Who needs a contractile actomyosin ring? The plethora of alternative ways to divide a protozoan parasite

Cytokinesis, or the division of the cytoplasm, following the end of mitosis or meiosis, is accomplished in animal cells, fungi and amoebae, by the constriction of an actomyosin contractile ring, comprising filamentous actin, myosin II and associated proteins. However, despite this being the best-studied mode of cytokinesis, it is restricted to the Opisthokonta and Amoebozoa, since members of other evolutionary supergroups lack myosin II and must, therefore, employ different mechanisms. In particular, parasitic protozoa, many of which cause significant morbidity and mortality in humans and animals as well as considerable economic losses, employ a wide diversity of mechanisms to divide, few, if any, of which involve myosin II. In some cases, cell division is not only myosin II-independent, but actin-independent too. Mechanisms employed range from primitive mechanical cell rupture (cytofission), to motility- and/or microtubule remodelling-dependent mechanisms, to budding involving the constriction of divergent contractile rings, to hijacking host cell division machinery, with some species able to utilise multiple mechanisms. Here, I review current knowledge of cytokinesis mechanisms and their molecular control in mammalian-infective parasitic protozoa from the Excavata, Alveolata and Amoebozoa supergroups, highlighting their often-underappreciated diversity and complexity. Billions of people and animals across the world are at risk from these pathogens, for which vaccines and/or optimal treatments are often not available. Exploiting the divergent cell division machinery in these parasites may provide new avenues for the treatment of protozoal disease

Hydroxyurea-induced synchronisation of bloodstream stage Trypanosoma brucei

Synchronisation of the <i>Trypanosoma brucei</i> cell cycle proved elusive for many years. A recent report demonstrated that synchronisation of procyclic form cells was possible following treatment with hydroxyurea. Here, that work is extended to the disease-relevant, mammalian-infective bloodstream stage trypanosome. Treatment of bloodstream stage Lister 427 <i>T. Brucei</i> cells growing <i>in vitro</i> with 10 μg ml<sup>−1</sup> hydroxyurea for 6 h led to an enrichment of cells in S phase. Following removal of the drug, cells proceeded uniformly through one round of the cell cycle, providing a much needed tool to enrich for specific cell cycle stages, in a manner similar to hydroxyurea treatment of procyclic form <i>T. brucei.</i&gt

Searching for novel cell cycle regulators in Trypanosoma brucei with an RNA interference screen

<b>Background</b><br /> The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes Human African Trypanosomiasis. Its cell cycle is complex and not fully understood at the molecular level. The T. brucei genome contains over 6000 protein coding genes with >50% having no predicted function. A small scale RNA interference (RNAi) screen was carried out in Trypanosoma brucei to evaluate the prospects for identifying novel cycle regulators.<p></p> <b>Results</b><br /> Procyclic form T. brucei were transfected with a genomic RNAi library and 204 clones isolated. However, only 76 RNAi clones were found to target a protein coding gene of potential interest. These clones were screened for defects in proliferation and cell cycle progression following RNAi induction. Sixteen clones exhibited proliferation defects upon RNAi induction, with eight clones displaying potential cell cycle defects. To confirm the phenotypes, new RNAi cell lines were generated and characterised for five genes targeted in these clones. While we confirmed that the targeted genes are essential for proliferation, we were unable to unambiguously classify them as cell cycle regulators.<p></p> <b>Conclusion</b><br /> Our study identified genes essential for proliferation, but did not, as hoped, identify novel cell cycle regulators. Screening of the RNAi library for essential genes was extremely labour-intensive, which was compounded by the suboptimal quality of the library. For such a screening method to be viable for a large scale or genome wide screen, a new, significantly improved RNAi library will be required, and automated phenotyping approaches will need to be incorporated.<p></p&gt

Giant FAZ10 is required for flagellum attachment zone stabilization and furrow positioning in Trypanosoma brucei

