael; Lwigale, Peter YunjuThe endoplasmic Reticulum (ER) is an important site for lipid synthesis, protein synthesis and transport. ER fusion is an essential process for its maintenance and biogenesis. Mutations in genes involved in this process cause Hereditary Spastic Paraplegia (HSP). These mutations are shown to affect intracellular trafficking and localization of membrane compartment. One of the important proteins causing early onset of HSP is Atlastin. Previous work in McNew lab at Rice University (Moss et al., 2011b) has shown that atlastin is involved in the homotypic fusion of the ER and the C-terminal cytoplasmic region of atlastin is essential for atlastin mediated fusion. During my studies presented in this thesis, I was able to demonstrate that the C-terminal cytoplasmic region of atlastin destabilizes lipid bilayers to facilitate fusion. The requirement of C-terminal cytoplasmic region is minimal when fusing two fluid (or unstable) lipid bilayers. The C-terminal cytoplasmic region of atlastin forms an amphipathic helix and mutations on the hydrophobic phase of the helix reduce fusion. These mutations are not dominant, as presence of full length atlastin on even one of the fusing lipid bilayers can significantly improve fusion during a heterotypic fusion reaction. Additionally, domain swaps between human atlastin-1 and drosophila atlastin show that the role of C-terminal cytoplasmic region is highly conserved. Also, during my research presented here in, I found that when the transmembrane region and C-terminal cytoplasmic region of human atlastin-1 were swapped with drosophila atlastin, it showed functional similarity. These results show that although atlastins in organisms play an important role in the ER fusion, there are likely species specific differences in how this is achieved. An understanding of atlastin mediated fusion should help in unraveling mechanisms of HSP pathogenesis and other disorders arising from dysfunctional ER.Item Cloning and sequencing ofpushover, a Drosophila gene affecting neuronal activity and glial morphology(1999) Richards, Stephen; Stern, Michaelpushover (push) is a mutation that causes neuronal hyperexcitability, uncoordination and male sterility. Here it is also shown that push mutant wandering third instar larvae exhibit segmental nerves that are slightly thickened compared to wild type controls. The additional thickness of these nerves is due to a thicker perineural glia layer surrounding the axon bundle of the nerve. From investigation of push mutant testes, it was determined that mature sperm are produced, but are immotile. The push gene was cloned and sequenced and encodes a 5322 amino acid protein with 12 putative transmembrane helices. Sequencing of the push gene in the push 1 mutant line revealed a premature stop codon at amino acid position 728 and in push2 a premature stop codon at position 883. In situ hybridization experiments show push is expressed in the embryonic CNS and in primary spermatocytes in the adult testes and suggest that push is not expressed in the perineural glia of wandering third instar larvae. Because push affects but is not expressed in these glia, it is hypothesized that push is involved in a signaling pathway connecting the motor neuron to the perineural glia.Item Control of perineurial glial growth in Drosophila melanogaster(2003) Yager, James Christopher; Stern, MichaelAlthough intercellular communication within peripheral nerves is critical to the structure and function of the nervous system, it is incompletely understood. Drosophila peripheral nerves comprise motor and sensory axons bundled by peripheral glia (Schwann cells) and wrapped by perineurial glia (perineurium). I have shown that growth of the perineurial glia is controlled by signaling pathways involving six genes: push, which encodes a large Zn2+ finger containing protein; amn which encodes a putative neuropeptide; Axs, which is suggested to encode a G-protein coupled receptor; ine, which encodes a putative neurotransmitter/osmolyte transporter; eag, which encodes a potassium channel; and NF1, which encodes neurofibromin and is the Drosophila ortholog of the human gene responsible for Neurofibromatosis type1. I provide evidence that neurofibromin, in accordance with its role as a Ras guanosine triphosphatase activating protein (Ras GAP), acts to down regulate Ras activity to control perineurial glial growth. My work suggests that loss of