We propose to use high resolution electron microscopy and image processing, and to combine them with biochemical, biophysical and immunological techniques to try and answer the following questions related to the molecular structure and interactions of a variety of cellular proteins or protein complexes: (1) what is the molecular architecture of keratin filaments (tonofilaments) isolated from various epithelial cells and how is it related to the molecular organization of intermediate-sized filaments in general? (2) how is differentiation (e.g. keratinization) of ipithelial cells manifested in the subunit composition and topography of their keratin filaments? (3) what are the differences of wild type and mutant beta actins isolated from malignant human fibroblasts with respect to their three-dimensional structure, filament polymerization properties and specific interactions with accessory proteins (e.g. myosin, tropomyosin, profilin, DNase I, capping proteins and gelation proteins)? (4) how are the motile properties and the structure and dynamics of the cytoskeleton of normal cells affected on microinjecting various amounts of these mutant actins into them? (5) where are various biochemically and immunologically characterized nuclear pore components located on a three-dimensional model obtained from the nuclear pore complex? (6) can we determine distinct structural states of the pore complex and corelate them with functionally distinct states mediating or regulating the exchange of molecular material between the nucleus and the cytoplasm? Answers to these and other questions will eventually contribute to a better understanding of the molecular basis of cellular mechanisms such as force generation, differentiation and dynamics of the cytoskeleton, and communication between the nucleus and the cytoplasm effected and controlled by constituents of the nuclear membrane. Last but not least, the combination of techniques proposed in this project may have a significant impact on future studies of molecular structure and function of other cellular proteins and protein complexes.
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