UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
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Item 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.Item EXPLORING THE ROLE OF NFκB HOMOLOGS IN AUTOPHAGIC CELL DEATH IN THE DROSOPHILA SALIVARY GLAND(2009) Ivory, Adrienne; Wu, Louisa P; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The innate immune response is an ancient, highly conserved means of defense against pathogens. An important mediator of innate immunity is the NF-κB (Nuclear Factor-Kappa B) family of transcription factors. Activation of immune-signaling pathways leads to the nuclear translocation of NFκB proteins which initiate the transcription of antimicrobial peptides (AMPs) that circulate and destroy microbes. In Drosophila, these AMPs are up-regulated during the destruction of larval salivary glands. Salivary gland cells are destroyed via autophagy during metamorphosis. This project sought to determine what, if any, role the NFκB transcription factors have in autophagic cell death. Using the Drosophila model, it was determined that a loss of AMP activity during metamorphosis results in a failure to completely degrade larval salivary glands, and this defect appears to be due to an inability to remove autophagic vacuoles. It is suggested that AMPs may serve to degrade the membranes of autophagic vacuoles.Item Implication of Matrix Metalloproteinases One and Two in Autophagic Cell Death in the Drosophila Salivary Gland(2006-12-06) Kobayashi, Marie; Baehrecke, Eric H; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Programmed cell death plays an important role in normal development. Defects in this process contribute to cancer. Matrix metalloproteinases (MMPs), zinc-dependant endopeptidases that cleave components of the extracellular matrix, are among the multiple protease types implicated in cell death. Here I provide evidence that MMPs function in Drosophila salivary gland cell death. Misexpression of the MMP inhibitor timp inhibited timely salivary gland cell death, while misexpression of mmp2 induced premature salivary gland cell death. mmp RNA interference was inconclusive because salivary gland persistence observed at 28°C was similar to fkh-GAL4 negative controls. MMPs and caspases might have an additive effect, since misexpression of timp and the caspase inhibitor p35 together enhanced salivary gland persistence compared to either timp or p35 misexpression alone. I also provide descriptive confocal microscopy of wild-type salivary glands using ?-Spectrin and the polarity marker Crumbs which suggest that polarity is lost during salivary gland cell death.