Trypanosoma Brucei Cells Research Articles

Phytochemical profiling and characterization of flavonoid derivatives from propolis sample and investigation of cytotoxic and antiprotozoal activities

Recently, the growth of consumer demand for functional foods with potential nutritional and health benefits led to rapid growth of analytical tools for profiling of bioactive metabolites and assure quality. Bee propolis is one of the most important bee products owing to its myriad health value. As a gummy exudate produced in beehives after harvesting from different plant species, bee propolis contains bioactive secondary metabolites. The current study aims to profiling the chemical composition of propolis samples from Nigeria using HPLC–UV-ELSD and with the aid of NMR-based analysis for assignment of metabolites classes abundant in Nigerian propolis. Red Nigerian propolis samples were subjected to phytochemical analysis using HPLC–UV-ELSD and NMR. Further chromatographic separation of promising fractions was performed by column chromatography and size exclusion chromatography. Screening of the antitrypanosomal and cytotoxic activities against Trypanosoma brucei and human leukemia cell lines (U937), respectively, was performed. The performance of LC–MS permitted identification of the different components from which 13 compound were identified and allowed combination of fractions to afford 9 fractions from which two isoflavonoids were isolated and identified using 1D and 2D NMR analysis with MS as isosativan and Medicarpin. Red Nigerian propolis crude extract showed the highest inhibitory activity at 6.5 µg/ml compared to moderate activity for the isolated compounds with MIC of 7.6 µg/ml and 12.1 µg/ml for medicarpin and isosativan, respectively. Moreover, the fraction RN-6 from the total extract showed the potent cytotoxic effect with IC50 = 26.5 µg/ml compared to standard diminazen which showed IC50 = 29.5 µg/ml.

Tractable Quinolone Hydrazides Exhibiting Sub-Micromolar and Broad Spectrum Antitrypanosomal Activities.

Nagana and Human African Trypanosomiasis (HAT), caused by (sub)species of Trypanosoma, are diseases that impede human and animal health, and economic growth in Africa. The few drugs available have drawbacks including suboptimal efficacy, adverse effects, drug resistance, and difficult routes of administration. New drugs are needed. A series of 20 novel quinolone compounds with affordable synthetic routes was made and evaluated in vitro against Trypanosoma brucei and HEK293 cells. Of the 20 compounds, 12 had sub-micromolar potencies against the parasite (EC50 values=0.051-0.57 μM), and most were non-toxic to HEK293 cells (CC50 values>5 μM). Two of the most potent compounds presented sub-micromolar activities against other trypanosome (sub)species (T. cruzi and T. b. rhodesiense). Although aqueous solubility is poor, both compounds possess good logD values (2-3), and either robust or poor microsomal stability profiles. These varying attributes will be addressed in future reports.

Analysis of the Leishmania mexicana promastigote cell cycle using imaging flow cytometry provides new insights into cell cycle flexibility and events of short duration.

Promastigote Leishmania mexicana have a complex cell division cycle characterised by the ordered replication of several single-copy organelles, a prolonged S phase and rapid G2 and cytokinesis phases, accompanied by cell cycle stage-associated morphological changes. Here we exploit these morphological changes to develop a high-throughput and semi-automated imaging flow cytometry (IFC) pipeline to analyse the cell cycle in live L. mexicana. Firstly, we demonstrate that, unlike several other DNA stains, Vybrant™ DyeCycle™ Orange (DCO) is non-toxic and enables quantitative DNA imaging in live promastigotes. Secondly, by tagging the orphan spindle kinesin, KINF, with mNeonGreen, we describe KINF's cell cycle-dependent expression and localisation. Then, by combining manual gating of DCO DNA intensity profiles with automated masking and morphological measurements of parasite images, visual determination of the number of flagella per cell, and automated masking and analysis of mNG:KINF fluorescence, we provide a newly detailed description of L. mexicana promastigote cell cycle events that, for the first time, includes the durations of individual G2, mitosis and post-mitosis phases, and identifies G1 cells within the first 12 minutes of the new cell cycle. Our custom-developed masking and gating scheme allowed us to identify elusive G2 cells and to demonstrate that the CDK-inhibitor, flavopiridol, arrests cells in G2 phase, rather than mitosis, providing proof-of-principle of the utility of IFC for drug mechanism-of-action studies. Further, the high-throughput nature of IFC allowed the close examination of promastigote cytokinesis, revealing considerable flexibility in both the timing of cytokinesis initiation and the direction of furrowing, in contrast to the related kinetoplastid parasite, Trypanosoma brucei and many other cell types. Our new pipeline offers many advantages over traditional methods of cell cycle analysis such as fluorescence microscopy and flow cytometry and paves the way for novel high-throughput analysis of Leishmania cell division.

