In Situ Actin Structures via Hybrid Cryo-electron Microscopy

Migration of cells is a fundamental part of life. Organisms require that their cells can move around, for example during development as a fetus or when the immune system must protect against invaders. How are cells able to move around so effectively? The answer is that they have evolved an elaborate system of force-generating biological machinery. At the front edge of a migrating cell there is a dense network of protein filaments called actin which continuously push outward so that the cell shape develops tentacle-like protrusions and a flattened adhesive lip which pulls the cell forward. Because cell movement is such a crucial aspect to life this biological machinery is very tightly regulated, and one way to do that is to produce additional proteins which can modify and direct the network as needed. While we know this happens, we have never directly observed many of these interactions with sufficient detail to understand precisely how these proteins are exerting their effects. This has hindered a complete understanding of how cells migrate, and has prevented attempts to target this machinery for drug treatment in cases where motility has gone awry, such as malignant metastasis. This research aims to visualize, at nearly atomic resolution and for the first time, key proteins which are orchestrating the migratory machinery. This will be done directly within the front edge of a migrating cell which has been flash frozen to preserve detail. Our main tool to accomplish this is an electron microscope operating at 300 kilovolts, which in ideal cases is capable of directly visualizing individual atoms. However, this is not an ideal case, as we will be trying to look into the front edge of a migrating cell. Since this is an environment of densely packed actin and motility machinery it will be a challenge to peer in to see much detail. Therefore, we will also need to develop and implement new ways of using the microscope which will allow us to view the interior of cells in stunning detail. Our approach is built upon a method called tomography, similar to a CT scan for a person, where the cell will be rotated while imaging in order to generate a 3D view of the inside of the cell. By taking advantage of this 3D information we can disentangle the dense filament network and identify positions of key proteins we are interested in. To help narrow our search in the dense network, we have already developed software to analyze the overall geometry and patterns within the filament network, allowing us to focus on areas where we suspect to find the proteins were looking for. Once we know where to look, we can use the microscope in a more conventional way to collect ultra-high resolution pictures of our target molecule. This work will yield the first ever highly detailed images of important proteins that direct actin-mediated cell motility, while also developing new electron microscopy methods for looking into cells which can be adopted by other labs around the world.

Cell migration is essential for development, immunity, and tissue maintenance. It is driven by dense networks of actin filaments at the front edge of cells, which generate protrusive and adhesive forces that move the cell forward. This machinery is tightly regulated by actin-binding proteins, yet many of their interactions have not been directly visualized at high resolution in crowded environments, limiting our understanding of cell motility in health and disease. The original goal of this project was to develop and apply cryo-electron microscopy and cryo-electron tomography methods, including cryoSPARTAN, to visualize actin-based motility machinery in near-native states. Tomography provides three-dimensional views of dense actin networks, allowing filament organization to be resolved and candidate regulatory proteins to be identified, supported by computational analysis of network geometry. In parallel, this work contributed to a published study on the Huntington's disease protein huntingtin (HTT). We showed that HTT directly binds and bundles actin filaments, organizing them into tightly packed arrays similar to those found at the leading edge of migrating cells. Using purified protein assemblies and cryo-EM tomography, we visualized HTT-actin interactions within crowded filament networks. Together, this work advances cryo-EM methodology for studying dense cytoskeletal systems and provides new structural insight into actin regulation, including the role of huntingtin in normal biology and disease.