neurofibromin leads to an increase in Ras activity in the peripheral glia that, in conjunction with loss of either Ine or Push, introduces a cell-nonautonomous signal that promotes growth of the perineurial glia. I have also found that Push does not act through Ras to control perineurial glial growth. My working hypothesis is that Amn acts through two separate pathways, one involving Push and the other involving neurofibromin, to inhibit perineurial glial growth. In this model, a separate pathway involving the substrate neurotransmitter of Ine promotes perineurial glial growth. I speculate that Ine may act to remove its substrate neurotransmitter from the extracellular space, thereby inhibiting the neurotransmitter from acting through its receptor to promote perineurial glial growth. Alternatively, Ine may control perineurial glial growth via its role as an osmolyte transporter. Eag may act to inhibit perineurial glial growth by repressing release of factors from the neurons or peripheral glia through maintaining these cells in a hyperpolarized state.Item Discovery of Rotavirus NSP2 Regulation of Lipid Droplet Formation and an Unrecognized Pathophysiological Mechanism of Rotavirus-induced Diarrhea(2020-04-14) Liu, zheng; Estes, Mary K; Crawford, Sue E; Stern, MichaelThe overall goal of my thesis is to reveal the mechanism of RV-mediated LD formation, and the role RV exploitation of LD biogenesis plays in RV pathogenesis. Across all age groups, diarrhea is one of the top five causes of death world¬wide; while among children younger than 5 years old, diarrheal diseases are the second leading cause of morbidity and mortality around the world (Liu et al., 2012). Rotaviruses (RV), calici¬viruses (particularly norovirus), astroviruses, and enteric adenoviruses are the four predominant causes of viral gastroenteritis (Zachos, 2016). The symptoms of RV infection, such as acute watery diarrhea, nausea, vomiting and low-grade fever, can last for several days, and cause dehydration (Bishop, 2009). The molecular mechanisms of RV-induced gastroenteritis are not completely clear yet. Currently, there are three proposed mechanisms (1) osmotic diarrhea due to malabsorption, (2) secretory diarrhea due to the effects of the rotavirus nonstructural protein NSP4 expression, and (3) activation of the enteric nervous system (ENS) (Crawford et al., 2017). These mechanisms may be interrelated. RV replicates and assembles immature viral particles in cytoplasmic compartments named viroplasms. Viroplasms contains both viral and cellular components. Viroplasms associate with lipid droplets (LDs), neutral lipids and LD-specific proteins have been detected on viroplasms. Disrupting LD formation prevents viroplasm assembly and RV replication (Cheung et al., 2010). Diacylglycerol acyltransferase (DGAT1) is the key cellular enzyme in triacylglycerol synthesis required for LD biogenesis. Recent research reported that loss-of-function mutations in DGAT1 gene lead to severe congenital diarrheal disease in young children (Gluchowski et al., 2017; Haas et al., 2012; van Rijn et al., 2018). Patients with DGAT1 deficiency experience symptoms, such as non-bloody diarrhea, vomiting, and dehydration, similar to what is seen in RV-infected patients. My thesis research results (see chapter 3) demonstrate that DGAT1 is degraded in RV-infected cells by a proteasome-mediated mechanism involving ubiquitinated RV nonstructural protein NSP2. RV infection induced DGAT1-silencing results in earlier formation and an increased number of viroplasm/LDs per cell that translates into a 4-5 fold increase in viral yield (p<0.05). These results suggest that RV-induced DGAT1 degradation may trigger LD biogenesis and promote viroplasm/LD formation and lead to DGAT1 deficiency in the small intestine. DGAT1 deficiency is a rare cause of neonatal diarrhea purportedly due to the altered trafficking of key ion transporters to the apical brush border of enterocytes. Confocal microscopy results demonstrated that RV-mediated DGAT1 degradation results in a similar loss of the apical brush border transporters sucrase isomaltase and the sodium hydrogen exchanger protein as in DGAT1-/- HIEs. However, western blot analysis revealed that expression of these and other proteins, ezrin, villin, E-cadherin, and JAM-1, was substantially reduced, indicating an alternative mechanism for diarrhea. RV-mediated DGAT1 degradation or DGAT1 deficiency does not lead to