Molecular Design of Filopodia, Cell's Sensory Organelles

描述（由申请人提供）：本提案的总体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的感觉和引导细胞器，丝状足的分子设计。丝状足在多种细胞过程中发挥着关键作用，包括神经元的导航和癌细胞的转移。该建议的概念框架是基于最近制定的丝状足起始的收敛延伸假说。这一假说认为，构成丝足结构和功能核心的肌动蛋白丝束是由树突状肌动蛋白网络重组形成的。重组是由丝状尖端复合体驱动的，它控制着长丝生长端的伸长和物理相互作用，并引发长丝束。我们建议研究该模型的具体元素，包括：(1)依赖Arp2/3的成核是否为丝状起始提供细丝；(2)丝状伪足持续长丝的分子机制；(3)倒钩端相互作用机理；(4)丝束形成的机制及其在丝状突起中的作用。提出的研究策略结合了亚细胞和超分子水平的结构、动态和功能方法。一般程序将是识别基本的分子参与者，确定它们在丝状足的超分子组织中的位置，分析它们的动力学，并确定它们在丝状足形成和细胞行为中的功能。这种策略独特地弥合了单个分子特性和整个细胞行为之间的差距。相关的光和铂复制电子显微镜将被用来联系结构和细胞行为。动力学研究将使用荧光显微镜、光漂白后荧光恢复和荧光共振能量转移技术。功能分析将主要基于新的RNA干扰方法。实验将使用特别有利的细胞模型系统：高度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足的形成机制，以及丝状足在细胞迁移、细胞间通讯和组织形态发生等发育和疾病的基本过程中的作用。

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.