| Summary: | The ubiquitin-like protein LC3 is a central component of the autophagy pathway where it functions in autophagosome formation and cargo selection. LC3 has an extensive network of interacting proteins, but little is known about their physiological regulation. To address this question, we first developed tools to quantitatively study protein complexes in living cells using diffusion-based methods. Toward this end we developed a freely accessible software program called FRAP-Toolbox that quantitatively analyzes fluorescence recovery after photobleaching (FRAP) data using the most up-to-date models for recoveries dominated by reactions, or diffusion. Next , we used several complementary fluorescence methodologies, including FRAP, to investigate how the LC3 protein interaction network is regulated by characterizing the size and stoichiometry of autophagosome independent LC3 in the cytoplasm of living cells and in cytoplasmic extracts. We found that a single soluble LC3 associates with a novel 500 kDa complex. LC3s association with these complexes is regulated by residues on LC3 that are important for binding to protein and mRNA, but not its lipid modification. Next, we developed tools to study LC3 complex formation in live cells. We found that LC3 associates with known interacting proteins in slowly diffusing complexes in both the cytoplasm and nucleus of living cells, and that these interactions can also be monitored using Förster resonance energy transfer. In addition, we developed an assay that can be more broadly used to study LC3 protein interactions by quantifying LC3's nucleocytoplasmic ratio both with and without co-overexpression of potential LC3 interacting proteins. Lastly, in order to gain novel insights into the nature of nuclear LC3 we investigated the regulation of its nuclear localization and dynamics. Through the studies in this dissertation, we uncovered several novel properties of LC3 and developed tools that will aid in future investigations of LC3 and other proteins in the autophagy pathway more generally.
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