Cell Biology & Molecular Genetics

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    Phagocytosis and signaling in the innate immune system
    (2012) Gonzalez, Elizabeth Anne Cates; Wu, Louisa P; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The innate immune response provides broad spectrum defense through germline encoded components. Many aspects of innate immunity, such as the activation of NFκB transcription factors and phagocytosis, are highly conserved within the animal kingdom. The innate immune response of the cow, in particular, is important due to the cow's agricultural value. A major proportion of acute disease in domestic cattle is caused by Gram-negative bacteria, which produce the outer membrane component lipopolysaccharide (LPS). LPS binds to Toll-like receptor (TLR) 4 and activates multiple signaling pathways, which have been well-studied in humans, but not in ruminants. Human myeloid differentiation-factor 88 (MyD88) and TIR-domain containing adaptor protein (TIRAP) are critical proteins in the LPS-induced NFκB and apoptotic signaling pathways in humans. We demonstrated through the expression of dominant negative constructs in bovine endothelial cells that both MyD88 and TIRAP activate NFκB in the cow. Additionally, bovine TIRAP was also shown to transduce LPS–induced apoptosis, indicating that multiple aspects of the TLR4–dependent signaling pathways are conserved between cows and humans. The model organism Drosophila melanogaster, was subsequently utilized to investigate the role of another branch of the innate immune response: phagocytosis. The extracellular fluid surrounding phagocytic cells in Drosophila has a high concentration of the amino acid glutamate. While glutamate has been well-characterized as a neurotransmitter, its effect, if any, on immune cells is largely unknown. We identified that a putative glutamate transporter in D. melanogaster, polyphemus (polyph), is critical to the fly's immune response. Flies with a disrupted polyph gene exhibit decreased phagocytosis of microbial-derived bioparticles but not of latex beads. Additionally, polyph flies show increased susceptibility to S.aureus infection, decreased induction of the antimicrobial peptide (AMP) Cecropin, increased melanization response, and increased ROS production. Glutamate transport has previously been shown to regulate the synthesis of the antioxidant glutathione. We demonstrate that a polyph–dependent redox system is necessary to maintain the immune cells' function against an infection. By utilizing two species, the cow and the fly, to study the innate immune system, we have gained unique and novel insights into NFκB activation and phagocytosis.
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    RNA SILENCING AND HIGHER ORDER CHROMATIN ORGANIZATION IN DROSOPHILA
    (2011) Moshkovich, Nellie; O'Brochta, David A; Lei, Elissa P; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Higher order chromatin organization influences gene expression, but mechanisms by which this phenomenon occurs are not well understood. RNA silencing, a conserved mechanism that involves small RNAs bound to an Argonaute protein, mediates gene expression via transcriptional or post-transcriptional regulation. Recently, a role for RNA silencing in chromatin has been emerging. In fission yeast, a major role of RNA interference (RNAi) is to establish pericentromeric heterochromatin. However, whether this mechanism is conserved throughout evolution is unclear. In Drosophila, a powerful model organism, there are multiple functionally distinct RNA silencing pathways. Previous studies have suggested the involvement of the Piwi-interacting RNA (piRNA) and endogenous small interfering RNA (endo-siRNA) pathways in heterochromatin formation in order to silence transposable elements in germline and somatic tissues, respectively, but direct evidence is lacking. We addressed whether the genomic locations generating these small RNAs may act as AGO-dependent platforms for heterochromatin recruitment. Our genetic and biochemical analyses revealed that heterochromatin is nucleated independently of endo-siRNA and piRNA pathways suggesting that RNAi-dependent heterochromatin assembly may not be conserved in metazoans. Chromatin insulators are regulatory elements characterized by enhancer blocking and barrier activity. Insulators form large nuclear foci termed insulator bodies that are tethered to the nuclear matrix and have been proposed to organize the genome into distinct transcriptional domains by looping out intervening DNA. In Drosophila, RNA silencing has been reported to affect nuclear organization of gypsy insulator complexes and formation of Polycomb repression bodies. Our studies revealed that AGO2 is required for CTCF/CP190-dependent Fab-8 insulator function independent of its catalytic activity or Dicer-2. Moreover, AGO2 associates with euchromatin but not heterochromatin genome-wide. Also, AGO2 associates physically with CP190 and CTCF, and mutation of CTCF, CP190, or AGO2 decreases chromosomal looping interactions and alters gene expression. We propose a novel RNAi-independent role for AGO2 in the nucleus. We postulate that insulator proteins recruit AGO2 to chromatin to promote or stabilize chromosomal interactions crucial for proper gene expression. Overall, our findings demonstrate novel mechanisms by which RNA silencing affects gene expression on the level of higher order chromatin organization.
