Use of Xenopus laevis Oocyte Nuclei and Nuclear Envelopes in Nucleocytoplasmic Transport Studies

Reiner Peters

Summary

In this chapter, two techniques for the analysis of transport through the nuclear pore complex are described. In the first technique, nuclei isolated manually from Xenopus laevis oocytes are used to measure the import kinetics of fluorescent substrates by confocal fluorescence microscopy. In the second technique, referred to as optical single transporter recording (OSTR), isolated Xenopus oocyte nuclei, perforated nuclei, or isolated nuclear envelopes are tightly bound to planar transparent substrates containing arrays of nanoscopic-to-microscopic cavities. Transport through membrane patches spanning these cavities is recorded by confocal microscopy. By these means, the transport through single nuclear pore complexes or populations of pore complexes can be quantitatively measured.

Key Words: Membrane transport kinetics; nuclear envelope; nuclear pore complex; nucleocytoplasmic transport; optical single transporter recording.

1. Introduction

A complete analysis of a membrane transporter usually requires identification and characterization in vivo; solubilization and characterization in vitro; expression in heterologous systems and reconstitution in spherical or planar lipid bilayers, functional characterization by electrical and optical techniques; and ultimately structural characterization at atomic resolution by crystallization and X-ray diffraction.

The nuclear pore complex (NPC) has resisted such strategies to a considerable extent. Sheer size and complexity (500-1000 protein molecules/NPC) are reasons for that, an intimate integration into the cellular network on both the structural and functional level is another. Fundamentally, difficulties in applying strategies developed for "normal" transporters to the NPC may relate to the fact that the NPC is not a normal transporter: It does not span a lipid bilayer separating topologically different phases but provides a gate between topologically identical phases, cytosol and karyosol. Only in cooperation with soluble transport factors does it achieve selectivity and directionality.

From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu Ā© Humana Press Inc., Totowa, NJ

In the beginning, nucleocytoplasmic transport could be studied exclusively by microinjection of live cells (1). Meanwhile, in vivo studies of nucleocytoplasmic transport have been much refined by a diversity of molecular genetic techniques including the green fluorescent protein (GFP) technology (2), photobleaching techniques (3), and cell fusion methods (4). Early attempts to employ isolated nuclei (5) or nuclear envelope vesicles (6) for nucleocytoplasmic transport studies were soon abandoned, leaving behind the widespread feeling that the NPC cannot be removed from the cellular context in functional form. "Artificial" nuclei, created by incubation of demembranized sperms with Xenopus egg extracts (7,8), played a crucial role in studies analyzing the assembly and disassembly of nuclear envelope and NPC but were less successful in transport studies.

A breakthrough was achieved in 1990 (9) by establishing conditions at which digi-tonin renders the plasma membrane permeable for macromolecules but does not affect nuclear envelope and NPC. Application of digitonin-permeabilized cells led to the discovery of karyopherins and Ran and thus triggered great progress. However, digi-tonin permeabilization yields little control over the nuclear content, a parameter of crucial importance in nuclear export studies.

In this chapter, two novel assays of nuclear transport are described. In the first assay (10), confocal fluorescence microscopy is employed to measure the import of fluorescent substrates into isolated Xenopus oocyte nuclei, thus proving that nuclei can be isolated in functional form. In the second assay, optical single transport recording (OSTR) (11) is employed to measure transport trough the NPC in isolated intact nuclei, perforated nuclei, or isolated nuclear envelopes. In OSTR, membranes are firmly attached to flat, transparent, solid substrates containing a dense arrays of microscopic or nanoscopic cavities, here referred to as test compartments (TCs). Transport across TC-spanning membrane patches is induced by changing the concentration of transport substrates in the OSTR chamber and monitored by recording the fluorescence of transport substrates in the TCs. As described elsewhere in more detail (12), OSTR permits analysis of all sorts of transporters, such as the very fast (e.g., ion channels) and the very slow (e.g., translocases, ABC pumps). Furthermore, it features a variable membrane patch size, substrate multiplexing, and parallel data acquisition and thus ideally complements electrical single-channel recording.

