Summary

The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell–cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar– or Vrp1–WASp-dependent Arp2/3 activation.

Introduction

Cell–cell adhesion molecules are crucial for the formation of organs and tissue structure during embryonic development and for their homeostasis. Cells express various cell adhesion molecules at their surface, which can either mediate homophilic or heterophilic cell–cell adhesions. Their intracellular tails interact with cytoplasmatic proteins involved in intracellular signaling. During syncytial muscle formation, mononucleated myoblasts must recognize and adhere before they fuse to form multinucleated muscles. In Drosophila, members of the immunoglobulin superfamily (IgSF) mediate the recognition and adhesion of founder cells (FCs) and fusion-competent myoblasts (FCMs) (Abmayr and Pavlath, 2012). FCs determine the position, size and epidermal attachment site of the muscle by the combinatorial expression of muscle identity genes and recruit FCMs. The IgSF molecule Dumbfounded (Duf; also known as Kirre) is exclusively expressed in FCs and functions redundantly with the IgSF molecule Roughest (Rst; also known as IrreC) (Ruiz-Gómez et al., 2000; Strünkelnberg et al., 2001). The extracellular domain of Duf binds to the Ig domain of the FCM-specific IgSF molecule Sticks and Stones (Sns) (Bour et al., 2000). Sns seems to be the main player in FCMs that mediates cell adhesion. The paralog of Sns named Hibris (Hbs) is also exclusively expressed in FCMs (Artero et al., 2001; Dworak et al., 2001). However, hbs mutants do not display any fusion defects and Hbs can rescue only a small amount of fusion in sns mutants (Shelton et al., 2009).

The IgSF molecules Duf, Rst and Sns are expressed in a ring-like structure at cell–cell contact points in FCs and FCMs (Kesper et al., 2007; Sens et al., 2010; Önel et al., 2011; Haralalka et al., 2011). In the center of this structure a dense F-actin focus forms (Kesper et al., 2007), predominantly in FCMs contacting an FC/growing myotube (Sens et al., 2010; Haralalka et al., 2011). In contrast, a thin sheath of F-actin is visible at cell–cell contact points in FCs/growing myotubes (Sens et al., 2010). In the absence of the cell adhesion molecules, F-actin foci fail to form (Richardson et al., 2007), indicating that they trigger the formation of F-actin foci. On a molecular level, recent studies have demonstrated that foci formation depends on the evolutionary conserved Arp2/3 complex (Massarwa et al., 2007; Richardson et al., 2007; Berger et al., 2008), which nucleates branched F-actin. The Arp2/3 complex becomes activated by two nucleation-promoting factors during myoblast fusion: Scar (Richardson et al., 2007; Berger et al., 2008; Gildor et al., 2009; Sens et al., 2010) and WASp (Massarwa et al., 2007; Schäfer et al., 2007). One open and intriguing question is how signaling from the cell adhesion molecules is linked to F-actin formation. Recent co-immunoprecipitation studies on non-muscle Drosophila S2 cells have shown that the SH2-SH3 adaptor protein Crk is able to bind to the intracellular domain of Sns and to the Drosophila WASp-interaction partner Vrp1 (Flybase; Berger et al., 2008) also known as Sltr (Kim et al., 2007) and Wip (Massarwa et al., 2007). Since Arp2/3-based actin polymerization is required in both myoblast types, forming a large actin focus in the FCM and a thin actin sheath in the FC, we have investigated signaling molecules that may be present in both cell types. Based on findings from mammalian Nephrins, we have investigated whether the SH2-SH3 adaptor protein Dock is involved in Drosophila myoblast fusion, and connects both Duf/Rst in the FCs and Sns/Hbs in the FCMs to downstream actin regulators.

Human Nephrins, Neph1 and Nephrin show 33% identity to Duf and Rst and 28% identity to Sns and Hbs (Gerke et al., 2003). They are involved in the formation of the slit diaphragm, a specialized podocyte cell–cell junction in the kidney essential for filtration of the blood (reviewed by Welsh and Saleem, 2010). Recent findings have demonstrated that the intracellular domain of Nephrin can bind to the Src-Homology 2 (SH2)/SH3 domain-containing adaptor protein Nck (Jones et al., 2006). In this study multiple YDxV sites were found in the intracellular domain of Nephrin that can interact with the SH2 domain of Nck.

Herein, we demonstrate that the Drosophila homolog of Nck, named Dreadlock (Dock), is required for myoblast fusion. Dock is expressed in both myoblast types and interacts genetically and biochemically with the Arp2/3 activators Scar, Vrp1 and WASp. Interaction studies in COS7 and Drosophila S2 cells further reveal that Dock binds to the intracellular domain of all Ig-domain proteins that are known to mediate the recognition and adhesion of FCs and FCMs. Surprisingly, we found that the cell adhesion proteins either bind to the SH2 domain (Hbs), the SH3 domains (Duf) or all domains of Dock (Sns and Rst). However, only duf, sns and hbs show a genetic interaction with dock.

Results

The SH2-SH3 adaptor protein Dock is expressed in FCs and FCMs

To define the requirement of the Drosophila SH2-SH3 adaptor protein Dock during myoblast fusion, we first studied the subcellular distribution of the Dock protein with an anti-Dock antibody (Clemens et al., 1996). The specificity of the Dock antibody was tested by the expression of a myristylated membrane-bound form of Dock that was driven in the Wingless (Wg) domain (Fig. 1A). Thereafter, we examined whether Dock is expressed in the somatic mesoderm. We observed ubiquitous expression of the Dock protein during embryogenesis (Fig. 1B,B′). The rP298 enhancer trap line expresses β-galactosidase in the nuclei of FCs (Nose et al., 1998). To mark myoblasts and distinguish between FCs and FCMs we labeled wild-type embryos carrying rP298-lacZ with β3-Tubulin, Dock and β-Gal antibodies (Fig. 1B). Dock is expressed in a punctuated manner in both myoblast types, FCs (Fig. 1B,B′ arrow) and FCMs (Fig. 1B,B′, arrowheads). Additionally, Dock seems to be expressed at muscle attachment sites (Fig. 1B,B′, asterisks).

Fig. 1.

Dock has a strong maternal component and is expressed in FCs and FCMs. (A) Lateral view of stage 10 embryo expressing UAS-dockmyr with wg–GAL4, probed with anti-Dock. (B) Higher magnification of rP298-lacZ-expressing wild-type embryo at stage 14, stained with anti-Dock (in green), anti-β3-Tubulin (in blue) to mark all myoblasts and anti-β-Gal (in red) to mark FCs. (B′) Same embryo showing anti-Dock antibody staining in green. Dock is expressed in FCs (arrow) and FCMs (arrowhead) and in muscle attachment sites (asterisks). (C,D) Lateral view of stage 16 embryos stained with anti- β3-Tubulin. (C) Homozygous dock04723 mutant embryo. (D) Homozygous dock04723 drke0A double mutant embryo. (E,E′) Lateral view of stage 15 embryos. (E) Homozygous dock-deficient embryo carrying the FC marker rP298-lacZ. (E′) Higher magnification of part of the embryo in E. Scale bars: 10 µm.

Fig. 1.

Dock has a strong maternal component and is expressed in FCs and FCMs. (A) Lateral view of stage 10 embryo expressing UAS-dockmyr with wg–GAL4, probed with anti-Dock. (B) Higher magnification of rP298-lacZ-expressing wild-type embryo at stage 14, stained with anti-Dock (in green), anti-β3-Tubulin (in blue) to mark all myoblasts and anti-β-Gal (in red) to mark FCs. (B′) Same embryo showing anti-Dock antibody staining in green. Dock is expressed in FCs (arrow) and FCMs (arrowhead) and in muscle attachment sites (asterisks). (C,D) Lateral view of stage 16 embryos stained with anti- β3-Tubulin. (C) Homozygous dock04723 mutant embryo. (D) Homozygous dock04723 drke0A double mutant embryo. (E,E′) Lateral view of stage 15 embryos. (E) Homozygous dock-deficient embryo carrying the FC marker rP298-lacZ. (E′) Higher magnification of part of the embryo in E. Scale bars: 10 µm.

The presence of Dock in myoblasts pointed us to investigate whether dock04723 null mutants display defects in myoblast fusion. We found that the muscle pattern of dock04723 null mutants is reminiscent of that seen in wild-type embryos (Fig. 1C). In podocytes, the SH2–SH3 adaptor proteins Nck and Grb2, which bind to the intracellular domain of phosphorylated Neph1, both cooperate to induce actin polymerization. Since dock null mutants show no mutant phenotype, we generated double mutants with dock and the Drosophila grb2 homolog drk by using the drke0A null allele. We expected to see fusion defects in dock drk double mutants if both genes cooperate during myoblast fusion. However, no fusion defects were observed (Fig. 1D).

Previously, we showed that zygotic wasp mutants show no fusion defects due to maternal wasp mRNA (Schäfer et al., 2007). The dock mRNA is also maternally contributed (Desai et al., 1999). For this reason we assessed whether maternal contributed dock might be sufficient to complete myogenesis. By using the dock deficiency Df(2L)ast2 we found that maternal Dock is detectable till stage 15 when myogenesis is almost completed (Fig. 1E,E′).

Dock binds Vrp1 via its SH3-1 and SH3-3 domain and WASp via all SH3 domains

The Dock protein contains three SH3 domains and a C-terminally located SH2 domain (Fig. 2A). The SH3 domains bind to proline-rich segments whereas SH2 domains bind to phosphotyrosine residues in a target protein (Li et al., 2001). WASp and its interaction partner Wip have been both identified to be downstream effectors of the mammalian Dock homolog Nck (Rivero-Lezcano et al., 1995; Quilliam et al., 1996; Antón et al., 1998). Drosophila possesses only one WASp and Wip protein called Vrp1. Like their mammalian homologs, Drosophila Vrp1 and WASp possess proline-rich sequences (Fig. 2A).

Fig. 2.

Dock binds Drosophila Vrp1 and WASp. (A) Schematic representation of the Dock, Vrp1 and WASp domain structure. WASp: src-homology domain 3 (SH3), src-homology domain 2 (SH2), WASp homology 1 domain (WH1), basic region (B), GTP-binding domain (GBD), verprolin homology (V), cofilin homology (C), acidic domain (A). Vrp1: proline-rich segment (PPP), WASp homology 2 domain (WH2), Dock: SH3-1, SH3-2, SH3-3, SH2. (B) Immunoblots of lysates and immunoprecipitates from transfected COS7 cells. Dock interacts with WASp in either the presence or absence of Vrp1 (lanes 4, 5, 8, 9). (C–E) Yeast two-hybrid assay. (C) Dock interacts with the proline-rich segments of Vrp1 and WASp (sectors 1, 3, 7 and 9). (D) The proline-rich segment of Vrp1 interacts with the SH3-1 and SH3-3 domain of Dock (sectors 1 and 5). (E) The proline-rich segment of WASp interacts with all SH3 domains of Dock (sectors 1, 3, 5 and 7).

