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. 2018 Sep 26;7:e37881. doi: 10.7554/eLife.37881

Figure 5. Pk3-interacting fragment of Par3 interferes with neural plate PCP.

Two dorsal blastomeres of 16 cell embryos were injected with RNAs encoding GFP-Pk3 (100 pg), HA-Vangl2 (25 pg) and mCherry (70 pg) without (A–A”) or with Par3[272-544] (0.5 ng) (B–B’’) or Par3[545-756] (0.5 ng) (C–C’’). Cells from embryos at stage 14 (St.14) with anteriorly polarized (arrows) and mislocalized (asterisks) GFP-Pk3 patches are shown. Anteroposterior (AP) axis of the neural plate is indicated. Scale bar, 20 µm. (D) Quantification of data in (A–C) shown as mean frequencies ± s. d. of polarized GFP-Pk3 patches in neuroepithelial cells. Total numbers of scored cells are shown above each bar; 5 to 25 cells were scored per embryo with five embryos taken for each experimental condition, statistical significance was determined by two-tailed Student’s t-test, p<0.001. Data are representative of two experiments. (E) Protein expression levels were assessed in stage 14 embryos by immunoblotting with anti-Myc, anti-GFP and anti-HA antibodies. Control, embryos injected with HA-Vangl2 and GFP-Pk3 RNAs without Par3 constructs, Uninj., uninjected embryos.

Figure 5.

Figure 5—figure supplement 1. Interaction of Pk3 and Par3 in Xenopus embryos is inhibited by Pk3 binding fragment of Par3.

Figure 5—figure supplement 1.

Interaction of Par3 and Pk3 was assessed by proximity biotinylation. Biotin and RNAs encoding FLAG-BL-Pk3, with or without Myc-Par3 RNA, 0.1 ng, and HA-Par3[272-544] or HA-Par3[545-756] were injected into the animal region of four- to-eight-cell embryos. Injected embryos were lysed at stage 13 for immunodetection of biotinylated proteins. Levels of Par3 biotinylation were assessed in Myc pulldowns. Biotinylation of HA-Par3[272-544] and HA[545-756] constructs was compared in embryo lysates. Only HA-Par3[272-544] was biotinylated. Protein levels are shown by immunoblotting with anti-biotin, anti-Myc and anti-HA antibodies.
Figure 5—figure supplement 2. Par3[272-544] construct does not affect Par3 localization.

Figure 5—figure supplement 2.

(A–C) Four animal blastomeres of 16 cell embryos were injected with Myc-Par3 RNA, 70 pg with or without 0.5 ng of HA-Par3[272-544] RNA. Injected embryos were cultured until stage 11, cryosectioned and immunostained for Myc (A, B). (A, B) Myc-Par3 with is enriched at the apical membrane. Scale bar, 10 µm. (C) Quantification of data in (A, C) shown as mean frequencies ± s. d. of superficial ectoderm cells with apically enriched Myc-Par3. Total numbers of scored cells are shown above each bar; 13 to 26 cells were scored per embryo with three embryos taken for each experimental condition, statistical difference determined by two-tailed Student’s t-test was not significant, p>0.05. Data are representative of two experiments.
Figure 5—figure supplement 3. The ability of Par3 to inhibit blastopore closure and body axis elongation is lost upon the disruption of Pk3 binding.

Figure 5—figure supplement 3.

Four-cell stage embryos were injected with Par3 constructs into the marginal zone of two dorsal blastomeres. (A–C) Vegetal views of stage 12 (St. 12) embryos injected with Par3∆∆ RNA (A, 0.5 ng), and wild type Par3 RNA (B, C, 0.5 ng). (D) Frequencies of mild (B) and severe (C) blastopore defects. (E) Representative images of stage 26 (St. 26) embryos injected with Par3 (0.5 ng) and Par3∆∆ (0.5 ng) RNAs. Par3-expressing embryos were shortened (top left) and often exhibited protruding endoderm due to incomplete blastopore closure (arrowhead). By contrast, Par3∆∆-injected embryos were similar to uninjected embryos. (F) Average length of embryos ± s. d. is shown. (G) Comparison of Par3 protein levels in injected stage 12 embryos by immunoblotting with anti-Myc antibodies. Each sample has been pooled from five embryos. Each lane was loaded with lysate amount equivalent to half-embryo.