In situ structural analysis of the flagellum attachment zone in Trypanosoma brucei using cryo-scanning transmission electron tomography

The flagellum of Trypanosoma brucei is a 20 micrometre-long organelle responsible for locomotion and cell morphogenesis. The flagellum attachment zone (FAZ) is a multi-protein complex whose function is to attach the flagellum to the cell body but also to guide cytokinesis. Cryo-transmission electron microscopy is a tool of choice to access the close-to-native structure of the FAZ. However, because of the large dimension of the cell body, the whole FAZ cannot be structurally studied in situ at high resolution in 3D using classical transmission electron microscopy approaches. In the present work, cryo-scanning transmission electron tomography, a new method capable of investigating thick cryo-fixed biological samples, has been used to study the structure and organisation of whole T. brucei cells at the bloodstream stage. The method allowed to visualise intracellular structures located deep inside the cells such as the nucleus and the nuclear envelope and to localise nuclear pore complexes. The organisation of the stick-like structure of the macromolecular protein complexes composing the FAZ filament is depicted from the posterior part to the anterior tip of the cell. This study provides new insights in the structure the FAZ filament.