Antibodies.

Rabbit polyclonal anti-cyclin Y (CycY) and anti–Sp1490-LRP6 (pLrp6) antibodies were as described (15). Other antibodies used were as follows: anti–α-tubulin (α-tub), anti-β-catenin (β-cat), and anti-ERK (Sigma); anti-LRP6 (C5C7) (Cell Signaling); anti–phospho-Histone 3-Ser10 (pH3) (Abcam); anti–polyubiquitin FK-1 (PolyUb) (ENZO Life Sciences); anti-Ube2c and anti-Cdk7 (Santa Cruz Biotechnology); and anti-Lys48–linked ubiquitin (anti-K48-Ub) (Millipore). Anti-Xenopus Aurora B (Aurkb) and anti-Xenopus Plk1/Plx1 (Plk1) were a kind gift from H. Funabiki, Rockefeller University, New York (31, 41). Anti-Xenopus Cyclin E was a kind gift from C. Bonne-Andrea, Centre de Recherche de Biochimie Macromoléculaire (CRBM), Montpellier, France (29). For Western blot, antibodies were diluted in Tris buffered saline with Tween 20 containing 5% (wt/vol) BSA and 1 mM EDTA.

Anti-PolyUb recognizes all ubiquitin chains. Anti–K48-Ub binds specifically to lysine-48–linked ubiquitin chains, which are the most common label for proteasomal degradation.

X. laevis Methods.

In vitro fertilization, embryo culture, staging, and microinjection were carried out as described (42). Morpholinos against ccny plus ccnyl1 (cycY), lrp6, and β-catenin have been described previously (15, 20, 21).

Stage 6 oocytes were manually defolliculated, injected equatorially with Morpholinos (MOs), and cultured a total of 48–72 h at 18 °C in oocyte culture medium (OCM) (18, 33). Oocyte maturation was stimulated with 1 μM progesterone in OCM for 12 h. Where indicated, oocytes were injected with MOs after defolliculation and fertilized using the host transfer technique (18). Fertilized eggs (showing a perivitelline space and no germinal vesicle spot) were selected and imaged under a Zeiss SteREO Discovery V20 microscope every minute until four-cell stage. To monitor a possible cleavage delay in experimental embryos, 10 host embryos were used as reference, and their average time point of first cleavage was set to 1. In Fig. 4 A–C, fertilized eggs were treated with 60 mM LiCl or mock treated (NaCl) for 1 h right after dejellying. In Fig. 2E, Middle and Right, oocytes were injected with preprolactin (PPL, as negative control), axin or dominant negative wnt11 (dnwnt11) mRNA and maturated with 1 μM progesterone in OCM for 12 h. In Fig. 4 D and E, one-cell-stage embryos were injected with recombinant dominant negative Ube2c protein (DN-Ube2C) (35), WT Ube2c, or buffer.

For Western blot analysis, whole Xenopus embryos, eggs, or oocytes were homogenized in Triton lysis buffer [1% Triton X-100, 50 mM Tris⋅HCl, pH 7.0, 150 mM NaCl, 10 mM NaF, 5 mM Na₃VO₄, 1 mM PMSF, 20 mM N-ethylmaleimide, and protease inhibitor mixture tablet (Roche)] at one embryo or oocyte per 10 μL, cleared with FREON followed by centrifugation (17,000 × g, 3 min at 4 °C), heated at 95 °C for 3 min with SDS loading buffer, and analyzed by SDS/PAGE. Eggs (Fig. 1 D and E) were treated with 5 μM A23187 in the presence of 100 µg/mL cycloheximide (Sigma) in combination with LiCl or NaCl (0.24 M unless otherwise indicated).

In Fig. 1E, for analysis of β-catenin polyubiquitination, eggs were lysed in lysis buffer II [30 mM Tris, pH 7.5, 150 mM NaCl, 10 mM NaF, 2 mM EDTA, 1 mM β-mercaptoethanol, 1× protease inhibitor mixture tablet (Roche), 20 μM MG-132, 10 mM N-ethylmaleimide, 0.8% Nonidet P-40, 6 M Urea] for 15 min at 4 °C. Lysates were cleared and diluted sixfold in IP Buffer [20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM NaF, 1× protease inhibitor mixture tablet (Roche), 25 μM MG-132, 10 mM N-ethylmaleimide, and 0.8% Nonidet P-40]. Proteins were immunoprecipitated using β-catenin antibody followed by Western blotting using anti-K48–linked ubiquitin antibody. Ubiquitination and proteasomal degradation in Xenopus oocytes have been previously reported (43).

In Fig. 1F, crude metaphase II and interphase egg extracts were prepared essentially as described (44). Briefly, Xenopus eggs were collected overnight in 1× Marc´s Modified Ringer´s solution (MMR, 5 mM Hepes, pH 7.5, 100 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 0.1 mM EGTA), dejellied in 2% cysteine solution [2% (wt/vol) l-Cysteine, 100 mM KCl, 1 mM MgCl₂, 100 μM CaCl₂, pH 7.8], washed extensively with XB buffer (100 mM KCl, 1 mM MgCl₂, 100 μM CaCl₂, 50 mM sucrose, and 10 mM Hepes, pH 7.5). To maintain eggs in metaphase II, XB buffer was supplemented with 5 mM EGTA and 1 mM MgCl₂. To release eggs into interphase, they were activated with A23187 Ca²⁺ ionophore (2 μg/mL, 5 min) before the washing steps. Washed eggs were packed [SW41 Ti rotor (Beckman), 2,000 rpm, 2 min, 16 °C] and then crushed [SW41 Ti rotor (Beckman), 11,000 rpm, 20 min at 4 °C]. The crude cytoplasm beneath the lipid plug was collected with an 18G needle, supplemented with protease inhibitors (Roche), 1 μg/mL cytochalasin D, 1 mM ATP, and 1 mM MgCl₂.

