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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 is down-regulated during in vitro chick skeletal myogenesis.Chick myogenic cells were grown for 24, 48 and 72-2. (A) Upper Western blot shows flotillin-2 reactivity and lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading. (B) Quantification of protein bands revealed a progressive decrease in the levels of flotillin-2 expression during skeletal muscle differentiation. *p<0.05; ANOVA followed by Tukey post hoc test versus 24-h group, n = 3.
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pone-0103990-g001: Flotillin-2 is down-regulated during in vitro chick skeletal myogenesis.Chick myogenic cells were grown for 24, 48 and 72-2. (A) Upper Western blot shows flotillin-2 reactivity and lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading. (B) Quantification of protein bands revealed a progressive decrease in the levels of flotillin-2 expression during skeletal muscle differentiation. *p<0.05; ANOVA followed by Tukey post hoc test versus 24-h group, n = 3.

Mentions: First, we analyzed the expression of flotillin-2 protein during chick in vitro myogenic differentiation. Chick myogenic cells were grown for 24, 48 and 72 hours, and cell culture extracts were analyzed by Western blot using an antibody against flotillin-2. Figure 1 shows that flotillin-2 is highly expressed in the first 24–48 hours of culture, and it is barely detectable in 72-h cultures. Chick myogenic cultures at 24 h are mainly composed of mononucleated cells, namely fibroblasts and myoblasts, while 48-h cultures contain fibroblasts, myoblasts and young multinucleated myotubes. Seventy-two-hour cultures are mainly composed of mature multinucleated myotubes (thicker and longer than those found in 48-h cultures), with a reduced number of fibroblasts and myoblasts. These results show that flotillin-2 is down-regulated during chick in vitro myogenesis and suggest the involvement of flotillin-2 in the initial steps of skeletal muscle differentiation. In contrast with these results obtained on chick muscle cells, it has been described previously that flotillin-2 is up-regulated during the differentiation of the mouse cell line C2C12 [9]. There are at least two possible explanations for these contradictory results: (i) differences between chick and mouse; and/or (ii) differences between primary cultures and immortalized cell lines. Primary cultures of chick skeletal muscle cells behave very differently from muscle cell lines (such as C2C12). Differentiation of muscle cell lines must be induced by external stimuli and the most common way is the reduction of serum in the culture medium. In contrast, chick primary myoblast cultures do not depend on variations in the amount of serum to differentiate. Skeletal myogenesis occurs in a robust and autonomous way in primary chick myoblast cultures, culminating with the formation of myotubes that can contain more than one hundred nuclei per cell.


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 is down-regulated during in vitro chick skeletal myogenesis.Chick myogenic cells were grown for 24, 48 and 72-2. (A) Upper Western blot shows flotillin-2 reactivity and lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading. (B) Quantification of protein bands revealed a progressive decrease in the levels of flotillin-2 expression during skeletal muscle differentiation. *p<0.05; ANOVA followed by Tukey post hoc test versus 24-h group, n = 3.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103990-g001: Flotillin-2 is down-regulated during in vitro chick skeletal myogenesis.Chick myogenic cells were grown for 24, 48 and 72-2. (A) Upper Western blot shows flotillin-2 reactivity and lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading. (B) Quantification of protein bands revealed a progressive decrease in the levels of flotillin-2 expression during skeletal muscle differentiation. *p<0.05; ANOVA followed by Tukey post hoc test versus 24-h group, n = 3.
Mentions: First, we analyzed the expression of flotillin-2 protein during chick in vitro myogenic differentiation. Chick myogenic cells were grown for 24, 48 and 72 hours, and cell culture extracts were analyzed by Western blot using an antibody against flotillin-2. Figure 1 shows that flotillin-2 is highly expressed in the first 24–48 hours of culture, and it is barely detectable in 72-h cultures. Chick myogenic cultures at 24 h are mainly composed of mononucleated cells, namely fibroblasts and myoblasts, while 48-h cultures contain fibroblasts, myoblasts and young multinucleated myotubes. Seventy-two-hour cultures are mainly composed of mature multinucleated myotubes (thicker and longer than those found in 48-h cultures), with a reduced number of fibroblasts and myoblasts. These results show that flotillin-2 is down-regulated during chick in vitro myogenesis and suggest the involvement of flotillin-2 in the initial steps of skeletal muscle differentiation. In contrast with these results obtained on chick muscle cells, it has been described previously that flotillin-2 is up-regulated during the differentiation of the mouse cell line C2C12 [9]. There are at least two possible explanations for these contradictory results: (i) differences between chick and mouse; and/or (ii) differences between primary cultures and immortalized cell lines. Primary cultures of chick skeletal muscle cells behave very differently from muscle cell lines (such as C2C12). Differentiation of muscle cell lines must be induced by external stimuli and the most common way is the reduction of serum in the culture medium. In contrast, chick primary myoblast cultures do not depend on variations in the amount of serum to differentiate. Skeletal myogenesis occurs in a robust and autonomous way in primary chick myoblast cultures, culminating with the formation of myotubes that can contain more than one hundred nuclei per cell.

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