Die Rolle von Drosophila Dynamin, Graf und der nicht-Rezeptor Tyrosinkinase Fes/Fer bei der Internalisierung von N-Cadherin in adhärierenden Founderzellen und fusionskompetenten Myoblasten

During embryogenesis of Drosophila melanogaster, two types of myoblasts founder cells and fusion-competent myoblasts fuse to form the later body wall musculature (Önel and Renkawitz-Pohl, 2009). Besides some proteins of the Immunoglobulin-Superfamily, also the calcium-dependent transmembrane protein N-Cadherin contributes to adhesion between the myoblasts. The later site of contact needs to be nearly free of proteins, so during the establishment of this zone N-Cadherin, which keeps membranes at a definite distance, is proposed to become internalized. Genetic interaction studies indicate that endocytosis is regulated by the Arf1-guanine-nucletide-exchange factor (GEF) Schizo (Dottermusch-Heidel et al., 2012). The aim of the present study was to analyze the mechanism by which N-Cadherin is endocytosed. By reviewing the literature, we identified proteins, which might be involved in the endocytosis of N-Cadherin. These proteins comprise the Dynamins, as well as Graf and the non-receptor tyrosinekinase Fps85D. Together with Arf1, Graf1 is involved in several endocytic processes in vertebrates (Doherty and Lundmark, 2009). Cell culture studies show that mCherry-Graf co-localizes with Schizo-eGFP and Arf1-eGFP in vesicular structures in Drosophila S2 cells. Furthermore, via live-imaging studies a co-localization between mCherry-Graf and N-CadherinTMintra-eGFP at the membranes of S2 cells were observed. A yeast two-hybrid assay revealed the interaction between N-Cadherinintra and Graf, for which the SH3-domain of Graf is essential. These results support the hypothesis that Graf is involved in the Schizo-mediated endocytosis of N-Cadherin. Besides, Schizo as well as Graf interact with the Scar/WAVE complex protein Abi in the yeast two-hybrid system. Abi might link N-Cadherin to the actin cytoskeleton. The non-receptor tyrosinekinase Fes/Fer/Fps is involved in the regulation of the adhesion strength of N-Cadherin-mediated adherence junctions in vertebrates (El Sayegh et al., 2005). Fps85D-mCherry, the fusion protein of the Drosophila orthologue, co-localizes both with N-CadherinTMintra-eGFP and with Schizo-eGFP and Arf1-eGFP in vesicular structures in cell culture. Moreover, a co-localization of Fps85D-mCherry and N-Cadherin were detected in the embryo at the membranes of myoblasts during fusion-relevant stages. Therefore, a function of Fps85D during the process of myoblast fusion seems to be possible. Furthermore, first interaction studies in the embryo indicate that there might be a genetic interaction between N-cadherin and fps85D and hypothesize that there might be a similar regulation as described in vertebrates.