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Myoblast fusion is a fundamental process during muscle formation of multicellular organisms. In Drosophila melanogaster the formation of somatic musculature occurs by the fusion of Founder Cells (FCs) and Fusion Competent Myoblasts (FCMs). After cell recognition and adhesion an adhesion ring is formed at the contact membranes, in which center an actin-rich core is arranged (Kesper et al., 2007). Life-imaging studies demonstrated the dynamic of these actin plaques. These are formed within 2 minutes and are dissolved shortly afterward. Mutant analyses show, that the Arp2/3-complex dependent actin polymerization is essential for myoblast fusion. Moreover based on the dynamic of actin plaques the depolymerization should also play an essential role. To study these F-actin based processes several approaches have been done.
The Arp2/3 dependent plaque formation is regulated by the NPFs Scar/Wave and WASP in both cell types. Mutations in Scar/Wave as well as in the WASP dependent Arp2/3 activation lead to reduction of actin plaques, but not to losing of plaques formation (this thesis and Sens et al., 2010). Therefore additional components should be involved in this process, such as the actinmonomere- binding protein Profilin/Chicadee (Chic). The performed dosage experiments and protein interaction studies in this work indicate Chic as a component of both Scar/Wave and WASP dependent actin polymerization. Additionally an Arp2/3 regulator Ena/Vasp was identified to play a role during the myoblast fusion as well as during the muscle attachment to the epidermis.
Another part of my work deals with the activation of Scar/Wave complex during myoblast fusion. These occurs merely trough the small GTPase Rac or results from a cooperation of Rac with components of the pentameric Scar/Wave complex. Drosophila genome harbors tree rac GTPases, while rac1 and rac2 play a redundant role during myoblast fusion. Expression of constitutive- active and dominant- negative constructs of Rac1 results in a strong fusion phenotype. Additionally double mutants of rac1 and rac2 show unfused myoblasts. Nevertheless in all mutants I could observe the formation of actin plaques. Moreover genetic and biochemical interaction studies in this thesis indicate an interaction of Rac with Chic, Scar/Wave, Sra1 and Kette. In order to detect activators and/or inactivators of both Rac GTPases, Yeast-Two-Hybrid screens were done with an activated form of Rac1 and Rac2. In this approach the Csk kinase and the actin binding protein WupA could be identified as potential interaction partners for Rac1.
The last part of this work deals with the investigation of actin-depolymerizing components, which are involved in the membrane breakdown during the fusion. For this reason mutations of depolymerizing factor Cofilin/Twinstar (Tsr) were analyzed. Neither the homozygous tsr mutants nor the expression of activated/ inactivated Tsr cause fusion defects. Therefore I analyzed an additional cofactor, Aip1/Flare (Flr), in respect of its mRNA expression and perform double mutants with tsr. The localization of flr transcript could be detected in embryonic somatic musculature as well as in ovaries, in testis and in imaginal discs. However flr single mutants show no muscle defects. In tsr; flr double mutants a weak muscle phenotype could be observed; that indicates a potential collaboration of both proteins during muscle formation. To sum up the results it can be postulated that Tsr acts in redundancy with additional depolymerizing proteins.