Article Figures & Data
Figures
- FIG. 1.
Expression of α19-giardin in trophozoites of G. lamblia. (A) RT-PCR on cDNA, total RNA, and RNA treated with DNase I as negative controls. (B) Western blots of trophozoite extract with pellet (P) and supernatant (S) fractions, each immunodecorated with α19-giardin antiserum. (C) Western blots of isolated flagella immunodecorated with antiserum directed against α1-giardin, α14-giardin, α19-giardin, and tubulin (Tub) as a charge control.
- FIG. 2.
Immunocytochemical localization of α19-giardin in trophozoites of G. lamblia. (A) Fixed and permeabilized trophozoites were incubated with antibodies against α19-giardin (1:1,000; then with Cy3-conjugated secondary antibodies at 1:100; red) and antitubulin (1:500; then with Cy2-conjugated secondary antibodies at 1:100; green); DIC, bright-field differential interference contrast. (B) Immunocytochemical localization of α19-giardin during G1 arrest of the trophozoites. Nuclear DNA is stained blue with DAPI. Scale bar, 10 μm.
- FIG. 3.
Membrane interaction of α19-giardin. (A) Extraction experiments of trophozoite membrane fraction with selected reagents. S, supernatant; P, pellet. For Triton X-114 partitioning detergent (D) and aqueous (A) phases are shown. (B) Reversible interaction of truncated α19-giardin with phospholipid (PL) vesicles in the absence or presence of Ca2+. The last pellet fraction was treated with an excess of EGTA (right two lanes). Controls were performed without phospholipids.
- FIG. 4.
Evidence of a myristoylation of α19-giardin. Western blot (A) and autoradiograms (B) of E. coli extracts after heterologous production of wild type α19-giardin (α19WT) and α19-giardin (G2A) (α19G2A), with or without gNMT in the presence of [3H]myristate. Western blots were immunodecorated with α19-giardin antiserum.
- FIG. 5.
Expression analysis of α19-giardin in transgenic trophozoites. (A) Expression of a single integrated copy of the α19-giardin-HA-tagged gene (wild type) product in transgenic cells with a chromosomally integrated copy of the pPacV-integ expression vector. Note the highly restricted localization of the protein (green) to the ventral flagellum pair. A total projection of the entire image stack (62 optical sections) is shown. (B) Overexpression of the α19-giardin-HA (G2A) mutant variant (green) in transgenic cells reveals its cytoplasmic distribution. A total projection of the entire image stack (46 optical sections) is shown. Nuclear DNA is stained blue with DAPI. DIC, bright-field (BF) differential interference contrast image. (C) Single frame of a time-lapse series of an actively attached cell expressing the α19-giardin::GFP reporter (green) localized in the ventral flagella. Note the typical wave form of the beating flagellum pair. The corresponding bright-field image is shown in the right panel. Scale bar, 10 μm. (See also supplemental material.)
- FIG. 6.
Evidence of palmitoylation of α19-giardin. (A) Localization of wild-type α19-giardin::GFP and α19-giardin::GFP (C3A) mutation in yeast. CUY2171 strains containing constructs expressing wild-type α19-giardin::GFP (α19-GFP) and α19-giardin::GFP (C3A) (α19C3A-GFP) were coexpressed in the presence and absence of Pfa3 and analyzed by fluorescence microscopy. (B) Cell fractionation and analyses by Western blotting in the presence and absence of Pfa3. P13, vacuolar fraction obtained after centrifugation at 13,000 × g; S100, soluble fraction obtained after centrifugation at 100,000 × g; Tal1, marker of a soluble protein; Vma6, marker of a vacuolar protein (each visualized by immunodecoration). (C) Determination of palmitoylation of α19-giardin constructs by biotin switch assay. Yeast cells were lysed, free cysteines were quenched with N-ethylmaleimide, and the extract was left untreated (−) or was treated (+) with hydroxylamine (HA) and biotin-BMMC. A portion (10%) of the lysate was precipitated with trichloroacetic acid. The remaining proteins were captured by pull-down assays with neutravidin and analyzed by Western blotting using anti-α19-giardin antibodies.
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Two channel time-lapse video sequences showing an actively attached cell expressing the α-19 giardin::GFP reporter localized
in the ventral flagella.
AVI Video file, 1.32 MB.
- Supplemental file 1
-
Two channel time-lapse video sequences showing an actively attached cell expressing the α-19 giardin::GFP reporter localized
in the ventral flagella.