The flagellum and flagellum attachment zone (FAZ) are important cytoskeletal structures in trypanosomatids, being required for motility, cell division and cell morphogenesis. Trypanosomatid cytoskeletons contain abundant high molecular mass proteins (HMMPs), but many of their biological functions are still unclear. Here, we report the characterization of the giant FAZ protein, FAZ10, in Trypanosoma brucei, which, using immunoelectron microscopy, we show localizes to the intermembrane staples in the FAZ intracellular domain. Our data show that FAZ10 is a giant cytoskeletal protein essential for normal growth and morphology in both procyclic and bloodstream parasite life cycle stages, with its depletion leading to defects in cell morphogenesis, flagellum attachment, and kinetoplast and nucleus positioning. We show that the flagellum attachment defects are probably brought about by reduced tethering of the proximal domain of the paraflagellar rod to the FAZ filament. Further, FAZ10 depletion also reduces abundance of FAZ flagellum domain protein, ClpGM6. Moreover, ablation of FAZ10 impaired the timing and placement of the cleavage furrow during cytokinesis, resulting in premature or asymmetrical cell division

The Trypanosoma brucei AIR9-like protein is cytoskeleton-associated and is required for nucleus positioning and accurate cleavage furrow placement

AIR9 is a cytoskeleton-associated protein in Arabidopsis thaliana with roles in cytokinesis and cross wall maturation, and reported homologues in land plants and excavate protists, including trypanosomatids. We show that the Trypanosoma brucei AIR9-like protein, TbAIR9, is also cytoskeleton-associated and colocalises with the subpellicular microtubules. We find it to be expressed in all life cycle stages and show that it is essential for normal proliferation of trypanosomes in vitro. Depletion of TbAIR9 from procyclic trypanosomes resulted in increased cell length due to increased microtubule extension at the cell posterior. Additionally, the nucleus was re-positioned to a location posterior to the kinetoplast, leading to defects in cytokinesis and the generation of aberrant progeny. In contrast, in bloodstream trypanosomes, depletion of TbAIR9 had little effect on nucleus positioning, but resulted in aberrant cleavage furrow placement and the generation of non-equivalent daughter cells following cytokinesis. Our data provide insight into the control of nucleus positioning in this important pathogen and emphasise differences in the cytoskeleton and cell cycle control between two life cycle stages of the T. brucei parasite

High-speed particle detection and tracking in microfluidic devices using event-based sensing

Visualising fluids and particles within channels is a key element of microfluidic work. Current imaging methods for particle image velocimetry often require expensive high-speed cameras with powerful illuminating sources, thus potentially limiting accessibility. This study explores for the first time the potential of an event-based camera for particle and fluid behaviour characterisation in a microfluidic system. Event-based cameras have the unique capacity to detect light intensity changes asynchronously and to record spatial and temporal information with low latency, low power and high dynamic range. Event-based cameras could consequently be relevant for detecting light intensity changes due to moving particles, chemical reactions or intake of fluorescent dyes by cells to mention a few. As a proof-of-principle, event-based sensing was tested in this work to detect 1 μm and 10 μm diameter particles flowing in a microfluidic channel for average fluid velocities of up to 1.54 m s−1. Importantly, experiments were performed by directly connecting the camera to a standard fluorescence microscope, only relying on the microscope arc lamp for illumination. We present a data processing strategy that allows particle detection and tracking in both bright-field and fluorescence imaging. Detection was achieved up to a fluid velocity of 1.54 m s−1 and tracking up to 0.4 m s−1 suggesting that event-based cameras could be a new paradigm shift in microscopic imaging

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

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 divisio

Sepsis: when a simple infection becomes deadly

The immune system plays a crucial role in maintaining a healthy body by working around the clock to recognize and respond to infection. Inflammation is part of the immune system’s protective response to an infection. The inflammatory response is incredibly powerful, so much so that it can damage the body’s cells if it is not tightly controlled. Sometimes, inflammation affects the whole body—this is called sepsis. The powerful and complex mechanisms in place to wipe out the infection can cause serious damage to healthy cells and tissues. Uncontrolled inflammation can cause irreversible damage to the body’s organs, such as the kidneys, eventually causing organs to shut down. If sepsis is not treated rapidly, it can lead to death. In this article, we describe the symptoms and diagnosis of sepsis and some of the current research being performed to better understand this dangerous process