Cell and Tissue Research

, Volume 348, Issue 3, pp 569–578 | Cite as

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

  • Raquel Vaz
  • Gabriel G. Martins
  • Sólveig Thorsteinsdóttir
  • Gabriela RodriguesEmail author
Regular Article


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.


Myoblast 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.

Supplementary material

Supplementary Movie S1

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)

Supplementary Movie S2

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)

Supplementary Movie S3

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)

Supplementary Movie S4

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)

441_2012_1364_Fig5_ESM.jpg (32 kb)
Supplementary Fig. S1

α5β1 is partially, but not exclusively, responsible for the elongationand alignment of C2C12 cells on fibronectin. C2C12 cultured for 2 days on fibronectin with20 μg/ml BMB5 (Chemicon*), an antibody reported specifically toblock fibronectin-α5β1 integrin interaction (b, c) or with bovine serum albumin (a). Culture with BMB5resulted in a mixture of patches of elongated and aligned cells (b) and patches of nonalignedcells with round nuclei (c). Bars 100 μm. *Vellón L, Royo F, Matthiesen R, Torres-Fuenzalida J, Lorenti A, Parada LA (2010)Functional blockade of α5β1 integrin induces scattering and genomic landscaperemodeling of hepatic progenitor cells. BMC Cell Biol 11:81. (JPG 32.1 KB)

441_2012_1364_MOESM5_ESM.tif (1.4 mb)
High resolution image file (TIF 1.44 MB)
441_2012_1364_Fig6_ESM.jpg (41 kb)
Supplementary Fig. S2

Fibronectin delays differentiation of C2C12 cells. To assess theinfluence of fibronectin on cell differentiation, C2C12 cells seeded on gelatine andfibronectin (yellow, orange, respectively) were cultured for 2, 4, 6 and 8 daysfollowed by fixation and immunolabeling for myogenin (F5D, D.S.H.B.) followed by thequantification of the myogenin-positive nuclei. A two-way analysis of variance showed thatthe time in culture influenced cell differentiation, although globally the matrix did notinfluence differentiation and no significant interaction between these two parameters wasdetected. By day 6, we detected a tendency for fewer myogenin-positive cells on fibronectinthan on gelatine and contrast analysis revealed that, by day 8, the number of myogenin-positivecells on fibronectin was significantly lower than in cultures grown on gelatine (columns mean values, error bars represent ±0.95 confidence intervals). (JPG 41.4 KB)

441_2012_1364_MOESM6_ESM.tif (3.4 mb)
High resolution image file (TIF 3.38 MB)


