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First published online April 3, 2008
doi: 10.1242/10.1242/jcs.022269

Journal of Cell Science 121, 1303-1313 (2008)
Published by The Company of Biologists 2008
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WASP and SCAR have distinct roles in activating the Arp2/3 complex during myoblast fusion

Susanne Berger1,*, Gritt Schäfer1,*, Dörthe A. Kesper1,{ddagger}, Anne Holz2, Therese Eriksson3, Ruth H. Palmer3, Lothar Beck4, Christian Klämbt5, Renate Renkawitz-Pohl1 and Susanne-Filiz Önel1,§

1 Fachbereich Biologie, Entwicklungsbiologie, Philipps-Universität Marburg, Karl-von-Frisch Str. 8, D-35043 Marburg, Germany
2 Institut für Allgemeine und Spezielle Zoologie, Stephanstr. 24, Justus-Liebig-Universität Giessen, D-35390 Giessen, Germany
3 UCMP, Umeå University, Building 6L, 90187 Umeå, Sweden
4 Fachbereich Biologie, Spezielle Zoologie, Philipps-Universität Marburg, Karl-von-Frisch Str. 8, D-35043 Marburg, Germany
5 Institut für Neurobiologie, Westfälische Wilhelms-Universität Münster, Badestr. 9, D-48149 Münster, Germany


Figure 1
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  		Fig. 1. schwächling is a new allele of Arp3. (A-F) Embryonic muscle pattern of stage-16 embryos stained with anti-β3-tubulin. (A) Wild-type. (B) Unfused myoblasts are attaching to growing myotubes in schwächling homozygous-mutant embryos (arrow). (C,D) The deficiency Df(3L)ZP1 shows a similar phenotype to schwächling (C) and also displays an unfused-myoblast phenotype in transheterogeneity to schwächling (D). (E,F) The EP-element insertion Arp66BEP3640 (E) only displays a fusion defect in embryos that are transheterozygous to schwächling (F, arrow). (G,H) In situ hybridizations with a labeled Arp3 antisense probe show that the Arp3 transcript is expressed in the mesoderm in stage 10 (G) and late stage 11 (H). (I) RT-PCR on RNA isolated from wild-type, Arp3schwächling–/+ and Arp3schwächling–/– embryos. Spliced Arp3 mRNA is present in all three lanes (509-bp product). The zygotically transcribed mRNA in Arp3schwächling–/+ and Arp3schwächling–/– embryos is unspliced (674 bp). (J) Schematic drawing of the Arp3 transcript. The ORF is denoted with shaded boxes. The donor/acceptor splice-site nucleotide sequence of intron 1 from wild-type embryos and Arp3schwächling mutant embryos is shown. schwächling mutants show a G-to-A transition in the acceptor splice site.

 

Figure 2
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  		Fig. 2. Arp3schwächling mutants stop myoblast fusion after precursor formation. (A-C) Anti-Eve stainings on stage-15 embryos visualize the nuclei of the DA1 muscle (arrow) and the pericardial cells (per). (A) The wild-type DA1 muscle contains up to 14 nuclei. (B) Since fusion is disturbed in homozygous Arp3schwächling mutant embryos, the DA1 muscle only contains approximately three nuclei. (C) The DA1 muscle in Arp3schwächling wasp3D3-035 double mutants is mainly mono-nucleated, showing that myoblasts fail to fuse completely. (D) Statistical analysis of the DA1 nuclei confirms that Arp3schwächling and wasp3D3-035 single mutants stop fusion after precursor formation. However, in Arp3schwächling wasp3 double mutants, no fusion takes place. (E,F) Electron microscopy studies on stage-14 wild-type embryos. (E) Assembly of the pre-fusion complex (arrow); (F) arrow marks the electron-dense plaque that is formed between two fusing myoblasts. N, cell nucleus. Magnifications: (E) 12,000x; (F) 21,000x.

 

Figure 3
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  		Fig. 3. wasp3D3-035 and wipD30 single mutants stop fusion during fusion-pore formation, whereas, in Arp3schwächling mutants, a fusion pore is formed. (A-D) Ultrathin sections of stage-14 embryos show myoblasts in the process of fusion (boxed area). (A'-D') Higher-magnification views of the boxed areas. (A,A') Wild type. (A') A forming fusion pore (arrows) and vesiculating membranes (arrowheads). (B) Myoblast fusing to a precursor cell in a wasp3D3-035 embryo. (B') The higher-magnification view shows that membranes are vesiculating. (C) In Arpschwächling mutant embryos, a trinucleated precursor cell is surrounded by several myoblasts. (C') A fusion pore has formed between the precursor cell and the FCM, but the FCM fails to integrate into the muscle. (D) Fusing myoblasts in a wipD30 (vrp1) embryo. (D') Vesiculating membranes. N, cell nucleus. Magnification: (A-D) 3600x; (A',D') 12,000x; (B',C') 21,000x.

 

Figure 4
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  		Fig. 4. Fusion is blocked in Arp3schwächling wasp3D3-035 double mutants. (A-C) Visualization of the somatic musculature of stage-16 embryos by staining for anti-β3-tubulin. (A'-C') Higher-magnification views. (A,A') Wild type. (B,B') wasp3D3-035 mutant embryo with unfused myoblasts. (C,C') The unfused-myoblast phenotype of the single mutants Arp3schwächling (see Fig. 1B) and wasp3D3-035 is enhanced in homozygous Arp3schwächling wasp3D3-035 double mutants (C', arrow).

