title: Myogenesis defect due to Toca-1 knockdown can be suppressed by expression of N-WASP.
abstract: 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 Cdc42-dependent actin assembly), is an adaptor protein which activates N-WASP in conjunction with Cdc42 to facilitate membrane invagination, endocytosis and actin cytoskeleton remodeling. Expression of Toca-1 in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated during differentiation. Knocking down Toca-1 expression in C2C12 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.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Bhawana
authors.initials: B
authors.lastname: George
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Neeraj
authors.initials: N
authors.lastname: Jain
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Pei
authors.initials: P
authors.lastname: Fen Chong
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Jun
authors.initials: J
authors.lastname: Hou Tan
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore. Electronic address: thirumaran@ntu.edu.sg.
authors.forename: Thirumaran
authors.initials: T
authors.lastname: Thanabalu
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015820:Cadherins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002450:Cell Communication / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002459:Cell Fusion; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D024510:Muscle Development / Q000187:drug effects; D017570:MyoD Protein / Q000378:metabolism; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism; D053778:Thiazolidines / Q000494:pharmacology; D016596:Vinculin / Q000378:metabolism; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D015820:Cadherins; D002352:Carrier Proteins; C000591411:FNBP1L protein, mouse; D017570:MyoD Protein; D053778:Thiazolidines; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D016596:Vinculin
keywords: Actin cytoskeleton; Arp2/3 complex; Cell adhesion; Cell migration; Membrane fusion; Myogenic differentiation