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-22Final 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: 2017-04-12Visualization Website: https://openorganelle.janelia.org/datasets/jrc_hela-22Publication Reference: Weigel et al., (2020)

细胞的功能多样性依赖于各类蛋白质精准转运至其对应的细胞器靶位点。对于占总蛋白约30%的分泌通路而言，介导首次转运（内质网（Endoplasmic Reticulum, ER）→高尔基体（Golgi Apparatus, Golgi））的转运载体的物理本质仍不明确。
我们利用全细胞聚焦离子束扫描电子显微镜结合冷冻结构照明显微镜，以及活细胞同步转运货物释放技术，为哺乳动物细胞的早期分泌区室提供了具备前所未有的各向同性分辨率的动态三维视图，并实现了蛋白质的精准定位。
研究发现，内质网并非仅依赖囊泡完成物质转运，而是生成了由连续脂质双分子层构成的复杂交织管状网络——即内质网出口位点（ER Exit Site, ERES），以此实现蛋白质的输出。
该转运网络可沿微管延伸数微米，同时仍通过细颈结构与内质网保持连接。
COPII包被复合体（COPII）定位于该细颈区域，动态调控蛋白质从内质网的转运入站；而COPI包被复合体（COPI）则作用于更远端的位置，伴随脱离后的管状结构加速运动，通过微管导向的转运过程前往与高尔基体融合。
样本：处于间期的过表达mApple-TNFα-RUSH的HeLa细胞，在添加50 μM生物素8分钟后进行冷冻固定。
实验方案：高压冷冻，随后在丙酮中以1%四氧化锇（OsO4）、0.1%乙酸铀（UA）及3%水的混合液进行冷冻替代树脂包埋，最终使用Eponate 12树脂完成包埋。
贡献：样本由奥布里·韦格尔（Aubrey Weigel，HHMI/Janelia）提供，成像前样品制备由格莱布·什滕格尔（Gleb Shtengel，HHMI/Janelia）完成，成像及后期处理由徐善川（C. Shan Xu，HHMI/Janelia）负责。
数据集ID：jrc_hela-22
最终体素尺寸（纳米）：8.0×8.0×8.0（X、Y、Z轴）
样本尺寸（微米）：83.36×8.46×33（X、Y、Z轴）
采集日期：2017年4月12日
可视化网站：https://openorganelle.janelia.org/datasets/jrc_hela-22
发表文献：韦格尔等（Weigel et al.），2020年