For in vitro β-catenin degradation (Fig. 1F, Bottom), interphase or metaphase II crude extracts were supplemented with 0.5 mg/mL recombinant ubiquitin (BostonBiochem), 100 μg/mL cycloheximide, in vitro-translated ³⁵S-Methionine–labeled β-catenin [Promega TNT(R) Coupled Reticulocyte Lysate kit; 1:10 dilution in extract], and 25 mM LiCl or NaCl (mock treatment). Destabilization of in vitro-translated ³⁵S-β-catenin in the metaphase II extract and the interphase extract was monitored at 0 h (input) and 3 h, in the presence (LiCl) or absence (NaCl) of Gsk3 inhibitor.

For Gsk3β quantification (Fig. 1F, Top), extracts were diluted 5× in their respective extraction buffers and centrifuged at 200,000 × g (Beckman, TLA200.2 rotor) for 2 h. Top cytosolic fraction was collected for Western blot analysis. Light and heavy membrane fractions, sandwiched between the cytoplasm and the glycogen pellet, were collected and passed through a sucrose cushion (extraction buffer containing 250 mM sucrose at 4 °C) to obtain the vesicular fraction and collected for Western blot analysis. Gsk3β protein levels in the cytosolic and vesicular fractions were quantified in triplicate in an LAS-3000 (Fujifilm).

Protein Array.

Eggs were collected overnight in 1× MMR solution. High quality eggs were pooled and washed in 0.5× MMR and dejellied in 2% cysteine solution (2% wt/vol l-Cysteine HCl in H₂O, pH 7.8). Eggs were washed once with 0.5× MMR followed by several washes in Lysis Buffer (LB, 10 mM Hepes, pH 7.7, 50 mM KCl, 2.5 mM MgCl₂, 50 mM sucrose, 50 μg/mL cycloheximide and 1 mM DTT). Washed eggs were first packed in the presence of 1 μg/mL cytochalasin D and protease inhibitors [SW41 Ti rotor (Beckman), 2,000 rpm, 2 min, 16 °C], followed by a crushing step [SW41 Ti rotor (Beckman), 11,000 rpm, 20 min at 16 °C to 4 °C]. Crude extracts were cooled on ice and supplemented with protease inhibitors, 1 μg/mL cytochalasin D, 100 μg/mL cycloheximide and an energy-regenerating mix giving 1 mM ATP, 1 mM MgCl₂, 7.5 mM creatine phosphate, and 30 μg/mL creatine phosphokinase. To obtain high-speed cytoplasm, crude extract was subjected to 2 h centrifugation at 200,000 × g, 4 °C. High speed extracts were further supplemented with 150 μM MG132, 1 μM ubiquitin-aldehyde (Santa Cruz), and 0.5 mg/mL ubiquitin. Extract dilution was roughly 75% and contained ∼11 mg/mL protein. Interphase was assessed by sperm nuclei swelling in crude extracts.

ProtoArray Human protein microarrays (ProtoArray 5.0 platform; Invitrogen) containing more than 9,000 proteins, were handled essentially as described (22). Briefly, the arrays were washed in TBST and blocked for 4 h at 4 °C using Microarray Blocking solution (Arrayit), followed by a wash in lysis buffer. Extracts (0.6 mL) were prewarmed to room temperature, treated with 120 mM LiCl or NaCl (Control), and incubated on the microarrays under coverslips at room temperature for 1 h. Microarrays were washed with TBST and incubated overnight at 4 °C with anti-polyubiquitin antibody FK-1 (4 μg/mL) (ENZO Life Sciences) diluted in TBST, washed again, and incubated with Alexa Fluor 680 anti-mouse antibody (Invitrogen) for 4 h. The microarrays were washed three times in TBST and two times in water, dried, and scanned in an Odyssey scanner (LI-COR) using the following parameters: channel 700; grid = 8 × 3, 21.17; intensity = 5; off-set = 0. ProtoArrays were analyzed using GenePix Pro (version 5.0.0.49; Molecular Devices), preprocessed following the manufacturer’s recommendations, and processed using a noise floor of 250 RFU and a cutoff of 512 relative fluorescence units. “Polyubiquitination hits” were defined as proteins whose anti-polyubiquitin immunofluorescence signal intensity showed a Z-factor above 0.2, replicate spot coefficient of variation (CV) below 0.5, and mean signal above the background noise floor. Retained for analysis were 3,873 proteins (Dataset S1). Candidates in which Gsk3 inhibition by LiCl reduced polyubiquitination in more than 1.8-fold (864) were further analyzed using STRING (string-db.org/) for known or inferred protein–protein interactions. The most prominent cluster, formed by mitotic effectors (blue lines), was depicted together with the fold change in polyubiquitination of its members, and indicated as a heat map.

Statistics.

For Western blots and embryonic cell cleavages, experiments were replicated at least three times, and representative images are shown. Figs. 3E and 4 C and E were analyzed with a Fisher’s exact test. Protein arrays in Fig. 2B were analyzed as described in Protein Array. Differences were considered to be statistically significant for P values of <0.05 (*), <0.01 (**), or <0.005 (***).