An exceptional opportunity is at hand for time-resolved structural analysis of motor molecules in muscle, to clarify their action as individual motors as well as in the balanced choreographic assemblies that animate whole muscles. Unexcelled paracrystalline order lets insect flight muscle (IFM) from the giant waterbug Lethocerus give distinctive X-ray diffraction (X-ray) for each physiological state, and give clearer thin-section electron microscope (EM) images of myosin crossbridges than any other muscle. Millisecond-timed snapshots of the myosin power stroke will integrate mechanical with structural data, coordinating EM 3D reconstructions (3-Ds) from quick-frozen fibers with time-slicing synchrotron X-ray diffraction. New methods for 3-Ds offer potent tools for analyzing crossbridge diversity in contracting muscle. 3-D EM tomography supports not only image averaging, but identification of non-averaged crossbridge structures, and 3-D classification of crossbridges by correspondence analysis. 3-Ds of the whole unit cell can simulate full 3-D Fouriers to check fidelity of thin-section Ems by comparison with native X-ray intensities. Crossbridge strain will be assessed by fitting myosin head atomic models into 3-Ds of 3-4 nm resolution, to see (e.g.) If motor action involves a 4-12 nm swing by the light-chain-domain lever-arm. Isometric contractions in IFM can be triggered for 1-2 s by stretch- activation, or longer and stronger by higher [Ca2+]. EM and X-ray comparisons between both active states (time resolution 10-50 ms) and control steady states of relaxed and rigor will be finished. Time-resolved snapshots of synchronized powerstrokes will be sought by perturbing isometric contractions with abrupt length change steps. Length steps elicit transient 2-10 ms responses in force and structure that indicate synchronized crossbridge action. Concerted deformation of strongly bound crossbridges will also be sought by quick-stretching IFM in rigor, ADP and AMPPNP states, where slower off-rates and regular crossbridge structure should favor uniform and conspicuous changes in shape and angle. The transition to rigor crossbridge form, considered the end-stage of the power stroke, will be imaged by freezing at 0.2s intervals during rapid rigorization after ATP removal, and also midway along fibers quick- frozen after being rigorized at one end while the other end stays relaxed or activated in an ATP buffer. Early ~pre-force~ stages in the crossbridge cycle will be sought by X-ray and 3-D EM of fully detached (MgATP-relaxed) and some weekly attached crossbridge states (glycol-AMPPNP cold or with Ca2+; ADP-A1F4(-) with Ca2+)
Showing the most recent 10 out of 36 publications