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    Characterization of Atg6 function in autophagy and growth control during Drosophila melanogaster development
    (2010) Hill, Jahda Hope; Wu, Louisa P; Baehrecke, Eric H; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The tumor suppressor Beclin 1 mitigates cell stress by regulating the lysosomal degradation pathway known as autophagy. This process involves formation of intracellular double-membraned vesicles, known as autophagosomes, which engulf proteins and damaged organelles and fuse with lysosomes, where the contents are degraded. It is unclear whether the function of Beclin 1 in autophagy is related to cell transformation in beclin 1+/- animals. Using the fruit fly, Drosophila melanogaster, I investigated the function of the Beclin 1 ortholog Atg6 in autophagy and growth control. Through transgenic experiments, I found that Atg6, like Beclin 1, induces autophagy by functioning in a complex consisting of the lipid kinase Vps34 and the serine–threonine kinase Vps15. I also generated a strong loss of function mutant, Atg61, and found that Atg6 is required for development. Atg6 mutant animals contained an excess of blood cells, which surrounded melanotic tumors prior to death. At the cellular level, Atg6 is required for autophagy and endocytosis, and cells lacking Atg6 accumulate high levels of the endoplasmic reticulum stress protein Hsc3 and the adaptor protein p62. I also showed that Atg6 mutant cells displayed mis-regulated nuclear localization of NF κB proteins, transcription factors whose downstream targets include regulators of innate immunity. Significantly, my results suggest that Atg6 may regulate growth independent of its function in autophagy, as mosaic loss of Atg6 in the eye resulted in over-representation of Atg6 mutant cells, a phenotype not shared by other autophagy gene mutant mosaics. Finally, through a collaborative effort, our lab identified a novel function for Atg6 in regulation of epithelial cell polarity. This finding is significant, as epithelial tumor cells are known to lose polarity during metastasis. Our studies have provided a significant contribution to the Beclin 1 field, by providing the first characterization of a Drosophila Atg6 mutant, and demonstrating its function in novel cellular processes.
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    Role and Regulation of Autophagy During Developmental Cell Death in Drosophila Melanogaster
    (2010) McPhee, Christina Kary; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Two prominent morphological forms of programmed cell death occur during development, apoptosis and autophagic cell death. Improper regulation of cell death can lead to a variety of diseases, including cancer. Autophagy is required for survival in response to starvation, but has also been associated with cell death. It is unclear how autophagy is regulated under specific cell contexts in multi-cellular organisms, and what may distinguish autophagy function during cell survival versus cell death. Autophagic cell death is characterized by cells that die in synchrony, with autophagic vacuoles in the cytoplasm, and phagocytosis of the dying cells is not observed. However, little is known about this form of cell death. Autophagic cell death is observed during mammalian development, during regression of the corpus luteum and involution of the mammary and prostate glands. Autophagic cell death is also observed during development of the fruitfly Drosophila melanogaster, during larval salivary gland cell death. Drosophila is an excellent genetic model system to study developmental cell death in vivo. Cells use two main catabolic processes to degrade and recycle cellular contents, the ubiquitin/proteasome system (UPS) and autophagy. Here I investigate the role of the UPS and autophagy in developmental cell death using Drosophila larval salivary glands as an in vivo model. Proteasome inhibitors are being used in anti-cancer therapies; however the cellular effects of proteasome inhibition have not been studied in vivo. Here I demonstrate that the UPS is impaired during developmental cell death in vivo. Taking a proteomics approach to identify proteins enriched in salivary glands during developmental cell death and in response to proteasome impairment, I identify several novel genes required for salivary gland cell death, including Cop9 signalsome subunit 6 and the engulfment receptor Draper. Here I show that the engulfment receptor Draper is required for salivary gland degradation. This is the first example of an engulfment factor that is autonomously required for self-clearance. Surprisingly, I find that Draper is cell-autonomously required for autophagy during cell death, but not for starvation-induced autophagy. Draper is the first factor to be identified that genetically distinguishes autophagy that is associated with cell death from cell survival.
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    THE RELATIONSHIP BETWEEN AUTOPHAGY, CELL SURVIVAL AND CELL DEATH IN A MODEL OF NEURODEGENERATION AND DEVELOPMENT.
    (2009) Batlevi, Yakup; Pick, Leslie; Baehrecke, Eric H; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The catabolic degradation of proteins is vital for the proper function and homeostasis of all cells. Autophagy is one of the major catabolic systems, and it is involved in processes that are as diverse as cell survival, cell death, immune reaction, cancer and neurodegeneration. Neurodegenerative diseases often have the pathology of protein accumulation in inclusions, but it is unclear whether these inclusions cause cell toxicity. Here I show that autophagy has protective functions in a model of a polyglutamine neurodegenerative disease in Drosophila. Inhibition of autophagy in this model enhances polyglutmine-induced degeneration, while activation of autophagy suppresses degeneration. Moreover, I observed similar protein aggregates in the larval salivary glands of a Drosophila dynein light chain mutant. This dynein light chain mutant is defective in autophagy, and their salivary glands fail to execute developmentally regulated programmed cell death. Ectopic activation of autophagy is sufficient to suppress the protein accumulation in dynein light chain mutant salivary glands. Both neurons and salivary glands are long-lived post-mitotic cells, and these cells are likely to have unique catabolic needs. Our data indicate that defects in catabolism are responsible for the neurodegenerative and salivary gland cell death defects that I observed, and could explain the association of autophagy with neurodegenerative diseases.