Polyaromatic Bis(indolyl)methane Derivatives with Antiproliferative and Antiparasitic Activity

Bis(indolyl)methanes (BIMs) are a class of compounds that have been recognized as an important core in the design of drugs with important pharmacological properties, such as promising anticancer and antiparasitic activities. Here, we explored the biological activity of the BIM core functionalized with different (hetero)aromatic moieties. We synthesized substituted BIM derivatives with triphenylamine, N,N-dimethyl-1-naphthylamine and 8-hydroxylquinolyl groups, studied their photophysical properties and evaluated their in vitro antiproliferative and antiparasitic activities. The triphenylamine BIM derivative 2a displayed an IC50 of 3.21, 3.30 and 3.93 μM against Trypanosoma brucei, Leishmania major and HT-29 cancer cell line, respectively. The selectivity index demonstrated that compound 2a was up to eight-fold more active against the parasites and HT-29 than against the healthy cell line MRC-5. Fluorescence microscopy studies with MRC-5 cells and T. brucei parasites incubated with derivative 2a indicate that the compound seems to accumulate in the cell’s mitochondria and in the parasite’s nucleus. In conclusion, the BIM scaffold functionalized with the triphenylamine moiety proved to be the most promising antiparasitic and anticancer agent of this series.

How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell.

ATP hydrolysis is required for the synthesis, transport and polymerization of monomers for macromolecules as well as for the assembly of the latter into cellular structures. Other cellular processes not directly related to synthesis of biomass, such as maintenance of membrane potential and cellular shape, also require ATP. The unicellular flagellated parasite Trypanosoma brucei has a complex digenetic life cycle. The primary energy source for this parasite in its bloodstream form (BSF) is glucose, which is abundant in the host's bloodstream. Here, we made a detailed estimation of the energy budget during the BSF cell cycle. As glycolysis is the source of most produced ATP, we calculated that a single parasite produces 6.0 x 1011 molecules of ATP/cell cycle. Total biomass production (which involves biomass maintenance and duplication) accounts for ~63% of the total energy budget, while the total biomass duplication accounts for the remaining ~37% of the ATP consumption, with in both cases translation being the most expensive process. These values allowed us to estimate a theoretical YATP of 10.1 (g biomass)/mole ATP and a theoretical [Formula: see text] of 28.6 (g biomass)/mole ATP. Flagellar motility, variant surface glycoprotein recycling, transport and maintenance of transmembrane potential account for less than 30% of the consumed ATP. Finally, there is still ~5.5% available in the budget that is being used for other cellular processes of as yet unknown cost. These data put a new perspective on the assumptions about the relative energetic weight of the processes a BSF trypanosome undergoes during its cell cycle.

Two New Cytotoxic Sesquiterpene-Amino Acid Conjugates and a Coumarin-Glucoside from Crossostephium chinense.

The Asteraceae family is a promising source of bioactive compounds, such as the famous Asteraceae plants Tanacetum cinerariifolium (pyrethrin) and Artemisia annua (artemisinin). As a result of our series of phytochemical studies of the subtropical plants, two novel sesquiterpenes, named crossoseamines A and B in this study (1 and 2, respectively), one undescribed coumarin-glucoside (3), and eighteen known compounds (4-21) were isolated from the aerial part of Crossostephium chinense (Asteraceae). The structures of isolated compounds were elucidated by spectroscopic methods, including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, HSQC, HMBC, and NOESY), IR spectrum, circular dichroism spectrum (CD), and high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS). All isolated compounds were evaluated for their cytotoxic activities against Leishmania major, Plasmodium falciparum, Trypanosoma brucei (gambiense and rhodesiense), and human lung cancer cell line A549 because of the high demand for the discovery of new drug leads to overcome the present side effects and emerging drug-resistant strains. As a result, the new compounds (1 and 2) showed significant activities against A549 (IC50, 1: 3.3 ± 0.3; 2: 12.3 ± 1.0 μg/mL), L. major (IC50, 1: 6.9 ± 0.6; 2: 24.9 ± 2.2 μg/mL), and P. falciparum (IC50, 1: 12.1 ± 1.1; 2: 15.6 ± 1.2 μg/mL).

