Penn Medicine

Yale Goldman, M.D., Ph.D.

Structure and periodicities of cross-bridges in relaxation, rigor and during contractions initiated by photolysis of caged Ca2+.

Lenart M, Murray JM, Franzini-Armstrong C, Goldman YE

Biophys. J. 71:2289-2306, 1996

Summed power spectra from Fourier transforms of multiple images of overlap zones. The summed power spectra are derived from 113 overlap zones at 220 ms after activation by photolysis of caged calcium. Note layer lines at both 43 and 37 nm spacings during contraction.

Ultra-rapid freezing and electron microscopy were used to directly observe ultra-structural details of the frog muscle fibers in rigor, relaxation and during force development initiated by laser photolysis of DM-nitrophen (a caged Ca2+). Longitudinal sections from relaxed fibers show helical tracks of the myosin heads on the surface of the thick filaments. Fibers frozen at ~13 ms, ~34 ms, and ~220 ms after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridges. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridges densities have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z line. This highly variable distribution is established very early during development of contraction. Cross-bridge density across the interfilament space is more uniform than in rigor, wherein the cross-bridges are more dense near the thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze periodicities 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 in the present data, presumably reflecting different sample processing and origins of contrast between the two methods. In relaxed fibers, OD patterns show strong meridional spots and layer lines up to the 6th order of the 43 nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, layer lines at 18, 24 and 36 nm indicate cross-bridge attachment along the thin filament helix. Following activation by photo-release of Ca2+, the 14.3 nm meridional spot is present, but the 2nd order meridional spot (22 nm) disappears. The myosin 43 nm layer line becomes less intense, and higher orders of 43 nm layer lines disappear. A 36 nm layer line is apparent by 13 ms and becomes progressively stronger while moving laterally away from the meridian of the pattern at later times indicating cross-bridges labeling the actin helix at decreasing radius.

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