Fig. 2.

Dock binds Drosophila Vrp1 and WASp. (A) Schematic representation of the Dock, Vrp1 and WASp domain structure. WASp: src-homology domain 3 (SH3), src-homology domain 2 (SH2), WASp homology 1 domain (WH1), basic region (B), GTP-binding domain (GBD), verprolin homology (V), cofilin homology (C), acidic domain (A). Vrp1: proline-rich segment (PPP), WASp homology 2 domain (WH2), Dock: SH3-1, SH3-2, SH3-3, SH2. (B) Immunoblots of lysates and immunoprecipitates from transfected COS7 cells. Dock interacts with WASp in either the presence or absence of Vrp1 (lanes 4, 5, 8, 9). (C–E) Yeast two-hybrid assay. (C) Dock interacts with the proline-rich segments of Vrp1 and WASp (sectors 1, 3, 7 and 9). (D) The proline-rich segment of Vrp1 interacts with the SH3-1 and SH3-3 domain of Dock (sectors 1 and 5). (E) The proline-rich segment of WASp interacts with all SH3 domains of Dock (sectors 1, 3, 5 and 7).

To address whether Drosophila Dock is involved in actin polymerization during myoblast fusion, we examined protein–protein interactions using co-immunoprecipitation and yeast-two hybrid assays. We transiently co-transfected COS7 cells with FLAG-tagged Dock, untagged Vrp1 and HA-tagged WASp (Fig. 2B, lanes 1–5 and 6–9). FLAG-tagged Dock, Vrp1 and HA-tagged WASp were immunoprecipitated by anti-FLAG (Fig. 2B, lanes 1–5). These data show that Dock forms a protein complex with Vrp1 and WASp. Moreover, co-immunoprecipitation of FLAG-tagged Dock, Vrp1 and HA-tagged WASp demonstrate that both Vrp1 and WASp are associated with Dock (Fig. 2B, lane 5). We confirmed these results by immunoprecipitating HA-tagged WASp and WASp associated proteins with anti-HA (Fig. 2B, lanes 6–9).

Next, we assessed which domains of Vrp1 and WASp mediate Dock binding by using the yeast two-hybrid system. We generated Vrp1 full length, WASp full length, Vrp1-PPP and WASp-PPP constructs that contain only the middle proline-rich region of Vrp1 and WASp, as marked in red in Fig. 2A, as well as Vrp1 and WASp deletion constructs lacking the proline-rich region. Binding of Dock to Vrp1 and WASp was only achieved when the middle proline-rich region of Vrp1 and WASp is present (Fig. 2C, sectors 1, 3, 7 and 9). In the absence of this proline-rich region, Dock fails to bind to Vrp1 and WASp (Fig. 2C, sectors 5 and 11). To determine which domains in Dock mediate binding to Vrp1 and WASp, we generated deletion constructs that lack only one of the SH3 domains (SH3-1, SH3-2 or SH3-3), all SH3 domains and only the SH2 domain. We found that only the SH3-1 and SH3-3 domains are essential for binding to Vrp1 (Fig. 2D, sectors 1, 3 and 5) and that all SH3 domains are required for binding to WASp (Fig. 2E, sectors 1, 3, 5 and 7).

Myoblast fusion in dock vrp1 and dock wasp double mutants is severely disturbed

We next examined whether dock, vrp1 and wasp interact genetically during myoblast fusion. Mutations in vrp1 cause severe fusion defects (Fig. 3A), while some fusion events still take place in vrp1 null mutants (Kim et al., 2007; Massarwa et al., 2007; Berger et al., 2008). The wasp3 mutation carries an intragenic deletion of 13 bp, leading to a truncated protein that lacks the VVCA domain of WASp (Schäfer et al., 2007). Because of a high maternal contribution, homozygous wasp3 mutants show a wild-type muscle pattern (Fig. 3C). We used the protein null allele vrp1f06715 (Berger et al., 2008) and the wasp3 allele (Ben-Yaacov et al., 2001) to generate double mutants with the dock null allele dock04723. In comparison to the single mutant phenotypes (Fig. 3A,C), we observed enhanced fusion defects in homozygous vrp1f06715 dock04723 (Fig. 3B) and wasp3 dock04723 double mutants (Fig. 3D). We further quantified the fusion defect of vrp1f06715 and dock04723 single and vrp1f06715 dock04723 and wasp3 dock04723 double mutants statistically (Table 1) by examining the number of nuclei of the segmental border muscle (SBM) with anti-Ladybird (Jagla et al., 1998).

Fig. 3.

dock vrp1 and dock wasp double mutants show enhanced fusion defects, but formation of F-actin foci and cytoplasmic continuity is achieved in vrp1 dock double mutants. (A–D) Lateral view of stage 16 embryos stained with anti-β3-Tubulin to visualize muscles and mononucleated myoblasts. (A) Homozygous vrp1f06715 single mutant embryo. (B) Homozygous vrp1f06715 dock04723 double mutant embryo with severe defects in myoblast fusion. (C) Homozygous wasp3 single mutant embryo. (D) Homozygous wasp3 dock04723 double mutant embryo. (E,F) Stage 15 embryos stained with phalloidin (red) and anti-β3-Tubulin (green). (E) Wild-type embryo showing a dense F-actin focus (arrow) in an FCM (white asterisks). (E′) Overlay with β3-Tubulin showing that the F-actin focus contacts a growing myotube. (F) Two FCMs contacting a growing myotube (gray and white asterisks). In one of them (white asterisks) a dense actin focus (arrow) is visible. The other one (gray asterisks) does not show an actin focus, but shows an expanded cell–cell contact. (F′) Overlay with β3-Tubulin. (G,G′) TEM analysis of fusing myoblasts in a stage 14 homozygous vrp1f06715 dock04723 double mutant embryo. (G) An FC is contacted by three FCMs. FCM 1 extends a filopodium towards the FC. (G′) Higher-magnification view of the boxed area in G showing that outer membranes of the FC and FCM 2 have fused (arrow) and that small discontinuities have formed between the apposing membranes. N, cell nucleus. Original magnification: (G) 3600×; (G′) 15,000×.

Fig. 3.

dock vrp1 and dock wasp double mutants show enhanced fusion defects, but formation of F-actin foci and cytoplasmic continuity is achieved in vrp1 dock double mutants. (A–D) Lateral view of stage 16 embryos stained with anti-β3-Tubulin to visualize muscles and mononucleated myoblasts. (A) Homozygous vrp1f06715 single mutant embryo. (B) Homozygous vrp1f06715 dock04723 double mutant embryo with severe defects in myoblast fusion. (C) Homozygous wasp3 single mutant embryo. (D) Homozygous wasp3 dock04723 double mutant embryo. (E,F) Stage 15 embryos stained with phalloidin (red) and anti-β3-Tubulin (green). (E) Wild-type embryo showing a dense F-actin focus (arrow) in an FCM (white asterisks). (E′) Overlay with β3-Tubulin showing that the F-actin focus contacts a growing myotube. (F) Two FCMs contacting a growing myotube (gray and white asterisks). In one of them (white asterisks) a dense actin focus (arrow) is visible. The other one (gray asterisks) does not show an actin focus, but shows an expanded cell–cell contact. (F′) Overlay with β3-Tubulin. (G,G′) TEM analysis of fusing myoblasts in a stage 14 homozygous vrp1f06715 dock04723 double mutant embryo. (G) An FC is contacted by three FCMs. FCM 1 extends a filopodium towards the FC. (G′) Higher-magnification view of the boxed area in G showing that outer membranes of the FC and FCM 2 have fused (arrow) and that small discontinuities have formed between the apposing membranes. N, cell nucleus. Original magnification: (G) 3600×; (G′) 15,000×.

Table 1.
Quantification of SBM and VA2 nuclei
Genotype Number of nuclei SBM* Number of nuclei VA2* Number of segments Number of embryos 
wild-type 6.9±0.7 (5–8) 9.0±0.98 (7–11) 6141 1913 
dock04723 6.3±0.8 (5–8)  13 
hbs459 6.2±0.67 (5–8)  38 
scarΔ37 4.47±1.59 (2–7)  51 12 
sns20-15 5.15±0.84 (3–7)  53 13 
vrp1f06715 4.95±0.96 (4–7)  42 11 
wasp3 4.6±0.49 (4–5)  15 
hbs459 vrp1f06715 2.95±1.84 (1–5)  45 12 
scarΔ37 dock04723 4.31±0.92 (3–6)  49 12 
vrp1f06715 dock04723 3.08±0.76 (2–4)  40 11 
wasp3; dock04723 4.04±0.71 (2–5)  23 
Mef2>duf-RNAi  7.37±0.99 (5–9) 52 13 
dock04723; Mef2>dock04723; duf-RNAi  6.4±0.74 (5–8) 15 
Genotype Number of nuclei SBM* Number of nuclei VA2* Number of segments Number of embryos 
wild-type 6.9±0.7 (5–8) 9.0±0.98 (7–11) 6141 1913 
dock04723 6.3±0.8 (5–8)  13 
hbs459 6.2±0.67 (5–8)  38 
scarΔ37 4.47±1.59 (2–7)  51 12 
sns20-15 5.15±0.84 (3–7)  53 13 
vrp1f06715 4.95±0.96 (4–7)  42 11 
wasp3 4.6±0.49 (4–5)  15 
hbs459 vrp1f06715 2.95±1.84 (1–5)  45 12 
scarΔ37 dock04723 4.31±0.92 (3–6)  49 12 
vrp1f06715 dock04723 3.08±0.76 (2–4)  40 11 
wasp3; dock04723 4.04±0.71 (2–5)  23 
Mef2>duf-RNAi  7.37±0.99 (5–9) 52 13 
dock04723; Mef2>dock04723; duf-RNAi  6.4±0.74 (5–8) 15 

Nuclei of the SBM muscle were visualized by anti-Dmef2 and anti-Ladybird stainings of stage 15/16 embryos. Nuclei of the VA2 muscle were visualized by anti-Dmef2 and anti-Slouch stainings of stage 15/16 embryos.

