MECHANISM OF ACTIN-BASED CELL MOTILITY

描述(逐字摘自申请人的摘要)：的总体目标 这项建议是为了了解动物细胞的爬行运动。这个 提案集中在爬行过程的一个方面，即分子 突出的机理。该提案的概念框架是 这种突出的能动性可以用最近的一部小说来表达 提出的假说称为树枝形核/阵列踏板模型。 这个模型规定，稳态突起是一个循环的结果 肌动蛋白细丝的分枝成核、伸长、封顶、脱枝和 解聚。 针对授权期的具体目标旨在测试要素 的树枝形核/阵列踏板假说，并评估 根据运动速度和运动速度设计运动机械 推力的大小。目标是：(1)建立以珠子为基础的 运动性分析；(2)评估Arp2/3复合体是否对 肌动蛋白细丝的成核和伸长之间的平衡；(3)评估 封端蛋白的功能是封端肌动蛋白细丝的带刺末端 全球而不是局部；(4)调查特权阶层的基础 工作界面上无盖端部的状态；(5)通过以下方式评估机构 哪种ADF/cofilin可加速肌动蛋白细丝的周转；以及(6)确定 交联蛋白对运动性持久性的贡献。 我们的研究战略将强调超分子的结合 结构方法、动态方法、模型系统方法和功能方法。实验是 提出了一种基于微珠的体外模型系统和体内良好的 细胞系统--两栖角质形成细胞和人类黑色素瘤细胞系。小说 方法和途径包括使用高分辨率铂复制品和 免疫电子显微镜，数字荧光散斑显微镜，珠基 运动性分析和激光光学捕获。这一结果将有助于 对肌动蛋白聚合驱动的突起机制的理解 细胞运动是正常和恶性的基本过程 转化的细胞。

DESCRIPTION (Verbatim from the applicant's abstract): The broad objective of this proposal is to understand the crawling motility of animal cells. The proposal is focused on one aspect of the crawling process, namely the molecular mechanism of protrusion. The conceptual framework underlying the proposal is that protrusive motility can be expressed in terms of a novel, recently formulated hypothesis termed the dendritic nucleation/array treadmilling model. This model stipulates that steady-state protrusion is the result of a cycle of branched nucleation of actin filaments, elongation, capping, debranching and depolymerization. The specific aims targeted for the grant period are designed to test elements of the dendritic nucleation/array treadmilling hypothesis and to evaluate the design of the motility machinery in terms of the velocity of motility and magnitude of pushing force. The aims are: (1) to establish a bead-based motility assay; (2) to evaluate whether the Arp2/3 complex is critical for the balance between nucleation and elongation of actin filaments; (3) to evaluate the concept that capping protein functions to cap actin filament barbed ends globally but not locally; (4) to investigate the basis for the privileged status of uncapped ends at the working interface; (5) to evaluate mechanisms by which ADF/cofilin accelerates actin filament turnover; and (6) to determine the contribution of cross-linking proteins to the persistence of motility. Our research strategy will emphasize a combination of supramolecular structural, dynamic, model system and functional approaches. Experiments are proposed in vitro with a bead-based model system and in vivo with favorable cell systems,--amphibian keratocytes and human melanoma cell lines. Novel methods and approaches include the use of high resolution platinum replica and immuno-electron microscopy, digital fluorescence speckle microscopy, bead-based motility assays and laser optical trapping. The results will contribute to an understanding of the mechanism of actin polymerization-driven protrusion and cell motility which are fundamental processes of normal and malignantly transformed cells.