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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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Cholesterol depletion enhances the expression of flotillin-2 protein and mRNA.Chick myogenic cells were grown 24(control, Ct). Cell culture extracts were analyzed in Western blot using an antibody against flotillin-2 (A). Lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading (A). Quantification of protein bands revealed a 40% increase in the levels of flotillin-2 expression after cholesterol depletion (B). RT-PCR analysis (for details, see Materials and Methods) of the expression of flotillin-2 in control and in MbCD-treated cells is shown in C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. Analysis of the expression of flotillin-2 shows a more than 2-fold increase in the levels of mRNA expression in MbCD-treated cells compared with control cells. *p<0.05; t test for unpaired samples, n = 3.
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pone-0103990-g011: Cholesterol depletion enhances the expression of flotillin-2 protein and mRNA.Chick myogenic cells were grown 24(control, Ct). Cell culture extracts were analyzed in Western blot using an antibody against flotillin-2 (A). Lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading (A). Quantification of protein bands revealed a 40% increase in the levels of flotillin-2 expression after cholesterol depletion (B). RT-PCR analysis (for details, see Materials and Methods) of the expression of flotillin-2 in control and in MbCD-treated cells is shown in C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. Analysis of the expression of flotillin-2 shows a more than 2-fold increase in the levels of mRNA expression in MbCD-treated cells compared with control cells. *p<0.05; t test for unpaired samples, n = 3.

Mentions: Since we found an increase in the number of flotillin-2 positive mononucleated cells after cholesterol depletion (Figure 9), we confirmed these data with the analysis of the expression of flotillin-2 mRNA and protein in chick myogenic cells. Chick myogenic cell cultures were treated at 24 h with 2 mM MbCD for 30 min, grown for the next 24 hours and subjected to a Western blot against flotillin-2. Results showed that MbCD induced a 40% increase in the expression of flotillin-2 protein as compared to untreated cells (Figure 11). We also analyzed the expression levels of flotillin-2 transcripts in control and MbCD-treated cells. Real time-PCR data revealed a more than 2-fold increase in the levels of flotillin-2 mRNA after cholesterol depletion (Figure 11). These results are in agreement with the data presented in Figure 9 showing that cholesterol depletion induced an increase in the number of flotillin-2-positive 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)

Cholesterol depletion enhances the expression of flotillin-2 protein and mRNA.Chick myogenic cells were grown 24(control, Ct). Cell culture extracts were analyzed in Western blot using an antibody against flotillin-2 (A). Lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading (A). Quantification of protein bands revealed a 40% increase in the levels of flotillin-2 expression after cholesterol depletion (B). RT-PCR analysis (for details, see Materials and Methods) of the expression of flotillin-2 in control and in MbCD-treated cells is shown in C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. Analysis of the expression of flotillin-2 shows a more than 2-fold increase in the levels of mRNA expression in MbCD-treated cells compared with control cells. *p<0.05; t test for unpaired samples, n = 3.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103990-g011: Cholesterol depletion enhances the expression of flotillin-2 protein and mRNA.Chick myogenic cells were grown 24(control, Ct). Cell culture extracts were analyzed in Western blot using an antibody against flotillin-2 (A). Lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading (A). Quantification of protein bands revealed a 40% increase in the levels of flotillin-2 expression after cholesterol depletion (B). RT-PCR analysis (for details, see Materials and Methods) of the expression of flotillin-2 in control and in MbCD-treated cells is shown in C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. Analysis of the expression of flotillin-2 shows a more than 2-fold increase in the levels of mRNA expression in MbCD-treated cells compared with control cells. *p<0.05; t test for unpaired samples, n = 3.
Mentions: Since we found an increase in the number of flotillin-2 positive mononucleated cells after cholesterol depletion (Figure 9), we confirmed these data with the analysis of the expression of flotillin-2 mRNA and protein in chick myogenic cells. Chick myogenic cell cultures were treated at 24 h with 2 mM MbCD for 30 min, grown for the next 24 hours and subjected to a Western blot against flotillin-2. Results showed that MbCD induced a 40% increase in the expression of flotillin-2 protein as compared to untreated cells (Figure 11). We also analyzed the expression levels of flotillin-2 transcripts in control and MbCD-treated cells. Real time-PCR data revealed a more than 2-fold increase in the levels of flotillin-2 mRNA after cholesterol depletion (Figure 11). These results are in agreement with the data presented in Figure 9 showing that cholesterol depletion induced an increase in the number of flotillin-2-positive 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