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- EMDB-13798: Cryo-electron tomogram of a podosome at the basal surface of an u... -

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Entry
Database: EMDB / ID: EMD-13798
TitleCryo-electron tomogram of a podosome at the basal surface of an unroofed primary human macrophage treated with cytochalasin D
Map dataCryo-electron tomogram of a podosome at the basal surface of an unroofed primary human macrophage treated with cytochalasin D
Sample
  • Organelle or cellular component: Podosome assembled at the basal surface of an unroofed human primary monocyte-derived macrophage treated with cytochalasin D
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsJasnin M
Funding support Germany, 3 items
OrganizationGrant numberCountry
Human Frontier Science Program (HFSP)RGP0035/2016 Germany
Foundation for Medical Research (France)FRM DEQ2016 0334894 Germany
German Research Foundation (DFG)ANR-DFG JA-3038/2-1 Germany
CitationJournal: Nat Commun / Year: 2022
Title: Elasticity of podosome actin networks produces nanonewton protrusive forces.
Authors: Marion Jasnin / Jordan Hervy / Stéphanie Balor / Anaïs Bouissou / Amsha Proag / Raphaël Voituriez / Jonathan Schneider / Thomas Mangeat / Isabelle Maridonneau-Parini / Wolfgang Baumeister ...Authors: Marion Jasnin / Jordan Hervy / Stéphanie Balor / Anaïs Bouissou / Amsha Proag / Raphaël Voituriez / Jonathan Schneider / Thomas Mangeat / Isabelle Maridonneau-Parini / Wolfgang Baumeister / Serge Dmitrieff / Renaud Poincloux /
Abstract: Actin filaments assemble into force-generating systems involved in diverse cellular functions, including cell motility, adhesion, contractility and division. It remains unclear how networks of actin ...Actin filaments assemble into force-generating systems involved in diverse cellular functions, including cell motility, adhesion, contractility and division. It remains unclear how networks of actin filaments, which individually generate piconewton forces, can produce forces reaching tens of nanonewtons. Here we use in situ cryo-electron tomography to unveil how the nanoscale architecture of macrophage podosomes enables basal membrane protrusion. We show that the sum of the actin polymerization forces at the membrane is not sufficient to explain podosome protrusive forces. Quantitative analysis of podosome organization demonstrates that the core is composed of a dense network of bent actin filaments storing elastic energy. Theoretical modelling of the network as a spring-loaded elastic material reveals that it exerts forces of a few tens of nanonewtons, in a range similar to that evaluated experimentally. Thus, taking into account not only the interface with the membrane but also the bulk of the network, is crucial to understand force generation by actin machineries. Our integrative approach sheds light on the elastic behavior of dense actin networks and opens new avenues to understand force production inside cells.
History
DepositionOct 29, 2021-
Header (metadata) releaseMay 11, 2022-
Map releaseMay 11, 2022-
UpdateJul 20, 2022-
Current statusJul 20, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13798.png

Downloads & links

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Map

FileDownload / File: emd_13798.map.gz / Format: CCP4 / Size: 814.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-electron tomogram of a podosome at the basal surface of an unroofed primary human macrophage treated with cytochalasin D
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
13.68 Å/pix.
x 248 pix.
= 3392.64 Å		13.68 Å/pix.
x 928 pix.
= 12695.04 Å		13.68 Å/pix.
x 928 pix.
= 12695.04 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 13.68 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-0.70812356 - 0.5773358
Average (Standard dev.)0.021176886 (±0.061159562)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions928928248
Spacing928928248
CellA: 12695.04 Å / B: 12695.04 Å / C: 3392.6401 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Podosome assembled at the basal surface of an unroofed human prim...

EntireName: Podosome assembled at the basal surface of an unroofed human primary monocyte-derived macrophage treated with cytochalasin D
Components
  • Organelle or cellular component: Podosome assembled at the basal surface of an unroofed human primary monocyte-derived macrophage treated with cytochalasin D

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Supramolecule #1: Podosome assembled at the basal surface of an unroofed human prim...

SupramoleculeName: Podosome assembled at the basal surface of an unroofed human primary monocyte-derived macrophage treated with cytochalasin D
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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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: 7.4
GridModel: Quantifoil / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE
DetailsCells were treated with cytochalasin D and then unroofed prior to vitrification
Cryo protectantNo
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Aurion / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
DetailsTilt-series were acquired using a dose-symmetric tilt scheme (Hagen et al. 2017) from -50 to 50 degrees with 2 degree steps.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 51 / Average exposure time: 4.0 sec. / Average electron dose: 3.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.25 µm / Nominal defocus min: 4.25 µm / Nominal magnification: 42000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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: 51

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