MYOSIN, ACCESSORY PROTEIN: ARRANGEMENT IN THICK FILAMENT

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We wish to study two particular aspects of A-band and thick filament structure. One of these deals with thick filament accessory proteins, such as M-, C- and H-proteins and titin. We plan to determine the relationship of the presence of these proteins to A-band (M-protein) adn thick filament (C-protein, H-protein, titin) integrity, both in vertebrate striated (chicken breast) muscle, where these proteins are known to exist, and in invertebrate long sarcomere striated muscles (Limulus, tarantula, scorpion, lobster, barnacle), where they have not yet been identified as components of the A-band. In this aspect of the study we will also investigate the effect of C-protein on the maintenance of the integrity of the synthetic filaments of invertebrate and nonmuscle myosin. We will also use light and electron microscopy and image analysis of myofibrils, isolated A-segments and thick filaments, and synthetic myosin aggregates, in combination with immunological localization techniques using monoclonal antibodies to study the presence, location and crossreactivity among these accessory proteins in different thick filaments. The second aspect of this proposal is to investigate the effects of environmental modifications such as pH, ionic strength (40-200mM). divalent cation concentration (Mg++ -1 to 8mM, Ca++ 0 to 1mM) and absence vs. presence of ATP on the order of the crossbridge array and the core paramyosin structure of isolated Limulus thick filaments. Here we hypothesize that changes from the native, relaxed condition will be readily identifiable and may mimic crossbridge positional changes during a contractile cycle,in situ. We will examine isolated filaments by electron microscopy optical diffraction and obtain definitive structural information by computer image analytic methods. These exeperiments will be compared with results obtained on the intact contractile apparatus in skinned Limulus fiber by Dr. John S. Wray, Max-Planck Institute for Medical Research, Heidelberg, West Germany. The results of these studies will provide insight into factors effective in the organization of both the thick filaments of the contractile apparatus into ordered A-bands and the position of myosin crossbriges in relation to the filament backbone, during muscle activity.