טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBar-Or Ella
SubjectThe Role of the KirreL3 Receptor in Motility and Fusion of
Myoblast Cells
DepartmentDepartment of Medicine
Supervisor Professor Eyal Bengal
Full Thesis textFull thesis text - English Version


Abstract


During myogenesis, mononucleated myoblasts withdraw from the cell cycle, initiate muscle specific gene expression, and subsequently fuse with one another to form nascent, multinucleated myofibers. This fusion process is an ordered set of specific cellular events: recognition, adhesion, alignment, and membrane union. Myoblast fusion in Drosophila is a dynamic relationship between two myoblast types: the founder cells and the fusion-competent myoblasts. Fusion process occurs in two steps. Initially, myoblasts fuse with one another to form nascent myotubes with a small number of nuclei. Next, additional rounds of fusion between myoblasts and nascent myotubes, resulting in the formation of mature myotubes. Studies have indicated that the fusion process is mediated by one of the cell-type-specific transmembrane receptor containing immunoglobulin domains which are conserved between Drosophila and zebrafish. One of these receptors is Kirrel, the zebrafish orthologue of Drosophila Duf.  In light of the convincing evidence of Kirrel involvement in myoblast fusion in both Drosophila and zebrafish, we hypothesized that Kirrel is also involved in processes leading to myoblast fusion in mammalians. In this project we have initiated a biochemical and functional analysis of Kirrel3 in myoblast and satellite cells culture models. By sh-mediated knockdown of Kirrel3 we found that Kirrel3 is necessary for cytoskeleton remodeling, satellite cells ability to elongate and to form membrane extensions, myoblast motility and formation of multi nuclear myotubes. Kirrel3 undergoes processing by metalloproteinases, which results in the shedding of its extracellular domain. In order to investigate the physiological function of the extra and intracellular domains, we constructed several deletion mutants of Kirrel3. The effects of these mutants on myoblast motility and fusion were analyzed by immunohistochemical staining and time-lapse analysis. The only mutant that appeared to affect cell fusion was a mutant lacking the intracellular domain of Kirrel3, which decreased the number of myotubes containing 4 or more nuclei. Kirrel3 intracellular domain is probably necessary to transmit a signal that triggers cytoskeletal rearrangements that contribute to processes leading to a successful myoblast fusion. We cannot make any significant conclusion from the time-lapse experiment with regards to the importance of Kirrel3's domains on myoblast motility due to inconsistent results. We have also found that Kirrel3 mutants were co expressed with MyoD and Myosin proteins and therefore may not affect cell differentiation. Still, in the absence of Kirrel3 protein the differentiation process may have been delayed.