Using Drosophila to Investigate v-ATPase-mediated Autolysosomal Acidification
Autophagy is a eukaryotic cellular recycling process, responsible for degradation of old or damaged cellular components within membrane-bound organelles called lysosomes. In autophagy, the lysosome, an acidic organelle, fuses with the autophagosome creating an acidic environment where degradation of cellular components occurs.
Under certain stress conditions such as starvation, activation of autophagy generates nutrients that can promote the survival of the cell. However, in some cases high levels of autophagy can promote cell death. Autophagy is implicated in a wide range of human health diseases, including cancer, neurodegeneration, and aging. Currently, not much is known about the mechanism behind autophagy, meaning autophagy based therapies are still in progress.
A key component of successful autophagy is the v-ATPase located on the lysosomal membrane. The v-ATPase is a hydrogen pump that transports H+ ions into the lysosome to make the organelle acidic for degradation to occur.
Although acidification of the lysosome is critical for its function, we know very little about how this process is regulated. For instance, we know that other ions, such as Ca2+, are involved in autophagy regulation, but its exact role is unknown. Knowledge about the mechanisms involved in autophagy regulation is important for our understanding of cellular function, and has the potential to be implemented into a wide range of human disease treatments/therapies.
Using the Drosophila melanogaster (fruit fly) larval fat body as a model for autophagy, I will use the following tools available in Drosophila to investigate the mechanism of autolysosomal acidification: 1) Atg8a-mCherry tracker line, a fluorescently-tagged protein that dissociates following fusion, 2) Cresyl Violet, a compound that stains acidified organelles within the cell, and 3) RNAi lines that allow depletion of v-ATPase subunits. By inhibiting the v-ATPase, the acidification of the autolysosome will not occur resulting in failed autophagy.
I hope to gain a deeper understanding of the role that lysosomal acidification plays in autophagy, in order to connect more pieces of the puzzle that is autophagy regulation.