Notes

1. The protease inhibitor PMSF is dissolved in ethanol and stored at 4°C. Trace amounts are added to LSB immediately before use.

Fig. 9. Visualization of the reversible calcium-mediated opening and closing of the nuclear baskets by time-lapse AFM. (A,B) The same NPCs with closed baskets (i.e., -Ca2+) (A) could be opened after addition of 0.1 mM Ca2+ (B). The opposite is true in C and D; the same NPCs with open baskets (i.e., +0.1 mM Ca2+) (C) could be closed after addition of 1 mM EGTA (i.e., -Ca2+) (D). Three corresponding NPCs in (A,B) and (C,D), respectively, are marked by arrowheads for better comparison. Scale bars: 100 nm. Reprinted with permission from ref. 18. © 1999 Elsevier.

Fig. 9. Visualization of the reversible calcium-mediated opening and closing of the nuclear baskets by time-lapse AFM. (A,B) The same NPCs with closed baskets (i.e., -Ca2+) (A) could be opened after addition of 0.1 mM Ca2+ (B). The opposite is true in C and D; the same NPCs with open baskets (i.e., +0.1 mM Ca2+) (C) could be closed after addition of 1 mM EGTA (i.e., -Ca2+) (D). Three corresponding NPCs in (A,B) and (C,D), respectively, are marked by arrowheads for better comparison. Scale bars: 100 nm. Reprinted with permission from ref. 18. © 1999 Elsevier.

Fig. 10. Imaging temperature-dependent plugging or unplugging of individual NPCs by time-lapse AFM. (A,B) The same area of cytoplasmic face of NPCs on NE area was imaged by AFM in contact mode at 4°C (A) and after warming the system to 25°C (B). Previously plugged NPCs (A) became unplugged on increasing the temperature (B). Four corresponding NPCs are marked by arrowheads for better comparison. Reprinted with permission from ref. 4. © 2003 Elsevier.

Fig. 10. Imaging temperature-dependent plugging or unplugging of individual NPCs by time-lapse AFM. (A,B) The same area of cytoplasmic face of NPCs on NE area was imaged by AFM in contact mode at 4°C (A) and after warming the system to 25°C (B). Previously plugged NPCs (A) became unplugged on increasing the temperature (B). Four corresponding NPCs are marked by arrowheads for better comparison. Reprinted with permission from ref. 4. © 2003 Elsevier.

2. Because most of the chemical fixatives used for EM are highly toxic (especially vapors of GA and OsO4), it is absolutely necessary to work in a ventilated hood. Not only is UA radioactive but also aqueous solutions are light sensitive. Hence, light-protected storage is required, either in dark flasks or in bottles that are wrapped in aluminum foil.

3. Because the diameter of an Eppendorf pipet tip is smaller than the diameter of both oocytes and isolated nuclei, the end of the tip has to be cut off, for example, with a sharp razor blade.

4. Adsorption of the NE to the support strongly depends on the physical properties of the support. If the grids are glow-discharged (i.e., in a reduced atmosphere of air yielding a net negative charge), NEs tend to absorb with their cytoplasmic face to the support so that their nuclear side is exposed for observation. Coating the grids with 0.1% poly-L-lysine has the same effect.

5. We prefer to perform all preparation steps on a grid. Changes of solution are followed by very quick touching of the edge of the EM grid with filter paper but without drying it completely. After blotting, immediately add a drop of the next solution. As an alternative, you may move the grids upside down from one solution drop to the next.

6. Proper freezing of the sample is crucial for obtaining good quality vitrified ice. To achieve this, the blotting time is critical: If it is too long, the sample becomes air dried, and the NPCs collapse before they are frozen. On the other hand, if the blotting time is too short, the water layer is too thick for freezing into a vitrified ice film. Instead, cubic ice is formed, which in turn causes mechanical damage (for more details, see ref. 10).

7. It is important from which side the grid is blotted. For a delicate sample such as the NE, the blotting should be done from the back side of the grid to minimize mechanical damage of the sample.

8. The principle of freeze-drying is that the vitrified ice is directly sublimated in the vacuum chamber of the freeze-fracture machine. Sublimation of vitreous ice occurs at temperatures ranging from -120 to -80°C at a vacuum of 2 x 10-7 torr or less.

9. This step is important for easier manipulation of the sample during its embedding into Epon resin. With a piece of filter paper, the excess water is removed, and a few drops of melted 2% agarose are added on top of the sample. When the agarose has hardened, small blocks containing sample are cut out of the agarose under a binocular microscope.

10. 0.1% BSA is used to block unspecific binding of the antibodies.

11. This section describes the use of a poly-L-lysine-coated silicon wafer as a stable support for imaging NEs by AFM. A carbon-coated copper grid is also a good support for the same purpose (18). However, the preparation of poly-L-lysine-coated silicon wafers is more straightforward, and both the cytoplasmic and the nuclear faces of the NE are relatively easily and reproducibly imaged by AFM on these wafers.

12. The step of rinsing silicon wafers with pure water after incubation with poly-L-lysine is important. Without that, it may cause distal openings of the nuclear baskets of NPCs because of the strong immobilization of NEs.

Acknowledgments

This work was supported by a National Center of Competence in Research (NCCR) program, Nanoscale Science by the Swiss National Science Foundation, and by research grants from the Swiss National Science Foundation (B. F.) and the Human Frontier Science Program (U. A.). Additional funds were provided by the Maurice E. Müller Foundation and by the Kanton Basel Stadt.

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