Cracking the Actin Nanomotor by 3D ElectronMicroscopy

The primary goal of this project is to determine the 3-dimensional architecture of actin filament arrays that function in actin-based motility. So why is this important? Actin filaments drive the movement of migrating cells during tissue development and repair and of tumour cells during metastasis. Pathogens, including Listeria monocytogenes and vaccinia virus use actin to propel themselves from infected cells to uninfected cells, out of sight of the immune system. Thus, resolving the mechanism of actin driven motility is essential to understand both normal and disease processes. Actin polymerization is initiated at membrane surfaces, in lamellipodia, by specific nucleators (the Arp2/3 complex and formins) and potentiated by other factors, such as Ena/VASP proteins. But how is the actin network of lamellipodia organized and cross-linked and what structural rearrangements accompany anchorage with the extracellular matrix, necessary for traction? Likewise, do pathogens use the rules of lamellipodia to move in cytoplasm? These questions can only be answered by acquiring 3-dimensional structural information about actin network architecture and the actin-membrane interface under conditions that faithfully preserve actin filaments. Three innovations are proposed to achieve these aims: 1) Deep stain electron microscope tomography: we recently discovered that for cells embedded in deep negative stain, three-dimensionality and actin filament substructure is preserved in lamellipodia. We will exploit this new technology to resolve lamellipodia architecture. 2) Nanowire driven motility. In a multidisciplinary endeavour, we will use nanowires (10nm in diameter) pushed by actin filaments in vitro for structural analysis of the actin motor. In parallel, we will apply the deep negative stain method/ 3D imaging to vaccinia virus infected cells to resolve the pathogen pushing machinery. 3) Correlated live cell imaging and cryo-electron tomography. We will develop a unique approach to catch cells, vitreously frozen in known phase of protrusive activity for structural analysis. New insights can be expected into the structural basis of actin-based motility and, in turn, into the fundamental processes underlying cell migration.

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 defense and tissue repair. Pathological processes, including tumor dissemination and atherosclerosis, likewise involve cell migration. A central player in these motile processes is the protein actin and our studies have focused on understanding the mechanisms by which actin produces movement. Actin is not however used exclusively by cells to move. Several bacterial and viral pathogens hijack the actin machinery of cells they infect to disseminate their infection. They do this by recruiting actin and co-factors to propel them in cytoplasm in a rocketing type regime, at the head of an actin comet tail, to jump from one cell to another, avoiding the surveillance of the immune system. The major aim of the project was to discover how cells use actin filaments to move. In this mission we exploited the new technology of electron microscope tomography (electron tomography), which allows resolution of the three dimensional arrangement of subcellular structures at nm resolution. Using a combination of light microscopy and electron tomography we have discovered 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 protein complexes that initiate and stabilize branch points in the network. Mathematical models have also been developed to simulate lamellipodia formation.To investigate the process of actin-dependent pathogen invasion we have focused on members of the baculovirus family of insect viruses, which are among the smallest pathogens propelled by actin, and thus best suited for high- resolution imaging. Like other pathogens that hijack the actin-based motile machinery, baculoviruses generate actin comet tails to move in infected cells and we have used electron tomography to resolve comet tail architecture. We have provided the first three dimensional images of comet tails, showing that propulsion is based on a fish-bone like array of actin filaments, with branch linkages between filaments like those found in lamellipodia. A mathematical model of pathogen movement has also been developed that simulates the observed structure of comet tails and the tracking movements of viruses in infected cells. 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 resolve the roles of different proteins and protein complexes in actin-based processes.