Fig. 2. Apical cytoskeletal polarization of tracheal cells in wild-type and shot mutant embryos. (A-L) 1 µm confocal sections of dorsal trunk tracheal cells in wild-type and shot³ mutant embryos. (A-C,G,H) Stage 15, wild type. (D-F,I,J) Stage 15, shot³. (A) Phalloidin staining of dorsal trunk tracheal cells reveals F-actin (red in C) apically concentrated around a lumen that extends through anastomosis sites. Arrowheads mark the basal surface. (B) Microtubules (green in C) detected with anti-tubulin also accumulate apically, though some cortical staining is also observed. (C) Merge of A and B. (D) F-actin accumulates apically around two lumens that end at an anastomosis site (bracket). (E) Tubulin staining is diffuse in tracheal cells, including those that form lumen. (F) Merge of D and E. (G) Actin-GFP is apically concentrated. (H) C-Shot L-GFP, a microtubule-associated protein, localizes apically. (I) Actin-GFP concentrates apically, but tracheal tubes dead end at anastomosis sites. (J) The C-Shot L-GFP distribution in tracheal cells is disorganized. (K) Stage 14, wild type. F-actin (red) accumulates apically in tracheal cells (membranes outlined in green with GAP43-GFP) and surrounds a lumen that is continuous through the fusion cells (blue). The fusion cells are compact and doughnut shaped. (L) Stage 14, shot³. F-actin (red) accumulates apically in tracheal cells (outlined in green) but the lumen does not extend through the fusion cells (blue). Arrows indicate apical sides of fusion cells. Scale bars: in F, 10 µm for A-F; in J, 10 µm for G-J; in L, 10 µm for K,L.