Expression of HaloTagged proteins in <i>G. intestinalis</i> and <i>T. vaginalis</i>.

<p>Western blot analyses of cellular fractions of <i>G. intestinalis</i> and <i>T. vaginalis</i> 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 <i>T. vaginalis</i> 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.</p

Live imaging of mitosomes and hydrogenosomes.

<p>Halo-TMR labeled organelles were followed in living cells. A) Labeled <i>G. intestinalis</i> cells were allowed to attach to the bottom of the well and directly observed while B) the labeled <i>T. vaginalis</i> 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.</p

Mitosomal and hydrogenosomal localization of HaloTagged proteins.

<p>Immunofluorescence analyses of <i>G. intestinalis</i> and <i>T. vaginalis</i> 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 <i>G. intestinalis</i> cells were fixed and labeled by anti-Tom40 specific polyclonal antibodies (green). B) TMR-Halo labeled <i>T. vaginalis</i> cells were fixed and decorated by anti-malic enzyme specific polyclonal antibodies (green). Nuclei were stained with DAPI (blue).</p