Fig. 1. Tube fusion proceeds through the formation and the maturation of a Shot containing track in fusion cells. (A) Schematic of morphological and cytoskeletal changes in fusion cells during fusion in an optical section along the plane of contact. Fusion cells (yellow) meet. They then form a new E-cadherin contact (blue) and initiate the assembly of a cytoskeletal track (red) that contains F-actin, Shot and microtubules (see Fig. 1B-K). This track matures, growing to span the two cells and guiding the invagination of the existing apical surfaces (light green) and associated cytoskeleton (pink). As the branches draw closer, the fusion cells also compress along the anteroposterior axis and elongate along the dorsoventral axis, bringing the existing apical surfaces closer. When the two apical surfaces meet, they fuse, leaving a ring of E-cadherin and associated cytoskeleton at the junction between the fusion cells. The two mature fusion cells are doughnut shaped. Other tracheal cells are wedge shaped. (B-K) Time lapse series of 0.5 µm confocal images of dorsal trunk tracheal cells in a live wild-type embryo expressing Shot L(A)-GFP in tracheal cells. Fusion sites are indicated by arrows, and are enlarged in F-J. Elapsed time is indicated in minutes (upper right corners). (B) At the start of the sequence (late stage 12), the tracheal branch tips are touching, but no track of Shot-GFP is visible at the future fusion site (arrow). (C) At 10 minutes, the anterior fusion cell accumulates Shot-GFP (arrowhead) along one side. (D) At 40 minutes, more Shot has begun to accumulate, and the track is centered more on the fusion cell contact. (D-G) A persistent track of Shot is visible from 40 minutes to 105 minutes. (F-I) The apical surfaces exhibit dynamic behavior (insets; concave arrow indicates an area that accumulates more Shot; the arrowheads indicate an area that loses and then regains Shot). (H) At 110 minutes, the apical surfaces of the two branches come closer together, forming a bottleneck structure. (I) At 115 minutes, a second track of Shot is clearly visible in this cross-section, indicating that the tubes are now joined. (J) The second track becomes darker, and the opening between the branches swells. A ring of Shot is visible (arrowhead, inset). (K) At 145 minutes, the opening assumes a diameter close to that of the adjacent branches. Scale bar: 10 µm. Anterior, upwards; dorsal, rightwards.

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