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

Während der Embryonalentwicklung in Drosophila melanogaster fusionieren zwei Typen von Myoblasten, Founderzellen und fusionskompetente Myoblasten, und bilden dadurch die spätere Körperwandmuskulatur (Önel und Renkawitz-Pohl, 2009). Dabei trägt neben den Proteinen der Immunoglobulin-Superfamilie auch...

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Bibliographic Details
Main Author: Braukmann, Carina
Contributors: Önel, Susanne-Filiz (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2016
Online Access:PDF Full Text
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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.