Fig. 1. Dynamic remodeling of the actin cytoskeleton during Drosophila myoblast fusion. Lateral views of stage 14 embryos. Phalloidin was used to label F-actin (red) in A-D. (A-A'') rP298-lacZ embryo stained with phalloidin and antibodies against ß-galactosidase to label FCs/myotubes (blue), and Lame duck to label FCMs (green). These images show the arrangement of myotubes and FCMs in one plane of focus and the occurrence of F-actin foci at this stage. F-actin is seen predominantly at the cell cortices. (B-B'') Higher magnification of A. F-actin foci form at the adhesion sites between FCs/myotubes and FCMs (arrowheads). For 3D reconstruction of an actin focus, see Movie 1 in the supplementary material. (C-C'') rP298-lacZ; twi-CD2 embryo stained with phalloidin and antibodies against β-galactosidase to label FCs/myotubes (blue), and CD2 to label mesoderm cell membranes (green). An actin focus is present in both the FC and the FCM, as evident by bisection of the focus with membrane staining (arrowhead). (D-D'') apME-GFP embryo stained with phalloidin and antibody against GFP to label the cytoplasm of apterous-expressing FCs/myotubes (green). GFP does not leak from the apterous-expressing myotube into the adherent FCM when the F-actin focus is present. (E-E'') Live twip-GFP-actin, apME-NLS-dsRed embryo. Each column of panels represents a time point from a timelapse sequence. Each image is an optical projection displaying 9 µm of the z-axis. The optical projection allows visualization of several cell layers simultaneously and tracking of all relevant cell movements. In this sequence, an actin focus (white arrowheads) forms at the adhesion site between an FCM and an apterous-labeled myotube. This focus dissolves, followed by fusion and addition of a labeled nucleus (yellow arrowhead) to the myotube. The nucleus of the fusing cell is indicated (asterisk). Additional actin accumulation in 468-second panel may represent a new actin focus forming. Scale bars: 20 µm in A; 5 µm in B-E.