Actin is found abundantly in all eukaryotic cells, where it plays a key role in contractile processes, and in the maintenance as well as the dynamics of cell shape and cytoplasmic consistency. In attempts to ultimately understand the molecular basis of these actin-based processes, we propose to use high resolution electron microscopy and three-dimensional (3D) image analysis and processing to develop a consistent 3D molecular model of the functionally important actin filament, and to investigate the structural basis of actin's specific interactions with itself and with various contractile, regulatory and cytoskeletal proteins. Using structural, proteinchemical, immunological and microinjection methods, we also propose to explore the role of mutant actins isolated from stably transformed human fibroblasts in neoplasia. Specifically, we propose to proceed as follows: (1) To determine a consistent 3D model of the actin molecule at the 1-2 nm resolution level from different types of negatively stained and frozen hydrated crystalline actin arrays. (2) To develop a consistent 3D molecular model of the actin filament from a variety of negatively stained and frozen hydrated actin filament arrays and to align molecular actin models within the resulting filament 3D reconstructions. (3) To map binding sites for contractile, regulatory and cytoplasmic proteins on the actin molecule via 3D structural analysis of stoichiometrically decorated crystalline actin arrays or from co-crystals induced from specific complexes between actin and actin-interacting proteins. (4) To structurally and functionally analyze several types of crosslinked actin dimers (a) to aid in developing a consistent molecular model of the actin filament (see 2) and (b) to understand their role in regulating actin polymerization. (5) To investigate the in vitro polymerization properties, 3D structure, and in situ distribution (i.e. via immunofluorescence with mutant actin-specific antibody) and interactions (i.e. via microinjection of mutant actin mixtures) of mutant beta-actins isolated from transformed human fibroblasts.
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