Myofibril structure of insect flight muscle (IFM) is being studied by coordinated use of electron microscopy, X-ray diffraction, quantitative microscopy and biochemical analysis, and emphasis on the structure and behavior of myosin crossbridges. Crossbridges are the primary movers in muscle contraction and most non-muscle motility, and their highly ordered lattice arrangement in IFM permits averaging methods to extract crossbridge structure indifferent states which can be related to different phases of their ratchet-like cycle. X-ray and optical diffraction criteria will guide our search for better fixation of actin and crossbridges by modifying glutaraldehyde with tannic acid, diamines, and polyethylene-glycol triazine. Quick-freeze and freeze-substitution methods will be optimized for thin- sectioning of stable states like rigor and relaxed before attempting the study of dynamic structural transitions. 3-D computer reconstructions from 10-25 nm sections, based on multiple tilt views and on oblique cross sections, will be completed for rigor and applied to a group of necleotide-induced states, to establish definitively the variation in size, shape and domain alignment of crossbridges. Computer modelling will explore how EM structure of actin and crossbridges of rigor can be reconciled with X-ray intensity modelling. EM-mechanical correlation studies of single fibers will characterize two new crossbridge configurations encountered in glycol-AMPPNP- induced progression of states, and will explore rigor induction and tension to examine the length of the power-stroke, strength of bridge attachment, and effects of crossbridge """"""""slippage"""""""". We expect to finish coordinated collaborative studies of crossbridge orientation by EM/X-ray and spin-labelling, and antibody localization of troponin within the crossbridge lattice. IN a different approach, quantitative microscopy (EM and interference), buoyant density banding in Percoll, and X-ray diffraction measurement of lattice spacing will be coordinated in studies of dry mass content of myofilaments and fibrils, to produce mass distribution profiles of insect and rabbit sarcomeres, ascertain absolute protein conon in sarcomere cross- bands, and to resolve an anomalous discrepancy between filament mass and whole fibril mass.
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