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Figure 1.Wnt/STOP operates in Xenopus oocytes and eggs in metaphase II. (A) Scheme of proposed Wnt signaling and protein polyubiquitination state in Xenopus oocytes and eggs (ovulated mature oocytes) as revealed in this study. Stage VI oocytes are arrested in prophase I (Pro. I). Progesterone (Prog.) matured oocytes and eggs are arrested in metaphase II (Meta. II). Fertilized or Ca2+ ionophore-released eggs progress to interphase. (B) Western blots of indicated proteins from Xenopus stage VI and progesterone-matured oocytes. alpha-tub, alpha-tubulin (loading control); beta-cat, beta-catenin; pH3, phosphorylated histone 3; pLrp6, phosphorylated Lrp6; PolyUb, polyubiquitination. (C) Western blots of indicated proteins from Xenopus matured oocytes injected with the indicated antisense Morpholinos (MO). alpha-tub, alpha-tubulin (loading control); beta-cat, beta-catenin; Co, control; cycY, cyclin Y; pH3, phosphorylated histone 3; pLrp6, phosphorylated Lrp6; PolyUb, polyubiquitination. (D and E) Western blots of indicated proteins from Xenopus eggs (naturally arrested in metaphase II) and treated with the calcium ionophore A23187 for the indicated times (minutes; D), or 25 min (E), in the presence of LiCl or NaCl (Control). (E) Analysis of the ubiquitination state of the β-catenin via immunoprecipitation (IP) followed by Western blotting (IB). α-tub, α-tubulin (loading control); β-cat, β-catenin; pH3, phosphorylated histone 3; pLrp6, phosphorylated Lrp6; PolyUb, polyubiquitination. (F) Gsk3 localization and activity in metaphase II and interphase eggs. (Top) Vesicular and cytoplasmic fractions were obtained from egg extracts, as described in Methods. Gsk3beta protein levels were determined by Western blot, and the ratio (vesicle/cytoplasm) is indicated. (Bottom) In vitro-translated 35S-beta-catenin was added to metaphase II extract and interphase extract at 0 h (input), and its degradation was monitored after 3 h, in the presence (LiCl) or absence (NaCl) of Gsk3 inhibitor. Note that higher vesicle-associated Gsk3 levels in metaphase II extracts are accompanied by greater beta-catenin stability.
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Figure 2. Gsk3 negatively regulates mitotic effectors in Xenopus extracts. (A) Scheme of the protein microarray experiment to assess Gsk3-dependent polyubiquitination. Interphase egg extracts were supplemented with ubiquitin, pretreated with LiCl or NaCl (Control), and then applied on protein arrays containing ∼9,000 proteins. Polyubiquitination of arrayed proteins (PolyUb) was monitored using anti-polyubiquitin antibodies and immunofluorescence detection using an array scanner. Signal intensities were transformed and are displayed as heat-map. (B) Distribution plot of polyubiquitination changes of 3,873 in vitro polyubiquitinated proteins after Gsk3 inhibition by LiCl (Log2[Control/LiCl]). Arrays were treated with 30 mM LiCl or 30 mM NaCl (Control). LiCl reduced polyubiquitination of 864 proteins >1.8-fold. Some candidates, associated with cell cycle progression and selected for further validation are indicated in the plot. (C) Cluster of mitotic associated proteins that are significantly regulated by LiCl in B. The functional network was determined using String 9.1 and color-coded based on the fold change in the microarray (Dataset S1). (D) Western blots of indicated proteins from Xenopus eggs (naturally arrested in metaphase II) and released into interphase with the Ca2+ ionophore A23187 for 25 min, treated with LiCl, or mock treated (NaCl). β-cat, β-catenin; cycE, cyclin E; Interph., interphase; M. II, metaphase II; pH3, phosphorylated histone 3. (E) Western blots of stage VI oocytes in Prophase I and Progesterone-matured (Prog.) oocytes in metaphase II. Oocytes were injected (Inj.) with Morpholinos (MO) or mRNA as indicated. Co, control; dnWnt11, dominant negative Wnt11; pH3, phosphorylated histone 3; PPL, preprolactin (negative control).
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Figure 3. Inhibiting Wnt/STOP induces cleavage arrest in Xenopus embryos. (A–F) Cell cycle progression in early Xenopus embryos upon maternal depletion of Wnt components. Co, control; cycY, cyclin gamma; beta-cat, B-catenin. (A) Experimental scheme of the Host Transfer experiment. Xenopus oocytes were Morpholino (MO) depleted as indicated, matured by progesterone and foster-mother transfer, and fertilized in vitro. Prog., progesterone. (B) Western blots of mature oocytes injected with the indicated Morpholinos. a-tub, alpha-tubulin (loading control); pH3, phosphorylated histone 3; pLrp6, phosphorylated Lrp6. (C) Proportion of the indicated morphant eggs recovered and fertilized after host transfer in D, respectively. n, number of initial mature oocytes. (D) Cell cycle progression in early Xenopus embryos upon maternal depletion of Wnt components. Fertilized eggs were filmed until four-cell stage, and the last frame is shown. Embryos arrested before the first or the second cleavage are marked with one or two asterisks, respectively. Eggs were labeled with a neutral color additive for identification before transfer in a foster mother. (E) Quantification of cleaving morphants from D. n, number of embryos. (F) Time required for the indicated morphants to reach the first cleavage. Note that many lrp6 and cycY morphants failed to cleave; thus, only the escapers were measured. n, number of embryos; n.s., not significant. (G) Control (Co) and beta-catenin (B-cat) morphants at tadpole stage upon maternal depletion.
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Figure 4. Inhibiting Wnt/STOP induces cleavage arrest through Gsk3 in Xenopus embryos. (A–C) Cleavage arrest in early Xenopus embryos depleted of Cyclin Y (cycY) can be rescued by Gsk3 inhibition. (A) Oocytes, eggs, and embryos were handled as described in Fig. 3D and treated with 60 mM LiCl to inhibit Gsk3 or control treated (NaCl) after fertilization. Fertilized eggs were filmed until four-cell stage, and the last frame is shown. (B) Fraction of the indicated morphant eggs recovered and fertilized after host transfer in A. n, number of initial mature oocytes. (C) Quantification of cleaving morphants from A. n, number of embryos. (D) Dominant negative Ube2c arrests cell cycle progression in early Xenopus embryos. Embryos were injected at one-cell stage with the indicated recombinant proteins and recorded at the four-cell stage. Embryos arrested before the first cleavage are marked with an asterisk. (E) Quantification of cleaving embryos from D. DN, DN-Ube2c; n, number of embryos; WT, Ube2c. (F) Wnt/beta-catenin and Wnt/STOP signaling in Xenopus.
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Fig. S1. Validation of Wnt/STOP targets in Xenopus. Quantification of the Western blots from Fig. 2D (A) and Fig. 2E (B). Protein levels were normalized to
the loading control (ERK).
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