MECHANISM OF ACTIN-BASED CELL MOTILITY

描述（来自申请人摘要的逐字逐句）： 这个建议是为了了解动物细胞的爬行运动。的 该建议集中在爬行过程的一个方面，即分子 突出机制。该建议所依据的概念框架是 最近， 公式化的假设被称为枝晶成核/阵列研磨模型。 该模型规定，稳态突起是一个循环的结果， 肌动蛋白丝的分支成核、伸长、帽化、去分支和 解聚 资助期内的具体目标旨在测试 枝晶形核/阵列球磨假设，并评估 根据运动速度设计运动机械， 推力的大小。其目的是：（1）建立一个基于微珠的 运动性测定;（2）评估Arp 2/3复合物是否是运动的关键。 肌动蛋白丝的成核和伸长之间的平衡;（3）评估 加帽蛋白的功能是将肌动蛋白丝的倒刺末端加帽， 全球性而非局部性;（4）调查特权的基础 工作界面上未加帽末端的状态;（5）通过 其中ADF/cofilin加速肌动蛋白丝的周转;以及（6）为了确定 交联蛋白对运动持久性的贡献。 我们的研究策略将强调超分子的组合， 结构、动态、模型系统和功能方法。实验 在体外提出了基于珠粒的模型系统，在体内提出了有利的 细胞系统，--两栖动物角膜细胞和人黑色素瘤细胞系。小说 方法和途径包括使用高分辨率铂复制品， 免疫电子显微镜，数字荧光散斑显微镜，珠基 运动性测定和激光光学捕获。结果将有助于 理解肌动蛋白聚合驱动突起的机制， 细胞运动是正常和恶性肿瘤的基本过程 转化细胞

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.