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Files in this Data Supplement:
Fig. S1. Dynamic actin-based behaviors of fusing myoblasts. Lateral views of stage 14 embryos. (A-B′′) rp298-lacZ, twi-CD2. Embryos stained with phalloidin to label F-actin (red) and antibodies against β-galactosidase to label FCs/myotubes (blue) and CD2 to label mesoderm cell membranes (green). (A) FCM extends filopdia (arrow) towards FC. (B) Tear-drop-shaped FCM (arrow) adhered to FC. (C-E′′) Live twip-GFP-actin embryos. Actin labeling is concentrated at the cell cortices and in cellular extensions such as lamellipodia and filopodia. The nucleus is evident as a non-labeled structure in the middle of the cells. Each panel represents a single time point from a timelapse sequence. Cell type is inferred from comparison to stage-matched fixed embryos stained with reagents to distinguish FCs/myotubes and FCMs. (C) FC for a segment border muscle prior to fusion extends lamellipodia (arrow) and filopoidium (arrowheads). (D) FCM extends lamellipodia (arrow) and a filopodium (arrowhead) prior to fusion. (E) FCMs adopt a tear-drop shape (arrow) prior to actin focus formation (arrowhead) and fusion. Scale bars: 5 μm.
Fig. S2. Measurement of the size of actin foci. Lateral views of stage 14 rp298-lacZ embryos stained with phalloidin to label F-actin (red) and with an antibody against β-galactosidase to label FCs/myotubes (blue). (A-A′′′) Wild-type (wt) embryo. In A′′, A′′′, B′, C′, D′′ and D′′′, the range indicator palette indicates signal as gray and white coloring, whereas background is labeled blue and oversaturation is labeled red. In A′′′, the overlay function has been used to measure the area of the actin focus. (B,B′) Same wild-type embryo as in A, but the actin signal has been oversaturated, as indicated by the red coloring of the range indicator (B′). Accurate measurement of the actin focus is not possible. (C,C′) Same wild-type embryos as in B, but the actin signal has been undersaturated, as indicated by the high amount of blue coloring. Again, accurate measurement of the actin focus is not possible. (D-D′′′) blow1 mutant embryo with proper settings. Focus size is larger than wild type. Scale bar: 5 μm.
Fig. S3. Foci formation is only detected during stages of myoblast fusion. Lateral views of twip-GFP-actin embryos. (A-C) Wild-type embryos only exhibit GFP-actin foci during fusion. No GFP-actin foci are present in stage 11 (A) or stage 16 (C) wild-type embryos, whereas several GFP-actin foci are seen in stage 14 embryos (arrows) while fusion is taking place. (D-F) GFP-actin foci persist in kette mutants. No GFP-actin is present in stage 11 ketteJ4-48 embryos (D). However, stage 14 ketteJ4-48 embryos contain many large GFP-actin foci (arrows in E, compare to B), and GFP foci are still seen in stage 16 ketteJ4-48 embryos (arrow in F, compare to C). Scale bar: 10 μm.
Fig. S4. GFP-actin labels F-actin structures. (A-A′′) Lateral view of stage 14 twip-GFP-actin embryo stained with phalloidin (red) and with an antibody against GFP (green). Phalloidin labels all filamentous actin, whereas GFP-actin labels all actin in the mesoderm. Actin foci colocalize as seen in the merged image (arrows). Hence, two different methods reveal the same structures. Scale bar: 10 μm.
Fig. S5. Single myoblast fusion event. Live stage 14 twip-GFP-actin, apME-NLS-GFP embryo. Each panel 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, allowing tracking of all relevant cell movements. Nuclei were identified by higher intensity of GFP label, as well as by comparison with embryos containing only the apME-NLS-GFP transgene. In this sequence, an actin focus (white arrowhead) forms at the site of adhesion between an FCM and an apterous-labeled myotube (nuclei marked with asterisks). This focus dissolves, followed by fusion and addition of an additional labeled nucleus (yellow arrowhead) to the myotube.
Movie 1. 3D reconstruction of actin focus. Lateral view of stage 14 rp298-lacZ embryo stained with phalloidin to label F-actin (red) and with antibodies against β-galactosidase to label FCs/myotubes (blue) and Lame duck to label FCMs (green). Image is a 3D reconstruction displaying four optical slices (2.15 μm) of the z axis. Specific cell labels are removed after the first frame to visualize the 3D structure of the actin focus. The focus extends across the cell cortices of an adherent FC and FCM.
Movie 2. Single myoblast fusion event. Movie of the timelapse sequence in Fig. 1E. Maximum optical projection representing 9 μm in total of a live twip-GFP-actin, apME-NLS-dsRed embryo. An actin focus (white arrow) forms at the site of adhesion between a FCM and an apME-NLS-dsRed labeled myotube. This focus dissolves, followed by fusion and addition of a dsRed-positive nucleus (yellow arrowhead) to the myotube. Frames are taken at 94-second intervals. Total sequence length is approximately 31 minutes.
Movie 3. Single myoblast fusion event. Movie of timelapse sequence in Fig. S5. Maximum optical projection representing 9 μm in total of a live twip-GFP-actin, apME-NLS-eGFP embryo. An actin focus (white arrow) forms at the site of adhesion between a FCM and an apME-NLS-eGFP labeled myotube. This focus dissolves, followed by fusion and addition of an eGFP-positive nucleus (yellow arrowhead) to the myotube. Frames were taken at 113-second intervals. Total sequence length is approximately 19 minutes.
Movie 4. Live analysis of fusion defect in kette mutant. Movie of timelapse sequence in Fig. 4A. Maximum optical projection representing 9 μm in total of a live twip-GFP-actin, apME-NLS-dsRed; ketteJ4-48 embryo. Actin foci (arrows) are larger than in wild type and do not dissolve, correlating with the block in fusion. Frames were taken at 129-second intervals. Total sequence length is approximately 32 minutes.
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