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 Rodrigues
Regular Article

Abstract

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.

Keywords

Myoblast behaviour Live imaging Extracellular matrix Integrins C2C12 cells 

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)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Raquel Vaz
    • 1
  • Gabriel G. Martins
    • 1
    • 2
  • Sólveig Thorsteinsdóttir
    • 1
    • 2
  • Gabriela Rodrigues
    • 1
    • 2
  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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