Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

International audienceMyoblasts 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

Flotillin-2 is found in pre-fusion myoblasts after cholesterol depletion.

<p>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>B</b> and <b>C</b>). Some cells were not treated and were fixed at 48 hours of culture (control, <b>A</b>). All cells were fixed with paraformaldehyde and stained with antibodies against desmin (<b>red</b>) and flotillin-2 (<b>green</b>) and with the nuclear dye DAPI (<b>blue</b>). Merged images are shown in <b>A–C</b>. Note an increase in the number of flotillin-2 positive-mononucleated cells in close contact with the membrane of multinucleated myotubes (arrows in <b>B</b> and <b>C</b>). Scale bar in <b>A</b> represents 20 µm.</p

Flotillin-2 distribution in multinucleated myotubes.

<p>Myogenic cells were grown for 72-flotillin-2 antibodies (green, <b>A</b> and <b>C</b>) and the nuclear dye DAPI (blue, <b>B</b> and <b>D</b>). Note that with methanol fixation it is possible to see that flotillin-2 is present in elongated dots in myotubes (arrows in <b>A</b> and <b>C</b>). Scale bar in <b>D</b> represents 20 µm.</p

Flotillin-2 distribution in myogenic cells during skeletal muscle differentiation.

<p>Myogenic cells were grown for 24(<b>A</b> and <b>B</b>) or 72 hours (<b>C</b>). Cells were fixed with paraformaldehyde and stained with antibodies against desmin (<b>red</b>, <b>A</b> and <b>C</b>) and flotillin-2 (<b>green</b>, <b>A, B</b> and <b>C</b>) and with the nuclear dye DAPI (<b>blue</b>, <b>A</b> and <b>C</b>). Merged images are shown in <b>A</b> and <b>C</b>. Note that with paraformaldehyde fixation it is possible to see flotillin-2 almost exclusively in mononucleated cells in vesicle-like structures (<b>B</b>) and nearly absent from myotubes (<b>A</b> and <b>C</b>). Scale bar in <b>A</b> and <b>C</b> represents 20 µm and in <b>B</b> represents 10 µm.</p

Brefeldin A induces a major reduction in the number of flotillin-2 containing vesicles.

<p>Chick myogenic cells were grown for 24(5 µg/ml) for 3 hours or with nocodazole (10 µg/ml) for 3 hours. After treatment, cells were grown in fresh culture medium for the next 3 hours. In images (<b>A–F</b>) cells were fixed with paraformaldehyde and stained with an antibody against flotillin-2 (<b>green, A,C,E</b>) and with the nuclear dye DAPI (<b>blue, B,D,F</b>). Note that while nocodazole have no effect in the presence and distribution of flotillin-2 positive vesicles (<b>C,D</b>), brefeldin A induced a major reduction in flotillin-2 containing vesicles (<b>E,F</b>). In images (<b>G–I</b>) cells were analyzed under phase contrast microscopy and superimposed with DAPI (blue). Scale bars represents 50 µm (in <b>A–F</b> and <b>G–I</b>).</p

Flotillin-2 is down-regulated during <i>in vitro</i> chick skeletal myogenesis.

<p>Chick myogenic cells were grown for 24, 48 and 72-2. (<b>A</b>) Upper Western blot shows flotillin-2 reactivity and lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading. (<b>B</b>) Quantification of protein bands revealed a progressive decrease in the levels of flotillin-2 expression during skeletal muscle differentiation. *p<0.05; ANOVA followed by Tukey post hoc test versus 24-h group, n = 3.</p

Cholesterol depletion enhances the expression of flotillin-2 protein and mRNA.

<p>Chick myogenic cells were grown 24(control, <b>Ct</b>). Cell culture extracts were analyzed in Western blot using an antibody against flotillin-2 (<b>A</b>). Lower Western blot shows α-tubulin reactivity of the same samples, and was used to normalize sample loading (<b>A</b>). Quantification of protein bands revealed a 40% increase in the levels of flotillin-2 expression after cholesterol depletion (<b>B</b>). RT-PCR analysis (for details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103990#s2" target="_blank">Materials and Methods</a>) of the expression of flotillin-2 in control and in MbCD-treated cells is shown in <b>C</b>. 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.</p

Distribution of flotillin-2 in human neonatal muscle cells.

<p>Human neonatal muscle cells were grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103990#s2" target="_blank">Materials and Methods</a> section. Cells were fixed with methanol and stained with an anti-flotillin-2 antibody (<b>green</b>, <b>A</b> and <b>B</b>) and the nuclear dye DAPI (<b>blue</b>, <b>A</b> and <b>B</b>). Merged images are shown in <b>A</b> and <b>B</b>. Note that flotillin-2 is present in human myoblasts and myotubes in vesicle-like structures (arrows in <b>A</b> and <b>B</b>). Scale bar in <b>A</b> represents 10 µm.</p

Transmission electron microscopy of chick fibroblastic cells.

<p>Chick myogenic cells were grown for 48(asterisks), well-preserved mitochondria (M), as well as several endoplasmic reticulum membranous profiles (ER) and a nucleus (N). Image shown in <b>b</b> is a higher magnification of a region of the image shown in <b>a</b>. Scale bar in <b>a</b> represents 1 µm and in <b>b</b> represents 500 <i>µ</i>m.</p

Cryo-immunogold EM labeling of flotillin-2 in myogenic cells.

<p>Chick myogenic cells were grown for 48-flotillin-2 antibody followed by a 10-<i>η</i>m gold-conjugated secondary antibody. Note that gold particles are present in vesicles (<b>a</b>) and associated with Golgi stacks (<b>b</b>). GC, Golgi complex. Scale bar in <b>a</b> represents 100 <i>η</i>m and in <b>b</b> represents 250 <i>η</i>m.</p