Date of Award

1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Yale E. Goldman

Abstract

In order to understand the contraction mechanism in vertebrate skeletal muscle, one must correlate the structural and mechanical details of the cross-bridge cycle on the millisecond time scale. Using electron microscopy, I investigated the structure of cross-bridges in fibers activated by photolysis of caged ca2+ and then ultra­rapidly frozen and freeze substituted with tannic acid and OsO4. Sections from relaxed fibers show helical tracks, presumed to be rnyosin heads, on the thick filament surface. Optical diffraction patterns show strong meridional spots and layer lines up to the 6th order of the 429 Å repeat, indicating preservation and resolution of periodic structures smaller than 100 Å . Following photo-release of ca2+, the myosin 1/429 Å-1 layer line becomes less intense, and higher orders disappear, both with a time course which precedes the rise in tension. Å 1/360 Å-1 layer line appears early in contraction ( 12-15 ms) and becomes stronger at later times. The intensity of the 1/143 Å_-1 meridional spot decreases initially and then increases to greater than its value in relaxed fibers, while it broadens six-fold laterally. The 1/430 Å-1 meridional spot is present during contraction but markedly weakened. The 1/215 Å-1 meridional spot is weak or absent. These results are consistent with time resolved X-ray diffraction data on the periodic structures within the fiber. The intensification of the 1/360 Å-1 layer line, with a concomitant decrease in the intensity of the 1/429 Å-1 layer line, supports the view that at least some cross­bridges decorate the thin filament during contraction with an act in based set of periodicities, but not to the same degree as is seen in rigor. The lateral spread of the 1/ 143 Å-1 meridional spot indicates a disorder of axial coherence among thick filaments during tension development. In sections along the (1,1) plane of activated fibers, the individual cross-bridges have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z-line. Fibers frozen at 12-15 ms, 30-35 ms, and 210-220 ms after photolysis all show surprisingly similar cross-bridges. Thus, a highly variable distribution of cross-bridge shapes and angles is established early in contraction.

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