Role of the RNA-binding protein ZC3H41 in the regulation of ribosomal protein messenger RNAs in trypanosomes

BackgroundTrypanosomes are single-celled eukaryotes that rely heavily on post-transcriptional mechanisms to regulate gene expression. RNA-binding proteins play essential roles in regulating the fate, abundance and translation of messenger RNAs (mRNAs). Among these, zinc finger proteins of the cysteine3histidine (CCCH) class have been shown to be key players in cellular processes as diverse as differentiation, regulation of the cell cycle and translation. ZC3H41 is an essential zinc finger protein that has been described as a component of spliced leader RNA granules and nutritional stress granules, but its role in RNA metabolism is unknown.MethodsCell cycle analysis in ZC3H41- and Z41AP-depleted cells was carried out using 4′,6-diamidino-2-phenylindole staining, microscopic examination and flow cytometry. The identification of ZC3H41 protein partners was done using tandem affinity purification and mass spectrometry. Next-generation sequencing was used to evaluate the effect of ZC3H41 depletion on the transcriptome of procyclic Trypanosoma brucei cells, and also to identify the cohort of mRNAs associated with the ZC3H41/Z41AP complex. Levels of 5S ribosomal RNA (rRNA) species in ZC3H41- and Z41AP-depleted cells were assessed by quantitative reverse transcription-polymerase chain reaction. Surface sensing of translation assays were used to monitor global translation.ResultsWe showed that depletion of the zinc finger protein ZC3H41 resulted in marked cell cycle defects and abnormal cell morphologies. ZC3H41 was found associated with an essential protein, which we named Z41AP, forming a stable heterodimer, and also with proteins of the poly(A)-binding protein 1 complex. The identification of mRNAs associated with the ZC3H41/Z41AP complex revealed that it is primarily composed of ribosomal protein mRNAs, and that binding to target transcripts is diminished upon nutritional stress. In addition, we observed that mRNAs encoding several proteins involved in the maturation of 5S rRNA are also associated with the ZC3H41/Z41AP complex. Finally, we showed that depletion of either ZC3H41 or Z41AP led to the accumulation of 5S rRNA precursors and a decrease of protein translation.ConclusionsWe propose that ZC3H41 and Z41AP play important roles in controlling the fate of ribosomal components in response to environmental cues.Graphical

Synchronization of Trypanosoma brucei by Counter-Flow Centrifugal Elutriation.

Centrifugal counter-flow elutriation is a non-invasive technique that separates cells based on their hydrodynamic volume in a specialized centrifugation chamber that allows the application of a counter-flow of buffer to oppose sedimentation. Here, we report a centrifugal counter-flow elutriation protocol for Trypanosoma brucei cells that is able to rapidly isolate highly enriched G1 subpopulations (>95%) of synchronized cells. The cells obtained are viable and proliferate without lag, allowing subsequent cell cycle phases to be obtained by continued culture. The synchronized cell cultures obtained by this process have uniform DNA content, a narrow size distribution, undergo synchronous division, and maintain synchrony into subsequent cell cycles.

Cloning and functional complementation of ten Schistosoma mansoni phosphodiesterases expressed in the mammalian host stages.

Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs.

Proteome-Wide Quantitative Phosphoproteomic Analysis of Trypanosoma brucei Insect and Mammalian Life Cycle Stages.

Mass spectrometry based proteomics allows for the identification and quantification of protein and phosphorylation site abundance on a proteome wide scale. Here we describe the metabolic labeling of cultured Trypanosoma brucei cells in either the bloodstream or procyclic life cycle stage using stable isotope labeling of amino acids in cell culture (SILAC), and the production of samples suitable for analysis by liquid chromatography tandem mass spectrometry. The protocols require little specialist equipment, and they typically enable quantification of over 4500 proteins and 9000 phosphorylation sites.

Organising the cell cycle in the absence of transcriptional control: Dynamic phosphorylation co-ordinates the Trypanosoma brucei cell cycle post-transcriptionally.

