Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Summary

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.

Abstract

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic phenomena, such as cytokinesis, mitotic spindle formation and assembly of the nucleus. Building upon early methods that imaged fixed extracts sampled at sparse time points, recent approaches image live extracts using time-lapse microscopy, revealing more dynamical features with enhanced temporal resolution. These methods usually require sophisticated surface treatments of the imaging vessel. Here we introduce an alternative method for live imaging of egg extracts that require no chemical surface treatment. It is simple to implement and utilizes mass-produced laboratory consumables for imaging. We describe a system that can be used for both wide-field and confocal microscopy. It is designed for imaging extracts in a 2-dimensional (2D) field, but can be easily extended to imaging in 3D. It is well-suited for studying spatial pattern formation within the cytoplasm. With representative data, we demonstrate the typical dynamic organization of microtubules, nuclei and mitochondria in interphase extracts prepared using this method. These image data can provide quantitative information on cytoplasmic dynamics and spatial organization.

Introduction

The cytoplasm constitutes the main volume of a cell and has a distinct organization. The ingredients of the eukaryotic cytoplasm can self-assemble into a wide range of spatial structures, such as microtubule asters and the Golgi apparatus, which in turn are dynamically arranged and turned over depending on the cell's identity and physiological state. Understanding the spatial organization of the cytoplasm and its link to cellular functions is thus important for understanding how the cell works. Xenopus laevis egg extracts have traditionally been used for bulk biochemical assays^{1,2,3,4,5,6,7,8}, but recent work establishes them as a powerful live imaging system for mechanistic studies of cytoplasmic structures and their cellular functions^{9,10,11,12,13,14,15,16,17,18}. These undiluted extracts preserve many structures and functions of the cytoplasm, while allowing direct manipulations of cytoplasmic contents not achievable in conventional cell-based models^19,20. This makes them ideal for characterizing cytoplasmic phenomena and dissecting their mechanistic underpinnings.

Existing methods for imaging extracts require chemical surface modifications, or fabrication of microfluidic devices. One coverslip-based method requires polyethylene glycol (PEG) passivation of glass coverslips²¹. A microemulsion-based method requires vapor deposition of trichloro(1H,1H,2H,2H-perfluorooctyl)silane on glass surfaces^22,23. Microfluidic-based systems allow precise control of the volume, geometry and composition of extract droplets, but require specialized microfabrication facilities^11,12,24.

Here we introduce an alternative method for imaging egg extracts that is easy to implement and utilizes readily available, low-cost materials. This includes preparation of an imaging chamber with a slide and a coverslip coated with fluorinated ethylene propylene (FEP) tape. The chamber can be used for imaging extracts with a variety of microscopy systems, including stereoscopes and upright and inverted microscopes. This method requires no chemical treatment of surfaces while achieving similar optical clarity obtained with existing glass-based methods discussed above. It is designed to image a layer of extracts with a uniform thickness across a 2D field, and can be easily extended to image a 3D volume of extracts. It is well suited for time-lapse imaging of collective cytoplasmic behavior over a large field of view.

We have used interphase-arrested egg extracts to demonstrate our imaging method. The extract preparation follows the protocol of Deming and Kornbluth¹⁹. Briefly, eggs naturally arrested in metaphase of meiosis II are crushed by a low speed spin. This spin releases the cytoplasm from meiotic arrest and allows the extract to proceed into interphase. Normally, cytochalasin B is added prior to the crushing spin to inhibit F-actin formation. However, it can be omitted if F-actin is desired. Cycloheximide is also added prior to the crushing spin to prevent the interphase extract from entering the next mitosis. The extracts are subsequently placed in the aforementioned imaging chambers and placed on a microscope. Finally, images are recorded over time at defined intervals by a camera connected to the microscope, producing time-lapse image series that capture the dynamical behavior of the extract in a 2D field.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Stanford University.

1. Preparation of slides and coverslips

1. Apply a layer of fluorinated ethylene propylene (FEP) adhesive tape to a glass slide with a roller applicator. Cut off excessive tape over the edges with a clean razor blade. Prepare FEP tape-coated coverslips in the same way (Figure 1A).
2. Apply a double-sided sticky imaging spacer to the FEP tape-coated side of the slide. Leave the protective liner on the top unpeeled (Figure 1A).
   ​NOTE: The slides and coverslips should be prepared before the experiment. They can be used immediately, or stored in boxes to prevent dust accumulation on surfaces. The well in the imaging spacer is 120 µm deep and has a diameter of 9 mm.

