Funktionelle Analysen zum Abbau von N-Cadherin während der Myoblastenfusion von Drosophila melanogaster durch Schizo und Abi und die Rolle der Drosophila nicht-Rezeptortyrosinkinasen Shark und Csk bei der Integrin-vermittelten Anheftung der Muskeln an die Epidermis

In Drosophila embryos, myoblast fusion is characterized by the fusion of two specific types of cells: founder cells (fc´s) and fusion-competent myoblasts (fcm´s). The recognition and adhesion of the myoblasts is mediated by adhesion proteins of the immunoglobulin superfamily (IgSF) and the cadherin superfamily. Before myoblasts can fuse, they have to reduce their distance, which is provided by a protein-free area. This requires that also cell adhesion proteins like N-Cadherin, which mediate the cell contact, are removed from the membrane. Genetic and biochemical analyses indicate that the removal of N-Cadherin is mediated by the Arf1 guanine nucleotide exchange factor (GEF) Schizo, the BAR-domain protein Graf1 and Abi, a regulator of F-actin polymerization. The first part of this thesis investigates if N-Cadherin is removed by Dynamin-independent Arf1-dependent endocytotic mechanisms and which role Abi plays in this process. Analyses of fluorescence intensities of N-Cadherin in Drosophila embryos and adult testis reveal that schizo mutants are not able to remove N-Cadherin. Particularly the loss of the Schizo Sec7 domain, which is important for the activation of the Arf1 GTPase, leads to an enrichment of N-Cadherin at the membranes. The overexpression of constitutively-active Schizo and Arf1 results in a decrease of N-Cadherin. The CLIC/GGEC endocytosis, which involves Arf1 and Graf1, is a possible mechanisms to remove N-Cadherin. Protein interactions reveal that activated Graf1, which misses the BAR domain, interacts with the intracellular domain of N-Cadherin. However, the presence of the BAR domain inhibits the interaction with N-Cadherinintra. graf1 mutants, generated by CRISPR/Cas9 in this thesis, show no fusion defects. This indicates that Graf1 is not essentiell for myoblast fusion. The physical interaction between schizo and abi during myoblast fusion implies at first a possible activation of the Scar/WAVE complex during the removal of N-Cadherin. However, the performed genetic interaction studies suggest an antagonistic effect of Abi and Schizo. In the second part of this thesis, several non-receptor tyrosine kinases (nRTKs) were examined for a possible function during myogenesis. Genetic interaction studies identified the nRTKs Shark and Csk. The loss of shark and csk leads to myoblast fusion defects and attachment defects of the muscles to the epidermis. The adhesion of the muscles to the epidermis is mediated by Integrin. The heterodimer αPS2βPS Integrin is expressed in muscle cells, and the αPS1βPS heterodimer in epidermal tendon cells. Analyses of the expression of βPS Integrin indicate a function of Shark and Csk during „inside-out“-Integrin signaling as βPS Integrin showed a decreased expression. Genetic interaction studies with the cell adhesion protein Sns, the WASp regulator Wip and Scar/WAVE additionally imply a function of the nRTK Csk during myoblast fusion as double mutants and gene dosage experiments show an increased amount of unfused myoblasts.