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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

Distribution of flotillin-2 in human neonatal muscle cells.Human neonatal muscle cells were grown as described in Materials and Methods section. Cells were fixed with methanol and stained with an anti-flotillin-2 antibody (green, A and B) and the nuclear dye DAPI (blue, A and B). Merged images are shown in A and B. Note that flotillin-2 is present in human myoblasts and myotubes in vesicle-like structures (arrows in A and B). Scale bar in A represents 10 µm.
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pone-0103990-g006: Distribution of flotillin-2 in human neonatal muscle cells.Human neonatal muscle cells were grown as described in Materials and Methods section. Cells were fixed with methanol and stained with an anti-flotillin-2 antibody (green, A and B) and the nuclear dye DAPI (blue, A and B). Merged images are shown in A and B. Note that flotillin-2 is present in human myoblasts and myotubes in vesicle-like structures (arrows in A and B). Scale bar in A represents 10 µm.

Mentions: Interestingly, flotillin-2 staining was found in vesicle-like structures within the cytoplasm of chick fibroblastic cells (Figures 2 and 3). Our data are consistent with the work published by Langhorst and colleagues [6] where they show that flotillin-2 is present in vesicles in subconfluent HeLa cell cultures. Flotillin-2 has been identified in different cellular compartments, including lipid rafts at the plasma membrane, lipid droplets [7], and vesicles of the endocytic pathway [8]. Flotillin-2 distribution varies among different cell types. Thus we next compared the distribution of flotillin-2 in chick muscle cells with that in human and mouse muscle cells. We labeled mouse myogenic cell line C2C12 and neonatal human muscle cells with flotillin-2 using both paraformaldehyde and methanol fixation. No flotillin-2 labeling was found after paraformaldehyde fixation in the mouse and human muscle cells, but a positive labeling was found after methanol fixation. Control experiments with no primary antibodies showed only a faint background staining in both mouse and human cells (data not shown). After methanol fixation, C2C12 myoblasts and neonatal human muscle cells showed flotillin-2 in a vesicle-like labeling (Figures 5 and 6). Flotillin-2 staining in the mouse and human muscle cells was similar to the distribution we found in chick fibroblasts (Figures 2 and 3). It is important to point out that we found differences in flotillin-2 distribution between chick primary myoblasts and mouse C2C12 myoblasts (compare Figures 2 and 5). Flotillin-2 was densely present in cytoplasmic vesicles in C2C12 cells, whereas little flotillin-2 labeling was found in chick primary myoblasts. Interestingly, Banning and colleagues [16] state that they were unable to find a single cell line that did not express flotillins, indicating that they may be essential for cultured cell lines. These results open an interesting question related to differences between myoblast cell lines and myoblast primary cultures. It is possible to speculate that flotillin-2 is preferentially expressed in proliferative cells.


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)

Distribution of flotillin-2 in human neonatal muscle cells.Human neonatal muscle cells were grown as described in Materials and Methods section. Cells were fixed with methanol and stained with an anti-flotillin-2 antibody (green, A and B) and the nuclear dye DAPI (blue, A and B). Merged images are shown in A and B. Note that flotillin-2 is present in human myoblasts and myotubes in vesicle-like structures (arrows in A and B). Scale bar in A represents 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103990-g006: Distribution of flotillin-2 in human neonatal muscle cells.Human neonatal muscle cells were grown as described in Materials and Methods section. Cells were fixed with methanol and stained with an anti-flotillin-2 antibody (green, A and B) and the nuclear dye DAPI (blue, A and B). Merged images are shown in A and B. Note that flotillin-2 is present in human myoblasts and myotubes in vesicle-like structures (arrows in A and B). Scale bar in A represents 10 µm.
Mentions: Interestingly, flotillin-2 staining was found in vesicle-like structures within the cytoplasm of chick fibroblastic cells (Figures 2 and 3). Our data are consistent with the work published by Langhorst and colleagues [6] where they show that flotillin-2 is present in vesicles in subconfluent HeLa cell cultures. Flotillin-2 has been identified in different cellular compartments, including lipid rafts at the plasma membrane, lipid droplets [7], and vesicles of the endocytic pathway [8]. Flotillin-2 distribution varies among different cell types. Thus we next compared the distribution of flotillin-2 in chick muscle cells with that in human and mouse muscle cells. We labeled mouse myogenic cell line C2C12 and neonatal human muscle cells with flotillin-2 using both paraformaldehyde and methanol fixation. No flotillin-2 labeling was found after paraformaldehyde fixation in the mouse and human muscle cells, but a positive labeling was found after methanol fixation. Control experiments with no primary antibodies showed only a faint background staining in both mouse and human cells (data not shown). After methanol fixation, C2C12 myoblasts and neonatal human muscle cells showed flotillin-2 in a vesicle-like labeling (Figures 5 and 6). Flotillin-2 staining in the mouse and human muscle cells was similar to the distribution we found in chick fibroblasts (Figures 2 and 3). It is important to point out that we found differences in flotillin-2 distribution between chick primary myoblasts and mouse C2C12 myoblasts (compare Figures 2 and 5). Flotillin-2 was densely present in cytoplasmic vesicles in C2C12 cells, whereas little flotillin-2 labeling was found in chick primary myoblasts. Interestingly, Banning and colleagues [16] state that they were unable to find a single cell line that did not express flotillins, indicating that they may be essential for cultured cell lines. These results open an interesting question related to differences between myoblast cell lines and myoblast primary cultures. It is possible to speculate that flotillin-2 is preferentially expressed in proliferative cells.

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