Expansion of Toxoplasma gondii cysts in vitro using enzymatically enhanced ultrastructure expansion microscopy

Session

Poster Session

Authors

Kseniia Bondarenko (2), Kayvon Pedram (1), Mathieu Gissot (3), Joanna Young (2)

Affiliations

1. Howard Hughes Medical Institute
2. University of Edinburgh
3. University of Lille

Keywords

Ultrastructure expansion microscopy, expansion microscopy, ExM, U-ExM, Toxoplasma, cyst, confocal, StcE

Abstract text

Expansion microscopy (ExM) is an innovative approach to achieve super-resolution images without using super-resolution microscopes, based on the physical expansion of the sample. The advent of ExM has unlocked super-resolution imaging for a broader scientific circle, lowering the cost and entry skill requirements to the field. One of its branches, ultrastructure ExM (U-ExM), has become popular among research groups studying Apicomplexan parasites, including the acute stage of Toxoplasma gondii infection. The chronic cyst-forming stage of Toxoplasma, however, resists U-ExM expansion, impeding precise protein localisation. Here, we solve the cyst’s resistance to denaturation required for successful U-ExM of the encapsulated parasites. As the cyst's main structural protein CST1 contains a mucin domain, we added an enzymatic digestion step using pan-mucinase StcE prior to the expansion protocol. This allowed full expansion of the cysts in fibroblasts and primary neuronal culture without interference with the epitopes of the cyst-wall associated proteins. Using StcE-enhanced U-ExM, we clarified the shape and location of the GRA2 protein important for establishing a normal cyst. Expanded cysts revealed GRA2 granules spanning across the cyst wall, with a notable presence observed outside on both sides of the CST1-positive layer. 

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