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

Sylvain Trépout

doi:10.1016/j.yjsbx.2020.100033

Abstract

The flagellum of Trypanosoma brucei is a 20 µm-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 structure of the FAZ in a close-to-native state. However, because of the large dimension of the cell body, the whole FAZ cannot be structurally studied in situ at the nanometre scale in 3D using classical transmission electron microscopy approaches. In the present work, cryo-scanning transmission electron tomography, a new method capable of investigating cryo-fixed thick biological samples, has been used to study whole T. brucei cells at the bloodstream stage. The method has been used to visualise and characterise the structure and organisation of the FAZ filament. It is composed of an array of cytoplasmic stick-like structures. These sticks are heterogeneously distributed between the posterior part and the anterior tip of the cell. This cryo-STET investigation provides new insights into the structure of the FAZ filament. In combination with protein structure predictions, this work proposes a new model for the elongation of the FAZ.

Highlights

Trypanosoma brucei is a unicellular parasite responsible for human African trypanosomiasis, known as sleeping sickness, occurring in sub-Saharan Africa (Büscher et al, 2017; Rotureau andVan Den Abbeele, 2013)
After immobilisation with formaldehyde, cells were deposited on electron microscopy grids, cryofixed in liquid ethane and imaged by cryo-Scanning Transmission Electron Tomography (STET)
The focus of this work is on the in situ ultrastructure and organisation of the flagellum attachment zone (FAZ) filament in whole T. brucei cells using cryo-STET

Summary

Introduction

This organism adopts different stages whose shape, intracellular organisation and metabolism vary during the complex life cycle in the insect vector or the mammalian host (bloodstream forms). Reverse genetic approaches such as RNA interference (Ngô et al, 1998), in situ tagging (Dean et al, 2015) and more recently CRISPR-Cas (Beneke et al, 2017) technologies are potent genetic tools to study gene function of fully sequenced T. brucei genome (Berriman et al, 2005; Sistrom et al, 2014). The sliding model explaining flagellum motility has been first proposed by Peter Satir in 1968

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Conclusion