Live Imaging of Mitosomes and Hydrogenosomes by HaloTag Technology

Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes

A novel Plasmodium falciparum SR protein is an alternative splicing factor required for the parasites' proliferation in human erythrocytes

International audienceMalaria parasites have a complex life cycle, during which they undergo significant biological changes to adapt to different hosts and changing environments. Plasmodium falciparum, the species responsible for the deadliest form of human malaria, maintains this complex life cycle with a relatively small number of genes. Alternative splicing (AS) is an important post-transcriptional mechanisms that enables eukaryotic organisms to expand their protein repertoire out of relatively small number of genes. SR proteins are major regulators of AS in higher eukaryotes. Nevertheless, the regulation of splicing as well as the AS machinery in Plasmodium spp. are still elusive. Here, we show that PfSR1, a putative P. falciparum SR protein, can mediate RNA splicing in vitro. In addition, we show that PfSR1 functions as an AS factor in mini-gene in vivo systems similar to the mammalian SR protein SRSF1. Expression of PfSR1-myc in P. falciparum shows distinct patterns of cellular localization during intra erythrocytic development. Furthermore, we determine that the predicted RS domain of PfSR1 is essential for its localization to the nucleus. Finally, we demonstrate that proper regulation of pfsr1 is required for parasite proliferation in human RBCs and over-expression of pfsr1 influences AS activity of P. falciparum genes in vivo

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

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

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