RUI: Tools and Approaches for Investigating the Basic Mechanisms of Autophagy

This research will advance our basic understanding of autophagy, a cellular clean-up and recycling system that helps to protect cells of many organisms – including humans – from stresses such as starvation, pathogens, and the accumulation of cellular debris. Results of this research could lead to improvements in crop yields as well as animal and human health. In addition to its scientific goals, this project will help create a diverse and technically literate workforce by providing a research-rich learning environment at Eastern Michigan University, a primarily undergraduate institution that serves a racially and socioeconomically diverse population of students. Twelve undergraduate and two master’s students will be supported to perform cutting-edge biochemical research for 1-2 years each. In addition, the project will help support semester-long, guided research experiences for ~24 undergraduate students taking a research-based senior-level biochemistry laboratory course. These two activities will provide students with the practical and intellectual skills needed to become the next generation of science, technology, and engineering professionals. This project uses yeast genetics to advance our understanding of the formation of the autophagosome, a double-membraned vesicle that envelops cellular cargo and delivers it to the vacuole (or, in metazoans, the lysosome) for degradation. Although previous research in Saccharomyces cerevisiae has established the proteins necessary for this process, much remains to be learned about how they function to carry it out. One central player is the Atg8 conjugation system, which attaches the small ubiquitin-like protein Atg8 to the lipid phosphatidylethanolamine, thus defining the forming autophagic membrane. Previous research has shown that the amount of Atg8 in a cell determines how large autophagosomes become, but not the number created. However, Atg7, which catalyzes the first step in the pathway leading to Atg8 conjugation, determines both the size and the number of autophagosomes. This project will investigate additional proteins in this pathway to resolve this conundrum and determine which branch of the pathway affects autophagosome number. The project will also investigate the molecular details of Atg11, a protein that organizes autophagosome formation around autophagic cargo by binding to and arranging core autophagy proteins. Mutational analysis of a region of Atg11 that preliminary data has shown to be crucial for binding to one of its key partners will demonstrate which specific residues are essential for this interaction. Finally, an improved mathematical simulation to allow more accurate estimation autophagosome size and number from transmission electron microscopy sections will be developed. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.