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Figure 1. Polarization of Pk3/Vangl2 complexes at the anterior cell cortex.(A) Experimental scheme. RNAs encoding GFP-Pk3 and mouse HA-Vangl2 (150 pg each) were injected into the animal-ventral region of four-cell embryos. Ectoderm was dissected from the ventral midline area of fixed embryos for imaging (red box). The anteroposterior (AP) and dorsoventral (DV) axes are indicated. (B, C) GFP fluorescence of stage 15 ectoderm expressing GFP-Pk3 alone (B) or with HA-Vangl2 (C). Arrows in C point to GFP-Pk3 at the anterior cortex. (D, E) Embryos mosaically-expressing GFP-Pk3 and HA-Vangl2 were fixed at stage 11 (D–D’’) or stage 15 (E–E’’). Staining of GFP and HA is shown as indicated. HA-Vangl2 polarization is poorly detected in some cells due to variation in protein levels. Scale bar, 20 µm. (F) A single cell expressing GFP-Pk3 and HA-Vangl2. Dashed line depicts the cell boundary. Pk3 orientation (white arrow) is defined as perpendicular to the line connecting the ends of the crescent (yellow bar). (G) Rose diagrams show the orientation of GFP-Pk3 crescents in the ventral ectoderm at the indicated stage. n, number of scored cells. (H) Summary of GFP-Pk3 polarization derived from data in (G). Each arrow in the polar plot displays the mean orientation of Pk3 crescents in a single embryo at stage 12.5 (blue) or stage 15 (red). Arrow length is 1 minus the circular variance around the mean. Data are representative of two independent experiments.DOI: http://dx.doi.org/10.7554/eLife.16463.002Figure 1—figure supplement 1. Different domains mediate Pk3 membrane recruitment and its polarization.(A) Domain structure of the full-length (FL) and truncated Pk3 proteins and the summary of their properties in ectodermal cells coexpressing Vangl2. Two properties were assessed: the ability to be recruited to the plasma membrane (PM), and the anterior polarization of Pk3/Vangl2 complexes. +/-, inefficient polarization of the ∆PET construct. (B) Embryos expressing mouse HA-Vangl2 and the indicated GFP-Pk3 proteins were fixed at stage 15 for imaging. Immunostaining of GFP in the epidermal ectoderm is shown. The red box illustrates the approximate position of the samples. Scale bar, 20 µm. (C) Embryos expressing HA-Vangl2 and the indicated GFP-Pk3 constructs were lysed at stage 15. Pk3 protein expression levels were assessed by immunoblotting with anti-GFP antibodies. Ponceau staining reflects protein loading (bottom panel).DOI: http://dx.doi.org/10.7554/eLife.16463.003 Image published in: Chu CW and Sokol SY (2016) © 2016, Chu et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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