Reconstitution Of β-catenin Degradation In Xenopus Egg Extract

Summary

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Abstract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways^1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus egg extract. One method is visually informative ([³⁵S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.

Introduction

Xenopus laevis egg extract has been used extensively to study many cell biological processes including cytoskeletal dynamics, nuclear assembly and import, apoptosis, ubiquitin metabolism, cell cycle progression, signal transduction, and protein turnover^1-17. The Xenopus egg extract system is amenable to the biochemical analysis of a legion of cellular processes because egg extract represents essentially undiluted cytoplasm that contains all the essential cytoplasmic components necessary to execute these processes and enable investigation. Large quantities of egg extract can be prepared at one time for biochemical manipulations that require large amounts of material (e.g., protein purification or high-throughput screening)^18-20. Another advantage is that the concentration of specific proteins in Xenopus egg extract can be precisely adjusted by addition of recombinant protein and/or immunodepletion of endogenous proteins in contrast to transfection of plasmid DNA where expression of the protein of interest is difficult to control. In addition, the lack of available recombinant proteins can be overcome by the addition of transcripts encoding the protein of interest, taking advantage of the freshly prepared Xenopus egg extract’s high capacity to translate exogenously added mRNA.

The regulation of protein degradation is critical for the control of many cellular pathways and processes²¹. Xenopus egg extract has been used extensively to study protein degradation as the system allows for multiple ways to monitor protein turnover without confounding influences of transcription and translation. The Wnt signaling pathway is a highly conserved signaling pathway that plays critical roles in development and disease. The turnover of β-catenin, the major effector of the Wnt pathway, is highly regulated, and an increased steady-state level of β-catenin is critical for the activation of Wnt target genes. The importance of β-catenin degradation is highlighted by the fact that mutations in the Wnt pathway that inhibit β-catenin degradation found in ~90% of all sporadic cases of colorectal cancer²². β-catenin degradation by components of the Wnt pathway can be faithfully recapitulated in Xenopus egg extract to study the mechanism of its turnover as well as to identify novel small molecule modulators of its degradation^{2,19,20,23-29} .

Methods for the preparation of Xenopus egg extract for studying the cell cycle have been described in previous JoVE publications^30-32. The current protocol describes a modification of these methods and is optimized for the degradation of [³⁵S]-radiolabeled β-catenin and luciferase-tagged β-catenin in Xenopus egg extract. The radiolabeled degradation assay allows for direct visualization of protein levels via autoradiography. [³⁵S]methionine is incorporated into the protein of interest using an in vitro translation reaction that can then be directly added to a degradation reaction. In addition, the radiolabeled protein turnover assay does not require an antibody against the protein of interest or an epitope tag, which can influence protein stability. Because even small changes in protein levels, as reflected in changes in the intensity of the radiolabeled protein band, are readily visualized by autoradiography, the [³⁵S]-radiolabeled degradation assay represents a very useful method for visualization of protein turnover².

Fusion of β-catenin to firefly luciferase (hereafter referred to as simply "luciferase") allows for precise and facile quantitative measurements of protein levels, in order to determine the kinetic properties of β-catenin turnover^19,20 . A major advantage of the luciferase assay is that it provides a strong quantitative system that is easily scaled up. The following protocol provides simple methods for assaying β-catenin degradation and a robust, efficient, and effective method for high-throughput screening of novel β-catenin modulators.

Protocol

1. Preparation of Xenopus Egg Extract

NOTE: Each frog yields approximately 1 ml of usable egg extract. Extracts from 10 frogs are typically prepared at one time, and the volume of buffer described below is for performing a 10 frog Xenopus egg extract prep. The buffer volume can be adjusted accordingly for larger or smaller preparations of egg extract. Preps generated in this manner consistently yield protein concentrations ≥ 50 mg/ml. The process of collecting eggs and processing them into extract is most efficient when conducted by two people. (For basic frog husbandry techniques, see Sive et al.³³).

