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

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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.

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Acknowledgements

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.

Author information

Correspondence to Gabriela Rodrigues.

Additional information

R.V. and this work were supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) project PTDC/BIA-BCM/67437/2006.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

Supplementary Fig. S1
figure5

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)

Supplementary Fig. S2
figure6

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)

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)

High resolution image file (TIF 1.44 MB)

High resolution image file (TIF 3.38 MB)

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Vaz, R., Martins, G.G., Thorsteinsdóttir, S. et al. Fibronectin promotes migration, alignment and fusion in an in vitro myoblast cell model. Cell Tissue Res 348, 569–578 (2012). https://doi.org/10.1007/s00441-012-1364-1

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Keywords

  • Myoblast behaviour
  • Live imaging
  • Extracellular matrix
  • Integrins
  • C2C12 cells