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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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Related in: MedlinePlus

Flotillin-2 distribution in myogenic cells during skeletal muscle differentiation.Myogenic cells were grown for 24(A and B) or 72 hours (C). Cells were fixed with paraformaldehyde and stained with antibodies against desmin (red, A and C) and flotillin-2 (green, A, B and C) and with the nuclear dye DAPI (blue, A and C). Merged images are shown in A and C. Note that with paraformaldehyde fixation it is possible to see flotillin-2 almost exclusively in mononucleated cells in vesicle-like structures (B) and nearly absent from myotubes (A and C). Scale bar in A and C represents 20 µm and in B represents 10 µm.
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pone-0103990-g002: Flotillin-2 distribution in myogenic cells during skeletal muscle differentiation.Myogenic cells were grown for 24(A and B) or 72 hours (C). Cells were fixed with paraformaldehyde and stained with antibodies against desmin (red, A and C) and flotillin-2 (green, A, B and C) and with the nuclear dye DAPI (blue, A and C). Merged images are shown in A and C. Note that with paraformaldehyde fixation it is possible to see flotillin-2 almost exclusively in mononucleated cells in vesicle-like structures (B) and nearly absent from myotubes (A and C). Scale bar in A and C represents 20 µm and in B represents 10 µm.

Mentions: Since chick primary myogenic cultures contain fibroblasts, myoblasts and multinucleated myotubes, we examined the distribution of flotillin-2 in the different types of cells. Primary cultures of chick skeletal muscle cells were grown for 24 and 72 hours, fixed with paraformaldehyde and immunolabeled for flotillin-2. Flotillin-2 was found predominantly in mononucleated cells and, interestingly, was almost absent from multinucleated myotubes (Figure 2). It is important to point out that mononucleated cells comprise both fibroblasts and myoblasts in chick primary myogenic cultures.


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 myogenic cells during skeletal muscle differentiation.Myogenic cells were grown for 24(A and B) or 72 hours (C). Cells were fixed with paraformaldehyde and stained with antibodies against desmin (red, A and C) and flotillin-2 (green, A, B and C) and with the nuclear dye DAPI (blue, A and C). Merged images are shown in A and C. Note that with paraformaldehyde fixation it is possible to see flotillin-2 almost exclusively in mononucleated cells in vesicle-like structures (B) and nearly absent from myotubes (A and C). Scale bar in A and C represents 20 µm and in B represents 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103990-g002: Flotillin-2 distribution in myogenic cells during skeletal muscle differentiation.Myogenic cells were grown for 24(A and B) or 72 hours (C). Cells were fixed with paraformaldehyde and stained with antibodies against desmin (red, A and C) and flotillin-2 (green, A, B and C) and with the nuclear dye DAPI (blue, A and C). Merged images are shown in A and C. Note that with paraformaldehyde fixation it is possible to see flotillin-2 almost exclusively in mononucleated cells in vesicle-like structures (B) and nearly absent from myotubes (A and C). Scale bar in A and C represents 20 µm and in B represents 10 µm.
Mentions: Since chick primary myogenic cultures contain fibroblasts, myoblasts and multinucleated myotubes, we examined the distribution of flotillin-2 in the different types of cells. Primary cultures of chick skeletal muscle cells were grown for 24 and 72 hours, fixed with paraformaldehyde and immunolabeled for flotillin-2. Flotillin-2 was found predominantly in mononucleated cells and, interestingly, was almost absent from multinucleated myotubes (Figure 2). It is important to point out that mononucleated cells comprise both fibroblasts and myoblasts in chick primary myogenic cultures.

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