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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Methods Mol Biol. 2022;2502:395–405. doi: 10.1007/978-1-0716-2337-4_25

Visualizing Nuclear Pore Complexes in Xenopus Egg Extracts

Sampada Mishra 1, Daniel L Levy 1,1
PMCID: PMC9219414  NIHMSID: NIHMS1814949  PMID: 35412252

Abstract

The nuclear pore complex (NPC) is the conduit in the nuclear envelope through which proteins and RNA are transported between the cytoplasm and nucleus. Xenopus egg extracts that support de novo assembly of nuclei have provided a robust system to study NPC structure and function because the biochemical composition of the extract can be easily manipulated. Here we describe how to assemble nuclei in Xenopus egg extract, how to visualize and analyze NPCs in both live and fixed samples, and different approaches to altering nucleocytoplasmic transport in extract.

Keywords: Xenopus egg extracts, NPC, nuclear pore complexes, spindown, mAb414, immunofluorescence, nuclear import

1. Introduction

The nucleus is a double membrane bound structure that houses the DNA of eukaryotic cells. At sites where the outer and inner nuclear membranes fuse are embedded multiprotein structures called nuclear pore complexes (NPCs). The proteins that constitute the NPC are generally referred to as nucleoporins (Nups) [1]. NPCs mediate protein transport into and out of the nucleus, as well as mRNA export. Although smaller molecules (< 40-60 kD) can freely diffuse through the NPC [2], transport of larger molecules is mediated by karyopherins, including importins that import proteins possessing a nuclear localization signal (NLS) and exportins that export nuclear export signal (NES)-containing proteins [3]. Disruption of NPCs and altering karyopherin levels and activities can affect nuclear transport [4–6]. Dictating the nucleocytoplasmic partitioning of proteins and RNA, NPCs regulate a wide range of activities, including nuclear structure and organization, DNA replication, transcription, and cell differentiation, to name a few [7–9].

Xenopus egg extract is a cell free system that is widely used to study nuclear morphology and NPCs. The eggs are arrested at metaphase II of meiosis, and cytoplasmic extracts derived from these eggs can be driven into interphase through calcium addition and blocked in interphase with cycloheximide that prevents cyclin synthesis [10]. As the extract is devoid of DNA, an exogenous source of DNA must be added to initiate nuclear assembly, usually demembranated Xenopus sperm chromatin [11]. Nuclei formed in Xenopus extract recapitulate nuclear structures and activities typical of in vivo nuclei, including nuclear envelopes with embedded NPCs, active nuclear import, nuclear expansion, and DNA replication [12–15]. In this protocol, we describe different ways to visualize NPCs in nuclei assembled in Xenopus egg extract by both live imaging and immunofluorescence, along with ways of altering nuclear transport through the NPC.

2. Materials

2.1. Nuclear formation

  1. 10x XB Buffer: 1 M KCl, 1 mM CaCl2, 10 mM MgCl2, 500 mM sucrose, 100 mM HEPES-KOH. Adjust the pH to 7.8, filter sterilize, and store at 4°C. Dilute to 1x with dH2O before use.

  2. Calcium stock solution: 10 mM CaCl2, 0.1 M KCl, 1 mM MgCl2. Store at −20°C in 500 μL aliquots.

  3. Cycloheximide stock solution: 10 mL of 1x XB buffer, 100 mg cycloheximide. Vortex to dissolve (warm slightly if necessary), syringe filter, and store at −20°C in 100 μL aliquots.

  4. Demembranated Xenopus sperm: see [16]

  5. Metaphase-arrested X. laevis egg extract: see [17]

  6. Energy mix: 190 mM creatine phosphate disodium, 25 mM ATP disodium salt, 25 mM MgCl2. Store at −20°C in 100 μL aliquots.

  7. Ethanol (70%)

  8. Oxygen scavenging mix: see [18]

  9. Sperm dilution buffer: 10 mM HEPES-KOH pH 7.8, 1 mM MgCl2, 100 mM KCl, 150 mM sucrose.

  10. Hoechst stock: 10 mg/mL Hoechst 33342 in dH2O. Store at −20°C in 40 μL aliquots.

  11. Nuclear Fix: Combine 125 μL 2 M sucrose, 12.5 μL 1 M HEPES-KOH pH 7.8, 250 μL 37% formaldehyde, 112 μL dH2O, and 0.5 μL Hoechst stock solution (10 mg/mL). This solution can be stored at room temperature and is stable for months.

  12. Valap sealant: Measure equal weights of vaseline, lanolin, and paraffin into a beaker. Melt using a hot plate set at a low heat setting. Stir to combine, generating a golden yellow mixture. The prepared sealant can be stored in capped jars at room temperature. Before use, the mixture should be warmed on a hot plate (see Note 1).