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Average number of nuclei ± standard deviation (range of nuclei numbers).

vrp1 dock double mutants are still able to form F-actin foci and achieve cytoplasmic continuity

A characteristic feature during myoblast fusion is the formation of an F-actin focus at cell–cell contact sites in FCMs (Kesper et al., 2007; Kim et al., 2007; Massarwa et al., 2007; Richardson et al., 2008; Sens et al., 2010; Haralalka et al., 2011). F-actin foci are highly dynamic structures that reach their maximum size within two minutes and dissolve shortly afterwards in less than one minute (Richardson et al., 2007). Interestingly, F-actin foci formation still occurs in mutants affecting Arp2/3-based actin polymerization. However, in some mutants the F-actin foci are enlarged and increased in number (Richardson et al., 2007). Only in scar vrp1 double mutants, in which myoblast fusion is almost completely blocked (Berger et al., 2008), the size of the F-actin focus is severely decreased (Sens et al., 2010). To characterize vrp1f06715 dock04723 double mutants in more detail, and to determine whether F-actin foci still form in homozygous double mutants, we visualized actin polymerization using phalloidin. In wild-type embryos, an F-actin focus forms at the site of contact between an FCM and a growing myotube (Fig. 3E,E′, arrows). The F-actin focus is still visible in vrp1f06715 dock04723 double mutants and occurs at the site of the FCM attaching to a growing myotube (Fig. 3F,F′, arrows). Furthermore, F-actin foci seem to be able to dissolve in FCMs, showing an expanded cell–cell contact site (Fig. 3F,F′, gray asterisks).

In Drosophila, myoblast fusion has been characterized at the ultrastructural level by transmission electron microscopy (TEM) analysis. Cell adhesion and alignment of myoblast cell membranes are accompanied by the appearance of electron-dense vesicles and plaques on both sites of the apposing membranes. The fusion of the apposing membranes can be followed by the initiation of cytoplasmic continuity (Doberstein et al., 1997; Massarwa et al., 2007; Berger et al., 2008). Additionally, recent studies using freeze substitution have observed the formation of finger-like protrusions in FCMs that project into the FC/growing myotube (Sens et al., 2010). TEM analyses on conventional fixed vrp1 mutants revealed that homozygous vrp1 mutants stop fusion when multiple discontinuities are visible at apposing membranes (Massarwa et al., 2007). The stronger fusion defect in vrp1f06715 dock04723 double mutants in comparison to vrp1f06715 single mutants, let us determine at which ultrastructural level vrp1f06715 dock04723 double mutants stop fusion. Using conventional TEM fixation methods, we found that FCMs in vrp1f06715 dock04723 double mutants are still able to form filopodia (Fig. 3G) and that fusion stops when discontinuities are visible at apposing membranes (Fig. 3G′, arrows).

Dock interacts with Hbs genetically, and biochemically via its SH2 domain

To address whether Dock links cell adhesion with actin polymerization in FCMs we generated dock hbs double mutants and observed severe fusion defects in those mutants (Fig. 4B,B′; Table 1).

Fig. 4.

Dock interacts with Hbs in FMCs through its SH2 domain. (A,B) Lateral view of stage 16 embryos stained with anti-β3-Tubulin. (A) Homozygous hbs459 mutant embryo. (A′) Higher-magnification view of part of A. (B) Homozygous hbs459 dock04723 mutant embryo. (B′) Higher-magnification view of part of B. (C) Dock interacts with the cytodomain of Hbs (lane 4). Lanes 1 and 5 show lysates from Dock and Hbsintra-transfected cells before immunoprecipitation with anti-HA. Lanes 2, 3, 6, 7 and 8 show lysates after immunoprecipitation and were either probed with anti-FLAG (lanes 2 and 3) or with anti-HA (lanes 6–8). (D) Schematic representation of Dock full-length in the N-YFP vector and Hbsintra in the C-YFP vector. (E–H) Transfected Drosophila S2 cells with (E) Hbsintra and the empty N-YFP vector, (F) Hbsintra and Dock-Fl, (G) Hbsintra and Dock lacking all SH3 domains and (H) Hbsintra and Dock lacking its SH2 domain. (I–L) Transfected Drosophila S2 cells with (I) mutated HbsT1088F and the empty N-YFP vector, (J) HbsT1088F and Dock-Fl, (K) HbsT1088F and Dock lacking all SH3 domains and (L) HbsT1088F and Dock lacking its SH2 domain. Scale bars: 5 µm. Red: Phalloidin, yellow: YFP.

Fig. 4.

Dock interacts with Hbs in FMCs through its SH2 domain. (A,B) Lateral view of stage 16 embryos stained with anti-β3-Tubulin. (A) Homozygous hbs459 mutant embryo. (A′) Higher-magnification view of part of A. (B) Homozygous hbs459 dock04723 mutant embryo. (B′) Higher-magnification view of part of B. (C) Dock interacts with the cytodomain of Hbs (lane 4). Lanes 1 and 5 show lysates from Dock and Hbsintra-transfected cells before immunoprecipitation with anti-HA. Lanes 2, 3, 6, 7 and 8 show lysates after immunoprecipitation and were either probed with anti-FLAG (lanes 2 and 3) or with anti-HA (lanes 6–8). (D) Schematic representation of Dock full-length in the N-YFP vector and Hbsintra in the C-YFP vector. (E–H) Transfected Drosophila S2 cells with (E) Hbsintra and the empty N-YFP vector, (F) Hbsintra and Dock-Fl, (G) Hbsintra and Dock lacking all SH3 domains and (H) Hbsintra and Dock lacking its SH2 domain. (I–L) Transfected Drosophila S2 cells with (I) mutated HbsT1088F and the empty N-YFP vector, (J) HbsT1088F and Dock-Fl, (K) HbsT1088F and Dock lacking all SH3 domains and (L) HbsT1088F and Dock lacking its SH2 domain. Scale bars: 5 µm. Red: Phalloidin, yellow: YFP.

To determine whether Hbs and Dock also interact at the protein level, we performed co-immunoprecipitation studies (Fig. 4C) and bimolecular fluorescence complementation (BiFC) assays. FLAG-tagged Dock and HA-tagged Hbsintra, containing only the cytodomain of Hbs, were transiently transfected into S2 cells. FLAG-tagged Dock was successfully immunoprecipitated with HA-tagged Hbsintra (Fig. 4C, lanes 2, 3 and 4). Lysates were immunoprecipitated with anti-HA antisera (Fig. 4C).

We then asked which domains are involved in mediating the interaction between Dock and Hbsintra by using the BiFC vectors N-YFP and C-YFP (Dottermusch-Heidel et al., 2012). The BiFC assay enables direct visualization of protein interactions in living cells (Kerppola et al., 2008). When co-expressed in S2 cells the non-fluorescent N-YFP and C-YFP fragments show no YFP fluorescence. A reconstitution of the split fluorescent protein only occurs when N-YFP and C-YFP are fused to interacting proteins. We cloned full-length Dock, Dock lacking all SH3 domains and Dock lacking the SH2 domain into the N-YFP vector. Hbsintra was cloned into the C-YFP vector (Fig. 4D). Drosophila S2 cells co-transfected with Hbsintra and the empty N-YFP vector did not show a reconstitution of the YFP signal (Fig. 4E). Only cells that were co-transfected with Hbsintra and Dock-Fl showed a YFP signal (Fig. 4F). The YFP signal was still observed in cells co-transfected with Hbsintra and Dock-ΔSH3, which lacks all SH3 domains (Fig. 4G). In contrast, no YFP reconstitution was observed in S2 cells co-transfected with Hbsintra and Dock-ΔSH2 (Fig. 4H). Based on these findings we concluded that the SH2 domain of Dock mediates interaction by binding to phosphorylated tyrosine residues within Hbsintra. The Nck SH2-binding site in Nephrin, the human ortholog of Hbs, has been identified as YDxV motif (Jones et al., 2006). Hbsintra contains six tyrosine residues that might become phosphorylated (Blom et al., 1999). However, only the tyrosine residue at amino acid position 1088 is a related YDxV motif. To map the Dock-interaction site in Hbs, we replaced this tyrosine residue with phenylalanine (Hbsintra T1088F). As a negative control, we co-transfected cells with mutated Hbsintra T1088F and the empty N-YFP vector (Fig. 4I). When Hbsintra T1088F was co-transfected with Dock-Fl, Dock-ΔSH3 or Dock-ΔSH2, reconstitution of the YFP signal failed (Fig. 4J,K,L), demonstrating that Y1088 is critical for Hbs–Dock interaction.

We then asked which domains are involved in mediating the interaction between Dock and Hbsintra by using the BiFC vectors N-YFP and C-YFP (Dottermusch-Heidel et al., 2012). The BiFC assay enables direct visualization of protein interactions in living cells (Kerppola et al., 2008). When co-expressed in S2 cells the non-fluorescent N-YFP and C-YFP fragments show no YFP fluorescence. A reconstitution of the split fluorescent protein only occurs when N-YFP and C-YFP are fused to interacting proteins. We cloned full-length Dock, Dock lacking all SH3 domains and Dock lacking the SH2 domain into the N-YFP vector. Hbsintra was cloned into the C-YFP vector (Fig. 4D). Drosophila S2 cells co-transfected with Hbsintra and the empty N-YFP vector did not show a reconstitution of the YFP signal (Fig. 4E). Only cells that were co-transfected with Hbsintra and Dock-Fl showed a YFP signal (Fig. 4F). The YFP signal was still observed in cells co-transfected with Hbsintra and Dock-ΔSH3, which lacks all SH3 domains (Fig. 4G). In contrast, no YFP reconstitution was observed in S2 cells co-transfected with Hbsintra and Dock-ΔSH2 (Fig. 4H). Based on these findings we concluded that the SH2 domain of Dock mediates interaction by binding to phosphorylated tyrosine residues within Hbsintra. The Nck SH2-binding site in Nephrin, the human ortholog of Hbs, has been identified as YDxV motif (Jones et al., 2006). Hbsintra contains six tyrosine residues that might become phosphorylated (Blom et al., 1999). However, only the tyrosine residue at amino acid position 1088 is a related YDxV motif. To map the Dock-interaction site in Hbs, we replaced this tyrosine residue with phenylalanine (Hbsintra T1088F). As a negative control, we co-transfected cells with mutated Hbsintra T1088F and the empty N-YFP vector (Fig. 4I). When Hbsintra T1088F was co-transfected with Dock-Fl, Dock-ΔSH3 or Dock-ΔSH2, reconstitution of the YFP signal failed (Fig. 4J,K,L), demonstrating that Y1088 is critical for Hbs–Dock interaction.