1. Egg Collection
   1. To prime the frogs, inject each female frog with 100 U of Pregnant Mare Serum Gonadotropin (PMSG) from a freshly made 250 U/ml stock. Use a 3 ml tuberculin syringe with a 27 G needle to inject subcutaneously, with the bevel of the needle up, into the dorsal lymph sac, which is located approximately 1 cm away from the midline from the notched discolorations along the length of the legs of the frog.
   2. Store primed frogs in water (plus 20 mM NaCl, see Sive et al.³³) at 18 °C for 5-10 days. For standing water tank systems, the animal density is approximately 4 L of water per female frog. NOTE: The minimum time required for priming to take effect is 5 days, and the effects of priming wear off after 10 days.
   3. Prepare 0.5x Marc’s Modified Ringers (MMR) solution from a 20x MMR stock. 20x MMR consists of 2 M sodium chloride, 40 mM potassium chloride, 40 mM calcium chloride, 20 mM magnesium chloride, and 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4.
   4. Set up buckets for all injected frogs (one frog per 4 L bucket). NOTE: Although more than one frog can be placed in the same bucket for egg collection, if one of the frogs lays predominantly poor quality eggs, a substantial amount of effort will be required to separate the poor quality eggs from those that are suitable for making extract. Thus, maximizing the number of frogs in the same tank to minimize the amount of buffer used for egg collect is not worth the risk.
   5. Inject 750 U Human Chorionic Gonadotropin (HCG) into the dorsal lymph sac of each frog using a 27 G needle as described in 1.1.1.
   6. Place each of the HCG-injected frogs into individual 4 L buckets containing 0.5x MMR cooled to 16 °C.
   7. Place the containers with the frogs in a 16 °C incubator to collect eggs O/N (15-16 hr). NOTE: Maintaining the proper temperature is critical for the entire procedure, from collecting eggs to preparing egg extract.
2. Dejellying Eggs
   NOTE: Eggs are covered with a jelly coat that must be removed prior to making extract. The likelihood of spontaneous lysis of the eggs increases as the time between egg laying and extract preparation increases. Thus, it is important to proceed through the following steps as rapidly as possible.
   1. Prepare 4 L of 1x MMR, 50 ml of 0.1x MMR, and 400 ml of 2% cysteine, pH 7.7, made in distilled water. Maintain all solutions at 16 °C.
   2. To expel additional eggs, gently squeeze the lower back and abdomen of the frog.
   3. Remove the frogs and the bulk of the MMR, to leave the eggs in approximately 1-200 ml of MMR in each bucket.
   4. Remove debris with a transfer pipette, and assess the quality of the eggs: high quality eggs are generally marked by a clear separation between the darkly pigmented animal hemisphere and the lightly colored vegetal hemisphere and have the highest dark-to-light contrast. Discard with a transfer pipette any eggs that appear stringy, mottled, or lysed (white and puffy) as they will decrease the overall quality of the extract. If >10% of the eggs are of poor quality, the entire batch should be discarded.
   5. Combine eggs into a 500 ml glass beaker and pour out as much MMR as possible while keeping eggs submerged.
   6. Rinse eggs by gentle swirling with twice the egg volume of MMR. Repeat twice, and remove any debris or obviously poor quality eggs.
   7. Add approximately 100 ml of 2% cysteine to the glass beaker, swirl gently to mix, and allow eggs to settle for 5 min at 16 °C. Pour off the cysteine. NOTE: Dejellying is marked by the gradual appearance of jelly coats floating above the eggs and the more compact packing of the eggs as they now occupy a smaller volume without the jelly coat.
   8. Add another 100 ml of 2% cysteine, gently swirl, wait 5 min, and then slowly pour off the cysteine. Repeat until eggs have become tightly compacted (usually by the third cysteine treatment). Note: If eggs are left too long in cysteine, they are prone to lysis. Similarly, dejellied eggs are fragile and prone to mechanical lysis if they are swirled too vigorously or if they are exposed to air. Once the eggs have been dejellied, it is important to rapidly proceed to the centrifugation steps.
   9. Pour off cysteine, and rinse away the jelly coat and other debris by gently washing eggs in 1x MMR. Pour off buffer carefully along the side of the beaker. Repeat twice or until MMR solution is no longer cloudy. While rinsing with 1x MMR continue to remove the bad eggs using a transfer pipette.
