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Keywords:

  • Cryo-FIB;
  • cytoplasmic space;
  • ER;
  • ER lumen;
  • topology;
  • 3D mapping

Summary

Mapping of a mammalian cell down to a feature size of 20–30 nm in 3D is a goal that will answer many questions concerning the connectivity (topology) of a Eukaryotic cell's traffic routes. These routes are defined and separated from one another by the protein-impregnated lipid membrane barrier of the endoplasmic reticulum (ER). We trace the routes from outside a live flash frozen buccal epithelial cell via gold (Au) labelled pores in the plasma membrane to the ER below and then through the cell as isosurfaces in 3D maps. The outer tubular ER with three-way branching changes to a sheet-like ER nearer the nucleus, and the cytoplasmic space between the ER membranes continues as a volume into the nuclear interior via the nuclear pores. We find some evidence that the last layer of the cytoplasmic ER membrane, also termed the outer nuclear membrane, has discrete gaps, so the ER lumen in these areas is continuous with the nuclear luminal domain and further, the inner nuclear membrane has small protrusions into the nucleus. The routes were established in live, unstained, unfixed, cells etched with a pAmp current of a focused ion beam (cryo-FIB) dual beam electron microscope, at −150°C, 1e-4Pa, and confirmed at 37°C in lipid-dye stained cells. The cryo-FIB etch of a cuboid of 2D planes, and its reconstruction into many 3D maps, takes only hours, facilitating the execution of experiments with comparative conditions in a few days.