2. Preparation and live imaging of interphase-arrested egg extracts

NOTE: The following protocol is adapted from Deming and Kornbluth¹⁹, Murray²⁰, and Smythe and Newport²⁵ with modifications. All steps should be performed at room temperature unless otherwise noted.

1. Three to ten days before egg collection, inject mature female Xenopus laevis frogs subcutaneously into the dorsal lymph sac with 100 IU of pregnant mare serum gonadotropin (PMSG).
2. Sixteen to eighteen hours prior to planned egg collection, inject the frogs from step 2.1 with 500 IU of human chorionic gonadotropin (hCG). Leave frogs at 18 °C in egg laying buffer (100 mM NaCl, 2 mM KCl, 1 mM MgSO₄·7H₂O, 2.5 mM CaCl₂·2H₂O, 0.5 mM HEPES, 0.1 mM EDTA, prepare as a 20x stock solution at pH 7.4 and dilute with clean frog tank water to 1x before use) until egg collection.
3. On the day of the experiment, collect eggs in a large glass Petri dish and assess egg quality. Discard any eggs that look like white puffy balls or appear in a string (Figure 1B). Examine the eggs under a stereoscope, keep the eggs with normal appearance (Figure 1C), and discard those with irregular or mottled pigment (Figure 1D).
   NOTE: This protocol works with eggs collected from a single frog, which typically lays 25 mL of eggs by 16 hours after hCG injection. Usually, a total of 3 to 6 frogs are induced by hCG, and the frog with the highest egg quality is chosen for the extract preparation experiment.
4. Transfer eggs to a 400 mL glass beaker, and remove as much egg laying buffer as possible by decanting.
5. Incubate the eggs in 100 mL of freshly prepared dejellying solution (2% w/v L-cysteine in water, adjust to pH 8.0 with NaOH) and gently swirl them periodically. After about 3 minutes, pour off the solution, and add 100 mL of fresh dejellying solution. Continue the incubation until the eggs are tightly packed (no space between eggs), but avoid leaving eggs in the dejellying solution for more than a total of 5 minutes.
6. Remove as much of the dejellying solution as possible by decanting, and wash the eggs in 0.25x MMR buffer (25 mM NaCl, 0.5 mM KCl, 0.25 mM MgCl₂, 0.5 mM CaCl₂, 0.025 mM EDTA, 1.25 mM HEPES, prepared as a 10x stock solution, adjusted to pH 7.8 with NaOH, and diluted in Milli-Q water before use) by adding the buffer, swirling the eggs, and then pouring off the buffer. Repeat a few times until a total of 1 L of the buffer is used for the washes.
7. Wash the eggs a few times with a total of 400 mL of egg lysis buffer (250 mM sucrose, 10 mM HEPES, 50 mM KCl, 2.5 mM MgCl₂, 1 mM DTT, made fresh and adjusted to pH 7.7 with KOH). Remove eggs with abnormal appearance using a Pasteur pipette between the washes.
   NOTE: Eggs with abnormal appearance refer to those that look like white puffy balls (Figure 1B), have mottled pigmentation (Figure 1D), are deteriorating with a growing white region (Figure 1E), or show darkened and contracted pigmented area in the animal hemisphere (Figure 1F).
8. Using a transfer pipette with its tip cut wide open, transfer the eggs to a 17 mL round-bottom centrifuge tube containing 1 mL of egg lysis buffer. Spin the tube in a clinical centrifuge at 400 x g for 15 seconds to pack the eggs.
9. Remove as much of the egg lysis buffer as possible from the top of packed eggs using a Pasteur pipette.
   NOTE: It is important to remove as much buffer from the packed eggs as possible, in order to minimize the dilution of the egg extract. Sometimes it is necessary to remove some loose eggs along with the residual buffer to accomplish this.
10. Determine the approximate volume of the packed eggs, and then add 5 µg/mL aprotinin, 5 µg/mL leupeptin, 5 µg/mL cytochalasin B, and 50 µg/mL cycloheximide directly on top of the packed eggs.
    NOTE: Aprotinin and leupeptin are protease inhibitors. Cytochalasin B inhibits actin polymerization, preventing the extract from contracting and gelling²⁶. Cycloheximide inhibits protein synthesis, thereby keeping the extract in the interphase of the cell cycle.
11. Crush the eggs by centrifuging the tube at 12,000 x g, 4 °C, for 15 minutes, in a swinging bucket rotor.
    NOTE: At the end of the centrifugation, the eggs should have ruptured and the lysate separated into three main layers: a yellow lipid layer on top, the cytoplasmic extract (also called crude extract) in the middle, and a dark dense layer containing the pigment granules at the bottom (Figure 1G).
12. Attach an 18-gauge needle to a syringe. With the needle tip bevel facing up, puncture the tube from the side at the bottom of the cytoplasmic layer, and recover the extract by drawing slowly.
    NOTE: Draw the cytoplasmic extract slowly to avoid the inclusion of contaminating content from the yellow lipid layer.
13. Transfer the recovered cytoplasmic extract to a new microcentrifuge tube and hold it on ice. Use the extract within 1 hour.
14. When ready to image, supplement the extract with desired reagents and fluorescence imaging probes.
    NOTE: Fluorescence imaging probes label specific cytoplasmic structures so that they can be visualized by a fluorescence microscope.
15. Remove the top protective liner from the imaging spacer on the slide prepared in step 1.2, and deposit approximately 7 µL of extract at the center of the well. Immediately apply the FEP tape-coated coverslip with the FEP side facing the extract to seal the well. Quickly proceed to imaging (Figure 1H,I).
16. Set the slide on an inverted or upright microscope with a motorized stage and a digital camera. Image the extracts at desired spatial positions and time intervals in both bright-field and fluorescence channels.
    NOTE: Typically, a 5x objective is used for imaging. The motorized stage enables automated image acquisition at multiple defined spatial positions. Bright-field microscopy visualizes cytoplasmic structures with different degrees of transparency. Fluorescence microscopy visualizes the cytoplasmic structures specifically labeled by the fluorescence imaging probes added in step 2.14. The camera records time-lapse images of these structures, thereby capturing the dynamics of cytoplasmic organization.

