Periodic Reporting for period 1 - ActinID (A molecular atlas of Actin filament IDentities in the cell motility machinery)
Reporting period: 2023-04-01 to 2025-09-30
Life depends on movement, and in higher organisms cell movement relies on the actin cytoskeleton and ABPs. However, we do not yet understand how ABPs are regulated to act in concerted fashion to orchestrate cell movement. This also limits our understanding how actin cytoskeleton remodelling regulates many other lifedefining cellular processes. Through novel developments for cryo-electron tomography and image processingwe will study for the first time all regulatory layers of actin cytoskeleton remodelling at molecular detail directly in situ, within the cell motility machinery of migratory cell protrusions. We will generate a visual proteome of the actin cytoskeleton, with our results also solving an outstanding question in the field: How do actin filaments with distinct functional properties emerge? The workflows we develop through ActinID will advance in situ structural biology to further increase the versatility and applicability of this powerful method.
ActinID will generate a molecular atlas of the cell motility machinery proteome in leading edge protrusions. Specifically, I will capitalize on our proven expertise in high-resolution cryo-ET of the actin cytoskeleton, as well as hardware and software development, to work on three objectives:
Objective 1: Large scale imaging of actin networks at single-filament resolution
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.
Objective 2: In situ visual proteomics of ABPs
Solve: high-resolution in situ structures of ABPs and F-actin and describe ABP quantity, ABP distributionand spatial correlation with their potential partners. This visual proteome will reveal how actin filament identities are regulated in an entire system.
Objective 3: Perturbation and functional analysis of the leading edge proteome
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.
Objective 1: Large scale imaging of actin networks at single-filament resolution
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.
Objective 2: In situ visual proteomics of ABPs
Solve: high-resolution in situ structures of ABPs and F-actin and describe ABP quantity, ABP distributionand spatial correlation with their potential partners. This visual proteome will reveal how actin filament identities are regulated in an entire system.
Objective 3: Perturbation and functional analysis of the leading edge proteome
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 transform our understanding of a highly conserved, multicomponent system: the actin cytoskeleton. Our work will advance both biology and methodology. Revealing ABP structures in situ in their biological context will go significantly beyond the current state-of-the-art. Our work will provide unprecedented insights into an emerging aspect of ABP regulation: how the complex interplay between biochemical, mechanical and geometrical aspects determines filament identities. This will help realign biochemical, structural and cell biological data and solve existing discrepancies in literature. Our results can provide further reaching answers beyond cell migration, as these concepts are also found in other cellular processes. Methodologically, our work will push the current limits of visual proteomics, an area of structural biology that has enormous potential to depict the molecular sociology of entire cellular landscapes. While we work in a specific biological context, our observations and methodological insights will be directly informative for other visual proteomics experiments. Specifically, our approach will take full advantage of the geometrical boundary conditions that dictate the functionality of a biological system, such as the actin cytoskeleton, and the activity of its components, to go beyond an isolated structural description.