Myogenesis defect due to Toca-1 knockdown can be suppressed by expression of N-WASP.

[wiskott-aldrich syndrome]

Skeletal muscle formation is a multistep process involving proliferation , differentiation , alignment and fusion of myoblasts to form myotubes which fuse with additional myoblast to form myofibers . Toca-1 (Transducer of Cdc 42 -dependent actin assembly ), is an adaptor protein which activates N-WASP in conjunction with Cdc 42 to facilitate membrane invagination , endocytosis and actin cytoskeleton remodeling . Expression of Toca-1 in mouse primary myoblasts and C 2 C 12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated during differentiation . Knocking down Toca-1 expression in C 2 C 12 cells (Toca-1 (KD ) cells ) resulted in a significant decrease in myotube formation and expression of shRNA-resistant Toca-1 in Toca-1 (KD ) cells rescued the myogenic defect , suggesting that the knockdown was specific and Toca-1 is essential for myotube formation . Toca-1 (KD ) cells exhibited elongated spindle-like morphology , expressed myogenic markers (MyoD and MyHC ) and localized N-Cadherin at cell periphery similar to control cells suggesting that Toca-1 is not essential for morphological changes or expression of proteins critical for differentiation . Toca-1 (KD ) cells displayed prominent actin fibers suggesting a defect in actin cytoskeleton turnover necessary for cell-cell fusion . Toca-1 (KD ) cells migrated faster than control cells and had a reduced number of vinculin patches similar to N-WASP (KO ) MEF cells . Transfection of N-WASP-expressing plasmid into Toca-1 (KD ) cells restored myotube formation of Toca-1 (KD ) cells . Thus , our results suggest that Toca-1 (KD ) cells have defects in formation of myotubes probably due to reduced activity of actin cytoskeleton regulators such as N-WASP . This is the first study to identify and characterize the role of Toca-1 in myogenesis .