Molecular Design of Filopodia, Cell's Sensory Organelles

描述（由申请人提供）：本提案的主要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指导细胞器，丝状伪足的分子设计。丝状伪足在多种细胞过程中发挥关键作用，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收敛伸长假说丝状伪足启动。这一假说指出，肌动蛋白丝束构成丝状伪足的结构和功能核心，是由树突状肌动蛋白网络的重组形成的。重组是由丝状伪足尖端复合体驱动的，该复合体控制丝状体生长末端的伸长和物理相互作用，并启动丝状体成束。我们建议研究该模型的具体要素，包括：（1）Arp 2/3依赖的成核是否为丝状伪足的起始提供丝状体;（2）丝状伪足中丝状体持续伸长的分子机制;（3）倒刺末端相互作用的机制;（4）丝状体成束的机制及其在丝状伪足突出中的作用。拟议的研究策略采用了结构，动态和功能的方法应用在亚细胞和超分子水平的组合。一般的程序将是确定必要的分子球员，以确定他们的位置在超分子组织的丝状伪足，分析他们的动力学，并建立他们的功能，丝状伪足的形成和细胞行为。这种策略独特地弥合了单个分子的特性与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与结构的细胞行为。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。功能测定将主要基于新的RNA干扰方法。实验将使用特别有利的细胞模型系统进行：高转移性小鼠黑素瘤细胞系和培养的神经元细胞。这些结果将有助于理解丝状伪足形成的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中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.