  1. Boettiger D, Enomoto-Iwamoto M, Yoon HY, Hofer U, Menko AS, Chiquet-Ehrismann R (1995) Regulation of integrin α5β1 affinity during myogenic differentiation. Dev Biol 169:261–272PubMedCrossRefGoogle Scholar
  2. Brand-Saberi B, Krenn V, Grim M, Christ B (1993) Differences in the fibronectin-dependence of migrating cell populations. Anat Embryol (Berl) 187:17–26CrossRefGoogle Scholar
  3. Brand-Saberi B, Gamel AJ, Krenn V, Müller TS, Wilting J, Christ B (1996) N-Cadherin is involved in myoblast migration and muscle differentiation in the avian limb bud. Dev Biol 178:160–173PubMedCrossRefGoogle Scholar
  4. Buckingham M (2006) Myogenic progenitor cells and skeletal myogenesis in vertebrates. Curr Opin Genet Dev 16:525–532PubMedCrossRefGoogle Scholar
  5. Burattini S, Ferri P, Battistelli M, Curci R, Luchetti F, Falcieri E (2004) C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. Eur J Histochem 48:223–233PubMedGoogle Scholar
  6. Cachaço AS, Pereira CS, Pardal RG, Bajanca F, Thorsteinsdóttir S (2005) Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse. Dev Dyn 232:1069–1078PubMedCrossRefGoogle Scholar
  7. Danen EHJ, Sonnenberg A (2003) Integrins in regulation of tissue development and function. J Pathol 200:471–480PubMedCrossRefGoogle Scholar
  8. Dhawan J, Rando TA (2005) Stem cells in postnatal myogenesis: molecular mechanisms of satellite cell quiescence, activation and replenishment. Trends Cell Biol 15:666–673PubMedCrossRefGoogle Scholar
  9. Disatnik MH, Rando TA (1999) Integrin-mediated muscle cell spreading. The role of protein kinase C in outside-in and inside-out signaling and evidence of integrin cross-talk. J Biol Chem 274:32486–32492PubMedCrossRefGoogle Scholar
  10. García AJ, Vega MD, Boettiger D (1999) Modulation of cell proliferation and differentiation through substrate-dependent changes in fibronectin conformation. Mol Biol Cell 10:785–798PubMedGoogle Scholar
  11. Goody MF, Henry CA (2010) Dynamic interactions between cells and their extracellular matrix mediate embryonic development. Mol Reprod Dev 77:475–488PubMedCrossRefGoogle Scholar
  12. Huttenlocher A, Lakonishok M, Kinder M, Wu S, Truong T, Knudsen KA, Horwitz AF (1998) Integrin and cadherin synergy regulates contact inhibition of migration and motile activity. J Cell Biol 141:515–526PubMedCrossRefGoogle Scholar
  13. Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11–25PubMedCrossRefGoogle Scholar
  14. Jansen KM, Pavlath GK (2008) Molecular control of mammalian myoblast fusion. Methods Mol Biol 475:115–133PubMedCrossRefGoogle Scholar
  15. Knudsen KA, Myers L, McElwee SA (1990) A role for the Ca2+-dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis. Exp Cell Res 188:175–184PubMedCrossRefGoogle Scholar
  16. Lan MA, Gersbach CA, Michael KE, Keselowsky BG, García AJ (2005) Myoblast proliferation and differentiation on fibronectin-coated self assembled monolayers presenting different surface chemistries. Biomaterials 26:4523–4531PubMedCrossRefGoogle Scholar
  17. Larsen M, Artym VV, Green JA, Yamada KM (2006) The matrix reorganized: extracellular matrix remodeling and integrin signaling. Curr Opin Cell Biol 18:463–471PubMedCrossRefGoogle Scholar
  18. Linask KK, Ludwig C, Han MD, Liu X, Radice GL, Knudsen KA (1998) N-cadherin/catenin-mediated morphoregulation of somite formation. Dev Biol 202:85–102PubMedCrossRefGoogle Scholar
  19. Martins GG, Rifes P, Amândio R, Rodrigues G, Palmeirim I, Thorsteinsdóttir S (2009) Dynamic 3D cell rearrangements guided by a fibronectin matrix underlie somitogenesis. PLoS One 4:e7429PubMedCrossRefGoogle Scholar
  20. May MJ, Entwistle G, Humphries MJ, Ager A (1993) VCAM-1 is a CS1 peptide-inhibitable adhesion molecule expressed by lymph node high endothelium. J Cell Sci 106:109–119PubMedGoogle Scholar
  21. Osses N, Brandan E (2002) ECM is required for skeletal muscle differentiation independently of muscle regulatory factor expression. Am J Physiol Cell Physiol 282:C383–C394PubMedGoogle Scholar
