In situ structural analysis reveals membrane shape transitions during autophagosome formation

Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.

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“…4 D and F ). Source data for all plots are deposited under https://zenodo.org/record/6607443 ( 76 ).…”

Section: Data Materials and Software Availabilitymentioning

confidence: 99%

In situ structural analysis reveals membrane shape transitions during autophagosome formation

Bieber

Capitanio

Erdmann

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…To approximate the number of phospholipids within the double-membranes of autophagosomes, we followed the calculations according to (Melia et al, 2020) with an intermembrane distance of 5 nm between the outer and inner membrane (Bieber et al, 2022).…”

Section: Quantitative Model For Autophagosome Biogenesismentioning

confidence: 99%

Parallel phospholipid transfer by Vps13 and Atg2 determines autophagosome biogenesis dynamics

Dabrowski

Tulli

Graef

2022

Preprint

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During autophagy, rapid membrane assembly expands small phagophores into large double-membrane autophagosomes. Theoretical modelling predicts the majority of autophagosomal phospholipids is derived from highly efficient non-vesicular phospholipid transfer (PLT) across phagophore-ER contacts (PERCS). Currently, the phagophore-ER tether Atg2 is the only PLT protein known to drive phagophore expansion in vivo. Here, our quantitative live-cell-imaging analysis reveals poor correlation between duration and size of forming autophagosomes and number of Atg2 molecules at PERCS of starving yeast cells. Strikingly, we find Atg2-mediated PLT is non-rate-limiting for autophagosome biogenesis, because membrane tether and PLT protein Vps13 localizes to the rim and promotes expansion of phagophores in parallel with Atg2. In the absence of Vps13, the number of Atg2 molecules at PERCS determines duration and size of forming autophagosomes with an apparent in vivo transfer rate of ~200 phospholipids per Atg2 molecule and second. We propose conserved PLT proteins cooperate in channeling phospholipids across organelle contact sites for non-rate-limiting membrane assembly during autophagosome biogenesis.

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“…Contacts with putative ER exit sites and Golgi cisternae and connections to endolysosome-related structures have also been observed (Biazik et al, 2015). Another recent study using cryo-electron tomography showed fine structures of the contacts of phagophores and autophagosomes with other organelles such as the vacuole, the ER, the nucleus, lipid droplets, and mitochondria in yeast cells (Bieber et al, 2022). However, electron tomography can capture images with a thickness of only several hundred nanometers, which is thinner than that of typical autophagosomes.…”

Section: Introductionmentioning

confidence: 97%

“…Because autophagosomes are formed de novo, their formation relies on the import of lipids and other components from other organelles. Previous reports showed that autophagosome formation undergoes in contact with the endoplasmic reticulum (ER) (Axe et al, 2008;Biazik et al, 2015;Bieber et al, 2022;Gudmundsson et al, 2022;Hayashi-Nishino et al, 2009;Uemura et al, 2014;Ye et al, 2022;Ylä-Anttila et al, 2009), ER exit sites (Biazik et al, 2015), mitochondria (Biazik et al, 2015;Bieber et al, 2022;Hailey et al, 2010), ERmitochondria contact sites (Hamasaki et al, 2013), the Golgi apparatus (Biazik et al, 2015;Nishida et al, 2009), lipid droplets (Bieber et al, 2022;Dupont et al, 2014;Ogasawara et al, 2020), endosomes (Biazik et al, 2015), and the nuclear envelope (Bieber et al, 2022;English et al, 2009). These organelles form contacts with phagophores and can communicate with them through vesicular or non-vesicular transfer.…”

Section: Introductionmentioning

confidence: 99%

Quantitative 3D correlative light and electron microscopy of organelle association during autophagy

Takahashi

Saito

Koyama-Honda

et al. 2022

Cell Struct. Funct.

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In macroautophagy, disk-shaped double-membrane structures called phagophores elongate to form cup-shaped structures, becoming autophagosomes upon closure. These autophagosomes then fuse with lysosomes to become autolysosomes and degrade engulfed material.Autophagosome formation is reported to involve other organelles, including the endoplasmic reticulum (ER) and mitochondria. Organelles are also taken up by autophagosomes as autophagy cargos. However, few studies have performed systematic spatiotemporal analysis of inter-organelle relationships during macroautophagy. Here, we investigated the organelles in contact with phagophores, autophagosomes, and autolysosomes by using threedimensional correlative light and electron microscopy with array tomography in cells starved 30 min. As previously reported, all phagophores associate with the ER. The surface area of phagophores in contact with the ER decreases gradually as they mature into autophagosomes and autolysosomes. However, the ER still associates with 92% of autophagosomes and 79% of autolysosomes, suggesting that most autophagosomes remain on the ER after closure and even when they fuse with lysosomes. In addition, we found that phagophores form frequently near other autophagic structures, suggesting the presence of potential hot spots for autophagosome formation. We also analyzed the contents of phagophores and autophagosomes and found that the ER is the most frequently engulfed organelle (detected in 65% of total phagophores and autophagosomes). These quantitative three-dimensional ultrastructural data provide insights into autophagosome-organelle relationships during macroautophagy.

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In situ structural analysis reveals membrane shape transitions during autophagosome formation

Cited by 11 publications

References 78 publications

In situ structural analysis reveals membrane shape transitions during autophagosome formation

In situ structural analysis reveals membrane shape transitions during autophagosome formation

Parallel phospholipid transfer by Vps13 and Atg2 determines autophagosome biogenesis dynamics

Quantitative 3D correlative light and electron microscopy of organelle association during autophagy

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