Principles and evolution of actin-nucleator complexes

The advance of a moving cell relies on the extension of cytoplasmic protrusions in the form of sheets termed lamellipodia and rods termed filopodia. The structural frameworks of lamellipodia and filopodia comprise actin filaments, organised in networks in lamellipodia and bundles in filopodia and extension is based on the polymerization of actin filaments nucleated at the cell membrane. This project aims to clarify the roles of different modulators of actin in the generation of lamellipodia networks. Specifically we will define the influence that these different factors have on the structural organization of lamellipodia by exploiting new advances in electron microscopy (electron tomography) that allow three dimensional visualization of actin filament arrays in situ. Electron tomography will be combined with correlated live cell imaging of cells in which the expression of individual proteins is manipulated to relate structure to function. The findings should shed new light on the process of nucleation and generation of actin filament arrays that drive cell movement.

There is no life without movement, at all levels of metazoan organization, from individual cells to the animal form. During development, individual cells migrate from the germ layers to lay down the body plan and in the adult organism migrating cells play key roles in immune defence and tissue repair. Pathological processes, including tumour dissemination and atherosclerosis, likewise involve cell migration. A central player in these motile processes is the protein actin. Actin polymerises in cells to produce filaments, which can interact with partner proteins to produce assemblies specialised either for pushing or pulling, both activities being required for cell movement. The major aim of the project was to provide new insights into how cells use actin filaments, together with different interacting partners to generate assemblies to push. Significant progress was made through studies within this priority programme by combining our expertise in electron tomography with the expertise of collaborating groups in molecular biology, biochemistry and genetics. Using a combination of light microscopy and electron tomography we have discovered, in structural terms, how actin filaments initiate and form the sheet-like regions (lamellipodia) that lead moving cells. In brief, lamellipodia are composed of networks of filaments generated by specific protein complexes that initiate and stabilize branch points in the network. By destabilizing the branching complex within cells, we have further shown that branched actin arrays are essential for pushing, but not for other processes of actin assembly, required to maintain cell structure. In addition to lamellipodia sheets, cells also extend bundles, called filopodia, whose role in cell movement is poorly understood. Further collaborative studies have unveiled proteins involved in filopodia turnover and have demonstrated a contribution of filopodia to the process of cell spreading, an essential step preceding locomotion. In parallel studies we have resolved the organization of actin comets generated by pathogens that hijack the actin machinery of cells they infect to spread their infection.Taken together, our findings on the organizations of actin filaments in different motile assemblies have contributed new insights into how the forces of actin polymerization are used to push in biological processes. Our lead in electron tomography of the cytoskeleton has also been exploited in several other collaborations to complement studies to resolve the roles of different proteins and protein complexes in actin-based processes.