Molecular Design of Filopodia, Cell's Sensory Organelles

描述(由申请人提供)：这项提案的广泛目标是了解细胞如何探索环境并以有意义的方式对其做出反应。该提案的具体重点是研究细胞感觉和引导细胞器丝状足的分子设计。丝状伪足在多种细胞过程中起着关键作用，包括神经元的导航和癌细胞的转移。该提案的概念框架是基于最近形成的丝足起始的收敛伸长假说。这一假说认为，肌动蛋白细丝束是由树突肌动蛋白网络重组形成的，它包括丝状基座的结构和功能核心。重组是由丝轴尖端复合体驱动的，它控制着丝状体生长末端的伸长和物理相互作用，并启动丝束。我们建议研究这一模型的特定元素，包括：(1)依赖Arp2/3的成核是否为丝轴起始提供细丝；(2)丝轴中持久细丝伸长的分子机制；(3)带刺末端相互作用的机制；(4)丝束形成的机制及其在丝轴伸出中的作用。拟议的研究策略结合了亚细胞和超分子水平上的结构、动力学和功能方法。一般的程序将是确定基本的分子成员，确定它们在丝状伪足的超分子组织中的位置，分析它们的动力学，并建立它们在丝状伪足形成和细胞行为中的功能。这一策略独特地弥合了单个分子的性质和整个细胞的行为之间的差距。相关的光和铂复制电子显微镜将被用来将结构与细胞行为联系起来。动力学研究将使用荧光显微镜、光漂白后的荧光恢复和荧光共振能量转移技术。功能分析将在很大程度上基于新的RNA干扰方法。实验将使用特别有利的细胞模型系统进行：高转移的小鼠黑色素瘤细胞系和培养的神经细胞。这些结果将有助于理解丝状伪足的形成机制，以及丝状伪足在细胞迁移、细胞间通讯和组织形态发生等发育和疾病基本过程中的作用。

DESCRIPTION (provided by applicant): The broad objective of this proposal is to understand how cells explore the environment and respond to it in a meaningful way. The specific focus of the proposal is on investigating the molecular design of the cell's sensory and guiding organelles, filopodia. Filopodia play critical roles in a variety of cellular processes including the navigation of neurons and metastasis of cancer cells. The conceptual framework of the proposal is based on the recently formulated convergent elongation hypothesis for filopodia initiation. This hypothesis states that the actin filament bundle comprising the structural and functional core of a filopodium is formed by reorganization of the dendritic actin network. Reorganization is driven by the filopodial tip complex, which controls elongation and physical interaction of filament growing ends and initiates filament bundling. We propose to investigate specific elements of this model including: (1) whether Arp2/3- dependent nucleation supplies filaments for filopodial initiation; (2) the molecular mechanism of persistent filament elongation in filopodia; (3) the mechanism of barbed end interaction; (4) the mechanism of filament bundling and its role in filopodial protrusion. The proposed research strategy employs a combination of structural, dynamic, and functional approaches applied at the subcellular and supramolecular levels. The general procedure will be to identify essential molecular players, to determine their place in the supramolecular organization of filopodia, to analyze their dynamics, and to establish their functions in filopodia formation and cell behavior. This strategy uniquely bridges the gap between properties of individual molecules and the behavior of a whole cell. Correlative light and platinum replica electron microscopy will be used to relate structure to cell behavior. Kinetic studies will use fluorescence microscopy, fluorescence recovery after photobleaching and fluorescence resonance energy transfer techniques. Functional assays will be largely based on the novel RNA interference approach. Experiments will be conducted using particularly favorable cell model systems: a highly metastatic mouse melanoma cell line and cultured neuronal cells. The results will contribute to an understanding of the mechanism of filopodia formation and the roles of filopodia in such fundamental processes in development and disease as cell migration, cell-cell communication, and tissue morphogenesis.