Hassan McQueen
Research Mentor(s): Lauren Surface
Department or Program: School of Dentistry
Authors: Hassan McQueen, Lauren Surface
Session: Session 2: 1:00pm-1:50pm
Poster: 29
Abstract
Lysosomes are a critical cellular organelle, required for eukaryotic cell function. The major role of lysosomes is to degrade unwanted macromolecules in the cell. Lysosomes are also known to send signals to other parts of the cell. Defects in lysosomal function can lead to Lysosomal Storage Disorders (LSD), whose symptoms include neurodegeneration, cancer, and other illnesses, emerging from abnormal storage of unwanted macromolecules. In the lysosome, there are two poorly characterized transmembrane proteins: ATRAID and SLC37A3. Our previous research has shown that these two proteins interact with each other, and that they are required for the effects of bisphosphonates (common osteoporosis drugs) in cells, in mice and likely in patients. Working at the Surface Lab at the School of Dentistry, we are interested in the endogenous function of these two proteins in the lysosome. The experiments outlined in this project are using wild-type and ATRAID and SLC37A3 knockout 293T cells to examine these questions. Using immunofluorescence and flow cytometry, we had previously determined that the number of lysosomes was higher in the knockout cells. However, when we examined intracellular lysosomal degradative activity via flow cytometry, we found that the knockout cells had significantly less activity than the wildtype cells, suggesting that both ATRAID and SLC37A3 are required for lysosomal function. One issue with analyses of these proteins’ function is that antibodies targeting these proteins have largely not worked effectively. Since our previous experiments suggested that these proteins are heavily glycosylated, we decided to deglycosylate cell lysates prior to analysis by western blotting to see if this improved antigen accessibility. Indeed, deglycosylation of cell lysates followed by acetone precipitation and then western blotting with ATRAID antibodies, clearly demonstrated the presence of ATRAID in deglycosylated cell lysates, which will open up future analyses. In addition, we want to determine how loss of these proteins affects lysosomal content. To do so, we are generating wildtype and knockout cells that express a tagged version of TMEM192 (a lysosomal protein) to isolate the lysosomes for a better understanding of their endogenous function. Together, these two proteins are believed to have a key role in lysosomal activity and these experiments will help us gain a better understanding of their specific functions.