   10. Perform a final gentle rinse with 30 ml 0.1x MMR, and gently pour off as much of the buffer as possible. Again, remove any obviously bad eggs.
3. Packing and Crushing Eggs by Centrifugation
   NOTE: The extract described below that is used for β-catenin degradation is a variant of the cytostatic factor extract (metaphase II-arrested). In contrast to the low-speed and high-speed extract used for cell cycle studies, intermediate speed extract works best for β-catenin degradation. Interphase extract similarly promotes robust β-catenin degradation although is more labor intensive to prepare.
   1. Add Leupeptin, Pepstatin, Aprotinin mixture (LPA, a protease inhibitor) at 10 µg/ml (diluted from a 10 mg/ml stock solution in DMSO) and Cytochalasin D at 20 µg/ml (diluted from a 10 mg/ml stock solution in DMSO) into the remaining 20 ml of 0.1x MMR.
   2. Add 0. x MMR containing LPA and Cytochalasin D to the washed eggs, swirl gently, and incubate for 5 min at 16 °C.
   3. Transfer eggs into 16 °C pre-chilled 50 ml centrifuge tubes, allow the eggs to settle, and remove residual buffer from the top. To prevent exposure to air, withdraw a small amount of buffer into the transfer pipette prior to withdrawing eggs for transfer. Continue to transfer additional eggs into the centrifuge tubes and remove residual buffer from the top of the tube until the eggs fill the centrifuge tube to the top, which will maximize the yield of extract.
   4. To pack the eggs, spin centrifuge tubes at 400 x g for 60 sec at 4 °C using a fixed angle rotor. Remove residual buffer from the top of the centrifuge tubes.
   5. For the crushing spin, spin tubes at 15,000 x g for 5 min at 4 °C.
4. Collecting Cytoplasmic Layer of Extract
   NOTE: The method described below differs from the classic method of puncturing the side of the centrifuge tube in order to collect the cytoplasmic layer. This protocol has been adapted for simplification and for use with particular centrifuge tubes, which are reusable. No noticeable differences in the kinetics of β-catenin degradation are found with either method. At this point extract should be kept cold during throughout the process, and all steps should be performed at 4 °C.
   1. Clear a hole in the lipid layer using P1000 pipette tip.
   2. Collect the cytoplasmic layer (between the dark pigmented layer and the light lipid layer) using a new P1000 pipette tip into clean pre-chilled centrifuge tubes (Figure 1). For high-quality extract that robustly degrades β-catenin, minimize the amount of pigmented and lipid layer that is withdrawn with the cytoplasmic layer.
   3. Spin extracted cytoplasmic layer at 15,000 x g for 10 min at 4 °C and again collect the cytoplasmic layer. Repeat the spin and extraction 1X. NOTE: The extract should be "straw colored." If there is substantial contamination with the pigmented and lipid layers at this point, one can repeat the spin one more time, although excessive spins will decrease the capacity of the extract to degrade β-catenin.
   4. Add LPA and Cytochalasin D to the extract at final concentrations of 10 µg/ml each. NOTE: Using the method described yields a fairly consistent total protein concentration of approximately 50 mg/ml (52.41 ± 5.40 mg/ml, n=3 separate preps) in Xenopus egg extracts.
   5. (Optional) For translation assays, add capped mRNA (0.1 mg/ml), RNAsin (1.5 U/ml), and energy regeneration mix (2.2.1) and incubate the reaction at RT for 2 hr (for further information see Salic et al.² and Sive et al.³³). Use translated extract immediately for β-catenin degradation assays or snap-freeze in liquid nitrogen for later use. NOTE: Freshly prepared extract has a high capacity to translate exogenously added mRNA, but unfortunately, this capacity is lost once the extract is frozen. Capped mRNA can be readily prepared using commercially available kits.
   6. Snap-freeze extract in liquid nitrogen. NOTE: Extracts are stored in small (200 µl) aliquots for single use because they rapidly lose their capacity to degrade β-catenin if refrozen. For long-term storage, extract can be stored in liquid nitrogen. For short-term storage, extract can be stored at -80 °C, although the capacity of extract to degrade β-catenin can be dramatically reduced with extended storage at -80 °C (longer than 2 months).

