Forum 05/13/2004

“Time-resolving myosin crossbridge dynamics by 3D EM of quick-frozen insect flight muscle”

M.K. Reedy, MD – Department of Cell Biology, Duke University Medical Center, Durham, NC

The beautifully ordered flight muscle of Lethocerus waterbugs keeps its normal mechanical, structural and nucleotide responses after glycerination and months of storage at -100 C. The myosin cross-bridge lattice gives clear 3D EM images and sharp fiber x-ray patterns.  We coordinate thin-section EM, X-ray and mechanics with the goal of enabling quick-frozen snapshots of myosin cross-bridges during the 2-4 ms of their concerted response to a sudden (0.1 ms) stretch or release.  Such snapshosts of the myosin powerstroke in situ will directly visualize the motor actions inferred from single-molecule motility assays, and from submillisecond time-resolved x-ray diffraction of frog muscle.Enroute to our goal, wecaptured the quick-frozen pattern of cycling, force-generation cross-bridges in non-shortening (isometric) contraction. To image load-induced strains, we’ve stretched rigor fibers, which are populated exclusively by non-cycling attached cross-bridges. 3D correspondence analysis of stretch-loaded rigor has identified different structural classes of myosin crossbridges. The class averages locate actin protomers exactly (for the first-time in muscle thin-sections), anchoring details of near-atomic modeling. The models reveal unexpected angles of the tethers (myosin S2) that connect the motors (myosin S1) to the thick filament backbone.  When these class averages are mapped back to their lattice origins, the superlattice pattern indicates a diffential distribution of strain.

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