global alterations in protein trafficking but instead results in loss of expression of proteins key to carbohydrate digestion, and water, electrolyte, and nutrient absorption. My results elucidate a new pathophysiological mechanism of RV-mediated and DGAT1 deficiency malabsorptive diarrhea. This research identified a previous unrevealed mechanism which contributes to the severe diarrheal disease induced by RV infection. This mechanism is not interrelated with the other proposed mechanisms, it is the first mechanism which recognizes the important roles of another viral protein (NSP2) rather than NSP4, and other cellular components (DGAT1) rather than Ca2+ signaling in RV-induced gastroenteritis. Since RV viroplasm requires LD biogenesis for particle assembly, additional research (see chapter 4) reports studies that explored the interaction between the viroplasm and LD-associated proteins. My results show that viroplasms are associated with a pool of LDs that are coated by PLIN1, but not PLIN2 or PLIN3. In addition, silencing PLIN1 by siRNA transfection or introducing homozygous mutation, significantly decreased the yield of RV in the cells by 50% to 90%, indicating the crucial role PLIN1 LDs may play in viroplasm formation. Co-Immunoprecipitation (co-IP) results show that PLIN1 physically interacts with both the viroplasm exclusive form of NSP2 (vNSP2) and the cytoplasmic dispersed form of NSP2 (dNSP2), whereas PLIN2 only interacts with dNSP2. In addition, more and larger PLIN1 coating LDs, which are also viroplasms, are observed in RV-infected cells. Almost no PLIN2 LD is observed in RV-infected cells, while many PLIN2 LDs can be seen in the neighboring uninfected cells. These results suggest PLIN2 may be degraded via its interaction with dNSP2 in a manner similar to how DGAT1 is degraded, whereas PLIN1 is stabilized in RV-infected cells. My western blot results show that the addition of MG132 inhibits the degradation of PLIN1 in mock- but not RV-infected cells, indicating RV infection inhibits PLIN1 turnover. Co-Immunoprecipitation results also show that similar amounts of both forms of NSP2 (d- and vNSP2) are pulled down by polyclonal PLIN1 antibody and phosphorylated specific monoclonal PLIN1 antibody, suggesting the phosphorylated PLIN1 is the isoform that interacts with both dNSP2 and vNSP2. Phosphorylated PLIN1 is reported to be exclusively located on LDs rather than other cellular components, such as the endoplasmic reticulum, peroxisomes, and mitochondria localized near LDs (Blanchette-Mackie et al., 1995). The interactions between phosphorylated PLIN1 and both d- and vNSP2 indicate viroplasm components are recruited to the surface of PLIN1 LDs, suggesting a scaffolding role of PLIN1 during viroplasm formation. Together, my research shows RV infection may reshape cellular lipid metabolism and other cellular pathways via the physical interaction between the two forms of the RV NSP2 protein and multiple cellular targets. Interaction with dNSP2 alone, which is K48- ubiquitinated and interacts with multiple units of the 26S proteasome, may promote the proteasome-mediated degradation of a cellular protein and the interaction with vNSP2 may stabilize or sequester the cellular proteins to viroplasm. We propose this is a previously unrecognized mechanism of RV-induced pathogenesis.Item Endogenous Nodal diverges Wnt signaling interpretation from definitive endoderm to posterior mesoderm in geometrically constrained human pluripotent cells.(2023-11-28) Ortiz Salazar, Miguel Ángel; Stern, Michael; Warmflash, AryehEmbryonic development is a dynamic and highly complex process. It demands the intricate coordination of several mechanisms and signaling pathways to regulate cell fate determination, tissue patterning, and functional integrity, ensuring the embryo’s proper formation until birth. One crucial signaling cascade in development is the Wnt pathway. It is essential during the early stages, as it directs gastrulation, the phase in which the embryo starts forming the germ layers, particularly the endoderm and mesoderm. The Wnt pathway also plays a role in establishing the anterior-to-posterior body axis and helps in elongating the body. However, the precise mechanisms of how Wnt coordinates these diverse stages yielding different outcomes remain not fully elucidated. In this work, we aimed to study the Wnt pathway and its role in cell fate decisions. We uncovered that a posterior-progenitor-like