Fibronectin promotes migration, alignment and fusion in an in vitro myoblast cell model

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.

[1]  D. Boettiger,et al.  Modulation of cell proliferation and differentiation through substrate-dependent changes in fibronectin conformation. , 1999, Molecular biology of the cell.

[2]  Kenneth M. Yamada,et al.  The matrix reorganized: extracellular matrix remodeling and integrin signaling. , 2006, Current opinion in cell biology.

[3]  Arnoud Sonnenberg,et al.  Integrins in regulation of tissue development and function , 2003, The Journal of pathology.

[4]  V. Krenn,et al.  Differences in the fibronectin-dependence of migrating cell populations , 2004, Anatomy and Embryology.

[5]  R. Kramer,et al.  Laminins promote the locomotion of skeletal myoblasts via the alpha 7 integrin receptor. , 1996, Journal of cell science.

[6]  J. Sanes.  Laminin, fibronectin, and collagen in synaptic and extrasynaptic portions of muscle fiber basement membrane , 1982, The Journal of cell biology.

[7]  J. Muschler,et al.  Integrin oL Subunit Ratios , Cytoplasmic Domains , and Growth Factor Synergy Regulate Muscle Proliferation and Differentiation , 2002 .

[8]  X. Liu,et al.  N-cadherin/catenin-mediated morphoregulation of somite formation. , 1998, Developmental biology.

[9]  Sudipto Roy,et al.  Myoblast fusion: when it takes more to make one. , 2010, Developmental biology.

[10]  E. Brandan,et al.  ECM is required for skeletal muscle differentiation independently of muscle regulatory factor expression. , 2002, American journal of physiology. Cell physiology.

[11]  A. Sonnenberg,et al.  Spatial and temporal expression of the β1D integrin during mouse development , 1997 .

[12]  Peter D Yurchenco,et al.  Basement membrane assembly, stability and activities observed through a developmental lens. , 2004, Matrix Biology.

[13]  J. Sanes,et al.  Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis , 1992, Cell.

[14]  D. Yaffe,et al.  Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle , 1977, Nature.

[15]  M. Goody,et al.  Dynamic interactions between cells and their extracellular matrix mediate embryonic development , 2010, Molecular reproduction and development.

[16]  M. Parsons,et al.  Collective Chemotaxis Requires Contact-Dependent Cell Polarity , 2010, Developmental cell.

[17]  G. Davis,et al.  3D Timelapse Analysis of Muscle Satellite Cell Motility , 2009, Stem cells.

[18]  A. Gamel,et al.  N-cadherin is involved in myoblast migration and muscle differentiation in the avian limb bud. , 1996, Developmental Biology.

[19]  G. Pavlath,et al.  Molecular control of mammalian myoblast fusion. , 2008, Methods in molecular biology.

[20]  J. Muschler,et al.  Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation , 1996, The Journal of cell biology.

[21]  M. Humphries,et al.  VCAM-1 is a CS1 peptide-inhibitable adhesion molecule expressed by lymph node high endothelium. , 1993, Journal of cell science.

[22]  A. Starzinski-Powitz,et al.  Involvement of M-cadherin in terminal differentiation of skeletal muscle cells. , 1995, Journal of cell science.

[23]  Benjamin G Keselowsky,et al.  Myoblast proliferation and differentiation on fibronectin-coated self assembled monolayers presenting different surface chemistries. , 2005, Biomaterials.

[24]  R. Matthiesen,et al.  Functional blockade of α5β1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells , 2010, BMC Cell Biology.

[25]  K. Mark,et al.  Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype , 1989 .

[26]  J. Lawton,et al.  Guidance of myogenic cell migration by oriented deposits of fibronectin. , 1983, Developmental biology.

[27]  D. Boettiger,et al.  Regulation of Integrin α5β1 Affinity during Myogenic Differentiation , 1995 .

[28]  Douglas W DeSimone,et al.  The extracellular matrix in development and morphogenesis: a dynamic view. , 2010, Developmental biology.

[29]  Richard O. Hynes,et al.  Integrins: Versatility, modulation, and signaling in cell adhesion , 1992, Cell.

[30]  T. Rando,et al.  Integrin-mediated Muscle Cell Spreading , 1999, The Journal of Biological Chemistry.

[31]  M. Buckingham.  Myogenic progenitor cells and skeletal myogenesis in vertebrates. , 2006, Current opinion in genetics & development.

[32]  J. Takagi,et al.  The RGD motif in fibronectin is essential for development but dispensable for fibril assembly , 2007, The Journal of cell biology.

[33]  Isabel Palmeirim,et al.  Dynamic 3D Cell Rearrangements Guided by a Fibronectin Matrix Underlie Somitogenesis , 2009, PloS one.

[34]  P. Ferri,et al.  C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. , 2004, European journal of histochemistry : EJH.

[35]  S. McElwee,et al.  A role for the Ca2(+)-dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis. , 1990, Experimental cell research.

[36]  S. Thorsteinsdóttir,et al.  Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse , 2005, Developmental dynamics : an official publication of the American Association of Anatomists.

[37]  Sólveig Thorsteinsdóttir,et al.  The extracellular matrix dimension of skeletal muscle development. , 2011, Developmental biology.

[38]  Z. Yablonka-Reuveni,et al.  The Skeletal Muscle Satellite Cell: The Stem Cell That Came in From the Cold , 2006, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[39]  T. Rando,et al.  Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment. , 2005, Trends in cell biology.

[40]  A. Walch,et al.  Cadherin-2 Controls Directional Chain Migration of Cerebellar Granule Neurons , 2009, PLoS biology.

[41]  A. Huttenlocher,et al.  Integrin and Cadherin Synergy Regulates Contact Inhibition of Migration and Motile Activity , 1998, The Journal of cell biology.