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

Abstract

12Autophagy is an essential physiological process by which eukaryotic cells degrade and recycle 13 cellular materials. Although the biochemical hierarchies of the mammalian autophagy pathway 14 have been identified, questions remain regarding the sequence, subcellular location, and 15 structural requirements of autophagosome formation. Here, we characterize the structural 16 organization of key components of the mammalian autophagic initiation machinery at ~20 nm 17 spatial resolution via three-color, three-dimensional super-resolution fluorescence microscopy. 18 We thus show that upon cell starvation, FIP200, a large structural protein of the ULK1 complex 19 with no direct yeast homolog, scaffolds the formation of cup-like structures located at SEC12-20 enriched remodeled ER-exit sites prior to LC3 lipidation. This cup scaffold, then, provides a 21 structural asymmetry to enforce the directional recruitment of downstream components, 22 including the Atg12-Atg5-Atg16 complex, WIPI2, and LC3, to the cup inside. Moreover, we 23 provide evidence that the early autophagic machinery is recruited in its entirety to these cup 24 structures prior to LC3 lipidation, and gradually disperses and dissociates on the outer face of the 25 phagophore membrane during elongation. We thus shed new light on the physical process of 26 mammalian autophagic initiation and development at the nanometer-scale. 28Macroautophagy, hereafter autophagy, is an essential catabolic process by which eukaryotic cells 29 degrade and recycle organelles and proteins 1-8 . The general mechanism of autophagy begins with 30 the activation of the ULK1 complex by mTORC1 in response to stresses. Subsequent recruitment 31 and activation of the PI3K complex, PtdIns3P effectors (WIPI proteins), and the Atg12-Atg5- 32Atg16 complex enable the lipidation of LC3, which drives the formation of a cup-shaped 33 membrane structure known as the isolation membrane, or phagophore. The phagophore engulfs 34 cargo and closes before fusing with lysosome for cargo degradation. Together, the phagophore 35 membrane with its associated protein machinery constitutes the autophagosome. 36Although the biochemical hierarchy of autophagic machinery has been studied 37 extensively 1,4 , open questions remain, especially regarding the spatiotemporal dynamics of 38 autophagosome formation 6-8 . In particular, although the overall mechanism of autophagy is 39 conserved across eukaryotes, autophagy initiation in yeast occurs at a single location (the pre-40 autophagosomal structure) whereas mammalian autophagosomes form throughout the cytosol, 41 likely scaffolded by the ER or related tubulovesicular networks 4,[9][10][11] . Meanwhile, many proteins 42 essential for autophagy in mammals have no direct homologs in yeasts 1,3,7 . Understanding the 43 functions of these proteins, particularly regarding their role in the structural organization of early 44 autophagosomes, is of critical importance. 45FIP200, also known as RB1CC1, is a component of the mammalian ULK1 complex, and 46 ...

ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer

“…On the other hand, the FIP200 dimer gives rise to a C shape, in contrast to the S shape of Atg17 ( Ragusa et al, 2012 ). The Atg17 S has been postulated to have a unique role in nucleating cup-shaped phagophores in bulk autophagy ( Bahrami et al, 2017 ) on the basis of first-principles physical membrane modeling, something that would not be achieved by a C. Cup-shaped FIP200 structures have been visualized in amino acid–starved U2OS cells by super-resolution microscopy ( Kenny et al, 2019 Preprint ), likely corresponding to phagophores observed in cells by EM ( Hayashi-Nishino et al, 2009 ; Ylä-Anttila et al, 2009 ). The ∼20-nm diameter of the FIP200 C shape is potentially compatible with localization on highly curved ATG9 vesicles or on the rim of the growing phagophore.…”

FIP200 organizes the autophagy machinery at p62-ubiquitin condensates beyond activation of the ULK1 kinase

“…In conclusion, although the components of the autophagy machinery can be recruited to p62 condensates, at least to some extent, independently of FIP200, the spatial organization of ATG13 and ULK1 as well as ATG9A was disturbed upon FIP200 deletion. The arrangement of FIP200 around p62 condensates is reminiscent of the localization of FIP200 and ATG13 in starvation induced autophagy (Karanasios et al, 2016;Kenny et al, 2019) and the frequently observed FIP200 protrusions might represent the sites where the autophagy machinery establishes contacts with the ER (Karanasios et al, 2016, Kenny et al, 2019. FIP200 might thus spatially organize ATG9A vesicles and the ULK1 complex allowing these components to come in contact with the PI3KC3-C1 for subsequent PI3P production.…”

“…and it appeared as a cup-shaped structure by super-resolution microscopy (Kenny, Chen et al, 2019). Recently, electron microscopy analysis of purified ULK1-ATG13-FIP200 complex, revealed that the N-terminal domain of FIP200 forms a C-shaped scaffold which organizes the other subunits of the complex (Shi, Yokom et al, 2020).…”

ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer

“…On the other hand, the FIP200 dimer gives rise to a C shape, in contrast to the S-shape of Atg17 (Ragusa et al, 2012). The Atg17 S has been postulated to have a unique role in nucleating cup-shaped phagophores in bulk autophagy (Bahrami et al, 2017) on the basis of first-principles physical membrane modeling, something that would not be achieved by a C. Cup-shaped FIP200 structures have been visualized in amino acid-starved U2OS cells by super-resolution microscopy (Kenny, 2019), likely corresponding to phagophores observed in cell by EM (Hayashi-Nishino et al, 2009;Yla-Anttila et al, 2009). The ~20 nm diameter of the FIP200 C shape is potentially compatible with localization on highly curved ATG9 vesicles or on the rim of the growing phagophore.…”