Structure and Dynamics of Filopodia Studied by Electron Cryo-Tomography and Single Molecule Fluorescence Imaging

Abstract

Filopodia are cellular projections around 100nm in diameter and usually several micrometres in length. They are composed of fascin-bundled actin filaments and the molecular motor myosin‑10 localises at the extreme tip. Filopodia are important in cell migration and in making cell-cell contacts and have been linked with roles in diverse areas of biology including angiogenesis, and path-finding properties of neuronal growth cones. Filopodia can be induced in model cell lines by overexpresion of myosin‑10 and live-cell imaging shows that they undergo stochastic cycles of extension and retraction. Myosin‑10 transports cargo along the actin filament bundle towards the filopodial tip, forming a complex of proteins which are responsible for governing growth, retraction and adhesion. We have used a combination of optical and electron microscopy to study the dynamics and ultra-structure of myosin‑10 induced filopodia. Electron cryo-tomography of flash-frozen vitrified mammalian cells revealed the arrangement of actin filaments within the filopodia. Single molecule imaging by total internal reflection fluorescence microscopy allowed movement of myosin‑10 with the filopodia to be quantified and super-resolution localisation gave structural and mechanical information about the filopodium that could be correlated to our electron tomographic images.