Biology subjects

Sunter, J.

Publications and source records attributed to Sunter, J..

10 recordsLinked to original sources

bioRxiv2026

Life without heterotrimeric kinesins: trypanosomatids use a combination of homodimeric kinesin-2 motors to drive intraflagellar transport

Heterotrimeric kinesin 2 is the canonical motor protein for anterograde intraflagellar transport (IFT), driving movement of protein complexes towards the tip of cilia and flagella. Here, we show that all members of the Euglenozoa group lack genes for heterotrimeric kinesins and instead possess a variable number of genes for two homodimeric kinesins termed KIN2A and KIN2B. When expressed in vitro, both Trypanosoma brucei kinesins form homodimers and move processively along brain microtubules, KIN2A being faster than KIN2B. Studies in T. brucei and Leishmania mexicana show anterograde and retrograde IFT of both kinesins, with KIN2A travelling throughout the whole length of the flagellum, while KIN2B is concentrated at its base. In the proximal portion of the flagellum, most KIN2B molecules travel without IFT proteins, except for a few particles that are associated with IFT proteins and reach the tip. Surprisingly, the absence of KIN2A has mild effects on IFT and flagellum assembly, whereas KIN2B is essential for both. Investigation of trypanosome flagella deprived of KIN2B revealed that IFT proteins do not access these flagella but that KIN2A can still circulate. We propose a division-of-labour model where KIN2B would be responsible for the import of IFT proteins while KIN2A would ensure most of the anterograde transport.

cell biology↗

bioRxiv2023

The C-terminus of CFAP410 forms a tetrameric helical bundle that is essential for its localization to the basal body

Cilia are antenna-like organelles protruding from the surface of many cell types in the human body. Defects in ciliary structure or function often lead to diseases that are collectively called ciliopathies. Cilia and flagella associated protein 410 (CFAP410) localizes at the basal body of cilia/flagella and plays essential roles in ciliogenesis, neuronal development, and DNA damage repair. It remains unknown how its specific basal body location is achieved. Multiple single amino acid mutations in CFAP410 have been identified in patients with various ciliopathies. One of the mutations, L224P, is located in the C-terminal domain (CTD) of human CFAP410 and causes severe spondylometaphyseal dysplasia, axial (SMDAX). However, the molecular mechanism for how the mutation causes the disorder remains unclear. Here, we report our structural studies on the CTD of CFAP410 from three distantly related organisms, Homo sapiens, Trypanosoma brucei, and Chlamydomonas reinhardtii. The crystal structures reveal that the three proteins all adopt the same conformation as a tetrameric helical bundle. Our work further demonstrates that the tetrameric assembly of the CTD is essential for the correct localization of CFAP410 in T. brucei, as the L224P mutation that disassembles the tetramer disrupts its basal body localization. Taken together, our studies reveal that the basal body localization of CFAP410 is controlled by the CTD and provide a mechanistic explanation for how the mutation L224P in CFAP410 causes ciliopathies in humans.

biochemistry↗

bioRxiv2023

3D electron microscopy of the Leishmania mexicana cell cycle: Patterns of organelle duplication and segregation and their implications for parasite biology

The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations and sub-pellicular microtubule organisation indicates that is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will be invaluable for future molecular cell biology analyses of these important parasites.

cell biology↗

bioRxiv2023

Discovery of essential kinetoplastid-insect adhesion proteins and their function in Leishmania-sand fly interactions

Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide1. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread2. One of the key life cycle stages is the haptomonad form, which is attached to the insect through its flagellum. This adhesion, which is conserved across kinetoplastid parasites, is implicated to have an important function within their life cycles and hence on disease transmission3-5. Here, we discovered kinetoplastid-insect adhesion proteins (KIAPs), which are localised in the attached haptomonad flagellum. Deletion of these KIAPs impaired cell adhesion in vitro and prevented Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. This result will provide important insights for a comprehensive understanding of the Leishmania life cycle.

microbiology↗

bioRxiv2023

Identification of 30 transition fibre proteins reveals a complex and dynamic structure with essential roles in ciliogenesis

Transition fibres are appendages that surround the distal end of mature basal bodies (also called distal appendages) and are essential for ciliogenesis, but only a small number of proteins have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 additional transition fibre proteins (TFPs) in the flagellated eukaryote Trypanosoma brucei and mapped the arrangement of the molecular components. We discovered TFPs recruited to the basal body pre- and post-initiation of ciliogenesis with differential expression of TFPs at the assembling new flagellum compared to the existing old flagellum. Knockdown by RNAi of 17 TFPs revealed 6 were necessary for ciliogenesis and a further 3 were necessary for normal flagellum length. We identified 9 TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work demonstrates that transition fibres are complex and dynamic in their composition throughout the cell cycle which relates to their essential roles in ciliogenesis and length regulation.

cell biology↗

bioRxiv2023

Unlocking Trypanosome Biology: A Comprehensive Protein-Tagging Toolkit for Localization and Functional Analysis

African trypanosomes are medically important parasites that cause Sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool for investigating protein localization and function. In a previous study, we developed two plasmids for rapid and reproducible protein tagging in trypanosomes, which enabled the localisation of all proteins in the trypanosome cell. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an expanded set of >100 vectors that utilizes universal primer annealing sequences, enabling protein tagging with a range of fluorescent and biochemical tags using five different selection markers. We evaluated the suitability of various fluorescent proteins for live cell imaging and determined their brightness and stability under different fixation conditions. Finally, we determined the optimal fluorescent protein for a set of specific experimental conditions demonstrating the utility of this toolkit.

