3DCELLART · Cytoskeleton architecture in host cells during Listeria infection using cryo-electron tomography

Bacterial infection into host cells is an important and highly active field of research. Understanding the interactions and the distribution of the host-cell scaffolding protein network during bacterial entry poses a major challenge. The molecular architecture of actin comet tails, filamentous structures assembled by internalized bacteria to move inside the host-cell cytoplasm and from cell-to-cell, remains unknown. Cryo-electron tomography (cryo-ET) is the most advanced method for visualizing the architecture of hydrated cells at a resolution better than 5 nm. Cryo-ET will be used to visualize the three dimensional (3D) cytoskeleton reorganization directly in eukaryotic cells infected by Listeria. Measurements will be performed at cryo-temperatures, on vitrified cell samples preserved in a close-to-life state. Specimen thickness limitations will be overcome by the use of the focused ion beam (FIB) micro-machining method, to obtain 500 nm thick samples as required for the collection of good data. Cryo-ET will be combined with correlative cryo-fluorescence microscopy, to localize the scaffolding components recruited during Listeria uptake and motility: host-cell actin, septin and clathrin. We expect to achieve nanometer resolution maps of the cell area of interest. The distribution and the ultrastructure of the cytoskeletal scaffold at Listeria entry and of Listeria actin comet tails will be provided. The work will provide unprecedented insight into cytoskeleton architecture during bacterial pathogenesis. The applicant obtained her PhD at a structural biology institute in France. Joining the Baumeister laboratory in Germany, and collaborating with the Cossart group, will allow her to address new challenging questions on the structural organisation of the cell, at unprecedented resolution. The acquired combination of skills at a world-class level will contribute significantly to her professional maturity, and to increase the competitiveness of European science.""