Skeletal muscle formation is a step-wise process of cell proliferation, differentiation and fusion of mono-nucleated myoblasts to multi-nucleated myotubes. The cellular steps involved in myotube formation are cell-extracellular matrix (ECM) adhesion, change of cell shape from fibroblast-like to elongated morphology, migration towards other fusing partners, cell-cell adhesion followed by membrane breakdown. Membrane reorganization and actin cytoskeleton remodelling are two essential factors in cellular steps during myogenic differentiation and fusion. IRSp53 (53 kDa Insulin Receptor Substrate protein) and Toca-1 (Transducer of Cdc42-dependent actin assembly) are members of BAR (BIN/amphiphysin!Rvs) domain proteins which co-ordinate membrane bending with actin cytoskeleton remodeling. BAR domain proteins work as scaffolds to change membrane curvature. IRSp53 through its I-BAR domain (Inverse BAR)/ IMD (IRSp53 and MIM (missing in metastases) homology Domain) can bend membrane in outward direction and Toca-1 through its F-BAR domain can bend membrane in inward direction. IRSp53 is well-known for inducing filopodia in concert with a number of proteins upon activation by small GTPases. Expression of IRSp53 is downregulated during the differentiation of C2C12 cells to myotubes. Knocking down the expression of IRSp53 using shRNA led to increase in the fusion index compared to cells transfected with the scrambled shRNA. In contrast, transfection of plasmid expressing IRSp53 led to inhibition of myogenic differentiation, decrease in ceii-ECM adhesion and increase in the induction of membrane projections. IRSp53 is an adaptor protein with an IMD/1-BAR domain located at the N-terminus, a GTPase Binding Domain (GBD) and a SH3 domain. Mutations which impaired the function of any of the three domains of IRSp53 abolished the ability of IRSp53 to inhibit myogenic differentiation. Expression of IRSp531MD alone inhibited myogeneis and reduced ceii-ECM adhesion . Therefore, IRSp531MD is the functional unit responsible for suppression of differentiation mediated by IRSp53 in C2C12 cells. Expression of another I-BAR domain protein IRTKS (Insulin receptor tyrosine kinase substrate ; also known as BAIAP2L 1) in C2C 12 cells also induced small membrane projections and inhibited myoblast fusion. Expression of Toca-1 , the F-BAR domain protein, in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated. Knocking down Toca-1 expression in C2C12 cells resulted in a significant decrease in myotube formation. Toca-1 knockdown cells displayed elongated morphology and expressed differentiation markers (MyoD and MyHC) suggesting there was no differentiation defect in Toca-1 knockdown cells. In addition, Toca-1 knockdown cells displayed slightly increased migration and decreased vinculin patches which is similar to N-WASP knockout fibroblasts. Moreover, expression of NWASP suppressed the fusion defect of Toca-1 KD C2C 12 cells suggesting that Toca-1 probably works via N- WASP in myoblast fusion. This study has provided molecular and functional insight into the role of BAR domain family of proteins in myogenic differentiation.

​Doctor of Philosophy (SBS)