Figure 1.
Expression of human Bax in induced Giardia trophozoites does not affect mitosomes.
A) Confocal microscopy analysis of the subcellular distribution of Bax in transgenic cells. Recombinant Bax (left panel, green) does not localize to mitosomes (middle panel, red) labeled with an anti-IscS rabbit antiserum as a mitosome marker. The signature central mitosome structure is indicated with an arrowhead. The merged images show a clear lack of co-localization. Nuclear DNA is stained with DAPI (blue). Inset: differential interference contrast (DIC) image. Scale bar: 5 µm. B) The central mitosome structure (Cm, arrowhead) is resolved as a tightly packed array of spherical organelles in electron microscopy of adherent cells. The subunits are indistinguishable from individual peripheral mitosomes (Pm, arrow). This organelle structure is unchanged in transgenic trophozoites expressing Bax. N, nucleus; Bb, basal bodies; Ax, axonemes; Vd, ventral disk.
Figure 2.
Expression of Bax is lethal for Giardia.
A) Induced cells expressing Bax stop dividing and die in the course of 6 hours. Inset: semi-quantitative RT-PCR analysis of Bax mRNA levels in transgenic cells pre induction (PI) and 4 hours after induction. B) Subcellular localization of CWPs in ESVs of encysting wild type cells labeled by a monoclonal anti-CWP antibody at 4 h post induction. C) Bax abolishes ESV formation in encysting cells and causes accumulation of CWPs in the cytoplasm and the nucleoplasm. Inset: differential interference contrast (DIC) image. Scale bar: 5 µm.
Figure 3.
CWP release from ESVs is Bax specific.
A) ESVs in encysting Bax-expressing cells treated with the membrane-permeable Bax-inhibiting peptide Ku70 are at least partially protected. Note the co-localization of Bax (green) with (partially intact) ESVs and significantly less cytoplasmic or nucleoplasmic CWP signal than in untreated cells (compare with FIG. 2C).16-May B) Deletion of the Bax C-terminus uncouples Bax targeting to ESV membranes and CWP release. BaxΔ22 lacks the C-terminal 22 amino acids of Bax and localizes to membranes of intact ESV. C, D) Electron micrographs of representative cells expressing Bax or BaxΔ22 at four hours post induction. C) Encysting trophozoite from the population expressing BaxΔ22 showing numerous ESVs with electron dense material (arrowheads). ESV and general compartment morphology are indistinguishable from wild type cells (not shown). Enlarged region shows an individual ESV. Peripheral vesicles underlying the plasma membrane are clearly visible. D) ESVs or organelle remnants are not present in surviving cell expressing Bax. Nuclei, and parts of the microtubule structures of the anterior flagella and significantly enlarged PVs are visible. N, nuclei. Scale bars: 2 µm.