Pooled shRNA Library Screening to Identify Enhancers of Lysosomal Exocytosis

• Main Author: So, Kathy S
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2016
• Subjects: Cellular biology
• ISBN: 9798557016087

Lysosomal exocytosis results in the removal of the lysosomal content by fusion of the lysosome with the plasma membrane, simultaneously relocating the lysosomal marker LAMP1 to the cell surface. Enhancing lysosomal exocytosis has been proposed as a promising therapeutic approach to many lysosomal storage disorders. We used a pooled shRNA screening approach to investigate the upstream effectors of this phenomenon in a high-throughput format. Defining exocytosis effectors will help to elucidate the biological pathways associated with lysosomal exocytosis and to identify targets that might enhance clearance. We hypothesize that shRNA knockdown of negative regulators of lysosomal exocytosis will cause an increase in LAMP1 on the plasma membrane and are potential targets for enhancing clearance. Conversely, knocking down activators of lysosomal exocytosis will cause a decrease in surface LAMP1. Hela cells were transduced with a pool of 27,500 shRNAs targeting 5,043 genes at approximately 100x library complexity. Cells were collected 7 days and 14 days after transduction and sorted by flow cytometry into High and Low surface LAMP1 populations. shRNA-associated barcodes were amplified from the cell’s gDNA and sequenced with the Illumina Miseq. shRNA barcodes mapping to 350 and 269 unique genes were found to be significantly over-represented in the High and Low surface LAMP1 populations, respectively. As expected, LAMP1 itself was among the strongest hits in the Lo-LAMP1 population. The Hi-LAMP1 genes were enriched for biological pathways such as clathrin-mediated endocytosis. Knockdown of genes involved in endocytosis has been reported to increase LAMP1 expression at the plasma membrane, which gives further confidence to the screening results. Our results demonstrate the utility of shRNA screens for helping us understand the biological mechanisms related to lysosomal exocytosis. In future studies, siRNA pools directed against selected hits from both the Hi- and Lo-LAMP1 populations will be validated using an imaging-based assay to evaluate surface LAMP1 expression in Hela cells and cholesterol clearance in Niemann-Pick C cells.