Autophagy is a catabolic mechanism by which cytoplasmic components are sequestered and transported by a double-membrane vesicle called an autophagosome to the lysosome for degradation. This recycling of organelles and macromolecules provides the cell with amino acids in times of nutrient deprivation though we do not fully know how the process is triggered or controlled. It is a highly regulated process in mammalian cells and its deregulation has been shown to contribute to multiple diseases. In order to find new regulators of mammalian autophagy, I performed a genome-wide screen using the Dharmacon human siRNA library in a stable human cell line expressing GFP-LC3, a specific marker for autophagosomal membranes. First I incubated the cells with the siRNA pools then I starved the cells of amino acids. This initiated the formation of GFP-LC3-labelled autophagosomes that I quantified using the Cellomics VTiScan microscope and accompanying software. I measured the effect of specific siRNA-mediated knock-down on multiple parameters including spot count. Accounting for cell death and normalising the data, I generated a Z-score for each siRNA pool and retested the best 500 autophagy-increasing and 500 autophagydecreasing siRNAs as above. The 190 strongest siRNA pools were deconvoluted leaving 20 hits that reproduced the phenotype with three or four out of four duplexes. These 20 hits were then assayed for endogenous LC3 lipidation in a different cell line and the ability of their siRNA to reduce mRNA levels was determined. Four increasers of GFP-LC3 spots increased endogenous LC3 lipidation, suggesting that these proteins are either negative regulators of autophagy or inhibit the maturation or degradation of autophagosomes. Five decreasers of GFP-LC3 spots also inhibited endogenous LC3 lipidation and I have characterised two of these proteins required for autophagy. SCOC colocalises with early autophagy markers and may be providing a scaffold for autophagy machinery. WAC, through its reported binding partners, may be playing a role in both the autophagic and ubiquitin/proteasome pathways.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:594295 |
| Date | January 2011 |
| Creators | McKnight, N. C. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1302281/ |
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