Protein structure and function in filopodia across scales

Cell migration is required for many physiological events, such as embryonic development, immune responses or wound healing. Deregulated cell motility is often associated with pathological conditions, including cancer metastasis. Improving our understanding of cell migration is therefore not only important for unravelling fundamental mechanisms in many different events, but also has direct implications in developing therapeutic strategies against pathological conditions. In order to move, cells require the formation of specialised structures composed of a dynamic and complex filament network, consisting of the protein actin. Among these structures are filopodia, thin finger-like protrusions extending from the cell periphery that are densely filled with bundled, parallel actin filaments. Filopodia have been reported to be sensory organelles involved in cell migration, but also viral infection. Despite their simple morphology, the spatial organisation of actin filaments within filopodia is regulated by a large number of proteins, but the exact function, formation and maintenance of filopodia has remained enigmatic. Several actin filament bundling proteins are found in filopodia that are important in arranging individual actin filaments into a higher-order structure to provide the actin network with the required stiffness and cohesion. Interestingly, these actin bundling proteins seem to perform highly redundant functions, but it is unclear why such a high level of redundancy seems necessary. Using a multimodal approach we want to link protein function and structure across scales, ranging from cellular morphology and movement to the protein ultrastructure level. This will allow us to decipher the functional redundancy and complexity of proteins required in filopodia. Our target proteins are the three main bundlers involved in filopodia in melanoma cells: fascin, Daam1 and fimbrin. First, we will address the importance and redundancy of these proteins via specific CRISPR/Cas9 mediated genetic depletion. Live imaging microscopy techniques followed by advanced cyro-electron tomography methods will reveal how this targeted interference influences filopodial morphology, dynamics and also how it changes filopodial ultrastructure. Our results will ultimately allow us to decipher the redundancy of different actin filament bundlers. We also aim to derive the 3D structure of actin-bundling proteins within filopodia in situ using a combination of cryo-ET and sophisticated image processing methods. These methods are unique in their ability to provide a physiological structural snapshot and will substantially add to our understanding of actin structural biology.

Many physiological and pathological conditions are linked to the regulation of cell motility within the organism. In order to move, cells often form peripheral structures, which are dependent on the interaction between different proteins in time and space. Filopodia are finger-like protrusions, which are extending beyond the cell body and have exploratory and environmental-sensing functions. Defective filopodia formation has been associated with cancer and viral interaction, including recently reported links to Coronavirus infection. In this project, we have attempted to reveal in detail the significance of a group of proteins, knowns as bundling proteins, to the initiation and maintenance of filopodia. These proteins are considered important for linking parallel actin filaments, which build a filopodium. However, the relative contribution of each of those proteins remains unclear. We have removed 3 distinct bundling proteins in cancer cells, in various combinations, and investigated how filopodia and cell migration are affected. Our investigations were performed both on cellular level (micrometer scale) using light microscopy and on ultrastructural level (nanometer scale) using cryo-electron microscopy (cryo-EM). Furthermore, we have also observed and quantified via live imaging microscopy the movement of cells. We demonstrate that, contrary to expectations, removal of bundling proteins leads to defects in filopodia, depending on which protein is affected, but does not completely eliminate the parallel orientation of their actin filaments. This implicates the possible involvement of other proteins for the maintenance of these finger-like projections. Our results revealed interesting correlations between the density of filopodia and cell migration and also how the modulation of other filamentous networks within the cell influences the formation of filopodia in the absence of bundling proteins. In the process of our investigation, we have developed workflows, which will be useful for the detailed ultrastructural characterization of cells in future projects, which can advance the fields of cell migration and cryo-EM. Overall, the outcome of this project will have implications for enhancing our understanding of cell motility in physiological and pathological conditions.