MECHANISM AND CONTROL OF MICROTUBULE ASSEMBLY

该提案的广泛目标是了解体内微管动力学的机制以及这种动态活动与重要细胞生物学功能之间的关系，包括细胞质的组织，细胞形式的产生，细胞分裂和细胞运动。 概念框架，基础的建议是，微管的组织和他们的营业额在细胞中的结果从细胞成分的相互作用。 两类重要的成分是：（1）作为成核和锚定结构的中心体和（2）分别影响负端和正端动态活性的细胞质因子。 在这个建议中，特别关注的是一个新的，最近制定的微管周转假说，我们称之为负端途径。本赠款期的具体目标旨在检验负端途径假设的要素，并确定其运作机制。 其目标是：（1）确定负末端稳定化是否依赖于成纤维细胞中的中心体而不依赖于上皮细胞中的中心体;（2）评估成纤维细胞和上皮细胞之间MT行为的差异是否受到细胞-细胞接触的调节;（3）确定MT负末端从中心体释放后的状态;（4）确定负端途径和正端动力学在MT转换过程中的相对贡献：（5）研究MT在体内的微铣削特性并确定正端因素的参与;和（6）开发一种体外MT微研磨系统作为发现负（和正）端因子的测定。我们的研究策略将采用动力学，结构，生物化学，分子生物学和细胞方法相结合。 新的方法和途径包括使用无中心体的细胞质作为微管研磨的测定系统;用于微管释放的无细胞系统;相关的数字荧光成像和复制电子显微镜;以及用于负和正末端因子的单微管标记。 这些结果将有助于理解细胞质组织的基本机制，这些机制是正常细胞和恶性转化细胞的维持、运动和分裂的基础。

The broad objectives of this proposal are to understand the mechanism of microtubule dynamics in vivo and the relationship between this dynamic activity and important cell biological functions including organization of the cytoplasm, the generation of cell form, cell division and cell locomotion. The conceptual framework, underlying the proposal is that the organization of microtubules and their turnover in cells result from an interplay of cellular components. Two important classes of component are: (1) the centrosome as a nucleating and anchoring structure and (2) cytoplasmic factors that effect the dynamic activity of the minus and plus ends, respectively. Of particular focus in this proposal will be a novel, recently formulated hypothesis for microtubule turnover which we call the minus-end pathway. The specific aims targeted for this grant period are designed to test elements of the minus-end pathway hypothesis and to determine the mechanism by which it operates. The aims are: (1) to determine whether minus end stabilization is dependent on the centrosome in fibroblasts but independent of the centrosome in epithelial cells; (2) to evaluate whether the difference in MT behavior between fibroblasts and epithelial cells is regulated by cell-cell contacts; (3) to determine the status of the MT minus end after its release from the centrosome; (4) to determine the relative contributions of the minus end pathway and plus-end dynamics to the process of MT turnover; (5) to investigate properties of MT treadmilling in vivo and determine the involvement of plus-end factors; and (6) to develop an in vitro MT treadmilling system as an assay for the discovery of minus (and plus) end factors. Our research strategy will employ a combination of kinetic, structural, biochemical, molecular biological and cellular approaches. Novel methods and approaches include the use of centrosome-free cytoplasts as an assay system for microtubule treadmilling; a cell-free system for microtubule release; correlative digital fluorescence imaging and replica electron microscopy; and single microtubule labeling for minus and plus end factors. The results will contribute to an understanding of basic mechanisms of cytoplasmic organization which underlie the maintenance, motility and division of normal as well as malignantly transformed cells.