Die Rolle von Blow und Drp1 bei der Auflösung des Präfusionskomplexes am Zell-Zell Kontakt während der Myoblastenfusion von Drosophila

In Drosophila, founder cell (FC) and fusion competent myoblasts (FCM) fuse during embryonic myogenesis to form the body wall musculature (Onel und Renkawitz-Pohl 2009). After the first fusion events an accumulation of electron-dense vesicles form the prefusion complex at the cell contact. A vesicularisation of opposing membranes occurs in order to incorporate the FCM into the FC/growing myotube. After recognition and adhesion a multimere signalling complex is established, called fusion-restricted myogenic adhesive structure (FuRMAS). One characteristic feature is the drastic reorganisation of F-actin at the cell-contact in FCM (Kesper et al. 2007; Onel und Renkawitz-Pohl 2009). The FCM-specific protein Blown fuse (Blow) is essential for myoblast fusion and is involved in the reorganisation on F-actin at the side of fusion (Jin et al. 2011; Schroter et al. 2004). Ultrastructural analysis of blow-Mutants show that they stop myoblast fusion during the establishment of the prefusion complex (Doberstein et al. 1997), whereas mutants of the F-actin reorganisation Wasp and Wip stop later during the vesicularisation of membranes (Berger et al. 2008; Massarwa et al. 2007). This raises the question, whether Blow coordinates f-actin formation and the release of electron-dense vesicles. A global yeast-2-hybrid screen identified Shark and Drp1 as newly potential interaction partner. In the present study the function of Shark and Drp1 during myoblast fusion was addressed. By using the yeast-2-hybrid, system the region of interaction between Shark and Blow could not be specified. Expression of shark-deletions missing either the Ank-repeats or both SH2-domains indicate that Shark is involved in the fusion process. Expression of UAS-shark Δ kin-mcherry in the adult flight muscles leading to distinct reduction in the average flight ability. This supports further the idea that Shark might be involved in myoblast fusion. Based on data of Syk the vertebrate homologue of Shark, during the B cell receptor (BCR) activation, it was analysed if there is a direct or genetic interaction between Shark and the IgSF Sns (Kroczek et al. 2010). Yeast-2-Hybrid analysis could not identify an interaction but doses experiments with the double mutants showed a genetic interaction. Indicating a similar function of Shark as shown for the BCR activation. The GTPase-domain of Drp1 could be shown in the yeast-2-hybrid system to be essential for the interaction between Drp1 and Blow. A dosage experiment on drp11, blow2-double mutants indicates a genetic interaction between Drp1 and Blow. The expression of 1 Einleitung 2 UAS-drp1 Δ GED-mcherry in contrast to UAS-drp1 Δ GTPase-mcherry in the embryo and the adult flight muscles leads to a stronger defect. In this study it was shown that the inhibition of Drp1 function in primary mouse myoblasts via incubation with Mdivi-1 results in a dose dependant drastic reduction in fusion-efficiency. Indicating further an involvement of Drp1 during fusion. Drp1 is a master regulator of mitochondrial division (Verstreken et al. 2005). A mitochondrial localisation study in sns20-15-, duf, rst-, sing22-, rols117-, ketteJ4-48 and blow2-mutants showed that mitochondria localise dynamic during fusion. During migration and early attachment mitochondria localise increased in the leading edge of the cell and the side of fusion, during late attachment the mitochondria vanished from the side of fusion. The loss of marf, a gene involved in mitochondrial trafficking, leads to a distinct fusion defect. This indicates that the transport of mitochondria seems to be important for the fusion. The expression of UAS-shibire-GED Δ PRR-mcherry indicates a role of the GTP hydrolysis during embryonic myogenesis. Rab4 and Rab11 play a role during mitochondrial homeostasis (Caza et al. 2014; Landry et al. 2014). An expression of a full-length (fl), as well as a potential dominant-negative and constitutive-active rab4- and rab11-construct in the embryo results in lightly defects. An evidence of a potentially contribution of Rab4 and Rab11 in the mitochondrial homeostasis