„Genetische und biochemische Analysen zur Regulation von N-Cadherin durch den Arf-GEF Schizo und zur Arp2/3-Komplex vermittelten F-Aktin Bildung während der Myoblastenfusion von Drosophila melanogaster“

In the development of multicellular organisms, cell-cell fusion is a rare, nevertheless elementary process. A well-studied example is the fusion of founder cells (FCs) with fusion-competent myoblasts (FCMs) during the formation of the somatic muscles of Drosophila melanogaster. At the beginning of this process, myoblasts must specifically recognize and adhere to each other. Subsequently, intracellular signaling pathways are activated resulting in the merger of the plasma membranes and the reorganization of the cytoplasm. All these steps involve actin-based processes. Mutants of the gene schizo (also called loner) show only unfused myoblasts, suggesting an essential function of this gene during fusion (Chen et al., 2003). Schizo is a guanine-nucleotide exchange factor (GEF) for Arf GTPases that are involved in secretory and endocytic vesicle transport, membrane organization and reorganization of the actin cytoskeleton. The present work is divided into two parts. In the first part analysis of the Schizo interaction partners N-Cadherin and CG12006 were performed. The second part deals with the reorganization of the actin cytoskeleton during myoblast fusion. In an attempt to learn more about the function of Schizo during myoblast fusion, we performed a yeast 2-hybrid screen that resulted in the identification of the Ca2+-dependent adhesion molecule N-Cadherin as a Schizo interaction partner (Diplomarbeit V. Groth, 2008). The focus of this work lies on the characterization of this interaction. CadN localizes to the cell membranes of FCs and FCMs in the early fusion-relevant stages. Surprisingly, the loss of CadN does not result in muscle defects. Interestingly, studies in vertebrate cell culture showed that cell adhesion molecules must be removed from the fusion site and that the cell fusion takes place in protein-free regions of the membrane. Genetic interaction studies described in this work suggest that Schizo acts as a negative regulator that initiates the internalization of CadN. Another hint for this is provided by live-imaging cell culture studies in which a co-localization of CadNTMintra-eGFP and Schizo-fl-mCherry in small vesicular structures can be observed. In this context, various endocytic pathways were analyzed. Based on the results of this work it is postulated that CadN must be removed from the membrane before the actual merger of the cell membranes can occur and that this is achieved by Schizo-mediated endocytosis of CadN. Another putative Schizo interaction partner is the α2-mannosyltransferase CG12006. Mannosyltransferases are involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors needed to anchor proteins without transmembrane domain to the cell surface. In-situ hybridizations in this study show the mesoderm-specific expression of CG12006. Interestingly, the RNAi-induced loss of CG12006 results in mild fusion defects, suggesting a relevance of the GPI anchor biosynthesis for myoblast fusion. Concerning the reorganization of the actin cytoskeleton during the fusion process, several actin regulators were analyzed in relation to myoblast fusion. Interestingly, a genetic interaction between Chickadee/Profilin and Wip during myoblast fusion was observed. The double mutant was characterized at the cellular and ultrastructural level. Furthermore, the nucleation-promoting factor Cortactin and Wip seem to interact genetically. Therefore, the studied actin regulators Chic and Cortactin seem to participate in the complex actin-regulatory mechanism during myoblast fusion.