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Department of Biological and Medical Sciences
Faculty of Health and Life Sciences
I contribute to the teaching on the undergraduate Microbiology and Genomic Medicine modules and supervise undergraduate research projects in the lab.
Currently in the lab I supervise 3 PhD students - Clare Halliday, Laura Smithson and Lauren Wilburn.
Mechanistic origins of cell organisation and architecture underlying parasite functions
In my lab, we use the flagellated eukaryotic parasites Trypanosoma brucei and Leishmania mexicana to understand the fundamental processes that define the cell organisation underlying parasite interactions with their hosts and vectors. The distinctive shape of trypanosomes and Leishmania is formed by a corset of cross-linked microtubules that are just beneath the cell membrane and both have a flagellum that provides the propulsive force enabling them to move. We focus on understanding the morphogenesis of cytoskeletal-membrane interfaces that contribute to i) cell and substrate attachments, ii) interaction with the insect vector and mammalian host. To do this we use a range of modern molecular cell biology techniques combined with both light end electron microscopy.
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.
UoA5: Biomedicine, Cell and Developmental Biology
Wellcome Trust, Collaborative Award – £2.3M (£337,000 to Oxford Brookes University), Apr 2021 – Mar 2026
Collaborative Award with Dr Eva Gluenz (University of Glasgow), Dr Richard Wheeler (University of Oxford), and Prof Jeremy Mottram (University of York) to define the molecular determinants required for Leishmania life cycle progression and virulence.
Royal Society, Newton International Fellowship - £99,000, Jun 2020 – May 2022
Post-doc fellowship for Brazilian researcher to study trypanosome flagellum assembly
Japan Society for Promotion of Science Overseas Fellowship - £90,000, Apr 2020 – Mar 2022
Post-doc fellowship for Japanese researcher to study Leishmania substrate attachment
Royal Society Research Grant - £17,800, Mar 2019 – Feb 2020
Identifying the molecular determinants of the interaction between the Leishmania metacyclic form and its host and vector
OBU GCRF Visiting Fellow Scheme – £7000, Aug 2018 – Oct 2018
To train visiting Brazilian post-doc in advanced techniques for the genetic manipulation of trypanosomatids
Wellcome Trust Biomedical Resource Grant - £709,168, Jan 2016 – Jan 2019
Genome scale genetic tagging and protein localisation in Trypanosoma brucei
co PI with Samuel Dean, Richard Wheeler, Mark Carrington, Sue Vaughan, Christiane Hertz-Fowler and Keith Gull
i) Assembly of cell-to-cell and cell-to-substrate attachment structures
The attachment of cells to either other cells or substrates is a fundamental attribute of eukaryotic cells, both microbial and metazoan, and is central to the development of multicellularity. Microbes in a ‘free-living’ environment attach to substrates, often in complex biofilms, to maintain themselves within a specific ecological niche. However, attachment is also central to parasitism and the trypanosome and Leishmania parasites are excellent systems to study attachment as they form both cell:cell and cell:substrate transmembrane attachment structures. They have a cell:cell junctional structure called the Flagellum Attachment Zone (FAZ) that connects the flagellum to the cell body. This structure is crucial for defining cell shape and form; we have defined its architecture and using innovative proteomic approaches, we discovered 8 proteins in the FAZ and showed that FAZ is assembled at a spatially distinct site to that of the flagellum. In addition, these parasites utilise their flagella to attach to substrates in their insect vectors, where they form an attachment plaque - a cell:substrate transmembrane attachment structure. Recently, we have begun to investigate the architecture and assembly of the attachment plaque in trypanosomes and Leishmania.
Alves AA et al., (2020) Control of assembly of extra-axonemal structures: the paraflagellar rod of trypanosomes. J Cell Sci. 133(10):jcs242271.
Sunter JD et al., (2015) Flagellum attachment zone protein modulation and regulation of cell shape in Trypanosoma brucei life cycle transitions. J Cell Sci. 128, 3117-3130. PMC4541047.
ii) Architecture of host-parasite interfaces
The flagellar pocket, present in microbes to mammalian cells, is a specialised region of the cell membrane at the base of the cilium/flagellum. In the trypanosome and Leishmania parasites it is the only site of endocytosis and exocytosis as well as the site of component sorting for flagellum construction. In concert with collaborators, we defined the detailed 3D structure of the Leishmania flagellar pocket and showed that it is remodelled during the life cycle, demonstrating that flagellar pocket architecture responds to changes in the extracellular environment and its functional requirements. In Leishmania we have shown that deletion of a single FAZ protein, FAZ5, alters flagellar pocket architecture and has a dramatic impact on parasite survival in vivo, illustrating the importance of understanding how cellular architecture is designed for pathogenicity. We have continued this work by undertaking a systematic screen of the role of FAZ proteins in flagellar pocket morphogenesis and function in Leishmania.
Halliday C et al., (2020) Role for the flagellum attachment zone in Leishmania anterior cell tip morphogenesis. PLoS Pathog. 16(10):e1008494.
Sunter JD et al., (2019) Leishmania flagellum attachment zone is critical for flagellar pocket shape, development in the sand fly, and pathogenicity in the host. Proc Natl Acad Sci U S A. 116:6351-6360.
We have strong links with research groups in Africa, primarily at the University of Ghana and we are combining our expertise to understand in detail the impact of animal African trypanosomiasis and develop new approaches to combat it.
My group regularly engages the public primarily through the Brookes Science Bazaar where we demonstrate how we can use microscopy to investigate the fascinating biology of parasites.
Cilia and flagella are required for cell motility and sensing the external environment and can vary in both length and stability. Stable flagella maintain their length without shortening and lengthening and are proposed to “lock” at the end of growth, but molecular mechanisms for this lock are unknown. We show that CEP164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei . CEP164C localizes to mature basal bodies of fully assembled old flagella, but not to growing new flagella, and basal bodies only acquire CEP164C in the third cell cycle after initial assembly. Depletion of CEP164C leads to dysregulation of flagellum growth, with continued growth of the old flagellum, consistent with defects in a flagellum locking mechanism. Inhibiting cytokinesis results in CEP164C acquisition on the new flagellum once it reaches the old flagellum length. These results provide the first insight into the molecular mechanisms regulating flagella growth in cells that must maintain existing flagella while growing new flagella.
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.