Expression and transport of SVSPct,
SVSPtm, and Ssec reporter proteins during Giardia
encystation. CWP protein is stained with Texas red (red), SAG1 with
FITC (green), and nuclei with DAPI (blue). In encysting trophozoites
containing large ESVs (A), SVSPct is localized on the surface membrane
(C) and the ER/nuclear envelope compartment. (B) Merged image and DAPI
staining. (D) Corresponding color-merged image of a newly developed
cyst <30 min after secretion of the cyst wall material. SVSPct is
still partially associated with the plasma membrane (arrows). (E–G)
Removal of the C-terminal pentapeptide from SVSPct (SVSPtm) leads to
accumulation of the reporter in internal compartments (G), presumably
ER/nuclear envelope (arrows in G), with no staining of the plasma
membrane. (F) Merged images and DAPI staining. ESVs develop normally
(E) and cyst formation is unimpaired (H, merged images). (I–M) Further
removal of the entire VSP transmembrane domain resulted in accumulation
of the now soluble reporter Ssec in the ER/nuclear envelope compartment
(L, ar-rows). (I) CWP signal alone. (K) Merged images and
DAPI staining. (M) Fully developed cyst showing Ssec trapped in
endomembrane compartments and nuclear envelope. Insets represent
differential interference contrast images of the corresponding cells.
Bars, 10 μm.