state is induced when the posterior signals, WNT3a and FGF8 are presented to human embryonic stem cell colonies in standard culture. However, when these colonies are geometrically constrained, colonies self- organize themselves into a structure composed of a definitive endoderm cell population surrounding and lifting an epiblast disk-like structure. These unprecedented colonies do not change cell fate when Wnt levels are increased, challenging the classic concentration- dependent morphogen mechanism. While studying this discrepancy, we were able to determine that TGF-ß pathway is responsible for maintaining the definitive endoderm cell fates. Only when this pathway was perturbed, cells were able to respond in a concentration-dependent manner to Wnt and acquire posterior mesoderm cell fates. Upon further inquiry, we were able to determine that specifically, Nodal is the main driver for endoderm cell fates. Finally, we determined that CHIR, a commonly used chemical Wnt activator, induces posterior cell fates in a concentration-dependent manner qualitatively different from induced WNT signaling dynamics and Nodal ligand production. Collectively, we demonstrate that cell fate decision-making is determined by the interplay between multiple pathways and not by the levels of a single pathway, highlighting the dynamic nature of development.Item Evidence that a mitochondrial death spiral underlies antagonistic pleiotropy(Wiley, 2017) Stern, MichaelThe antagonistic pleiotropy (AP) theory posits that aging occurs because alleles that are detrimental in older organisms are beneficial to growth early in life and thus are maintained in populations. Although genes of the insulin signaling pathway likely participate in AP, the insulin-regulated cellular correlates of AP have not been identified. The mitochondrial quality control process called mitochondrial autophagy (mitophagy), which is inhibited by insulin signaling, might represent a cellular correlate of AP. In this view, rapidly growing cells are limited by ATP production; these cells thus actively inhibit mitophagy to maximize mitochondrial ATP production and compete successfully for scarce nutrients. This process maximizes early growth and reproduction, but by permitting the persistence of damaged mitochondria with mitochondrial DNA mutations, becomes detrimental in the longer term. I suggest that as mitochondrial ATP output drops, cells respond by further inhibiting mitophagy, leading to a further decrease in ATP output in a classic death spiral. I suggest that this increasing ATP deficit is communicated by progressive increases in mitochondrial ROS generation, which signals inhibition of mitophagy via ROS-dependent activation of insulin signaling. This hypothesis clarifies a role for ROS in aging, explains why insulin signaling inhibits autophagy, and why cells become progressively more oxidized during aging with increased levels of insulin signaling and decreased levels of autophagy. I suggest that the mitochondrial death spiral is not an error in cell physiology but rather a rational approach to the problem of enabling successful growth and reproduction in a competitive world of scarce nutrients.Item High-content behavioral profiling reveals neuronal genetic network modulating Drosophila larval locomotor program(BioMed Central, 2017) Aleman-Meza, Boanerges; Loeza-Cabrera, Mario; Peña-Ramos, Omar; Stern, Michael; Zhong, WeiweiAbstract Background Two key questions in understanding the genetic control of behaviors are: what genes are involved and how these genes interact. To answer these questions at a systems level, we conducted high-content profiling of Drosophila larval locomotor behaviors for over 100 genotypes. Results We studied 69 genes whose C. elegans orthologs were neuronal signalling genes with significant locomotor phenotypes, and conducted RNAi with ubiquitous, pan-neuronal, or motor-neuronal Gal4 drivers. Inactivation of 42 genes, including the nicotinic acetylcholine receptors nAChRα1 and nAChRα3, in the neurons caused significant movement defects. Bioinformatic analysis suggested 81 interactions among these genes based on phenotypic pattern similarities. Comparing the worm and fly data sets, we found that these genes were highly conserved in having neuronal expressions and locomotor phenotypes. However, the genetic interactions were not conserved for ubiquitous profiles, and may be mildly conserved for the