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Differences in the expression and distribution of flotillin-2 in chick, mice and human muscle cells.

Possidonio AC, Soares CP, Portilho DM, Midlej V, Benchimol M, Butler-Browne G, Costa ML, Mermelstein C - PLoS ONE (2014)

Bottom Line: Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation.Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes.These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Diferenciação Muscular e Citoesqueleto, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.

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Flotillin-2 distribution in multinucleated myotubes.Myogenic cells were grown for 72-flotillin-2 antibodies (green, A and C) and the nuclear dye DAPI (blue, B and D). Note that with methanol fixation it is possible to see that flotillin-2 is present in elongated dots in myotubes (arrows in A and C). Scale bar in D represents 20 µm.
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pone-0103990-g004: Flotillin-2 distribution in multinucleated myotubes.Myogenic cells were grown for 72-flotillin-2 antibodies (green, A and C) and the nuclear dye DAPI (blue, B and D). Note that with methanol fixation it is possible to see that flotillin-2 is present in elongated dots in myotubes (arrows in A and C). Scale bar in D represents 20 µm.

Mentions: Since we found very little flotillin-2 staining in myotubes after paraformaldehyde fixation (Figures 2 and 3), we decided to rule out the possibility of fixation artifacts by testing other fixation protocols. Methanol fixation showed flotillin-2 concentrated in elongated dots in the subsarcolemmal region of 72-h chick multinucleated myotubes (Figure 4).


Differences in the expression and distribution of flotillin-2 in chick, mice and human muscle cells.

Possidonio AC, Soares CP, Portilho DM, Midlej V, Benchimol M, Butler-Browne G, Costa ML, Mermelstein C - PLoS ONE (2014)

Flotillin-2 distribution in multinucleated myotubes.Myogenic cells were grown for 72-flotillin-2 antibodies (green, A and C) and the nuclear dye DAPI (blue, B and D). Note that with methanol fixation it is possible to see that flotillin-2 is present in elongated dots in myotubes (arrows in A and C). Scale bar in D represents 20 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126691&req=5

pone-0103990-g004: Flotillin-2 distribution in multinucleated myotubes.Myogenic cells were grown for 72-flotillin-2 antibodies (green, A and C) and the nuclear dye DAPI (blue, B and D). Note that with methanol fixation it is possible to see that flotillin-2 is present in elongated dots in myotubes (arrows in A and C). Scale bar in D represents 20 µm.
Mentions: Since we found very little flotillin-2 staining in myotubes after paraformaldehyde fixation (Figures 2 and 3), we decided to rule out the possibility of fixation artifacts by testing other fixation protocols. Methanol fixation showed flotillin-2 concentrated in elongated dots in the subsarcolemmal region of 72-h chick multinucleated myotubes (Figure 4).

Bottom Line: Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation.Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes.These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Diferenciação Muscular e Citoesqueleto, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.

Show MeSH
Related in: MedlinePlus