The dynamics of F-actin plugs/foci within the FuRMAS requires regulated F-actin polymerization and depolymerization. Indeed, to date, there is an accumulating body of evidence to show that the precise regulation of actin polymerization is crucial for successful myoblast fusion (Schröter et al.,2004; Kesper et al.,2007; Kim et al.,2007; Massarwa et al.,2007; Richardson et al.,2007; Berger et al.,2008). Actin-related proteins like the Arp2/3 complex initiate the formation of new F-actin filaments by anchoring new filaments to a pre-existing actin network (reviewed by Pollard,2007). During myoblast fusion, the underlying activation of the Arp2/3 complex is differentially regulated in fcs and fcms and in the first and second fusion phase (Fig. 4; Table 1). Rho, Rac, and Cdc42 are small GTPases, which are involved in many actin-dependent processes (reviewed by Etienne-Manneville and Hall,2002). Cdc42 seems not to play a major role in myoblast fusion (Schäfer et al.,2007) in contrast to Rac (see below). The first actin regulators that were identified to be important for myoblast fusion were the small rac-GTPases rac1, rac2 (Luo et al.,1994; Doberstein et al.,1997; Hakeda-Suzuki et al.,2002), and kette, the Drosophila homologue of Nap1/Hem-2 (Schröter et al.,2004). Interestingly, kette interacts genetically with the fcm-specifically expressed gene blow (Schröter et al.,2004). Fusion mutants such as kette have distinct fusion interruption at the ultrastructural level (see Ultrastructural Features of Myoblast Fusion section).
The small Rac-GTPase and the vertebrate homologue of Kette Nap-1/Nap-125 are known to be involved in activating the Arp2/3 regulator SCAR/WAVE. A protein complex including Nap-125 keeps SCAR/WAVE in an inactive state, whereas the binding of Rac activates SCAR/WAVE, which in turn stimulates the Arp2/3 complex (Takenawa and Suetsugu,2007). Mbc is required for myoblast fusion (Rushton et al.,1995; Erickson et al.,1997) and is thought to be the GEF that activates Rac (Côté and Vuori,2002; see Fig. 4). Indeed, Mbc and Rac GTPases colocalize with F-actin plugs (Richardson et al.,2007). The stimulation of the Arp2/3 complex by the nucleation-promoting factors such as SCAR/WAVE and WASP is achieved through three short evolutionary conserved motifs that are also known as the VCA module: The V motif interacts with actin monomers and the CA motif can bind the Arp2/3 complex and actin. In Drosophila, immunhistochemical studies suggest that Kette is also required for the correct localization of SCAR/WAVE (Richardson et al.,2007). Embryos that lack zygotic scar/wave activity show a weak fusion defect (Richardson et al.,2007; Berger et al.,2008), a phenotype that can be enhanced by the additional reduction of maternal scar/wave (Richardson et al.,2007). Besides SCAR/WAVE, the Wiskott-Aldrich Syndrome protein WASP is required for myoblast fusion. Analysis of a new wasp allele wasp3D3-035 that lacks the CA domain of the VCA module and acts as a dominant-negative mutation revealed that this domain is essential for correct myoblast fusion (Schäfer et al.,2007). Double mutant experiments with Arp3 and wasp mutant embryos as well as the loss of maternal and zygotic WASP activity imply that WASP is only required during the second fusion phase (Massarwa et al.,2007; Berger et al.,2008). Moreover, cell type–specific rescue experiments indicate that WASP is only required in fcms (Schäfer et al.,2007); however, this issue needs further clarification as Massarwa et al. (2007) reported contradictory results. In addition, WASP is known to form a complex with its fcm-specifically expressed interaction partner Verprolin 1 (Vrp1) (also known as D-Wip or Solitary) (Massarwa et al.,2007; Kim et al.,2007), and homozygous vrp1 null mutants display arrested fusion after precursor formation. On the ultrastructural level, Kim et al (2007) observed mislocalization of the prefusion complex and accumulation of vesicles, and thus proposed that F-actin regulation is required to target vesicles to the membrane; however, the membranes appear to remain intact. In contrast, Massarwa et al. (2007) and Berger et al. (2008) observed membrane vesiculation in vrp1 mutants and found no evidence of accumulation of vesicles. The reason for these differing findings remains to be clarified, e.g., different fixation methods (high-pressure freezing; chemical fixation) could give different results. In wasp mutants, the prefusion complex is also correctly formed and no vesicle accumulation is observed (Berger et al.,2008; Massarwa et al.,2007). Interestingly, wasp mutants exhibit a highly similar phenotype to vrp1 mutants (Berger et al.,2008; Massarwa et al.,2007), a finding that fits well with the fact that WASP and Vrp1 are interacting proteins. To clarify the role of SCAR/WAVE, WASP, and Vrp1 during myoblast fusion, scar/wave vrp1 double mutants were analysed and found to display no evidence of fusion taking place. Taken together, the evidence strongly suggests that SCAR/WAVE and Vrp1 control the first fusion phase, whereas SCAR/WAVE, WASP, and Vrp1 are all required for the second fusion phase (Berger et al.,2008). This in turn suggests that Arp2/3 is activated independently of WASP in the first fusion phase. Indeed, it was proposed that vertebrate Wip could interact with Cortactin to induce actin polymerization via the Arp2/3 complex (Kinley et al.,2003). However, whether this is also the case for myoblast fusion remains to be investigated.
The Arp2/3 complex consists of 7 proteins: 2 actin-related proteins, namely Arp2 and Arp3, as well as 5 further subunits (ArpC1–5). Mutations in the Arp3 and ArpC1 genes have been shown to disrupt fusion (Massarwa et al.,2007; Richardson et al.,2007; Berger et al.,2008). Molecular players that regulate F-actin branching during the first and second fusion phase in a cell-type-specific manner are summarized in Figure 4. Interestingly, on the ultrastructural level, myoblast fusion in wasp and vrp1 mutants is disrupted at a different step than in Arp3schwächling mutants. In detail, wasp and vrp1 mutants stop fusion during membrane vesiculation (Massarwa et al.,2007; Berger et al.,2008), whereas Arp3schwächling mutants stop fusion after membrane remnants have been removed and a fusion pore has been formed, but the fcm is not integrated into the growing myotube (Berger et al.,2008). From these data, a model has been proposed that suggests that the enlargement of the fusion pore depends on Arp2/3-mediated F-actin branching. Membrane vesiculation and breakdown, however, seem to be controlled by other factors, e.g., a so far unknown fusogene (a protein that fuses membranes). We strongly suggest the presence of co-ordinated pathways leading to fusion pore formation and expansion, and also due to the complexity of the intracellular domain of SNS, the presence of multiple pathways at least in fcms (Kocherlakota et al.,2008). Finally, to add weight to this notion, biochemical experiments have provided evidence to suggest that SNS interacts with Crk, a SH2-SH3 type of adaptor protein, and that Vrp1 mediates the formation of a Crk-Vrp1-WASP complex in fcms (Kim et al.,2007).