Howe, AJ; (2016) Structure and dynamics of mammalian filopodia by electron cryo-tomography and single molecule fluorescence. Doctoral thesis , UCL (University College London).

Abstract

Filopodia are cellular projections around 100nm in diameter and usually several micrometres in length. They are composed of filamentous actin fibres and have the molecular motor myosin10 localised at the tip. Filopodia are involved in processes of cell migration and have been linked with roles in diverse areas including angiogenesis, and the extension and path finding properties of neuronal growth cones. Filopodia can be induced in model cell lines by myosin10 overexpression, and live cell imaging shows they undergo stochastic cycles of extension and retraction. Myosin10 binds and transports cargo along bundled actin fibres to the tips of filopodia, forming a complex of proteins which is responsible for governing growth, retraction and adhesion. Electron cryo-tomography can be used to determine the native three dimensional ultrastructure of filopodia. These results reveal the arrangement and packing of actin filaments in the filopodia of rapidly frozen vitrified mammalian cells. Single molecule Total Internal Reflection Fluorescence Microscopy (TIRFM) of fluorescently tagged myosin10 constructs shows the dynamics of whole filopodia and intrafilopodial trafficking of individual myosins, with super-resolution microscopy allowing for the precise localisation of molecules.

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