Corinna Benz

Malleable mitochondrion of Trypanosoma brucei.

The importance of mitochondria for a typical aerobic eukaryotic cell is undeniable, as the list of necessary mitochondrial processes is steadily growing. Here, we summarize the current knowledge of mitochondrial biology of an early-branching parasitic protist, Trypanosoma brucei, a causative agent of serious human and cattle diseases. We present a comprehensive survey of its mitochondrial pathways including kinetoplast DNA replication and maintenance, gene expression, protein and metabolite import, major metabolic pathways, Fe-S cluster synthesis, ion homeostasis, organellar dynamics, and other processes. As we describe in this chapter, the single mitochondrion of T. brucei is everything but simple and as such rivals mitochondria of multicellular organisms.

Nuclear DBF-2-related Kinases Are Essential Regulators of Cytokinesis in Bloodstream Stage Trypanosoma brucei

Nuclear DBF-2-related (NDR) kinases are essential regulators of cell cycle progression, growth, and development in many organisms and are activated by the binding of an Mps One Binder (MOB) protein partner, autophosphorylation, and phosphorylation by an upstream STE20 family kinase. In the protozoan parasite, Trypanosoma brucei, the causative agent of human African trypanosomiasis, the NDR kinase, PK50, is expressed in proliferative life cycle stages and was shown to complement a yeast NDR kinase mutant cell line. However, the function of PK50 and a second NDR kinase, PK53, in T. brucei has not been determined to date, although trypanosome MOB1 is known to be essential for cytokinesis, suggesting the NDR kinases may also be involved in this process. Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited. This led to a deregulation of the cell cycle and cell death and provides genetic validation of these kinases as potential novel drug targets for human African trypanosomiasis. Recombinant active PK50 and PK53 were produced and biochemically characterized. Both enzymes autophosphorylated, were able to trans-phosphorylate generic kinase substrates in vitro, and were active in the absence of phosphorylation by an upstream kinase. Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo. Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases.

Cytokinesis in Bloodstream Stage Trypanosoma brucei Requires a Family of Katanins and Spastin

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell division.

Modulation of flagellum attachment zone protein FLAM3 and regulation of the cell shape in Trypanosoma brucei life cycle transitions.

ABSTRACT The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure – the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms. Summary: Trypanosoma brucei FLAM3 is a flagellar FAZ protein. Its depletion leads to a reduction in FAZ length, which has different consequences depending on the life cycle stage of the parasite.

Identification and Functional Characterisation of CRK12:CYC9, a Novel Cyclin-Dependent Kinase (CDK)-Cyclin Complex in Trypanosoma brucei

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.

Roles of the Nfu Fe–S targeting factors in the trypanosome mitochondrion

Iron-sulphur clusters (ISCs) are protein co-factors essential for a wide range of cellular functions. The core iron-sulphur cluster assembly machinery resides in the mitochondrion, yet due to export of an essential precursor from the organelle, it is also needed for cytosolic and nuclear iron-sulphur cluster assembly. In mitochondria all [4Fe-4S] iron-sulphur clusters are synthesised and transferred to specific apoproteins by so-called iron-sulphur cluster targeting factors. One of these factors is the universally present mitochondrial Nfu1, which in humans is required for the proper assembly of a subset of mitochondrial [4Fe-4S] proteins. Although most eukaryotes harbour a single Nfu1, the genomes of Trypanosoma brucei and related flagellates encode three Nfu genes. All three Nfu proteins localise to the mitochondrion in the procyclic form of T. brucei, and TbNfu2 and TbNfu3 are both individually essential for growth in bloodstream and procyclic forms, suggesting highly specific functions for each of these proteins in the trypanosome cell. Moreover, these two proteins are functional in the iron-sulphur cluster assembly in a heterologous system and rescue the growth defect of a yeast deletion mutant.

Cell cycle synchronisation of Trypanosoma brucei by centrifugal counter-flow elutriation reveals the timing of nuclear and kinetoplast DNA replication

We report an optimised centrifugal counter-flow elutriation protocol for the rapid and direct isolation of G1 cell cycle synchronised populations of both the procyclic and bloodstream form stages of Trypanosoma brucei that yields viable and proliferative cells. The high quality of the synchronisation achieved can be judged by the uniform DNA content, narrow size distribution, synchronous division, and the maintenance of synchronicity into subsequent cell cycles. We show that early-eluting fractions represent different G1 subpopulations that progress through the cell cycle with distinct temporal profiles post-elutriation, as exemplified by the observation of the maturation of a second flagellar basal body in late G1 phase, DNA replication in S phase, and dimethylation of histone H3 in mitosis/cytokinesis. We use our temporal observations to construct a revised model of the relative timing and duration of the nuclear and kinetoplast cell cycle that differs from the current model.

Dynamin-like proteins in Trypanosoma brucei: A division of labour between two paralogs?

Dynamins and dynamin-like proteins (DLPs) belong to a family of large GTPases involved in membrane remodelling events. These include both fusion and fission processes with different dynamin proteins often having a specialised function within the same organism. Trypanosoma brucei is thought to have only one multifunctional DLP (TbDLP). While this was initially reported to function in mitochondrial division only, an additional role in endocytosis and cytokinesis was later also proposed. Since there are two copies of TbDLP present in the trypanosome genome, we investigated potential functional differences between these two paralogs by re-expressing either protein in a TbDLP RNAi background. These paralogs, called TbDLP1 and TbDLP2, are almost identical bar a few amino acid substitutions. Our results, based on cell lines carrying tagged and RNAi-resistant versions of each protein, show that overexpression of TbDLP1 alone is able to rescue the observed endocytosis and growth defects in the mammalian bloodstream form (BSF) of the parasite. While TbDLP2 shows no rescue in our experiments in BSF, this might also be due to lower expression levels of the protein in this life stage. In contrast, both TbDLP proteins apparently play more complementary roles in the insect procyclic form (PCF) since neither TbDLP1 nor TbDLP2 alone can fully restore wildtype growth and morphology in TbDLP-depleted parasites.

Dynamic regulation of the Trypanosoma brucei transferrin receptor in response to iron starvation is mediated via the 3’UTR

The bloodstream form of the parasite Trypanosoma brucei obtains iron from its mammalian host by receptor-mediated endocytosis of host transferrin through its own unique transferrin receptor (TbTfR). Expression of TbTfR rapidly increases upon iron starvation by post-transcriptional regulation through a currently undefined mechanism that is distinct from the mammalian iron response system. We have created reporter cell lines by fusing the TbTfR 3′UTR or a control Aldolase 3’UTR to reporter genes encoding GFP or firefly Luciferase, and inserted the fusions into a bloodstream form cell line at a tagged ribosomal RNA locus. Fusion of the TbTfR 3’UTR is sufficient to significantly repress the expression of the reporter proteins under normal growth conditions. Under iron starvation conditions we observed upregulation of the TbTfR 3’UTR fusions only, with a magnitude and timing consistent with that reported for upregulation of the TbTfR. We conclude that the dynamic regulation of the T. brucei transferrin receptor in response to iron starvation is mediated via its 3’UTR, and that the effect is independent of genomic location.