STRUCTURE AND PERIODICITIES OF CROSS-BRIDGES IN RELAXATION IN RIGOR, AND DURING CONTRACTIONS INITIATED BY PHOTOLYSIS OF CAGED CA2+

Ultra-rapid freezing and electron microscopy were used to directly obs erve structural details of frog muscle fibers in rigor, in relaxation, and during force development initiated by laser photolysis of DM-nitr ophen (a caged Ca2+). Longitudinal sections from relaxed fibers show h elical tracks of the myosin heads on the surface of the thick filament s. Fibers frozen at similar to 13, similar to 34, and similar to 220 m s after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridge dispositions. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridge de nsities have a wide range of shapes and angles, perpendicular to the f iber axis or pointing toward or away from the Z line. This highly vari able distribution is established very early during development of cont raction. Cross-bridge density across the interfilament space is more u niform than in rigor, wherein the cross-bridges are more dense near th e thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze peri odicities of structures within the overlap regions of the sarcomeres. Most aspects of these patterns are consistent with time resolved x-ray diffraction data from the corresponding states of intact muscle, but some features are different, presumably reflecting different origins o f contrast between the two methods and possible alterations in the str ucture of the electron microscopy samples during processing. In relaxe d fibers, OD patterns show strong meridional spots and layer lines up to the sixth order of the 43-nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, l ayer lines at 18, 24, and 36 nm indicate cross-bridge attachment along the thin filament helix. After activation by photorelease of Ca2+, th e 14,3-nm meridional spot is present, but the second-order meridional spot (22 nm) disappears. The myosin 43-nm layer line becomes less inte nse, and higher orders of 43-nm layer lines disappear. A 36-nm layer l ine is apparent by 13 ms and becomes progressively stronger while movi ng laterally away from the meridian of the pattern at later times, ind icating cross-bridges labeling the actin helix at decreasing radius.