neuronal profiles. Unexpectedly, our data also revealed a possible motor-neuronal control of body size, because inactivation of Rdl and Gαo in the motor neurons reduced the larval body size. Overall, these data established a framework for further exploring the genetic control of Drosophila larval locomotion. Conclusions High content, quantitative phenotyping of larval locomotor behaviours provides a framework for system-level understanding of the gene networks underlying such behaviours.Item In vivo analysis of gain-of-function mutations in the Drosophila eag-encoded potassium ion channel(2006) Cardnell, Robert John Gunn; Stern, MichaelNeuronal Na+ and K+ channels elicit currents in opposing directions and thus have opposing effects on neuronal excitability. Mutations in genes encoding Na+ or K+ channels often interact genetically, leading either to phenotypic suppression or enhancement for genes with opposing or similar effects on excitability respectively. For example, the effects of mutations in Shaker (Sh), which encodes a K+ channel subunit, are suppressed by loss of function mutations in the Na+ channel structural gene para, but enhanced by loss of function mutations in a second K + channel encoded by eag. Here I characterize three novel mutations that suppress the effects of a Sh mutation on behavior and neuronal excitability. Recombination mapping localized the mutations to the eag locus, and I used sequence analysis to determine that two of the mutations are caused by a single amino acid substitution (G297E) in the S2-S3 linker of Eag. Because these novel eag mutations confer opposite phenotypes to eag loss of function mutations, I suggest that eag G297E causes an eag gain of function phenotype. I hypothesize that the G297E substitution may cause premature, prolonged or constitutive opening of the Eag channels by favoring the "unlocked" state of the channel. The third mutation has two amino acid substitutions in Eag (A259V and E762V) and may also be a gain of function allele of eag . Interestingly, these mutations appear to manifest their most obvious phenotypes under conditions that prolong the action potential.Item Negative Feedback Mechanisms Regulating Neurotransmitter Release at the Drosophila Neuromuscular Junction(2012) Lin, Chun-Jen (Curtis); Stern, MichaelHomeostasis is an indispensable phenomenon in the maintenance of living organisms. Genetic defects which disrupt negative feedback processes can impact homeostatic regulation, potentially resulting in disease. To uncover the molecular mechanisms governing these and other diseases potentially related to defective homeostasis, I used the Drosophila neuromuscular junction as a model system. I characterized two potential mechanisms that regulate homeostasis within the nervous system. First, in Drosophila larval motor neurons, ligand activation of Drosophila metabotropic glutamate receptor A (DmGluRA) mediates a Phosphoinositide 3-kinase (PI3K)-dependent downregulation of neuronal activity, but the mechanism by which mGluR activates PI3K remains incompletely understood. Here, I identified Ca 2+ /Calmodulin-dependant protein kinase II (CaMKII) and the Focal adhesion kinase (DFak) as critical intermediates in the DmGluRA-dependent activation of PI3K at Drosophila motor nerve terminals. I found that transgene-induced CaMKII inhibition or the DFak CG1 null mutation each block the ability of glutamate application to activate PI3K in larval motor nerve terminals, whereas transgene-induced CaMKII activation increases PI3K activity in motor nerve terminals in a DFak-dependent manner, even in the absence of glutamate application. I conclude that the activation of PI3K by DmGluRA is mediated by CaMKII and DFak. Second, I observed that Push, a putative E3-ubiquitin ligase and Ca 2+ /Calmodulin binding protein, regulates both neurotransmitter release and retrograde signaling in the Drosophila neuromuscular junction. I found that RNAi-mediated Push inhibition in the neuron increases but, in the muscle decreases, neurotransmitter release. Similar results were obtained from RNAi knock down of PLCβ and IP3R, which mediates Ca 2+ release from the endoplasmic reticulum. I conclude that Push mediation of the ubiquitin proteasome system may be important in the regulation of PLCβ/IP3R-mediated intracellular Ca 2+ release, and that this Ca 2+ release in the neuron inhibits neurotransmitter release, but in the muscle activates neurotransmitter