TUBULIN FOLDING AND MICROTUBULE DYNAMICS

微管，微管蛋白及其相关蛋白的聚合物， 是真核细胞质的基本组织者。它们是动态 结构：在与细胞周期相比短的时间跨度内， 他们反复地分离，然后重新组装， 它们的组成蛋白质。这一行为，受到监管的一个 多种已知因素，影响细胞分化， 细胞内转运和确定细胞形状。 其机制 需要调节微管分子的亲和力， 彼此之间，以及彼此之间的联系和分离。 的 这个持续项目的长期目标是测量和 解释这些亲和力和比率，从而有助于 了解微管蛋白如何参与形成和重塑 动态细胞骨架 现在建议延长该项目的 研究微管蛋白的单个多肽链的折叠的范围， 提供了一种技术手段， 重组来源，并开始学习如何折叠，并可能 重折叠，可能会影响微管结构和动力学。 最近 研究发现，一种分子伴侣，CCT， 辅因子，促进和管理微管蛋白的折叠。 拟 调动这些新知识， 功能微管蛋白，并研究其组装成微管。 通过 利用变性的重组α-和β-链， E.大肠杆菌，并通过使用伴侣系统在体外折叠它们，我们 目的：（1）了解体外折叠反应的动力学 并利用这些信息来提高活性折叠的产量 蛋白质，（2）研究后修饰微管蛋白的折叠 以探索修饰对折叠的影响，并了解 CCT系统既具有重折叠功能，又具有新折叠功能 合成链;（3）启动组装动力学研究 以及微管的分解，其中包含新折叠的微管蛋白。 这些结果应该有助于将分子生物学和 对细胞骨架这一不可或缺的部分的功能研究， 从而有助于最终了解其细胞功能， 无论是健康还是疾病。

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.