Sns interacts genetically with Dock and Vrp1

We next examined zygotic mutant combinations of sns4.3, a hypomorphic allele with a point mutation in the FNIII domain and the dock null allele dock04723 (Fig. 5A–E). We did not observe a dramatic enhancement of the myoblast fusion defects. However, quantification of the unfused myoblasts in confocal Z series of four abdominal hemisegments revealed a statistically significant increase of 30–40% in the number of unfused myoblasts in embryos homozygous for sns4.3 when either one or both alleles of dock were mutant (Fig. 5F). Furthermore, we generated a sns vrp1 double mutant carrying the hypomorphic sns allele sns20-15 (Paululat et al., 1995) and the vrp1 null allele wipD30 (Massarwa et al., 2007). Homozygous double mutants possess a stronger myoblast fusion defect (Fig. 5H,I) than sns20-15 (Fig. 5G,I) and vrp1 single mutants (Fig. 5I).

Fig. 5.

sns interacts genetically with dock and vrp1, and Dock colocalizes with Sns and Duf at cell–cell contact points. (A–E) Lateral view of stage 16 embryos stained with anti-myosin heavy chain. White spots indicate the unfused myoblasts, as identified by analysis of individual sections of the confocal Z-series. (A) Homozygous sns4.3 embryo. (B) Embryo homozygous for sns4.3 and heterozygous for dock04723. (C) Embryo homozygous for sns4.3 and dock04723. (A′,B′,C′) Internal confocal sections of the above projections, in which unfused myoblasts (arrows) are most apparent. Multinucleated muscles are indicated by arrowheads. (D) Embryo homozygous for dock04723 and heterozygous for sns4.3. (E) Embryo homozygous for dock04723. (F) Quantification of unfused myoblasts. Error bars show the s.e.m. (G,H) Lateral view of stage 16 embryos stained with anti-β3-Tubulin. (G) Homozygous sns20-15 mutant embryo with unfused myoblasts (arrowhead) and multinucleated muscles (arrow). (H) Homozygous sns20-15 wipD30 double mutant embryos with severe fusion defects (arrow and arrowhead). (I) Quantification of fusions in G and H, determined by the number of nuclei in the Ladybird-expressing muscle. (J–N) Primary myoblast cultures 14 hours after plating showing an FCM that contacts a growing myotube. (J) DIC optic. (K) Overlay. Cultures were stained for Sns (red), Dock (green) and Duf (blue; L-N). (L) Sns is expressed on the site of the FCM. (M,N) Dock and Duf are, like Sns, expressed at cell–cell contact points.

Fig. 5.

sns interacts genetically with dock and vrp1, and Dock colocalizes with Sns and Duf at cell–cell contact points. (A–E) Lateral view of stage 16 embryos stained with anti-myosin heavy chain. White spots indicate the unfused myoblasts, as identified by analysis of individual sections of the confocal Z-series. (A) Homozygous sns4.3 embryo. (B) Embryo homozygous for sns4.3 and heterozygous for dock04723. (C) Embryo homozygous for sns4.3 and dock04723. (A′,B′,C′) Internal confocal sections of the above projections, in which unfused myoblasts (arrows) are most apparent. Multinucleated muscles are indicated by arrowheads. (D) Embryo homozygous for dock04723 and heterozygous for sns4.3. (E) Embryo homozygous for dock04723. (F) Quantification of unfused myoblasts. Error bars show the s.e.m. (G,H) Lateral view of stage 16 embryos stained with anti-β3-Tubulin. (G) Homozygous sns20-15 mutant embryo with unfused myoblasts (arrowhead) and multinucleated muscles (arrow). (H) Homozygous sns20-15 wipD30 double mutant embryos with severe fusion defects (arrow and arrowhead). (I) Quantification of fusions in G and H, determined by the number of nuclei in the Ladybird-expressing muscle. (J–N) Primary myoblast cultures 14 hours after plating showing an FCM that contacts a growing myotube. (J) DIC optic. (K) Overlay. Cultures were stained for Sns (red), Dock (green) and Duf (blue; L-N). (L) Sns is expressed on the site of the FCM. (M,N) Dock and Duf are, like Sns, expressed at cell–cell contact points.

We further examined the localization of Dock, by comparison to Sns and Duf in primary myoblasts, which provide better resolution of expression at points of cell contact. Dock colocalizes with Sns and Duf at points of cell–cell contact between primary FCs and FCMs (Fig. 5J–N). In summary, these data support a model in which Sns interacts with Dock to promote Vrp1–WASp-based actin polymerization during myoblast fusion.

Dock interacts biochemically via its SH3 domains with Sns

To assess whether Sns interacts biochemically with Dock and to define the regions that are critical for binding, we combined co-immunoprecipitation studies with BiFC assays. First, Drosophila S2 cells were transiently transfected with HA-tagged Sns and FLAG-tagged Dock. FLAG-tagged Dock was efficiently immunoprecipitated with HA-tagged full-length Sns (Fig. 6A, lanes 1 and 2). The Dock binding ability of HA-tagged Sns was significantly reduced (Fig. 6A, lane 3) when using a mutated form of Sns that lacks all phosphotyrosine residues and the two consensus PxxP motifs, and is compromised in its ability to rescue sns mutant embryos (Kocherlakota et al., 2008). To map the interaction domains within Dock, S2 cells were co-transfected with full-length HA-tagged Sns and a fragment containing only the FLAG-tagged Dock SH2 domain or the three SH3 domains. Our data demonstrate that both the SH2 and SH3 domains of Dock, in isolation, can bind to Sns (Fig. 6A, lanes 4–7). To evaluate regions of Sns that are critical for this binding, we utilized an HA-tagged intracellular domain of Sns. In contrast to full-length Sns, which bound to the Dock SH2 domain, Snsintra does not interact with FLAG-tagged Dock SH2 (data not shown). This finding is consistent with the possibility that phosphorylation of tyrosines in the intracellular domain requires full-length Sns at least in S2 cells. In contrast, full-length FLAG-tagged Dock and FLAG-tagged Dock containing all three SH3 domains co-immunoprecipitated efficiently with Snsintra (Fig. 6B, lanes 8–10). This interaction is reduced to near background levels upon mutation of the two Sns consensus PxxP sites (Fig. 6B, lanes 11–13) and is in line with functional studies showing that Sns lacking all cytodomain tyrosines and the two consensus PxxP motifs severely reduced rescue of sns mutant embryos (Kocherlakota et al., 2008). We interpret these results to suggest that additional non-consensus SH3 binding sites must be present within Sns, and find that binding of Snsintra and Dock-SH3 is further reduced by mutation of non-PxxP sites in Sns (Fig. 6B, lanes 14–16). These data suggest that Sns and Dock can interact through either the Dock SH2 or Dock SH3 domains, and that the latter binding relies primarily if not exclusively on consensus PxxP sites in Sns.

Fig. 6.

Full length Sns interacts with the SH2 and SH3 domains of Dock, but Snsintra only interacts with the SH3 domain of Dock. (A,B) Immunoblots of lysates and immunoprecipitates of transfected Drosophila S2 cells. (A) Sns full-length interacts Dock full-length (lane 2), the SH2 domain (lane 4) and the SH3 domains (lane 6) of Dock. The deletion of the 14 tyrosine residues and the two consensus PxxP motifs in Snsintra reduces Dock binding (lane 3). (B) Snsintra binds to Dock full-length (lane 9) and to the SH3 domains of Dock (lanes 10, 14). The binding is reduced when the two consensus PxxP motifs in Snsintra are deleted (lanes 12, 13 and 15). Additional mutation of the non PxxP sites in Snsintra 2xPxxP further reduces binding (lane 16). (C) Schematic representation of Dock full-length in the N-YFP vector and Snsintra in the C-YFP vector. (D–L) BiFC assay on transfected S2 cells with (D) Snsintra and Dock-Fl, (E) Snsintra and Dock lacking its SH2 domain, (F) Snsintra and Dock lacking all SH3 domains, (G) Snsintra missing the two consensus PxxP motifs and Dock-Fl, (H) Snsintra 2xPxxP and Dock lacking its SH2 domain, (I) Snsintra 2xPxxP and Dock lacking all SH3 domains, (J) Snsintra missing 14 mutated phosphotyrosine residues and the two PxxP motifs and Dock-Fl, (K) SnsintraF14-2xPxxP and Dock lacking its SH2 domain, (L) SnsintraF14-2xPxxP and Dock lacking all SH3 domains. Scale bars: 5 µm.

Fig. 6.

Full length Sns interacts with the SH2 and SH3 domains of Dock, but Snsintra only interacts with the SH3 domain of Dock. (A,B) Immunoblots of lysates and immunoprecipitates of transfected Drosophila S2 cells. (A) Sns full-length interacts Dock full-length (lane 2), the SH2 domain (lane 4) and the SH3 domains (lane 6) of Dock. The deletion of the 14 tyrosine residues and the two consensus PxxP motifs in Snsintra reduces Dock binding (lane 3). (B) Snsintra binds to Dock full-length (lane 9) and to the SH3 domains of Dock (lanes 10, 14). The binding is reduced when the two consensus PxxP motifs in Snsintra are deleted (lanes 12, 13 and 15). Additional mutation of the non PxxP sites in Snsintra 2xPxxP further reduces binding (lane 16). (C) Schematic representation of Dock full-length in the N-YFP vector and Snsintra in the C-YFP vector. (D–L) BiFC assay on transfected S2 cells with (D) Snsintra and Dock-Fl, (E) Snsintra and Dock lacking its SH2 domain, (F) Snsintra and Dock lacking all SH3 domains, (G) Snsintra missing the two consensus PxxP motifs and Dock-Fl, (H) Snsintra 2xPxxP and Dock lacking its SH2 domain, (I) Snsintra 2xPxxP and Dock lacking all SH3 domains, (J) Snsintra missing 14 mutated phosphotyrosine residues and the two PxxP motifs and Dock-Fl, (K) SnsintraF14-2xPxxP and Dock lacking its SH2 domain, (L) SnsintraF14-2xPxxP and Dock lacking all SH3 domains. Scale bars: 5 µm.

We further examined the binding domains of both proteins in vivo using the BiFC assay (Fig. 6C). In agreement with our previous co-immunoprecipitation experiments (Fig. 6A, lane 2), S2 cells transfected with the Snsintra and Dock full-length (Dock-Fl) showed a reconstitution of the YFP signal (Fig. 6D). The reconstitution of YFP was still observed when we used DockΔSH2 instead of the Dock-Fl (Fig. 6E). However, no YFP signal was detectable with the DockΔSH3 deletion construct that lacks all SH3 domains (Fig. 6F). As with the above co-transfection studies, these data suggest that Snsintra alone is unable to interact with Dock SH2, possibly indicating that this isolated domain cannot be phosphorylated on tyrosine. These data also confirm that Dock interaction with Sns can be mediated through the Dock SH3 via proline-rich motifs in Sns. Consistent with the above studies, the YFP signal was still reconstituted using Snsintra2xPxxP (Fig. 6G–I) and SnsintraF14-2xPxxP (Fig. 6J–L).

