Figure 1.
Expression of HaloTagged proteins in G. intestinalis and T. vaginalis.
Western blot analyses of cellular fractions of G. intestinalis and T. vaginalis transformants expressing GiIscU-Halo and TvFtx-Halo fusions, respectively. A) GiIscU-Halo was detected by specific anti-IscU polyclonal antibodies in cell lysate and high-speed pellet (HSP). Two bands in these fractions represent the nuclear encoded (GiIscU) and episomally encoded HaloTag fusion (GiIscU-Halo). B) TvFtx-Halo product was detected by anti-HA monoclonal antibodies in T. vaginalis cellular fractions. The fusion protein was found exclusively in cell lysate and in hydrogenosomes. The upper panels demonstrate the protein profile on the coomassie stained SDS-PAGE gel. Lys-lysate, Cyt-cytosol, HSP-high-speed pellet, Hyd-hydrogenosomes.
Figure 2.
Mitosomal and hydrogenosomal localization of HaloTagged proteins.
Immunofluorescence analyses of G. intestinalis and T. vaginalis transformants expressing GiIscU-Halo and TvFtx-Halo fusion, respectively. Cells were incubated with TMR-Halo ligand (red), washed and fixed for immunofluorescence analysis. A) TMR-Halo labeled G. intestinalis cells were fixed and labeled by anti-Tom40 specific polyclonal antibodies (green). B) TMR-Halo labeled T. vaginalis cells were fixed and decorated by anti-malic enzyme specific polyclonal antibodies (green). Nuclei were stained with DAPI (blue).
Figure 3.
Live imaging of mitosomes and hydrogenosomes.
Halo-TMR labeled organelles were followed in living cells. A) Labeled G. intestinalis cells were allowed to attach to the bottom of the well and directly observed while B) the labeled T. vaginalis cells were mounted in 2% agarose and then submitted to microscopy. Five different snapshots in time are shown. The original movies are part of the supplementary data.