2. Preparing Extract for β-catenin Degradation Assay

1. Depletion from Xenopus Extract
   NOTE: A major advantage of Xenopus extract is the capacity to readily deplete components of a pathway and precisely add back a defined amount of a protein in order to determine its dose-dependent effects.
   1. Use freshly prepared Xenopus egg extract or quickly thaw frozen extract and place on ice. Perform all manipulations in the cold.
   2. Add extract to 1/10 the volume of pelleted antibody or affinity beads (e.g., 20 ml pelleted beads to 200 ml extract). In order to minimize dilution of the extract, withdraw as much liquid from the beads as possible before addition of the extract using gel loading tips with long, tapered tips.
   3. Rotate extract-bead mix at 4 °C for 1 hr.
   4. Spin extract-bead mix at 12,600 x g in microfuge at 4 °C for 30 sec. Alternatively, if magnetic beads are used, apply magnetic field to collect beads.
   5. Transfer depleted extract to a fresh microfuge tube on ice. Be careful not to transfer any beads with the extract.
   6. Confirm efficiency of depletion by immunoblotting both depleted extract and beads.
   7. Prepare extract for β-catenin degradation assay as described in 2.2.
2. Optimizing Xenopus Extract for β-catenin Degradation
   NOTE: β-catenin degradation in Xenopus egg extract is an energy-dependent process that quickly depletes the endogenous ATP stores. Consequently, an energy regeneration system is required to maintain robust β-catenin degradation.
   1. Prepare a 20x energy regeneration (ER) mix consisting of 150 mM creatine phosphate, 20 mM ATP, 600 µg/ml creatine phosphokinase, and 20 mM MgCl₂. ER should be aliquoted and stored at -80 °C. Avoid repeated freeze/thaw cycles by using small frozen aliquots.
   2. Quickly thaw Xenopus egg extract by rubbing the frozen tube between hands. Place the tube on ice just before all of the extract has melted.
   3. Add 10 µl of energy regeneration mix (20x ER) into an aliquot (200 μl) of Xenopus egg extract. Mix thoroughly by quickly flicking the tube and pulse vortexing. Pulse-spin and immediately place on ice.
   4. (optional) The turnover of β-catenin can be slightly enhanced in Xenopus egg extract by addition of ubiquitin (1.25 mg/ml final). Cycloheximide (0.1 mg/ml final) can also be added to minimize translation of endogenous transcripts.
   5. Aliquot the appropriate volumes for degradation assay into pre-chilled microfuge tubes on ice. For radiolabeled β-catenin degradation assays, withdraw 2-5 ml extract for each time point.

3. Radiolabeled β-catenin degradation assay in Xenopus egg extract

NOTE: All steps should be performed on ice unless otherwise indicated.

1. Preparing Radiolabeled β-catenin
   1. Prepare freshly in vitro-synthesized [³⁵S]methionine-radiolabeled protein using commercially available kits. NOTE: Generating ³⁵S-labeled (half-life 87 days) proteins are easily and efficiently produced using commercially available in vitro-coupled transcription-translation kits. It is important that the translated protein is sufficiently labeled such that changes in protein turnover can be readily visualized.
   2. To confirm successful radiolabeling, perform SDS-PAGE/autoradiography with 0.5 µl of the translated protein. Quantify the intensity of the radiolabeled β-catenin band using ImageJ, ImageQuant, or an alternative quantitative software program. NOTE: The radiolabeled β-catenin protein band should be clearly visible on film within a few hours (4-6 hr).
   3. Snap-freeze the radiolabeled protein in liquid nitrogen for storage until use. Note: Prolonged storage (>2 months) and multiple freeze/thaw (greater than 2) can severely impact the capacity of the radiolabeled β-catenin to degrade robustly in Xenopus egg extract. Typically, the stability of radiolabeled proteins declines significantly after 1 month of storage at -80 °C; thus, relatively freshly prepared radiolabeled β-catenin gives best results in these degradation assays.
2. Performing β-catenin Degradation Assay
   1. Add 1-3 µl (depending on the strength of the radiolabeled band signal) of in vitro-translated β-catenin (and other proteins, small molecules, etc. that are being tested) into 20 µl of Xenopus reaction mix on ice. Mix thoroughly by quickly flicking the tube and a short pulse of vortexing; this is an important step as Xenopus egg extract is very viscous, and incomplete mixing will affect the consistency of the results. Pulse spin and place on ice.
   2. Start the β-catenin degradation reaction by shifting the tubes to RT.
   3. At the designated time point, remove 1-5 ml of the sample and mix immediately with SDS sample buffer (5x volume) to stop the reaction. To make sure the degradation reaction is completely terminated, flick tube several times and vortex vigorously.
   4. Perform SDS-PAGE/autoradiography. Run 1 ml equivalents (~50 mg of protein) of the extract for each time point/lane. Degradation of β-catenin in Xenopus egg extract should be evidenced by the time-dependent decrease in intensity of the radiolabeled β-catenin band Figure 2. Quantify results using ImageJ, ImageQuant, or other preferred imaging software if necessary.
   5. (optional) Soak SDS-polyacrylamide gel in fixing solution (10% acetic acid and 30% methanol in distilled water) prior to drying to decrease background radioactivity and increase the quality of the image.

4. β-catenin-luciferase Degradation Assay in Xenopus Egg Extract

Perform all steps on ice unless otherwise indicated.

