ASSEMBLY AND DISASSEMBLY OF CYTOPLASMIC MICROTUBULES

微管是真核细胞中普遍存在的组成部分， 细胞质的组织者，并作为基质和指南， 胞内转运 在这些活动中，他们 反复分解成它们的组成蛋白质， 由这些蛋白质重组而成 这种动态行为， 受多种因素调节，影响细胞分化， 细胞内转运和确定细胞形状。 了解 并操纵它，从根本上掌握了 微管分子的相互作用是必要的， 这些分子结合和解离的速率。 这 项目的总体目标是抓住这一点， 将单个速率常数和平衡常数转化为一种机制 微管组装和拆卸的描述。 其具体目标 有四个。 第一，定量使用视频增强（VE） 微分干涉对比（DIC）显微镜测量， 了解新发现的 组装/拆卸微管。 这种变化必须揭示 生长端的基本功能特性。 二是 通过视频增强DIC和荧光显微镜研究 微管相关蛋白和分子马达影响稳定性 微管。 对实际机制的了解相对较少 这一重要的细胞骨架调节手段通过其发挥作用。 第三，表征微管蛋白缓慢但有意义的交换， 微管壁和溶液之间的亚基。 第四， 应用折叠催化剂，亲环蛋白和“分子伴侣” 类，试图使微管蛋白的多肽折叠 体外链。 成功的重折叠将打开一条通道， 分子生物学和功能研究这一重要组成部分， 细胞骨架

Microtubules, ubiquitous components of eukaryotic cells, act as organizers of the cytoplasm and as substrates and guides for intracellular transport. In the course of these activities, they repeatedly dissociate into their constituent proteins and are then reassembled from those proteins. This dynamic behavior, subject to regulation by a variety of factors, affects cellular differentiation, intracellular transport and determination of cell shape. To understand and manipulate it, a fundamental grasp of the affinities of the microtubule's molecules for each other is needed, as well as knowledge of the rates at which those molecules associate and dissociate. This project's overall goal is to get that grasp and to integrate the individual rate constants and equilibrium constants into a mechanistic description of microtubule assembly and disassembly. Its particular aims are four. First, to make quantitative use of video-enhanced (VE) differential-interference-contrast (DIC) microscopy to measure and understand the newly discovered variability of rates of assembly/disassembly of microtubules. This variability must reveal fundamental functional properties of the growing end. Second, to investigate by video-enhanced DIC and fluorescence microscopy how microtubule-associated proteins and molecular motors affect the stability of microtubules. Relatively little is known about the actual mechanism by which this important means of cellular cytoskeletal regulation acts. Third, to characterize the slow but meaningful exchange of tubulin subunits between the wall of the microtubule and the solution. Fourth, to apply folding catalysts, of the cyclophilin and "molecular chaperone" classes, in an attempt to bring about folding of tubulin's polypeptide chains in vitro. Successful refolding would .open a path between molecular biological and functional studies of this important part of the cytoskeleton.