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- EMDB-61093: Cryo-electron tomography of optogenetically-induced lamellipodia -

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Basic information

Entry
Database: EMDB / ID: EMD-61093
TitleCryo-electron tomography of optogenetically-induced lamellipodia
Map data
Sample
  • Cell: lamellipodia in COS-7 cells
Keywordsactin cytoskeleton / plasma membrane / lamellipodia / STRUCTURAL PROTEIN
Biological speciesChlorocebus aethiops (grivet)
Methodelectron tomography / cryo EM
AuthorsInaba H / Imasaki T / Aoyama K / Yoshihara S / Takazaki H / Kato T / Goto H / Mitsuoka K / Nitta R / Nakata T
Funding support Japan, 13 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21H05254 Japan
Japan Society for the Promotion of Science (JSPS)18K19328 Japan
Japan Society for the Promotion of Science (JSPS)18K15002 Japan
Japan Society for the Promotion of Science (JSPS)20K16105 Japan
Japan Society for the Promotion of Science (JSPS)22K06219 Japan
Japan Society for the Promotion of Science (JSPS)22K06809 Japan
Japan Society for the Promotion of Science (JSPS)22K20680ZA Japan
Japan Society for the Promotion of Science (JSPS)23K14178ZA Japan
Japan Society for the Promotion of Science (JSPS)23K23823 Japan
Japan Society for the Promotion of Science (JSPS)23K06674 Japan
Japan Agency for Medical Research and Development (AMED)JP21gm161003 Japan
Japan Science and TechnologyJPMJFR214K Japan
Japan Science and TechnologyJPMJMS2024 Japan
Citation
Journal: iScience / Year: 2025
Title: Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics.
Authors: Hironori Inaba / Tsuyoshi Imasaki / Kazuhiro Aoyama / Shogo Yoshihara / Hiroko Takazaki / Takayuki Kato / Hidemasa Goto / Kaoru Mitsuoka / Ryo Nitta / Takao Nakata /
Abstract: Lamellipodia are sheet-like protrusions essential for cell migration and endocytosis, but their ultrastructural dynamics remain poorly understood because conventional electron microscopy lacks ...Lamellipodia are sheet-like protrusions essential for cell migration and endocytosis, but their ultrastructural dynamics remain poorly understood because conventional electron microscopy lacks temporal resolution. Here, we combined optogenetics with cryo-electron tomography (cryo-ET) to visualize the actin cytoskeleton and membrane structures during lamellipodia formation with temporal precision. Using photoactivatable-Rac1 (PA-Rac1) in COS-7 cells, we induced lamellipodia formation with a 2-min blue light irradiation, rapidly vitrified samples, and analyzed their ultrastructure with cryo-ET. We obtained 16 tomograms of lamellipodia at different degrees of extension from three cells. These revealed small protrusions with unbundled actin filaments, "mini filopodia" composed of short, bundled actin filaments at the leading edge, and actin bundles running nearly parallel to the leading edge within inner regions of lamellipodia, suggesting dynamic reorganizations of the actin cytoskeleton. This approach provides a powerful framework for elucidating the ultrastructural dynamics of cellular processes with precise temporal control.
#1: Journal: Biorxiv / Year: 2024
Title: Cryo-electron tomography of the actin cytoskeleton and membrane organization during lamellipodia formation using optogenetics.
Authors: Inaba H / Imasaki T / Aoyama K / Yoshihara S / Takazaki H / Kato T / Goto H / Mitsuoka K / Nitta R / Nakata T
History
DepositionAug 6, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateJun 18, 2025-
Current statusJun 18, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61093.png

Downloads & links

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Map

FileDownload / File: emd_61093.map.gz / Format: CCP4 / Size: 490.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
14.85 Å/pix.
x 148 pix.
= 2197.563 Å		14.85 Å/pix.
x 1132 pix.
= 16808.389 Å		14.85 Å/pix.
x 768 pix.
= 11403.571 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: 14.8484 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-2449.042500000000018 - 2015.326000000000022
Average (Standard dev.)3.1189637 (±103.952194000000006)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin305255-82
Dimensions1132768148
Spacing7681132148
CellA: 11403.571 Å / B: 16808.389 Å / C: 2197.5632 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : lamellipodia in COS-7 cells

EntireName: lamellipodia in COS-7 cells
Components
  • Cell: lamellipodia in COS-7 cells

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Supramolecule #1: lamellipodia in COS-7 cells

SupramoleculeName: lamellipodia in COS-7 cells / type: cell / ID: 1 / Parent: 0 / Details: PA-Rac1 induced lamellipodia
Source (natural)Organism: Chlorocebus aethiops (grivet) / Organ: kidney

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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
VitrificationCryogen name: ETHANE
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: SIGMA-Aldrich / Diameter: 20 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.44 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 4.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD (ver. 4.11.24) / Number images used: 61
CTF correctionType: NONE

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