טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMubariki Raeda
SubjectThe Adhesion Molecule Kirrel3/Neph2: Regulator of
Morphological Changes Required for Myoblast Fusion
DepartmentDepartment of Medicine
Supervisor Professor Eyal Bengal
Full Thesis textFull thesis text - English Version


Abstract

During myogenesis, mononucleated myoblasts withdraw from the cell-cycle and differentiate into myocytes that subsequently fuse to form multinucleated myotubes which express muscle specific proteins. This fusion process is an ordered set of specific cellular events that include cell morphological changes, migration, recognition, adhesion, alignment and membrane union.   

Myoblasts fusion in Drosophila occurs between two cell types, muscle founder cells (FCs) and fusion competent myoblasts (FCMs). FCs express Dumbfounded/Kirre on their plasma membrane which is responsible for organizing myoblasts fusion to form the body wall muscle. In Zebrafish, Kirre's homolog, Kirrel, is expressed in myoblasts of the fast twitch lineage and is essential for muscle precursor fusion into syncytia. The mammalian homologs of the above receptors belong to the Neph/Kirrel family, are expressed in many tissues and function as adhesion molecules. Family members of Neph/Kirrel participate in tight interactions between kidney podocytes and are involved in neural synapses formation. One family member, Nephrin plays a role in secondary myoblasts fusion.  A recent study performed in our lab indicated that Kirrel3/Neph2 is involved in events that lead to myoblast fusion. Kirrel3 is expressed in a MyoD-dependent manner at the tips of elongated myocytes prior to fusion. We showed that Kirrel3 undergoes proteolytic cleavage which is mediated by the proteasome in myoblasts. Kirrel3-depleted myoblasts did not elongate, nor migrated in a directed manner or fused to create multinucleated myofibers. Our current hypothesis is that Kirrel3 is a key adhesion and signaling molecule which coordinates actin and membrane remodeling that are required for myoblasts' fusion. In this study, I aimed to investigate Kirrel3 cleavage at the plasma membrane and to explore the intracellular signaling events downstream of Kirrel3 that lead to myoblasts elongation.

First, I demonstrate that Kirrel3 undergoes proteolytic cleavage at the plasma membrane where two cleavage events occur; the first at the intracellular domain and the second at the extracellular domain of the receptor.

Second, I investigated the intracellular signaling pathway downstream of Kirrel3. I found that Kirrel3 knockdown in differentiating myoblasts elevates the levels of active RhoA. Based on this finding, I investigated the role of two intracellular RhoA inhibitors, Graf1; a GTPase Activating Protein (GAP) of RhoA and RhoE, a small GTPase protein. I found that Graf1 or RhoE knockdown by shRNA prevented myoblasts elongation, but did not significantly affect differentiation. In addition, knockdown of each of these proteins led to the expression of higher GTP-RhoA levels. I present evidence that Kirrel3 and Graf1 physically interact and that this interaction is mediated by the intracellular domain of Kirrel3. These results indicate that Kirrel3 inhibits RhoA activity via Graf1 and RhoE enabling the elongation of myocytes.

Last, I began investigating the possible role of Kirrel3 in muscle regeneration, by monitoring the expression of Kirrel3 RNA and protein in cardiotoxin-injured mice muscle. I found that Kirrel3 is transiently expressed during regeneration, indicating that Kirrel3 may have a role in the regeneration process.

Overall, these results indicate that Kirrel3 is necessary for the events leading to myoblast cell fusion in mammals.