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- EMDB-25485: In situ cryo-electron tomography reveals local cellular machineri... -

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Entry
Database: EMDB / ID: EMD-25485
TitleIn situ cryo-electron tomography reveals local cellular machineries for axon branch development (premature branch)
Map dataTomogram of premature axon branch of hippocampal neuron.
Sample
  • Cell: Primary hippocampal neuron from mouse
KeywordsIn situ / cryo-ET / cryo-EM / tomography / Axon branch / Neuron / actin / microtubules / cytoskeleton / ER / ribosome / mitochondria / branching / hippocampus / CELL ADHESION
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsNedozralova H / Basnet N / Ibiricu I / Bodakuntla S / Biertumpfel C / Mizuno N
Funding support Germany, 5 items
OrganizationGrant numberCountry
European Research Council (ERC)724209 Germany
Max Planck Society Germany
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI) Germany
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS) Germany
Boehringer Ingelheim Fonds (BIF) Germany
CitationJournal: J Cell Biol / Year: 2022
Title: In situ cryo-electron tomography reveals local cellular machineries for axon branch development.
Authors: Hana Nedozralova / Nirakar Basnet / Iosune Ibiricu / Satish Bodakuntla / Christian Biertümpfel / Naoko Mizuno /
Abstract: Neurons are highly polarized cells forming an intricate network of dendrites and axons. They are shaped by the dynamic reorganization of cytoskeleton components and cellular organelles. Axon ...Neurons are highly polarized cells forming an intricate network of dendrites and axons. They are shaped by the dynamic reorganization of cytoskeleton components and cellular organelles. Axon branching allows the formation of new paths and increases circuit complexity. However, our understanding of branch formation is sparse due to the lack of direct in-depth observations. Using in situ cellular cryo-electron tomography on primary mouse neurons, we directly visualized the remodeling of organelles and cytoskeleton structures at axon branches. Strikingly, branched areas functioned as hotspots concentrating organelles to support dynamic activities. Unaligned actin filaments assembled at the base of premature branches accompanied by filopodia-like protrusions. Microtubules and ER comigrated into preformed branches to support outgrowth together with accumulating compact, ∼500-nm mitochondria and locally clustered ribosomes. We obtained a roadmap of events supporting the hypothesis of local protein synthesis selectively taking place at axon branches, allowing them to serve as unique control hubs for axon development and downstream neural network formation.
History
DepositionNov 21, 2021-
Header (metadata) releaseMar 9, 2022-
Map releaseMar 9, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_25485.png

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Map

FileDownload / File: emd_25485.map.gz / Format: CCP4 / Size: 1.5 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram of premature axon branch of hippocampal neuron.
Voxel sizeX=Y=Z: 21.84 Å
Density
Minimum - Maximum-10.516254 - 10.742527000000001
Average (Standard dev.)0.20828003 (±0.51485527)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-42
Dimensions928928464
Spacing928928464
CellA: 20267.52 Å / B: 20267.52 Å / C: 10133.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z21.8421.8421.84
M x/y/z928928464
origin x/y/z0.0000.0000.000
length x/y/z20267.52020267.52010133.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00-42
NC/NR/NS928928464
D min/max/mean-10.51610.7430.208

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Supplemental data

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Sample components

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Entire : Primary hippocampal neuron from mouse

EntireName: Primary hippocampal neuron from mouse
Components
  • Cell: Primary hippocampal neuron from mouse

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Supramolecule #1: Primary hippocampal neuron from mouse

SupramoleculeName: Primary hippocampal neuron from mouse / type: cell / ID: 1 / Parent: 0 / Details: Premature axon branch
Source (natural)Organism: Mus musculus (house mouse) / Strain: CD-1 / Organ: brain / Tissue: hippocampal neurons

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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: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Detailsembryonic neurons grown on gold EM grid
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 26000
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Bioquantum
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsPhase plate
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 90.0 e/Å2 / Details: 90 e-/A^2 total dose per tilt series
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 61

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