Results

Xenopus laevis egg extracts can be used to study the self-organization of the cytoplasm during interphase. Figure 2A shows results from a successful experiment. We supplemented interphase-arrested extracts with demembranated Xenopus laevis sperm nuclei¹⁹ at a concentration of 27 nuclei/µL and 0.38 µM purified GST-GFP-NLS^27,28,

Discussion

Xenopus laevis egg extracts have emerged as a powerful model system for imaging-based studies of various subcellular structures^{10,14,15,16,17,18,21,31,32,33,}

Materials

Name	Company	Catalog Number	Comments
17 ml centrifuge tube	Beckman Coulter	337986
22x22 mm square #1 cover glass	Corning	284522
Aprotinin	MilliporeSigma	10236624001	Protease inhibitor
Cycloheximide	MilliporeSigma	01810	Protein synthesis inhibitor
Cytochalasin B	MilliporeSigma	C6762	Actin polymerization inhibitor
Female Xenopus laevis frogs	Nasco	LM00535MX
Fluorescent HiLyte 488 labeled tubulin protein	Cytoskeleton, Inc.	TL488M-A	For visualizing the microtubule cytoskeleton
Fluorescent HiLyte 647 labeled tubulin protein	Cytoskeleton, Inc.	TL670M-A	For visualizing the microtubule cytoskeleton
Fluorinated ethylene propylene (FEP) optically clear tape	CS Hyde company	23-FEP-2-5
Glass Pasteur pipette	Fisher Scientific	13-678-20C
Human chorionic gonadotropin (hCG)	MilliporeSigma	CG10
Imaging spacer	Electron Microscopy Sciences	70327-8S
Leupeptin	MilliporeSigma	11017101001	Protease inhibitor
Microscope slides	Fisher Scientific	12-518-100B
Mineral oil	MilliporeSigma	330760
MitoTracker Red CMXRos	Thermo Fisher Scientific	M7512	For visualizing mitochondria
Pregnant mare serum gonadotropin (PMSG)	BioVendor	RP1782725000
Roller applicator	Amazon	B07HMBJSP8	For applying the FEP tape to the glass slides and coverslips
Single-edged razor blades	Fisher Scientific	12-640	For removing excessive FEP tape
Transfer pipette	Fisher Scientific	13-711-7M