Appendix 2: Movie Clips is supplied as a Zip Archive and will need to be unzipped before viewing.The mechanism of force transfer from contracting sarcomeres to the membrane and endomysium of striated muscle fibres is unclear. The caveolar complex array in striated muscle membranes is a local concentration of cholesterol, sphingomyelin, signalling molecules and the protein caveolin-3. Immunofluorescence microscopy of caveolin-3 in the membrane reveals a regular pattern of fluorescent nodes arranged in longitudinal and transverse rows. The primary aim of this study was to analyse this pattern and how caveolin-3 behaves during contraction. Dynamic imaging and Fast Fourier Transforms (FFTs) were used to study force transmission across the fibre membrane. \ud This pattern was studied in frozen sections of both shortened and rest-length striated muscle fibres. Direct and FFT measurements of spacings between these nodes demonstrated significant reductions in longitudinal measurements in shortened muscle when compared to rest-length muscle. Caveolin-3 nodes lay in register with underlying actin bands in both muscle states, and co-localised with dystrophin. Caveolin-3 was not detectable in C2C12 myoblasts. During differentiation expression became detectable at 2 days. Caveolin-3 was present during myoblast fusion, before forming the regular pattern on the membrane from days 4-5. Fibres became contractile after 5-6 days of development. By 12 days, muscle fibres are 1-2 mm long, multinucleated myotubes with evidence of the caveolin-3 immunofluorescence pattern seen in mature fibres. Knockdown of caveolin-3 expression greatly reduced the number of differentiated myotubes at 12 days. This pattern was not demonstrated in contracting myotubes, possibly owing to lack of permeability to the antibody. \ud The results are consistent with the hypothesis that the force of contraction is transferred across the whole membrane rather than at fibre distal ends
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