Fig. 5. The myosin folding intermediates exhibit partially folded motor domains. C2C12 cells expressing GFP-myosin were fixed either in 4% paraformaldehyde (A-C,G-I), or 100% methanol (D-F), and labeled with anti-myosin mAb, F59. (A) In paraformaldehyde-fixed cells the GFP-myosin is found in striated myofibrils and in folding intermediates (green). (B) The mAb F59 labels the intermediates, but not the mature myofibrils of infected or uninfected myotubes (red). (C) The color merged image shows a green myotube surrounded by yellow and red labeled cells containing the myosin folding intermediate. The arrow in B and C marks an uninfected cell that contains F59-stained folding intermediates. Methanol fixation partially denatures cellular proteins and exposes the F59 epitope in the striated myofibrils. (D) The GFP-myosin is assembled predominantly in striated myofibrils in the myotubes of this 96-hour culture. (E) F59 uniformly labels all the myosin-containing structures in the methanol fixed sample, resulting in a complete overlap with the GFP-myosin as seen in the color merged image (F). The arrow in E and F mark an uninfected cell that contains an F59 stained folding intermediate. (G-I) Uninfected C2C12 myocytes, 48 hour after transfer to differentiation medium, were paraformaldehyde fixed and stained with mAb F59 (green), anti-Hsp90 (red) and DAPI (blue). The filamentous and globular folding intermediates stain with both F59 and anti-Hsp90 in this projection of a 3D confocal microscopy volume dataset. The magnification in A-F is the same.