Additional Funding Sources
The project described was supported by the Center of Excellence in Biomedical Research through the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant Nos. P20GM109095 and P20GM103408 and the National Science Foundation S-STEM Gateway Scholarships in Biological Sciences under Grant Award No. DUE-1644233. The project was further supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant No. P20GM103408.
Abstract
Autophagy is molecular machinery for “self-digestion” in cells. It is a highly conserved process for cell survival in response to stressors such as starvation, growth factor deprivation, and pathogen infection. Autophagy is a unique membrane trafficking process whereby double-layered membranes are formed to engulf parts of the cytoplasm for degradation. The origin of the autophagosome membrane and how its formation is initiated remain open questions after more than 50 years of investigation, and it is still not well understood how the membranes grow and expand to form the autophagosome. A number of lipids have been identified in the autophagosome membrane, including phosphoinositides and phosphatidylethanolamines. However, the complete lipid composition of autophagosome precursors and of completed autophagosomes is not known. This knowledge gap hinders the in-depth understanding of the role of lipids in autophagy. The objective of the proposed project is to elucidate lipid composition of autophagosomes. In this project, HeLa-Difluo™ hLC3 cells, a commercially available autophagy reporter cell line, were cultured to 80% confluency and treated with rapamycin to stimulate autophagy. Autophagosome membranes were isolated via immunoprecipitation using an anti-GFP antibody immunocomplex bound to magnetic beads. Lipid components from the isolated membrane were extracted and analyzed by MALDI. The results of this project contribute to the complete elucidation of lipid composition of the autophagosome membrane and the role of lipids in autophagy.
Uncovering the Origins of Autophagosomes: How Cells Recycle Waste
Autophagy is molecular machinery for “self-digestion” in cells. It is a highly conserved process for cell survival in response to stressors such as starvation, growth factor deprivation, and pathogen infection. Autophagy is a unique membrane trafficking process whereby double-layered membranes are formed to engulf parts of the cytoplasm for degradation. The origin of the autophagosome membrane and how its formation is initiated remain open questions after more than 50 years of investigation, and it is still not well understood how the membranes grow and expand to form the autophagosome. A number of lipids have been identified in the autophagosome membrane, including phosphoinositides and phosphatidylethanolamines. However, the complete lipid composition of autophagosome precursors and of completed autophagosomes is not known. This knowledge gap hinders the in-depth understanding of the role of lipids in autophagy. The objective of the proposed project is to elucidate lipid composition of autophagosomes. In this project, HeLa-Difluo™ hLC3 cells, a commercially available autophagy reporter cell line, were cultured to 80% confluency and treated with rapamycin to stimulate autophagy. Autophagosome membranes were isolated via immunoprecipitation using an anti-GFP antibody immunocomplex bound to magnetic beads. Lipid components from the isolated membrane were extracted and analyzed by MALDI. The results of this project contribute to the complete elucidation of lipid composition of the autophagosome membrane and the role of lipids in autophagy.