Intermediate filaments (IF), microfilaments, microtubules and their accessory proteins make up the cytoskeleton, a complex structure that plays a major role in the maintenance of cell form. In this project, we will examine the structure and assembly properties of the IF proteins in muscle and nerve cells using chemical and high resolution morphological techniques. We will chemically synthesize a number of peptides whose amino acid sequences are selected from the sequences of desmin and the neurofilament (NF) triplet polypeptides and then use them to examine the functional role of the protein domains from which they are taken. For desmin, we will use the peptides directly as competitive inhibitors of filament assembly, as measured by a new fluorescence assay that we have developed. Concomitantly, images of the arrested assembly products at molecular resolution will be generated using the quick-freeze, deep-etch technique for electron microscopy (EM). By examining how the subunits are prevented from fitting into the IF we hope to be able to understand how the subunits are packed in a normal filament. Polyclonal antisera will be generated against the NF-derived synthetic peptides, which are taken from the C-terminal extensions of the triplet, NF-L, NF-M and NF-H. The antibodies will be used in immunoblotting and immunolabeling experiments to test the hypothesis that the C-terminal extensions make up the NF crossbridges and to localize the putative phosphorylation site of NF-M. We will also determine, by fluorescence energy transfer experiments, whether IF subunits are structurally polar or apolar, and by two ultrastructural approaches if they assemble unidirectionally or bidirectionally. To explore the notion that IF may be more dynamic than commonly though, we will look for exchange of desmin subunits between formed filaments in vitro and follow the fate of fluorescently labeled desmin subunits after microinjection into developing cardiac and skeletal muscle cells. Our long term goal is to map the roles played by the various domains of the IF protein molecule in filament structure and eventually to modify those of functional interest. This may allow us to insert modified IF protein molecules into the cytoskeleton as a means to gain better understanding of cytoskeletal morphogenesis. Knowledge at this level may contribute to the understanding of cardiac and skeletal muscle morphogenesis at higher levels of organization and thus lead to more effective prevention of heart and other muscle diseases.
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