FCM-specific F-actin foci are invasive and the WASP complex is required for foci invasion. (A–F) Confocal images of stage 14 wild-type embryos showing horizontal (A–D) and perpendicular (E and F) pairs of founder cell/myotube and FCM. Boxed areas in A and C are magnified in B and D, respectively. Founder cells are outlined in A and C and FCMs in A–D. (A and B) An F-actin focus invading a founder cell. Embryo double labeled with α-Duf (red) and phalloidin (green). Arrowhead indicates the inward curvature on the founder cell membrane. (C and D) Membrane rearrangements at the invasive tip of an F-actin focus. Embryo expressing membrane-targeted mCherry-CAAX in all muscle cells (with twi-GAL4) labeled with α-mCherry (red) and phalloidin (green). Arrowhead indicates the invasive tip of the FCM-specific F-actin focus. (E and F) Two examples of F-actin foci encircled by cell adhesion molecules. Embryo triple labeled with α-Duf (red), α-Sns (FCM; enriched at sites of fusion; blue), and phalloidin (green). (G) Schematic drawing of the asymmetric muscle cell adhesion junction. Before fusion, an F-actin focus (green oval) forms at the tip of the FCM (right) and invades the apposing founder cell (left) to create a cup-shaped dimple. The inner wall of the cup is lined with Sns (blue), and the outer wall with Duf (red). Depending on the angle at which the FCM invasion is viewed by confocal microscopy, the cell adhesion molecules can appear as a U-shaped dimple cupping a portion of the actin focus (hatched) in a horizontal section (A–D) or overlapping rings encircling the actin focus in a perpendicular section (E and F). Numbers show average actin foci size (1.7 µm²), diameter of the adhesive rings (1.2 µm), and depth of invasion (0.3–1.9 µm). (H) Ultrastructural details of an invasive F-actin focus. An FCM (pseudo-colored pink) projects multiple F-actin–enriched invasive fingers into a binucleated myotube in a stage 13 wild-type (wt) embryo fixed by HPF/FS. Serial sections of this invasive structure are shown in Fig. S3 A. The F-actin–enriched areas within the FCMs (boundary marked by dashed green lines) are identified by their light gray coloration and lack of ribosomes and intracellular organelles. Although actin filaments (7-nm diameter) are difficult to be fixed and visualized by HPF/FS (or conventional chemical fixation) EM, magnified inset shows faint actin filaments (arrowheads) within an invasive finger. (I–K) F-actin foci fail to invade properly in sltr mutant embryos. F-actin–enriched fingers in FCMs in stage 14 sltr embryos either folded upon each other without extending toward the apposing founder cell (I and J; 8/10 actin foci analyzed show this phenotype), or appear wider and shorter than wild type (K; 2/10). Magnified inset in I shows faint actin filaments (arrowhead), as well as a portion of the founder cell membrane (arrows) pulled into the FCM territory by the folded fingers, which may account for the extensive colocalization between founder cell markers (Duf and Ants) and phalloidin staining in sltr mutant embryos revealed by confocal microscopy (Fig. 3 D and Fig. S1, B and E; Kim et al., 2007). n: founder cell/myotube nuclei (H–K). Bars: (A and C) 10 µm; (B, D, E, and F) 5 µm; (H–K) 500 nm.