2. Materials

1. Fresh sample of Xenopus laevis ovary.

2. Amphibian Ringer's: 88 mM NaCl, 1 mM KCl, 0.8 mM MgSO4, 1.4 mM CaCl2, 5 mM HEPES, pH 7.4.

3. Stereomicroscope with "cold" (glass fiber) light source.

4. Small glass dishes, 30-mm diameter, 5-mm rim height, for Xenopus oocyte isolation.

5. Forceps for Xenopus oocyte isolation (e.g., Dumont no. 5).

6. Pasteur pipets, tip diameter larger than 1.5 mm, for transfer of Xenopus oocytes from one dish to another and the like.

7. Mock 3, a mock intracellular medium buffered to 3 [lM free Ca2+, 90 mM KCl, 10 mM NaCl, 2 mM MgCl2, 0.1 mM CaCl2, 1.0 mM N-(2-hydroxyethyl)ethylene-diaminetriacetic acid, 10 mM HEPES, pH 7.3.

8. Transfer pipet: a normal microliter pipet set to a volume of approx 3 ||L with a tip diameter increased to approx 2 mm.

9. Glass capillary with a tip briefly exposed to a flame to form a blunt and bent end, similar in shape to a hockey club, for manual purification of isolated Xenopus oocyte nuclei and their attachment to TC arrays.

10. Plastic dishes (Falcon 353004) for construction of microchambers or OSTR chambers.

11. Drills, 2.5- and 3.5-mm diameter, for creation of microchambers or OSTR chamber, respectively.

12. Sandpaper, finest grade.

13. Cover slips, 15 x 15 mm, for creation of microchambers and OSTR chambers.

14. Transport solution 1 (see ref. 10), for studying import into isolated intact nuclei: 0.5 ||M PK4 (a P-galactosidase fusion protein containing a nuclear localization sequence, NLS) or 0.5 ||M GG-NLS (a fusion protein of GFP, GST, and an NLS); 0.5 ||M karyopherin-a; 0.5 ||M karyopherin-P1; 2 ||M Texas-red-labeled 70-kDa dextran (TRD70); energy mix (final concentrations of 2 mM adenosine triphosphate [ATP], 25 mM phosphocreatine, 30 U/mL creatine phosphokinase, 200 |M GTP; 20 g/L bovine serum albumin (BSA); in mock 3.

15. Preformed TC arrays, custom made by excimer laser irradiation of approx 100-| m thick polycarbonate, may be ordered from Bartels Mikrotechnik, Dortmund, Germany. These arrays are available with TC diameters, depths, and pitches between approx 5 and 100 |m

16. Eastman Instant Adhesive no. 910 (SERVA, Heidelberg, Germany).

17. Brush for applying adhesive.

18. Polycarbonate track-etched membrane filters, type Cyclopore TransparentĀ® (Whatman, Maidstone, Kent, UK), for creating random TC arrays. These filters are available with pore diameters of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 3.0, 5.0, and 8.0 |m. Filter thickness varies between 20 |m (0.1-|lm pore diameter) and 12 |m (8.0-|lm pore diameter), pore density between 200 pores/(100 x 100) |m2 (0.1-|m pore diameter) and 10 pores/(100 x 100) |m2 (8.0-|lm pore diameter).

19. Double-sticky tape, referred to as optically clear adhesive, type 8141, 3M Company.

20. Ultrasound bath.

21. Stainless steel pins no. 26002-10 and holder no. 26018-17, Fine Science Tools, Heidelberg, Germany.

22. Transport solution 2 for OSTR measurements (see ref. 13): 4 |M Alexa488-labeled NTF2 or 4 |M GFP, 100 mM sucrose in mock 3.

23. GELoader Tips (Eppendorf, Hamburg, Germany), which are very fine plastic pipet tips for injection of transport substrate into OSTR chamber.

24. Confocal laser scanning system based on an inverted microscope.

25. Software for image processing (e.g., ImageJ).

3. Methods

3.1. Confocal Fluorescence Microscopic Measurement of Nucleocytoplasmic Transport Using Isolated Xenopus Oocyte Nuclei

3.1.1. Isolation of Xenopus Oocyte Nuclei

1. Use two fine forceps to remove a stage VI oocyte (~1.2-mm diameter; Fig. 1A) from a piece of Xenopus ovary (see Notes 1 and 2).

2. Use a Pasteur pipet to transfer the oocyte into a small glass dish (Subheading 2., item 4) containing mock 3 at ambient temperature.