Dock interacts with Duf and Rst at the protein level

In primary isolated myoblasts Dock colocalizes with the cell adhesion molecule Duf (Fig. 6A,C–E). Mammalian Neph1 is related to Drosophila Duf and Rst and co-operates with Nephrin to induce actin polymerization in kidney podocytes (Garg et al., 2007). Since F-actin is present at cell–cell contact points of both myoblast populations in Drosophila (Kesper et al., 2007; Sens et al., 2010; Haralalka et al., 2011), we also investigated whether Dock interacts with the Drosophila NEPH1 orthologs Duf and Rst. Our yeast two-hybrid data indicate that Dock binds to the cytodomain of Duf (Dufintra; Fig. 7A) via its SH3 domains (Fig. 7B). The cytodomain of Rst (Rstintra) did not show an interaction with Dock (data not shown). To verify these results, we performed a BiFC assay by co-transfecting Drosophila S2 cells with the constructs shown in Fig. 7C and with Dock constructs. Co-transfected S2 cells with Dufintra and Dock full length showed a reconstitution of the YFP signal (Fig. 7D). This reconstitution failed only with a Dock deletion construct that lacks all SH3 domains (Fig. 7F), but not when the SH2 domain was deleted (Fig. 7E). The cytodomain of Duf possesses a small proline-rich region to which the SH3 domains of Dock could bind. We therefore generated a DufΔ810-956aa deletion construct that lacks this region and tested it for interaction with Dock full length. No reconstitution of the YFP signal was observed (Fig. 7G), suggesting that the interacting region is located in this fragment.

Fig. 7.

Dock interacts with Dufintra through its SH3 domains and with Rstintra through its SH2 and SH3 domains. (A,B) Yeast two-hybrid assay. (A) Dufintra interacts with Dock full-length. (B) All SH3 domains mediate the interaction between Dufintra and Dock full-length (sectors 1, 3, 5 and 7). (C) Schematic representation of Dock full-length in the N-YFP vector, and Dufintra and Rstintra in the C-YFP vector. (D–K) BiFC assay on transfected Drosophila S2 cells with (D) Dufintra and Dock-Fl, (E) Dufintra and Dock lacking its SH2 domain, (F) Dufintra and Dock lacking all SH3 domains, (G) Dufintra containing an intracellular deletion of amino acids 810–956 and Dock-Fl, (H) Rstintra and Dock-Fl, (I) Rstintra and Dock lacking its SH2 domain, (J) Rstintra and Dock lacking all SH3 domains, (K) Rstintra and empty N-YFP vector. Scale bars: 5 µm.

Fig. 7.

Dock interacts with Dufintra through its SH3 domains and with Rstintra through its SH2 and SH3 domains. (A,B) Yeast two-hybrid assay. (A) Dufintra interacts with Dock full-length. (B) All SH3 domains mediate the interaction between Dufintra and Dock full-length (sectors 1, 3, 5 and 7). (C) Schematic representation of Dock full-length in the N-YFP vector, and Dufintra and Rstintra in the C-YFP vector. (D–K) BiFC assay on transfected Drosophila S2 cells with (D) Dufintra and Dock-Fl, (E) Dufintra and Dock lacking its SH2 domain, (F) Dufintra and Dock lacking all SH3 domains, (G) Dufintra containing an intracellular deletion of amino acids 810–956 and Dock-Fl, (H) Rstintra and Dock-Fl, (I) Rstintra and Dock lacking its SH2 domain, (J) Rstintra and Dock lacking all SH3 domains, (K) Rstintra and empty N-YFP vector. Scale bars: 5 µm.

We next investigated whether Rst is a potential Dock interaction partner. Rstintra is able to bind to Dock (Fig. 7H). Interestingly, our data indicate that Dock binds Rst via its SH2 and SH3 domains (Fig. 7I,J,K, control). To correlate the observed protein interactions with myoblast fusion, we also performed double mutant experiments. To generate duf; dock double mutants we crossed a duf-RNAi line in the background of dock04723 null mutants. Mesoderm-specific knockdown of duf was achieved using the Mef-GAL4 driver line (Fig. 8A,B). The knockdown of duf with Mef–GAL4 in a wild-type background did not induce any fusion defects (Fig. 8A). In contrast, the knockdown of duf with Mef–GAL4 in a dock04723 mutant background severely disturbed muscle formation (Fig. 8B). A quantitative analysis of the number of nuclei of the SBM showed no fusion defects. Only when we quantitatively analyzed the ventral VA2 muscle that contains up to 9 nuclei (Beckett and Baylies, 2007) the number of nuclei within this muscle was clearly reduced (Table 1). rst; dock double mutants used the loss of function allele rst6, which carries a 98 bp deletion in the cytodomain (Ramos et al., 1993). If rst and dock cooperate during myoblast fusion, we expected a severe defect in myoblast fusion. However, the muscle pattern of rst6; dock double mutants is reminiscent of rst6 single mutants (Fig. 8C,D). From these data, we cannot clearly deduce that the binding of Dock to Rst is essential for myoblast fusion.

Fig. 8.

dock interacts genetically with duf and scar. (A–F) Lateral view of stage 16 embryos stained with anti-β3-Tubulin. (A) The expression of duf-RNAi in all myoblasts in a wild-type background. (B) Expression of duf-RNAi in all myoblasts in a dock mutant background. (C) Homozygous rst6 mutant embryo. (D) rst6; dock04723 double mutant embryo. (E) Homozygous scarΔ37 mutant embryo. (F) Homozygous scarΔ37 dock04723 embryo with unfused myoblasts. (G) Schematic representation of the Scar domain structure: basic region (B), proline-rich segment (PPP), verprolin homology (V), cofilin homology (C) and acidic (A) domains. Dock full length interacts with Scar full length (sector 2), but not with the proline-rich segment of Scar (sector 3).

Fig. 8.

dock interacts genetically with duf and scar. (A–F) Lateral view of stage 16 embryos stained with anti-β3-Tubulin. (A) The expression of duf-RNAi in all myoblasts in a wild-type background. (B) Expression of duf-RNAi in all myoblasts in a dock mutant background. (C) Homozygous rst6 mutant embryo. (D) rst6; dock04723 double mutant embryo. (E) Homozygous scarΔ37 mutant embryo. (F) Homozygous scarΔ37 dock04723 embryo with unfused myoblasts. (G) Schematic representation of the Scar domain structure: basic region (B), proline-rich segment (PPP), verprolin homology (V), cofilin homology (C) and acidic (A) domains. Dock full length interacts with Scar full length (sector 2), but not with the proline-rich segment of Scar (sector 3).

Dock interacts with Scar to mediate myoblast fusion

Since Scar has been proposed to be responsible for the formation of a thin sheath of actin in FCs (Sens et al., 2010), we also investigated whether Dock may interact with Scar during myoblast fusion. Zygotic mutant combinations of the scar null allele scarΔ37 (Zallen et al., 2002) and the null allele dock04723 were examined for myoblast fusion defects. scarΔ37 single mutants show a weak fusion phenotype (Fig. 8E; Table 1); unfused myoblasts were clearly visible in scarΔ37 dock04723 double mutants (Fig. 8F).

Drosophila Scar possesses like Vrp1 and WASp a proline-rich segment (Fig. 8G, indicated in red). To assess whether Scar serves as a direct interaction partner for Dock we performed a yeast two-hybrid assay. Interestingly, we observed that Scar full-length binds to Dock (Fig. 8G, sector 2). However, this interaction does not seem to depend on the proline-rich region of Scar (Fig. 8G, sector 3).

Discussion

In response to extracellular signals, cell surface receptors must interact with intracellular signaling pathways to induce a cellular response. In this study we have identified the SH2–SH3 adaptor protein Dock as a new component in myoblast fusion. Based on protein interaction studies, immunostaining results and genetic interaction data, we suggest that Dock transfers the signal from the cell surface receptors to the actin cytoskeleton in FCs and FCMs.

Conserved signaling components between Nephs and Nephrins and their Drosophila orthologs

Nephs, Nephrins and their Drosophila orthologs are transmembrane receptors belonging to the immunoglobulin superfamily. Their extracellular domains can interact in trans, forming homodimers and heterodimers, and thus can serve as ligand-receptor pairs (Barletta et al., 2003; Galletta et al., 2004). The function of Nephs and Nephrins has been associated with the maintenance of the kidney filtration barrier (Tryggvason, 2001) and, despite their structural similarities with Drosophila orthologs, it initially appeared that they fulfill different functions during organ formation in vertebrates and insects. However, recent studies have demonstrated a conserved function for Nephs and Nephrins during vertebrate myoblast fusion (Srinivas et al., 2007; Sohn et al., 2009), as well as for their orthologs in Drosophila nephrocytes (Weavers et al., 2009; Zhuang et al., 2009).

Arp2/3-based actin polymerization is essential for both myoblast fusion (reviewed by Schejter and Baylies, 2010; Önel et al., 2011; Abmayr and Pavlath, 2012) and podocyte formation (Welsch et al., 2001). Since previous studies report that the intracellular domain of Neph1 and Nephrin become tyrosine phosphorylated, a modification that is required for their interaction with the SH2–SH3 adaptor proteins Nck and Grb2 (Jones et al., 2006; Garg et al., 2007), we assessed the role of the Drosophila homolog of Nck during myoblast fusion. Because Drosophila dock, drk (corresponding to Nck, Grb2) double mutants did not show any fusion defects, we concluded that the Drosophila homologs of Nck and Grb2 do not cooperate during myoblast fusion as they do during actin polymerization in mammalian podocytes (Garg et al., 2007). However, our biochemical and genetic data in this study support the notion that Dock functions in both myoblast types by binding to different motifs in Duf, Hbs and Sns (Fig. 9).

Fig. 9.

A model for Dock interactions with cell adhesion molecules and Scar, Vrp1–WASp-dependent actin regulation. In FCs Dock interacts with the proline-rich region within the cytodomain of the cell adhesion molecule Duf via its SH3 domains. This probably promotes Scar-dependent F-actin formation. In contrast, in FCMs Dock binds to the cytodomain of two cell adhesion molecules – Hbs and Sns. Thereby, the SH2 domain of Dock binds to phosphorylated T1088 within the cytodomain of Hbs and the SH3 domains to the cytodomain of Sns. These interactions might promote Scar- as well as Vrp1–WASp-dependent F-actin formation.

