, Volume 65, Issue 5, pp 725–735 | Cite as

Extracellular matrix is required for muscle differentiation in primary cell cultures of larval Mytilus trossulus (Mollusca: Bivalvia)

  • Vyacheslav DyachukEmail author
Original Research


Components of the extracellular matrix may modulate the growth factor effects that play important roles in the proliferation and differentiation of precursor cells. We developed an in vitro cultivation protocol for cells of the larval marine bivalve Mytilus trossulus to study the role that extracellular matrix components may play in myodifferentiation and replication-mediated DNA synthesis using immunofluorescence and confocal laser scanning microscopy. Here, we demonstrate that the extracellular matrix regulates the expression of muscle proteins, leading to their assembly and the terminal muscle differentiation of larval cells during cultivation. We further show that the myogenesis process progresses in cells cultivated on fibronectin, carbon or poly-l-lysine but is inhibited in cells grown on a collagen carpet. Consistent with a decrease in muscle protein expression in cells cultivated on collagen, we demonstrate an increase in the number of BrdU-positive cells in comparison with cells cultured on other substrates during the entire cultivation period. Moreover, we demonstrate that the matrix-dependent myogenic differentiation of larval mussel cells is reversible. Round-shaped cells cultivated on collagen were able to differentiate into muscle cells after reseeding on fibronectin, carbon or poly-l-lysine. In addition, cells cultured on collagen and then transplanted to fibronectin exhibited distinct cross-striation and contractile activity. Taken together, our data suggest that the extracellular matrix participates in the regulation of the proliferation and myodifferentiation of mussel trochophore progenitor cells and validate novel approaches for successfully culturing cells from bivalves over extended periods.


Cell culture Mussel Myogenic differentiation ECM 



This work was supported by the Russian Foundation for Basic Research (grant no. 13-04-00946); the study was partly performed at the “CHROMAS” center (St. Petersburg State University, Russia) and used technical resources of the “Vostok” Marine Biological Station and the core facility of IMB FEB RAS. V. Dyachuk received financial support from the Organizing Committee to attend the Marine Invertebrate Cell Culture symposium.

Conflict of interest

There is absolutely no conflict of interest in this manuscript.


