Architectural dynamics of actin-based motility

The motility of cells is integral to life and involves the regulated assembly and disassembly of the actin cytoskeleton. A process essential to motility is protrusion, which is mediated by the formation of thin sheets of cytoplasm termed lamellipodia. Protrusion itself is based on the polarised polymerisation of actin filaments, coupled with their structuring into networks and bundles. Current interest focuses on establishing how this is achieved in molecular and structural terms. Resolving the structural basis of protrusion requires electron microscopy to define filament arrangements, filament-filament interactions and the localisation of associated structuring and signalling complexes. However, due to differing results yielded by the application of alternative preparative methods for electron microscopy there is current controversy about actin filament organisation and hence about the basis of protrusion. Methods of electron microscopy must therefore be applied that obviate steps which potentially introduce artefacts in filament organisation. In pilot experiments we have shown the potential of cryo electron microscopy for resolving actin filaments in the cytoskeleton. The present project aims to exploit this method to visualize lamellipodia architecture and to localise associated molecules in actin networks. The results of this work should lead to a better understanding of actin-based cell motility.

The aim of this project was to gain new insights into how cells move. The forces that drive cell movement arise through the controlled reorganization of the filament framework referred to as the "actin cytoskeleton". The actin cytoskeleton is constructed from actin filaments that have the ability to "push", by polymerization, or to "pull" by interacting with the contractile protein myosin. In the first part of this project, we addressed the question of how filaments push at the cell front and focused on elucidating the three dimensional organization of the protruding regions of cells called lamellipodia. To avoid the potential distortions introduced by processing methods for conventional electron microscopy, we focused efforts on obtaining structural information using the emerging technique of cryo-electron tomography (cryo-ET), in which cells are rapidly frozen and observed directly in vitreous ice. By recording multiple images at different angles of specimen tilt, the three dimensional organization of filaments can be obtained using reprojection algorithms. This approach required the development of procedures for growing suitable cell models on carrier films for cryo-ET, for correlated light microscopy and electron microscopy and for assaying freezing protocols. Since a microscope equipped for cryo-ET does not yet exist in Austria, we performed microscopy in the European Molecular Biology Laboratory in Heidelberg, where this technique became fully available in Summer 2004. Extensive tests showed that living cells are readily lysed by current blotting procedures used to remove excess liquid prior to rapid freezing. Cryo-ET was therefore performed, in the first instance, on cells that had been arrested by chemical fixation and then frozen. A series of tomograms has been collected and image analysis is now in progress. In a second part, we applied the techniques developed for correlative light and electron microscopy to the analysis of actin filament reorganizations associated with substrate adhesion in motile cells. Cells expressing fluorescent "adhesion proteins" were monitored by high-resolution fluorescence microscopy, chemically fixed and imaged by electron microscopy after negative staining. The findings show that the lamellipodium plays a key role in adhesion assembly. Manuscripts describing this work are in preparation.