  13. Epifluorescence microscope with DAPI, GFP/FITC, and RFP/TRITC filters

  14. Cotton tipped applicators

  15. 1.5 ml Eppendorf tubes

  16. Hot plate

  17. Kimwipes

  18. Water bath set at 16-20°C

  19. Wide bore pipette tips (tip opening ~1.5 mm for 200 μl pipette tips) (see Note 2)

  20. 22 mm × 22 mm coverslips and glass slides (see Note 3)

2.2. Imaging of nuclear pore complexes

  1. 10x ELB: 1.25 M sucrose, 250 mM KCl, 12.5 mM MgCl2, 50 mM HEPES pH 7.8, filter sterilize, store at 4°C. Dilute to 1x with dH2O before use.

  2. Wash buffer: 0.1% NP-40 in 1x PBS

  3. GST-GFP-NLS or GST-mCherry-NLS recombinant protein: see [6,19]. Our stock concentrations range from 2-28 mg/ml.

  4. Nuclear cushion: 1x XB, 0.2 M sucrose, 25% glycerol. Prepare 500 ml using dH2O. Filter sterilize and store at 4°C.

  5. Fixing solution: 1x ELB, 15% glycerol, 2.6% paraformaldehyde.

  6. PBS-3% BSA (bovine serum albumin): 3% BSA dissolved in 1x PBS.

  7. Anti-nucleoporin antibodies (e.g. mAb414) (see Note 4)

  8. Fluorescently-labeled secondary antibodies (e.g. Alexa Fluor)

  9. mAb414 labeled with Alexa Fluor

  10. Dish of methanol containing a coverslip holder at −20°C

  11. Round coverslips (12 mm)

  12. Round bottom glass tubes (15 ml)

  13. Conical and cylindrical inserts for tubes (see Note 5)

  14. Parafilm

  15. Petri dishes

  16. Vectashield

  17. Nail polish

3. Methods

3.1. Nuclear formation in extract

  1. Prepare demembranated sperm and fresh metaphase-arrested Xenopus egg extract as described previously [17,16].

  2. Aliquot 100 μl of freshly prepared extract into a 1.5 ml microfuge tube using wide pore pipette tips. Keep on ice (see Note 6).

  3. Add 1.5 μl cycloheximide stock, 6 μl calcium stock, and 2 μl energy mix. Gently tap to mix or invert several times for larger volumes (see Notes 7 and 8).

  4. Add demembranated sperm such that there are ~1000 sperm/μl of extract and gently tap to mix or invert for larger volumes (see Note 9).

  5. Incubate the reaction in a 16-20°C water bath. Gently tap the reaction tube every 15 minutes to redistribute the nuclei that tend to settle to the bottom of the tube.

  6. To track progression of nucleus formation, place 4 μl of the reaction on a glass slide using a wide bore pipette tip (see Note 10), add 2 μl of nuclear fix, and mix with a pipette tip. Overlay with a coverslip and seal with warmed valap using a cotton applicator.

  7. Image using an epifluorescence microscope. By Hoechst staining, the sperm will initially appear as condensed S-shaped structures. Over time, the sperm chromatin will decondense. The formation of round structures is indicative of successful nucleus formation. At later time points as the nuclei grow larger, the chromatin will take on a more string-like appearance [20].

  8. The most stringent test for nucleus formation is nuclear import and retention of a fluorescently labeled import cargo, such as GST-GFP-NLS (Fig 1a). Mix 4 μl of the reaction with an appropriate volume of GST-GFP-NLS stock solution (e.g. 0.5 μl) on a glass slide, mix well with a pipette tip, overlay with a coverslip, seal with valap, and image using an epifluorescence microscope. If nuclei have assembled with intact nuclear envelopes, GST-GFP-NLS will be imported through NPCs and accumulate within the nuclei (see Note 11 and 12). It is also possible to measure nuclear import kinetics [20].

Figure 1: Imaging of nuclei assembled in Xenopus egg extract.

Figure 1:

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(a) Live nuclei 90 minutes after initiating nuclear assembly import and retain GST-GFP-NLS, indicating they are intact. (b) Wide-field imaging of nuclei at different times points after initiating nuclear assembly. Nuclei were fixed and stained with mAb414 to visualize NPCs. (c) Confocal image of NPCs on the surface of a nucleus assembled in X. laevis egg extract. Scale bar = 25 μm.

3.2. Visualization of nuclear pore complexes

3.2.1. Immunofluorescence of NPCs

  1. Combine 500 μl freshly prepared fixing solution with 30 μl extract containing assembled nuclei in a 1.5 ml microfuge tube. Invert immediately several times to mix and then place on a rotator at room temperature for 15 minutes (see Notes 13 and 14) (Fig 2a–b).

