SIM showed an abundant Jonah(1) lectin localized to the ectocyst layer of mature cyst walls, while abundant Luke(2), Luke(3), and Leo lectins localized to the endocyst layer and ostioles.

A. A Jonah(1) lectin with a single CAA domain, which was tagged with GFP and expressed under its own promoter in transfected A. castellanii, localized to the ectocyst layer (yellow arrows) of the wall of mature cysts. WGA labeled both ectocyst and endocyst layers, while CFW labeled the endocyst layer (pink arrows) and ostioles (turquoise arrows). B. Leo-GFP with two 8-Cys domains, which was also expressed under its own promoter, was present in the endocyst layer and sharply outlined the ostioles. Luke(2)-GFP with two CBM49s (C) and Luke(3) with three CBM49s (D), each expressed under its own promoter, were also present in the endocyst layer and outlined conical ostioles of mature cysts. When expressed under a GAPDH promoter, Jonah(1)-GFP localized to the ectocyst layer (E), while Luke(2)-GFP localized to the endocyst layer and ostioles (F). CFW (blue) consistently labeled the endocyst layer, often labeled the ostioles (A to D), and rarely labeled the ectocyst layer (A and E). In this experiment, WGA (red) consistently labeled the ectocyst layer, often labeled the endocyst layer (C, D, and F), and often labeled ostioles (C, D, and F). A to F. Scale bars are 2 μm.