  1. Beningo KA, Lo CM, Wang YL (2002) Flexible polyacrylamide substrata for the analysis of mechanical interactions at cell-substratum adhesions. Methods Cell Biol 69:325–339CrossRefGoogle Scholar
  2. Bray MA, Sheehy SP, Parker KK (2008) Sarcomere alignment is regulated by myocyte shape. Cell Motil Cytoskeleton 65:641–651CrossRefGoogle Scholar
  3. Buckingham M (1994) Molecular biology of muscle development. Cell 78:15–21CrossRefGoogle Scholar
  4. Burke RD (1999) Invertebrate integrins: structure, function, and evolution. Int Rev Cytol 191:257–284CrossRefGoogle Scholar
  5. 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 232:1069–1078CrossRefGoogle Scholar
  6. Cukierman E, Pankov R, Stevens DR, Yamada KM (2001) Taking cell-matrix adhesions to the third dimension. Science 294:1708–1712CrossRefGoogle Scholar
  7. Deroanne CF, Lapiere CM, Nusgens BV (2001) In vitro tubulogenesis of endothelial cells by relaxation of the coupling extracellular matrix-cytoskeleton. Cardiovasc Res 49:647–658CrossRefGoogle Scholar
  8. Dlugosz AA, Antin PB, Nachmias VT, Holtzer H (1984) The relationship between stress fiber-like structures and nascent myofibrils in cultured cardiac myocytes. J Cell Biol 99:2268–2278CrossRefGoogle Scholar
  9. Duband JL, Dufour S, Hatta K, Takeichi M, Edelman GM, Thiery JP (1987) Adhesion molecules during somitogenesis in the avian embryo. J Cell Biol 104:1361–1374CrossRefGoogle Scholar
  10. Edmondson DG, Olson EN (1993) Helix-loop-helix proteins as regulators of muscle-specific transcription. J Biol Chem 268:755–758Google Scholar
  11. Elangbam CS, Qualls CW Jr, Dahlgren RR (1997) Cell adhesion molecules—update. Vet Pathol 34:61–73CrossRefGoogle Scholar
  12. Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126:677–689CrossRefGoogle Scholar
  13. Gullberg D, Ekblom P (1995) Extracellular matrix and its receptors during development. Int J Dev Biol 39:845–854Google Scholar
  14. Hanselmann R, Smolowitz R, Gibson D (2000) Identification of proliferating cells in hard clams. Biol Bull 199:199–200CrossRefGoogle Scholar
  15. Har-El R, Tanzer ML (1993) Extracellular matrix. 3: Evolution of the extracellular matrix in invertebrates. FASEB J 7:1115–1123Google Scholar
  16. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110:673–687CrossRefGoogle Scholar
  17. Ishii N, Wadsworth WG, Stern BD, Culotti JG, Hedgecock EM (1992) UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans. Neuron 9:873–881CrossRefGoogle Scholar
  18. Katz BZ, Yamada KM (1997) Integrins in morphogenesis and signaling. Biochimie 79:467–476CrossRefGoogle Scholar
  19. Kim C, Ye F, Ginsberg MH (2011) Regulation of integrin activation. Annu Rev Cell Dev Biol 27:321–345CrossRefGoogle Scholar
  20. Krieger M, Scott MP, Matsudaira PT, Lodish HF, Darnell JE, Zipursky L, Kaiser C, Berk A (2004) Molecular cell biology, 5th edn. W.H. Freeman, New YorkGoogle Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly the head of bacteriophage T4. Nature 227:680–682CrossRefGoogle Scholar
  22. Larsen M, Artym VV, Green JA, Yamada KM (2006) The matrix reorganized: extracellular matrix remodeling and integrin signaling. Curr Opin Cell Biol 18:463–471CrossRefGoogle Scholar
  23. Lecroisey C, Sealat L, Gieseler K (2007) The C. elegans dense body: anchoring and signaling structure of the muscle. J Muscle Res Cell Motil 28:79–87CrossRefGoogle Scholar
  24. Marigómez I, Lekube X, Cancio I (1999) Immunochemical localisation of proliferating cells in mussel digestive gland tissue. Histochem J 31:781–788CrossRefGoogle Scholar
  25. Martin-Bermudo MD, Dunin-Borkowski OM, Brown NH (1998) Modulation of integrin activity is vital for morphogenesis. J Cell Biol 141:1073–1081CrossRefGoogle Scholar
  26. McDonald KA, Horwitz AF, Knudsen KA (1995) Adhesion molecules and skeletal myogenesis. Semin Dev Biol 6:105–116CrossRefGoogle Scholar
  27. Mercer KB, Flaherty DB, Miller RK, Qadota H, Tinley TL, Moerman DG, Benian GM (2003) Caenorhabditis elegans UNC-98, a C2H2 Zn finger protein, is a novel partner of UNC-97/PINCH in muscle adhesion complexes. Mol Biol Cell 14:2492–2507CrossRefGoogle Scholar
