Fig. 5. A mutation in the E-cadherin juxtamembrane dominantly blocks tracheal tube fusion and disrupts microtubule tracks. Anterior, leftwards; dorsal, upwards (except in P-T where anterior is upwards and dorsal is rightwards. (A-D) Stage 15 embryos stained with mAb 2A12. (A) The lumen in a stage 15 wild-type embryo expressing wild-type E-cadherin in tracheal cells is continuous at fusion sites in the dorsal trunk (arrowheads) and lateral trunk (arrows). Scale bar: 10 µm in A-D. (B) Expression of -arm E-cadherin in tracheal cells does not detectably affect fusion (arrowheads, arrows). (C) Expression of AAA-JXT E-cadherin in tracheal cells blocks fusion in the dorsal (arrowheads) and lateral (arrows), as well as at the dorsal midline (not shown). (D) Expression of E-cadherin mutant in the GGG juxtamembrane sequence in all cells (Pacquelet et al., 2003) has no effect on tracheal fusion or development (arrowheads, arrows). (E) Drosophila S2 cells expressing HA epitope-tagged p120 (red, E') and AAA-JXT mutant E-cadherin (green, E''). E-cadherin and p120 colocalize at cell contacts (arrows). F) Stage 14 embryo expressing wild-type E-cadherin in tracheal cells. Scale bar: 10 µm in F-H,J,K. E-cadherin is localized largely in adherens junctions (arrowhead). (G) Stage 14 embryo expressing arm E-cadherin in tracheal cells. Adherens junctions appear normal (arrowhead). Somewhat more E-cadherin is found outside the adherens junctions than when wild-type is overexpressed in tracheal cells. (H) Stage 14 embryo expressing AAA-JXT E-cadherin in tracheal cells. E-cadherin is delocalized. (I) Western blot with anti-E-cadherin (Oda et al., 1994) revealing the relative amounts of NP40 soluble (S) and pelleted (P) E-cadherin in wild-type Oregon R embryos expressing no additional (OreR) E-cadherin or wild-type E-cadherin (+WT), AAA-JXT E-cadherin, or arm E-cadherin in tracheal cells. (J) Same embryo as in F. Fusion has occurred and F-actin accumulates apically in fusion cells (arrowhead). (K) Same embryo as in H. F-actin accumulates at the fusion site, but in an aggregate (arrowhead). (L-O) Stage 13 embryo expressing AAA-JXT E-cadherin in tracheal cells. Scale bar: 10 µm in L-O. (L) F-actin weakly accumulates at the fusion site (arrowhead). (M) GAP-43 GFP labels the membranes of the fusion cells (arrowhead), which are elongated. (N) A weak track of Shot is visible at the fusion cell contact (arrowhead). (O) Merge of L-N. F-actin, red; GFP, green; Shot, blue. (P-T) Frames from videos of fusion in AAA-JXT mutant embryos. Minutes elapsed, lower left. Scale bar: 10 µm in P-T. (P) A weak Shot fusion track (arrow) is visible at the start of the first sequence. Apical surfaces appear open (arrowheads). (Q,R) The track changes little even after 50 (Q) or 160 (R) minutes, becoming only moderately more intense. The apical surfaces remain open (arrowheads). (S) At 170 minutes in a second sequence, over 1 hour after fusion occurs in wild type, the track persists and the apical surfaces remain open (arrowheads). (T) At 170 minutes in a third sequence, the existing apical surfaces draw closer together after a track forms and shrinks (not shown), but remain blind-ended (arrowheads). (U) Microtubules in dorsal trunk tracheal cells form fusion tracks (arrowheads) in a stage 13 wild-type embryo. Scale bar: 10 µm in U,V. (V) The microtubule track is broken (left arrowhead) or missing in fusion cells (right arrowhead) in a stage 13 embryo expressing AAA-JXT E-cadherin in tracheal cells. (W-Y) A late stage 13 embryo expressing AAA-JXT E-cadherin under the control of hairy-GAL4. Segments expressing the mutant transgene (arrows). (W) Shot fails to accumulate in fusion tracks (arrowheads). (X) E-cadherin contacts fail to form between fusion cells (arrowheads). Adherens junctions appear abnormally arranged in segments expressing the transgene. However, E-cadherin remains largely in adherens junctions (arrows). Scale bar: 10 µm in W-Y. (Y) Merge of W and X. Shot, red; E-cad, green.

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