Coupling Auxin-Inducible Degron System with Ultrastructure Expansion Microscopy to Accelerate the Discovery of Gene...

Ultrastructure expansion microscopy (U-ExM) is an emerging technique allowing the localization of proteins and cellular structures, at a level of resolution only distinguishable previously via immunoelectron microscopy. U-ExM, as its name indicates, is based on the physical expansion of the sample in the three dimensions without altering its internal features. The proteins of interest are later immunostained for their detection. To accelerate the discovery of gene function in the protozoan parasite Toxoplasma gondii, U-ExM can be coupled to the auxin-inducible degron system (mAiD system). This pipeline led to the subcellular localization of the gene product at unprecedented resolution and simultaneously assessed the consequences of conditional gene disruption. In this chapter, we explain the specific U-ExM protocol used for T. gondii tachyzoite samples and provide non-trivial advice and tips to successfully perform the experiments.