Autophagy is a catabolic pathway required for the recycling of cytoplasmic materials. To define the mechanisms underlying autophagy it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of cell lines expressing HaloTagged autophagy factors from their endogenous loci, we analyzed the abundance, single-molecule dynamics, and autophagosome association kinetics of autophagy proteins involved in autophagosome biogenesis. We demonstrate that autophagosome formation is inefficient and ATG2-mediated tethering to donor membranes is a key commitment step in autophagosome formation. Furthermore, our observations support the model that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles, and that the ULK1 complex and PI3-kinase form a positive feedback loop required for autophagosome formation. Finally, we demonstrate that the duration of autophagosome biogenesis is ∼110 s. In total, our work provides quantitative insight into autophagosome biogenesis and establishes an experimental framework to analyze autophagy in human cells.

中文翻译：

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自噬的定量分析揭示了 ATG9 和 ATG2 在自噬体形成中的作用


自噬是细胞质物质回收所需的分解代谢途径。为了定义自噬的机制，定量表征活细胞中自噬因子的动态行为至关重要。使用一组从其内源基因座表达 HaloTagged 自噬因子的细胞系，我们分析了参与自噬体生物发生的自噬蛋白的丰度、单分子动力学和自噬体关联动力学。我们证明自噬体形成效率低下，ATG2 介导的供体膜束缚是自噬体形成的关键承诺步骤。此外，我们的观察结果支持这样的模型：吞噬细胞是由移动 ATG9 囊泡上自噬因子的积累启动的，并且 ULK1 复合物和 PI3 激酶形成自噬体形成所需的正反馈环。最后，我们证明自噬体生物发生的持续时间约为 110 秒。总的来说，我们的工作提供了对自噬体生物发生的定量见解，并建立了分析人类细胞自噬的实验框架。