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- EMDB-19194: In situ cryo-electron tomogram of an autophagosome in the project... -

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Basic information

Entry
Database: EMDB / ID: EMD-19194
TitleIn situ cryo-electron tomogram of an autophagosome in the projection of an iPSC-derived neuron #2
Map dataTomogram of an autophagosome containing ER captured in the projection of an iPSC-derived neuron. Tomogram denoised with cryo-CARE with a model trained on this same tomogram.
Sample
  • Organelle or cellular component: autophagosome containing membrane cargo captured in situ in the peripheral projections of iPSC-derived neurons grown a EM grid
KeywordsAutophagy / ERphagy / Degradation / Neuron / Cargo / Microtubules / ER / Autophagosome / CYTOSOLIC PROTEIN
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsHoyer MJ / Capitanio C / Smith IR / Paoli JC / Bieber A / Jiang Y / Paulo JA / Gonzalez-Lozano MA / Baumeister W / Wilfling F ...Hoyer MJ / Capitanio C / Smith IR / Paoli JC / Bieber A / Jiang Y / Paulo JA / Gonzalez-Lozano MA / Baumeister W / Wilfling F / Schulman BA / Harper WJ
Funding support United States, 1 items
OrganizationGrant numberCountry
Aligning Science Across Parkinsons (ASAP)ASAP-000282 United States
CitationJournal: Nat Cell Biol / Year: 2024
Title: Combinatorial selective ER-phagy remodels the ER during neurogenesis.
Authors: Melissa J Hoyer / Cristina Capitanio / Ian R Smith / Julia C Paoli / Anna Bieber / Yizhi Jiang / Joao A Paulo / Miguel A Gonzalez-Lozano / Wolfgang Baumeister / Florian Wilfling / Brenda A ...Authors: Melissa J Hoyer / Cristina Capitanio / Ian R Smith / Julia C Paoli / Anna Bieber / Yizhi Jiang / Joao A Paulo / Miguel A Gonzalez-Lozano / Wolfgang Baumeister / Florian Wilfling / Brenda A Schulman / J Wade Harper /
Abstract: The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle ...The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of the ER and the role of individual ER-phagy receptors is limited. Here we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodelling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodelling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both the magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodelling and versatile genetic toolkit provide a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.
History
DepositionDec 20, 2023-
Header (metadata) releaseFeb 7, 2024-
Map releaseFeb 7, 2024-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19194.png

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Map

FileDownload / File: emd_19194.map.gz / Format: CCP4 / Size: 2.8 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram of an autophagosome containing ER captured in the projection of an iPSC-derived neuron. Tomogram denoised with cryo-CARE with a model trained on this same tomogram.
Voxel sizeX=Y=Z: 11.72 Å
Density
Minimum - Maximum-20.884499999999999 - 13.985004999999999
Average (Standard dev.)0.15032952 (±0.8427676)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions10241024712
Spacing10241024712
CellA: 12001.28 Å / B: 12001.28 Å / C: 8344.641 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : autophagosome containing membrane cargo captured in situ in the p...

EntireName: autophagosome containing membrane cargo captured in situ in the peripheral projections of iPSC-derived neurons grown a EM grid
Components
  • Organelle or cellular component: autophagosome containing membrane cargo captured in situ in the peripheral projections of iPSC-derived neurons grown a EM grid

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Supramolecule #1: autophagosome containing membrane cargo captured in situ in the p...

SupramoleculeName: autophagosome containing membrane cargo captured in situ in the peripheral projections of iPSC-derived neurons grown a EM grid
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Strain: iPSC KOLF2.0_AAVS-TREG3-NGN2 / Tissue: induced-Neurons, DIV 18 / Location in cell: neuronal projections

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 45 sec. / Details: Grid coated with Matrigel
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV
DetailsiPSC-derived iNeurons on the grid were transduced at Day 12 with Lentiviruses carrying mCherry-LC3B and TEX264-GFP and plunged at DIV 18.
Cryo protectantnone
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: TFS Selectris X
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 2.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 50

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