Marion Jasnin - Resolving Arp2/3 complex-mediated branch nucleation in space and time using cryo-electron tomography

In this talk, I will demonstrate the potential of cryo-electron tomography (cryo-ET) for time-resolved in situ structural biology research. To illustrate this, I will use our ongoing study of native actin assemblies. Actin plays a pivotal role in numerous cellular processes by forming and disassembling highly dynamic, ordered structures. Nevertheless, our understanding of how the molecular components of the actin machinery collaborate within cells to produce nanonewton force- generating actin systems, such as podosomes in human macrophages or those involved in endocytosis in yeast, remains limited. I will demonstrate how cryo-ET can be employed to reveal the polarity of actin filaments at the monomer level within podosomes in human macrophages. This approach provides valuable insights into podosome assembly dynamics. It can also be used to recreate the spatial and temporal distribution of Arp2/3 complex-mediated branch junctions in both human macrophage podosomes and in yeast during clathrin-mediated endocytosis. To achieve this, we analyzed the spatial orientation of branch junctions in our cellular tomograms using high-resolution template matching and subtomogram averaging. Based on this information, we simulated successive generations of actin filament branches. We validated our model predictions by comparing the generations assigned by our model with those visualized in our data. Finally, I will introduce CryoAll: a Vision Transformer model trained on cryo-ET data that can segment various cellular structures, including cytoskeletal elements. Classical methods would fail to do this due to the density and complexity of these actin networks. Our integrative approach therefore reveals the assembly mechanisms of native actin networks at the molecular level, helping us to understand the underlying force generation mechanisms. Proposing time-resolved in situ cryo-electron tomography, this work opens up new avenues in the field of 4D structural biology.