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.

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.

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.

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

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 , which, using immunoelectron microscopy, we show localizes to the intermembrane staples in the FAZ intracellular domain.

The remarkable Dr Robertson.

Muriel Robertson (1883-1973) was a pioneering protozoologist who made a staggering number of important contributions to the fields of parasitology, bacteriology and immunology during her career, which spanned nearly 60 years. These contributions were all the more remarkable given the scientific and social times in which she worked. While Muriel is perhaps best known for her work on the life cycle and transmission of the African trypanosome, Trypanosoma brucei, which she carried out in Uganda at the height of a major Sleeping Sickness epidemic, her work on the Clostridia during the First and Second World Wars made significant contributions to the understanding of anaerobes and to the development of anti-toxoid vaccines, and her work on the immunology of Trichomonas foetus infections in cattle, carried out in collaboration with the veterinarian W.

The streamlined genome of Phytomonas spp. relative to human pathogenic kinetoplastids reveals a parasite tailored for plants.

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp.

Regulators of Trypanosoma brucei cell cycle progression and differentiation identified using a kinome-wide RNAi screen.

The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs) involved in the control of cellular proliferation and differentiation. To address this, a T.

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.

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 colocalizes 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.

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.

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.

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

Background: 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.

Hydroxyurea-induced synchronisation of bloodstream stage Trypanosoma brucei.

Synchronisation of the Trypanosoma brucei 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.

Cytokinesis in trypanosomatids.

The process of cytokinesis, where the cytoplasm of one cell is divided to produce two daughter cells, is intricate in trypanosomatids because of the requirement to replicate and segregate a number of single copy organelles, including the nucleus, kinetoplast, Golgi apparatus, and flagellum. Identifying regulators of the three stages of cytokinesis, initiation, furrow ingression, and abscission is complicated by the fact that cell division in trypanosomatids is easily perturbed and aberrant cells are readily produced during functional characterization of gene products. In this review, we discuss direct and indirect effects on cytokinesis, using Trypanosoma brucei as a model.

Trypanosoma brucei Polo-like kinase is essential for basal body duplication, kDNA segregation and cytokinesis.

Polo-like kinases (PLKs) are conserved eukaryotic cell cycle regulators, which play multiple roles, particularly during mitosis. The function of Trypanosoma brucei PLK was investigated in procyclic and bloodstream-form parasites. In procyclic trypanosomes, RNA interference (RNAi) of PLK, or overexpression of TY1-epitope-tagged PLK (PLKty), but not overexpression of a kinase-dead variant, resulted in the accumulation of cells that had divided their nucleus but not their kinetoplast (2N1K cells).

Cell cycle regulation in Trypanosoma brucei.

Cell division is regulated by intricate and interconnected signal transduction pathways that precisely coordinate, in time and space, the complex series of events involved in replicating and segregating the component parts of the cell. In Trypanosoma brucei, considerable progress has been made over recent years in identifying molecular regulators of the cell cycle and elucidating their functions, although many regulators undoubtedly remain to be identified, and there is still a long way to go with respect to determining signal transduction pathways. However, it is clear that cell cycle regulation in T.

Trypanosoma brucei MOB1 is required for accurate and efficient cytokinesis but not for exit from mitosis.

Two MOB1 genes, MOB1-A and MOB1-B, were identified in Trypanosoma brucei. MOB1-A of T. brucei was shown to form a complex with TbPK50, a functional homologue of the Schizosaccharomyces pombe protein kinase Orb6, and immune precipitated MOB1-A exhibited histone H1 protein kinase activity.

The Trypanosoma brucei cyclin, CYC2, is required for cell cycle progression through G1 phase and for maintenance of procyclic form cell morphology.

CYC2 is an essential PHO80-like cyclin that forms a complex with the cdc2-related kinase CRK3 in Trypanosoma brucei. In both procyclic and bloodstream form T. brucei, knock-down of CYC2 by RNA interference (RNAi) led to an accumulation of cells in G(1) phase.

The cell cycle of parasitic protozoa: potential for chemotherapeutic exploitation.

There is an urgent need to develop new drugs against eukaryotic parasitic protozoa such as Plasmodium, Trypanosoma and Leishmania, which cause the diseases malaria, trypanosomiasis and the leishmaniases respectively. The biology of these organisms has many unusual facets that might be exploited for drug design, and the recent availability of parasite genome sequence data has facilitated the search for novel drug targets. Here we review current understanding of the cell cycle in these parasites and show that important structural and functional differences exist between parasite and mammalian cell cycle control machineries and signal transduction pathways, which might be utilised for rational drug design.

Stage-specific differences in cell cycle control in Trypanosoma brucei revealed by RNA interference of a mitotic cyclin.

African trypanosomes have a tightly coordinated cell cycle to effect efficient segregation of their single organelles, the nucleus, flagellum, and kinetoplast. To investigate cell cycle control in trypanosomes, a mitotic cyclin gene (CYC6) has been identified in Trypanosoma brucei. We show that CYC6 forms an active kinase complex with CRK3, the trypanosome CDK1 homologue, in vivo.