Figure 2

Mapping the Buc-binding motif in zfVasa (zfVasa-BBM). (A) Schematic illustration of the systematic truncation of zfVasa (dark grey) with a helicase core containing the N-terminal (blue) and C-terminal (light rose) RecA-like domains. Numbers left to the colored bars indicate the corresponding amino acids. (B) Quantification of fluorescent-positive embryos based on the microinjection of mRNA encoding the different combinations of BiFC Buc and zfVasa constructs. The data presented are averaged from three independent experiments. The Y-axis represents the percentage of fluorescent embryos, and the X-axis shows the injected constructs. Error bars represent the standard deviation of the mean. Average fluorescent-positive embryos ≥ 60% denoted as ‘++’, average fluorescent-positive embryos ≤ 60% shown as ‘+’, and embryos with no fluoresce denoted as ‘-‘. (D–K) Confocal images of live embryos at 3 hpf (hours post-fertilization) after the injection of zfVasa constructs with wild-type Buc. (C) The imaging area is boxed in red, as indicated in the cartoon on the left. This region is outlined with a white dashed line (D–K). Injection of wild-type zfVasa showed a fluorescent signal (D; 77 ± 3.0%, n = 96). After splitting zfVasa, zfVasa-IDR (amino acids 1–277 and labeled in orange on top of (A)) did not show fluorescence (E; 0 ± 0%, n = 43), but zfVasa-HC (amino acids 278–715 and labeled in orange on top of (A)) showed a fluorescent signal (F; 70 ± 4.0%, n = 85). From the three constructs of zfVasa-HC, the construct containing the amino acids 278–495 did not show an interaction signal (G; 0 ± 0%, n = 57). Besides, the construct containing the amino acids 496–623 showed more fluorescent embryos (H; 64 ± 4.0%, n = 76) than the other construct containing amino acids 624–715 (I; 14 ± 5.0%, n = 55). The zfVasa construct containing the amino acids 600–625 isolated a minimum peptide, which interacted with Buc (J; 69 ± 8.2%, n = 64). No fluorescent signal was observed after removing amino acids 600–625 in full-length zfVasa (zfVasaΔ (600–625)) (K; 0 ± 0%, n = 71). (L) SDS-PAGE (15%) stained with Coomassie Brilliant Blue. GST pull-down assay performed with recombinant GST-Buc-VBM (expected molecular weight approximately 32 kDa) and zfVasa (residues 227–670; expected molecular weight approximately 56 kDa). Protein markers (in kDa) are indicated in the middle. Compared to the control input samples (lanes 1–3), the pull-down (lanes 5–7) GST-Buc-VBM brings down zfVasa, suggesting that both fragments directly bind to each other. Scale bar 100 µm. Test statistics: Student’s t-test, **** = 0.0001. ns. = nonsignificant.