The cell division cycle of the unicellular eukaryote Trypanosome brucei is tightly regulated despite the paucity of transcriptional control that results from the arrangement of genes in polycistronic units and lack of dynamically regulated transcription factors. To identify the contribution of dynamic phosphorylation to T. brucei cell cycle control we have combined cell cycle synchronisation by centrifugal elutriation with quantitative phosphoproteomic analysis. Cell cycle regulated changes in phosphorylation site abundance (917 sites, average 5-fold change) were more widespread and of a larger magnitude than changes in protein abundance (443 proteins, average 2-fold change) and were mostly independent of each other. Hierarchical clustering of co-regulated phosphorylation sites according to their cell cycle profile revealed that a bulk increase in phosphorylation occurs across the cell cycle, with a significant enrichment of known cell cycle regulators and RNA binding proteins (RBPs) within the largest clusters. Cell cycle regulated changes in essential cell cycle kinases are temporally co-ordinated with differential phosphorylation of components of the kinetochore and eukaryotic initiation factors, along with many RBPs not previously linked to the cell cycle such as eight PSP1-C terminal domain containing proteins. The temporal profiles demonstrate the importance of dynamic phosphorylation in co-ordinating progression through the cell cycle, and provide evidence that RBPs play a central role in post-transcriptional regulation of the T. brucei cell cycle. Data are available via ProteomeXchange with identifier PXD013488.

Tomographic Collection of Block-Based Sparse STEM Images: Practical Implementation and Impact on the Quality of the 3D Reconstructed Volume.

The reduction of the electron dose in electron tomography of biological samples is of high significance to diminish radiation damages. Simulations have shown that sparse data collection can perform efficient electron dose reduction. Frameworks based on compressive-sensing or inpainting algorithms have been proposed to accurately reconstruct missing information in sparse data. The present work proposes a practical implementation to perform tomographic collection of block-based sparse images in scanning transmission electron microscopy. The method has been applied on sections of chemically-fixed and resin-embedded Trypanosoma brucei cells. There are 3D reconstructions obtained from various amounts of downsampling, which are compared and eventually the limits of electron dose reduction using this method are explored.

Preliminary Results Indicate the Anti-Proliferative Effect of HIV-1 Protease Inhibitors on Trypanosoma brucei Cells can be Due to the Non-Specific Targeting of Metallo-and Cysteine-Proteases

Background: Initial studies have confirmed the efficacy of protease inhibitors in the treatment of Trypanosoma cruzi, Plasmodium falciparum, and Leishmania major. However, studies on efficacy and specific protease inhibition of HIV-1 protease inhibitors on Trypanosoma brucei cells remained untouched. The objective of the current study was to determine the efficacy of two HIV-1 protease inhibitors, ritonavir, and saquinavir, in Trypanosoma brucei proliferation and to determine if these HIV-1 protease inhibitors target the activity of the Trypanosoma brucei major proteases. Methods: Time dependency test at variable increasing concentrations, motile cell counts, alamarBlue® cell proliferation/viability assay and zymography were among the methods applied. Results: Both ritonavir (IC50=12.23 ± 0.33 µM) and saquinavir (IC50=11.49 ± 0.31 µM) effectively inhibited Trypanosoma brucei cells proliferation. The major proteases identified in these cells were the cysteine (~29 kDa Mr) and metallo- (~66 kDa Mr) proteases. Protein band densitometry results showed a statistically significant (Pvalue< 0.05) inhibition in metallo-and cysteine-proteases’ activity in Trypanosoma brucei cells. Conclusion: The results suggest that RTV and SQV showed an anti-proliferative effect in Trypanosoma brucei cells possibly due to the non-specific targeting of the cysteine-and metalloprotease activities of the parasite.

Base excision repair plays an important role in the protection against nitric oxide- and in vivo-induced DNA damage in Trypanosoma brucei

Uracil-DNA glycosylase (UNG) initiates the base excision repair pathway by excising uracil from DNA. We have previously shown that Trypanosoma brucei cells defective in UNG exhibit reduced infectivity thus demonstrating the relevance of this glycosylase for survival within the mammalian host. In the early steps of the immune response, nitric oxide (NO) is released by phagocytes, which in combination with oxygen radicals produce reactive nitrogen species (RNS). These species can react with DNA generating strand breaks and base modifications including deaminations. Since deaminated cytosines are the main substrate for UNG, we hypothesized that the glycosylase might confer protection towards nitrosative stress. Our work establishes the occurrence of genotoxic damage in Trypanosoma brucei upon exposure to NO in vitro and shows that deficient base excision repair results in increased levels of damage in DNA and a hypermutator phenotype. We also evaluate the incidence of DNA damage during infection in vivo and show that parasites recovered from mice exhibit higher levels of DNA strand breaks, base deamination and repair foci compared to cells cultured in vitro. Notably, the absence of UNG leads to reduced infectivity and enhanced DNA damage also in animal infections. By analysing mRNA and protein levels, we found that surviving UNG-KO trypanosomes highly express tryparedoxin peroxidase involved in trypanothione/tryparedoxin metabolism. These observations suggest that the immune response developed by the host enhances the activation of genes required to counteract oxidative stress and emphasize the importance of DNA repair pathways in the protection to genotoxic and oxidative stress in trypanosomes.