  28. Naganuma T, Degnan BM, Horikoshi K, Morse DE (1994) Myogenesis in primary cell cultures from larvae of the abalone, Haliotis rufescens. Mol Marine Biol Biotechnol 3:131–140Google Scholar
  29. Odintsova NA, Khomenko AV (1991) Primary cell culture from embryos of the Japanise scallop Mizuchopecten yessoensis (Bivalvia). Cytotechnol 6:49–54CrossRefGoogle Scholar
  30. Odintsova NA, Plotnikov SV, Karpenko AA (2000) Isolation and partial characterization of myogenic cells from mussel larvae in vitro. Tissue Cell 32:417–424CrossRefGoogle Scholar
  31. Odintsova NA, Dyachuk VA, Nezlin LP (2010) Muscle and neuronal differentiation in primary cell culture of larval Mytilus trossulus (Mollusca: Bivalvia). Cell Tissue Res 339:625–637CrossRefGoogle Scholar
  32. Pelham RJ Jr, Wang Y (1997) Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci USA 94:13661–13665CrossRefGoogle Scholar
  33. Plotnikov SV, Karpenko AA, Odintsova NA (2003) Comparative characteristic of Mytilus muscle cells developed in vitro and in vivo. J Exp Zool Part A 298:77–85CrossRefGoogle Scholar
  34. Sanger JW, Kang S, Siebrands CC, Freeman N, Du A, Wang J, Stout AL, Sanger JM (2005) How to build a myofibril. J Muscle Res Cell Motil 26:343–354CrossRefGoogle Scholar
  35. Schwarzbauer J (1999) Basement membranes: putting up the barriers. Curr Biol 9:R242–R244CrossRefGoogle Scholar
  36. Serpentini A, Ghayor C, Poncet JM, Hebert V, Galéra P, Pujol JP, Boucaud-Camou E, Lebel JM (2000) Collagen study and regulation of the de novo synthesis by IGF-I in hemocytes from the gastropod mollusc, Haliotis tuberculata. J Exp Zool 287:275–284CrossRefGoogle Scholar
  37. Shelud’ko NS, Matusovskaya GG, Permyakova TV, Matusovsky OS (2004) Twitchin, a thick-filament protein from molluscan catch muscle, interacts with F-actin in a phosphorylation-dependent way. Arch Biochem Biophys 432:269–277CrossRefGoogle Scholar
  38. Shelud’ko NS, Tuturova KP, Permyakova TV, Plotnikov SV, Orlova AA (1999) A novel thick filament protein in smooth muscles of bivalvia mollusks. Comp Biochem Physiol 122:277–285Google Scholar
  39. Taipale J, Keski-Oja J (1997) Growth factors in the extracellular matrix. FASEB J 11:51–59Google Scholar
  40. Towbin H, Gordon J, Staehelin T (1979) Electroforetic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354CrossRefGoogle Scholar
  41. Urbano JM, Domínguez-Giménez P, Estrada B, Martín-Bermudo MD (2011) PS integrins and laminins: key regulators of cell migration during Drosophila embryogenesis. PLoS ONE 6:e23893CrossRefGoogle Scholar
  42. Velleman SG (2002) Role of the extracellular matrix in muscle growth and development. J Anim Sci 80:E8–E13Google Scholar
  43. von der Mark K, Ocalan M (1989) Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype. Differentiation 40:150–157CrossRefGoogle Scholar
  44. Williams BD, Waterston RH (1994) Genes critical for muscle development and function in Caenorhabditis elegans identified through lethal mutations. J Cell Biol 124:475–490CrossRefGoogle Scholar
  45. Wolfstetter G, Holz A (2012) The role of LamininB2 (LanB2) during mesoderm differentiation in Drosophila. Cell Mol Life Sci 69:267–282CrossRefGoogle Scholar
  46. Yablonka-Reuveni Z, Paterson BM (2001) MyoD and myogenin expression patterns in cultures of fetal and adult chicken myoblasts. J Histochem Cytochem 49:455–462CrossRefGoogle Scholar
  47. Yeung T, Georges PC, Flanagan LA, Marg B, Ortiz M, Funaki M, Zahir N, Ming W, Weaver V, Janmey PA (2005) Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskeleton 60:24–34CrossRefGoogle Scholar
  48. Yurchenco PD, Amenta PS, Patton BL (2004) Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol 22:521–538CrossRefGoogle Scholar
  49. Zagris N, Chung AE, Stavridis V (2000) Differential expression of laminin genes in early chick embryo. Int J Dev Biol 44:815–818Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.A.V. Zhirmunsky Institute of Marine BiologyFar Eastern Branch of the Russian Academy of SciencesVladivostokRussia

Personalised recommendations