During the first stage of encystation, SIM showed Jonah(1)-GFP and glycopolymers labeled by WGA and GST-CBM49 were present in dozens of distinct vesicles.

A. After 3 hr encystation, Jonah(1)-GFP (green), which was expressed under its own promoter, was present in dozens of small vesicles. WGA (red) labeled fewer but larger vesicles, which did not overlap with those containing Jonah(1)-GFP (see merge). B. After 6 hr encystation, glycopolymers labeled by GST-CBM49 (red) were present in dozens of vesicles that did not overlap with those labeled by WGA (green). C. After 9 hr encystation, glycopolymers labeled with GST-CBM49 and WGA were again very abundant in vesicles that did not overlap. CFW was not visible in vesicles of organisms encysting for 3 to 6 hr, but CFW (blue) labeled the surface of encysting protists at 9 hr. A single ostiole, which was small and circular (turquoise arrow), was present on the surface of one encysting protist, while ostioles were absent from the other organisms. When expressed under its own promoter, neither Luke(2)-GFP nor Leo-GFP was present in the first stage of encystation. A to C. Scale bars are 2 μm.