Microtubules, polymers of alphabeta-tubulin and its associated proteins, are essential organizers of the eukaryotic cytoplasm. They are dynamic structures: within a time span short in comparison to the cell cycle, they repeatedly dissociate into, and then reassemble themselves from, their constituent proteins. This behavior, subject to regulation by a variety of known factors, affects cellular differentiation, intracellular transport and determination of cell shape. Its mechanism requires modulation of the affinities of the microtubule's molecules for each other and of the rates at which they associate and dissociate. The long-term goal of this continuing project has been to measure and interpret these affinities and rates and thus to contribute to understanding how tubulin participates in formation and remodeling of the dynamic cytoskeleton. It is now proposed to extend the project's scope to study the folding of tubulin's individual polypeptide chains, both to provide a technical means to obtain active tubulin from recombinant sources and to begin to learn how folding, and possibly refolding, may influence microtubular structure and dynamics. Recent discoveries have shown that a molecular chaperone, CCT, with its cofactors, facilitates and governs tubulin's folding. It is proposed to mobilize this new knowledge to generate homogeneous and well-defined functional tubulin, and to study its assembly into microtubules. By making use of denatured recombinant alpha-and beta-chains produced in E. coli, and by using the chaperone system to fold them in vitro, we intend: (1) to understand the kinetics of the in vitro folding reaction and to use the information to increase the yield of active folded protein, (2) to study folding of post-translationally modified tubulins to probe the influence of modifications on folding and to learn if the CCT system can serve a refolding function as well as folding newly synthesized chains; (3) to initiate study of the dynamics of assembly and disassembly of microtubules which incorporate newly folded tubulins. The results should help to bring together the molecular biological and functional studies of this indispensable part of the cytoskeleton and thus contribute to eventual understanding of its cellular functions, both in health and in disease.
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