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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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Brefeldin A induces a major reduction in the number of flotillin-2 containing vesicles.Chick myogenic cells were grown for 24(5 µg/ml) for 3 hours or with nocodazole (10 µg/ml) for 3 hours. After treatment, cells were grown in fresh culture medium for the next 3 hours. In images (A–F) cells were fixed with paraformaldehyde and stained with an antibody against flotillin-2 (green, A,C,E) and with the nuclear dye DAPI (blue, B,D,F). Note that while nocodazole have no effect in the presence and distribution of flotillin-2 positive vesicles (C,D), brefeldin A induced a major reduction in flotillin-2 containing vesicles (E,F). In images (G–I) cells were analyzed under phase contrast microscopy and superimposed with DAPI (blue). Scale bars represents 50 µm (in A–F and G–I).
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pone-0103990-g010: Brefeldin A induces a major reduction in the number of flotillin-2 containing vesicles.Chick myogenic cells were grown for 24(5 µg/ml) for 3 hours or with nocodazole (10 µg/ml) for 3 hours. After treatment, cells were grown in fresh culture medium for the next 3 hours. In images (A–F) cells were fixed with paraformaldehyde and stained with an antibody against flotillin-2 (green, A,C,E) and with the nuclear dye DAPI (blue, B,D,F). Note that while nocodazole have no effect in the presence and distribution of flotillin-2 positive vesicles (C,D), brefeldin A induced a major reduction in flotillin-2 containing vesicles (E,F). In images (G–I) cells were analyzed under phase contrast microscopy and superimposed with DAPI (blue). Scale bars represents 50 µm (in A–F and G–I).

Mentions: In order to test the hypothesis that flotillin 2-containing vesicles somehow are involved in chick myoblast fusion, we try to disrupt intracellular vesicles with two agents that have been described to be able to disorganize intracellular vesicles: brefeldin A and nocodazole (Figure 10). Our results show that while nocodazole have no effect in the presence and distribution of flotillin-2 positive vesicles, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles (Figure 10). Quantification of myoblast fusion index showed a 13% decrease in chick myoblast fusion after brefeldin A treatment (Table 1). The fungal macrocyclic lactone brefeldin A has been shown to inhibit protein trafficking in the endomembrane system of eukariotic cells [19], by specifically blocking the intracellular transport of protein from endoplasmic reticulum to the Golgi apparatus. In agreement with our data, Ichikawa and colleagues [20] described that brefeldin A inhibited myotube formation in the mouse C2C12 cell line. We can conclude from our data that brefeldin A treatment is related both to a decrease in the number of flotillin-2 containing vesicles and to a decrease in myoblast fusion in chick 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)

Brefeldin A induces a major reduction in the number of flotillin-2 containing vesicles.Chick myogenic cells were grown for 24(5 µg/ml) for 3 hours or with nocodazole (10 µg/ml) for 3 hours. After treatment, cells were grown in fresh culture medium for the next 3 hours. In images (A–F) cells were fixed with paraformaldehyde and stained with an antibody against flotillin-2 (green, A,C,E) and with the nuclear dye DAPI (blue, B,D,F). Note that while nocodazole have no effect in the presence and distribution of flotillin-2 positive vesicles (C,D), brefeldin A induced a major reduction in flotillin-2 containing vesicles (E,F). In images (G–I) cells were analyzed under phase contrast microscopy and superimposed with DAPI (blue). Scale bars represents 50 µm (in A–F and G–I).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103990-g010: Brefeldin A induces a major reduction in the number of flotillin-2 containing vesicles.Chick myogenic cells were grown for 24(5 µg/ml) for 3 hours or with nocodazole (10 µg/ml) for 3 hours. After treatment, cells were grown in fresh culture medium for the next 3 hours. In images (A–F) cells were fixed with paraformaldehyde and stained with an antibody against flotillin-2 (green, A,C,E) and with the nuclear dye DAPI (blue, B,D,F). Note that while nocodazole have no effect in the presence and distribution of flotillin-2 positive vesicles (C,D), brefeldin A induced a major reduction in flotillin-2 containing vesicles (E,F). In images (G–I) cells were analyzed under phase contrast microscopy and superimposed with DAPI (blue). Scale bars represents 50 µm (in A–F and G–I).
Mentions: In order to test the hypothesis that flotillin 2-containing vesicles somehow are involved in chick myoblast fusion, we try to disrupt intracellular vesicles with two agents that have been described to be able to disorganize intracellular vesicles: brefeldin A and nocodazole (Figure 10). Our results show that while nocodazole have no effect in the presence and distribution of flotillin-2 positive vesicles, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles (Figure 10). Quantification of myoblast fusion index showed a 13% decrease in chick myoblast fusion after brefeldin A treatment (Table 1). The fungal macrocyclic lactone brefeldin A has been shown to inhibit protein trafficking in the endomembrane system of eukariotic cells [19], by specifically blocking the intracellular transport of protein from endoplasmic reticulum to the Golgi apparatus. In agreement with our data, Ichikawa and colleagues [20] described that brefeldin A inhibited myotube formation in the mouse C2C12 cell line. We can conclude from our data that brefeldin A treatment is related both to a decrease in the number of flotillin-2 containing vesicles and to a decrease in myoblast fusion in chick 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