Since its discovery, autophagy has been implicated in prevalent diseases that occur with advanced age including cancer, neurological dementias, and cardiovascular health. As we age, autophagy becomes defective, and key structures called autophagosomes are unable to sequester and degrade defective cellular material effectively. On the other hand, tumors can hijack autophagosome biogenesis leading to a poor patient prognosis. The exact pathogenesis of these conditions remains poorly understood. To actualize the potential of autophagy in the clinic, it is important to define the mechanisms governing autophagosome biogenesis, so that one day we can understand and correct the key defects that are currently plaguing patients.Autophagosomes are created on an as needed basis; usually they are made in response to cellular stresses such as nutrient depletion or intracellular organelle dysfunction. Cells starved of amino acids, downregulate mTOR resulting in cell cycle arrest and the initiation of autophagy. Intriguingly, while the amino acid withdrawal is uniform across all cells, they have a highly varied response, suggesting a complex and misunderstood regulatory pathway. I set out to create novel tools and methods to quantitatively define the signals regulating autophagosome biogenesis. I develop a pipeline for autophagy foci analysis. I pair this novel methodology with endogenously tagged autophagy factors to provide paradigm changing insights into the regulation of autophagosome biogenesis.For the first time, we define absolute protein abundances of the autophagy pathway. Both ATG2A and ULK1 are far lower expressed than other autophagy proteins and are likely purposefully regulated in this manner. Intriguingly, the ULK1 complex is thought to be constitutive with its other subunits, however, ATG13 was ~9 fold higher than ULK1, it is likely that ULK1 is the limiting subunit within the complex.Live-cell imaging of endogenously tagged factors revealed that ~80% of the population of foci were short ~30 seconds or less. Dual-tracking of short foci with ATG8 family members and P62 showed that these structures rarely colocalize, suggesting that the phagophores are not progressing to mature autophagosomes. Diffusion coefficient analysis of foci identifies two gaussian fitted populations in all autophagy factors, but ATG2. Dual-tracking of ATG2A and ATG13 shows that double positive foci are longer lived, while non-colocalized ATG2A foci were short lived, adding further evidence that many foci never mature into autophagosomes.Knockout of ATG9A abrogated ATG2A foci, but intriguingly Halo-ATG9A, rarely formed foci. Imaging of Halo-ATG9A differed substantially from previous reports where ATG9A foci transiently interacted at autophagosomes. We rectified this difference by imaging Halo-ATG9A over-time and showed that it accumulates in the lysosome and demonstrated that previous interactions were lysosomal ATG9A interacting with autophagosomes.
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