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First published online 14 November 2007
doi: 10.1242/dev.010678

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SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodeling at the site of myoblast fusion

¹ Program in Developmental Biology, Sloan Kettering Institute, New York, NY 10021, USA.
² Weill Graduate School at Cornell Medical School, New York, NY 10021, USA.

View larger version (95K): [in this window] [in a new window]   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.

View larger version (78K): [in this window] [in a new window]   Fig. 2. Roles of fusion proteins in actin remodeling. Lateral views of stage 14 rp298-lacZ embryos stained with phalloidin to label F-actin (red) and antibody against β-galactosidase to label FCs/myotubes (blue), except E, which is stained with an antibody against Slouch to label a subset of FCs/myotubes (blue). Actin foci are indicated by arrowheads. Scale bar: 5 µm. One optical slice is shown for each mutant. For details of focus size determination, see Materials and methods and Fig. S2 in the supplementary material. (A) Wild type. (B,C) Class 1: no, or few foci. (B) snsXB3 embryo. (C) rolsT627 embryo. A focus of wild-type size is occasionally seen (arrowhead). (D) Class 2: wild-type actin foci: lonerT1032 embryo. (E-H) Class 3: enlarged actin foci. (E) Rac1J11, Rac2, mtl embryo. (F) ketteJ4-48 embryo. (G) blow1 embryo. (H) mbcC1 embryo.

View larger version (74K): [in this window] [in a new window]   Fig. 3. Localization of fusion machinery with actin focus. Lateral views of stage 14 rp298-lacZ embryos stained with phalloidin to label F-actin (red) and antibodies against β-galactosidase to label FCs/myotubes (blue), and Sns (green, A-A''), Rols (green, B-B''), Loner (green, C-C''), Rac1 (green, D-D''), Kette (green, E-E''), Blow (green, F-F'') or Mbc (green, G-G''). Channels are shown separately and then merged. Sns, Rols, Kette, Blow and Mbc protein colocalize with F-actin foci (arrowheads, A,B,E,F,G), whereas Loner does not (C). Rac partially overlaps with the F-actin foci (D). Scale bar: 5 µm.

View larger version (84K): [in this window] [in a new window]   Fig. 4. Kette regulates actin foci dissolution during Drosophila myoblast fusion. (A-A'') Lateral views of live stage 14 twip-GFP-actin, apME-NLS-dsRed; ketteJ4-48 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, allowing visualization of several cell layers simultaneously and tracking of all relevant cell movements. Large actin foci form (arrowheads) but do not dissolve as in wild type (compare with Fig. 1E). No incorporation of new red nuclei is seen, consistent with the kette fusion block. Scale bar: 5 µm. (B) Actin foci persist significantly longer in ketteJ4-48 null mutants compared to wild type. Focus duration (mean±s.d.): wild type=10.9±6.9 minutes, ketteJ4-48=36.0±17.6 minutes. This difference is significant by a two-tail unpaired Student's t-test (P<0.0001, n=25 foci).

View larger version (88K): [in this window] [in a new window]   Fig. 5. Localization of fusion machinery in different mutant classes. Lateral views of stage 14 rp298-lacZ embryos stained with phalloidin to label F-actin (red) and an antibody against β-galactosidase to label FCs/myotubes (blue). Channels are shown separately and merged. Scale bar: 5 µm. (A-C'') Embryos stained with an antibody against Blow (green). (A) Class 1: snsXB3 mutant. Blow no longer has a polarized localization (compare with Fig. 3F) and instead is distributed cortically in FCMs. (B) Class 2: lonerT1032 mutant. Blow localization overlaps with the actin focus (arrowhead, compare with Fig. 3F). (C) Class 3: ketteJ4-48 mutant. Blow is polarized and distributed throughout large actin accumulations (arrowhead). (D-F'') Embryos stained with antibody against Rols to label FCs/myotubes (green). (D) ketteJ4-48 mutant. (E) blow1 mutant. (F) mbcC1 mutant. Dotted lines indicate FC/myotube membranes and were drawn based on Rols localization. In each case, enlarged actin foci localize across both the myotube and FCM.

View larger version (79K): [in this window] [in a new window]   Fig. 6. SCAR and Arp2/3 are required for myoblast fusion and regulate actin foci dissolution during fusion. (A-D) Lateral views of stage 16 embryos stained with antibody against myosin heavy chain to visualize body wall muscles. Scale bar: 20 µm. (A) Wild-type embryo. (B) SCAR37 embryo. Approximately 10-20 free myoblasts (arrowhead) are seen in each hemisegment indicating a myoblast fusion defect. (C) Embryo from SCARk13211 germline clones with reduced levels of maternal and zygotic SCAR protein. Increased numbers of free myoblasts (arrowhead) are seen, along with thinner muscles, indicating a more severe myoblast fusion defect. (D) Arp3EP3640 embryo. These embryos display a myoblast fusion defect, with approximately 10-20 free myoblasts (arrowhead) seen in each hemisegment. (E-J) Lateral views of stage 14 embryos stained with phalloidin to label F-actin (red). F-actin labels the foci as well as cortical actin. Scale bar: 5 µm. (E) SCAR37 embryo. Actin foci (arrowhead) appear larger than in wild type. The focus shown is an example of the larger foci seen in these mutants, although the average focus size is similar to wild type (Table 1). (F) SCARk13211 germline clone embryo with reduced levels of maternal and zygotic SCAR protein. Large accumulations of F-actin have formed at the site of adhesion between FC/myotubes and FCMs (arrowhead). (G) Arp3EP3640 embryo. Actin foci appear larger than in wild type (arrowhead). The focus shown is an example of the larger foci seen in these mutants, although the average focus size is similar to wild type (Table 1). (H-I'') Embryos stained with antibodies against β-galactosidase to label FCs/myotubes (blue) and SCAR (green). (H) rP298-lacZ embryo. SCAR protein partially colocalizes with F-actin foci (arrowhead) in both FCMs and FCs/myotubes. (I) rP298-lacZ; ketteJ4-48 embryo. SCAR protein is virtually undetectable in this mutant background. Residual protein is mislocalized (compare with H). (J-J'') SCARk13211 germline clone embryo stained against Rols (green). Dotted line indicates FC/myotube membrane and was drawn based on Rols localization. Rols partially overlaps with actin focus (arrowhead), indicating that enlarged actin foci localize across both FCs/myotubes and FCMs.

View larger version (46K): [in this window] [in a new window]   Fig. 7. Model of Drosophila myoblast fusion. Updated model of myoblast fusion based on the known fusion genes following this work. Those proteins that colocalize with the F-actin foci are colored yellow, those that do not are purple. The localization of Rac partially overlaps with actin foci. Solid arrows between proteins indicate well-established biochemical interactions, while dashed arrows indicate genetic and/or suggested, but unsubstantiated, biochemical interactions. See Discussion for details.

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