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Figure 1
Mapping of the Vasa-binding motif in Buc (Buc-VBM). (A–C) Illustration of the BiFC assay. (A) Venus protein (yellow) is split into two nonfluorescent parts, Venus N-terminus (VN; rose) and Venus C-terminus (VC; blue) (B) Fusion of zfVasa (grey) and Buc (green) with VN and VC fragments, respectively. (C) Interaction of zfVasa and Buc (horizontal red lines) reconstitute a functional Venus protein forming a bimolecular fluorescent complex. (D) Schematic illustration of the systematic truncation of Buc (green). Numbers left to the colored bars indicate the corresponding amino acids. Interactions are marked by a ‘+’, while no interactions as ‘-’. Buc-VBM is indicated in pink. (E) Quantification of the fluorescent-positive embryos based on different combinations of the BiFC Buc and zfVasa constructs. The data presented are averaged from three independent experiments. The Y-axis represents a percentage of the average fluorescent embryos, and the X-axis represents injected constructs. (G–N) Confocal images of live embryos at 3 hpf (hours post-fertilization) after the injection of Buc constructs with wild-type zfVasa. The imaging area is boxed in red, as indicated in the cartoon on the left (F). This region is outlined with a white dashed line (G–N). The injection of wild-type Buc showed a fluorescent signal (G; 82 ± 3.9%, n = 58). After splitting Buc into Buc-NTD (amino acids 1–362) and Buc-CTD (amino acids 363–639), only the Buc-CTD show fluorescence (I; 77 ± 5.7%, n = 79), and there is no florescence with Buc-NTD (H; 0 ± 0%, n = 105). Splitting Buc-CTD into two parts, the construct containing the amino acids 363–400 showed fluorescent embryos (J; 72 ± 4.1%, n = 79) but not the amino acids 401–639 (K; 0 ± 0%, n = 77). Interestingly, the highly conserved domain of Buc (amino acids 372–394) displayed fluorescence (L; 66 ± 2.8%, n = 69). No fluorescent signal was observed after deleting amino acids 372–394 in full-length Buc (BucΔ (372–394) (M; 0 ± 0%, n = 71) and BucΔ (363–400) (N; 0 ± 0%, n = 64). (O) Multiple sequence alignment revealed that amino acids 372–394 (labeled on the top of the sequence) are highly conserved in the Buc homolog in Xenopus and the Xvelo and Buc homologs in chickens, in addition to Buc orthologs in other teleost species. Amino acids are colored based on the ClustalX color code. Hydrophobic amino acids are (Alanine (A), Isoleucine (I), Leucine (L), Methionine (M), Phenylalanine (F), Tryptophan (W), and Valine (V)) colored in blue. Positively charge amino acids ((Lysine (K) and Arginine (R)) are colored in red. Negatively charge amino acids ((Aspartic acid (D) and Glutamic acid (E)) are colored in magenta. Polar amino acids ((Asparagine (N), Glutamine (Q), Serine (S), and Threonine (T)) are colored in green. Aromatic amino acids ((Histidine (H) and Tyrosine (Y)) are colored in cyan. Cystine is colored in pink. Glycine is colored in orange. Proline is colored in yellow. Protein sequences for Buc orthologs were retrieved from the Ensembl database (Ensembl Release 104), except the Buc homolog in Xenopus and the Xvelo and Buc homologs in chickens, which were retrieved from Xenobase Version 5.3.0 and the NCBI protein database, respectively [38,39]. The protein sequences used for multiple sequence alignment were: ENSDARP00000125536; Zebrafish (Danio rerio), ENSLOCP00000015864; Spotted gar (Lepisosteus oculatus), ENSELUP00000012527; Northern pike (Esox luciusI), ENSSFOP00015022504; Asian bonytongue (Scleropages formosus), ENSIPUP00000003130; Channel catfish (Ictalurus punctatus), ENSAMXP00000053358; Mexican tetra (Astyanax mexicanus-2), ENSPKIP00000037210; Paramormyrops (Paramormyrops kingsleyae ),ENSPNAP00000033609; Red-bellied piranha (Pygocentrus nattereri),ENSHHUP00000031919; Huchen (Hucho hucho), ENSAMXP00005041590; Pachon cavefish (Astyanax mexicanus-1), ENSDCDP00000002250; Denticle herring (Denticeps clupeoides), ENSCHAP00000009659; Atlantic herring (Clupea harengus), ENSECRP00000007749; Reedfish (Erpetoichthys calabaricus), XB-GENE-5934753; Xenopus (Xenopus laevis), {"type":"entrez-protein","attrs":{"text":"XP_040546153.1","term_id":"2024370216","term_text":"XP_040546153.1"}}XP_040546153.1; and chicken (Gallus gallus). Test statistics: Student’s t-test, **** = 0.0001. ns. = nonsignificant. Error bars represent the standard deviation of the mean. Scale bar 100 µm.