Fig. 5. Vasa C-terminus is responsible for localized translation on the spindle.

a A schematic diagram of Vasa deletion or mutation constructs tested in this study. VasaC1-3 series are the constructs mutated for the Vasa-C-terminal regions (see also Supplementary Fig. S7 for detail). b Live imaging of the 8–16-cell embryonic cell injected with each of the Vasa mutants fused to GFP. Vasa’s spindle localization was lost in Vasa-C-terminal mutants (e.g., Vasa-ΔC-term, Vasa-C1-3, and Vasa-C3), yet not in other mutants except for Vasa-7F that lacks all of the conserved motifs important for DEAD-box helicase activities (Supplementary Fig. S7). c Localized translation on the spindle was measured by the OPP signal level in each embryo injected with each of the Vasa mutants along with Vasa morpholino antisense oligo to knock down endogenous Vasa activity. The OPP signal appears on the spindle when the introduced Vasa mutants are functional. A whole Z-section of the cell of interest (1 μm per slice; ~35 slices in total) was imaged, analyzed, and presented as a maximum 2D-projection. The whole embryo images are presented in Supplementary Fig. S7. d Vasa C-terminus deletion or mutation resulted in the reduced signal of localized translation on the spindle. The relative OPP intensity level on the spindle versus in the cytoplasm was obtained as follows: The average signal level of three detection ROIs was obtained per embryo. A total of ~27 embryos were analyzed in this manner per sample group and the average value was presented in the graph. () indicates the total number of ROIs measured for each sample group. All experiments were performed at least three independent times. Adjusted p-value <0.0001. n = 81, 45, 36, 48, 48, 36, 27, 45, 33, 27, 18, 33 from the left-right columns of graph (d). The box plot defined by two box lines indicates the 25th and 75th percentile, respectively, with a centerline at the 50th percentile and minima and maxima whiskers. Two-way ANOVA was used for all statistical analyses in this figure. ****p < 0.0001. Columns represent means ± SD or SEM. All scale bars = 10 μm. Source data are provided as a Source Data file.