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Myotube Formation on Micro-patterned Glass: Intracellular organization and protein distribution in C2C12 skeletal muscle cells
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
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2008 (English)In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 56, no 10, 881-892 p.Article in journal (Refereed) Published
Abstract [en]

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 peri-nuclear 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.

Place, publisher, year, edition, pages
2008. Vol. 56, no 10, 881-892 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:su:diva-25088DOI: 10.1369/jhc.2008.951228OAI: oai:DiVA.org:su-25088DiVA: diva2:198853
Available from: 2008-05-14 Created: 2008-05-07 Last updated: 2011-03-16Bibliographically approved
In thesis
1. Cell Behavior and the Role of Profilin
Open this publication in new window or tab >>Cell Behavior and the Role of Profilin
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Profilin is a key regulator of the microfilament system. It binds to actin monomers in a 1:1 complex, forming the profilin:actin complex, which is the major precursor of actin for filament formation in vivo. The distribution of profilin has been studied in a variety of cells. It is present not only in the cytoplasm but also in the nucleus. In the cytoplasm, it is evenly distributed in a dotted pattern, which is concentrated at the edge of advancing lamellipodia and in the perinuclear region. In the nucleus, it is localized to Speckles and Cajal bodies. However, the distribution of the profilin:actin has not been possible to establish due to the lack of specific reagents. In this thesis I present the localization of the profilin:actin complex and demonstrate the importance of profilin during cell migration.

The distribution of the profilin:actin complex was studied using affinity purified antibodies generated against a covalently coupled variant of profilin:actin in colocalization experiments with VASP and the Arp2/3 complex. In both cases, close co-distribution with profilin:actin was found. In order to study the role of profilin in vivo in migratory cells, I used the siRNA-technique to deplete profilin from motile mouse melanoma B16 cells. The particular cell line employed expressed actin fused to green fluorescent protein, which enabled imaging of live cells. Upon profilin-deficiency severe effects on cell behavior were observed, e.g. the cells lacked the ability to form characteristic broad lamellipodia at advancing edges, instead small protruding structures were generated and extended with a significantly reduced rate compared to control cells. Observations were also made suggesting that profilin regulates the expression of actin in mammalian cells.

A new experimental system for studies of myoblast fusion and subsequent myotube formation in vitro was also established during these studies. This will facilitate systematic studies of molecular processes connected to muscle development.

Place, publisher, year, edition, pages
Stockholm: Wenner-Grens institut för experimentell biologi, 2008. 53 p.
National Category
Cell Biology
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-7687 (URN)978-91-7155-677-6 (ISBN)
Public defence
2008-06-05, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 13:00
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Available from: 2008-05-14 Created: 2008-05-07Bibliographically approved

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