Molecular Mechanisms of Autophagy and its Cellular Functions
Autophagy is an essential process in cells whereby, paradoxically, destruction of cellular components is necessary for cellular renewal, homeostasis, and survival under stresses ranging from starvation to infection. Substrates of autophagy, either bulk cytoplasmic contents or selectively targeted proteins and organelles, are engulfed in a growing double-membraned structure known as the phagophore. The phagophore matures and closes to form into the autophagosome, whose contents are degraded upon fusion of the autophagosome with the vacuole or lysosome. Autophagy occurs in all eukaryotes and many components of the autophagic machinery are conserved among yeast, animals, and plants. Many open questions remain as to how these individual components mechanistically work together to orchestrate autophagy, as well as how autophagy then functions in specific cellular contexts. Here, I study two protein complexes that are necessary for autophagy initiation, the Atg1 kinase complex and the phosphatidylinositol 3-kinase complex. In the first study, I use super-resolution, quantitative imaging of live cells coupled with structure-based mutational analysis to show that the Atg1 C-terminal domain has an essential function in autophagosome expansion that is downstream and separate from its Atg13-dependent role in autophagy initiation. The Atg1 C terminus is strikingly dynamic in the absence of Atg13, a phenomenon of unknown significance given that Atg1 and Atg13 have been thought to function in complex. The identification of an Atg13-independent role for the Atg1 suggests that these dynamics may be important for Atg1 function, particularly in autophagosome maturation. The second study examines the remarkable dynamics of the VPS34 kinase domain in context of the autophagic phosphatidylinositol 3-kinase complex. Using electron microscopy and both in vitro and in vivo assays of autophagy activity, we show that the dynamic dislodging of the VPS34 kinase domain is essential for autophagy initiation and that this movement is sterically inhibited by the VPS15 scaffold. Taking a step back from mechanistic intricacies and examining the ever-growing functions of autophagy in cellular context, in the last chapter I show that autophagy functions in organelle biogenesis. Specifically, autophagy degrades the protein OFD1 at centriolar satellites and promotes growth of the primary cilia, a sensory organelle. We identify a previously unknown substrate of autophagy and show that autophagic degradation of OFD1 can induce the formation of primary cilia in cancer cells, setting the stage for further investigations of the function of primary cilia and autophagy in cancer and other ciliopathies. Thus, this work spans studies of the molecular mechanisms of autophagy as well as its cellular functions.