Linking IgSF-mediated cell adhesion with Arp2/3-based actin polymerization during Drosophila myoblast fusion

The somatic musculature of Drosophila is analogous to vertebrate skeletal muscles and is generated by the fusion of mononucleate myoblasts. Muscle fusion in Drosophila involves two distinct cell populations, founder cells (FCs) and fusion-competent myoblasts (FCMs). The recognition and adhesion of b...

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1. Verfasser: Balasankara Reddy, Kaipa
Beteiligte: Önel, Susanne (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2011
Biologie
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Zusammenfassung:The somatic musculature of Drosophila is analogous to vertebrate skeletal muscles and is generated by the fusion of mononucleate myoblasts. Muscle fusion in Drosophila involves two distinct cell populations, founder cells (FCs) and fusion-competent myoblasts (FCMs). The recognition and adhesion of both myoblast types is mediated by members of the Immunoglobin superfamily (IgSF) that are expressed myoblast type specifically: Duf and Rst are expressed in FCs. Sns, Hbs and Rst are present in FCMs. The heterophilic interaction of these Ig-domain proteins leads to signal activation and results in the formation of F-actin at the sites of cell-cell contact. Duf and Rst serve in functional redundancy in FCs. The formation of new actin filaments at existing filaments is regulated by the actin-related protein complex (Arp)2/3. The Arp2/3 complex is a multiprotein complex consisting of seven subunits including Arp2 and Arp3. The complex becomes activated by nucleation promoting factors (NPFs), i.e the Wiskott-Aldrich syndrome protein (WASP) and the suppressor of CAMP-receptor (SCAR). Additionally, WASP is activated by the WASP-interacting protein Verprolin1 (WIP). These proteins act together in the WASP-WIP complex. Although WASP and SCAR are activated differently to induce Arp2/3-mediated actin polymerization, they share a common proline-rich region, which is known to bind to SH3 domain containing proteins. In this study I have addressed two questions. First, I have tried to elucidate the redundant nature of Duf and Rst in FCs. The intracellular domain of Rst and Duf contains three conserved domains, i.e a PADVI, SAIYGNPYLR and NSLLPPLPP domain. Expression of RstΔPADVI in all myoblasts or exclusively in FCs as well as FCMs impaired myoblast fusion. This indicates that the PADVI domain in the intracellular domain of Rst plays an important role during myoblast fusion. To identify Rst specific interaction partners I further performed a yeast two-hybrid screen and identified Actin57B, Papilin and Nidogen as possible Rst interaction partner. Second, I investigated the role of the SH2-SH3 adopter Dreadlock (Dock) in myoblast fusion. Dock is expressed in FCs and FCMs. In vertebrates, the homologue of Dock - called Nck - links cell adhesion during podocyte formation with actin cytoskeleton rearrangement by binding to the intracellular domain of Nephrin. Nephrin is a homologue of Drosophila Sns and Hbs. Biochemical data provided in this study show that Dock is able to bind to the intracellular domain of Sns, Hbs, Duf and Rst and to the proline-rich region of the actin regulators WASP and WIP via its SH3 domains. Interestingly, I found that the SH2 domain of Dock binds to phosphorylated tyrosine at position 1089 in the intracellular domain of Hbs. The SH3 domains of Dock can bind to Sns and Duf and all SH2 and SH3 domains are required to bind to Rst. To demonstrate that these protein interactions are relevant for myoblast fusion, I carried out double mutant experiments. I could show that dock interacts genetically with the FC-specific gene rols, which encodes for an adaptor protein that binds to the intracellular domain of Duf, and with the FCM-specific cell adhesion molecule hbs. These data indicate that Dock functions in both myoblast populations during myoblast fusion and serves as a linker to transfer the fusion signal from the cell adhesion molecules Duf and Hbs to the actin cytoskeleton, e.g. by interacting with WASP and WIP.
DOI:https://doi.org/10.17192/z2012.0046