1. Preparing β-catenin-luciferase
   1. Synthesize non-radiolabeled, luciferase-tagged β-catenin using the transcription-translation coupled system with complete amino acid mix.
   2. Confirm production of the luciferase-tagged β-catenin by measuring luciferase activity from 0.5-1 µl of the reaction. Assess background luminescence by measuring luminescence from an untranslated reaction mix. NOTE: Multiple commercial kits are available for measuring luciferase activity. Long-lived luminescence, however, works particularly well for the degradation assay.
2. Performing β-catenin-luciferase Degradation Assay
   1. Thaw and prepare Xenopus egg extract as in 2.2.
   2. Add in vitro-translated β-catenin-luciferase fusion (from 4.1) into prepared Xenopus reaction mix (from 2.2) on ice and mix well as in 3.2.1. NOTE: The activity of the β-catenin luciferase that is added to the extract is typically between 20 - 50,000 relative luminescence units (RLU)/ml of extract (based on measurements obtained from 4.1.2). Starting signal should be approximately 100,000 RLU (2-5 ml of the in vitro-translated β-catenin-luciferase fusion).
   3. Shift the extract to RT to start the degradation reaction.
   4. Remove an aliquot of the reaction at the indicated time and snap-freeze in liquid nitrogen. NOTE: Triplicate samples are typically removed for analysis for each time point. Frozen extract can be stored at -80 °C until they are ready to be analyzed.
   5. Thaw samples ice, transfer samples to standard white 96-well plates on ice, and process for luciferase activity.

Results

A schematic of β-catenin degradation in Xenopus egg extract is shown in Figure 2A. ³⁵S-labeled β-catenin was incubated in Xenopus egg extract, aliquots (1 ml extract equivalent) were removed at the appropriate times, and samples were subjected to SDS-PAGE followed by autoradiography. β-catenin degradation by components of the Wnt pathway is mediated by the ubiquitin-proteasome system², and degradation of [³⁵S]-radiolabeled β-catenin i...

Discussion

Xenopus egg extract is a robust biochemical system for investigating β-catenin turnover. The concentration of β-catenin in Xenopus egg extract is ~25 nM ². Under optimal conditions, the egg extract is capable of degrading β-catenin at a rate of 50-100 nM/hr and is half-maximal at 200 nM²⁴. There are several critical steps for successful reconstitution of β-catenin degradation using Xenopus egg extract. These include 1) generating high quality X...

Materials

Name	Company	Catalog Number	Comments
Pregnant mare serum gonadotropin (PMSG)	ProSpec	hor-272-A	Reconstituted with distilled water before use.
Human chorionic gonadotropin	Sigma	CG10-10VL
Potassium chloride	Fisher	BP366-1
Sodium chloride	Research Products International	S23020-5000.0
Magnesium chloride	Fisher	BP214-500
Calcium chloride	Acros Organics	AC42352-5000
HEPES	Fisher	BP310-1
Cysteine	Acros Organics	AC17360-1000
Leupeptin	Sigma	L2884-10MG
Aprotinin	Sigma	A1153-10MG
Pepstatin	Sigma	P4265-5MG
Cytochalasin B	Sigma	C8273-10MG
3 ml syringe: Luer Lock tip	Becton Dickinson	309657
27 G needle	Becton Dickinson	305109
96 well solid white polystyrene microplate, round bottomed	Corning	3605
Steady-Glo luciferase assay system	Promega	E2520	Store long-term at -80 °C, can store for up to 1 month at -20 °C
TNT Sp6 coupled reticulocyte lysate system	Promega	L4600
TNT Sp6 high-yield wheat germ protein expression system	Promega	L3260	Generally higher yield than reticulocyte lysate
EasyTag Express protein labeling mix [S35]	Perkin Elmer	NEG772007MC
Creatine phosphate	Sigma	27920-5G
ATP	Sigma	A2383-5G
Creatine phosphokinase	Sigma	C3755-35KU
Dimethyl sulfoxide (DMSO)	Sigma	D8418-50ML
Dual-Glo luciferase assay system	Promega	E2920	Same storage conditions as Steady-Glo
50 ml Centrifuge tubes	Fisher Scientific	0556214D
Sorvall SS-34 fixed angle rotor	Thermo Scientific	28020
115 V 50/60 Hz Minicentrifuge	Fisher Scientific	05-090-128
mMessage mMachine Sp6 kit	Ambion	AM1340
Anti-firefly luciferase antibody	Abcam	ab16466
Anti-GSK3 antibody	BD Transduction Laboratories	610201
FLUOStar Optima	BMG Labtech
Sorvall RC-6 Plus centrifuge	Thermo Scientific
16 °C Incubator	Percival Scientific