A new reporter cell line for studies with proteasome inhibitors in Trypanosoma brucei

A Trypanosoma brucei cell line is described that produces a visual readout of proteasome activity. The cell line contains an integrated transgene encoding an ubiquitin-green fluorescent protein (GFP) fusion polypeptide responsive to the addition of proteasome inhibitors. A modified version of T. brucei ubiquitin unable to be recognized by deubiquitinases (UbG76V) was fused to eGFP and constitutively expressed. The fusion protein is unstable but addition of the proteasome inhibitor lactacystin stabilizes it and leads to visually detectable GFP. This cell line can be widely used to monitor the efficiency of inhibitor treatment through detection of GFP accumulation in studies involving proteasome-mediated proteolysis, screening of proteasome inhibitors or other events related to the ubiquitin-proteasome pathway.

Metacyclic VSG expression site promoters are recognized by the same general transcription factor that is required for RNA polymerase I transcription of bloodstream expression sites

Infectious metacyclic Trypanosoma brucei cells develop in the salivary glands of tsetse flies. A critical aspect of the developmental program leading to acquisition of infectivity is the synthesis of a variant surface glycoprotein (VSG) coat. Metacyclic VSG genes are transcribed from a set of specialized VSG expression sites (ESs) that differ from bloodstream VSG ESs by being monocistronic, being significantly shorter, lacking long stretches of 70-bp repeats, and having distinct promoter sequences. Both metacyclic and bloodstream VSG ESs are transcribed by the multifunctional T. brucei RNA polymerase I (Pol I), however the factor that recognizes the divergent metacyclic VSG ES promoters and recruits Pol I during the development to infectious cells remains unknown. We used an in vitro assay to show that the promoters for both metacyclic and bloodstream VSG ESs are recognized by the same class I transcription factor A (CITFA). This general Pol I transcription initiation factor was previously shown to be essential for the transcription of bloodstream VSG genes, procyclin genes and rRNA genes, and was demonstrated to have distinct binding affinities for these three types of promoters. We now show that differences in the sequence of individual metacyclic VSG ESs promoters determine different affinities for CITFA.

High-throughput Gene Tagging in Trypanosoma brucei.

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in &lt;em&gt;Trypanosoma brucei&lt;/em&gt;

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

Phosphorylation of eIF2α on Threonine 169 is not required for Trypanosoma brucei cell cycle arrest during differentiation

The trypanosome life cycle consists of a series of developmental forms each adapted to an environment in the relevant insect and/or mammalian host. The differentiation process from the mammalian bloodstream form to the insect-midgut procyclic form in Trypanosoma brucei occurs in two steps in vivo. First proliferating ‘slender' bloodstream forms differentiate to non-dividing ‘stumpy' forms arrested in G1. Second, in response to environmental cues, stumpy bloodstream forms re-enter the cell cycle and start to proliferate as procyclic forms after a lag during which both cell morphology and gene expression are modified. Nearly all arrested cells have lower rates of protein synthesis when compared to the proliferating equivalent. In eukaryotes, one mechanism used to regulate the overall rate of protein synthesis involves phosphorylation of the alpha subunit of initiation factor eIF2 (eIF2α). The effect of eIF2α phosphorylation is to prevent the action of eIF2B, the guanine nucleotide exchange factor that activates eIF2 for the next rounds of initiation. To investigate the role of the phosphorylation of eIF2α in the life cycle of T. brucei, a cell line was made with a single eIF2α gene that contained the phosphorylation site, threonine 169, mutated to alanine. These cells were capable of differentiating from proliferating bloodstream form cells into arrested stumpy forms in mice and into procyclic forms in vitro and in tsetse flies. These results indicate that translation attenuation mediated by the phosphorylation of eIF2α on threonine 169 is not necessary for the cell cycle arrest associated with these differentiation processes.

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

ABSTRACTThe 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.