  2. In the meantime, prepare spindown tubes (Fig 2c). For each sample, outfit a 15 ml round bottom tube with the conical-shaped adaptor on the bottom topped with the cylindrical, grooved adapter. Add 5 ml of nuclear cushion to the tube and then drop in a clean round coverslip so that it lays flat on the adapter (see Note 15).

  3. Carefully layer the solution containing fixed nuclei on top of the nuclear cushion using wide pipette tips (Fig. 2d). Minimize mixing of the sample containing nuclei with the cushion. Centrifuge at 16°C in a swinging bucket rotor at 1000g for 15 minutes.

  4. Insert the tip of an aspirator firmly into the groove on the cylindrical adapter to aspirate out the nuclear cushion and remove the cylindrical adapter from the tube (Fig. 2e).

  5. Carefully remove the coverslip using forceps. Take note of the orientation of the coverslip as the nuclei are spun down onto the topside of the coverslip (see Note 16).

  6. Place the coverslips in a coverslip holder emersed in −20°C methanol for 5 minutes (see Notes 17 and 18) (Fig 2f).

  7. Transfer the coverslips to a humid incubator using forceps, nucleus side up (Fig. 2g). Wash the coverslips twice with ~300 μl wash buffer. Carefully moving the tip of an aspirator around the edge of the coverslip is a convenient way to remove buffer between washes (see Notes 19 and 20).

  8. Block the nuclei using ~75 μl PBS-3% BSA per coverslip for 1 hour at room temperature (see Note 21).

  9. Remove the blocking solution and overlay 75 μl of primary antibody diluted appropriately in PBS-3% BSA. A typical antibody used for this purpose is mAb414 at 1 μg/ml (see Note 4). Incubate 1 hour at room temperature or overnight at 4°C (see Notes 22 and 23).

  10. Wash briefly 5-7 times with wash buffer.

  11. Overlay 75 μl of fluorescently-labeled secondary antibody diluted to a concentration of 2 μg/ml in PBS-3% BSA. For mAb414 we use an Alexa Fluor labeled anti-mouse secondary antibody. Incubate for 1 hour at room temperature.

  12. Wash briefly 5-7 times with wash buffer. Dilute the Hoechst stock 1:1000 in PBS-3% BSA and overlay 75 μl onto each coverslip. Incubate for 5 min at room temperature (see Note 24).

  13. Wash briefly 5-7 times with wash buffer and remove excess solution. Mount the coverslip (nucleus side facing down) onto 3 μl of vectashield on a glass slide (Fig. 2h).

  14. Seal the coverslip with nail polish (Fig. 2i). Visualize NPC staining using an epifluorescence microscope and appropriate filters depending upon the fluorophores used. Slides can be stored at 4°C for at least several weeks (see Note 25).

Figure 2: Spindown procedure to perform nucleus immunofluorescence.

Figure 2:

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(a) Extract with asssembled nuclei (yellow). (b) Nuclei are added to fixing solution (blue) and the tube is rotated on a rotator. (c) Assemble a spindown tube by dropping the conical-shaped insert into a 15 ml Corex tube followed by the grooved cylindrical adapter. (d) The tube is filled with nuclear cushion and a round coverslip is laid on top of the cylindrical adaptor. Extract containing fixed nuclei is carefully layered on top of the nuclear cushion. The tube is centrifuged at 1000g for 15 min at 16°C. (e) The nuclear cushion is removed by aspiration by placing the aspirator tip in the groove of the cylindrical adapter. (f) The coverslip is carefully transferred to a coverslip holder in a dish of −20°C methanol using forceps and incubated for 5 minutes. (g) The coverslip is then laid nucleus side up on a piece of parafilm in a petri dish and washed with PBS-0.1% NP-40. Immunofluorescence is performed as described in the protocol. (h) A drop of vectashield is placed on a glass slide and the coverslip is laid nucleus side down on top of the vectashield, minimizing the introduction of air to avoid bubbles. (i) The coverslip is then sealed to the slide with nail polish and imaged by epifluorescence microscopy. Diagrams were created with Biorender.com.

3.2.2. Live visualization of NPCs

  1. Assemble nuclei in extract as described in 3.1. Add 5 μg/ml mAb414 that has been directly labeled with Alexa Fluor to the extract reaction.

  2. Aliquot 7 μl of the reaction on a glass slide, overlay with a coverslip, seal with valap, and observe using an epifluorescence microscope.

3.2.3. Quantification of NPCs

  1. Based on wide-field imaging (Fig. 1b), NPC staining intensity can be quantified using image analysis software like ImageJ or Metamorph. After performing background correction, appropriately threshold the image and measure either total intensity or average intensity (i.e. total intensity divided by area) for each nucleus.

