Eva Gluenz

The cell cycle of Leishmania: morphogenetic events and their implications for parasite biology

The cell cycle is central to understanding fundamental biology of Leishmania, a group of human‐infective protozoan parasites. Leishmania have two main life cycle morphologies: the intracellular amastigote in the mammalian host and the promastigote in the fly. We have produced the first comprehensive and quantitative description of a Leishmania promastigote cell cycle taking a morphometric approach to position any cell within the cell cycle based on its length and DNA content. We describe timings of cell cycle phases and rates of morphological changes; kinetoplast and nucleus S phase, division and position, cell body growth and morphology changes, flagellum growth and basal body duplication. We have shown that Leishmania mexicana undergoes large changes in morphology through the cell cycle and that the wide range of morphologies present in cultures during exponential growth represent different cell cycle stages. We also show promastigote flagellum growth occurs over multiple cell cycles. There are clear implications for the mechanisms of flagellum length regulation, life cycle stage differentiation and trypanosomatid division in general. This data set therefore provides a platform which will be of use for post‐genomic analyses of Leishmania cell biology in relation to differentiation and infection.

The heart of darkness: growth and form of Trypanosoma brucei in the tsetse fly.

The first description of African trypanosomes was made over a century ago. The importance of the tsetse in transmission and cyclic development of trypanosomes was discovered soon afterwards, and has been the focus of numerous studies since. However, investigation of trypanosomes in tsetse flies requires high resource investment and unusual patience; hence, many facets of trypanosome biology in the tsetse remain to be characterised despite the long history of research. Here, current knowledge and questions about some of the developmental changes in trypanosomes that occur in tsetse flies are summarised, along with recent technical advances that can now be used to provide some answers.

Beyond 9+0: noncanonical axoneme structures characterize sensory cilia from protists to humans

The intracellular amastigote stages of parasites such as Leishmania are often referred to as aflagellate. They do, however, possess a short axoneme of cryptic function. Here, our examination of the structure of this axoneme leads to a testable hypothesis of its role in the cell biology of pathogenicity. We show a striking similarity between the microtubule axoneme structure of the Leishmania mexicana parasite infecting a macrophage and vertebrate primary cilia. In both, the 9‐fold microtubule doublet symmetry is broken by the incursion of one or more microtubule doublets into the axoneme core, giving rise to an architecture that we term here the 9v (variable) axoneme. Three‐dimensional reconstructions revealed that no particular doublet initiated the symmetry break, and moreover it often involved 2 doublets. The tip of the L. mexicana flagellum was frequently intimately associated with the macrophage vacuole membrane. We propose that the main function of the amastigote flagellum is to act as a sensory organelle with important functions in host‐parasite interactions and signaling in the intracellular stage of the L. mexicana life cycle.— Gluenz, E., Höög, J. L., Smith, A. E., Dawe, H. R., Shaw, M. K., Gull, K. Beyond 9+0: noncanonical axoneme structures characterize sensory cilia from protists to humans. FASEB J. 24, 3117–3121 (2010). www.fasebj.org

The Kinetoplast Duplication Cycle in Trypanosoma brucei Is Orchestrated by Cytoskeleton-Mediated Cell Morphogenesis

ABSTRACT The mitochondrial DNA of Trypanosoma brucei is organized in a complex structure called the kinetoplast. In this study, we define the complete kinetoplast duplication cycle in T. brucei based on three-dimensional reconstructions from serial-section electron micrographs. This structural model was enhanced by analyses of the replication process of DNA maxi- and minicircles. Novel insights were obtained about the earliest and latest stages of kinetoplast duplication. We show that kinetoplast S phase occurs concurrently with the repositioning of the new basal body from the anterior to the posterior side of the old flagellum. This emphasizes the role of basal body segregation in kinetoplast division and suggests a possible mechanism for driving the rotational movement of the kinetoplast during minicircle replication. Fluorescence in situ hybridization with minicircle- and maxicircle-specific probes showed that maxicircle DNA is stretched out between segregated minicircle networks, indicating that maxicircle segregation is a late event in the kinetoplast duplication cycle. This new view of the complexities of kinetoplast duplication emphasizes the dependencies between the dynamic remodelling of the cytoskeleton and the inheritance of the mitochondrial genome.