release via a retrograde signal.Item Peroxisome Biogenesis in Drosophila melanogaster: Protein Trafficking, Lipid Metabolism, and Muscle Function(2013-12-02) Faust, Joseph; McNew, James A.; Bartel, Bonnie; Diehl, Michael R.; Stern, Michael; Bennett, Matthew R.Peroxisomes are ubiquitous organelles required for many essential functions, such as fatty acid metabolism. Defects in peroxisome biogenesis cause a spectrum of human diseases known as peroxisome biogenesis disorders (PBDs). These devastating diseases lack effective therapies and it is unclear how peroxisome dysfunction causes the disease state. Animal models are needed to understand the connection between peroxisome biology and animal physiology. The fruit fly, Drosophila melanogaster, has recently become an important animal model in the study of peroxisomes. We have identified the major peroxisomal proteins and pathways in flies and examined peroxisomal protein trafficking. We have found that fruit fly peroxisomes share many features in common with higher animals, but display some important differences. Flies appear to have lost one of the pathways used in other organisms to target proteins to the peroxisomal matrix. Also some proteins are dually localized to peroxisomes and the cytoplasm likely through a weak interaction with the protein machinery that brings peroxisomal proteins into the organelle. We have also generated fly mutants with impaired peroxisome biogenesis and shown that peroxisomes are required for normal development and lipid metabolism. Flies with impaired peroxisome biogenesis also show defects in multiple processes that depend on muscle function, such as locomotion. PBD patients also display muscle defects, but it is thought to be a secondary effect of neuronal dysfunction. We propose that peroxisome loss in humans, like in flies, may directly affect muscle physiology, possibly by disrupting energy metabolism. Understanding the role of peroxisomes in fly physiology and specifically in muscle cells may reveal novel aspects of PBD etiology.Item Peroxisomes Are Required for Lipid Metabolism and Muscle Function in Drosophila melanogaster(Public Library of Science, 2014) Faust, Joseph E.; Manisundaram, Arvind; Ivanova, Pavlina T.; Milne, Stephen B.; Summerville, James B.; Brown, H.Alex; Wangler, Michael F.; Stern, Michael; McNew, James A.Peroxisomes are ubiquitous organelles that perform lipid and reactive oxygen species metabolism. Defects in peroxisome biogenesis cause peroxisome biogenesis disorders (PBDs). The most severe PBD, Zellweger syndrome, is characterized in part by neuronal dysfunction, craniofacial malformations, and low muscle tone (hypotonia). These devastating diseases lack effective therapies and the development of animal models may reveal new drug targets. We have generated Drosophila mutants with impaired peroxisome biogenesis by disrupting the early peroxin gene pex3, which participates in budding of pre-peroxisomes from the ER and peroxisomal membrane protein localization. pex3 deletion mutants lack detectible peroxisomes and die before or during pupariation. At earlier stages of development, larvae lacking Pex3 display reduced size and impaired lipid metabolism. Selective loss of peroxisomes in muscles impairs muscle function and results in flightless animals. Although, hypotonia in PBD patients is thought to be a secondary effect of neuronal dysfunction, our results suggest that peroxisome loss directly affects muscle physiology, possibly by disrupting energy metabolism. Understanding the role of peroxisomes in Drosophila physiology, specifically in muscle cells may reveal novel aspects of PBD etiology.Item Ras signaling in either prothoracic gland cells or cholinergic neurons of Drosophila melanogaster regulates fly size(2007) Caldwell, Philip E.; Stern, MichaelBody size in multicellular organisms is determined by the integartion of two factors: the rate of growth and the duration of growth. In most animals, the rate of growth is controlled cell autonomously by the insulin-stimulated Pi3 kinase (Pi3K) pathway. However, the duration of growth is controlled in a more complex manner that involves endocrine factors that act cell non-autonomously. For example, in insects such as Drosophila, the duration of each larval phase is regulated by the timing of release of the molting hormone ecdysone from the prothoracic gland (PG). The molecular mechanisms by which the rate of growth and the