Fibronectin promotes migration, alignment and fusion in an in vitro myoblast cell model
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Myogenesis is a complex process in which committed myogenic cells differentiate and fuse into myotubes that mature into the muscle fibres of adult organisms. This process is initiated by a cascade of myogenic regulatory factors expressed upon entry of the cells into the myogenic differentiation programme. However, external signals such as those provided by the extracellular matrix (ECM) are also important in regulating muscle differentiation and morphogenesis. In the present work, we have addressed the role of various ECM substrata on C2C12 myoblast behaviour in vitro. Cells grown on fibronectin align and fuse earlier than cells on laminin or gelatine. Live imaging of C2C12 myoblasts on fibronectin versus gelatine has revealed that fibronectin promotes a directional collective migratory behaviour favouring cell-cell alignment and fusion. We further demonstrate that this effect of fibronectin is mediated by RGD-binding integrins expressed on myoblasts, that N-cadherin contributes to this behaviour, and that it does not involve enhanced myogenic differentiation. Therefore, we suggest that the collective migration and alignment of cells seen on fibronectin leads to a more predictable movement and a positioning that facilitates subsequent fusion of myoblasts. This study highlights the importance of addressing the role of fibronectin, an abundant component of the interstitial ECM during embryogenesis and tissue repair, in the context of myogenesis and muscle regeneration.
KeywordsMyoblast behaviour Live imaging Extracellular matrix Integrins C2C12 cells
We are grateful to the members of our group for helpful discussions and to our laboratory rotation students, Márcio Madureira and Ana Rita Leitoguinho, for help with RT-PCR. The MF20, F5D and MNCD2 antibodies developed by D.A. Fishman, by W.E. Wright and M. Takeichi and by H. Matsunami, respectively, were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA52242.
Time-lapse movie of C2C12 cells on gelatine with startingconfluence of 60%. Total film length: 10h50min. Colour scale corresponds to time. (AVI 7.32 mb)
Time-lapse movie of C2C12 cells on fibronectin with startingconfluence of 60%. Total film length: 10h50min. Colour scale corresponds to time. (AVI 7.60 mb)
Time-lapse movie of C2C12 cells on gelatine with startingconfluence of 90%. Total film length: 12h15min. Colour scale corresponds to time. (AVI 8.61 mb)
Time-lapse movie of C2C12 cells on fibronectin with startingconfluence of 90%. Total film length: 12h15min. Colour scale corresponds to time. (AVI 8.79 mb)