Determination of genetic interactions required for dystrophin-dystroglycan function and regulation in a Drosophila model of muscular dystrophy.

In humans genetically diverse forms of muscular dystrophy (MD) are associated with a disrupted Dystrophin-Glycoprotein Complex (DGC). The DGC consists of dystrophin, dystroglycans, sarcoglycans, sarcospan, syntrophins and dystrobrevin. Characterization of the Drosophila melanogaster DGC shows that the fly retains all essential components of DGC, but with substantially less diversity. Similarly to humans, mutations in the fly Dystrophin (Dys), Dystroglycan (Dg) and Sarcoglycan genes cause reduced mobility, shortened lifespan and age-dependent muscle degeneration. In addition, mutations in Dys and Dg affect neurotransmitter release in the neuromuscular junctions and cause the axon migration defects. Furthermore, Dys and Dg are important for the establishment of cell polarity. Since disruption of the connection between the components of DGC causes muscular dystrophy in humans, I decided to study the interaction between two major components of the DGC, Dys and Dg, in more detail. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPxY motif has been established as a binding site for Dys WW-domain. Now my in vitro studies indicate that both Drosophila Dg PPxY motifs, WWbsI and WWbsII can bind to the WW domain of Dys and phosphorylation of the tyrosine of each PPxY motif abolishes Dg-Dys binding, suggesting a possible regulatory mechanism. Moreover, I have shown that both WW binding sites are required for maintaining the Dg function in vivo for the establishment of oocyte polarity. Additionally, the sequence comparison of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation at these sites. Therefore, it has been proposed that the presence of the two WW binding sites in Dg secures the essential interaction between Dg and Dys and might further provide additional regulation of the cytoskeletal rearrangements involving DGC. To reveal new components that interact with DGC or regulate its function I used a Drosophila model for MD and performed genetic screens to identify dominant modifiers of Dg and Dys related mutant phenotypes. In the primary large scale screen I have looked for modifications of easily score-able phenotype such as alterations in the posterior crossvein. Using this screening strategy I have found modifiers that belong to different functional groups: genes involved in the muscle function, neuronal/cell migration and motor function factors as well as cytoskeletal components and members of the TGF-beta, EGFR and Notch signaling pathways. In order to shed light on the mechanisms of muscle degeneration caused by Dys and Dg down-regulation the secondary screen in muscle tissue was performed. As a result, the modifiers that suppressed/enhanced muscle degeneration phenotype were identified. Further studies of identified components showed their requirement in either muscle or nervous tissue, where specific interaction with the DGC components may occur. The novel components that contribute to DGC-dependent muscle maintenance are being analyzed.