Super-resolution microscopy unveils FIP200-scaffolded, cup-shaped organization of mammalian autophagic initiation machinery

Abstract

Autophagy is an essential physiological process by which eukaryotic cells degrade and recycle cellular materials. Although the biochemical hierarchies of the mammalian autophagy pathway have been identified, questions remain regarding the sequence, subcellular location, and structural requirements of autophagosome formation. Here, we characterize the structural organization of key components of the mammalian autophagic initiation machinery at [~]20 nm spatial resolution via three-color, three-dimensional super-resolution fluorescence microscopy. We thus show that upon cell starvation, FIP200, a large structural protein of the ULK1 complex with no direct yeast homolog, scaffolds the formation of cup-like structures located at SEC12-enriched remodeled ER-exit sites prior to LC3 lipidation. This cup scaffold, then, provides a structural asymmetry to enforce the directional recruitment of downstream components, including the Atg12-Atg5-Atg16 complex, WIPI2, and LC3, to the cup inside. Moreover, we provide evidence that the early autophagic machinery is recruited in its entirety to these cup structures prior to LC3 lipidation, and gradually disperses and dissociates on the outer face of the phagophore membrane during elongation. We thus shed new light on the physical process of mammalian autophagic initiation and development at the nanometer-scale.