MECHANISM AND CONTROL OF MICROTUBULE ASSEMBLY

这一建议的主要目的是了解体内微管动力学的机制，以及这种动态活动与重要的细胞生物学功能之间的关系，包括细胞质的组织、细胞形态的产生、细胞分裂和细胞运动。该提议的基本概念框架是，微管的组织及其在细胞中的周转是细胞组成部分相互作用的结果。两类重要的成分是：(1)中心体作为成核和锚定结构；(2)分别影响负端和正端动态活性的细胞质因子。在这项提议中特别关注的将是一个新的，最近形成的关于微管周转的假说，我们称之为负端通路。这一授权期的具体目标旨在测试负末端通路假说的要素，并确定其运作机制。其目的是：(1)确定负端稳定化是否依赖于成纤维细胞的中心体，而不依赖于上皮细胞的中心体；(2)评估成纤维细胞和上皮细胞之间MT行为的差异是否受细胞-细胞接触的调节；(3)确定MT从中心体释放后的状态；(4)确定负端通路和正端动力学在MT周转过程中的相对贡献；(5)研究MT的体内特性并确定正端因子的参与；以及(6)开发一种体外MT跑步机系统，作为发现负(和正)终末因子的检测方法。我们的研究战略将采用动力学、结构、生化、分子生物学和细胞方法的组合。新的方法和途径包括使用无中心体的细胞质作为微管踏板磨削的测试系统；无细胞微管释放系统；相关的数字荧光成像和复制电子显微镜；以及单微管标记的负端因子和正端因子。这些结果将有助于理解细胞质组织的基本机制，这些机制是正常和恶性转化细胞维持、移动和分裂的基础。

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.