Fig. 8. The F-actin- and the microtubule-binding domains of Shot proteins redundantly promote lumen formation in fusion cells. These cells occupy anastomosis sites in the dorsal (arrows) and lateral (concave arrows) trunks and at the dorsal midline (long arrows). (A-D) Rescue activity of Shot L(A)-GFP and derivatives in stage 16 shot3 mutant embryos. Shot L(A)-GFP (schematic in A) contains an N-terminal F-actin binding domain (orange), central plakin-like (yellow) and spectrin repeat domains (black), and a C-terminal domain that binds microtubules via the GAS2 motif (blue). The gray X is used to indicate domains missing in Shot L(A)-GFP derivatives (schematics in B-D). (A) Lateral view. Tracheal expression of Shot L(A)-GFP restores lumen formation in fusion cells in the dorsal and lateral trunks. (B) Dorsal view. Tracheal expression of actin-binding defective Shot L(C)-GFP restores lumen formation in fusion cells in the dorsal trunk and at the dorsal midline. No gaps in the dorsal trunk were observed in ten rescued embryos, whereas all mutant embryos have multiple gaps. (C) Tracheal expression of a Shot L(A)-GFP derivative that lacks the microtubule binding GAS2 motif restores lumen formation in dorsal trunk and midline fusion cells. No gaps in the dorsal trunk were observed in ten rescued embryos. (D) Tracheal expression of a Shot L(A)-GFP fusion that lacks both cytoskeletal interaction domains does not restore lumen formation in dorsal trunk or midline fusion cells. (E-H) 1 µm confocal sections through the middle of the dorsal trunk of stage 15 wild-type embryos expressing Shot-GFP fusions. All Shot-GFP fusions produced green fluorescence when expressed in embryos and were expressed at comparable levels, approx. five- to tenfold higher than that of endogenous Shot proteins. GFP fusions were visualized with anti-GFP to enhance signal detection. (E) Shot L(A)-GFP accumulates apically in tracheal cells. (F) Shot L(C)-GFP accumulates apically, but is less apically concentrated than Shot L(A)-GFP. (G) Shot L(A)-GFP-
GAS2 accumulates apically, but is less apically concentrated than Shot L(A)-GFP. (H) Shot L(C)-GAS2-GFP molecules lack both the F-actin- and the microtubule-binding domains, and are cortically and cytoplasmically distributed. Scale bars: in D, 25 µm for A-D; in H, 25 µm for E-H.
