Journal: Proc Natl Acad Sci U S A / Year: 2023 Title: In situ snapshots along a mammalian selective autophagy pathway. Authors: Meijing Li / Ishita Tripathi-Giesgen / Brenda A Schulman / Wolfgang Baumeister / Florian Wilfling / Abstract: Selective macroautophagy (hereafter referred to as autophagy) describes a process in which cytosolic material is engulfed in a double membrane organelle called an autophagosome. Autophagosomes are ...Selective macroautophagy (hereafter referred to as autophagy) describes a process in which cytosolic material is engulfed in a double membrane organelle called an autophagosome. Autophagosomes are carriers responsible for delivering their content to a lytic compartment for destruction. The cargo can be of diverse origin, ranging from macromolecular complexes to protein aggregates, organelles, and even invading pathogens. Each cargo is unique in composition and size, presenting different challenges to autophagosome biogenesis. Among the largest cargoes targeted by the autophagy machinery are intracellular bacteria, which can, in the case of range from 2 to 5 μm in length and 0.5 to 1.5 μm in width. How phagophores form and expand on such a large cargo remains mechanistically unclear. Here, we used HeLa cells infected with an auxotrophic to study the process of phagophore biogenesis using in situ correlative cryo-ET. We show that host cells generate multiple phagophores at the site of damaged -containing vacuoles (SCVs). The observed double membrane structures range from disk-shaped to expanded cup-shaped phagophores, which have a thin intermembrane lumen with a dilating rim region and expand using the SCV, the outer membrane of , or existing phagophores as templates. Phagophore rims establish different forms of contact with the endoplasmic reticulum (ER) via structurally distinct molecular entities for membrane formation and expansion. Early omegasomes correlated with the marker Double-FYVE domain-Containing Protein 1 (DFCP1) are observed in close association with the ER without apparent membrane continuity. Our study provides insights into the formation of phagophores around one of the largest selective cargoes.
A: 13066.24 Å / B: 13066.24 Å / C: 1422.08 Å α=β=γ: 90.0 °
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Supplemental data
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Sample components
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Entire : HeLa cells infected with Salmonella
Entire
Name: HeLa cells infected with Salmonella
Components
Cell: HeLa cells infected with Salmonella
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Supramolecule #1: HeLa cells infected with Salmonella
Supramolecule
Name: HeLa cells infected with Salmonella / type: cell / ID: 1 / Parent: 0
Source (natural)
Organism: Homo sapiens (human)
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Experimental details
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Structure determination
Method
cryo EM
Processing
electron tomography
Aggregation state
cell
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Sample preparation
Buffer
pH: 7
Vitrification
Cryogen name: ETHANE-PROPANE
Cryo protectant
10% Glycerol
Sectioning
Focused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 15 nA / Focused ion beam - Duration: 20 sec. / Focused ion beam - Temperature: 91 K / Focused ion beam - Initial thickness: 1000 nm / Focused ion beam - Final thickness: 150 nm Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is FEI Aquilos FIB. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.
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Electron microscopy
Microscope
TFS KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 120.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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Movie
Controller
Open data
Basic information
Map data
Sample
Homo sapiens (human)
Authors
Germany, 1 items 
Citation
Structure visualization
Downloads & links
EMDB map data format
emd_16421.png
http://ftp.pdbj.org/pub/emdb/structures/EMD-16421
Z (Sec.)
Y (Row.)
X (Col.)
Sample components
Processing
Electron microscopy
FIELD EMISSION GUN
