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Department of Biological and Medical Sciences
Faculty of Health and Life Sciences
The diseases - leishmaniasis and trypanosomiasis
Both Leishmania and trypanosomes have a complex life cycle in which they are transmitted by an insect vector between mammalian hosts. Leishmania causes a range of different diseases from the severe and fatal visceral leishmaniasis to the self-healing cutaneous leishmaniasis and is found throughout the tropics and into the southern Mediterranean. Trypanosoma brucei on the other hand is restricted to sub-Saharan Africa, where it causes the fatal disease African sleeping sickness.
Understanding cell morphogenesis
The distinctive shape of Leishmania and trypanosomes is formed by a corset of cross-linked microtubules that are just beneath the cell membrane. Both organisms have a flagellum that provides the propulsive force that enables them to move and unusually this flagellum is laterally attached to the side of the cell body. In Leishmania this attachment region is very short, whereas in trypanosomes the flagellum is attached for the majority of its length. The attachment of the flagellum is mediated by a large complex cytoskeletal structure called the Flagellum Attachment Zone, which forms a seam in the microtubule array. Assembly of the Flagellum Attachment Zone is a key step in cell morphogenesis and disruption of this process leads to dramatic changes in cell shape and form. The aim of the lab is to understand the role of the Flagellum Attachment Zone from its individual components to how they interact and assemble and how this influences cell morphology. To do this we use a range of modern molecular cell biology techniques combined with both light end electron microscopy.
The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum. These are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenesis of the anterior cell tip. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division.
Eukaryotic flagella are complex microtubule based organelles and in many organisms there are extra axonemal structures present, including the outer dense fibres of mammalian sperm and the paraflagellar rod (PFR) of trypanosomes. Flagellum assembly is a complex process occurring across three main compartments, the cytoplasm, the transition fibre-transition zone, and the flagellum. It begins with translation of protein components, followed by their sorting and trafficking into the flagellum, transport to the assembly site and then incorporation. Flagella are formed from over 500 proteins; the principles governing axonemal component assembly are relatively clear. However, the coordination and sites of extra-axonemal structure assembly processes are less clear. We have discovered two cytoplasmic proteins in T. brucei that are required for PFR formation, PFR assembly factors 1 and 2. Deletion of either PFR-AF1 or PFR-AF2 dramatically disrupted PFR formation and caused a reduction in the amount of major PFR proteins. The presence of cytoplasmic factors required for PFR formation aligns with the concept of processes occurring across multiple compartments to facilitate axoneme assembly and this is likely a common theme for extra-axonemal structure assembly.
The Leishmania lysosome has an atypical structure, consisting of an elongated vesicle filled tubule running along the anterior-posterior axis of the cell, which is termed the multi-vesicular tubule (MVT) lysosome. Alongside the MVT lysosome are one or more microtubules, the lysosomal microtubule(s). Previous work indicated there were cell cycle related changes to MVT lysosome organisation; however, these only provided snapshots and did not connect the changes to the lysosomal microtubule(s) or lysosomal function. Using mNeonGreen tagged cysteine peptidase A and SPEF1 as markers of the MVT lysosome and lysosomal microtubule(s) we examined the dynamics of these structures through the cell cycle. Both the MVT lysosome and lysosomal microtubule(s) elongated at the beginning of the cell cycle before plateauing and then disassembling in late G2 before cytokinesis. Moreover, the endocytic rate in cells where the MVT lysosome and lysosomal microtubule(s) had disassembled was extremely low. The dynamic nature of the MVT lysosome and lysosomal microtubule(s) parallels that of the Trypanosoma cruzi cytostome/cytopharynx, which also has a similar membrane tubule structure with associated microtubules. As the cytostome/cytopharynx is an ancestral feature of the kinetoplastids, thissuggeststhat the Leishmania MVT lysosome and lysosomal microtubule(s) is a reduced cytostome/cytopharynx-like feature.
Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new-flagellum and the old-flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new-flagellum daughter in particular re-modelling rapidly and extensively in early G1. This re-modelling process involves an increase in cell body, flagellum, and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old-flagellum daughter undergoes a different G1 re-modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that two daughters of a proliferative division of T. brucei are non-equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non-equivalence.
Leishmania kinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical for Leishmania pathogenicity. The Leishmania FP has a bulbous region posterior to the FP collar, and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host-parasite interactions. Deletion of the FAZ protein FAZ5 clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: mutants were unable to develop late stage infections in sand flies and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity.
The kinetoplastids Trypanosoma brucei and Leishmania mexicana are eukaryotes with a highly structured cellular organisation that is reproduced with great fidelity in each generation. The pattern of signal from a fluorescently tagged protein can define the specific structure/organelle that this protein localises to, and can be extremely informative in phenotype analysis in experimental perturbations, life cycle tracking, post-genomic assays and functional analysis of organelles. Using the vast coverage of protein subcellular localisations provided by the TrypTag project, an ongoing project to determine the localisation of every protein encoded in the T. brucei genome, we have generated an inventory of reliable reference organelle markers for both parasites that combines epifluorescence images with a detailed description of the key features of each localisation. We believe this will be a useful comparative resource that will enable researchers to quickly and accurately pinpoint the localisation of their proteins of interest and will provide cellular markers for many types of cell biology studies. We see this as another important step in the post-genomic era analyses of these parasites, in which ever expanding datasets generate numerous candidates to analyse. Adoption of these reference proteins by the community is likely to enhance research studies and enable better comparison of data.
Flagella have multiple functions that are associated with different axonemal structures. Motile flagella typically have a 9+2 arrangement of microtubules, whereas sensory flagella normally have a 9+0 arrangement. Leishmania exhibits both of these flagellum forms and differentiation between these two flagellum forms is associated with cytoskeletal and cell shape changes. We disrupted flagellum elongation in Leishmania by deleting the intraflagellar transport (IFT) protein IFT140, and examined the effects on cell morphogenesis. Δift140 cells have no external flagellum, having only a very short flagellum within the flagellar pocket. This short flagellum had a collapsed 9+0 (9v) axoneme configuration reminiscent of that in the amastigote, and was not attached to the pocket membrane. Although amastigote-like changes occurred in the flagellar cytoskeleton, the cytoskeletal structures of Δift140 cells retained their promastigote configurations, as examined by fluorescence microscopy of tagged proteins and serial electron tomography. Thus, Leishmania promastigote cell morphogenesis does not depend on the formation of a long flagellum attached at the neck. Furthermore, our data show that disruption of the IFT system is sufficient to produce a switch from the 9+2 to the collapsed 9+0 (9v) axonemal structure; echoing the process that occurs during the promastigote to amastigote differentiation.
The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is nonconventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNAbinding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes.