ASSEMBLY AND DISASSEMBLY OF CYTOPLASMIC MICROTUBULES

微管，真核细胞中普遍存在的成分，起着 细胞质的组织者以及作为底物和引导物的 细胞内运输。在这些活动中，他们 反复分解成它们的组成蛋白，然后 由这些蛋白质重组而成。此动态行为受 受多种因素的调节，影响细胞分化， 细胞内转运和细胞形状的确定。要理解 并操纵它，从根本上把握 微管分子之间的相互作用是必要的，还需要了解 这些分子结合和解离的速度。这 项目的总体目标是掌握这一点，并将 将个体速率常数和平衡常数转化为机械论 描述微管的组装和拆卸。它的特殊目标 有四个人。第一，量化使用视频增强(VE) 差分干涉衬度(DIC)显微镜测量和 理解新发现的变异率 微管的组装/拆卸。这种可变性必须揭示 生长端的基本功能特性。第二，到 用视频增强DIC和荧光显微镜研究如何 微管相关蛋白和分子马达影响稳定性 微管。人们对其实际机制知之甚少。 这一细胞细胞骨架调节的重要手段就是通过这种方式发挥作用的。 第三，描述缓慢但有意义的微管蛋白交换 微管壁和溶液之间的亚基。第四, 应用亲环素和“分子伴侣”的折叠催化剂 类，试图实现微管蛋白多肽的折叠 试管中的链条。成功的重新折叠将打开一条路径 这一重要部分的分子生物学和功能研究 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.