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- EMDB-50110: Electron tomogram of ER-nuclear envelope junction of HeLa cell in... -
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Basic information
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Title | Electron tomogram of ER-nuclear envelope junction of HeLa cell in early telophase | |||||||||
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![]() | Endoplasmic reticulum / Nuclear envelope / Organelle contact site / Membrane junction / CELL CYCLE | |||||||||
Biological species | ![]() | |||||||||
Method | electron tomography / cryo EM / negative staining | |||||||||
![]() | Bragulat-Teixidor H / Otsuka S | |||||||||
Funding support | ![]()
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![]() | ![]() Title: The endoplasmic reticulum connects to the nucleus by constricted junctions that mature after mitosis. Authors: Helena Bragulat-Teixidor / Keisuke Ishihara / Gréta Martina Szücs / Shotaro Otsuka / ![]() ![]() Abstract: Junctions between the endoplasmic reticulum (ER) and the outer membrane of the nuclear envelope (NE) physically connect both organelles. These ER-NE junctions are essential for supplying the NE with ...Junctions between the endoplasmic reticulum (ER) and the outer membrane of the nuclear envelope (NE) physically connect both organelles. These ER-NE junctions are essential for supplying the NE with lipids and proteins synthesized in the ER. However, little is known about the structure of these ER-NE junctions. Here, we systematically study the ultrastructure of ER-NE junctions in cryo-fixed mammalian cells staged in anaphase, telophase, and interphase by correlating live cell imaging with three-dimensional electron microscopy. Our results show that ER-NE junctions in interphase cells have a pronounced hourglass shape with a constricted neck of 7-20 nm width. This morphology is significantly distinct from that of junctions within the ER network, and their morphology emerges as early as telophase. The highly constricted ER-NE junctions are seen in several mammalian cell types, but not in budding yeast. We speculate that the unique and highly constricted ER-NE junctions are regulated via novel mechanisms that contribute to ER-to-NE lipid and protein traffic in higher eukaryotes. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 14.7 GB | ![]() | |
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Header (meta data) | ![]() ![]() | 9 KB 9 KB | Display Display | ![]() |
Images | ![]() | 232.8 KB | ||
Filedesc metadata | ![]() | 3.9 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
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Voxel size | X=Y=Z: 4.51 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
-Entire : HeLa Kyoto cell
Entire | Name: HeLa Kyoto cell |
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Components |
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-Supramolecule #1: HeLa Kyoto cell
Supramolecule | Name: HeLa Kyoto cell / type: cell / ID: 1 / Parent: 0 |
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Source (natural) | Organism: ![]() |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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![]() | electron tomography |
Aggregation state | cell |
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Sample preparation
Buffer | pH: 7.4 Details: DMEM without Riboflavin and Phenol Red, containing 10% FBS, 1% Pen/Strep, and 50 nM SiR-DNA |
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Staining | Type: NEGATIVE / Material: Uranyl acetate and lead citrate |
Sugar embedding | Material: Agar 100 Epoxy resin Details: Frozen cells were substituted in 0.1% uranyl acetate (UA), 2% Osmium tetroxide and 5% H2O in acetone following this temperature ramp: -90 C to -80 C for 10 hours, -80 C to -30 C for 10 ...Details: Frozen cells were substituted in 0.1% uranyl acetate (UA), 2% Osmium tetroxide and 5% H2O in acetone following this temperature ramp: -90 C to -80 C for 10 hours, -80 C to -30 C for 10 hours, -30 C for 4 hours, -30 C to 0 C for 6 ours, 0 C to 20 C for 4 hours, 20 C for 5-6 hours. |
Vitrification | Cryogen name: NITROGEN |
High pressure freezing | Instrument: OTHER Details: High pressure freezing chamber was 1 mm thick, 6.0 mm diameter, with central cavities 5.0 mm x 5.0 mm x 25 um deep. The chamber had been in contact with 1-hexadecene.. The value given for _ ...Details: High pressure freezing chamber was 1 mm thick, 6.0 mm diameter, with central cavities 5.0 mm x 5.0 mm x 25 um deep. The chamber had been in contact with 1-hexadecene.. The value given for _em_high_pressure_freezing.instrument is Leica EM ICE. This is not in a list of allowed values {'LEICA EM PACT', 'LEICA EM PACT2', 'BAL-TEC HPM 010', 'OTHER', 'LEICA EM HPM100', 'EMS-002 RAPID IMMERSION FREEZER'} so OTHER is written into the XML file. |
Cryo protectant | 20% Ficoll-PM400 |
Sectioning | Ultramicrotomy - Instrument: Leica Ultracut UCT / Ultramicrotomy - Temperature: 298 K / Ultramicrotomy - Final thickness: 250 |
Fiducial marker | Manufacturer: Cytodiagnostics / Diameter: 15 nm |
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Electron microscopy
Microscope | FEI TECNAI F20 |
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Image recording | Film or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.2 µm |
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Image processing
Final reconstruction | Algorithm: BACK PROJECTION / Number images used: 110 |
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