Overexpressed Diversin localizes to the centrosome in Xenopus ectoderm. (A) Experimental scheme. (B-E) Two animal blastomeres of eight-cell embryos were injected with 0.5 ng of Diversin-RFP (Div-RFP; B-D) or HA-Diversin (HA-Div; E) RNA alone or with 0.15 ng of CAAX-GFP RNA. At stage 10, the injected embryos were fixed for immunostaining on cryosections or used to prepare ectodermal explants. (B,C) In ectodermal explants, Div-RFP was distributed as a single dot in interphase cells (B) or two dots in mitotic cells (C). (D,E) In cryosections, Div-RFP was distributed in ectodermal cells at early gastrula stage as one or two dots per cell and colocalized with γ-tubulin (E). (D) CAAX-GFP stained with anti-GFP antibodies marks the cell membrane. RFP and DAPI co-staining is shown (B,C) or a merged image (D″). Metaphase plates (C,D″) or centrosomes (E) are indicated by arrowheads. Scale bars: 20 μm. Scale bar in B also applies to C.

Non-uniform membrane recruitment of Diversin by Fz. (A-C) Animal cap explants were prepared from stage-10 embryos injected as described in Fig. 1A with the following RNAs: Div-RFP (0.5 ng), Fz8 (1 ng), Stbm-CFP (0.5 ng), CAAX-GFP (0.1 ng) and Fz8-GFP (1 ng). Diversin was recruited to the cell membrane in a non-uniform manner by Fz8 (A,C; arrowheads), but not by Stbm (B). The distribution of Fz8-GFP differed from the localization of Diversin (C). Note the uniform membrane localization of CAAX-GFP, Stbm-CFP and Fz8-GFP (A-C). Scale bar: 20 μm.

Different Diversin domains play distinct roles in Diversin subcellular localization. (A) Diversin constructs and summary of the Fz8 recruitment data. Ank, ankyrin repeats (grey bar); M, middle region; C, C-terminal region; cc, coiled-coil motif. (B) Western analysis of stage-10.5 embryo lysates demonstrates similar expression levels of Diversin constructs. Embryos were injected with the following RNAs: HA-Div-RFP (0.5 ng), HA-ΔM-RFP (0.25 ng), HA-ΔAnk-RFP (0.25 ng) and HA-ΔC-RFP (2 ng). β-tubulin (βTub) controls loading. Molecular weight markers are indicated. Uninj., un-injected. (C-J) Animal cap explants from embryos injected with Diversin RNAs as in B (C-F) or co-injected with Fz8 RNA (1 ng; G-J). In the absence of Fz8, ΔAnk is present in many puncta (D), ΔM in nucleoplasm and nuclear puncta (E), and ΔC in the nucleus (F). Upon coexpression of Fz8, ΔC and full-length Diversin were recruited to the cell cortex (G,J), whereas ΔAnk and ΔM were not (H,I). Note the uniform membrane localization of ΔC (J). Scale bar: 20 μm.

Centrosomal localization of Diversin was disrupted by the C-terminal construct. (A) Scheme showing the C-terminal construct of Diversin (Div-C). Ank, ankyrin repeats (grey bar); M, middle region; C, C-terminal region; cc, coiled-coil motif. (B-D) Cryosections from embryos injected with 0.5 ng HA-Diversin RNA and/or 1 ng RFP-Div-C RNA. Staining with anti-HA (B,C) and anti-Diversin (D) antibodies revealed the disruption of the centrosomal localization of HA-Diversin by Div-C. Scale bar: 20 μm.

Diversin is translocated into cortical puncta in response to Wnt proteins. Embryos were co-injected with Diversin-RFP RNA (0.5 ng) and either Wnt3a or Wnt5a RNA (1 ng each) as described in Fig. 1A. Cryosections were co-stained with anti-γ-tubulin. Diversin-RFP overlaps, but does not completely colocalize with γ-tubulin, a centrosome marker (A-A″). Upon coexpression with Wnt3a (B) or Wnt5a (C), Diversin fluorescence is redistributed to cytoplasmic and cortical puncta (arrowheads in B,B″,C,C″), whereas γ-tubulin staining is not affected. DAPI co-staining is shown in merged images on the right. Scale bar: 20 μm.

Colocalization of Diversin and Dsh. (A,B) Animal cap explants were prepared from stage-10 embryos injected with the following RNAs: Div-RFP (0.5 ng), Dsh-GFP (0.15 ng) and Fz8 (1 ng). Diversin colocalized with Dsh to cytoplasmic puncta (A, arrowheads), whereas Fz8 caused a non-uniform distribution of both Diversin and Dsh on the cell membrane (B). Scale bar: 20 μm.

Diversin constructs with a defect in centrosomal localization are unable to promote β-catenin degradation. (A) HA-Diversin-RFP constructs with deletions in the middle and in the C-terminal region. (B-D) Cryosections prepared from embryos injected with the following RNA: HA-Diversin-RFP (0.3 ng), HA-ΔM4 (0.15 ng) or HA-ΔC11 (2 ng). Sections were immunostained with anti-γ-tubulin antibodies. ΔM4 and ΔC11 reveal mostly nuclear and peri-membrane, rather than centrosomal, localization. (B-D) Diversin colocalized with γ-tubulin (arrowheads). Scale bar: 20 μm. (E) Levels of β-catenin in embryonic animal caps injected with the following RNAs: FLAG–β-catenin (30 pg), Wnt3a (10 pg), HA-Diversin-RFP (0.5 ng), HA-ΔM4 (0.25 ng) and HA-ΔC11 (2 ng). Wnt3a-dependent β-catenin stabilization was inhibited by wild-type Diversin, but not by ΔM4 or ΔC11. Diversin constructs are expressed at similar levels, β-tubulin is a loading control. Uninj., un-injected.

Div-C diminishes Wnt inhibitory activity of the wild-type Diversin. (A) Div-C suppresses the inhibitory effect of wild-type Diversin on β-catenin stabilization. RNAs injected were: Wnt3a (10 pg), HA-Diversin-RFP (0.5 ng), Div-C–RFP (1 ng) and FLAG–β-catenin (30 pg). (A,C) After protein separation and transfer, transfer membranes were cut at approximately the 80-kD band level and separately probed with anti-HA antibody to detect overexpressed wild-type Diversin and with anti-Diversin antibody to detect Div-C (which has no HA tag). β-tubulin is a loading control. (B) Div-C diminishes the inhibitory effects of Diversin on Siamois reporter activation by Wnt3a. Luciferase assay with pSia-Luc DNA (20 pg) upon co-injection of 10 pg of Wnt3a RNA with 0.5 ng of HA-Div-RFP and 1 ng of Div-C-RFP into animal ventral blastomere of eight-cell embryos. Four groups of six embryos were collected at stage 10 for the reporter assay. Means ± s.d. are shown. (C) Diversin and Div-C expression levels in embryos used for B. (D) Model for Diversin function at the centrosome. Centrosomal Diversin promotes β-catenin degradation. Upon Wnt signaling, Diversin is redistributed to punctate structures at the cell cortex and the cytoplasm, resulting in stabilized β-catenin and increased target gene transcription.