  2. Based on confocal imaging on the surface of nuclei (Fig. 1c), NPC density can be quantified. The number of NPCs is counted within a defined area of nuclear envelope and divided by the area to obtain NPC density. This density can also be multiplied by total nuclear surface area to obtain the total number of NPCs per nucleus. NPC counting can be performed manually or using image analysis software.

3.3. Altering nuclear import

  1. Antibodies against specific importins or Nups can be used for immunodepletion from extract [21,6,22]. Alternatively, the antibody can be added directly to extract to inhibit the protein of interest [6]. In the case of immunoinhibition, it is generally best to first dialyze the antibody into 1x XB buffer.

  2. Import can be more generally inhibited by adding dominant negative importin fragments (e.g. 30 μM of importin β binding domain of importin α) [5,19], 0.2 mg/ml wheat germ agglutinin that interacts with glycosylated Nups and effectively plugs the NPC [23], the small molecule importazole that inhibits importin β [24], or RanQ69L protein that is constitutively GTP bound.

  3. Nuclear import can be increased by adding recombinant karyopherins to the extract [25,6,19] (see Note 26).

Acknowledgements

We thank Pan Chen for her comments on the protocol. Research in the Levy lab is supported by the National Institutes of Health/National Institute of General Medical Sciences (R35GM134885 and P20GM103432) and the American Cancer Society (RSG-15-035-01-DDC).

Footnotes

1.

Do not overheat the Valap. If it turns brown it should be discarded.

2.

Normal pipette tips can be used as an alternative by cutting the tips off to make a wider opening.

3.

Use 70% ethanol to clean coverslips and glass slides before use. Dry with kimwipes.

4.

The antibody mAb414 is often used to detect NPCs because it recognizes multiple Nups containing FG-repeats. Other primary antibodies that recognize specific Nups can be used as appropriate [26–28].

5.

The adapters may need to be custom made (Fig. 2c). Design and schematics available upon request.

6.

It is best to use the extract within 3-4 hours of preparation.

7.

Thaw the cycloheximide and calcium stock solutions at room temperature 10-15 minutes before use. The energy mix should be thawed on ice.

8.

Do not vortex or vigorously mix the delicate extract as this can inactivate the extract.

9.

If clumping or unequal mixing of sperm with extract is observed, it is sometimes helpful to first dilute the sperm with sperm dilution buffer or extract prior to addition.

10.

Using wide bore pipette tips helps to minimize damage to nuclei during pipetting.

11.

For long timelapse imaging of nuclei, add oxygen scavenging mix to the extract to minimize photodamage [18].

12.

The greatest source of variability in these experiments is the quality of the egg extract and demembranated sperm. If nuclei fail to assemble after 45 minutes, discard the extract and prepare a new extract with eggs collected from different frogs. If nucleus formation continuously fails or nuclear morphology is always aberrant, prepare a new batch of demembranated sperm with adequate detergent treatment.

13.

Endogenous nuclei can also be isolated from X. laevis embryos [29,17], and NPCs can be similarly visualized and analyzed using the same protocol.

14.

Longer fixation times are acceptable.

15.

It is usually better to drop in the coverslip after adding the nuclear cushion. Swinging the tubes slightly will help the coverslip sink and lie on top of the cylindrical insert.

16.

It is helpful to orient the top of the coverslips facing the same direction in the coverslip holder, so it is easy to remember which side contains the nuclei.

17.

Only remove the methanol from the freezer just before use.

18.

Omit this step if your antibody is known to be incompatible with methanol post-fixation. As an alternative, permeabilize with PBS-0.1% Triton.

19.

Take care not to place the tip of the aspirator directly on top of the coverslip as this might disturb the nuclei.

20.

For the humid incubator, we place a sheet of parafilm in a petri dish and surround the parafilm with moistened kimwipes. The coverslips are placed on the parafilm. The dish is covered and incubated in the dark at room temperature. It is particularly important that incubation with fluorescently-labeled secondary antibody is performed in the dark.

21.

Blocking can also be performed overnight at 4°C.

22.

If GST-GFP-NLS was added to the reaction, most of the GST-GFP-NLS signal will leak out of the nucleus after permeabilization, so this import substrate is best visualized live.

23.

The antibody dilution and incubation time may need to be optimized. For weak signals, the incubation time can be increased (e.g. overnight at 4°C) and/or the antibody dilution decreased. For precious antibody samples, volumes as small as 25-30 μl can be used.

24.

Instead of performing a separate Hoechst staining, the secondary antibody solution can be supplemented with a 1:1000 dilution of Hoechst stock solution.

25.

It is worth noting that Xenopus NPCs have also been visualized by electron microscopy and super-resolution imaging [30,31,15,32].

26.

CRM1-mediated nuclear export is negligible in Xenopus egg extract [33].

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