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. ? ?
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