Xenbase Image ID: 76378

Figure 5. Rspo3 Requires Clathrin-Mediated Endocytosis to Induce Phospho-JNK (A) Confocal microscopy of dissociated animal cap cells treated with hRspo3DC-SNAP549 protein for 1 hr (orange, arrowheads in CoMo). 4-cell stage embryos were injected animally with membrane-bound Venus mRNA (green) and indicated Morpholinos (Mo) (20 ng of Sdc4; 20 ng of Fz7; 5 ng of Wnt5a). The average number of vesicles per cell is indicated in the graph. Data with standard error of the mean are shown. (B) Confocal microscopy of Dvl-GFP (green) in stage 8 Xenopus animal caps. 4-cell stage embryos were injected animally with indicated Mo and/or mRNAs together with Dvl-GFP mRNA. (C and D) Confocal microscopy of animal cap (AC) cells from embryos injected at 4-cell stage with indicated Mo or mRNA (C) or treated with endocytosis inhibitors (D) as described in Experimental Procedures. Dissociated AC cells, embryos were injected animally with membrane-bound Venus mRNA (green); ACs were explanted and dissociated at stage 8, incubated for 1 hr with hRspo3DC-SNAP549 (red), fixed, and analyzed. AC tissue, whole stage 8 ACs were treated either with Protein A (top two panels) or recombinant hRspo3DC-streptag-PrA2 (Rspo3 treatment) and immunostained with anti-pJNK antibody, as described in Experimental Procedures. Cells and tissues were counter-stained with Hoechst (blue). Note that clathrin inhibitorreated cells (AP2m2 Mo, MDC) bind and cluster Rspo3 at the surface but fail to internalize the protein and to induce phospho-JNK. Larger Image Printer Friendly View

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