Perturbation of phosphatidylethanolamine synthesis affects mitochondrial morphology and cell-cycle progression in procyclic-form Trypanosoma brucei.

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are the two major constituents of eukaryotic cell membranes. In the protist Trypanosoma brucei, PE and PC are synthesized exclusively via the Kennedy pathway. To determine which organelles or processes are most sensitive to a disruption of normal phospholipid levels, the cellular consequences of a decrease in the levels of PE or PC, respectively, were studied following RNAi knock‐down of four enzymes of the Kennedy pathway. RNAi against ethanolamine‐phosphate cytidylyltransferase (ET) disrupted mitochondrial morphology and ultrastructure. Electron microscopy revealed alterations of inner mitochondrial membrane morphology, defined by a loss of disk‐like cristae. Despite the structural changes in the mitochondrion, the cells maintained oxidative phosphorylation. Our results indicate that the inner membrane morphology of T. brucei procyclic forms is highly sensitive to a decrease of PE levels, as a change in the ultrastructure of the mitochondrion is the earliest phenotype observed after RNAi knock‐down of ET. Interference with phospholipid synthesis also impaired normal cell‐cycle progression. ET RNAi led to an accumulation of multinucleate cells. In contrast, RNAi against choline‐/ethanolamine phosphotransferase, which affected PC as well as PE levels, caused a cell division phenotype characterized by non‐division of the nucleus and production of zoids.

A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids

One of the first steps in understanding a proteins function is to determine its localization; however, the methods for localizing proteins in some systems have not kept pace with the developments in other fields, creating a bottleneck in the analysis of the large datasets that are generated in the post-genomic era. To address this, we developed tools for tagging proteins in trypanosomatids. We made a plasmid that, when coupled with long primer PCR, can be used to produce transgenes at their endogenous loci encoding proteins tagged at either terminus or within the protein coding sequence. This system can also be used to generate deletion mutants to investigate the function of different protein domains. We show that the length of homology required for successful integration precluded long primer PCR tagging in Leishmania mexicana. Hence, we developed plasmids and a fusion PCR approach to create gene tagging amplicons with sufficiently long homologous regions for targeted integration, suitable for use in trypanosomatids with less efficient homologous recombination than Trypanosoma brucei. Importantly, we have automated the primer design, developed universal PCR conditions and optimized the workflow to make this system reliable, efficient and scalable such that whole genome tagging is now an achievable goal.

The Limits on Trypanosomatid Morphological Diversity

Cell shape is one, often overlooked, way in which protozoan parasites have adapted to a variety of host and vector environments and directional transmissions between these environments. Consequently, different parasite life cycle stages have characteristic morphologies. Trypanosomatid parasites are an excellent example of this in which large morphological variations between species and life cycle stage occur, despite sharing well-conserved cytoskeletal and membranous structures. Here, using previously published reports in the literature of the morphology of 248 isolates of trypanosomatid species from different hosts, we perform a meta-analysis of the occurrence and limits on morphological diversity of different classes of trypanosomatid morphology (trypomastigote, promastigote, etc.) in the vertebrate bloodstream and invertebrate gut environments. We identified several limits on cell body length, cell body width and flagellum length diversity which can be interpreted as biomechanical limits on the capacity of the cell to attain particular dimensions. These limits differed for morphologies with and without a laterally attached flagellum which we suggest represent two morphological superclasses, the ‘juxtaform’ and ‘liberform’ superclasses. Further limits were identified consistent with a selective pressure from the mechanical properties of the vertebrate bloodstream environment; trypanosomatid size showed limits relative to host erythrocyte dimensions. This is the first comprehensive analysis of the limits of morphological diversity in any protozoan parasite, revealing the morphogenetic constraints and extrinsic selection pressures associated with the full diversity of trypanosomatid morphology.

