This sections uses ultrastructural approaches to test for motions of the myosin head and thus provides the basis for modeling the force bearing transition in the myosin cross bridge activity cycle. Implementation of rapid freezing techniques, in combination with caged compounds has provided the exceptional opportunity for time resolved structural analysis of myosin motors in the context of the intact myofibril. We propose to use rapid freezing in combination with rapid stretch and release protocols to synchronize cross bridges in frog fibers and to correlate their structural states and disposition with tension development. Image analysis will be used to dissect the contribution of various regions of the myosin head to the shape changes during tension development. We will also take advantage of the unexcelled paracrystalline order of insect flight muscle (IFM) in a collaborative exploration of myosin cross bridges, that will eventually lead to 3-D EM tomography and 3-D classification of cross-bridges by correspondence analysis (with Dr. M.K Reedy). Cross bridges will be either trapped in relaxed (MgATP-relaxed), early """"""""pre-force"""""""" states (glycol- AMPPNP cold or with Ca2+; ADP-AlF/4 with Ca2+), strong binding states (rigor) or following a rapid stretch of fully activated fibers. A second component of the project explores structure and function of myosin heavy chain isoforms and of the regulatory light chain in the processes of myofibril assembly and contraction in Drosophila. Collaborations within the program project allow a fruitful combination of genetic and transgenic approaches with functional and structural assays. A novel myosin, myosin rod protein or MRP, in which the catalytic and actin binding head region is substituted by an N terminal extension homologous to that of a light chain, offers a unique opportunity for tested whether this extension allows a direct, tethering interaction with actin. The structural effects of genetic and transgenic manipulations that vary the amount of MRP normally expressed in special flight muscle will be assessed by a battery of techniques, from thin sectioning of intact muscles to rotary shadowing and negative staining of isolated filaments, complemented by diffraction analysis. Ultrastructure of myofibrils and thick filaments from the indirect flight muscle (IFM) of transgenic flies will serve as basis for assessing effects of misexpression of various proteins on the stretch activation responses and on the specific myofibril architecture of these muscles. The IFMs will be induced to express myosin heavy chains with alternative exon 11S belonging to other muscles without stretch activation properties, over a null background.
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