Figure 4.
N-Rok::vhhGFP4Viand N-Rok::dGBP1 are optimized variants of synthetic N-Rok. (a) Panels show lateral views of fixed sqh_Sqh::GFP embryos at stage 13–14 (dorsal closure) expressing N-Rok::vhhGFP4Vi in the engrailed domain (visualized by co-expression of mCherry-nls). Embryos were stained with anti-phospho-Sqh/MRLC antibody. The panel on the right shows the magnification of the respective Sqh::GFP and p-Sqh images for each of the presented embryos. White arrows point to the actomyosin cable around the dorsal hole, yellow arrows point to Sqh::GFP and p-Sqh foci. (b) Dorsal closure was used to compare the functionality of N-Rok::vhhGFP4Vi to N-Rok::vhhGFP4ZH-86Fb (Fig. 3 a). Panels show stills from live-imaging with dorsal views of the developing sqh_Sqh::GFP embryos at stages 13/14–16 (dorsal closure) expressing N-Rok::vhhGFP4Vi in the engrailed domain (visualized by co-expression of mCherry-nls). Please note that both in a and b the “control” images are the same as on Figs. 2 d and 3 a, respectively, to have a single reference image for all N-Rok variants used. (c) Schematic illustration of the synthetic kinase with destabilized GFP nanobody (dGBP1) work concept. In a similar way as in Fig. 1 a, a synthetic kinase uses GFP to bring a constitutively active kinase domain (Rok KD) in close proximity to a fluorescent fusion substrate protein (target). The persistence of the kinase domain around the fluorescent fusion protein achieves efficient phosphorylation (P) of the target. In the absence of the GFP-target, dGBP1 nanobody is destabilized, and the whole nanobody-kinase fusion protein is targeted for degradation. (d) Panels show lateral views of stage 13–14 (dorsal closure) fixed sqh_Sqh::GFP or control yw embryos expressing N-Rok::dGBP1ZH-86Fb in the engrailed domain (visualized by co-expression of mCherry-nls). Embryos were stained with an anti-phospho-Sqh/MRLC antibody. The panel on the right shows the magnification of the respective Sqh::GFP and p-Sqh images. Yellow arrows point to Sqh::GFP and p-Sqh foci. (e) Dorsal closure was used to compare the functionality of N-Rok::dGBP1ZH-86Fb to N-Rok::vhhGFP4ZH-86Fb (Fig. 3 a) and N-Rok::vhhGFP4Vi (b). Panels show stills from live-imaging with dorsal views of the developing sqh_Sqh::GFP or control yw embryos expressing N-Rok::dGBP1 in the engrailed domain (visualized by co-expression of mCherry-nls). For every embryo genotype, the number of analyzed embryos is indicated (n). For N-Rok::dGBP1ZH-86Fb the n number is given as a proportion of embryos in which clear p-Sqh clumps corresponding to Sqh::GFP foci were visible to the total number of embryos included in the analysis. Scale bar, 50 μm in b and e; 20 μm in a and d; in the zoomed panel, 10 μm.

N-Rok::vhhGFP4 Vi and N-Rok::dGBP1 are optimized variants of synthetic N-Rok. (a) Panels show lateral views of fixed sqh_Sqh::GFP embryos at stage 13–14 (dorsal closure) expressing N-Rok::vhhGFP4Vi in the engrailed domain (visualized by co-expression of mCherry-nls). Embryos were stained with anti-phospho-Sqh/MRLC antibody. The panel on the right shows the magnification of the respective Sqh::GFP and p-Sqh images for each of the presented embryos. White arrows point to the actomyosin cable around the dorsal hole, yellow arrows point to Sqh::GFP and p-Sqh foci. (b) Dorsal closure was used to compare the functionality of N-Rok::vhhGFP4Vi to N-Rok::vhhGFP4ZH-86Fb (Fig. 3 a). Panels show stills from live-imaging with dorsal views of the developing sqh_Sqh::GFP embryos at stages 13/14–16 (dorsal closure) expressing N-Rok::vhhGFP4Vi in the engrailed domain (visualized by co-expression of mCherry-nls). Please note that both in a and b the “control” images are the same as on Figs. 2 d and 3 a, respectively, to have a single reference image for all N-Rok variants used. (c) Schematic illustration of the synthetic kinase with destabilized GFP nanobody (dGBP1) work concept. In a similar way as in Fig. 1 a, a synthetic kinase uses GFP to bring a constitutively active kinase domain (Rok KD) in close proximity to a fluorescent fusion substrate protein (target). The persistence of the kinase domain around the fluorescent fusion protein achieves efficient phosphorylation (P) of the target. In the absence of the GFP-target, dGBP1 nanobody is destabilized, and the whole nanobody-kinase fusion protein is targeted for degradation. (d) Panels show lateral views of stage 13–14 (dorsal closure) fixed sqh_Sqh::GFP or control yw embryos expressing N-Rok::dGBP1ZH-86Fb in the engrailed domain (visualized by co-expression of mCherry-nls). Embryos were stained with an anti-phospho-Sqh/MRLC antibody. The panel on the right shows the magnification of the respective Sqh::GFP and p-Sqh images. Yellow arrows point to Sqh::GFP and p-Sqh foci. (e) Dorsal closure was used to compare the functionality of N-Rok::dGBP1ZH-86Fb to N-Rok::vhhGFP4ZH-86Fb (Fig. 3 a) and N-Rok::vhhGFP4Vi (b). Panels show stills from live-imaging with dorsal views of the developing sqh_Sqh::GFP or control yw embryos expressing N-Rok::dGBP1 in the engrailed domain (visualized by co-expression of mCherry-nls). For every embryo genotype, the number of analyzed embryos is indicated (n). For N-Rok::dGBP1ZH-86Fb the n number is given as a proportion of embryos in which clear p-Sqh clumps corresponding to Sqh::GFP foci were visible to the total number of embryos included in the analysis. Scale bar, 50 μm in b and e; 20 μm in a and d; in the zoomed panel, 10 μm.

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