  22. Rieger S, Senghaas N, Walch A, Köster RW (2009) Cadherin-2 controls directional chain migration of cerebellar granule neurons. PLoS Biol 7:e1000240PubMedCrossRefGoogle Scholar
  23. Rochlin K, Yu S, Roy S, Baylies MK (2010) Myoblast fusion: when it takes more to make one. Dev Biol 341:66–83PubMedCrossRefGoogle Scholar
  24. Rosen GD, Sanes JR, LaChance R, Cunningham JM, Roman J, Dean DC (1992) Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis. Cell 69:1107–1119PubMedCrossRefGoogle Scholar
  25. Rozario T, DeSimone DW (2010) The extracellular matrix in development and morphogenesis: a dynamic view. Dev Biol 341:126–140PubMedCrossRefGoogle Scholar
  26. Ruoslahti E (1991) Integrins. J Clin Invest 87:1–5PubMedCrossRefGoogle Scholar
  27. Sanes JR (1982) Laminin, fibronectin, and collagen in synaptic and extrasynaptic portions of muscle fiber basement membrane. J Cell Biol 93:442–451PubMedCrossRefGoogle Scholar
  28. Sastry SK, Lakonishok M, Thomas DA, Muschler J, Horwitz AF (1996) Integrin α subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation. J Cell Biol 133:169–184PubMedCrossRefGoogle Scholar
  29. Siegel AL, Atchison K, Fisher KE, Davis GE, Cornelison DD (2009) 3D timelapse analysis of muscle satellite cell motility. Stem Cells 27:2527–2538PubMedCrossRefGoogle Scholar
  30. Takahashi S, Leiss M, Moser M, Ohashi T, Kitao T, Heckmann D, Pfeifer A, Kessler H, Takagi J, Erickson HP, Fässler R (2007) The RGD motif in fibronectin is essential for development but dispensable for fibril assembly. J Cell Biol 178:167–178PubMedCrossRefGoogle Scholar
  31. Theveneau E, Marchant L, Kuriyama S, Gull M, Moepps B, Parsons M, Mayor R (2010) Collective chemotaxis requires contact-dependent cell polarity. Dev Cell 19:39–53PubMedCrossRefGoogle Scholar
  32. Thorsteinsdóttir S, Deries M, Cachaço AS, Bajanca F (2011) The extracellular matrix dimension of skeletal muscle development. Dev Biol 354:191–207PubMedCrossRefGoogle Scholar
  33. Turner DC, Lawton J, Dollenmeier P, Ehrismann R, Chiquet M (1983) Guidance of myogenic cell migration by oriented deposits of fibronectin. Dev Biol 95:497–504PubMedCrossRefGoogle Scholar
  34. van der Flier A, Gaspar AC, Thorsteinsdóttir S, Baudoin C, Groeneveld E, Mummery CL, Sonnenberg A (1997) Spatial and temporal expression of the β1D integrin during mouse development. Dev Dyn 210:472–486PubMedCrossRefGoogle Scholar
  35. Vellón L, Royo F, Matthiesen R, Torres-Fuenzalida J, Lorenti A, Parada LA (2010) Functional blockade of α5β1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells. BMC Cell Biol 11:81PubMedCrossRefGoogle Scholar
  36. von der Mark K, Öcalan M (1989) Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype. Differentiation 40:150–157PubMedCrossRefGoogle Scholar
  37. Yaffe D, Saxel O (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270:725–727PubMedCrossRefGoogle Scholar
  38. Yao CC, Ziober BL, Sutherland AE, Mendrick DL, Kramer RH (1996) Laminins promote the locomotion of skeletal myoblasts via the alpha 7 integrin receptor. J Cell Sci 109:3139–3150PubMedGoogle Scholar
  39. Yurchenco PD, Amenta PS, Patton BL (2004) Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol 22:521–538PubMedCrossRefGoogle Scholar
  40. Zammit PS, Partridge TA, Yablonka-Reuveni Z (2006) the skeletal muscle satellite cell: the stem cell that came in from the cold. J Histochem Cytochem 54:1177–1191PubMedCrossRefGoogle Scholar
  41. Zeschnigk M, Kozian D, Kuch C, Schmoll M, Starzinski-Powitz A (1995) Involvement of M-cadherin in terminal differentiation of skeletal muscle cells. J Cell Sci 108:2973–2981PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Raquel Vaz
    • 1
  • Gabriel G. Martins
    • 1
    • 2
  • Sólveig Thorsteinsdóttir
    • 1
    • 2
  • Gabriela Rodrigues
    • 1
    • 2
    Email author
  1. 1.Centro de Biologia Ambiental/Departamento de Biologia Animal, Faculdade de CiênciasUniversidade de LisboaLisboaPortugal
  2. 2.Instituto Gulbenkian de CiênciaOeirasPortugal

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