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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 is found in pre-fusion myoblasts after cholesterol depletion.Immunofluorescence of flotillin-2, desmin and DAPI in cultures treated with methyl-β-cyclodextrin (MbCD) showing flotillin-2 expression in mononucleated cells that are fusing with myotubes. Chick myogenic cells were grown for 24 hours, treated with 2 mM MbCD for 30 min and grown for the next 24 hours (B and C). Some cells were not treated and were fixed at 48 hours of culture (control, A). All cells were fixed with paraformaldehyde and stained with antibodies against desmin (red) and flotillin-2 (green) and with the nuclear dye DAPI (blue). Merged images are shown in A–C. Note an increase in the number of flotillin-2 positive-mononucleated cells in close contact with the membrane of multinucleated myotubes (arrows in B and C). Scale bar in A represents 20 µm.
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pone-0103990-g009: Flotillin-2 is found in pre-fusion myoblasts after cholesterol depletion.Immunofluorescence of flotillin-2, desmin and DAPI in cultures treated with methyl-β-cyclodextrin (MbCD) showing flotillin-2 expression in mononucleated cells that are fusing with myotubes. Chick myogenic cells were grown for 24 hours, treated with 2 mM MbCD for 30 min and grown for the next 24 hours (B and C). Some cells were not treated and were fixed at 48 hours of culture (control, A). All cells were fixed with paraformaldehyde and stained with antibodies against desmin (red) and flotillin-2 (green) and with the nuclear dye DAPI (blue). Merged images are shown in A–C. Note an increase in the number of flotillin-2 positive-mononucleated cells in close contact with the membrane of multinucleated myotubes (arrows in B and C). Scale bar in A represents 20 µm.

Mentions: Interestingly, after cholesterol depletion it was possible to observe an increase in the number of flotillin-2-positive mononucleated cells in close contact with the cell membrane of multinucleated myotubes (Figure 9). Previously, our group has shown that cholesterol depletion by 2 mM MbCD induces an increase in myoblast proliferation and in myoblast fusion in chick primary cultures of skeletal muscle cells [2], [17]. It has been shown by Langhorst and colleagues [6] that cholesterol extraction by 12 mM MbCD reduces flotillin-2 vesicle trafficking in HeLa cells. Flotillins have been implicated in membrane trafficking and in the recruitment of specific molecules to cell contact sites. Regulated cycling of membrane proteins enables cells to rapidly modulate protein levels at the plasma membrane. Changes in the cycling rate of a vesicular pathway will immediately affect the distribution of its associated proteins between intracellular compartments and the plasma membrane [18].


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 found in pre-fusion myoblasts after cholesterol depletion.Immunofluorescence of flotillin-2, desmin and DAPI in cultures treated with methyl-β-cyclodextrin (MbCD) showing flotillin-2 expression in mononucleated cells that are fusing with myotubes. Chick myogenic cells were grown for 24 hours, treated with 2 mM MbCD for 30 min and grown for the next 24 hours (B and C). Some cells were not treated and were fixed at 48 hours of culture (control, A). All cells were fixed with paraformaldehyde and stained with antibodies against desmin (red) and flotillin-2 (green) and with the nuclear dye DAPI (blue). Merged images are shown in A–C. Note an increase in the number of flotillin-2 positive-mononucleated cells in close contact with the membrane of multinucleated myotubes (arrows in B and C). Scale bar in A represents 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103990-g009: Flotillin-2 is found in pre-fusion myoblasts after cholesterol depletion.Immunofluorescence of flotillin-2, desmin and DAPI in cultures treated with methyl-β-cyclodextrin (MbCD) showing flotillin-2 expression in mononucleated cells that are fusing with myotubes. Chick myogenic cells were grown for 24 hours, treated with 2 mM MbCD for 30 min and grown for the next 24 hours (B and C). Some cells were not treated and were fixed at 48 hours of culture (control, A). All cells were fixed with paraformaldehyde and stained with antibodies against desmin (red) and flotillin-2 (green) and with the nuclear dye DAPI (blue). Merged images are shown in A–C. Note an increase in the number of flotillin-2 positive-mononucleated cells in close contact with the membrane of multinucleated myotubes (arrows in B and C). Scale bar in A represents 20 µm.
Mentions: Interestingly, after cholesterol depletion it was possible to observe an increase in the number of flotillin-2-positive mononucleated cells in close contact with the cell membrane of multinucleated myotubes (Figure 9). Previously, our group has shown that cholesterol depletion by 2 mM MbCD induces an increase in myoblast proliferation and in myoblast fusion in chick primary cultures of skeletal muscle cells [2], [17]. It has been shown by Langhorst and colleagues [6] that cholesterol extraction by 12 mM MbCD reduces flotillin-2 vesicle trafficking in HeLa cells. Flotillins have been implicated in membrane trafficking and in the recruitment of specific molecules to cell contact sites. Regulated cycling of membrane proteins enables cells to rapidly modulate protein levels at the plasma membrane. Changes in the cycling rate of a vesicular pathway will immediately affect the distribution of its associated proteins between intracellular compartments and the plasma membrane [18].

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