Fig. 9.

A model for Dock interactions with cell adhesion molecules and Scar, Vrp1–WASp-dependent actin regulation. In FCs Dock interacts with the proline-rich region within the cytodomain of the cell adhesion molecule Duf via its SH3 domains. This probably promotes Scar-dependent F-actin formation. In contrast, in FCMs Dock binds to the cytodomain of two cell adhesion molecules – Hbs and Sns. Thereby, the SH2 domain of Dock binds to phosphorylated T1088 within the cytodomain of Hbs and the SH3 domains to the cytodomain of Sns. These interactions might promote Scar- as well as Vrp1–WASp-dependent F-actin formation.

Diverse signaling cascades activating Arp2/3 polymerization in FCs and FCMs

Two actin nucleation-promoting factors are required during myoblast fusion to activate the Arp2/3 complex and promote F-actin sheath and foci formation in FCs and FCMs: WASp and Scar. Here we show that Dock biochemically interacts with Scar, Vrp1 and WASp (Fig. 9). The functional relevance of these biochemical interactions is also supported by genetic interaction studies with dock, scar, vrp1 and wasp. A direct interaction of Dock with mammalian Wip and WASp has been suggested in earlier studies (Quilliam et al., 1996; Antón et al., 1998). However, we found that the binding domains between mammalian and Drosophila Nck/Dock differ. Whereas the SH3-2 domain of Nck is essential for binding mammalian Wip (Antón et al., 1998) and the SH3-3 domain of Nck binds mammalian WASp (Quilliam et al., 1996), our study revealed that the SH3-1 and SH3-3 domain are required for binding to Drosophila Vrp1 and all SH3 domains are necessary for the interaction with Drosophila WASp.

A direct interaction between Dock and Scar has not been reported yet. Mammalian Nck has been identified as an interaction partner for the Nck-associated protein 1 (Nap1) (Kitamura et al., 1996), which is known as kette in Drosophila. Nap1/Kette is a member of the Scar complex that regulates Scar activity during Arp2/3-based actin polymerization (reviewed by Pollard, 2007). The activation of the Scar complex might be achieved by binding to activate Rac and Nck. However, the nature of the Scar complex regulation is still not clear yet and remains a matter of debate. The loss of rac1, 2 and kette disturbs Drosophila myoblast fusion (Hakeda-Suzuki et al., 2002; Schröter et al., 2004; Gildor et al., 2009). Rac (Vasyutina et al., 2009) and Nap1 (Nowak et al., 2009) are also essential for mammalian myoblast fusion, supporting the notion of a conserved function for Nck/Dock in mammalian and Drosophila myoblast fusion.

Our data further show that F-actin foci in FCMs are still present in dock vrp1 double mutants. This might be due to the presence of the SH2–SH3 adaptor protein Crk. Crk has been shown to bind the FCM-specific cell adhesion molecule Sns and Vrp1 by co-immunoprecipitation studies using Drosophila non-muscle S2 cells (Kim et al., 2007). Additionally, Crk is able to bind the FCM-specific protein Blow via its SH2 domain, which has been reported to compete with WASp for Vrp1 binding (Jin et al., 2011). Taking these data into consideration, it is possible that Dock may act independently of Crk in FCs, whereas it cooperates with Crk in FCMs to mediate actin polymerization.

Materials and Methods

Fly stocks

The dock04723 null allele, the deficiency Df(2R)ast2, the drke0A null allele, the rst6 allele and wingless–GAL4 were obtained from the Bloomington Stock Center. The UAS–duf RNAi line (Dietzl et al., 2007) was ordered from the Vienna Drosophila RNAi Center. The hbs459 allele was kindly provided by Mary Baylies (Sloan-Kettering Institute, New York). UAS–dockmyr was obtained from Sven Bogdan (Münster University). Eyal Schejter (Weizmann Institute of Science, Rehovot) kindly provided the wipD30 allele. The protein null allele vrp1f06715 was previously described in Berger et al. (Berger et al., 2008). The sns20-15 allele was previously described as rost15 by Paululat et al. (Paululat et al., 1995). The sns4.3 allele has not been previously described. This EMS-induced point mutation changes V1003E. The Mef2–GAL4 driver line has been described by Ranganaykulu et al. (Ranganaykulu et al., 1998). All crosses were performed at 25°C using standard procedures.

Immunohistochemistry

Embryos were collected at 25°C and processed as described (Hummel et al., 1997). Drosophila S2 cells and primary myoblast cultures were fixed in 4% F-PBS at 25°C, permeabilized in 0.1% Triton X-100 in PBS for 2.5 minutes and blocked in 5% BSA in PBS for 30 minutes. As primary antibodies we used the anti-β3-Tubulin antibody (1∶10,000) (Buttgereit et al., 1996; Leiss et al., 1988), anti-Dock antibody (1∶1000) (Clemens et al., 1996), anti-β-Gal antibody (1∶3000) (Cappel Laboratories), anti-Sns antibody (1∶1000) (Haralalka et al., 2011), anti-Duf antibody (1∶1000) (Galletta et al., 2004), anti-Ladybird (1∶500) (a gift from Krzysztof Jagla, Faculté de Médicine, France) and anti-Slouch (1∶200) (a gift from Manfred Frasch, Universität Erlangen, Germany). Cy2- and Cy3-conjugated secondary antibodies were purchased from Dianova. Embryos used for phalloidin staining were fixed for 30 minutes in 4% F-PBS and devitellinized by hand after fixation. Embryos were incubated in phalloidin–TRITC (1∶500; Sigma-Aldrich), anti-β3-Tubulin and anti-β-Gal antibodies overnight. Drosophila S2 cells were stained in phalloidin–TRITC (1∶20; Sigma-Aldrich) for 15 minutes after fixation. Primary myoblasts and myosin stained embryos were incubated with primary antibody over night at 4°C and secondary antibody for 3 hours at 25°C. Fluorescence images were acquired using a Leica TCSsp2 or Zeiss LSM-510 META confocal scanning microscope.

Quantification of fusion

For quantification of fusion in sns and dock recombinants, embryos were fluorescently stained for myosin heavy chain as described above. Comparable confocal Z-series were cropped to include four abdominal hemisegments between A2 and A6. Samples were de-identified and unfused myoblasts hand counted in Zeiss LSM software, as indicated by white spots in Fig. 5A–E. For statistical analysis, P-values were determined using the Student's t-test, and P-values of <0.0001 were interpreted to indicate statistically significant differences. Error was determined as standard error of the mean (s.e.m.).

DNA constructs

All constructs used in COS7 cells studies were generated in the pcDNA3.1 vector. Dock-FLAG included full-length Dock followed by three copies of a FLAG tag. In HA-WASp, full-length WASp is preceded by one HA tag. Full-length Vrp1 was untagged. For Drosophila S2 co-immunoprecipitation studies, UAS-Sns-HA has been described (Kocherlakota et al., 2008). UAS-Sns[F14-2xPxxP]-HA combines site-directed point mutations that change all 14 cytoplasmic tyrosine residues to alanine (UAS-SnsF14-HA; (Kocherlakota et al., 2008) and site directed mutations that change the cytoplasmic consensus PxxP proline rich regions (UAS-Sns2xPxxP) (Kocherlakota et al., 2008) into one construct. For Dock constructs with a single FLAG tag, full-length Dock, the Dock SH2 domain or all three Dock SH3 domains were cloned into the Gateway pTWF vector (Invitrogen). For studies using copper-inducible genes in pRmHa3, Sns-intra-HA includes AA1108–1482 followed by a single HA tag. Sns-intra[2xPxxP]-HA was generated by replacing the region of the wild-type parent with the 2xPxxP sequence above and previously described (Kocherlakota et al., 2008). Sns-Intra[RRKK-2xPxxP]-HA was generated by site-directed mutagenesis of the previous pRmHA3 construct to change the cytoplasmic RRKK sequence to alanines. pRmHa3-Dock-2xflag was generated by PCR amplification of full-length Dock from TWF-Dock above, using oligos that added EcoRI and NheI restriction sites. This fragment was subcloned into pRmHA3. Dock-SH3(3)2xFLAG was generated by PCR amplification of the Dock SH3 domain region (amino acid 1–279) and subcloning into pRmHA3.

Immunoprecipitation and western blotting

For studies in Fig. 2B, COS7 cells were grown in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and 5% CO2 at 37°C and transiently transfected with Effectene Transfection Reagent (Qiagen) as directed by the manufacturer. After 48 hours, cells were washed with PBS and lysed by addition of lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP40, 50 mM PMSF, 1 µg/ml leupeptin and pepstatin]. After 10 minutes, the lysate was collected, passed through a 25 gauge needle 12 times, and clarified by centrifugation at 15,000 g for 15 minutes. 800 µg of lysate was incubated with a 40 µl slurry of anti-HA or anti-FLAG beads (Sigma-Aldrich) for 5 hours at 4°C. Beads were washed 4× 10 minutes in washing buffer [20 mM Tris-HCl (pH7.5), 250 mM NaCl, 1 mM EDTA and 0.5% NP40], and then boiled for 5 minutes in 2× Laemmli buffer.

For studies in Fig. 4C, Drosophila S2 cells were cultured in Drosophila Schneider's medium (Invitrogen) containing 10% fetal bovine serum at 25°C, and transiently transfected using TransFectin Lipid Reagent (Bio-Rad). Transfected S2 cells were centrifuged at 13,000 g for three minutes at 4°C. Cell pellets were resuspended in lysis buffer. Cells were centrifuged at 13,000 g for 10 minutes and pellet was collected. 50 µl of 2× Laemmli buffer was added to the 50 µl of the supernatant and boiled for 10 minutes, this was used as Input. For HA-Hbsintra and Dock-FLAG co-IP, 50 µl of anti-HA (Sigma-Aldrich) agarose beads were taken and washed twice with 750 µl of IP buffer and the rest of the supernatant was added to the anti-HA agarose beads for 2–3 hours at 4°C. The mixture was spun down for 30 seconds at 2000 rpm and washed in cold IP buffer and boiled in 2× Laemmli buffer.