duration of growth are integrated remain poorly understood. To help shed light in this area, I have investigated the intracellular signaling events that regulate ecdysone release in the Drosophila PG. I have found that expressing activated Ras, or the targets of Ras signaling Raf or Pi3K, in the PG reduces fly size and accelerates larval development via precocious synthesis and release of ecdysone. In contrast, expression of dominant-negative (dn) Ras, Raf, or Pi3K increases fly size and prolongs larval development via delayed synthesis and release of ecdysone. These results indicate that Ras-Raf and Pi3K signaling act in the PG to regulate the duration of growth by altering the timing of ecdysone synthesis and release. Conversely, I have found that expressing dn-Ras or dn-Raf, but not dn-Pi3K, in cholinergic neurons increases fly size and prolongs larval development, whereas, expression of activated Ras or Raf, but not Pi3K, in cholinergic nerurons decreases fly size, but delays larval development. Inhibition of insulin signaling in flies, via chromosomal loss-of-function mutations, also decreases fly size and delays development, raising the possibility that Ras-Raf signaling in cholinergic neurons may affect fly size by controlling the rate of growth via systemic insulin signaling.Item Ras-dependent and Ras-independent effects of PI3K in Drosophila motor neurons(2012) Johnson, Cassidy Brown; Stern, MichaelThe lipid kinase PI3K plays key roles in cellular responses to activation of receptor tyrosine kinases or G protein coupled receptors such as the metabotropic glutamate receptor (mGluR). Activation of the PI3K catalytic subunit p110 occurs when the PI3K regulatory subunit p85 binds to phosphotyrosine residues present in upstream activating proteins. In addition, Ras is uniquely capable of activating PI3K in a p85-independent manner by binding to p110 at amino acids distinct from those recognized by p85. Because Ras, like p85, is activated by phosphotyrosines in upstream activators, it can be difficult to determine if particular PI3K-dependent processes require p85 or Ras. Here we ask if PI3K requires Ras activity for either of two different PI3K-regulated processes within Drosophila larval motor neurons. To address this question, we determined the effects on each process of transgenes and chromosomal mutations that decrease Ras activity, or mutations that eliminate the ability of PI3K to respond to activated Ras. We found that PI3K requires Ras activity to decrease motor neuron excitability, an effect mediated by ligand activation of the single Drosophila mGluR DmGIuRA. In contrast, the ability of PI3K to increase synaptic bouton number is Ras independent. These results suggest that distinct regulatory mechanisms underlie the effects of PI3K on distinct phenotypic outputs. We additionally found that the glutamate-activation of DmGIuRA initiates ERK signaling; however the signaling intermediates linking DmGIuRA to this kinase cascade are unknown.Item Regulation of Innate Immune Cells(2012-09-05) Maharjan, Anu; Stern, Michael; Bennett, George N.; Braam, Janet; Grande-Allen, K. JaneImmune cells such as neutrophils and monocytes enter tissues after tissue damage and clear cell debris to allow repair cells such as fibroblasts to close the wound. Monocytes also differentiate into fibroblast-like cells called fibrocytes to mediate wound healing, similar to fibroblasts. However, in abnormal wound healing such as acute respiratory distress syndrome (ARDS) and fibrosing diseases, the accumulation of immune cells such as neutrophils or fibrocytes become detrimental to health. In ARDS, neutrophils accumulate in the lungs and causes additional damage by producing reactive oxygen species (ROS). In fibrosing diseases, increased fibrocyte differentiation is one of the causes that increase extracellular matrix deposition, which leads to severe scar tissue build up. Since there are no effective treatments for ARDS or fibrosing diseases, understanding the regulation of neutrophil activation or fibrocyte differentiation could be helpful to develop new effective therapies. The Gomer lab has found several factors that either promote or inhibit fibrocyte differentiation. The pro-fibrotic cytokines such as IL-4 and IL-13 potentiate fibrocyte differentiation while the plasma protein serum amyloid P (SAP), crosslinked IgG, and the pro-inflammatory cytokines IFN-γ and IL-12 inhibit fibrocyte differentiation. In