ActinID · A molecular atlas of Actin filament IDentities in the cell motility machinery

The actin cytoskeleton is essential to cellular function and hence to life. It is regulated not only via a complex synergistic and competitive interplay between actin-binding proteins (ABP), but also by filament biochemistry and filament geometry. Both the biochemical identity of an actin filament as well as the geometrical identity of actin networks are defined by ABP activity, while biochemistry and geometry also regulate ABP recruitment. However, it is still unknown how actin filament biochemistry, geometry and ABP activity jointly define actin cytoskeleton remodelling in a dynamic system such as cell migration. Resolving this critical relationship would enable a better understanding of the multitude of cellular processes that ultimately depend on actin.ActinID will tackle this question by advancing in situ structural biology to generate a molecular atlas of the actin cytoskeleton proteome in migratory cell protrusions. Our overarching aims are to:1) Develop: cryo-electron tomography workflows to achieve 3D imaging of entire actin networks in cellular protrusions at single-filament resolution, combined with detailed quantitative analysis. This will provide the ground truth on how dynamic filament geometries steer directional cell movement.2) Solve: high-resolution in situ structures of ABPs and F-actin and describe ABP quantity, ABP distribution and spatial correlation with their potential partners. This visual proteome will reveal how actin filament identities are regulated in an entire system.3) Study: the reciprocal regulation of ABP activity, filament geometry and biochemistry via (genetic) manipulation of ABPs using integrative cell and structural biology experiments, and relate this to cell migration characteristics.ActinID will be transformative for our understanding of actin cytoskeleton regulation, while also advancing the potential of in situ structural biology to go beyond isolated structural descriptions of biological systems.