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Myotube Formation on Micro-patterned Glass: Intracellular Organization and Protein Distribution in C2C12 Skeletal Muscle Cells

Daniel L. Yamamoto, Robert I. Csikasz, Yu Li, Gunjana Sharma, Klas Hjort, Roger Karlsson and Tore Bengtsson

Department of Physiology (DLY,RIC,TB) and Department of Cell Biology (YL,RK), Arrhenius Laboratories, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden, and Ångströmslaboratoriet, Uppsala University, Uppsala, Sweden (GS,KH)

Correspondence to: Tore Bengtsson, Department of Physiology, Arrhenius Laboratories E5, The Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden. E-mail: tore.bengtsson{at}zoofys.su.se. Co-corresponding author: Roger Karlsson. E-mail: roger.karlsson{at}cellbio.su.se

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. Studies of myocyte differentiation, cell fusion, and muscle repair are limited by an appropriate in vitro muscle cell culture system. We developed a novel cell culture technique [two-dimensional muscle syncytia (2DMS) technique] that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. This technique is based on UV lithography–produced micro-patterned glass on which conventionally cultured C2C12 myoblasts proliferate, align, and fuse to neatly arranged contractile myotubes in parallel arrays. Combining this technique with fluorescent microscopy, we observed alignment of actin filament bundles and a perinuclear distribution of glucose transporter 4 after myotube formation. Newly formed myotubes contained adjacently located MyoD-positive and MyoD-negative nuclei, suggesting fusion of MyoD-positive and MyoD-negative cells. In comparison, the closely related myogenic factor Myf5 did not exhibit this pattern of distribution. Furthermore, cytoplasmic patches of MyoD colocalized with bundles of filamentous actin near myotube nuclei. At later stages of differentiation, all nuclei in the myotubes were MyoD negative. The 2DMS system is thus a useful tool for studies on muscle alignment, differentiation, fusion, and subcellular protein localization. (J Histochem Cytochem 56:881–892, 2008)

Key Words: skeletal muscle in vitro culturing • MyoD • glucose transporter 4 • F-actin • myogenesis • cell fusion • differentiation

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