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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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Cryo-immunogold EM labeling of flotillin-2 in myogenic cells.Chick myogenic cells were grown for 48-flotillin-2 antibody followed by a 10-ηm gold-conjugated secondary antibody. Note that gold particles are present in vesicles (a) and associated with Golgi stacks (b). GC, Golgi complex. Scale bar in a represents 100 ηm and in b represents 250 ηm.
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pone-0103990-g008: Cryo-immunogold EM labeling of flotillin-2 in myogenic cells.Chick myogenic cells were grown for 48-flotillin-2 antibody followed by a 10-ηm gold-conjugated secondary antibody. Note that gold particles are present in vesicles (a) and associated with Golgi stacks (b). GC, Golgi complex. Scale bar in a represents 100 ηm and in b represents 250 ηm.

Mentions: In order to confirm the presence of flotillin-2 in vesicular structures in chick fibroblasts, we studied the subcellular distribution of flotillin-2 after cryo-immunogold labeling in electron microscopy. Gold particles were found in the Golgi apparatus and in the membrane of vesicles found in the cytoplasm of chick fibroblastic cells (Figure 8). The presence of flotillin-2 in vesicles and in the Golgi suggests a role of flotillin-2 in the secretory pathway in chick muscle fibroblasts. Further studies are necessary to elucidate the possible involvement of flotillin-2-positive vesicles in the secretion of specific extracellular signaling molecules by fibroblastic cells. It is important to point out that we did not observe gold particles at the cell membrane of chick fibroblasts after the cryo-immunogold labeling in electron microscopy.


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)

Cryo-immunogold EM labeling of flotillin-2 in myogenic cells.Chick myogenic cells were grown for 48-flotillin-2 antibody followed by a 10-ηm gold-conjugated secondary antibody. Note that gold particles are present in vesicles (a) and associated with Golgi stacks (b). GC, Golgi complex. Scale bar in a represents 100 ηm and in b represents 250 ηm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103990-g008: Cryo-immunogold EM labeling of flotillin-2 in myogenic cells.Chick myogenic cells were grown for 48-flotillin-2 antibody followed by a 10-ηm gold-conjugated secondary antibody. Note that gold particles are present in vesicles (a) and associated with Golgi stacks (b). GC, Golgi complex. Scale bar in a represents 100 ηm and in b represents 250 ηm.
Mentions: In order to confirm the presence of flotillin-2 in vesicular structures in chick fibroblasts, we studied the subcellular distribution of flotillin-2 after cryo-immunogold labeling in electron microscopy. Gold particles were found in the Golgi apparatus and in the membrane of vesicles found in the cytoplasm of chick fibroblastic cells (Figure 8). The presence of flotillin-2 in vesicles and in the Golgi suggests a role of flotillin-2 in the secretory pathway in chick muscle fibroblasts. Further studies are necessary to elucidate the possible involvement of flotillin-2-positive vesicles in the secretion of specific extracellular signaling molecules by fibroblastic cells. It is important to point out that we did not observe gold particles at the cell membrane of chick fibroblasts after the cryo-immunogold labeling in electron microscopy.

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