this thesis, I have now shown that additional factors such as toll-like receptor 2 (TLR2) agonists and low molecular weight hyaluronic acid (LMWHA) inhibit fibrocyte differentiation, while high molecular weight hyaluronic acid (HMWHA) potentiate fibrocyte differentiation. The accumulation of neutrophils in the lungs is one of the major factors that debilitate the health of a patient in ARDS. Since neutrophils have Fc receptors, I examined the effect of SAP on neutrophil spreading, adherence, activation, and accumulation. SAP inhibits neutrophil spreading induced by cell debris and TNF-α induced adhesion, but SAP is unable to have any effect on classic neutrophil adhesion molecules or the production of hydrogen peroxide. SAP inhibits neutrophil accumulation in the lungs of bleomycin-injured mice. There is an exciting possibility of using SAP as a therapeutic agent to treat ARDS.Item The Drosophila inebriated/rosA transporter: Dual roles in the control of neuronal excitability and osmotic stress response(2000) Huang, Xi; Stern, MichaelMembers of the Na+/Cl- dependent neurotransmitter transporter family perform the re-uptake of neurotransmitter released into synapses and thus control the magnitude and duration of synaptic transmission. The importance of these molecules in the nervous system is further demonstrated by the fact that Na+/Cl- dependent neurotransmitter transporters are targets of psychoactive drugs such as cocaine and Prozac, and are linked to diseases such as Autism. In addition, studies in cultured kidney cells have shown that these molecules also accumulate osmolytes and maintain normal cellular structure and function under hypertonic stress. Inhibition of neurotransmitter transporters in the kidney can cause kidney failure. In this thesis is described the positional cloning of the Drosophila inebriated(ine)/rosA gene that encodes a putative Na+/Cl- neurotransmitter transporter. Mutations in this gene cause increased neuronal excitability at the neuromuscular junction and photoreceptor cells, as well as increased sensitivity to a hypertonic environment. The ine/ rosA gene produces two proteins that differ only in their N-terminal intracellular domain. One form, ine/rosA-l, contains an additional unique sequence of 313 amino acids at the N-terminus compared to the second form, ine/rosA-s. The two transcripts of this gene have very similar distribution patterns in Drosophila embryos. Functional studies on the ine/ rosA gene include various attempts to identify the substrate(s) for the ine/rosA transporter and the localization of the ine/rosA products. Overexpression of the ine/rosA-s cDNA in the Malpighian tubules, the Drosophila analog of mammalian kidney, enhanced Drosophila salt resistance. These studies have established that the ine/ rosA gene plays a vital role in the regulation of both neuronal excitability and water and salt metabolism, and have laid a foundation for further elucidation of neurotransmitter transporter function that may lead to new targets and clues for the treatment of disorders in the nervous system and the kidney.Item The Drosophila melanogaster attP40 docking site and derivatives are insertion mutations of msp-300(Public Library of Science, 2022) Graaf, Kevin van der; Srivastav, Saurabh; Singh, Pratibha; McNew, James A.; Stern, MichaelThe ɸC31 integrase system is widely used in Drosophila melanogaster to allow transgene targeting to specific loci. Over the years, flies bearing any of more than 100 attP docking sites have been constructed. One popular docking site, termed attP40, is located close to the Nesprin-1 orthologue msp-300 and lies upstream of certain msp-300 isoforms and within the first intron of others. Here we show that attP40 causes larval muscle nuclear clustering, which is a phenotype also conferred by msp-300 mutations. We also show that flies bearing insertions within attP40 can exhibit decreased msp-300 transcript levels in third instar larvae. Finally, chromosomes carrying certain “transgenic RNAi project” (TRiP) insertions into attP40 can confer pupal or adult inviability or infertility, or dominant nuclear clustering effects in certain genetic backgrounds. These phenotypes do not require transcription from the insertions within attP40. These results demonstrate that attP40 and insertion derivatives act as msp-300 insertional mutations. These findings should be considered when interpreting data from attP40-bearing flies.