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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 mainly expressed in fibroblasts and weakly expressed in myoblasts.Myogenic cells were grown for 24(A and B). Cells were fixed with paraformaldehyde and stained with antibodies against MyoD (red, A and B) and flotillin-2 (green, A, and B) and with the nuclear dye DAPI (blue, B). Merged images are shown in A and B. Note that flotillin-2 is highly expressed in MyoD-negative cells and weakly expressed in MyoD-positive cells (A and B). Scale bar in B represents 20 µm.
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pone-0103990-g003: Flotillin-2 is mainly expressed in fibroblasts and weakly expressed in myoblasts.Myogenic cells were grown for 24(A and B). Cells were fixed with paraformaldehyde and stained with antibodies against MyoD (red, A and B) and flotillin-2 (green, A, and B) and with the nuclear dye DAPI (blue, B). Merged images are shown in A and B. Note that flotillin-2 is highly expressed in MyoD-negative cells and weakly expressed in MyoD-positive cells (A and B). Scale bar in B represents 20 µm.

Mentions: To determine whether flotillin-2 positive cells were fibroblasts and/or myoblasts in chick myogenic cultures, we triple-labeled 24-h cells with an anti-chick MyoD antibody [10], an anti-flotillin-2 antibody, and the nuclear dye DAPI. The results show that MyoD-negative cells were highly labeled for flotillin-2, while MyoD-positive cells were weakly labeled for flotillin-2 (Figure 3). These data indicate that in chick myogenic cultures flotillin-2 is mainly expressed in fibroblasts and weakly expressed in myoblasts. We found that both round and bipolar MyoD-positive-myoblasts were weakly positive for flotillin-2. These data suggest that flotillin-2 is expressed in rounded chick myoblasts prior to their elongation and fusion, and that somehow flotillin-2 is redistributed and/or down-regulated after myoblast fusion into myotubes. These results are in agreement with the work of Volonté and co-workers [9], who showed that flotillin-2 is virtually absent from mature skeletal muscle tissues (diaphragm and psoas muscle) of mice. Draeger and colleagues [15] also showed that profound structural rearrangements occur during skeletal muscle maturation including a striking decrease in membrane lipid segregation and a down-regulation of raft-associated proteins.


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 mainly expressed in fibroblasts and weakly expressed in myoblasts.Myogenic cells were grown for 24(A and B). Cells were fixed with paraformaldehyde and stained with antibodies against MyoD (red, A and B) and flotillin-2 (green, A, and B) and with the nuclear dye DAPI (blue, B). Merged images are shown in A and B. Note that flotillin-2 is highly expressed in MyoD-negative cells and weakly expressed in MyoD-positive cells (A and B). Scale bar in B represents 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103990-g003: Flotillin-2 is mainly expressed in fibroblasts and weakly expressed in myoblasts.Myogenic cells were grown for 24(A and B). Cells were fixed with paraformaldehyde and stained with antibodies against MyoD (red, A and B) and flotillin-2 (green, A, and B) and with the nuclear dye DAPI (blue, B). Merged images are shown in A and B. Note that flotillin-2 is highly expressed in MyoD-negative cells and weakly expressed in MyoD-positive cells (A and B). Scale bar in B represents 20 µm.
Mentions: To determine whether flotillin-2 positive cells were fibroblasts and/or myoblasts in chick myogenic cultures, we triple-labeled 24-h cells with an anti-chick MyoD antibody [10], an anti-flotillin-2 antibody, and the nuclear dye DAPI. The results show that MyoD-negative cells were highly labeled for flotillin-2, while MyoD-positive cells were weakly labeled for flotillin-2 (Figure 3). These data indicate that in chick myogenic cultures flotillin-2 is mainly expressed in fibroblasts and weakly expressed in myoblasts. We found that both round and bipolar MyoD-positive-myoblasts were weakly positive for flotillin-2. These data suggest that flotillin-2 is expressed in rounded chick myoblasts prior to their elongation and fusion, and that somehow flotillin-2 is redistributed and/or down-regulated after myoblast fusion into myotubes. These results are in agreement with the work of Volonté and co-workers [9], who showed that flotillin-2 is virtually absent from mature skeletal muscle tissues (diaphragm and psoas muscle) of mice. Draeger and colleagues [15] also showed that profound structural rearrangements occur during skeletal muscle maturation including a striking decrease in membrane lipid segregation and a down-regulation of raft-associated proteins.

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