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Isotropic 3D electron microscopy data of HeLa cell treated with BFA for correlative light and electron microscopy studies (jrc_hela-bfa)

posted on 22.12.2020, 20:06 by Aubrey Weigel, Chi-Lun Chang, Gleb Shtengel, C. Shan Xu, David P. Hoffman, Melanie Freeman, Nirmala Iyer, Jesse Aaron, Satya Khuon, John Bogovic, Wei Qiu, Harald Hess, Jennifer Lippincott-Schwartz

Cellular versatility depends on the accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by ~30% of all proteins), the physical nature of the vessel conducting the first portage (ER→Golgi) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with unprecedented isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (the ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to ER by a thin neck. COPII localizes to the neck region and dynamically regulates cargo entry from ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

Sample: Interphase HeLa cell overexpressing mEmerald-Sec23 frozen 6 min post 10 µM BFA addition.

Protocol: High pressure freezing, freeze-substitution resin embedding with 1% OsO4 0.1% UA 3% H2O in acetone; resin embedding in Eponate 12.

Contributions: Sample provided by Aubrey Weigel and Chi-Lun Chang (HHMI/Janelia), prepared for imaging by Gleb Shtengel (HHMI/Janelia) and Melanie Freeman (UC Berkeley), with imaging and post-processing by C. Shan Xu (HHMI/Janelia).

Dataset ID: jrc_hela-bfa

Final voxel size (nm): 8.0 x 8.0 x 8.0 (X, Y, Z)

Dimensions (µm): 65 x 4.8 x 51.6 (X, Y, Z)

Acquisition date: 2019-10-15

Visualization Website:

Publication Reference: Weigel et al., (2020)


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