For studies in Fig. 6, Drosophila S2 cells were cultured in SFX medium (serum-free insect cell culture medium, HyClone) at 25°C, and transiently transfected using the Effectene Transfection Reagent (Qiagen). For Sns/Dock interaction studies genes were cloned into the pUAST vector. After transfection cells were washed with PBS and resuspended in crosslinking solution [1.5 mM dithiobis-succinimidyl propionate (DSP) in PBS] for 60 minutes. The samples were rewashed with PBS. Cell pellets were resuspended in lysis buffer [250 mM NaCl, 20 mM Tris-HCl (pH7.5), 0.5% NP40, 2 mM EDTA, 1× protease inhibitor cocktail (cOmplete, EDTA-free, Roche) and Na3VO4]. Lysate was passed through a 25 gauge needle 10 times, centrifuged at 13,000 g for 15 minutes at 4°C and the supernatant collected. For each sample, 500–1000 µg of cell lysate was incubated with 20 µl of 50% slurry of anti-HA beads (Sigma-Aldrich) overnight at 4°C. Beads were washed with Buffer A [20 mM Tris-HCl (pH 8.0), 250 mM NaCl, 1 mM EDTA, 0.5% NP40, protease inhibitor cocktail and Na3VO4] 2× 10 minutes and washed 2× 10 minutes with Buffer B [20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM EDTA, 0.5% NP40, protease inhibitor cocktail and Na3VO4] for 10 minutes. Beads were resuspended in loading buffer and processed as above. For transfections involving genes cloned into the RmHa3 vector (Fig. 6B), expression was induced with 0.5 mM CuSO4 20–24 hours after transfection. Cells were washed in PBS and pellets were resuspended in lysis buffer, and passed through a 25 gauge needle 10 times. Cells were centrifuged at 13,000 g for 15 minutes at 4°C. Equal volumes of supernatant and 2× Laemmli buffer were boiled for 5 minutes and used as input. 10 µl anti-FLAG resins (EZview; Sigma-Aldrich) was mixed with 1 mg of lysate for 4 hours at 4°C. The beads were washed with lysis buffer 4× 10 minutes at 4°C, and processed in 2× Laemmli buffer as above. Proteins were analyzed in immunoblots using anti-HA (1∶5000; Roche) or anti-FLAG (1∶5000; Sigma) using ECL plus.

Yeast two-hybrid assay

The yeast two-hybrid assay was performed using the Matchmaker LexA two-hybrid system (Clontech) according to the manufacturer's instructions. scar full-length, scar containing the proline-rich region (aa 388–491), vrp1 full-length, vrp1 lacking the proline-rich region (aa 348–635) and only containing the proline-rich region as well as wasp full-length, wasp lacking the proline-rich region (aa 310–415) and only containing the proline-rich region were amplified by PCR and cloned into pGilda. As PCR templates the scar cDNA SD02991, vrp1 cDNA GH25793 and the wasp cDNA RE12101 were used. Furthermore, the intracellular domains of Duf and Rst were amplified from the duf cDNA (Galletta et al., 2004) and the rst cDNA RE01586 (DGRC) and cloned into pGilda. dock and dock deletions were amplified from the LD42588 cDNA and cloned into pB42AD.

Acknowledgements

We thank Renate Renkawitz-Pohl and Christine Dottermusch-Heidel for fruitful discussions. We are grateful to Sabina Huhn, Tim Fries and Helga Kisselbach-Heckmann, Claude Shelton, Shufei Zhuang, Elspeth Pearce and Erin Katzfey for technical advice and assistance.

Funding

This work was supported by the Deutsche Forschungsgemeinschaft [grant number OE311/4-1 to S.-F.Ö.], the Graduate School 1216 award [grant number to S.-F.Ö.]; the European Molecular Biology Organization [grant number ASTF 284-2007 to G.S.]; the DFG Priority Programme (grant number SPP 1464 to S.B.); the Stowers Institute for Medical Research and the National Institutes of Health [grant number R01-AR44274 to S.M.A.]. Deposited in PMC for release after 12 months.