 

Figure 5
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  		Fig. 5. The Arp2/3 regulators SCAR and the WASP-Vrp1 complex act in concert to regulate myoblast fusion in Drosophila. All embryos were stained with anti-β3-tubulin. (A-C) Lateral views of stage-16 embryos. (A) Homozygous scark13811 embryos hardly show fusion defects. (B) Unfused myoblasts (arrow) are observed in homozygous scark13811 mutant embryos that lack one copy of wasp3. (C) scark13811 wasp3 double-mutant embryos display lots of unfused myoblasts (arrow). (D-G) Dorsal views of stage-14/15 embryos. (D) Wild type. (E) Embryos that are homozygous for scar{Delta}37 display a weak fusion defect (arrow). Some muscles are missing (arrowhead) and dorsal closure is delayed. (F) vrp1f06715 mutant embryos stop fusion after precursor formation. (G) scar{Delta}37 vrp1f06715 double mutants exhibit a strong myoblast-fusion defect.

 

Figure 6
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  		Fig. 6. Vrp1 is expressed in discrete foci, but becomes localized in the filopodia upon cell-cell contact. (A-C) Stage-13/14 embryos stained with anti-Vrp1 antibody. (A'-C') Higher-magnification views of the boxed areas in A-C. (A) Embryo carrying the enhancer-trap insertion rP298 stained with anti-β-galactosidase and anti-Vrp1. Ventral view of a stage-14 embryo. (A') In round myoblasts, Vrp1 can be detected in discrete foci (asterisks). However, in FCMs that have formed a filopodium, Vrp1 is enriched at the tip of the FCM, which attaches to a growing myotube (arrow points towards the tip of a filopodium and the arrowhead marks a dinucleated precursor). Note that the precursor cell (arrowhead) also shows a weak expression of Vrp1. (B) sns20-23-null mutant embryo at stage 13; lateral view. (B') Vrp1 is still present in discrete foci (asterisks); however, no localization of Vrp1 to the tip of a filopodium was observed. (C) Ventral view of a stage-14 wasp3D3-035 Arp3schwächling double-mutant embryo. (C') Vrp1 can be found in round myoblasts in discrete foci (asterisk) and in the tip of a filopodium (arrow). Scale bars: 10 µm.

 

Figure 7
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  		Fig. 7. Duf is localized correctly in wasp3D3-035 and Arp3schwächling mutants, suggesting that assembly of the FuRMAS occurs. (A-D) Anti-Duf staining on stage-15/16 embryos. (A) In a wild-type embryo, Duf is expressed on the site of the FC/growing myotube as a ring (frontal view, arrowhead). The asterisks mark FCMs attaching to a growing myotube (lateral view). (B) Three FCMs attach to a growing myotube (asterisks, lateral view) in an Arp3schwächling embryo. Duf is present on the site of the FC/growing myotube. (C) In a wasp3D3-035 embryo, the asterisks once again highlight FCMs (lateral view) that attach to growing myotubes expressing Duf. (D) In Arp3schwächling wasp3D3-035 double-mutant embryos, Duf is also expressed and concentrated at the site of cell-cell contact. Three FCMs can be seen to attach to a Duf-expressing cell (asterisks). Scale bars: 10 µm.

 

Figure 8
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  		Fig. 8. A modified model for myoblast fusion in Drosophila. (A) Actin regulates the expansion of the FuRMAS and integrates the FCM into the growing myotube. Blow is localized to the middle of the ring that expresses Sns in FCMs and Duf in FCs. Our data suggest that Vrp1 is also present in this location, inducing the branching of F-actin in concert with WASP. The correct localization of these components (Kesper et al., 2007Go) is required for the formation of a fusion pore, and hence their presence guarantees successful fusion. Transmission electron microscopy studies suggest that wasp and vrp1 mutants are capable of forming a pore. In Arp3 (encoding a subunit of the Arp2/3 complex) mutants, fusion is disrupted after fusion-pore formation. Therefore, we propose that F-actin is required for the integration of FCMs into the FC/growing myotube. (B) Molecular model for regulating F-actin branching during the first and the second step of myoblast fusion. Duf and Sns form a ring in FCs and FCMs, respectively. The fusion pore will be formed in the middle of the ring (indicated in red). We postulate that the SCAR-Kette complex regulates F-actin formation during the first fusion step. The formation of F-actin during the first fusion step might additionally require the activity of Vrp1. During the second fusion step, the signal from the outer membrane in FCs becomes translated by Rols7, which is involved in the localization of Drosophila Titin (Menon and Chia, 2001Go). Our results suggest that F-actin becomes regulated differently in FCs and FCMs. The activity of the actin regulator WASP might be required in FCMs, in which it acts together with Vrp1. We propose that the actin regulators SCAR and Kette are also involved during the second fusion step, because Kette and WASP act antagonistically during the fusion process. Functions of the proteins involved during the first and second fusion step are described in Table 1.

 

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© The Company of Biologists Ltd 2008