Fig. 1. Isolation and purification of Xenopus oocyte nuclei for nucleocytoplasmic transport measurements. A stage VI Xenopus oocyte (A) was manually dissected. The nucleus was (B) isolated, (C) cleaned of adhering yolk particles, and (D) deposited in a microchamber. From ref. 10.

3. Place the dish on the stage of a stereomicroscope and adjust the illuminating glass fibers such that the light beams enter the dish almost horizontally. This yields a darkfield effect, facilitating visualization of isolated nuclei otherwise virtually invisible.

4. At approx 16x total magnification, grip the oocyte at the equator by forceps. Tear the oocyte gently apart. Try to spot the nucleus, which initially is visible only as a hole in the yolk (see Notes 3 and 4). Gently isolate the nucleus.

5. Dip the tip of the transfer pipet into mock 3 and extrude the air from the pipet tip (see Note 5). Aspirate the nucleus. Place another small glass dish containing fresh mock 3, not contaminated by Ringer's solution and yolk, on the stage of the stereomicroscope and release the nucleus into mock 3.

6. Further purify the nucleus from adhering yolk particles (Fig. 1C) by repeatedly touching the nucleus with the blunt end of a microcapillary (Subheading 2., item 9). Isolation and purification of a nucleus should be completed within 1 to 2 min.

3.1.2. Preparation of Microchambers

1. Drill a hole of 2.5-mm diameter into the bottom of a tissue culture dish. Remove ridges using sandpaper. Use a jet of compressed air to remove debris and dust.

2. Attach a regular cover slip to the bottom of the dish using any glue appropriate. We prepare a batch of approx 20 chambers at a time, which lasts for many experiments because each microchamber can be used several times.

3.1.3. Deposition of an Isolated Nucleus in a Microchamber

1. Fill a microchamber with mock 3 (requires ~5 ||L). Mock 3 should be kept at ambient temperature (see Note 5).

2. Employing the transfer pipet, aspirate an isolated clean nucleus prepared in advance according to Subheading 3.1.1. (but see Note 2). Deposit the nucleus in the microchamber (Fig. 1C). The nucleus will sink to the bottom of the microchamber and rest on the cover slip (Fig. 1D).

3.1.4. Addition of Transport Solution

1. Aspirate 10 |L transport solution 1 into a GELoader tip.

2. Dip the GELoader tip into the microchamber and place it on top of the oocyte nucleus. Slowly expel the transport solution. Because of its BSA content, the transport solution will sink to the bottom of the microchamber. Mock 3 will be displaced and gather at the top of the microchamber.

3. After addition of import solution, aspirate surplus of mock 3 from the top of the microchamber.

3.1.5. Recording of Transport Kinetics

1. Mount the microchamber on the stage of a confocal laser scanning microscope. Visualize the nucleus in through-light using a 10x objective. Bring the largest perimeter of the nucleus into the focal plane. Adjust laser power, filter settings, multiplier voltages, con-focal aperture, and so on so that the brightness of both transport substrate (NLS protein) and control substrate (TRD70) is about equal.

2. Start scanning and acquire scans at intervals, properly resolving import kinetics (Fig. 2). The nucleus should be perfectly impermeable for the control substrate. If that is not the case, the nuclear envelope may have been damaged during isolation and purification of the nucleus. The experiment has then to be discarded.

3. Alternatively, to reduce the time lag between substrate addition and confocal scanning, adjust the confocal microscope in advance using a test specimen. Place the microchamber on the stage of the confocal microscope, start scanning, and inject transport solution into the OSTR chamber (see Note 6).

Fig. 2. Measurement of nucleocytoplasmic transport using isolated Xenopus oocyte nuclei. In a microchamber, an isolated Xenopus oocyte nucleus was incubated with transport solution 1 containing an NLS protein (P4K) and a control substrate (TRD70). Scans were taken after addition of the transport solution at indicated times. From ref. 10.

Fig. 2. Measurement of nucleocytoplasmic transport using isolated Xenopus oocyte nuclei. In a microchamber, an isolated Xenopus oocyte nucleus was incubated with transport solution 1 containing an NLS protein (P4K) and a control substrate (TRD70). Scans were taken after addition of the transport solution at indicated times. From ref. 10.

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