References

Abmayr
S. M.
,
Pavlath
G. K.
(
2012
).
Myoblast fusion: lessons from flies and mice.
Development
139
,
641
656
.
Antón
I. M.
,
Lu
W. G.
,
Mayer
B. J.
,
Ramesh
N.
,
Geha
R. S.
(
1998
).
The Wiskott-Aldrich syndrome protein-interacting protein (WIP) binds to the adaptor protein Nck.
J. Biol. Chem.
273
,
20992
20995
.
Artero
R. D.
,
Castanon
I.
,
Baylies
M. K.
(
2001
).
The immunoglobulin-like protein Hibris functions as a dose-dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling.
Development
128
,
4251
4264
.
Barletta
G. M.
,
Kovari
I. A.
,
Verma
R. K.
,
Kerjaschki
D.
,
Holzman
L. B.
(
2003
).
Nephrin and Neph1 co-localize at the podocyte foot process intercellular junction and form cis hetero-oligomers.
J. Biol. Chem.
278
,
19266
19271
.
Beckett
K.
,
Baylies
M. K.
(
2007
).
3D analysis of founder cell and fusion competent myoblast arrangements outlines a new model of myoblast fusion.
Dev. Biol.
309
,
113
125
.
Ben–Yaacov
S.
,
Le Borgne
R.
,
Abramson
I.
,
Schweisguth
F.
,
Schejter
E. D.
(
2001
).
Wasp, the Drosophila Wiskott-Aldrich syndrome gene homologue, is required for cell fate decisions mediated by Notch signaling.
J. Cell Biol.
152
,
1
13
.
Berger
S.
,
Schäfer
G.
,
Kesper
D. A.
,
Holz
A.
,
Eriksson
T.
,
Palmer
R. H.
,
Beck
L.
,
Klämbt
C.
,
Renkawitz–Pohl
R.
,
Önel
S. F.
(
2008
).
WASP and SCAR have distinct roles in activating the Arp2/3 complex during myoblast fusion.
J. Cell Sci.
121
,
1303
1313
.
Blom
N.
,
Gammeltoft
S.
,
Brunak
S.
(
1999
).
Sequence and structure-based prediction of eukaryotic protein phosphorylation sites.
J. Mol. Biol.
294
,
1351
1362
.
Bour
B. A.
,
Chakravarti
M.
,
West
J. M.
,
Abmayr
S. M.
(
2000
).
Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion.
Genes Dev.
14
,
1498
1511
.
Buttgereit
D.
,
Paululat
A.
,
Renkawitz–Pohl
R.
(
1996
).
Muscle development and attachment to the epidermis is accompanied by expression of beta 3 and beta 1 tubulin isotypes, respectively. Int J
Dev. Biol.
40
,
189
196
.
Clemens
J. C.
,
Ursuliak
Z.
,
Clemens
K. K.
,
Price
J. V.
,
Dixon
J. E.
(
1996
).
A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.
J. Biol. Chem.
271
,
17002
17005
.
Desai
C. J.
,
Garrity
P. A.
,
Keshishian
H.
,
Zipursky
S. L.
,
Zinn
K.
(
1999
).
The Drosophila SH2-SH3 adapter protein Dock is expressed in embryonic axons and facilitates synapse formation by the RP3 motoneuron.
Development
126
,
1527
1535
.
Dietzl
G.
,
Chen
D.
,
Schnorrer
F.
,
Su
K-C.
,
Barinova
Y.
,
Fellner
M.
,
Gasser
B.
,
Oppel
S.
,
Scheiblauer
S.
(
2007
).
A genome-wide transgenic RNAi library for conditional gene inactivation in
Drosophila. Nature
448
,
151
U1
.
Doberstein
S. K.
,
Fetter
R. D.
,
Mehta
A. Y.
,
Goodman
C. S.
(
1997
).
Genetic analysis of myoblast fusion: blown fuse is required for progression beyond the prefusion complex.
J. Cell Biol.
136
,
1249
1261
.
Dottermusch–Heidel
C.
,
Groth
V.
,
Beck
L.
,
Oenel
S. F.
(
2012
).
The Arf-GEF Schizo/Loner regulates N-cadherin to induce fusion competence of Drosophila myoblasts.
Dev. Biol.
368
,
18
27
. j.ydbio.2012.04.031
Dworak
H. A.
,
Charles
M. A.
,
Pellerano
L. B.
,
Sink
H.
(
2001
).
Characterization of Drosophila hibris, a gene related to human nephrin.
Development
128
,
4265
4276
.
Galletta
B. J.
,
Chakravarti
M.
,
Banerjee
R.
,
Abmayr
S. M.
(
2004
).
SNS: adhesive properties, localization requirements and ectodomain dependence in S2 cells and embryonic myoblasts.
Mechanisms of Development.
121
,
1455
1468
.
Garg
P.
,
Verma
R.
,
Nihalani
D.
,
Johnstone
D. B.
,
Holzman
L. B.
(
2007
).
Neph1 cooperates with nephrin to transduce a signal that induces actin polymerization.
Mol. Cell. Biol.
27
,
8698
8712
.
Gerke
P.
,
Huber
T. B.
,
Sellin
L.
,
Benzing
T.
,
Walz
G.
(
2003
).
Homodimerization and heterodimerization of the glomerular podocyte proteins nephrin and NEPH1.
J. Am. Soc. Nephrol.
14
,
918
926
.
Gildor
B.
,
Massarwa
R.
,
Shilo
B. Z.
,
Schejter
E. D.
(
2009
).
The SCAR and WASp nucleation-promoting factors act sequentially to mediate Drosophila myoblast fusion.
EMBO Rep.
10
,
1043
1050
.
Hakeda–Suzuki
S.
,
Ng
J.
,
Tzu
J.
,
Dietzl
G.
,
Sun
Y.
,
Harms
M.
,
Nardine
T.
,
Luo
L.
,
Dickson
B. J.
(
2002
).
Rac function and regulation during Drosophila development.
Nature
416
,
438
442
.
Haralalka
S.
,
Shelton
C.
,
Cartwright
H. N.
,
Katzfey
E.
,
Janzen
E.
,
Abmayr
S. M.
(
2011
).
Asymmetric Mbc, active Rac1 and F-actin foci in the fusion-competent myoblasts during myoblast fusion in Drosophila.
Development
138
,
1551
1562
.
Hummel
T.
,
Schimmelpfeng
K.
,
Klämbt
C.
(
1997
).
Fast and efficient egg collection and antibody staining from large numbers of Drosophila strains.
Dev. Genes Evol.
207
,
131
135
.
Jagla
T.
,
Bellard
F.
,
Lutz
Y.
,
Dretzen
G.
,
Bellard
M.
,
Jagla
K.
(
1998
).
ladybird determines cell fate decisions during diversification of Drosophila somatic muscles.
Development
125
,
3699
3708
.
Jin
P.
,
Duan
R.
,
Luo
F.
,
Zhang
G.
,
Hong
S. N.
,
Chen
E. H.
(
2011
).
Competition between Blown fuse and WASP for WIP binding regulates the dynamics of WASP-dependent actin polymerization in vivo.
Dev. Cell
20
,
623
638
.
Jones
N.
,
Blasutig
I. M.
,
Eremina
V.
,
Ruston
J. M.
,
Bladt
F.
,
Li
H. P.
,
Huang
H. M.
,
Larose
L.
,
Li
S. S. C.
,
Takano
T.
et al.  (
2006
).
Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes.
Nature
440
,
818
823
.
Kerppola
T. K.
2008
).
Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.
Annu. Rev. Biophys.
37
,
465
487
.
Kesper
D. A.
,
Stute
C.
,
Buttgereit
D.
,
Kreisköther
N.
,
Vishnu
S.
,
Fischbach
K. F.
,
Renkawitz–Pohl
R.
(
2007
).
Myoblast fusion in Drosophila melanogaster is mediated through a fusion-restricted myogenic-adhesive structure (FuRMAS).
Dev. Dyn.
236
,
404
415
.
Kim
S.
,
Shilagardi
K.
,
Zhang
S. L.
,
Hong
S. N.
,
Sens
K. L.
,
Bo
J.
,
Gonzalez
G. A.
,
Chen
E. H.
(
2007
).
A critical function for the actin cytoskeleton in targeted exocytosis of prefusion vesicles during myoblast fusion.
Dev. Cell
12
,
571
586
.
Kitamura
T.
,
Kitamura
Y.
,
Yonezawa
K.
,
Totty
N. F.
,
Gout
I.
,
Hara
K.
,
Waterfield
M. D.
,
Sakaue
M.
,
Ogawa
W.
,
Kasuga
M.
(
1996
).
Molecular cloning of p125Nap1, a protein that associates with an SH3 domain of Nck.
Biochem. Biophys. Res. Commun.
219
,
509
514
.
Kocherlakota
K. S.
,
Wu
J-M.
,
McDermott
J.
,
Abmayr
S. M.
(
2008
).
Analysis of the cell adhesion molecule sticks-and-stones reveals multiple redundant functional domains, protein-interaction motifs and phosphorylated tyrosines that direct myoblast fusion in Drosophila melanogaster.
Genetics
178
,
1371
1383
.
Leiss
D.
,
Hinz
U.
,
Gasch
A.
,
Mertz
R.
,
Renkawitz–Pohl
R.
(
1988
).
Beta-3 Tubulin expression characterizes the differentiating mesodermal germ layer during Drosophila embryogenesis.
Development
104
,
525
531
.
Li
W.
,
Fan
J.
,
Woodley
D. T.
(
2001
).
Nck/Dock: an adapter between cell surface receptors and the actin cytoskeleton.
Oncogene
20
,
6403
6417
.
Massarwa
R.
,
Carmon
S.
,
Shilo
B. Z.
,
Schejter
E. D.
(
2007
).
WIP/WASp-based actin-polymerization machinery is essential for myoblast fusion in Drosophila.
Dev. Cell
12
,
557
569
.
Nose
A.
,
Isshiki
T.
,
Takeichi
M.
(
1998
).
Regional specification of muscle progenitors in Drosophila: the role of the msh homeobox gene.
Development
125
,
215
223
.
Nowak
S. J.
,
Nahirney
P. C.
,
Hadjantonakis
A. K.
,
Baylies
M. K.
(
2009
).
Nap1-mediated actin remodeling is essential for mammalian myoblast fusion.
Journal of Cell Science
121
,
3282
3293
.
Önel
S-F.
,
Dottermusch
C.
,
Sickmann
A.
,
Buttgereit
D.
,
Renkawitz–Pohl
R.
(
2011
).
Role of the Actin Cytoskeleton Within FuRMAS During Drosophila myoblast fusion and first functionally conserved factors in vertebrates.
In
Cell Fusions: Regulation and Control
(ed.
Larsson
L-I
), pp.
138
170
.
Dordrecht, NL
:
Springer
.
Paululat
A.
,
Burchard
S.
,
Renkawitz–Pohl
R.
(
1995
).
Fusion from myoblasts to myotubes is dependent on the rolling stone gene (rost) of Drosophila.
Development
121
,
2611
2620
.
Pavenstädt
H.
,
Kriz
W.
,
Kretzler
M.
2003
).
Cell biology of the glomerular podocyte.
Physiol. Rev.
83
,
253
307
.
Pollard
T. D.
(
2007
).
Regulation of actin filament assembly by Arp2/3 complex and formins.
Annu. Rev. Biophys. Biomol. Struct.
36
,
451
477
.
Quilliam
L. A.
,
Westwick
J. K.
,
Der
C. J.
,
Lambert
Q. T.
(
1996
).
Cloning and characterization of proteins that bind the SH3 domains of the Nck adaptor protein.
FASEB J.
10
,
A998
.
Ramos
R. G. P.
,
Igloi
G. L.
,
Lichte
B.
,
Baumann
U.
,
Maier
D.
,
Schneider
T.
,
Brandstätter
J. H.
,
Fröhlich
A.
,
Fischbach
K. F.
(
1993
).
The irregular chiasm C-roughest locus of Drosophila, which affects axonal projections and programmed cell death, encodes a novel immunoglobulin-like protein.
Genes Dev.
7
,
2533
2547
.
Richardson
B. E.
,
Beckett
K.
,
Nowak
S. J.
,
Baylies
M. K.
(
2007
).
SCAR/WAVE and Arp2/3 are crucial for cytoskeletal remodeling at the site of myoblast fusion.
Development
134
,
4357
4367
.
Rivero–Lezcano
O. M.
,
Marcilla
A.
,
Sameshima
J. H.
,
Robbins
K. C.
(
1995
).
Wiskott-Aldrich syndrome protein physically associates with Nck through Src homology 3 domains.
Mol. Cell. Biol.
15
,
5725
5731
.
Ruiz–Gómez
M.
,
Coutts
N.
,
Price
A.
,
Taylor
M. V.
,
Bate
M.
(
2000
).
Drosophila dumbfounded: a myoblast attractant essential for fusion.
Cell
102
,
189
198
.
Schäfer
G.
,
Weber
S.
,
Holz
A.
,
Bogdan
S.
,
Schumacher
S.
,
Müller
A.
,
Renkawitz–Pohl
R.
,
Önel
S. F.
(
2007
).
The Wiskott-Aldrich syndrome protein (WASP) is essential for myoblast fusion in Drosophila.
Dev. Biol.
304
,
664
674
.
Schejter
E. D.
,
Baylies
M. K.
(
2010
).
Born to run: creating the muscle fiber.
Curr. Opin. Cell Biol.
22
,
566
574
.
Schröter
R. H.
,
Lier
S.
,
Holz
A.
,
Bogdan
S.
,
Klämbt
C.
,
Beck
L.
,
Renkawitz–Pohl
R.
(
2004
).
kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila.
Development
131
,
4501
4509
.
Sens
K. L.
,
Zhang
S. L.
,
Jin
P.
,
Duan
R.
,
Zhang
G. F.
,
Luo
F. B.
,
Parachini
L.
,
Chen
E. H.
(
2010
).
An invasive podosome-like structure promotes fusion pore formation during myoblast fusion.
J. Cell Biol.
191
,
1013
1027
.
Shelton
C.
,
Kocherlakota
K. S.
,
Zhuang
S.
,
Abmayr
S. M.
(
2009
).
The immunoglobulin superfamily member Hbs functions redundantly with Sns in interactions between founder and fusion-competent myoblasts.
Development
36
,
1159
1168
.
Sohn
R. L.
,
Huang
P.
,
Kawahara
G.
,
Mitchell
M.
,
Guyon
J.
,
Kalluri
R.
,
Kunkel
L. M.
,
Gussoni
E.
(
2009
).
A role for nephrin, a renal protein, in vertebrate skeletal muscle cell fusion.
Proc. Natl. Acad. Sci. USA
106
,
9274
9279
.
Srinivas
B. P.
,
Woo
J.
,
Leong
W. Y.
,
Roy
S.
(
2007
).
A conserved molecular pathway mediates myoblast fusion in insects and vertebrates.
Nat. Genet.
39
,
781
786
.
Strünkelnberg
M.
,
Bonengel
B.
,
Moda
L. M.
,
Hertenstein
A.
,
de Couet
H. G.
,
Ramos
R. G. P.
,
Fischbach
K. F.
(
2001
).
rst and its paralogue kirre act redundantly during embryonic muscle development in Drosophila.
Development
128
,
4229
4239
.
Sugie
A.
,
Umetsu
D.
,
Yasugi
T.
,
Fischbach
K. F.
,
Tabata
T.
2010
).
Recognition of pre- and postsynaptic neurons via nephrin/NEPH1 homologs is a basis for the formation of the Drosophila retinotopic map.
Development
137
,
3303
3313
.
Tryggvason
K.
(
2001
).
Nephrin: role in normal kidney and in disease.
Adv. Nephrol. Necker Hosp.
31
,
221
234
.
Vasyutina
E.
,
Martarelli
B.
,
Brakebusch
C.
,
Wende
H.
,
Birchmeier
C.
(
2009
).
The small G-proteins Rac1 and Cdc42 are essential for myoblast fusion in the mouse.
Proc. Natl. Acad. Sci. USA
106
,
8935
8940
.
Weavers
H.
,
Prieto–Sánchez
S.
,
Grawe
F.
,
Garcia–López
A.
,
Artero
R.
,
Wilsch–Bräuninger
M.
,
Ruiz–Gómez
M.
,
Skaer
H.
,
Denholm
B.
(
2009
).
The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm.
Nature
457
,
322
326
.
Welsch
T.
,
Endlich
N.
,
Kriz
W.
,
Endlich
K.
(
2001
).
CD2AP and p130Cas localize to different F-actin structures in podocytes.
Am. J. Physiol. Renal Physiol.
281
,
F769
F777
.
Welsh
G. I.
,
Saleem
M. A.
(
2010
).
Nephrin-signature molecule of the glomerular podocyte?
J. Pathol.
220
,
328
337
.
Zallen
J. A.
,
Cohen
Y.
,
Hudson
A. M.
,
Cooley
L.
,
Wieschaus
E.
,
Schejter
E. D.
(
2002
).
SCAR is a primary regulator of Arp2/3-dependent morphological events in Drosophila.
J. Cell Biol.
156
,
689
701
.
Zhuang
S. F.
,
Shao
H. J.
,
Guo
F. L.
,
Trimble
R.
,
Pearce
E.
,
Abmayr
S. M.
(
2009
).
Sns and Kirre, the Drosophila orthologs of Nephrin and Neph1, direct adhesion, fusion and formation of a slit diaphragm-like structure in insect nephrocytes.
Development
136
,
2335
2344
.