Thick myofilament mass determination by electron scattering measurements with the scanning transmission electron microscope

Summary An accurate value for mass/length of thick myofilaments is required to establish a limit for the maximum number of myosin molecules per crossbridge repeat. The mass/length of the crossbridge regions of desalted thick myofilaments from insect flight muscle (Lethocerus andMusca) and rabbit psoas has been measured with a computer-linked STEM by comparing the electron scattering signal per unit length of unstained thick filaments with that from TMV particles in the same image. Filament preparation was aided by limited digestion of myofibrils to remove Z bands using calcium-activated factor (CAF) from rabbit skeletal muscle; SDS gels showed that this selective protease spared myosin and tended to spare paramyosin but removed C protein.Lethocerus filaments prepared by the CAF procedure were 20–25% heavier per unit length than those prepared by conventional (simple) shearing, and retained a clear and generally uniform 14.5 nm crossbridge repeat by negative staining.We have expressed mass/length of thick filaments as myosin equivalents (mol. wt 0.470 × 10⁶) per crown (that is, the 14.5 nm insect or 14.3 nm vertebrate repeat along thick filaments). TMV standards, calculated to weigh 0.1304×10⁶ daltons nm^–1 and thus equivalent to 4.02 myosins per 14.5 nm, were uniform to ±3%s.d. for 73 particles after normalizing means for each different image field. After subtracting the known paramyosin content from insect measurements (11% for waterbug, 2% for the housefly), but making no C protein correction to rabbit measurements, the following results were obtained:Lethocerus (all) 4.19±0.50 (243 filaments);Lethocerus (CAF prepns) 4.40±0.44 (145 filaments);Musca (all CAF) 4.14±0.37 (57 filaments); rabbit (all CAF) 2.86±0.34 (75 filaments).These values favour the lowest integral number of myosins per crown among currently competing models of thick filament structure. The rabbit value agrees with several previous estimates, including the STEM measurements of Lamvik, which indicated three rather than four myosins/crown. The insect flight myofilament value of four forces re-evaluation of previous estimates by quantitative gels and quantitative microscopy of whole fibrils which had favoured six myosins/crown.