Structural asymmetry and discrete nucleic acid subdomains in the Trypanosoma brucei kinetoplast

The mitochondrial genome of Trypanosoma brucei is contained in a specialized structure termed the kinetoplast. Kinetoplast DNA (kDNA) is organized into a concatenated network of mini and maxicircles, positioned at the base of the flagellum, to which it is physically attached. Here we have used electron microscope cytochemistry to determine structural and functional domains involved in replication and segregation of the kinetoplast. We identified two distinct subdomains within the kinetoflagellar zone (KFZ) and show that the unilateral filaments are composed of distinct inner and outer filaments. Ethanolic phosphotungstic acid (E‐PTA) and EDTA regressive staining indicate that basic proteins and DNA are major constituents of the inner unilateral filaments adjoining the kDNA disc. This evidence for an intimate connection of the unilateral filaments in the KFZ with DNA provides support for models of minicircle replication involving vectorial export of free minicircles into the KFZ. Unexpectedly however, detection of DNA in the KFZ throughout the cell cycle suggests that other processes involving kDNA occur in this domain. We also describe a hitherto unrecognized, intramitochondrial, filamentous structure rich in basic proteins that links the kDNA discs during their segregation and is maintained between them for an extended period of the cell cycle.

Histone H1 Plays a Role in Heterochromatin Formation and VSG Expression Site Silencing in Trypanosoma brucei

The African sleeping sickness parasite Trypanosoma brucei evades the host immune system through antigenic variation of its variant surface glycoprotein (VSG) coat. Although the T. brucei genome contains ∼1500 VSGs, only one VSG is expressed at a time from one of about 15 subtelomeric VSG expression sites (ESs). For antigenic variation to work, not only must the vast VSG repertoire be kept silent in a genome that is mainly constitutively transcribed, but the frequency of VSG switching must be strictly controlled. Recently it has become clear that chromatin plays a key role in silencing inactive ESs, thereby ensuring monoallelic expression of VSG. We investigated the role of the linker histone H1 in chromatin organization and ES regulation in T. brucei. T. brucei histone H1 proteins have a different domain structure to H1 proteins in higher eukaryotes. However, we show that they play a key role in the maintenance of higher order chromatin structure in bloodstream form T. brucei as visualised by electron microscopy. In addition, depletion of histone H1 results in chromatin becoming generally more accessible to endonucleases in bloodstream but not in insect form T. brucei. The effect on chromatin following H1 knock-down in bloodstream form T. brucei is particularly evident at transcriptionally silent ES promoters, leading to 6–8 fold derepression of these promoters. T. brucei histone H1 therefore appears to be important for the maintenance of repressed chromatin in bloodstream form T. brucei. In particular H1 plays a role in downregulating silent ESs, arguing that H1-mediated chromatin functions in antigenic variation in T. brucei.

Blocking Variant Surface Glycoprotein Synthesis in Trypanosoma brucei Triggers a General Arrest in Translation Initiation

Background The African trypanosome Trypanosoma brucei is covered with a dense layer of Variant Surface Glycoprotein (VSG), which protects it from lysis by host complement via the alternative pathway in the mammalian bloodstream. Blocking VSG synthesis by the induction of VSG RNAi triggers an unusually precise precytokinesis cell-cycle arrest. Methodology/Principal Findings Here, we characterise the cells arrested after the induction of VSG RNAi. We were able to rescue the VSG221 RNAi induced cell-cycle arrest through expression of a second different VSG (VSG117 which is not recognised by the VSG221 RNAi) from the VSG221 expression site. Metabolic labeling of the arrested cells showed that blocking VSG synthesis triggered a global translation arrest, with total protein synthesis reduced to less than 1–4% normal levels within 24 hours of induction of VSG RNAi. Analysis by electron microscopy showed that the translation arrest was coupled with rapid disassociation of ribosomes from the endoplasmic reticulum. Polysome analysis showed a drastic decrease in polysomes in the arrested cells. No major changes were found in levels of transcription, total RNA transcript levels or global amino acid concentrations in the arrested cells. Conclusions The cell-cycle arrest phenotype triggered by the induction of VSG221 RNAi is not caused by siRNA toxicity, as this arrest can be alleviated if a second different VSG is inserted downstream of the active VSG221 expression site promoter. Analysis of polysomes in the stalled cells showed that the translation arrest is mediated at the level of translation initiation rather than elongation. The cell-cycle arrest induced in the presence of a VSG synthesis block is reversible, suggesting that VSG synthesis and/or trafficking to the cell surface could be monitored during the cell-cycle as part of a specific cell-cycle checkpoint.