Fig. 2

Phospho-mutations of S815 regulate photoreceptor OS localization of Kif17. a 5 dpf larvae previously injected at the one cell stage with one of three different transgenic constructs under control of the TaCP promoter for expression in cone photoreceptors: Kif17-GFP (left), phospho-mimetic Kif17(S815D)-GFP (middle), and phospho-deficient Kif17(S815A)-GFP (right) were stained with Hoechst (blue) to label nuclei and blue cone opsin (red) to label cone OS (COS). A z-series of confocal images was taken with 0.2 μm steps through the depth of the photoreceptor OS. Shown are montages of sequential images in the z-series revealing that Kif17(S815D)-GFP exhibits significant accumulation in the OS while Kif17(S815A)-GFP accumulates at the base of the OS. Note that in cone photoreceptors, the ciliary axoneme extends along the side of the opsin-containing discs. Scale bar is 1 μm. b Models depicting structure of the photoreceptor OS imaged in Fig. 2a. There appears to be two subsets of GFP localization: GFP localizing along the length of the OS (top) and GFP accumulating at the base of the OS (bottom). c Quantification of the frequency of OS accumulation of Kif17-GFP (n = 5 total injected larvae: 3 stained for green opsin, 2 stained for blue opsin; 60 cells), Kif17(S815D)-GFP (n = 7: 4 green, 3 blue; 62 cells), and Kif17(S815A)-GFP (n = 7: 4 green, 3 blue; 126 cells) when co-labeled with either green opsin (square) or blue opsin (circle). d Line intensity analysis of the GFP signal for Kif17-GFP (pink line, n = 5 total injected larvae: 3 stained for green opsin, 2 stained for blue opsin; 17 cells), Kif17(S815D)-GFP (purple line, n = 5: 3 green, 2 blue; 17 cells), and Kif17(S815A)-GFP (red line, n = 5: 3 green, 2 blue; 25 cells). Two-way ANOVA was performed to determine significance in the different interaction between GFP signal intensity pattern and transgene (p < 0.0001, ****)