cell biology↗

bioRxiv2022

Structural studies of cilia and flagella associated protein 410 (CFAP410) reveal its bimodular organization with an N-terminal LRR motif and a C-terminal tetrameric helical bundle

Cilia and flagella associated protein 410 (CFAP410) is a protein localized at the basal body of cilia/flagella and plays essential roles in ciliogenesis. Multiple single amino acid mutations in CFAP410 have been identified in patients. However, the molecular mechanism for how the mutations cause these disorders remains poorly understood due to a lack of high-resolution structures of the protein. Our studies demonstrate that CFAP410 adopts a bimodular architecture. We have previously reported our structural studies on the C-terminal domain (CTD) of CFAP410 from various organisms. Here we report a 1.0-[A] resolution crystal structure of the N-terminal domain (NTD) of Trypanosoma brucei CFAP410. We further examined how the disease-causing mutations in this domain may affect the folding and structural stability of CFAP410. Our results suggest that the single-residue mutations in the CFAP410-NTD cause human diseases by destabilizing the structure that subsequently disrupts its interaction with other partners.

biochemistry↗

bioRxiv2021

Monoallelic antigen expression in trypanosomes requires a stage-specific transcription activator

Monoallelic expression of a single gene family member underpins a molecular "arms race" between many pathogens and their host, through host monoallelic immunoglobulin and pathogen monoallelic antigen expression. In Trypanosoma brucei, a single, abundant, variant surface glycoprotein (VSG) covers the entire surface of the bloodstream parasite1 and monoallelic VSG transcription underpins their archetypal example of antigenic variation. It is vital for pathogenicity, only occurring in mammalian infectious forms1. Transcription of one VSG gene is achieved by RNA polymerase I (Pol I)2 in a singular nuclear structure: the expression site body (ESB)3. How monoallelic expression of the single VSG is achieved is incompletely understood and no specific ESB components are known. Here, using a protein localisation screen in bloodstream parasites, we discovered the first ESB-specific protein: ESB1. It is specific to VSG-expressing life cycle stages where it is necessary for VSG expression, and its overexpression activates inactive VSG promoters. This showed monoallelic VSG transcription requires a stage-specific activator. Furthermore, ESB1 is necessary for Pol I recruitment to the ESB, however transcript processing and inactive VSG gene exclusion ESB sub-domains do not require ESB1. This shows that the cellular solution for monoallelic transcription is a complex factory of functionally distinct and separably assembled sub-domains.

microbiology↗

bioRxiv2020

Role for the flagellum attachment zone in the resolution of cell membrane morphogenesis during Leishmania cell division

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 and 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 morphogenetic resolution of the anterior cell tip during cell division. 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 in membrane organisation at the anterior cell tip, resulting in cells that late in division were connected to each other by a membranous bridge structure between their flagella. These changes had a great impact in vivo with the FAZ2 null mutant unable to develop and proliferate in sand flies and causing 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. Author summaryLeishmania are parasites that cause leishmaniasis in humans with symptoms ranging from mild cutaneous lesions to severe visceral disease. The life cycle of these parasites alternates between the human host and the sand fly vector, with distinct forms in both. These different forms have different cell shapes that are adapted for survival in these different environments. Leishmania parasites have an elongated cell shape with a flagellum extending from one end and this shape is due to a protein skeleton beneath the cell membrane. This skeleton is made up of different units one of which is called the flagellum attachment zone (FAZ), that connects the flagellum to the cell body. We have found that one of the proteins in the FAZ called FAZ2 is important for generating the shape of the cell at the point where the flagellum exits the cell. When we deleted FAZ2 we found that the cell membrane at the tip of the was distorted resulting in unusual connections between the flagella of different cells. We found that the disruption to the cell shape reduces the ability of the parasite to infect mice and develop in the sand fly, which shows the importance of the parasite shape.

cell biology↗

bioRxiv2019

A key regulatory protein for flagellum length control in stable flagella

Cilia and flagella are highly conserved microtubule-based organelles that have important roles in cell motility and sensing [1]. They can be highly dynamic and short lived such as primary cilia or Chlamydomonas [2] or very stable and long lived such as those in spermatozoa [3] photoreceptors [4] or the flagella of many protist cells [3,4]. Although there is a wide variation in length between cell types, there is generally a defined length for a given cell type [1]. Many unicellular flagellated and ciliated organisms have an additional challenge as they must maintain flagella/cilia at a defined length whilst also growing new flagella/cilia in the same cell. It is not currently understood how this is achieved. A grow-and-lock model was proposed for the maintenance of stable flagella where a molecular lock is applied to prevent flagellum length change after assembly [5]. The molecular mechanisms of how this lock operates are unknown, but could be important in cells where an existing flagellum must be maintained whilst a new flagellum assembles. Here we show that Cep164C contributes to the locking mechanism at the base of the flagellum in Trypanosoma brucei. It is only localised on the transition fibres of basal bodies of fully assembled flagella and missing from assembling flagella. In fact, basal bodies only acquire Cep164C in the third cell cycle after they assemble in trypanosomes. Depletion leads to dysregulation of flagellum growth with both longer and shorter flagella; consistent with defects in a flagellum locking mechanism. By controlling delivery of components into the old assembled flagellum, maintenance of stable flagella can occur but limits further growth. This offers an important explanation for how many eukaryotic unicellular cells maintain their existing flagella whilst growing new ones before these cells divide. This work also reveals additional regulatory roles for Cep164 in eukaryotic organisms.

cell biology↗