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Isotropic 3D electron microscopy data of HeLa cell overexpressing TNFa-RUSH cargo for correlative light and electron microscopy studies (jrc_hela-22)
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 mApple-TNFa-RUSH, frozen 8 min post 50 µM biotin 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 (HHMI/Janelia), prepared for imaging by Gleb Shtengel (HHMI/Janelia), with imaging and post-processing by C. Shan Xu (HHMI/Janelia).
Dataset ID: jrc_hela-22
Final voxel size (nm): 8.0 x 8.0 x 8.0 (X, Y, Z)
Dimensions (µm): 83.36 x 8.46 x 33 (X, Y, Z)
Acquisition date: 2017-04-12
Visualization Website: https://openorganelle.janelia.org/datasets/jrc_hela-22
Publication Reference: Weigel et al., (2020)