THICK AND INTERMEDIATE FILAMENTS IN STRIATED MUSCLE

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The aim of these studies is to understand in detail the structure of two kinds of filaments in striated muscle--the myosin-containing, thick filaments and the desmin-containing intermediate filaments. The filaments will be studied at three levels of structural organization--the subunits of assembly, the filaments themselves and, in the case of thick filaments, the filament lattice. A unique morphological approach--quick-freezing followed by freeze-etching and rotary replication--will be used to prepare samples for electron microscopy. To study thick filament and crossbridge structure, intact muscle fibers as well as fractionated thin filaments with an "in vivo" complement of crossbridges will be quick-frozen, examined by electron microscopy and analyzed by image processing techniques. The location of the light chains in the myosin molecule will also be determined morphologically and verified using monoclonal antibodies directed against light chain epitopes. The assembly of intermediate filaments from their building blocks (protofilaments) will be studied using in vitro systems. The effects of two monoclonal antibodies to desmin, known to bind to protofilament ends, upon the polymerization of desmin will be assessed quantitatively as well as by electron microscopy. The possible existence of assembly intermediates will be probed by assembling purified vimentin protofilaments at sub-physiological salt concentrations, isolating the intermediate species and testing their ability to modulate further IF polymerization. Rate constants and directionality of polymerization will be determined. Information obtained in these studies will help to understand the molecular events during muscular contraction, and how muscle contraction is coordinated at the level of the cytoskeleton. The elucidation of muscle intermediate filament structure will also likely be of general applicability to other subclasses of filaments in epithelial cells and cells of the nervous system, since all intermediate filament proteins are now known to share homologous amino acid sequences.